identifier
stringlengths
11
32
pdf_url
stringlengths
17
4.62k
lang
stringclasses
120 values
error
stringclasses
1 value
title
stringlengths
2
500
source_name
stringlengths
1
435
publication_year
float64
1.9k
2.02k
license
stringclasses
3 values
word_count
int64
0
1.64M
text
stringlengths
1
9.75M
https://openalex.org/W2947448204
https://digital.csic.es/bitstream/10261/240118/1/Cucumber_Montes_PV_Art2019.pdf
English
null
Cucumber mosaic virus infection as a potential selective pressure on Arabidopsis thaliana populations
PLOS pathogens
2,019
cc-by
12,980
Editor: Hui-Shan Guo, Institute of Microbiology, CHINA Editor: Hui-Shan Guo, Institute of Microbiology, CHINA Received: January 17, 2019 Accepted: May 1, 2019 Published: May 28, 2019 Copyright: © 2019 Montes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2019 Montes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was funded by grants BFU2015-64018-R (Plan Estatal de I+D+i, MINECO, Spain) to FGA, and BIO2016-75754-P (Agencia Estatal de Investigacio´n, Spain and FEDER, UE) to CAB. NM was in receipt of a FPI contract (BES- 2009-026698) from MEC, Spain. The funders had no role in study design, data collection and Nuria Montes1¤, Carlos Alonso-BlancoID2, Fernando Garcı´a-ArenalID1* Nuria Montes1¤, Carlos Alonso-BlancoID2, Fernando Garcı´a-ArenalID1* 1 Centro de Biotecnologı´a y Geno´mica de Plantas (UPM-INIA), and E.T.S.I. Agrono´mica, Alimentaria y de Biosistemas, Campus de Montegancedo, Universidad Polite´cnica de Madrid, Pozuelo de Alarco´n (Madrid), Spain, 2 Departamento de Gene´tica Molecular de Plantas, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas (CNB-CSIC), Campus Universidad Auto´noma, Cantoblanco, Madrid, Spain a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: Fisiologı´a Vegetal, Departamento Ciencias Farmace´uticas y de la Salud, Facultad de Farmacia, Universidad San Pablo-CEU, Boadilla del Monte (Madrid), Spain and Servicio de Reumatologı´a, Hospital Universitario de la Princesa, Instituto de Investigacio´n Sanitaria (IIS-IP), Madrid, Spain. * fernando.garciaarenal@upm.es OPEN ACCESS It has been proposed that in wild ecosystems viruses are often plant mutualists, whereas agroecosystems favour pathogenicity. We seek evidence for virus pathogenicity in wild eco- systems through the analysis of plant-virus coevolution, which requires a negative effect of infection on the host fitness. We focus on the interaction between Arabidopsis thaliana and Cucumber mosaic virus (CMV), which is significant in nature. We studied the genetic diver- sity of A. thaliana for two defence traits, resistance and tolerance, to CMV. A set of 185 indi- viduals collected in 76 A. thaliana Iberian wild populations were inoculated with different CMV strains. Resistance was estimated from the level of virus multiplication in infected plants, and tolerance from the effect of infection on host progeny production. Resistance and tolerance to CMV showed substantial genetic variation within and between host popula- tions, and depended on the virus x host genotype interaction, two conditions for coevolution. Resistance and tolerance were co-occurring independent traits that have evolved indepen- dently from related life-history traits involved in adaptation to climate. The comparison of the genetic structure for resistance and tolerance with that for neutral traits (QST/FST analyses) indicated that both defence traits are likely under uniform selection. These results strongly suggest that CMV infection selects for defence on A. thaliana populations, and support plant-virus coevolution. Thus, we propose that CMV infection reduces host fitness under the field conditions of the wild A. thaliana populations studied. Citation: Montes N, Alonso-Blanco C, Garcı´a- Arenal F (2019) Cucumber mosaic virus infection as a potential selective pressure on Arabidopsis thaliana populations. PLoS Pathog 15(5): e1007810. https://doi.org/10.1371/journal. ppat.1007810 RESEARCH ARTICLE Cucumber mosaic virus infection as a potential selective pressure on Arabidopsis thaliana populations RESEARCH ARTICLE Introduction It is commonly accepted that hosts and pathogens coevolve [1]. This concept rests on the assumption that pathogens are virulent parasites, defining virulence as the negative impact of infection on the host fitness. As a consequence, hosts will evolve defences to limit pathogen infection, or to compensate for its costs [2]. In plants, the two major defences against patho- gens are resistance, defined as the ability of the host to limit infection and/or parasite multipli- cation and tolerance, which limits the fitness effect of a given parasite burden, i.e., specifically decreases virulence [3,4]. As host defences may reduce the parasite’s fitness, hosts and parasites may coevolve, coevolution being the process of reciprocally adaptive genetic change in two or more species [1]. Evidence for host pathogen coevolution is not abundant. For plants it derives mostly from studies of agroecosystems in which the pathogen evolves in response to the deployment of resistance in the host population [5]. These studies have provided the bases for theory on host-pathogen coevolution, including the gene-for-gene model of host-pathogen interaction [6,7]. However, coevolution requires certain conditions to be met [1]: i) genetic variation in the relevant host (e.g., resistance, tolerance) and pathogen (e.g., infectivity, viru- lence) traits; ii) reciprocal effects of the relevant traits of the interaction on the fitness of host and pathogen; iii) dependence of the outcome of the host-pathogen interaction on the combi- nation of host and pathogen genotypes involved. These conditions must be analysed in wild systems, in which the host may evolve in response to environmental pressures, including path- ogen infection, at odds with agricultural systems. Evidence for plant-pathogen coevolution from wild pathosystems is limited to a few instances, all involving fungal, oomycete or bacterial pathogens [8,9]. To our knowledge, plant-virus coevolution has not been demonstrated in any wild system. In fact, it has been proposed that viruses often would be mutualistic symbionts, rather than pathogens, in wild plant ecosystems [10–12], and it has been shown that virus infection may be detrimental or positive for the host depending on the environment [13,14]. Hence the inter- est in seeking evidence about whether viruses are plant pathogens in wild ecosystems and viruses and plants co-evolve, or if virus virulence is the result of the specific conditions of agroecosystems. analysis, decision to publish, or preparation of the manuscript. analysis, decision to publish, or preparation of the manuscript. ecosystems and, consequently, plants have evolved defences against virus infection. To test this hypothesis, we studied the genetic diversity of Arabidopsis thaliana for two defence traits, resistance and tolerance, to Cucumber mosaic virus (CMV) at a regional scale in the Iberian Peninsula. Resistance and tolerance to CMV showed substantial genetic variation within and between host populations, and depended on the virus x host genotype interaction, two conditions for coevolution. Resistance and tolerance were inde- pendent traits that co-occurred at the population and regional scales, and that have evolved independently from other adaptive life-history traits. Analyses also indicated that resistance and tolerance are likely under selection, most likely due to virus infection. These results support a hypothesis of plant-virus coevolution and contribute to demon- strate that plant viruses may be virulent parasites of plants in wild ecosystems. Competing interests: The authors have declared that no competing interests exist. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Author summary Plant-virus coevolution has not been demonstrated in any wild system, and it has been proposed that viruses often would be mutualistic symbionts, rather than pathogens, in wild plant ecosystems. We analyse if viruses are virulent pathogens of plants in wild 1 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations analysis, decision to publish, or preparation of the manuscript. Virus infection as a selective pressure on Arabidopsis wild populations (CMV). A. thaliana is an annual semelparous species with two distinct developmental periods: the vegetative growth period, producing a rosette of leaves, and the reproductive period in which the inflorescence grows, new flowers are produced continuously, and older flowers develop into siliques [24]. It is a cosmopolitan species, with a broad native distribution in Eur- asia and Africa [25–27]. The Iberian Peninsula has been shown to contain the largest A. thali- ana diversity in Eurasia due to its colonization from different refugia after the last glaciations [26–29]. In Iberia, A. thaliana occurs in a variety of habitats, and substantial genetic variation has been described, within and among populations, for relevant adaptive traits including phe- nological traits like flowering time and seed dormancy [30–33]. Although wild populations of A. thaliana have been shown to contain ample genetic and phenotypic diversity for responses to herbivores and pathogens [34–38], the diversity for traits related to plant-virus interactions has not been systematically analysed. CMV is an RNA virus with the broadest host range including about 1,200 species in more than 100 plant families. CMV is horizontally transmitted by many species of aphids in a non- persistent manner, and through the seed with efficiencies that depend on the genotypes of CMV and the plant species [39]. In A. thaliana, seed transmission rates vary between 2 and 8% [40, 41]. CMV isolates are highly diverse and have been classified into subgroups IA, IB and II, based on the nucleotide sequence similarity of their genomic RNAs [39,42]. Analyses of the incidence of five viruses in six wild A. thaliana populations from central Spain during 10 years showed that CMV was most prevalent, up to 80% according to popula- tion and year [43,44], indicating that the A. thaliana–CMV interaction is significant in nature. As in other hosts monitored in the Iberian Peninsula, Subgroup IA isolates are most prevalent [44–46]. Our group has analysed the role of resistance and tolerance in this interaction. The infection of 21 wild genotypes of A. thaliana representing the variation of the species in Eur- asia with three CMV strains, showed that quantitative resistance to CMV depended on the interaction between host genotype x virus strain, and was a host trait with moderate to high heritability [47]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Introduction Reports of negative effects of virus infection in wild plants in their natural habitats are not abundant [e.g., [15–22] and indicate that effects may largely depend on site or host population [23], but the genetic variation of defence and virulence has not been analysed in these systems. To analyse plant-virus coevolution in wild ecosystems we have chosen the system Arabidop- sis thaliana L. Heynh. (Brassicaceae)-Cucumber mosaic virus (Cucumovirus, Bromoviridae), PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 2 / 24 Virus infection as a selective pressure on Arabidopsis wild populations PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virulence, estimated as the effect of infection on viable seed production, did not correlate with virus load, due to host genotype x virus strain-specific tolerance and, again, tolerance was a host trait with moderate to high heritability [47]. Interestingly, tolerance was positively correlated with the length of post-embryonic development (life span) of the host genotypes [47], and was due, at least in part, to host life-history trait modification upon infec- tion: long life span genotypes delayed flowering upon infection and re-allocated resources from vegetative growth to reproduction, thus decreasing the effects of infection on progeny production, i.e., attaining tolerance [48]. It remains to be shown that defence polymorphisms result from the selection applied by CMV infection, and not by any other environmental factor known to modulate plant developmental architecture and phenology, such as life span, flower- ing time and plant size, which are known to have a role in adaptation of A. thaliana to the abi- otic environment [49–51]. In this work we study the genetic diversity of A. thaliana for resistance and tolerance to CMV at a regional scale in the Iberian Peninsula. To this end, we exploit a collection of 76 nat- ural populations covering the wide ecological, environmental and genetic diversities of A. thaliana in this region [32,49]. We address the following questions: i) Which is the amount of genetic diversity for resistance and tolerance to different CMV genotypes? ii) Are the geo- graphic and environmental climatic patterns of resistance and tolerance similar or different from those of related adaptive life history traits of A. thaliana? iii) Are resistance and tolerance traits under natural selection? Addressing these questions is crucial to determine if CMV infec- tion has a negative impact on its host fitness under natural field conditions and, consequently, if there is coevolution between A. thaliana and CMV. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 3 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Table 1. Values (mean and range of variation), heritability and autocorrelation, of life history and defence traits to CMV in the Iberian population of A. thaliana. Autocorrelation Traita nb Mean±SEc Min-Maxd h2 b e Moran's I f Distanceg RW 76 0.50±0.04 0.04–1.80 0.96 0.43–0.85 219.31 IW 76 1.83±0.06 0.66–3.48 0.71 0.41–0.89 204.69 SW 76 0.85±0.02 0.31–1.23 0.61 0.23–0.33 153.47 GP 76 117.72±0.80 103.40–140.71 0.92 0.43–0.59 191.75 LP 76 184.26±0.76 170.80–198.00 0.72 ns ns Resistance to Cdc-CMVh 76 19.28±1.37 0.56–56.28 0.81 ns ns Resistance to Lro-CMVh 76 7.53±0.71 0.26–27.83 0.78 ns ns Tolerance to Cdc-CMVi 76 0.35±0.02 0.03–0.77 0.70 ns ns Tolerance to Lro-CMVi 76 0.51±0.02 0.06–0.79 0.54 ns ns ation), heritability and autocorrelation, of life history and defence traits to CMV in the Iberian population of A. thaliana a: Traits are: Rosette weight (RW), inflorescence without seed weight (IW) and viable seed weight (SW), expressed in g, and growth period (GP) and life span (LP), expressed in days. b: number of individuals. c: Mean value and standard error of at least 5 replicated plants. c: Mean value and standard error of at least 5 replicated plants. e: Broad sense heritability expressed as percentage of genetic variation. e: Broad sense heritability expressed as percentage of genetic variation. f: Values of significant values of Moran's index (P<0.001). g: Geographic distance in km, showing significant values of Moran’s index. h: Resistance is expressed as virus accumulation (μg of virus RNA g fresh leaf weight-1). i: Tolerance is expressed as effect of infection on viable seed production (SWi/SWm). ns: non-significant. https://doi.org/10.1371/journal.ppat.1007810.t001 the variance (24.6% for A. thaliana genotype, 38.9% for virus isolate and 24.5% for their inter- action). On average, Cdc-CMV accumulated to higher levels than Lro-CMV (19.28±1.37 and 7.53±0.71 μg virus RNA g fwt-1, respectively) (Fig 2 and Table 1). Virus accumulation in the host plant showed high heritability for both CMV isolates, with an average of 0.8 (Table 1), heritability being defined as the genetic component of the variance of the trait. Tolerance was estimated from the effect of virus infection on progeny production. Since CMV infection does not affect seed viability or the weight of single seeds in a large number of A. Genetic variation for resistance and tolerance to CMV in A. thaliana To estimate the genetic diversity for resistance and tolerance to CMV, 76 A. thaliana wild genotypes collected in different natural populations from the Iberian Peninsula (Fig 1 and S1 Table) were assayed. We consider here a population as the set of A. thaliana plants growing in a specific geographical site. Plants were inoculated after eight-week vernalisation, using two CMV isolates from Iberian A. thaliana populations, Cdc-CMV and Lro-CMV. All 76 geno- types were systemically infected by both CMV isolates, no immunity or hypersensitive resis- tance reaction being detected. Resistance was estimated from the levels of virus multiplication, quantified as virus RNA accumulation. RNA accumulation varied between 0.26 and 56.28 μg of virus RNA g fwt-1 (Table 1, Fig 2 and S2 and S3 Tables), variation significantly depending on the A. thaliana genotype (F75,816 = 2.98, P<10−4), the virus isolate (F1,816 = 113.30, P<10−4) and the interac- tion virus isolate x host genotype (F75,816 = 75.82, P<10−4), which together explained 88% of Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population locations. Circles with asterisk indicate the 12 populations used for within/between population analyses, their names appearing next to them. https://doi.org/10.1371/journal.ppat.1007810.g001 Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population loca populations used for within/between population analyses, their names appearing next to them. Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population locations. Circles with asterisk indicate the 12 populations used for within/between population analyses, their names appearing next to them. Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population locations. Circles with asterisk indicate the 12 populations used for within/between population analyses, their names appearing next to them. https://doi.org/10.1371/journal.ppat.1007810.g001 https://doi.org/10.1371/journal.ppat.1007810.g001 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 4 / 24 thaliana genotypes [14,47,52], tolerance was estimated as the ratio of seed weight in infected to mock-inoculated plants (SWi/SWm), which varied between 0.03 and 0.79 (Table 1, Fig 2, S2 and S3 Tables). As for resistance, tolerance significantly depended on A. thaliana genotype (F76,816 = 3.10, P<10−4), virus isolate (F1,816 = 71.40, P<10−4) and their interaction (F75,816 = 5.18, P<10−4), which together explained 73% of the variance (26.9% for A. thaliana genotype, 25.0% for virus isolate and 21.2% for their interaction). Tolerance to Cdc-CMV (0.35±0.02) was lower than tolerance to Lro-CMV (0.51±0.02) (Fig 2 and Table 1). Tolerance in the host showed medium to high heritability, between 0.70 and 0.54 for Cdc-CMV and Lro-CMV, respectively. No significant relationship was detected between virus RNA accumulation and SWi/SWm across genotypes nor within genotypes (r-0.11, P0.358). Together, results show that natural populations of A. thaliana contain substantial but independent genetic variation for resistance and tolerance to CMV. scence without seed weight (IW) and viable seed weight (SW), expressed in g, and growth period (GP) and life span (LP), https://doi.org/10.1371/journal.ppat.1007810.t001 a: Traits are: Rosette weight (RW), inflorescence without seed weight (IW) and viable seed weight (SW), expressed in g, and growth period (GP) and life span (LP), expressed in days PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Genetic diversity and geographic or climatic patterns of life history traits in A. thaliana Tolerance to CMV in A. thaliana is related to the host life history traits, as tolerance is due in part to a reallocation of resources from growth to reproduction, which depends on the allome- try of the vegetative to reproduction organs, (SW+IW)/RW [48]. In the 76 wild genotypes, resource reallocation upon infection by both CMV isolates also depended on plant allometry, being more efficient in genotypes with lower (SW+IW)/RW (F1,76>14.58, P<10−4), and the effect of infection by both CMV isolates on RW correlated positively with LP and RW of mock-inoculated plants (r0.29, P0.022). Neither resistance nor tolerance correlated with viable seed production of mock-inoculated controls (r0.02, P0.540). We then analysed several life history traits related with growth and phenology in eight-week vernalised mock-inoculated plants of the 76 wild genotypes. Rosette weight (RW), inflorescence without seeds weight (IW) and seed weight (SW), growth period (GP) and life-span (LP) signifi- cantly differed between genotypes (F75,3999.23, P<10−4) (S2 and S3 Tables). Heritability of these traits was high, between 0.61 and 0.96 (Table 1). Overall, A. thaliana populations display a large genetic variation for the analysed life history traits, as in previous works [33,49,50]. g g y y p Rosette weight (RW), inflorescence without seeds weight (IW) and seed weight (SW) signif- icantly differed between genotypes (F75,3999.23, P<10−4) (S2 Table). To determine if life his- tory traits related to tolerance showed similar or different geographic patterns than CMV defences, we analysed their autocorrelation. All growth and phenological traits showed signifi- cant spatial autocorrelation up to 153 km (Table 1). Furthermore, we analysed the relationship between these life history traits and climate using Dutilleul’s t-test, univariate SAR models and Mantel tests (S4 Table). Overall, RW, IW, SW and GP, but not LP, were positively correlated with altitude and negatively correlated with most climatic variables, including annual mean, minimal and maximal temperature, and precipitation seasonality (Fig 3 and S4 Table). These analyses showed A. thaliana genotypes from higher altitude, lower temperatures and higher precipitation seasonality flowered later, developed larger rosettes and inflorescences and pro- duced more seeds. Moreover, Mantel tests showed that genetic distance was positively corre- lated with IW, SW, GP (r>0.15, P<0.008) and marginally with RW (r = 0.08, P = 0.078) but not with LP (r<-0.02, P>0.700). In contrast to CMV defence traits, life history traits related with resource allocation vary according to the climatic environment where populations evolved. Virus infection as a selective pressure on Arabidopsis wild populations Finally, we analysed if there is a relationship between the genetic diversity for CMV defence traits and the overall genetic diversity of the A. thaliana wild genotypes estimated from neutral markers (250 SNPs). Mantel tests between pair-wise genetic distances estimated from neutral markers and pair-wise differences for virus RNA accumulation or SWi/SWm did not detect any significant correlation in relation to Cdc-CMV or Lro-CMV (r-0.04, P0.403). Genetic diversity and geographic or climatic patterns of life history traits in A. thaliana Therefore, resistance and tolerance to CMV show different evolutionary histories than life history traits, likely reflecting distinct abiotic and biotic environmental selective forces acting on each group of traits. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Geographic and climatic patterns of A. thaliana resistance and tolerance to CMV To determine if there is a geographic pattern for the genetic diversity for CMV resistance or tolerance, we first analysed the spatial autocorrelation of both variables. Neither virus 5 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations accumulation nor SWi/SWm showed significant spatial autocorrelation at any geographic scale Fig 2. Variation for resistance and tolerance to CMV in A. thaliana. Frequency distributions are for accumulation (μg RNA g fresh leaf weight-1) of (A) Cdc-CMV and (B) Lro-CMV RNA, and of the effect of infection by (C) Cdc-CMV and (D) to Lro-CMV on seed production. https://doi.org/10.1371/journal.ppat.1007810.g002 Fig 2. Variation for resistance and tolerance to CMV in A. thaliana. Frequency distributions are for accumulation (μg RNA g fresh leaf weight-1) of (A) Cdc-CMV and (B) Lro-CMV RNA, and of the effect of infection by (C) Cdc-CMV and (D) to Lro-CMV on seed production. Fig 2. Variation for resistance and tolerance to CMV in A. thaliana. Frequency distributions are for accumulation (μg weight-1) of (A) Cdc-CMV and (B) Lro-CMV RNA, and of the effect of infection by (C) Cdc-CMV and (D) to Lro-CMV accumulation nor SWi/SWm showed significant spatial autocorrelation at any geographic scale (P>0.050) (Table 1). Second, to find if these defence traits might be associated with the climate, we analysed their relationship with climatic variables from the original local populations. Neither the accu- mulation of any of the two CMV isolates, nor the SWi/SWm ratio in plants infected by any of them, significantly correlated with any of the analysed abiotic variables (see Methods and S4 Table) according to Dutilleul’s t-tests (r-0.30, P0.039), SARs (F7.185, P0.009), or Man- tel tests (r-0.12, P0.019) (S4 Table). 6 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 A. thaliana population differentiation for resistance and tolerance to CMV To quantify the distribution of genetic diversity for CMV defence traits within and among A. thaliana populations we analysed ten randomly sampled individual plants (henceforth named as “individuals”) from 10 or 12 Iberian populations, which were tested for their resistance and tolerance to two CMV isolates. One isolate from an Iberian population of A. thaliana (Cdc- CMV) and a reference isolate (Fny-CMV) were chosen because they had been used in previous work [43,47,48]. Since CMV resistance and tolerance depend on the environment [14,48,53], PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 7 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Virus infection as a selective pressure on Arabidopsis wild populations Fig 3. Relationships between life-history traits and geographic or climatic factors. Correlations are shown (A, B, C) for rosette weight (RW), (D, E, F) inflorescence weight (IW), (G, H, I) seed weight (SW) and (J, K, L) growth period (GP) with altitude, annual mean temperature and precipitation seasonality. Values are means of at least five replicates per plant genotype. Fig 3. Relationships between life-history traits and geographic or climatic factors. Correlations are shown (A, B, C) for rosette weight (RW), (D, E, F) inflorescence weight (IW), (G, H, I) seed weight (SW) and (J, K, L) growth period (GP) with altitude, annual mean temperature and precipitation seasonality. Values are means of at least five replicates per plant genotype. Fig 3. Relationships between life-history traits and geographic or climatic factors. Correlations are shown (A, B, C) for rosette weight (RW), (D, E, F) inflorescence weight (IW), (G, H, I) seed weight (SW) and (J, K, L) growth period (GP) with altitude, annual mean temperature and precipitation seasonality. Values are means of at least five replicates per plant genotype. https://doi.org/10.1371/journal.ppat.1007810.g003 two experiments were performed with different vernalisation period lengths, as vernalisation affects life history traits relevant to tolerance such as rosette size, rosette leaf number, flowering time [33,49,54], and seed germination [55]. An eight-week vernalisation treatment simulated a cold winter, whereas a four-week vernalisation simulated a mild winter, as often occur across years in the original population locations. In both experiments, all individuals were systemi- cally infected by both CMV isolates. The two experiments yielded similar results. For clarity only results of the long vernalisation treatment experiment are presented in the text, but results of the short-vernalisation are shown in S5 Table. Virus accumulation varied considerably among individuals within populations (Fig 4, Table 2 and S6 Table). The average virus accumulation in each population ranged from 2.62 to 32.02 μg virus RNA g fwt-1. The heritability of virus accumulation varied between 0.23 and 0.90 depending on CMV isolate and host population (Table 2). Virus accumulation significantly depended on the virus isolate (F1,884 = 67.88, P<104), the A. thaliana population (F9,884 = 2.40, P = 0.059) (Fig 4 and Table 2), the A. thaliana individual nested to population (F88,884 = 2.54, P<10-4), and on the interactions CMV isolate x A. thaliana population (F9,884 = 4.11, P<10-4) and CMV isolate x A. thaliana individual nested to population (F88,884 = 7.08, P<10-4). CMV isolate, A. thaliana individual nested to population and their interaction explained 49.7, 3.3 and 14.6% of the variance, respectively, while A. thaliana population and the interaction CMV iso- late x population explained 7.0 and 5.5%. The average accumulation was higher for Cdc-CMV than for Fny-CMV (18.61±2.76 and 7.37±1.74 μg virus RNA g fwt-1, respectively). Besides, aver- age values of Cdc-CMV accumulation over individuals and populations correlated significantly, or marginally, with the corresponding values of Fny-CMV accumulation (rs = 0.32, P = 0.001; rs = 0.60, P = 0.067, respectively), indicating that, in general, individuals and populations that were more resistant to Cdc-CMV were also more resistant to Fny-CMV. CMV tolerance also showed substantial variation among individuals within populations, SWi/SWm values ranging between 0.02 and 0.93 for Cdc-CMV, and between 0.03 and 0.81, for Fny-CMV-infected plants (Fig 4, Table 2 and S6 Table). Average SWi/SWm values in each pop- ulation varied from 0.16 to 0.47 (Fig 4 and Table 2), indicating a lower range of variation among than within populations. Heritability of tolerance varied between 0.10 and 0.80 depending on CMV isolate and host population (Table 2). SWi/SWm varied significantly depending on virus isolate (F1,884 = 5.30, P = 0.046), A. thaliana population (F9,884 = 2.40, P = 0.059) (Fig 4 and Table 2), A. thaliana individual nested within population (F88,884 = 2.445, P = 0.023) and the interaction CMV isolate x A. thaliana individual (F88,884 = 2.65, P<10−4). A. thaliana individual and the interaction CMV isolate x A. thaliana individual explained a larger proportion of SWi/SWm variance (32.2%, 16.1%, respectively) than CMV isolate or A. thaliana population (1.6%, 6.4%, respectively). When averaged over all individuals, tolerance to Cdc- CMV (0.36±0.06) and Fny-CMV (0.32±0.06) were similar. Average values of SWi/SWm in Cdc-CMV-infected plants correlated across individuals and populations with those of Fny- CMV-infected plants (rs = 0.73, P = 0.016; rs = 0.60, P<10−4, respectively). Thus, as for resis- tance, the individuals and populations more tolerant to Cdc-CMV were also, in general, more tolerant to Fny-CMV. However, values of virus accumulation and SWi/SWm did not correlate over individuals for any CMV isolate (rs0.09, P0.129). Together, these results indicate that A. thaliana defences against CMV infection depend on the host genetic variation determining the specific defences. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations Virus infection as a selective pressure on Arabidopsis wild populations PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 8 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 9 / 24 Virus infection as a selective pressure on Arabidopsis wild populations PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 10 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 10 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Fig 4. Variation for resistance and tolerance to CMV within and among wild A. thaliana populations. Frequency distributions of resistance (virus accumulation, μg RNA g fresh leaf weight-1) and tolerance (SWi/SWm) to Cdc-CMV (blue) and Fny-CMV (red). Three-letter code for each population is as in Fig 1. Fig 4. Variation for resistance and tolerance to CMV within and among wild A. thaliana populations. Frequency distributions of resistance (virus accumulation, μg RNA g fresh leaf weight-1) and tolerance (SWi/SWm) to Cdc-CMV (blue) and Fny-CMV (red). Three-letter code for each population is as in Fig 1. https://doi.org/10.1371/journal.ppat.1007810.g004 https://doi.org/10.1371/journal.ppat.1007810.g004 Comparison of genetic differentiation among A. thaliana populations between quantitative traits and neutrals markers To find out if CMV defence traits of A. thaliana might be under selection, we compared the genetic differentiation among populations for CMV resistance and tolerance, with that for neutral genetic variation (Fig 5). Two-hundred and fifty genome-wide SNPs, distinguished 74 different genotypes among the 120 individuals analysed (S7 Table) and were used to calculate FST values. As in the previous section, analyses are based on the results of the long vernalisation treatment experiment, analyses based on the short-vernalisation treatments gave similar results and are shown in S5 Table. The estimated average genetic differentiation among populations for neutral markers was 0.58 (95%CI = 0.54–0.62), which is presumed to reflect the demographic history of the popula- tions. Genetic differentiation among populations for quantitative traits was estimated by QST values, leading to a similar average value of 0.31 (95%CI = 0.22–0.52) for accumulation of both CMV isolates. The average QST estimated for SWi/SWm was 0.18 (95%CI = 0.12–0.35) for Cdc- CMV and 0.10 (95%CI = 0.06–0.21) for Fny-CMV-infected plants. Therefore, the genetic vari- ation of A. thaliana for both defence traits is distributed mostly within populations. QST values for resistance and tolerance to Cdc-CMV and Fny-CMV were significantly smaller than FST values (Fig 5), thus indicating that A. thaliana populations are genetically less differentiated for resistance and tolerance to two CMV isolates than for neutral markers. Furthermore, we analysed if FST and QST values followed a pattern of isolation-by-distance. Mantel tests detected a significant correlation between FST values and geographic distance between pairs of populations (r = 0.50, P<10−4), a likely result of their demographic history. By contrast, no significant correlation was found between the pair-wise QST values for virus accumulation or SWi/SWm, for any CMV isolate, and their geographic distances (r0.24, P0.258). Therefore, factors other than demography contribute to the population differentia- tion patterns observed for resistance and tolerance to CMV. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Discussion Host-pathogen coevolution determines the dynamics and genetics of infectious disease, and may shape the genetic structure of host and pathogen populations [1]. Understanding this topic, central in pathology and evolutionary biology, requires knowledge on the genetics of defence and pathogenicity, and the dynamics of their change in populations [1]. The abun- dance of theoretical analyses of host-pathogen coevolution (e.g., [1,56,57]) is not matched by a similar amount of empirical and experimental studies. While there is abundant information compatible with host-pathogen coevolution in plant systems (e.g. [58]), it mostly derives from crops, in which the genetic composition of the host plant is manipulated by humans. Studies from wild systems, in which the genetic composition of host and pathogen populations may be determined by reciprocal selection, are much scarcer, particularly for plant-virus interactions [5,59]. To address this question we challenged A. thaliana individuals collected from a high number of local populations with different CMV isolates. We chose to inoculate plants mechanically rather than by aphid transmission, which is the natural means of horizontal transmission [39]. Mechanical inoculation ensures a high rate of infection and minimises inoc- ulum dose effects on virus accumulation, as opposed to aphid transmission, which is highly PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 11 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations Table 2. Values (mean and range of variation) and heritability of defence traits to CMV in Iberian populations of A. thalianaa. Population Resistance to Cdc-CMVb Resistance to Fny-CMVb Tolerance to Cdc-CMVc Tolerance to Fny-CMVc Agu Mean±SEd 32.02±3.64 11.85±2.84 0.43±0.07 0.36±0.07 Min-Maxe 7.98–45.64 2.16–26.11 0.14–0.78 0.08–0.74 nf 10 10 10 10 h2 b g 0.74 0.90 0.49 0.44 Bis Mean±SEd 17.05±2.31 10.91±1.11 0.34±0.04 0.37±0.07 Min-Maxe 9.37–30.04 3.31–14.14 0.12–0.48 0.03–0.71 nf 10 10 10 10 h2 b g 0.74 0.58 0.38 0.8 Gra Mean±SEd 15.77±1.68 6.24±1.15 0.40±0.07 0.26±0.05 Min-Maxe 6.69–24.45 2.42–13.97 0.12–0.84 0.04–0.54 nf 10 10 10 10 h2 b g 0.66 0.68 0.64 0.741 Leo Mean±SEd 22.36±1.90 9.48±1.40 0.32±0.07 0.22±0.05 Min-Maxe 15.45–32.05 3.59–16.29 0.05–0.74 0.03–0.46 nf 10 10 10 10 h2 b g 0.62 0.79 0.61 0.77 Mer Mean±SEd 17.58±2.56 11.55±2.35 0.16±0.04 0.19±0.06 Min-Maxe 5.37–36.76 3.89–24.15 0.04–0.46 0.05–0.67 nf 10 10 10 10 h2 b g 0.79 0.89 0.32 0.67 Moc Mean±SEd 15.49±2.25 5.15±0.43 0.35±0.04 0.32±0.05 Min-Maxe 4.08–24.43 2.92–7.07 0.13–0.49 0.12–0.57 nf 10 10 10 10 h2 b g 0.71 0.34 0.27 0.47 Pob Mean±SEd 14.91±1.71 2.62±0.60 0.36±0.07 0.33±0.05 Min-Maxe 5.33–22.55 0.94–7.01 0.02–0.59 0.07–0.57 nf 9 9 9 9 h2 b g 0.52 0.23 0.74 0.10 Pra Mean±SEd 15.04±2.15 4.78±0.93 0.47±0.09 0.41±0.07 Min-Maxe 5.19–24.98 1.99–10.26 0.06–0.93 0.04–0.68 nf 9 9 9 9 h2 b g 0.635 0.63 0.55 0.66 Qui Mean±SEd 15.23±2.34 6.21±0.78 0.47±0.06 0.46±0.06 Min-Maxe 6.26–27.61 3.87–11.85 0.20–0.69 0.24–0.81 nf 10 10 10 10 h2 b g 0.68 0.34 0.64 0.52 San Mean±SEd 20.00±2.48 4.16±1.46 0.36±0.06 0.32±0.05 Min-Maxe 11.38–38.89 0.08–12.18 0.11–0.66 0.10–0.57 nf 10 10 10 10 h2 b g 0.69 0.81 0.47 0.32 Average of populations Mean±SE 18.53±1.65 7.25±0.30 0.37±1.09 0.31±0.02 Min-Max 14.91–32.02 2.62–11.85 0.16–0.47 0.22–0.46 h2 b g 0.60 0.62 0.51 0.55 (Continued) Values (mean and range of variation) and heritability of defence traits to CMV in Iberian populations of A. thalianaa. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 12 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Table 2. (Continued) Population Resistance to Cdc-CMVb Resistance to Fny-CMVb Tolerance to Cdc-CMVc Tolerance to Fny-CMVc Over populations h2 b g 0.77 0.85 0.57 0.55 a: Values are for plants inoculated after an eight-week vernalisation period. b: Resistance is expressed as virus accumulation (μg of virus RNA g fresh leaf weight-1). c: Tolerance is expressed as effect of infection on viable seed production (SWi/SWm). d: Mean ± SE: Mean value and standard error (SE) of at least 5 replicated plants. e: Minimum and maximum values of the trait. f: number of individuals g: Broad sense heritability expressed as percentage of genetic variation. https://doi.org/10.1371/journal.ppat.1007810.t002 inefficient for CMV [39, 40]. Moreover, there is no information on the aphid species that transmit CMV in A. thaliana populations in central Spain. Also, A. thaliana genotypes were assayed under common controlled conditions, which are not necessarily the same as in the field. The challenge with different CMV strains of 185 individuals collected in 76 A. thaliana local populations from the Iberian Peninsula, showed large differences in quantitative resis- tance, as estimated from the level of virus multiplication in infected plants. Similarly, large dif- ferences were found for tolerance to CMV, estimated as the effect of infection on host progeny production. A large part of the variation of resistance and tolerance was explained by the virus isolate, in agreement with previous results showing that CMV isolates vary largely in multipli- cation rate and virulence in A. thaliana [41,47,48,53]. Variation for resistance and tolerance Fig 5. Genetic differentiation among wild A. thaliana populations for CMV resistance and tolerance traits, or for neutral markers. Fig shows values of quantitative genetic differentiation (QST) for resistance (squares) and tolerance (triangles), to Cdc-CMV (blue) or Fny-CMV (red), and for neutral genetic differentiation (FST) (black circle), among ten A. thaliana populations. 95%CI are indicated. https://doi.org/10.1371/journal.ppat.1007810.g005 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations occurred at all analysed spatial scales: among individuals from a local population, among local populations, and within the whole Iberian Peninsula region. These conclusions held for assays conducted in different environments, a result to be underscored, as resistance and tolerance of A. thaliana to CMV can be modulated by the abiotic environment [14]. The observed variation in resistance and tolerance had a significant genetic component, as they showed medium to high heritability values (0.23–0.81 for resistance, and 0.10–0.70 for tolerance) depending on the spatial scale of the analysis and the isolate of CMV. Thus, our results show genetic variation for presumably defence traits in the host population, a condition for host-pathogen coevolu- tion. Regardless of spatial scales, our data also show that resistance and tolerance significantly depend on the interactions between virus genotype and host genotype, another condition for host-pathogen coevolution. It has been reported that A. thaliana genotypes showing high tolerance to CMV have a long life span, and that tolerance is, at least in part, the result of resource re-allocation from vegeta- tive growth to reproduction, which is more efficient in long-lived genotypes [14,47,48,52]. It also has been shown that these life history and phenological traits have evolved as (direct or indirect) responses to climatic conditions [33,49,50,60]. Accordingly, the analysis of 76 A. thaliana genotypes from different Iberian populations showed that resource reallocation upon infection depended on the allometric ratio (SW+IW)/RW, and the effect of CMV infection on vegetative growth correlated positively with LP and RW of mock-inoculated plants. Life span, vegetative growth and seed production in non-infected plants were correlated with climatic variables. In contrast, the genetic variation for CMV multiplication in the infected host, and for the effect of CMV infection on seed production, was unrelated to those climatic factors. Therefore, the CMV defence traits have evolved, at least partly, independently from those other adaptive traits which have evolved in response to climate. Accordingly, the evolution of defence traits is not the result of A. thaliana responses to climate. These differential evolution- ary histories strongly suggest that these traits are true resistance and tolerance defence responses that may have evolved in response to CMV infection (see below). This conclusion also agrees with the fact that resistance and tolerance are virus-specific traits of A. thaliana, and not unspecific responses to the stress of virus infection [52]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Fig 5. Genetic differentiation among wild A. thaliana populations for CMV resistance and tolerance traits, or for neutral markers. Fig shows values of quantitative genetic differentiation (QST) for resistance (squares) and tolerance (triangles), to Cdc-CMV (blue) or Fny-CMV (red), and for neutral genetic differentiation (FST) (black circle), among ten A. thaliana populations. 95%CI are indicated. Fig 5. Genetic differentiation among wild A. thaliana populations for CMV resistance and tolerance traits, or for neutral markers. Fig shows values of quantitative genetic differentiation (QST) for resistance (squares) and tolerance (triangles), to Cdc-CMV (blue) or Fny-CMV (red), and for neutral genetic differentiation (FST) (black circle), among ten A. thaliana populations. 95%CI are indicated. https://doi.org/10.1371/journal.ppat.1007810.g005 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 13 / 24 Virus infection as a selective pressure on Arabidopsis wild populations CMV defence traits is that CMV infection plays a role as selective factor. Although we cannot discard that the analysed defence traits may have evolved in response to selection from other pathogens or pests, several arguments make a strong case for selection pressure due to CMV infection: i) the high prevalence of CMV (up to 80%) in wild A. thaliana populations in the Ibe- rian peninsula [43,44]; ii) the fact that the analysed defence traits are virus-specific in A. thaliana [52]; and iii) the lack of correlation between CMV multiplication and virulence [47], virus mul- tiplication being highly dependent on virus genotype and environment [14,47]. Selection for resistance to pathogens has been best documented in populations of Linum marginale in response to the fungus Melampsora lini, of Plantago lanceolata in response to the fungus Podo- sphaera plantaginis, or of A. thaliana in response to the oomycete Hyaloperonospora arabidopsi- dis or the bacterium Pseudomonas syringae [37,68,69,72–74,78,82,83]. Our results extend these observations to plant-virus interactions. If defence in A. thaliana against CMV is under selec- tion, a corollary is that CMV is a virulent pathogen of this plant under natural conditions, a rele- vant conclusion that contributes significantly to understanding plant-virus interactions. Th b d f d f d l h h g y g p The observed pattern of genetic diversity for resistance and tolerance to CMV, higher within than between populations, can be explained by the features of the pathosystem. CMV is ubiquitous in Iberia [45,84] and has been found in all monitored wild populations of A. thali- ana, prevalence differing among populations and years [43]. Also, our present and past results [47,48] show that CMV isolates differ in virulence to A. thaliana, and that virulence is modu- lated by environmental factors as diverse as temperature, light intensity or host plant density [14,53].Variation in the genetic composition of CMV populations would also result in varia- tion for CMV infection-associated selection, as the outcome of the interaction depends on the A. thaliana and CMV genotypes involved. These factors would explain the maintenance of genetic variation in defence traits within host populations and the limited differentiation among populations. Furthermore, this explanation suggests that resistance and tolerance to CMV involve fitness penalties for A. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Thus, our study shows sub- stantial genetic variation for resistance and tolerance to CMV within and between populations of A. thaliana. Genetic variation within or/and between populations for resistance to a variety of pathogens has been reported for a limited number of wild plants, including Amphicarpaea bracteata, Eucalyptus globulus, Podophyllum peltatum, Linum marginale, Silene latifolia, Pha- seolus vulgaris, Plantago laceolata or A. thaliana [61–78]. Analyses of the variation for plant tolerance to pathogens are much rarer [47,75,79], and none of them has analysed within popu- lation variation. All these studies refer to resistance or tolerance to fungi, oomycetes or bacte- ria, and the only report we are aware of on variation for resistance to a virus, is our previous analysis in the A. thaliana-CMV system [43], which involved a much more limited sample of host populations. The analyses in this study also showed that resistance and tolerance display different evolu- tionary histories than neutral genetic variation, since QST values for resistance and tolerance are lower than FST values. Accordingly, resistance and tolerance are traits likely under uniform selection, i.e., a selection which favours a higher diversity of traits within than among popula- tions. This conclusion is supported by results for two CMV isolates and in different environ- mental conditions. Also, neutral genetic differentiation follows a pattern of isolation by distance, which is not detected for the genetic differentiation for resistance or tolerance. By contrast, similar analyses have previously suggested diversifying selection on the genetic varia- tion for quantitative traits such as flowering time, leaf number, specific leaf area or leaf succu- lence in A. thaliana [32,80,81]. The most parsimonious explanation for uniform selection on PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 14 / 24 Plant material and environmental data Two different sets of A. thaliana samples from the Iberian Peninsula were analysed. First, 76 accessions or wild genotypes collected from different populations were selected to cover the genetic and environmental diversity of the species in that region [30,49] (Fig 1 and S1 Table). This collection spanned 800 x 700 km, populations being spaced in the average 384.9±3.7 (20.2–1,038.1 km). Altitudes ranged from 123 to 1,670 m above sea level. Each sample was genetically different based on previous SNP genotyping and genome sequences [51,96]. Sec- ond, ten individuals plants (individuals) randomly sampled from 12 of these populations were selected for intra and inter-population analyses (Fig 1 and S1 Table). Samples from eight of these populations have been previously genotyped for 250 genome-wide SNPs that were segre- gating in these populations [30,32]. For the remaining four populations (Bis, Mer, Moc, Pob) 10 individuals/population were genotyped in this study for the same set of SNPs. The climatic information from the locations of A. thaliana populations was obtained from the digital climatic atlas of the Iberian Peninsula at 1 km2 resolution [50,97]. Thirty-three vari- ables were used, related to temperature, precipitation and solar radiation (S3 Table). In addi- tion, 19 bioclimatic variables derived by combination of annual trends, seasonality and extreme conditions were also included (www.worldclim.org). Altitude was also analysed as a proxy for climate. Annual mean temperature of the populations ranged 6.1–17.4˚C (12.5±0.3) and annual precipitation ranged 405.7–1695.8 mm (753.8±33.9) (S8 Table). All accessions or individuals used in this study were propagated by selfing during two gen- erations by the single seed descent procedure, in a glasshouse supplemented with lamps to pro- vide a long-day photoperiod. This allowed reducing residual heterozygosity that might contain some wild individuals but also removing any potential maternal and grand-mother effects. Seeds were stratified (darkness, 4ºC) for 7 days before germination at 25/20ºC day/night, 16 h light. Ten day-old seedlings were transferred to 4ºC, 8 h light, for vernalisation during 4 or 8 weeks, depending on the experiment. After vernalisation, plants were transplanted to 0.43 L pots and returned to the greenhouse, where they were kept (25/20ºC day/night, 16 h light) until the end of the experiment. Virus infection as a selective pressure on Arabidopsis wild populations with variation for climatic factors, in contrast to variation for other adaptive life-history traits of A. thaliana. In addition, we found evidence that these two defence traits are under uniform selection. The results of this study are compatible with CMV infection acting as a selection pressure for defence on populations of A. thaliana and, hence, we propose that CMV infection likely reduces the host fitness under the field conditions of the analysed wild A. thaliana popu- lations, although field experiments would be required to prove this fact. The results presented here also show that some of the conditions for coevolution are met in the system A. thaliana- CMV, but more work on the virus side is necessary to prove if coevolution occurs. These results raise two challenging questions: what are the mechanisms that maintain polymor- phisms for resistance and tolerance within A. thaliana populations, and what is the negative impact of CMV infection on the host in nature and how does such an impact vary according to field conditions. thaliana, which would hinder fixation of resistance/toler- ance alleles and would contribute to the maintenance of defence polymorphisms within popu- lations [85]. We have not found evidence for such costs under the assayed conditions, as neither resistance nor tolerance were negatively correlated with viable seed production of mock-inoculated controls. However, fitness costs might not be detectable under our experi- mental conditions, and/or might be unveiled under the less favourable environment of the field. Another interesting result is that resistance and tolerance to CMV co-occur in wild A. thali- ana populations. Theory predicts that resources being limited, hosts would not invest in both resistance and tolerance, which would be mutually exclusive defences. The conditions that should favour the evolution of resistance or tolerance have been much modelled, and a nega- tive correlation between both traits across host genotypes is expected [4,86,87]. We found no correlation between resistance and tolerance in any experiment, indicating that they evolve as independent traits. It has been proposed that resistance and tolerance could coexist if costs and benefits of each defence were different and non-additive [88–91]. Models also propose that tolerance alleles should become fixed in host populations, which would not be polymor- phic for this trait under most assumptions [91–93]. A report on the tolerance to CMV in Mimulus guttatus conforms to these predictions, as tolerance had no costs, but showed little genetic variation [94,95]. On the contrary, our results do not agree with model predictions, as we found large genetic variation for tolerance and evidence for coexistence of resistance and tolerance. In conclusion, this work shows that in A. thaliana there is genetic variation, within and among populations, for defences to CMV that result in lower virus multiplication or in lower impact of infection on the plant fitness. Genetic variation for defence to CMV is not associated PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 15 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Quantification of CMV multiplication Virus multiplication in plants was estimated from virus RNA accumulation as described in Paga´n et al., (2014) [41]. Briefly, at fifteen days post-inoculation 0.01 g fresh weight (fwt) of leaf tissue was harvested from four different systemically infected leaves. Nucleic acids were extracted from the pooled leaf tissue using TRI-reagent (Sigma-Aldrich, St Louis, MO, USA). Virus RNA was then quantified by dot-blot hybridization with 32P-labelled RNA probes com- plementary to nucleotides 1933–2215 of Fny-CMV RNA3 (GeneBank Acc. No. D10538). In each blot, internal standards for Fny-CMV, Cdc-CMV or Lro-CMV RNA were included as a two-fold dilution series (1–0.001 μg) of purified virion RNA in nucleic acid extracts from non- inoculated plants. Mock-inoculated samples served as negative controls. Nucleic acid extracts were blotted at different dilutions to ensure that hybridization signal was on the linear portion of the RNA concentration-hybridization curve. As loading controls, parallel membranes were hybridized with a cDNA probe of β-tubulin chain 2 (TUB2) mRNA of A. thaliana (1086–1568 nt, GeneBank Acc. No. NM_125664.4). Virus infection as a selective pressure on Arabidopsis wild populations named At-CMV in [43]. Isolates were multiplied in Nicotiana clevelandii, Fny-CMV from transcripts of cDNA clones and Cdc-CMV and Lro-CMV from biological clones derived from local lesions in Chenopodium quinoa. Virions were purified as in [99]. A. thaliana plants were mechanically inoculated at the five-leaf stage (stage 1.05, [100]) with 15 μl of sap from infected N. clevelandii leaves in 0.01 M phosphate buffer pH 7.0, 0.2% sodium diethyldithiocarbamate. Fifteen μl of buffer were applied to mock-inoculated controls. The (unkown) virus concentra- tion in leaf sap ensured infection of 100% of inoculated plants. Each treatment (virus-inocu- lated or buffer mock-inoculated) involved at least five replicated plants from each original sample, that is at least five plants derived from the same genotype or individual. All plants in each experiment were grown in a completely randomized design. Quantification of life-history traits and tolerance to CMV Rosette weight was used to estimate vegetative growth effort, inflorescence plus seed weight to estimate total reproductive effort, and seed weight to estimate progeny production [101]. Pre- vious work has shown that CMV infection does not affect seed viability, nor the weight of indi- vidual seeds, in a broad range of A. thaliana genotypes [14,47,52]. Plants were harvested at complete senescence and dry weight of rosettes (rosette weight, RW), inflorescence structures without seeds (inflorescence weight, IW) and seeds (seed weight, SW) were measured sepa- rately (g). Two phenological parameters of A. thaliana life cycle were quantified: Growth period (GP) and life-span (LP) were measured as the time (days) between planting seedlings in soil and opening of the first flower (GP), or complete senescence (LP). Tolerance was mea- sured by the effect of virus infection on progeny production: SWi/SWm, where i and m denote infected and mock inoculated plants, respectively [48]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus isolates and inoculations Three subgroup IA CMV isolates were used, Fny-CMV, Cdc-CMV and Lro-CMV, which dif- fer in the sequence of their genomic RNAs in about 1% of positions. Fny-CMV is a well-char- acterized reference isolate [98]. Cdc-CMV and Lro-CMV were isolated from field-infected A. thaliana plants of the Cdc and Cho populations, respectively, in 2008 and 2011, Cdc-CMV was PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 16 / 24 Virus infection as a selective pressure on Arabidopsis wild populations genotype (nested within populations) as random factors. The 95% confidence intervals (95% CI) for QST values were estimated as P S2ðn1Þ w2 n1  s2  S2ðn1Þ w2 n1 h i ¼ 0:95 [106]. Genetic differentia- tion for neutral markers was estimated as FST [107] using the analysis of molecular variance (AMOVA) as implemented in ARLEQUIN v3.5.1.2 [108]. AMOVA were performed using multilocus genotypes for 250 segregating SNPs [30,32] and their significances were estimated from 1,000 permutations. The relationships between Euclidean geographical distance and FST or QST values among population pairs were determined by Mantel correlation test using PASSaGE v.2 [109] with 1,000 permutations. Genetic distances between individuals were calculated as the proportion of allelic differences over the total number of alleles in the corresponding set of polymorphic loci, using GGT v. 2.0 [110]. Statistical analyses Differences in RW, IW, SW, GP, LP, virus accumulation or tolerance to CMV, according to host individual/genotype and virus isolate, were analysed by general linear models (GLM) con- sidering host individual/genotype as a random factor, and virus isolate as a fixed factor. Differ- ences in RW, IW, SW, GP, LP, virus accumulation or tolerance to CMV according to population, host individual/genotype, and virus isolate, were analysed by GLM considering iso- late as a fixed factor, and population and individual/genotype nested to population, as random factors. Relationships between values of different traits were tested using Spearman’s correlation test. GLMs and Spearman’s correlation tests were performed using SPSS 20 software package. Spatial autocorrelation patterns of environmental variables, life-history traits, virus accu- mulation and tolerance to virus, were analysed using correlograms [111] generated with PAS- SaGE v.2. For each variable, Moran’s I autocorrelation coefficients [112] were calculated and their significance tested from 1,000 permutations. Correlation between pairs of environmental variables, between pairs of different traits and between environmental variable and different traits were tested with Dutilleul’s modified t-test using SAM v.4 [113,114]. Simultaneous auto- regressive models (SAR) [115] were performed to test the relationship between environmental variables and different traits using SAM v.4. Bonferroni correction was applied for multiple comparisons. The relationships between environmental variables and life-history or defence traits were analysed by partial Mantel tests controlling for the location of populations given by the geo- graphic distance matrix using PASSaGE v.2. For that, matrices of euclidean distances were derived for each environmental variable and phenotypic trait and significance was evaluated from 1,000 permutations. Genetic analyses Broad sense heritability of each trait was estimated as h2 b = VG/(VG+VE), where VG is the among-genotypes or among-populations variance component and VE is the residual variance. Variance components were determined using the REML method [102] of SPSS 20 package (SPSS Inc., Chicago, USA). Genetic differentiation between populations for quantitative traits was measured by QST values [103], estimated as VB/(VB+VW) [104,105], where VB is the between-population vari- ance and VW is the within-population variance. VB and VW were estimated by the REML method from a nested analysis of variance performed using population and individual or PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 17 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Acknowledgments Antolı´n Lo´pez Quiro´s and Miguel A´ngel Mora provided excellent technical support. Author Contributions Conceptualization: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Conceptualization: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Formal analysis: Nuria Montes. Funding acquisition: Fernando Garcı´a-Arenal. Funding acquisition: Fernando Garcı´a-Arenal. Investigation: Nuria Montes, Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Writing – original draft: Nuria Montes. Investigation: Nuria Montes, Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Writing – original draft: Nuria Montes. Writing – review & editing: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Writing – review & editing: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Virus infection as a selective pressure on Arabidopsis wild populations S4 Table. Coefficients of the correlation/regression between values of mock-inoculated A. thaliana life-history or defence to CMV traits, and those of the environmental variables of the corresponding population obtained by SARs, Correlation with Dutilleul´s correction, and Mantel test. A threshold significance value (α = 0.0014) was set after applying Bonferro- ni’s correction (1– [1– a] 1/n) for multiple comparisons. (XLSX) S5 Table. Mean, minimum and maximum values of resistance and tolerance to CMV of A. thaliana individuals inoculated after a four-week vernalisation period. Mean, minimum and maximum values, heritability and QST of resistance and tolerance to CMV for twelve A. thaliana populations are also shown. (XLSX) S6 Table. Mean values of resistance and tolerance to CMV for A. thaliana individuals inoc- ulated after an eight-week vernalisation period. (XLSX) S6 Table. Mean values of resistance and tolerance to CMV for A. thaliana individuals inoc- ulated after an eight-week vernalisation period. (XLSX) ulated after an eight-week vernalisation period. (XLSX) S7 Table. Distribution of genotypes (H) and individuals (lower case three letter codes) in 12 analysed A. thaliana populations. (XLSX) S8 Table. Values (mean and range of variation) of the environmental variables used in the study. Values are the averages of 76 population. S7 Table. Distribution of genotypes (H) and individuals (lower case three letter codes) in 12 analysed A. thaliana populations. (XLSX) S8 Table. Values (mean and range of variation) of the environmental variables used in the S7 Table. Distribution of genotypes (H) and individuals (lower case three letter codes) in 12 analysed A. thaliana populations. (XLSX) S8 Table. Values (mean and range of variation) of the environmental variables used in the study. Values are the averages of 76 population. (XLSX) PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Supporting information S1 Table. Geographic origin and location of Iberian A. thaliana populations. (XLSX) S2 Table. GLM analysis of values of life history traits of mock-inoculated A. thaliana plants, of tolerance to CMV, and of virus accumulation, using "host genotype" as a random factor. For each trait, heritability (h2 b), mean, minimum and maximum values, are shown. (XLSX) S3 Table. Values of climatic variables for 76 Iberian A. thaliana populations, and for the phenotypic traits of representative individuals. (XLSX) S3 Table. Values of climatic variables for 76 Iberian A. thaliana populations, and for the phenotypic traits of representative individuals. (XLSX) 18 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations 6. Brown JKM, Tellier A. Plant-Parasite Coevolution: Bridging the Gap between Genetics and Ecology. Annu Rev Phytopathol. 2011; 49: 345–367. https://doi.org/10.1146/annurev-phyto-072910-095301 PMID: 21513455 7. Brown JKM. Durable resistance of crops to disease: a Drawinian perspective. Annu Rev Phytopathol. 2015; 53: 513–539. https://doi.org/10.1146/annurev-phyto-102313-045914 PMID: 26077539 8. Salvaudon L, Giraud T, Shykoff JA. Genetic diversity in natural populations: a fundamental component of plant–microbe interactions. Curr Opin Plant Biol. 2008; 11: 135–143. https://doi.org/10.1016/j.pbi. 2008.02.002 PMID: 18329329 9. Burdon JJ, Thrall PH. Coevolution of Plants and Their Pathogens in Natural Habitats. Science. 2009; 324: 755–756. https://doi.org/10.1126/science.1171663 PMID: 19423818 10. Roossinck MJ. Symbiosis versus competition in plant virus evolution. Nat Rev Microbiol. 2005; 3: 917–924. https://doi.org/10.1038/nrmicro1285 PMID: 16322740 11. Roossinck MJ, Garcı´a-Arenal F. Ecosystem simplification, biodiversity loss and plant virus emer- gence. Curr Opin Virol. 2015; 10: 56–62. https://doi.org/10.1016/j.coviro.2015.01.005 PMID: 25638504 12. Fraile A, Garcı´a-Arenal F. Environment and evolution modulate plant virus pathogenesis. Curr Opin Virol. 2016; 17: 50–56. https://doi.org/10.1016/j.coviro.2016.01.008 PMID: 26827164 13. Xu P, Chen F, Mannas JP, Feldman T, Sumner LW, Roossinck MJ. Virus infection improves drought tolerance. New Phytol. 2008; 180: 911–921. https://doi.org/10.1111/j.1469-8137.2008.02627.x PMID: 18823313 14. Hily JM, Poulicard N, Mora MA´ , Paga´n I, Garcı´a-Arenal F. Environment and host genotype determine the outcome of a plant-virus interaction: from antagonism to mutualism. New Phytol. 2016; 209: 812– 822. https://doi.org/10.1111/nph.13631 PMID: 26365599 15. Maskell LC, Raybould AF, Cooper JI, Edwards ML, Gray AJ. Effects of turnip mosaic virus and turnip yellow mosaic virus on the survival, growth and reproduction of wild cabbage (Brassica oleracea). Ann Appl Biol. 1999; 135: 401–407. 16. Funayama S, Terashima I, Yahara T. Effects of virus infection and light environment on population dynamics of Eupatorium makinoi (Asteraceae). Am J Bot. 2001; 88: 616–622. PMID: 11302846 17. Power AG, Mitchell CE. Pathogen spillover in disease epidemics. Am Nat. 2004; 164: S79–S89. https://doi.org/10.1086/424610 PMID: 15540144 18. Malmstrom CM, Hughes CC, Newton LA, Stoner CJ. Virus infection in remnant native bunchgrasses from invaded California grasslands. New Phytol. 2005; 168: 217–230. https://doi.org/10.1111/j.1469- 8137.2005.01479.x PMID: 16159335 19. Malmstrom CM, Stoner CJ, Brandenburg S, Newton LA. Virus infection and grazing exert counteract- ing influences on survivorship of native bunchgrass seedlings competing with invasive exotics. J Ecol. 2006; 94: 264–275. https://doi.org/10.1111/j.1365-2745.2006.01101.x PMID: 18784788 20. Prendeville HR, Tenhumberg B. Pilson D. Effects of virus on plant fecundity and population dynamics. New Phytol. 2014; 202: 1346–1356. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 References 1. Woolhouse MEJ, Webster JP, Domingo E, Charlesworth B, Levin BR. Biological and biomedical impli- cations of the co-evolution of pathogens and their hosts. Nat Genet. 2002; 32: 569–577. https://doi. org/10.1038/ng1202-569 PMID: 12457190 2. Agnew P, Koella JC, Michalakis Y. Host life history responses to parasitism. Microb Infect. 2000; 2: 891–896. 3. Råberg L. How to Live with the Enemy: Understanding Tolerance to Parasites. PLoS Biol. 2014; 12: e1001989. https://doi.org/10.1371/journal.pbio.1001989 PMID: 25369060 4. Paga´n I, Garcı´a-Arenal F. Tolerance to plant pathogens: theory and experimental evidences. Int J Mol Sci. 2018; 19: 810. 5. Fraile A, Garcı´a-Arenal F. The coevolution of plants and viruses: Resistance and pathogenicity. Adv Virus Res. 2010; 76: 1–32. https://doi.org/10.1016/S0065-3527(10)76001-2 PMID: 20965070 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 19 / 24 Virus infection as a selective pressure on Arabidopsis wild populations 28. The 1001 Genomes Consortium. 1,135 Genomes reveal the global pattern of polymorphism in Arabi- dopsis thaliana. Cell. 2016; 166: 481–491. https://doi.org/10.1016/j.cell.2016.05.063 PMID: 27293186 29. Lee CR, Svardal H, Farlow A, Exposito-Alonso M, Ding W, Novikova P, et al. On the post-glacial spread of human commensal Arabidopsis thaliana. Nat Commun. 2017; 8: 14458. https://doi.org/10. 1038/ncomms14458 PMID: 28181519 30. Pico´ FX, Mendez-Vigo B, Martinez-Zapater JM, Alonso-Blanco C. Natural genetic variation of Arabi- dopsis thaliana is geographically structured in the Iberian Peninsula. Genetics. 2008; 180: 1009– 1021. https://doi.org/10.1534/genetics.108.089581 PMID: 18716334 31. Pico´ FX. Demographic fate of Arabidopsis thaliana cohorts of autumn-and spring-germinated plants along an altitudinal gradient. J Appl Ecol. 2012; 100: 1009–1018. 32. Me´ndez-Vigo B, Gomaa NH, Alonso-Blanco C, Pico´ FX. Among- and within-population variation in flowering time of Iberian Arabidopsis thaliana estimated in field and glasshouse conditions. New Phy- tol. 2013; 197: 1332–1343. https://doi.org/10.1111/nph.12082 PMID: 23252608 33. Vidigal DS, Marques AC, Willems LA, Buijs G, Me´ndez-Vigo B, Hilhorst HW, et al. Altitudinal and cli- matic associations of seed dormancy and flowering traits evidence adaptation of annual life cycle tim- ing in Arabidopsis thaliana. Plant Cell Environ. 2016; 39: 1737–1748. https://doi.org/10.1111/pce. 12734 PMID: 26991665 34. Mauricio R, Rausher MD. Experimental manipulation of putative selective agents provides evidence for the role of natural enemies in the evolution of plant defense. Evolution. 1997; 51: 1435–1444. https://doi.org/10.1111/j.1558-5646.1997.tb01467.x PMID: 28568624 35. Jorgensen TH, Emerson BC. Functional variation in a disease resistance gene in populations of Arabi- dopsis thaliana. Mol Ecol. 2008; 17: 4912–4923. https://doi.org/10.1111/j.1365-294X.2008.03960.x PMID: 19140981 36. Huard-Chauveau C, Perchepied L, Debieu M, Rivas S, Kroj T, Kars I, et al. An atypical kinase under balancing selection confers broad-spectrum disease resistance in Arabidopsis. PLoS Genet. 2013; 9: e1003766. https://doi.org/10.1371/journal.pgen.1003766 PMID: 24068949 37. Karasov TL, Kniskern JM, Gao L, DeYoung BJ, Ding J, Dubiella U, et al. The long-term maintenance of a resistance polymorphism through diffuse interactions. Nature. 2014; 512: 436–440. https://doi. org/10.1038/nature13439 PMID: 25043057 38. Brachi B, Meyer CG, Villoutreix R, Platt A, Morton TC, Roux F, et al. Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana. Proc Natl Acad Sci USA. 2015; 112: 4032–4037. https://doi.org/10.1073/pnas.1421416112 PMID: 25775585 39. Jacquemond M. Cucumber mosaic virus. Adv Virus Res. 2012; 84: 439–504. https://doi.org/10.1016/ B978-0-12-394314-9.00013-0 PMID: 22682176 40. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 https://doi.org/10.1111/nph.12730 PMID: 24571200 21. Fraile A, McLeish M, Paga´n I, Gonza´lez-Jara P, Piñero D, Garcı´a-Arenal F. Environmental heteroge- neity and the evolution of plant-virus interactions: Viruses in wild pepper populations. Virus Res. 2017; 15: 68–76. 22. Rodrı´guez-Nevado C, Montes N, Paga´n I. Ecological Factors Affecting Infection Risk and Population Genetic Diversity of a Novel Potyvirus in Its Native Wild Ecosystem. Front Plant Sci. 2017; 8: 1958. https://doi.org/10.3389/fpls.2017.01958 PMID: 29184567 23. Pallett DW, Thurston MI, Cortina-Borja M, Edwards ML, Alexander M, Mitchell E, et al. The incidence of viruses in wild Brassica rapa ssp. sylvestris in southern England. Ann Appl Biol. 2002; 141: 163– 170. 24. Ausin I, Alonso-Blanco C, Martinez-Zapater JM. Environmental regulation of flowering. Int J Dev Biol. 2005; 49: 689–705. https://doi.org/10.1387/ijdb.052022ia PMID: 16096975 25. Hoffmann MH. Biogeography of Arabidopsis thaliana (L.) Heynh. (Brassicaceae) J Biogeogr. 2002; 29: 125–134. 26. Brennan AC, Me´ndez-Vigo B, Haddioui A, Martı´nez-Zapater JM, Pico´ FX, Alonso-Blanco C. The genetic structure of Arabidopsis thaliana in the south-western Mediterranean range reveals a shared history between North Africa and southern Europe. BMC Plant Biol. 2014; 14: 17. https://doi.org/10. 1186/1471-2229-14-17 PMID: 24411008 27. Durvasula A, Fulgione A, Gutaker RM, Alacakaptan SI, Flood PJ, Neto C, et al. African genomes illu- minate the early history and transition to selfing in Arabidopsis thaliana. Proc Natl Acad Sci USA. 2017; 114: 5213–5218. https://doi.org/10.1073/pnas.1616736114 PMID: 28473417 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 20 / 24 Virus infection as a selective pressure on Arabidopsis wild populations 48. Paga´n I, Alonso-Blanco C, Garcı´a-Arenal F. Host Responses in Life-History Traits and Tolerance to Virus Infection in Arabidopsis thaliana. PLoS Pathog. 2008; 4: e1000124. https://doi.org/10.1371/ journal.ppat.1000124 PMID: 18704166 49. Me´ndez-Vigo B, Pico´ FX, Ramiro M, Martinez-Zapater JM, Alonso-Blanco C. Altitudinal and Climatic Adaptation Is Mediated by Flowering Traits and FRI, FLC, and PHYC Genes in Arabidopsis. Plant Physiol. 2011; 157: 1942–1955. https://doi.org/10.1104/pp.111.183426 PMID: 21988878 50. Manzano-Piedras E, Marcer A, Alonso-Blanco C, Pico´ FX. Deciphering the Adjustment between Envi- ronment and Life History in Annuals: Lessons from a Geographically-Explicit Approach in Arabidopsis thaliana. PLoS ONE. 2014; 9: e87836. https://doi.org/10.1371/journal.pone.0087836 PMID: 24498381 51. Tabas-Madrid D, Me´ndez-Vigo B, Arteaga N, Marcer A, Pascual-Montano A, Weigel D, et al. Genome-wide signatures of flowering adaptation to climate temperature: regional analyses in a highly diverse native range of Arabidopsis thaliana. Plant Cell Environ. 2018; 41: 1806–1820. https://doi.org/ 10.1111/pce.13189 PMID: 29520809 52. Shukla A, Paga´n I, Garcı´a-Arenal F. Effective tolerance based on resource reallocation is a virus-spe- cific defence in Arabidopsis thaliana. Mol Plant Pathol. 2018; 19: 1454–1465. https://doi.org/10.1111/ mpp.12629 PMID: 29027740 53. Paga´n I, Alonso-Blanco C, Garcı´a-Arenal F. Differential tolerance to direct and indirect density-depen- dent costs of viral infection in Arabidopsis thaliana. PLoS Pathog. 2009; 5: e1000531. https://doi.org/ 10.1371/journal.ppat.1000531 PMID: 19649316 54. Bjo¨rn HK, Gavin RS, Nienhuis J. Effects of photoperiod and vernalization on the number of leaves at flowering in 32 Arabidopsis thaliana (Brassicaceae) ecotypes. Am J Bot. 1993; 80: 646–648. 55. Auge GA, Leverett D, Edwards BR, Donohue K. Adjusting phenotypes via within- and across-genera- tional plasticity. New Phytol. 2017; 216: 343–349. https://doi.org/10.1111/nph.14495 PMID: 28262950 56. Agrawal A, Lively CM. Infection genetics: gene-for-gene versus matching-alleles models and all points in between. Evol Ecol Res. 2002; 4: 79–90. 57. Agrawal A, Lively CM. Modelling infection as a two-step process combining gene-for-gene and match- ing-allele genetics. Proc R Sci. 2003; 270: 323–34. 58. Sacrista´n S, Garcı´a-Arenal F. The evolution of virulence and pathogenicity in plant pathogen popula- tions. Mol Plant Pathol. 2008; 9: 369–384. https://doi.org/10.1111/j.1364-3703.2007.00460.x PMID: 18705877 59. Alexander H, Mauck K, Whitfield A, Garrett K, Malmstrom C. Plant-virus interactions and the agro-eco- logical interface. Eur J Plant Pathol. 2014; 138: 529–547. 60. Bergelson J, Roux F. Towards identifying genes underlying ecologically relevant traits in Arabidopsis thaliana. Nat Rev Genet. 2010; 11: 867–879. https://doi.org/10.1038/nrg2896 PMID: 21085205 61. Parker MA. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Hily JM, Garcı´a A, Moreno A, Plaza M, Wilkinson MD, Fereres A, Fraile A, Garcı´a-Arenal F. The Rela- tionship between Host Lifespan and Pathogen Reservoir Potential: An Analysis in the System Arabi- dopsis thaliana-Cucumber mosaic virus. PLoS Pathog. 2014; 10: e1004492. https://doi.org/10.1371/ journal.ppat.1004492 PMID: 25375140 41. Paga´n I, Montes N, Milgroom MG, Garcı´a-Arenal F. Vertical Transmission Selects for Reduced Viru- lence in a Plant Virus and for Increased Resistance in the Host. PLoS Pathog. 2014; 10: e1004293. https://doi.org/10.1371/journal.ppat.1004293 PMID: 25077948 42. Palukaitis P, Garcı´a-Arenal F. Cucumoviruses. Adv Virus Res. 2003; 62: 241–323. PMID: 14719367 43. Paga´n I, Fraile A, Ferna´ndez-Fueyo E, Montes N, Alonso-Blanco C, Garcı´a-Arenal F. Arabidopsis thaliana as a model for the study of plant-virus co-evolution. Philos Trans R Soc Lond, B, Biol Sci. 2010; 365: 1983–1995. https://doi.org/10.1098/rstb.2010.0062 PMID: 20478893 44. Montes N. Arabidopsis thaliana como modelo de estudio de la coevolucio´n planta-virus Thesis, Uni- versidad Polite´cnica de Madrid (UPM). 2016; Available from: https://doi.org/10.20868/UPM.thesis. 42853. 45. Fraile A, Alonso-Prados JL, Aranda MA, Bernal JJ, Malpica JM, Garcı´a-Arenal F. Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus. J Virol. 1997; 71: 934–940. PMID: 8995610 46. Bonnet J, Fraile A, Sacrista´n S, Malpica JM, Garcı´a-Arenal F. Role of recombination in the evolution of natural populations of Cucumber mosaic virus, a tripartite RNA plant virus. Virology. 2005; 332: 359– 368. https://doi.org/10.1016/j.virol.2004.11.017 PMID: 15661167 47. Paga´n I, Alonso-Blanco C, Garcı´a-Arenal F. The Relationship of Within-Host Multiplication and Viru- lence in a Plant-Virus System. PLoS ONE. 2007; 2: e786. https://doi.org/10.1371/journal.pone. 0000786 PMID: 17726516 21 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations 70. Kaltz O, Shykoff JA. Local adaptation in host–parasite systems. Heredity. 1998; 81: 361–370. 71. de Meaux J, Mitchell-Olds T. Evolution of Plant Resistance at the Molecular Level: Ecological Context of Species Interactions. Heredity. 2003; 91: 345–52. https://doi.org/10.1038/sj.hdy.6800342 PMID: 14512948 72. Laine AL. Resistance variation within and among host populations in a plant-pathogen metapopula- tion-implications for regional pathogen dynamics. J. Ecol. 2004; 92: 990–1000. 73. Laine AL. Spatial scale of local adaptation in a plant-pathogen. J Evol Biol. 2005; 18: 930–938. https:// doi.org/10.1111/j.1420-9101.2005.00933.x PMID: 16033565 74. Laine AL. Evolutionary of host resistance: looking for coevolutionry hotspot at small spatial scales. Phi- los Trans R Soc Lond B Biol Sci. 2006; 273: 267–273. 75. Salvaudon L, He´raudet V, Shykoff JA. Parasite-host fitness trade-offs change with parasite identity: genotype-specific interactions in a plant-pathogen system. Evolution. 2005; 59: 2518–2524. PMID: 16526500 76. Salvaudon L, Heraudet V, Shykoff JA. Arabidopsis thaliana and the Robin Hood parasite: a chivalrous oomycete that steals fitness from fecund hosts and benefits the poorest one?. Biol. Lett. 2008; 4: 526–529. https://doi.org/10.1098/rsbl.2008.0332 PMID: 18682359 77. Kniskern JM, Barrett LG, Bergelson J. Maladaptation in wild populations of the generalist plant patho- gen Pseudomonas syringae. Evolution. 2011; 65: 818–830. https://doi.org/10.1111/j.1558-5646. 2010.01157.x PMID: 21044058 78. Salvaudon L, Shykoff JA. Variation in Arabidopsis developmental responses to oomycete infection: resilience vs changes in life history traits. New Phytol. 2013; 197: 919–926. https://doi.org/10.1111/ nph.12073 PMID: 23231447 79. Kover PX, Schaal BA. Genetic variation for disease resistance and tolerance among Arabidopsis thali- ana accessions. Proc Natl Acad Sci USA. 202; 99: 11270–11274. 80. Mitchell-Olds T. Arabidopsis thaliana and its wild relatives: a model system for ecology and evolution. Trends Ecol Evol. 2011; 16: 693–700. 81. Luo Y, Widmer A, Karrenberg. The roles of genetic drift and natural selection in quantitative trait diver- gence along an altitudinal gradient in Arabidopsis thaliana. Heredity. 2015; 114: 220–228. https://doi. org/10.1038/hdy.2014.89 PMID: 25293874 82. Jakob K, Goss EM, Araki H, Van T, Kreitman M, Bergelson J. Pseudomonas viridiflava and P. syrin- gae—Natural Pathogens of Arabidopsis thaliana. Mol Plant Microbe Interact. 2002; 15: 1195–1203. https://doi.org/10.1094/MPMI.2002.15.12.1195 PMID: 12481991 83. Goss EM, Bergelson J. Variation in resistance and virulence in the interaction between Arabidopsis thaliana and a bacterial pathogen. Evolution. 2006; 60: 1562–1573. PMID: 17017057 84. Alonso-Prados JL, Aranda MA, Malpica JM, Garcı´a-Arenal F, Fraile A. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Disease impact and local genetic diversity in the clonal plant Podophyllum peltatum. Evo- lution. 1989; 43: 540–547. https://doi.org/10.1111/j.1558-5646.1989.tb04250.x PMID: 28568395 62. Jarosz AM, Burdon JJ. Predominance of a single major gene for resistance to Phakopsora pachyrhizi and Glycine argyrea: Phenotypic and genetic patterns. Heredity. 1990; 64: 347–353. 63. Jarosz AM, Burdon JJ. Host-pathogen interactions in natural populations of Linum marginale and Mel- ampsora lini. II. Local and regional variation in patterns of resistance and racial structure. Evolution. 1991; 45: 1618–27. https://doi.org/10.1111/j.1558-5646.1991.tb02667.x PMID: 28564135 64. Antonovics J, Thrall PH, Jarosz A, Stratton D. Ecological genetics of metapopulations: the Silene– Ustilago plant–pathogen system. In: Real L, ed. Ecological genetics. Princeton, US, Princeton Univer- sity Press; 1994. pp 146–170. 65. Burdon JJ, Jarosz AM. Host–pathogen interactions in natural populations of Linum marginale and Mel- ampsora lini. I. Patterns of resistance and racial variation in a large host population. Evolution. 1991; 45: 205–217. https://doi.org/10.1111/j.1558-5646.1991.tb05278.x PMID: 28564067 66. Hamilton MG, Williams DR, Tilyard PA, Pinkard EA, Wardlaw TJ, Glen M, et al. A latitudinal cline in dis- ease resistance of a host tree. Heredity. 2013; 110: 372–379. https://doi.org/10.1038/hdy.2012.106 PMID: 23211794 67. Stahl EA, Dwyer G, Mauricio R, Kreitman M, Bergelson J. Dynamics of disease resistance polymor- phism at the Rpm1 locus of Arabidopsis. Nature. 1999; 400: 667–671. https://doi.org/10.1038/23260 PMID: 10458161 68. Thrall PH, Burdon JJ, Young A. Variation in resistance and virulence among demes of a plant host- pathogen metapopulation. J. Ecol. 2001; 89: 736–748. 69. Thrall PH, Burdon JJ. Evolution of gene-for-gene systems in metapopulations: the effect of spatial scale of host and pathogen dispersal. Plant Pathol. 2002; 51: 169–184. 22 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations 94. Carr DE, Murphy JF, Eubanks MD. The susceptibility and response of inbred and outbred Mimulus guttatus to infection by Cucumber mosaic virus. Evol Ecol. 2003; 17: 85–103. 95. Carr DE, Murphy JF, Eubanks MD. Genetic variation and covariation for resistance and tolerance to Cucumber mosaic virus in Mimulus guttatus (Phrymaceae): a test for costs and constraints. Heredity. 2006; 96: 29–38. https://doi.org/10.1038/sj.hdy.6800743 PMID: 16189544 96. Gomaa NH, Montesinos-Navarro A, Alonso-Blanco C, Pico´ FX. Temporal variation in genetic diversity and effective population size of Mediterranean and subalpine Arabidopsis thaliana populations. Mol Ecol. 2001; 20: 3540–3554. 97. Ninyerola M, Pons X, Roure JM. Atlas Clima´tico Digital de la Penı´nsula Ibe´rica. Metodologı´a y aplica- ciones en bioclimatologı´a y geobota´nica. Universidad Auto´noma de Barcelona: Bellaterra; 2005. 98. Palukaitis P, Roossinck MJ, Dietzgen RG, Francki RIB. Cucumber Mosaic Virus. Adv Virus Res. 1992; 41: 281–348. PMID: 1575085 99. Lot H, Marrou J, Quiot JB, Esvan C. Contribution à l’e´tude du virus de la mosaïque du concombre (CMV). Me´thode de purification rapide du virus. Ann Phytopathol. 1975, 4: 25–38. 100. Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Go¨rlach J. Growth stage- based phenotypic analysis of Arabidopsis: A model for high throughput functional genomics in plants. Plant Cell. 2001; 13: 1499–1510. https://doi.org/10.1105/TPC.010011 PMID: 11449047 101. Thompson K, Stewart AJA. The Measurement and Meaning of Reproductive Effort in Plants. Am Nat. 1981; 117: 205–211. 102. Lynch M, Walsh B. Genetics and analysis of quantitative traits. Sunderland, MA, USA: Sinauer Asso- ciates Inc; 1998. 103. Spitze K. Population structure in Daphnia obtusa: quantitative genetic and allozymic variation. Genet- ics. 1993; 135: 367–374. PMID: 8244001 104. Bonnin I, Prosperi JM, Olivieri I. Genetic markers and quantitative genetic variation in Medicago trun- catula (Leguminosae): A comparative analysis of population structure. Genetics. 1996; 143: 1795– 1805. PMID: 8844165 105. Le Corre V. Variation at two flowering time genes within and among populations of Arabidopsis thali- ana: comparison with markers and traits. Mol Ecol. 2005; 14: 4181–4192. https://doi.org/10.1111/j. 1365-294X.2005.02722.x PMID: 16262868 106. Quinn GP, Keough MJ. Experimental Design and Data Analysis for Biologists. UK: Cambridge Uni- versity Press; 2002. 107. Weir BS, Cockerham CC. Estimating F-statistics for the analysis of population structure. Evolution. 1984; 38: 1358–1370. https://doi.org/10.1111/j.1558-5646.1984.tb05657.x PMID: 28563791 108. Excoffier L, Lischer HEL. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Satellite RNA of Cucumber mosaic virus spreads epidemically in natural populations of its helper virus. Phytopathology. 1998; 88: 520–524. https://doi.org/10.1094/PHYTO.1998.88.6.520 PMID: 18944903 85. Antonovics J, Thrall P. The cost of resistance and the maintenance of genetic polymorphism in host- parasite systems. Philos Trans R Soc Lond B Biol Sci. 1994; 257: 105–110. 86. Herms DA, Mattson WJ. The dilemma of plants: To grow or defend. Q Rev Biol. 1992; 67: 283–335. 87. Manzaneda AJ, Prasad KVSK, Mitchell-Olds T. Variation and fitness costs for tolerance to different types of herbivore damage in Boechera stricta genotypes with contrasting glucosinolate structures. New Phytol. 2010; 188: 464–477. https://doi.org/10.1111/j.1469-8137.2010.03385.x PMID: 20663059 88. Mauricio R, Rausher MD, Burdick DS. Variation in the defense strategies of plants: are resistance and tolerance mutually exclusive?. Ecology. 1997; 78: 1301–1311. 89. Boots M, Bowers RG. Three mechanisms of host resistance to microparasites–avoidance, recovery and tolerance–show different evolutionary dynamics. J Theor Biol. 1999; 201: 13–23. https://doi.org/ 10.1006/jtbi.1999.1009 PMID: 10534432 90. Restif O, Koella JC. Shared control of epidemiological traits in a coevolutionary model of host-parasite interactions. Am Nat. 2003; 161: 827–836. https://doi.org/10.1086/375171 PMID: 12858269 91. Restif O, Koella JC. Concurrent Evolution of Resistance and Tolerance to Pathogens. Am Nat. 2004; 164: E90–E102. https://doi.org/10.1086/423713 PMID: 15459887 92. Roy BA, Kirchner JW. Evolutionary dynamics of pathogen resistance and tolerance. Evolution. 2000; 54: 51–63. PMID: 10937183 93. Miller MR, White A, Boots M. The evolution of host resistance: tolerance and control as distinct strate- gies. J Theor Biol. 2005; 236: 198–207. https://doi.org/10.1016/j.jtbi.2005.03.005 PMID: 16005309 23 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019 Arlequin suite ver 3.5: A new series of programs to perform population genet- ics analyses under Linux and Windows. Mol Ecol Resour. 2010; 10: 564–567. https://doi.org/10.1111/ j.1755-0998.2010.02847.x PMID: 21565059 109. Rosenberg MS, Anderson CD. PASSaGE: pattern analysis, spatial statistics and geographic exege- sis. Version 2. Methods Ecol Evol. 2011; 2: 229–232. 110. van Berloo R. GGT 2.0: versatile software for visualization and analysis of genetic data. J. Hered. 2008; 99: 232–236. https://doi.org/10.1093/jhered/esm109 PMID: 18222930 111. Sokal RR, Oden NL. Spatial autocorrelation in biology 1. Methodology. Biological Journal of the Lin- nean Society, 10: 199–228. 112. Moran PAP. Notes on continuous stochastic phenomena. Biometrika. 1950; 37: 17–23. PMID: 15420245 113. Dutilleul P, Clifford P, Richardson S, Hemon D. Modifying the t test for assessing the correlation between two spatial processes. Biometrics. 1993; 49: 305–314. 114. Rangel TF, Diniz-Filho JAF, Bini LM. SAM: A comprehensive aplication for Spatail Analysis in Macroe- cology. Ecography. 2010; 33: 1–5. 115. Beale CM, Lennon JJ, Yearsley JM, Brewer MJ, Elston DA. Regression analysis of spatial data. Ecol Lett. 2010; 13: 246–264. https://doi.org/10.1111/j.1461-0248.2009.01422.x PMID: 20102373 24 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810 May 28, 2019
https://openalex.org/W2127478103
https://wjso.biomedcentral.com/counter/pdf/10.1186/s12957-015-0477-x
English
null
Three cases of sporadic meningioangiomatosis with different imaging appearances: case report and review of the literature
World journal of surgical oncology
2,015
cc-by
4,507
CASE REPORT Open Access * Correspondence: cjr.zhangyunting@vip.163.com †Equal contributors Department of Radiology, Tianjin Medical University General Hospital, No. 154, Anshan Dao Road, Heping District, Tianjin 300052, People’s Republic of China © 2015 Sun et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sun et al. World Journal of Surgical Oncology (2015) 13:89 DOI 10.1186/s12957-015-0477-x Sun et al. World Journal of Surgical Oncology (2015) 13:89 DOI 10.1186/s12957-015-0477-x WORLD JOURNAL OF SURGICAL ONCOLOGY Abstract Background: Meningioangiomatosis (MA) is a rare meningiovascular malformation or hamartomatous lesion in the central nervous system. Radiographic findings of MA may show a variety of characteristics according to different histological components. We present three cases of sporadic MA with different imaging appearances in an attempt to identify specific imaging characteristics. Case presentation: In case 1, an irregular hyperdense solid mass was localized in the left middle cranial fossa, demonstrating low and equal signal intensity on T1-weighted imaging (T1WI; TR/TE 2,048.9 ms/26.1 ms), high signal intensity with multiple flow void effect on T2-weighted imaging (T2WI; TR/TE 4,000 ms/106.4 ms), and significant and homogeneous enhancement on post-contrast magnetic resonance imaging (MRI). In case 2, the lesion in the right insular lobe showed a cystic-mural nodule pattern. The cystic content demonstrated similar density or signal intensity as cerebrospinal fluid, while the mural nodule demonstrated equal density or signal intensity on computed tomography (CT) and MRI. On post-contrast MRI, the mural nodule showed significant enhancement, but the cystic wall and content showed no enhancement. In case 3, a remarkably enhanced solid nodule was found in the cortex of the left parietal lobe with multiple small cysts surrounding it. This nodule showed low signal intensity on T2WI and diffusion-weighted imaging (DWI; TR/TE 6,000 ms/96.8 ms, b = 1,000 s/mm2). The preoperative diagnoses of the above three cases were meningioma, hemangioblastoma, and ganglioglioma. However, all were pathologically diagnosed as MA. Conclusion: The presented cases demonstrate that MA may present with solid and cystic imaging patterns, which may include large cystic-mural nodules and small intra- and extra-cystic patterns. Although MA imaging diagnoses are difficult, several MRI signs may include specific characteristics, such as a flow void effect on T2WI and separating cysts in the cystic MA (as shown in our cases), gyriform hyperintensity on T2-fluid attenuated inversion recovery (FLAIR) sequence, and susceptibility artifacts on T2 gradient echo (GRE) sequences (as found in the literature). Keywords: Meningioangiomatosis, Magnetic resonance imaging, Flow void effect, Cystic meningioangiomatosis and meningovascular proliferation interwoven with bands of fibrous connective tissue. The radiographic findings of MA may demonstrate a variety of characteristics accord- ing to different histological components. In this study, we present three cases of sporadic MA with different imaging appearances and attempt to identify specific characteris- tics to help in preoperative diagnoses. In addition, the relevant medical literature was reviewed. Three cases of sporadic meningioangiomatosis with different imaging appearances: case report and review of the literature hihua Sun†, Fei Jin†, Jing Zhang, Yue Fu, Wei Li, Hong Guo and Yunting Zhang* Zhihua Sun†, Fei Jin†, Jing Zhang, Yue Fu, Wei Li, Hong Guo and Yunting Zhang* Background Meningioangiomatosis (MA) is a rare meningiovascular malformation or hamartomatous lesion in the central nervous system, which was first described by Bassoe and Nuzum [1] and then was named by Worster-Drought et al. [2]. MA may occur sporadically or in association with neurofibromatosis (NF) type 2. The pathological characteristics of MA include leptomeningeal calcification * Correspondence: cjr.zhangyunting@vip.163.com † Case presentation Case 1 * Correspondence: cjr.zhangyunting@vip.163.com †Equal contributors Department of Radiology, Tianjin Medical University General Hospital, No. 154, Anshan Dao Road, Heping District, Tianjin 300052, People’s Republic of China A 73-year-old female patient had a history of binocular diplopia for 1 week. Physical examination showed a Page 2 of 6 Sun et al. World Journal of Surgical Oncology (2015) 13:89 limitation in abduction movement in the left eye. A head computed tomography (CT) scan showed an irregular mixed hyperdense mass in the left middle cranial fossa (Figure 1a). On magnetic resonance imaging (MRI), the lesion demonstrated low and equal signal intensity on T1-weighted imaging (T1WI; TR/TE 2,048.9 ms/26.1 ms) and high signal intensity with multiple flow void effect on T2-weighted imaging (T2WI; TR/TE 4,000 ms/106.4 ms) (Figure 1b,c). On post-contrast MRI, the lesion showed significant and homogeneous enhancement after gado- linium diethylenetriamine pentaacetate (Gd-DTPA) was administered. The margin between the lesion and the adjacent brain cortex was well demarcated, and there was no obvious mass effect. The preoperative diagnosis was meningioma. tumor was firm, pink, and enveloped and had a general blood supply. The base was located in the side wall of the cavernous sinus and sphenoid crest. Microscopically, there was extensive fibroblastic proliferation and an in- creased number of vessels surrounded by meningothelial cells (Figure 1e,f). Pathological diagnosis was MA. Case 2 A 23-year-old male patient presented with left hemia- nesthesia for more than 2 years. Physical examination showed hypesthesia and slight ataxia on the left side, and a decrease of graphics and two-point discrimination sensation. A cystic-mural nodule lesion was localized in the right insular lobe on head plain CT (Figure 2a). The cystic portion had low density with 9 Hu, and the mural nodule was isodense with 35 Hu. The cystic content demonstrated similar signal intensity as cerebrospinal fluid (CSF), while the mural nodule demonstrated iso- signal intensity on T1WI, T2WI, and diffusion-weighted imaging (DWI; TR/TE 6,000 ms/96.8 ms, b = 1,000 s/mm2) (Figure 2b,c,d). On post-contrast MRI, the mural nodule showed significant enhancement, but the cystic wall and content demonstrated no enhancement (Figure 2e). The adjacent brain parenchyma and sulci were compressed and deformed, but there was no edema around the tumor. The preoperative diagnosis was hemangioblastoma. The patient underwent a left temporal craniotomy, and complete removal of the tumor was performed. The Figure 1 Solid meningioangiomatosis. (a) CT scan showed an irregular mixed high-density mass in the left middle cranial fossa. (b) On T1WI, the lesion demonstrated low and equal signal intensity. (c) On T2WI, the lesion showed high signal intensity with a multiple flow void effect. (d) On post-contrast MRI, the lesion showed significant and homogeneous enhancement. (e, f) Microphotography of specimens showed extensive fibroblastic proliferation and an increased number of vessels surrounded by meningothelial cells. The patient underwent a right temporal craniotomy. Upon operation, approximately 50 ml of a yellow trans- parent cyst fluid was extracted from the cystic tumor. The purple mural nodule was approximately 8 mm in diameter with fibrous adhesion to the right insular cortex. Pathological examination demonstrated a classic MA appearance (Figure 2f). Case 3 (a, b) DWI and T2WI demonstrated a low signal intensity nodule in the left parietal cortex and multiple small cysts surrounding it. (c) On post-contrast MRI, the nodule was remarkably enhanced. (d) Microscopically, fibroblast-like spindle cells were arranged in a spiral shape around multiple vessels, and the cortical neurons were entrapped within the lesion. accompanied by NF has been shown to mostly occur in the frontal lobe (35%) [6,7]. Atypical locations have also been reported [8], including the third ventricle, thalamus, cerebral peduncle, and brain stem. Multiple MA lesions accompanied by NF have occurred in 35% of patients, but the incidence of sporadic MA was only 13% [9]. Figure 2 Cystic meningioangiomatosis with a cystic-mural Figure 2 Cystic meningioangiomatosis with a cystic-mural nodule pattern. (a) CT scan showed a cystic-mural nodule lesion in the right insular lobe. (b-d) In non-enhanced MRI, the cystic content demonstrated similar signal intensity as cerebrospinal fluid (CSF), while the mural nodule demonstrated iso-signal intensity on T1WI, T2WI, and DWI. (e) On post-contrast MRI, the mural nodule demonstrated significant enhancement, while the cystic wall and content showed no enhancement. (f) Pathological examination showed perivascular spindle-cell proliferation. Figure 2 Cystic meningioangiomatosis with a cystic-mural nodule pattern. (a) CT scan showed a cystic-mural nodule lesion in the right insular lobe. (b-d) In non-enhanced MRI, the cystic content demonstrated similar signal intensity as cerebrospinal fluid (CSF), while the mural nodule demonstrated iso-signal intensity on T1WI, T2WI, and DWI. (e) On post-contrast MRI, the mural nodule demonstrated significant enhancement, while the cystic wall and content showed no enhancement. (f) Pathological examination showed perivascular spindle-cell proliferation. The pathogenesis of MA remains unclear. Proposed hypotheses include hamartoma with degenerative changes, leptomeningeal meningioma with invasion in adjacent brain tissue, and cortical vascular malformation. MA was formerly classified as a neurocutaneous syndrome, and some cases associated with NF supported the theory of a hamartoma [10]. Although not all cases have a meningeal component and malignancy characteristics are typically absent, the association between meningioma in rare cases and molecular aberrations regarding the NF2 gene in both lesions suggests that MA may be correlated with meningioma. Kim et al. [11] found that the meningioma- tosis portions of meningiomatosis-meningioma have loss of heterozygosity for the 22q12 locus in 28.6% (2/7) of coexisting cases of meningiomatosis-meningioma, whereas each pure meningiomatosis harbors one loss of hetero- zygosity at either 22q12 or 9p21. Case 3 A 9-year-old girl suffered from an involuntary convul- sion in her right limb for 1 month. Physical examination showed no positive signs. On non-enhanced MRI, a solid nodule 11 mm in diameter was found in the cortex of the left parietal lobe, demonstrating low signal intensity on DWI and T2WI (Figure 3a,b). Multiple small cysts were around the nodule. There was a slight mass effect and surrounding edema. After Gd-DTPA administration, the nodule was remarkably enhanced (Figure 3c). The preoperative diagnosis was ganglioglioma. The patient underwent a left parietal craniotomy, and the tumor was completely removed. The tumor was pur- ple with an abundant blood supply, and it was unclearly demarcated from adjacent brain tissue. Microscopically, fibroblast-like spindle cells were arranged in spiral shape around multiple vessels, and cortical neurons were en- trapped within the lesion (Figure 3d). The pathological diagnosis was MA. Figure 1 Solid meningioangiomatosis. (a) CT scan showed an irregular mixed high-density mass in the left middle cranial fossa. (b) On T1WI, the lesion demonstrated low and equal signal intensity. (c) On T2WI, the lesion showed high signal intensity with a multiple flow void effect. (d) On post-contrast MRI, the lesion showed significant and homogeneous enhancement. (e, f) Microphotography of specimens showed extensive fibroblastic proliferation and an increased number of vessels surrounded by meningothelial cells. Page 3 of 6 Sun et al. World Journal of Surgical Oncology (2015) 13:89 Figure 2 Cystic meningioangiomatosis with a cystic-mural nodule pattern. (a) CT scan showed a cystic-mural nodule lesion in the right insular lobe. (b-d) In non-enhanced MRI, the cystic content demonstrated similar signal intensity as cerebrospinal fluid (CSF), while the mural nodule demonstrated iso-signal intensity on T1WI, T2WI, and DWI. (e) On post-contrast MRI, the mural nodule demonstrated significant enhancement, while the cystic wall and content showed no enhancement. (f) Pathological examination showed perivascular spindle-cell proliferation. Figure 3 Cystic meningioangiomatosis with a multiple microcystic pattern. (a, b) DWI and T2WI demonstrated a low signal intensity nodule in the left parietal cortex and multiple small cysts surrounding it. (c) On post-contrast MRI, the nodule was remarkably enhanced. (d) Microscopically, fibroblast-like spindle cells were arranged in a spiral shape around multiple vessels, and the cortical neurons were entrapped within the lesion. Figure 3 Cystic meningioangiomatosis with a multiple microcystic pattern. Case 3 The theory of cortical vascular malformation corresponded to the histopatho- logical characteristic of MA. Cortical vascular malformation induced the perivascular meningothelial proliferation of cells from vessel walls or pluripotent arachnoid cap cells in Discussion MA can be divided into two categories: sporadic or asso- ciated with NF type 2. Sporadic meningioangiomatosis usually occurs in young adults or children who present with seizures or headaches. In contrast, NF2-associated cases are usually asymptomatic and discovered post- mortem. MA has a slight male predominance [3], but according to some authors [4], it has no sex predilection. Meningioma is the most commonly associated neoplasm, and other associated abnormalities include cerebral hemor- rhage, oligodendroglioma, arteriovenous malformations, cerebral softening, and meningeal hemangiopericytoma [5]. MA was mostly localized in the supratentorial region, particularly in the temporal or frontal lobes. Sporadic MA has mainly been reported in the temporal lobe (33%) followed by the frontal lobe (25%), but MA MA was mostly localized in the supratentorial region, particularly in the temporal or frontal lobes. Sporadic MA has mainly been reported in the temporal lobe (33%) followed by the frontal lobe (25%), but MA Sun et al. World Journal of Surgical Oncology (2015) 13:89 Page 4 of 6 Sun et al. World Journal of Surgical Oncology (2015) 13:89 Virchow-Robin spaces. Leptomeninges and arachnoid cap cells normally surround blood vessels as they penetrate the cortex. Conceivably, chronic leptomeningeal stimula- tion by underlying cortical lesions could result in MA histopathological changes. MR images was iso- to hypointense on T1-weighted im- ages and hypo- to hyperintense on T2-weighted images. In case 1, multiple flow void effect could be observed within MA on T2WI. To our knowledge, this characteristic was first described for MA. This effect corresponded to a pre- dominantly vascular histological type, that is, proliferating vessels. Contrast-enhanced images also showed various types of enhancement patterns ranging from mild to strong and even no enhancement, but remarkable enhancement was the most common pattern with a prevalence of 79.6% [14]. The mass effect was none or slight. Surrounding edema was always absent, but adjacent subcortical white matter was occasionally hyperintense on T2WI resulting from edema or gliosis [15]. Histopathologically, MA has been regarded as a hamartomatous, reactive, or neoplasic lesion originating from multivariate cells, such as meningothelial, fibroblas- tic, myofibroblastic, smooth muscle, or pluripotent cells [12]. The characteristics of MA include leptomeningeal proliferation accompanied by calcification or psammoma body formation, perivascular cuffs of spindle-shaped fibroblast-like cells, whorls, or bands of meningothelial cells in association with sharply demarcated intracortical plaques of proliferating small vessels. Discussion In our three cases, all had the specific histological characteristics mentioned above in the solid portion, but calcification was found only in case 1. Other histological abnormalities included gliosis, neuronal dysplasia, cystic degeneration of white matter, and vascular hyalinization. Some specific signs on MRI may be helpful for MA imaging diagnoses. Gyriform hyperintense on T2-fluid attenuated inversion recovery (FLAIR) sequence has been reported to be the most prominent characteristic for sporadic MA on MR imaging, which can be attributed to a thickened cortex with proliferating leptomeningeal vessels interwoven with bands of fibrous connective tissue [13]. Calcification may be ignored on routine T1WI and T2WI, but it produces a susceptibility artifact on T2 gradient echo (GRE) sequences [14]. Chronic hemosiderin from cavernous malformations also showed susceptibility artifacts; thus, T2 GRE sequences may only be thought of as a reference imaging modality for MA diagnoses. Magnetic resonance spectrum (MRS) can aid in the evaluation of brain lesions by analyzing the spectrum of metabolites present in the area of study. Rokes et al. [16] have reported a case of MA with a distinct choline (Cho) peak and a decreasing N-acetyl aspartate (NAA) peak on MRS, which suggested a proliferating tumor of presumed non-neuronal origin. The high Cho peak could have been a reflection of the proliferation of meningothelial cells and/or fibroblasts that formed the concentric cuffs sur- rounding blood vessels in the cortex. However, the high Cho peak could have also been observed under other conditions, for example, high-grade brain tumors [17]. Immunohistochemical staining of MA was relatively unhelpful for diagnosis. Positive vimentin expression reflected the proliferation of blood vessels and perivas- cular fibroblast-like cells in almost all cases. However, the results of other immunohistochemical markers, such as epithelial membrane antigen (EMA), S-100 protein, and glial fibrillary acidic protein (GFAP), were variable in the literature [10]. According to different percentages of perivascular cell proliferation and cortical vascular proliferation, MA may be broadly classified into predominantly cellular or vascular types [13]. Predominantly cellular MA has moderate to high cellularity such as that found in cases 2 and 3 (Figures 2f and 3d). In the central portion of the lesion, MA cells emerge from the perivascular location and infiltrate the cortex. Approximately 90% of reported cases have cortical invasion, but the proliferating cells have no significant atypia, mitoses, or necrosis. Cho: choline; CSF: cerebrospinal fluid; CT: computed tomography; DNET: dysembryoplastic neuroepithelial tumor; DWI: diffusion-weighted imaging; EMA: epithelial membrane antigen; FLAIR: fluid attenuated inversion Authors’ information Differential diagnoses for MA are not easy due to variable radiological appearances. Extra-axial tumors, such as meningiomas, are the most common differential diagnosis. Intra-axial tumors in the superficial cortex, such as oligodendroglioma, ganglioglioma, and dysembryoplas- tic neuroepithelial tumor (DNET), are also frequently considered [24]. However, for cystic MA, we thought that differential tumors should include some cystic-mural tumors with non-enhanced cystic walls, such as heman- gioblastomas, pilocystic astrocytomas, and pleomorphic xanthoastrocytomas. The MA cysts are relatively sepa- rated from solid masses or nodule, which may be helpful for differential diagnoses. Other non-tumor diseases, that is, arteriovenous malformation, cavernous hemangioma, and granuloma, are occasionally misdiagnosed as MA. ZS, FJ, JZ, YF, WL, and YZ are radiologists. HG is an imaging engineer. Competing interests Th h d l h p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Consent Written informed consent was obtained from the patient for the publication of this case report and any accompany- ing images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. 14. Kashlan ON, LaBorde DV, Davison L, Saindane AM, Brat D, Hudgins PA, et al. Meningioangiomatosis: a case report and literature review emphasizing diverse appearance on different imaging modalities. Case Rep Neurol Med. 2011;2011:361203. 15. Meltzer CC, Liu AY, Perrone AM, Hamilton RL. Meningioangiomatosis: MR imaging with histopathologic correlation. AJR Am J Roentgenol. 1998;170:804–5. 15. Meltzer CC, Liu AY, Perrone AM, Hamilton RL. Meningioangiomatosis: MR imaging with histopathologic correlation. AJR Am J Roentgenol. 1998;170:804–5. Conclusions The cases in this study demonstrated that MA may present with solid or cystic imaging patterns. Imaging diagnoses for MA were difficult due to non-specific characteristics. Several signs on MRI may be helpful, such as gyriform hyperintensity on T2-FLAIR sequences, susceptibility artifacts on T2 GRE sequence, and flow void effects on T2WI as in our case. Cysts in cystic MA demonstrate variable appearances in location, number, and size, but separation from solid masses or nodules may be one of its specific characteristics. 8. Kollias SS, Crone KR, Ball Jr WS, Prenger EC, Ballard ET. Meningioangiomatosis of the brain stem. Case report. J Neurosurg. 1994;80:732–5. 9. Kim SH, Yoon SH, Kim JH. A case of infantile meningioangiomatosis with a separate cyst. J Korean Neurosurg Soc. 2009;46:252–6. 9. Kim SH, Yoon SH, Kim JH. A case of infantile meningioangiomatosis with a separate cyst. J Korean Neurosurg Soc. 2009;46:252–6. 10. Wiebe S, Munoz DG, Smith S, Lee DH. Meningioangiomatosis. A comprehensive analysis of clinical and laboratory features. Brain. 1999;122:709–26. 10. Wiebe S, Munoz DG, Smith S, Lee DH. Meningioangiomatosis. A comprehensive analysis of clinical and laboratory features. Brain. 1999;122:709–26. 11. Kim NR, Cho SJ, Suh YL. Allelic loss on chromosomes 1p32, 9p21, 13q14, 16q22, 17p, and 22q12 in meningiomas associated with meningioangiomatosis and pure meningioangiomatosis. J Neurooncol. 2009;94:425–30. 12. Kim WY, Kim IO, Kim WS, Cheon JE, Yeon KM. Meningioangiomatosis: MR imaging and pathological correlation in two cases. Pediatr Radiol. 2002;32:96–8 13. Yao Z, Wang Y, Zee C, Feng X, Sun H. Computed tomography and magnetic resonance appearance of sporadic meningioangiomatosis correlated with pathological findings. J Comput Assist Tomogr. 2009;33:799–804. Acknowledgements We thank Sun Cuiyun who provided pathological figures of the cases. This work was supported by National Natural Science Foundation of China (NSFC, no. 81201152). We thank Sun Cuiyun who provided pathological figures of the cases. This work was supported by National Natural Science Foundation of China (NSFC, no. 81201152). Received: 1 July 2014 Accepted: 22 January 2015 Received: 1 July 2014 Accepted: 22 January 2015 References 1. Bassoe P, Nuzum F. Report of a case of central and peripheral neurofibromatosis. J Nerv Ment Dis. 1915;42:785–96. 2. Worster-Drought C, Dickson WEC, McMenemy WH. Multiple meningeal and perineural tumors with analogous changes in the glia and ependyma. Brain. 1937;60:85–117. 3. Arcos A, Serramito R, Santín JM, Prieto A, Gelabert M, Rodriguez-Osorio X, et al. Meningioangiomatosis: clinical-radiological features and surgical outcome. Neurocirugía. 2010;21:461–6. Surgical resection is important not only for seizure control but also for pathological diagnosis. Most MAs are completely removed without recurrence. However, long-term seizures disappeared in only 43% of patients after operation, and antiepileptic drug administration was required in greater than 70% of patients [10,25]. 4. Tacconi L, Thom M, Symon L. Cerebral meningioangiomatosis: case report. Surg Neurol. 1997;48:255–60. 5. Chen YY, Tiang XY, Li Z, Luo BN, Huang Q. Sporadic meningioangiomatosis- associated atypical meningioma mimicking parenchymal invasion of brain: a case report and review of the literature. Diagn Pathol. 2010;5:39–45. 6. Omeis I, Hillard VH, Braun A, Benzil DL, Murali R, Harter DH. Meningioangiomatosis associated with neurofibromatosis: report of 2 cases in a single family and review of the literature. Surg Neurol. 2006;65:595–603. 7. Abdulazim A, Samis Zella MA, Rapp M, Gierga K, Langen KJ, Steiger HJ, et al. Meningioangiomatosis in a patient with progressive focal neurological deficient-case report and review of literature. Br J Neurosurg. 2013;27(2):253–5. Authors’ contributions SZ wrote the initial manuscript, and JF corrected grammatical errors in the text. ZJ, FY, and LW made the imaging diagnoses for the cases, and GH modified the figures. ZY revised the manuscript, and all authors read and approved the final manuscript. Discussion Predomin- antly vascular MA contains thick-walled, hyalinized, and calcified blood vessels with minimal perivascular cell proliferation, which is observed in case 1 (Figure 1e,f). Hemorrhage was commonly found in this subtype. We presumed that the MA imaging appearances could be divided into two patterns: solid and cystic. Solid MA was more common than cystic MA and demonstrated a higher probability of calcification and remarkable enhance- ment in most cases. Less than ten cases with cystic MA have been reported [18-23], including cases 2 and 3, although the total number of reported MA cases is approximately 120. Cysts were localized within or around solid tumors, one or multiple, and demonstrated micro- or macrocysts. The exact mechanism underlying MA cyst development remains controversial. Park et al. [22] specu- lated that cysts were due to the accumulation of CSF within lesions in a manner similar to the mechanism of cyst formation in cystic meningiomas. Supported evidence included cysts that were eccentric to the solid mass and It has been observed that there are no specific MA characteristics upon MRI or CT; thus, correct preopera- tive MA diagnoses were difficult. MA has always been misdiagnosed as meningioma, oligodendroglioma, gang- lioglioma, metastasis, or arteriovenous malformation. With regards to the location of lesions, they may manifest as intra-axial or extra-axial lesions with well- or ill-defined margins because of the cortex invasion. CT scans show that lesions exhibit different densities or even normal appearance, but calcification may be a specific indication for MA. The probability of calcification is variable, and the highest observed was 89.6% [14]. The signal intensity in Sun et al. World Journal of Surgical Oncology (2015) 13:89 Page 5 of 6 Page 5 of 6 adjacent to sulci. However, communication between cysts and the subarachnoid space has not been confirmed in MA [20]. Another potential mechanism is enlarged perivascular spaces in which CSF gradually accumulates, eventually resulting in the formation of cysts. In our patient, pathologic examination demonstrated that no tumor cells were present in the cyst wall. The cyst was separated from the solid portion of the tumor with no communication between the cyst and subarachnoid space. Therefore, we assumed that enlarged perivascular space may be a major reason for forming cystic MA. recovery; GFAP: glial fibrillary acidic protein; GRE: gradient echo; MA: meningioangiomatosis; MRI: magnetic resonance imaging; MRS: magnetic resonance spectrum; NAA: N-acetyl aspartate; NF: neurofibromatosis; T1WI: T1-weighted imaging; T2WI: T2-weighted imaging. 16. Rokes C, Ketonen LM, Fuller GN, Weinberg J, Slopis JM, Wolff JE. Imaging and spectroscopic findings in meningioangiomatosis. Pediatr Blood Cancer. 2009;53:672–4. 12. Kim WY, Kim IO, Kim WS, Cheon JE, Yeon KM. Meningioangiomatosis: MR imaging and pathological correlation in two cases. Pediatr Radiol. 2002;32:96–8 15. Meltzer CC, Liu AY, Perrone AM, Hamilton RL. Meningioangiomatosis: MR imaging with histopathologic correlation. AJR Am J Roentgenol. 1998;170:804–5. Sun et al. World Journal of Surgical Oncology (2015) 13:89 Abbreviations Ch h l CS 16. Rokes C, Ketonen LM, Fuller GN, Weinberg J, Slopis JM, Wolff JE. Imaging and spectroscopic findings in meningioangiomatosis. Pediatr Blood Cancer. 2009;53:672–4. 16. Rokes C, Ketonen LM, Fuller GN, Weinberg J, Slopis JM, Wolff JE. Imaging and spectroscopic findings in meningioangiomatosis. Pediatr Blood Cancer. 2009;53:672–4. Page 6 of 6 Page 6 of 6 17. Sibtain NA, Howe FA, Saunders DE. The clinical value of proton magnetic resonance spectroscopy in adult brain tumours. Clin Radiol. 2007;62:109–19. 18. Fedi M, Kalnins RM, Shuey N, Fitt GJ, Newton M, Mitchell LA. Cystic meningioangiomatosis in neurofibromatosis type 2: an MRI-pathological study. Br J Radiol. 2009;82:e129–32. 19. Wang Y, Gao X, Yao ZW, Chen H, Zhu JJ, Wang SX, et al. Histopathological study of five cases with sporadic meningioangiomatosis. Neuropathology. 2006;26:249–56. 20. Kobayashi H, Ishii N, Murata J, Saito H, Kubota KC, Nagashima K, et al. Cystic meningioangiomatosis. Pediatr Neurosurg. 2006;42:320–4. 21. Kuchelmeister K, Richter HP, Kepes JJ, Schachenmayr W. Case re 21. Kuchelmeister K, Richter HP, Kepes JJ, Schachenmayr W. Case rep microcystic meningioma in a 58-year-old man with multicystic meningioangiomatosis. Neuropathol Appl Neurobiol. 2003;29:170– 22. Park MS, Suh DC, Choi WS, Lee SY, Kang GH. Multifocal meningioangiomatosis: a report of two cases. AJNR Am J Neuroradiol. 1999;20:677–80. 22. Park MS, Suh DC, Choi WS, Lee SY, Kang GH. Multifocal 23. Li P, Cui G, Wang Y, Geng M, Wang Z. Multicystic meningioangiomatosis. BMC Neurol. 2014;14:32. 24. Feng R, Hu J, Che X, Pan L, Wang Z, Zhang M, et al. Diagnosis and surgical treatment of sporadic meningioangiomatosis. Clin Neurol Neurosurg. 2013;115(8):1407–14. 25. Seo DW, Park MS, Hong SB, Hong SC, Suh YL. Combined temporal and frontal epileptogenic foci in meningioangiomatosis. Eur Neurol. 2003;49:184–6. 25. Seo DW, Park MS, Hong SB, Hong SC, Suh YL. Combined temporal and frontal epileptogenic foci in meningioangiomatosis. Eur Neurol. 2003;49:184–6. Abbreviations Ch h l CS Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W2091057801
https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/1471-2334-13-102
English
null
The development and validation of dried blood spots for external quality assurance of syphilis serology
BMC infectious diseases
2,013
cc-by
5,193
Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Open Access © 2013 Smit et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The development and validation of dried blood spots for external quality assurance of syphilis serology Pieter W Smit1,2*, Thomas van der Vlis1, David Mabey2, John Changalucha3, Julius Mngara3, Benjamin D Clark2,3, Aura Andreasen2,4, Jim Todd2,3, Mark Urassa3, Basia Zaba2 and Rosanna W Peeling2 Abstract Background: Syphilis causes up to 1,500,000 congenital syphilis cases annually. These could be prevented if all pregnant women were screened, and those with syphilis treated with a single dose of penicillin before 28 weeks gestation. In recent years, rapid point-of-care tests have allowed greater access to syphilis screening, especially in rural or remote areas, but the lack of quality assurance of rapid testing has been a concern. We determined the feasibility of using dried blood spots (DBS) as specimens for quality assurance of syphilis serological assays. Methods: We developed DBS extraction protocols for use with Treponema pallidum particle agglutination assay (TPPA), Treponema pallidum haemagglutination assay (TPHA) and an enzyme immunoassay (EIA) and compared the results with those using matching plasma samples from the same patient. Results: Since DBS samples showed poor performance with TPHA and EIA (TPHA sensitivity was 50.5% (95% confidence interval: 39.9–61.2%) and EIA specificity was 50.4% (95% CI: 43.7–57.1%), only the DBS TPPA was used in the final evaluation. DBS TPPA showed an sensitivity of 95.5% (95% CI: 91.3–98.0%) and a specificity of 99.0% (95% CI: 98.1–99.5%) compared to TPPA using plasma samples as a reference. Conclusion: DBS samples can be recommended for use with TPPA, and may be of value for external quality assurance of point-of-care syphilis testing. Keywords: Dried blood spots, Syphilis, Treponema pallidum, DBS, Sensitivity, Evaluation therefore less suitable than point-of-care tests (POCT) for use in rural or remote locations. POCT screening tests for syphilis that are sensitive and specific in detecting trepo- nemal antibodies are now available [3]. The main advantages of POCTs are that they are easy to use, can be stored at room temperature, and can be used with whole blood, collected with a finger prick. The Global Report on Preterm Birth and Stillbirth and modelling studies have identified syphilis POCTs testing and treatment as an ur- gent priority for reducing perinatal morbidity and mortality [4-6]. Many countries have therefore started to scale up the use of POCTs in prenatal screening programmes for syphilis, but the lack of suitable methods for external qual- ity assurance (EQA) is a serious concern. * Correspondence: Pieter.smit@lshtm.ac.uk 1Leiden Cytology and Pathology Laboratory, Leiden, The Netherlands 2London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK Full list of author information is available at the end of the article Background It is estimated that the burden of congenital syphilis is large, with 1.5 million cases per year worldwide [1]. These could be prevented if all pregnant women were screened and treated with a single dose of benzathine penicillin be- fore 28 weeks gestation [2]. Syphilis testing is usually done using laboratory based assays such as Treponema pallidum haemagglutination assay (TPHA), Treponema pallidum particle agglutination assay (TPPA), rapid plasma reagin (RPR), or enzyme Immunoassay (EIA). These tests need to be used with serum or plasma samples and require a cen- trifuge, shaker, and refrigeration for the reagents. They are Most QA methods have been developed to monitor the quality of tests performed in the laboratory, and are not designed for monitoring POCT usage by healthcare Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Page 2 of 6 Page 2 of 6 workers at remote locations [7]. Dried Blood Spots (DBS) have been suggested to be a suitable EQA meth- odology for HIV POCTs, since they are easily collected, require minimal training and can be sent at ambient temperature for retesting at a centralised laboratory [8]. DBS samples have been used in prevalence studies for syphilis serology, but without prior validation of the methodology [9-11]. DBS samples have been evaluated with TPPA [12], TPHA [13], and an in-house EIA which is not commercially available [14]. The TPHA used in the study by Backhouse et al. is no longer commercially available. The fourteen year old TPPA protocol used by Coates et al. did not include a control for biologically re- active samples, and the final testing concentration of DBS eluate was more diluted than with plasma, poten- tially leading to reduced sensitivity for samples with low antibody titres [12]. The objectives of this study were to develop and validate DBS protocols for use with com- mercially available syphilis diagnostic assays, and to de- termine their performance in syphilis serological assays using plasma samples as a reference. clinicians and transported to the NIMR laboratory in Mwanza. EDTA blood samples were bar-coded in the field to ensure anonymous testing. Within 24 hours, the blood samples were centrifuged and plasma was stored in 3 aliquots (1 mL each) at −20°C. From all subjects participating in the serosurvey who opted for VCT, 1,645 samples were randomly selected for this study. All syphilis serology assays were performed according to manufacturer’s directions. Procedures For the community based HIV study, DBS samples (Proteinsaver 903 filter paper, Whatman, GE healthcare, USA) were collected by finger prick, air dried at ambient temperature for at least 3 hours and stored with desiccants in individual ziplock bags. DBS samples were transported at ambient temperature and upon arrival at the laboratory stored at −20°C. After participants donated a DBS sample, they were invited to opt in for VCT. Whole blood samples were collected from consenting participants who opted in for VCT by trained Background The positive and negative controls supplied with the kits were used with every run. The technicians were blinded to other test results and TPHA and TPPA results were read by two trained la- boratory technicians. All tests were performed in at the NIMR laboratory, which participates in the WHO EQA programs for TPPA and Rapid plasma Reagin (RPR) tests. An active syphilis case is defined as RPR positive, confirmed by a treponemal test. The results were entered using the laboratory information management system. The protocol development and evaluation was divided into two phases. In the first phase, we determined the feasibility of using DBS with TPHA, TPPA and EIA using 464 samples. During DBS protocol development, care was taken to ensure an equivalent DBS sample in- put was used for each serological method, compared to plasma. In the second phase, the serological tests that work best with DBS were selected and validated with 1,181 samples (Figure 1). Plasma samples were used as the reference standard. The RPR test was performed on plasma samples that gave discrepant results between DBS and plasma samples to determine if any active syphilis cases might have been missed. Active syphilis in- fection was defined as a positive TPPA and RPR test. Samples were prospectively collected from July through September 2010 and were tested until March 2011. Dur- ing phase 1, the preliminary evaluation of different syph- ilis serological methods with DBS samples, TPPA, TPHA and EIA were all performed from the same DBS eluate as only one DBS spot was available. As a reference method, TPPA, TPHA and EIA were performed on matching plasma samples in parallel. To ensure blinded reading in the laboratory, plasma samples were tested before DBS samples. Additionally, the three tests on DBS were performed separately from each other, to maintain blindness to other test results. Research setting The Kisesa open cohort is a well-established on-going community-based study covering six villages in northern Tanzania. The cohort study conducts regular demographic surveillance on HIV prevalence and incidence [15]. Sexual behaviour data and HIV status are collected by surveys for which all adults aged 15 years or older are eligible to par- ticipate. Study participants that opted for voluntary coun- selling and testing (VCT) were offered HIV and syphilis POCT (SD Bioline, USA) performed by trained and experienced technicians. If the VCT participant was trepo- nemal antibody positive by POCT, free medical treatment was provided according to the Tanzanian government recommendations, and all those positive for HIV were re- ferred to Tanzanian care-and treatment centres. The study was approved by the Medical Research Coordinating Committee of the National Institute for Medical Research in Tanzania (NIMR) and the ethical committee of the London School of Hygiene and Tropical Medicine. RPR Quantitative RPR (BD Macro-vue RPR, Beckton Dickinson, Sparks MD, USA) was performed according to manufacturer’s protocols using plasma samples by trained laboratory technicians. Data analysis The sensitivity, specificity and confidence intervals were calculated according to standard methods. The agree- ment between various methods was tabulated. Microsoft Excel (Microsoft, USA) and the statistical software Stata 11 (StataCorp LP, Texas, USA) were used for analysis of the results. EIA Plasma aliquots were brought to room temperature and 50 μl were used to test for treponemal antibodies by En- zyme Immuno Assay (EIA) (Lab21 Syphilis Total Anti- body EIA, Lab21 healthcare, Kentford, UK). EIA plasma tests were performed according to manufacturer’s protocols by trained laboratory technicians. A DBS EIA protocol was developed in collaboration with the developer of the assay. 40 μl DBS eluate was added and incubated for two hours at 37°C. Plates were washed five times using an automated washer, 50 μl con- jugate was added, shaken and incubated for 30 minutes at 37°C, washed five times, 50 μl substrate was added and incubated for 30 minutes at room temperature while kept in the dark. 50 μl stop solution was added and the wells were read as Optical Density (OD) 450/620 nm using an automated reader (DTX 800, Beckman Coulter, USA) with cut-off limits calculated according to the in- struction manual. The results were then entered directly into the laboratory information management system. Figure 1 Study design diagram. The number of matching plasma and DBS samples used for each phase of the study are given in the boxes on the left and the number of matching DBS and plasma samples tested for each of the assays are given in the boxes on the right. RPR has been performed on plasma, not on DBS. in 100 μl Phosphate buffered Saline (PBS) with 0.05% Tween80 in a clean 96 flat wells plate, shaken for 2 - minutes and eluted overnight at 4°C. Upon the next day, the plate was shaken 2 minutes and brought to room temperature. 25 μl sample dilution buffer was added to the first column of a clean 96 U-shaped plate, 25 μl DBS eluate was added and mixed thoroughly. 25 μl of the mixture was transferred to a second column and 25 μl sensitized particles were added to column one, 25 μl unsensitized particles to column two. Plates were covered and incubated for at least two hours at room temperature on a vibration free surface, before result in- terpretation. Discordant results between the two technicians were recorded as indeterminate. This proto- col allows TPPA and HIV serological tests to be performed from one DBS spot. TPPA A total of 1,645 plasma aliquots were brought to room temperature and 25 μl were used to test for treponemal antibodies by TPPA (Fujirebio, Tokyo, Japan), according to manufacturer’s protocols, by laboratory technicians who routinely perform TPPA on plasma samples. For DBS TPPA testing, the protocol was adjusted as follows. A 6 mm disk was manually punched and eluted Page 3 of 6 Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 1645 matching plasma and DBS samples Phase 1 464 matching samples TPPA-464 samples tested EIA-282 samples tested TPHA-445 samples tested Phase 2 1181 matching samples TPPA RPR–1171 plasma samples tested –1181 samples Figure 1 Study design diagram. The number of matching plasma and DBS samples used for each phase of the study are given in the boxes on the left and the number of matching DBS and plasma samples tested for each of the assays are given in the boxes on the right. RPR has been performed on plasma, not on DBS. For DBS TPHA testing, the following protocol was developed; 25 μl of DBS eluate (obtained as described above) was added to 25 μl sample diluent, mixed and divided over two wells (25 μl each). 75 μl test or control cells were added and incubated for 1 hour. For both TPHA and TPPA protocols, the final DBS sample elu- tion volume was kept comparable with plasma sample volume. Discordant results between the two technicians were recorded as indeterminate. matching plasma and DBS samples Phase 1 464 matching samples TPHA During the first phase of the project, protocols for DBS samples were developed for TPPA, (DBS TPPA) TPHA (DBS TPHA) and EIA (DBS EIA). 464 DBS samples were tested with TPPA, TPHA, and EIA. Table 1 shows the sensitivity and specificity of all three syphilis serology tests using DBS samples compared to plasma samples. Plasma aliquots were brought to room temperature and 25 μl were used to test for treponemal antibodies by TPHA (Lab21 syphilis TPHA, Lab21 healthcare, Kentford, UK). TPHA plasma tests were performed according to manufacturer’s protocols by trained laboratory technicians. Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Page 4 of 6 Table 1 Preliminary evaluation: performance of three syphilis serological assays using Dried Blood Spots compared to plasma Plasma as reference (using the same assay) Positive samples detected Negative samples detected Sensitivity (95% CI) * Specificity (95% CI) * DBS TPPA† (n=463) 82/96 363/367 85.4% 98.9% (76.7–91.8%) (97.2–99.7%) DBS EIA (n=282) 53/56 114/226 94.6% 50.4% (85.1–98.9%) (43.7–57.1%) DBS TPHA‡ (n=445) 46/91 353/354 50.5% 99.7% (39.9–61.2%) (98.4–100%) n= samples. * 95% confidence interval. † 1 TPPA DBS indeterminate excluded. ‡ 19 TPHA indeterminate results excluded. Table 1 Preliminary evaluation: performance of three syphilis serological assays using Dried Blood Spots compared to plasma * * performance of three syphilis serological assays using Dried Blood Spots compared to the 34 indeterminate results for DBS TPPA, 30 (88.3%) were negative and 4 (11.7%) were positive for TPPA using plasma samples. Out of the 34 indeterminate results, 15 samples were deemed indeterminate because of discord- ant readings by two technicians and 16 samples were called indeterminate by both technicians. Of the 179 TPPA positive plasma samples, 66 were RPR positive (Table 3). DBS detected 66 out of 67 samples that were plasma TPPA and RPR positive. The one discordant result was DBS TPPA indeterminate. Unfortunately it was not possible to retest the 10 false positive DBS samples since insufficient sample eluate was available by the time plasma and DBS results were compared. To obtain the titration of the eight false negative DBS samples, matching plasma samples were retested with quantitative TPPA and quanti- tative RPR, as shown in Table 4. Four plasma samples were negative when retested with TPPA, suggesting a bor- derline sample or false positive reading when initially tested. TPHA The DBS EIA was discontinued before the end of phase 1 because of the many false positive results (specificity 50.4%). The TPHA was also excluded from further testing because of low sensitivity (50.6%). To improve sensitivity of DBS TPPA, technicians were trained to use a lower cut-off for interpretation of the DBS TPPA test results, based on the agglutination patterns seen in phase 1. Reading was adjusted by altering the ag- glutination positive and negative thresholds applied for DBS samples because of the higher background compared to plasma samples (Figure 2). Phase 2: Evaluation of DBS samples in comparison to matching plasma samples For the final evaluation of DBS TPPA, 1,181 matching DBS and plasma samples were included. The average age was 31.9 years (range 15–84) and 760 participants were fe- male (64%). For 8 persons, no data on gender or age was recorded and for another 3 persons no data on gender was available. Out of the 1,181 samples, 179 (15.2%) plasma samples tested positive by TPPA (Table 2). Exclud- ing 34 indeterminate results, DBS TPPA showed a sensi- tivity of 95.5% (95% CI: 91.3–98.0%) and a specificity of 99.0% (95% CI: 98.1–99.5%) compared to TPPA plasma as the reference method. The DBS TPPA reading adjustments incorporated in phase 2 resulted in a 10.1% increase in sensitivity without compromising specificity. An overall agreement of 98.7% between the two readers was found. No non-specific reactivity (positive with unsensitized particles) with DBS samples was detected. Of Discussion This potentially makes DBS a suitable sample for EQA of POCTs in remote settings. DBS samples showed excellent sensitivity for the detec- tion of active syphilis It should be noted the study took place in Tanzania. Although tests were performed and stored according to the manufacturer’s recommendations, potential environ- mental effects by transporting and using the kits under tropical conditions could not be completely ruled out. Additionally, only one DBS spot was available per sam- ple which restricted the ability to retest discordant test results or develop appropriate procedures for indeter- minate samples. Unfortunately, we were not able to obtain acceptable performance for the use of DBS samples with the Syph- ilis Total Antibody EIA and Lab21 Syphilis TPHA. The EIA false positive results were primarily caused by a high background, possibly due to substances eluted from the filter paper and whole blood that adhered non- specifically to the wells. The TPHA false negative results were most likely caused by a reduced sensitivity when using DBS samples. DBS samples tested with TPPA gave a sensitivity of 95.5% and specificity of 99.0% compared to plasma samples. Because TPPA is an agglutination assay, experience in reading results is essential and therefore training is ne- cessary. 34 DBS samples (3%) were marked as indeter- minate due to difficulty in interpreting the results or because of discordant reading by two technicians. DBS samples can potentially be used for quantitation with TPPA, although it would require evaluation against titers obtained with plasma samples. Because of the subjectiv- ity, it is recommended that TPPA should be read by two readers. Of the eight samples that were false negative by DBS TPPA, four were negative when retested with quantita- tive TPPA on plasma samples and four were false negatives, of which two had relatively high TPPA titres (1/320 and 1/640). We tested all plasma samples using the RPR assay to determine if any of the false negative samples were from women with active syphilis, defined as being RPR and TPPA positive. Since only one false negative DBS sample was positive for RPR, TPPA using DBS TPPA has been used as a surveillance tool in a few studies [9,11,12] that used a protocol developed by Coates et al. [12]. The DBS TPPA protocol developed in this study is an improvement to the protocol developed by Coates et al. Discussion We developed and validated protocols for the use of DBS samples with various syphilis serological assays. DBS samples can be recommended for use with TPPA, and may be of value for external quality assurance of point-of-care syphilis testing. When finger-prick blood has been obtained to perform syphilis POC testing, DBS can be used to collect blood for EQA purposes directly afterwards. Finger-prick blood spotted onto filter paper can be stored and shipped at room temperature, Figure 2 DBS TPPA test result of seven patients. Test outcome (−= negative, + −= indeterminate, and + = positive) was used in phase 2 of this study. Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Page 5 of 6 Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Page 5 of 6 Table 2 Correlation between detection of Treponema pallidum antibodies by plasma TPPA and DBS TPPA (n=1147) TPPA plasma Positive Negative Total DBS TPPA Positive 169 10 179 Negative 8 960 968 Total 177 970 1147* sensitivity of DBS against plasma 95.5% (95% CI: 91.3–98,0%). specificity of DBS against plasma 99.0% (95% CI: 98.1–99.5%). * excluding 34 indeterminate results. Table 2 Correlation between detection of Treponema pallidum antibodies by plasma TPPA and DBS TPPA Table 4 Rapid Plasma Reagin (RPR) results on 8 plasma samples with false negative DBS TPPA results Sample DBS TPPA* RPR† BBI99P N N N BBI792 N N N BBI6GG N N N BBI5YI N N N BBI8ZC N 1/80 N BBI6LP N 1/160 N BBI6K6 N 1/320 N BBI6NZ N >1/640 1/64 N= negative. * Quantitative TPPA. † Quantitative RPR. Table 4 Rapid Plasma Reagin (RPR) results on 8 plasma samples with false negative DBS TPPA results sensitivity of DBS against plasma 95.5% (95% CI: 91.3–98,0%). specificity of DBS against plasma 99.0% (95% CI: 98.1–99.5%). * excluding 34 indeterminate results. sensitivity of DBS against plasma 95.5% (95% CI: 91.3–98,0%). specificity of DBS against plasma 99.0% (95% CI: 98.1–99.5%). * excluding 34 indeterminate results. allowing the samples to be transported to a central la- boratory where retesting can be done. The reduction in required materials, no cold chain requirements and min- imal training of personnel at clinic level, decreases costs considerably in comparison to standard blood collection by venepuncture [16]. Additionally, the stability of human antibodies stored on DBS has been shown to be resilient to ambient temperatures for months [17-19]. Discussion as we adjusted the elution so that the sample input into the TPPA assays from DBS and plasma are comparable. We also included unsensitized particles in the procedure to control for biologically re- active samples. Table 3 RPR titres of TPPA DBS positive samples Table 3 RPR titres of TPPA DBS positive samples Table 3 RPR titres of TPPA DBS positive samples RPR titre N=179 Negative 113 1/1 13 1/2 19 1/4 13 1/8 7 1/16 4 1/32 6 1/64 1 >1/128 3 Author details 1 1Leiden Cytology and Pathology Laboratory, Leiden, The Netherlands. 2London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK. 3National Institute for Medical Research, NIMR, Mwanza, Tanzania. 4Mwanza Intervention Trials Unit, Mwanza, Tanzania. doi:10.1186/1471-2334-13-102 Cite this article as: Smit et al.: The development and validation of dried blood spots for external quality assurance of syphilis serology. BMC Infectious Diseases 2013 13:102. Received: 6 August 2012 Accepted: 21 February 2013 Published: 26 February 2013 Received: 6 August 2012 Accepted: 21 February 2013 Published: 26 February 2013 Competing interest The authors declare that they have no competing interests. Competing interest The authors declare that they have no competing interests. Acknowledgements We are grateful to the study participants, the staff of Kisesa cohort study, the laboratory staff of the National Institute for Medical Research, Mwanza, and Mathilde E. Boon for their participation in this project. This study was funded by a grant to the UNICEF/UNDP/World Bank/WHO Special Programme on Research and Training in Tropical Diseases from the Bill & Melinda Gates Foundation (OPP 47697). The EIA and TPHA kits were kindly donated by the manufacturer for this evaluation. 18. Ferraz AS, Belo EF, Coutinho LM, Oliveira AP, Carmo AM, Franco DL, Ferreira T, Yto AY, Machado MS, Scola MC, et al: Storage and stability of IgG and IgM monoclonal antibodies dried on filter paper and utility in Neisseria meningitidis serotyping by Dot-blot ELISA. BMC Infect Dis 2008, 8:30. 18. Ferraz AS, Belo EF, Coutinho LM, Oliveira AP, Carmo AM, Franco DL, Ferreira T, Yto AY, Machado MS, Scola MC, et al: Storage and stability of IgG and IgM monoclonal antibodies dried on filter paper and utility in Neisseria meningitidis serotyping by Dot-blot ELISA. BMC Infect Dis 2008, 8:30. Foundation (OPP 47697). The EIA and TPHA kits were kindly donated by the manufacturer for this evaluation. 19. Behets F, Kashamuka M, Pappaioanou M, Green TA, Ryder RW, Batter V, George JR, Hannon WH, Quinn TC: Stability of human immunodeficiency virus type 1 antibodies in whole blood dried on filter paper and stored under various tropical conditions in Kinshasa, Zaire. J Clin Microbiol 1992, 30(5):1179–1182. Authors’ contribution 13. Backhouse JL, Lee MH, Nesteroff SI, Hudson BJ, Hamilton PA: Modified indirect hemagglutination test for detection of treponemal antibodies in finger-prick blood. J Clin Microbiol 1992, 30(3):561–563. PWS initiated the study, developed laboratory protocols and drafted the manuscript. TV developed protocols and drafted the manuscript. DM and RWP provided supervision throughout the study and made major contributions to editing the manuscript. JT was responsible for sample collection, sample process and revision of the manuscript before submission. JM and JC provided supervision for laboratory work, handling and extraction of the data. BDC provided guidance on the data analysis and participated in the interpretation of results. AA, BZ and MU planned sample collection and collaborated in writing of the manuscript. All authors read and approved the final manuscript. 14. Stevens R, Pass K, Fuller S, Wiznia A, Noble L, Duva S, Neal M: Blood spot screening and confirmatory tests for syphilis antibody. J Clin Microbiol 1992, 30(9):2353–2358. 15. Tazama project. http://www.tazamaproject.org. 16. De Castro Toledo Jr AC, Januario JN, Rezende RMS, Siqueira AL, De Mello BF, Fialho EL, Ribeiro RA, Da Silva HL, Pires EC, Simoes TC, et al: Dried blood spots as a practical and inexpensive source for human immunodeficiency virus and hepatitis C virus surveillance. Memorias do Instituto Oswaldo Cruz 2005, 100(4):365–370. 16. De Castro Toledo Jr AC, Januario JN, Rezende RMS, Siqueira AL, De Mello BF, Fialho EL, Ribeiro RA, Da Silva HL, Pires EC, Simoes TC, et al: Dried blood spots as a practical and inexpensive source for human immunodeficiency virus and hepatitis C virus surveillance. Memorias do Instituto Oswaldo Cruz 2005, 100(4):365–370. 17. Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J, Abeku T, Bousema T, Ghani AC, et al: Dried blood spots as a source of anti-malarial antibodies for epidemiological studies. Malar J 2008, 7:195. doi:10.1186/1475-2875-7-195. 17. Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J, Abeku T, Bousema T, Ghani AC, et al: Dried blood spots as a source of anti-malarial antibodies for epidemiological studies. Malar J 2008, 7:195. doi:10.1186/1475-2875-7-195. Conclusions As prenatal screening for syphilis using POCTs becomes widely implemented, an EQA method appropriate for use with blood collected by a finger prick must be developed to assure the proficiency of POC testing in rural or remote areas. The objectives of this study were to develop and validate DBS protocols for use with com- mercially available syphilis diagnostic assays, and to Smit et al. BMC Infectious Diseases 2013, 13:102 http://www.biomedcentral.com/1471-2334/13/102 Page 6 of 6 Page 6 of 6 determine their performance in syphilis serological assays using plasma samples as a reference. Based on the high sensitivity and specificity of DBS TPPA compared to plasma TPPA, DBS can be recommended for use with TPPA. Our study also showed the importance of training laboratory technicians in performing and reading the DBS TPPA, even when they are already trained in plasma TPPA. We obtained a 10.1% increase in sensitivity when technicians were more experienced in interpreting DBS TPPA agglutinations. rapid syphilis tests into an antenatal syphilis screening programme in Mwanza, Tanzania. Sex Transm Infect 2006, 82(Suppl 5):v38–v43. 6. Aledort JE, Ronald A, Rafael ME, Girosi F, Vickerman P, Le Blancq SM, Landay A, Holmes K, Ridzon R, Hellmann N, et al: Reducing the burden of sexually transmitted infections in resource-limited settings: the role of improved diagnostics. Nature 2006, 444(Suppl 1):59–72. g pp 7. Plate DK: Evaluation and implementation of rapid HIV tests: the experience in 11 African countries. AIDS Res Hum Retroviruses 2007, 23(12):1491–1498. 8. Chaillet P, Zachariah R, Harries K, Rusanganwa E, Harries AD: Dried blood spots are a useful tool for quality assurance of rapid HIV testing in Kigali, Rwanda. Trans R Soc Trop Med Hyg 2009, 103(6):634–637. 9. Dada Y, Milord F, Frost E, Manshande JP, Kamuragiye A, Youssouf J, Khelifa M, Pepin J: The Indian Ocean paradox revisited: HIV and sexually transmitted infections in the Comoros. Int J STD AIDS 2007, 18(9):596–600. References 1. Schmid GP, Stoner BP, Hawkes S, Broutet N: The need and plan for global elimination of congenital syphilis. Sex Transm Dis 2007, 34(7 Suppl):S5–S10. 1. Schmid GP, Stoner BP, Hawkes S, Broutet N: The need and plan for global elimination of congenital syphilis. Sex Transm Dis 2007, 34(7 Suppl):S5–S10. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Abbreviations DBS D i d bl d 10. Gregson S, Mason PR, Garnett GP, Zhuwau T, Nyamukapa CA, Anderson RM, Chandiwana SK: A rural HIV epidemic in Zimbabwe? Findings from a population-based survey. Int J STD AIDS 2001, 12(3):189–196. DBS: Dried blood spots; TPPA: Treponema pallidum particle agglutionation; TPHA: Treponema pallidum haemagglutination assay; EIA: Enzyme immunoassay; RPR: Rapid plasma reagin; VCT: Voluntary counselling and testing; OD: Optical density; POCT: Point of care test; QA: Quality assurance. 11. Hesketh T, Li L, Ye X, Wang H, Jiang M, Tomkins A: HIV and syphilis in migrant workers in eastern China. Sex Transm Infect 2006, 82(1):11–14. 11. Hesketh T, Li L, Ye X, Wang H, Jiang M, Tomkins A: HIV and syphilis in migrant workers in eastern China. Sex Transm Infect 2006, 82(1):11–14. 12. Coates GL, Guarenti L, Parker SP, Willumsen JF, Tomkins AM: Evaluation of the sensitivity and specificity of a Treponema pallidum dried blood spot technique for use in the detection of syphilis. Trans R Soc Trop Med Hyg 1998, 92(1):44. Submit your next manuscript to BioMed Central and take full advantage of: 2. Watson-Jones D, Gumodoka B, Weiss H, Changalucha J, Todd J, Mugeye K, Buve A, Kanga Z, Ndeki L, Rusizoka M, et al: Syphilis in pregnancy in Tanzania. II. The effectiveness of antenatal syphilis screening and single- dose benzathine penicillin treatment for the prevention of adverse pregnancy outcomes. J Infect Dis 2002, 186(7):948–957. y p and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit • Convenient online submission • Thorough peer review 3. Mabey D, Peeling RW, Ballard R, Benzaken AS, Galban E, Changalucha J, Everett D, Balira R, Fitzgerald D, Joseph P, et al: Prospective, multi-centre clinic-based evaluation of four rapid diagnostic tests for syphilis. Sex Transm Infect 2006, 82(Suppl 5):v13–v16. 4. Victora CG, Rubens CE, Group GR: Global report on preterm birth and stillbirth (4 of 7): delivery of interventions. BMC Pregnancy Childbirth 2010, 10(Suppl 1):S4. 5. Vickerman P, Peeling RW, Terris-Prestholt F, Changalucha J, Mabey D, Watson-Jones D, Watts C: Modelling the cost-effectiveness of introducing
https://openalex.org/W2811465081
https://dergipark.org.tr/en/download/article-file/484528
English
null
Effect of Tilia tomentosa methanolic extract on growth performance, digestive enzyme activity, immune response and haematological indices of common carp (Cyprinus carpio)
Marine science and technology bulletin
2,018
cc-by
6,749
Ahmed Alhadi Almabrok1,2, Iman Daw Amhamed1,2, Gamaia Ali Mohamed1,2, Soner Bilen2, Tarek Abdalsalam Salem Altief1,2* Ahmed Alhadi Almabrok1,2, Iman Daw Amhamed1,2, Gamaia Ali Mohamed1,2, Soner Bilen2, Tarek Abdalsalam Salem Altief1,2* 1 Kastamonu University, Institute of Science, Department of Aquaculture, Kastamonu, Turkey 2 Kastamonu University, Faculty of Fisheries and Aquaculture, Department of Aquaculture Kastamonu, Turkey Keywords: Tilia tomentosa Common carp Growth Haematology Digestive enzyme activity Immune indices Please cite this paper as follows: Almabrok, A.A., Amhamed, I.D., Mohamed, G.A., Bilen, S., Altief, T.A.S. (2018). Effect of Tilia tomentosa methanolic extract on growth performance, digestive enzyme activity, immune response and haematological indices of common carp (Cyprinus carpio). Marine Science and Technology Bulletin, 7(1): 12-20. Also it considered as the most growing food produce industry with an average growth rate more than 7.7% per year through the last decades, the majority of aquaculture production are come from Asia (Gjedrem et al., 2012). Approximately 600 aquatic species are raised in captivity in around 190 countries for produce in fish culture system of varying input intensity * Corresponding author E-mail address: telhasy@yahoo.com (T.A.S. Altief) Mar. Sci. Tech. Bull. (2018) 7(1): 12-20 e-ISSN: 2147-9666 info@masteb.com Mar. Sci. Tech. Bull. (2018) 7(1): 12-20 e-ISSN: 2147-9666 info@masteb.com http://dergipark.gov.tr/masteb http://www.masteb.com/ A B S T R A C T This study was conducted to determine the effect of dietary supplementation with Tilia tomentosa on the growth performance, digestive enzyme activity, haematological indices and nonspecific immune indices of juvenile common carp (Cyprinus carpio). Fish with an average weight of 4.35 ± 0.16 g were fed a diet supplemented with an aqueous methanolic extract of T. tomentosa at a dose of 0% (control), 0.01%, 0.05% or 0.1% for 45 days. The final weight, weight gain and specific growth rate were observed to be significantly higher for the 00.1% and 0.1% groups compared with the control group (P < 0.05). The feed conversion ratio was significantly decreased in the 0.05% and 0.1% groups compared with the control (P < 0.05). The activities of various digestive enzymes (amylase, lipase and trypsin) were also measured and no significant differences were observed compared to the control (P > 0.05). The mean cell volume of the 0.01% group was significantly increased compared to the control (P < 0.05) and increased lysozyme activity was observed in the 0.05% and 0.1% groups. Respiratory burst activity was significantly increased (P < 0.05) on days 15 and 30 for the 0.1% and 0.05% groups, respectively. No differences were observed for myeloperoxidase activity among the four groups. These results suggest that aqueous methanolic extract of T. tomentosa has a growth-promoting and immunostimulatory effect on common carp. Received: 04.05.2018 Received in revised form: 23.05.2018 Accepted: 23.05.2018 Available online: 30.06.2018 Preparation of T. tomentosa Extract The plants were collected from the Kastamonu province in the north of Turkey and extracted using a methanol extraction method (Pakravan et al., 2012) with some modifications (Bilen et al., 2016) as follows: All ripe parts of the fruit except the seeds were ground in a mechanical grinder to a fine powder. Every 50 g of the ground plant were mixed with 1 L of 40% methanol (Sigma-Aldrich) and the mixture was allowed to stand at room temperature for three days with brief shaking once a day to mix. The extract was then filtered through filter paper (Whatman filter paper No. 1) and the filtrate was collected and evaporated in a rotary evaporator at 55–65 °C to remove the alcohol from the fruit extract. The final crude product was dissolved in distilled water and kept in a flask at 4 °C for later experiments. 𝑊𝐺(%) = 100 × [ (𝐹𝑖𝑛𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡−𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡) 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡 ] 𝑆𝐺𝑅 (𝑤𝑒𝑖𝑔ℎ𝑡 % 𝑑−1) = 100 × [𝑙𝑛(𝐹𝑖𝑛𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡) −𝑙𝑛(𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡) 𝐸𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝐷𝑎𝑦𝑠 ] 𝐹𝐶𝑅= 𝐹𝑒𝑒𝑑 𝐼𝑛𝑡𝑎𝑘𝑒 (𝑔) 𝑊𝐺 (𝑔) 𝑆𝑅(%) = 100 × [ 𝐹𝑖𝑛𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ] 𝐹𝐶𝑅= 𝐹𝑒𝑒𝑑 𝐼𝑛𝑡𝑎𝑘𝑒 (𝑔) 𝑊𝐺 (𝑔) 𝑆𝑅(%) = 100 × [ 𝐹𝑖𝑛𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ] In these formulae, WG indicates weight gain, SGR indicates specific growth rate, FCR indicates feed conversion ratio, and SR indicates survival rate. and technological sophistication (Aklakur et al., 2015). 4.35 ± 0.16 g were obtained from a commercial fish farm in Antalya, Turkey. The fish were transported to the Faculty of Fisheries and Aquaculture, Kastamonu University, Turkey. The 480 carp were randomly divided into four groups of three replicates each (i.e. four groups of three aquariums, 12 aquariums in total) with 40 fish per aquarium. The fish were acclimatised for two weeks before the experiment started. During acclimation, the fish were fed with a commercial diet twice a day. During the experimental period, the fish were fed the commercial diet supplemented with T. tomentosa extract at the following percentages (w/w): 0%, 0.01%, 0.05%, or 0.1%. The fish were fed by hand with the experimental diet for 45 days to satiation twice a day (at 9 am and 4 pm). The fish were maintained under a natural photoperiod (12-h dark/12-h light). The water quality parameters were checked daily and were within the accepted margins throughout the experiment (dissolved oxygen, 6.8– 7.2 mg/L; pH, 7.7–8.5; water temperature, 25–28 °C). During last decades there has been a continuous growth of aquaculture industries all over the world and such intensive production would experience disease problems. Infectious diseases that occur as sporadic events in wild fish populations may cause high mortalities when appearing in intensive fish farming (Gudding et al., 1999). Enhancing the immune system of cultured fish appears to be the most promising method for preventing disease. Fish typically depend on nonspecific immune mechanisms to a much greater extent than most animals (Chakrabarti and Vasudeva, 2006). Medicinal plants contain phytochemicals, which exhibit biological activities such as the prevention of chronic degenerative diseases (Fukumoto and Mazza, 2000). Herbal medicines have been used in aquaculture to promote growth, improve the immune system, and stimulate the appetite and fight against microbes and other stressors, due to the presence of various active compounds like flavanoids, alkaloids, phenolics, pigments, steroids, terpenoids and essential oils (Citarasu, 2010). Recently, immunostimulants of herbal origin have been shown to boost the disease resistance of fish towards a number of diseases by improving their nonspecific and specific defence mechanisms (Harikrishnan et al. 2011). Indeed, many herbs have been reported to enhance the immune response of fish (Khondoker et al, 2016). Sample Collection Every 15 days of the feeding trial (i.e. on days 15, 30 and 45), three fish per aquarium (a total of nine carp from each experimental group) were randomly chosen, anaesthetised by phenoxyethanol at 0.01 mL/L and individually weighed and sampled. The kidney tissues were collected and transferred individually to 1.5 mL RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) for direct assay of the immunological parameters. Blood was collected on day 45 of the feeding trials from the caudal vein using heparinised syringes in EDTA-tubes, and later used for the direct assay of the haematological parameters. Samples of the intestine were also collected on day 45 of the feeding trials. These samples were cleaned to remove waste and all visible fat and then stored at −80 °C for the digestive enzyme assay. The Tilia tomentosa extracts contained flavonols (quercetin, kaempferol, apigenin derivatives) as principal components with the exception of a single commercial extract with hydroxycinnamic acids as the most abundant metabolites (İIeri et al., 2015). T. tomentosa is used as a medicinal plant in Turkish folk medicine (Baser et al., 2005). The present study was carried out to determine whether the T. tomentosa extract influences the digestive enzyme activity, growth, haematological parameters, or activity of the nonspecific immune response of the common carp (Cyprinus carpio). Material and Methods The weight of each fish was individually measured at the beginning and end of the study. The growth performance was calculated according to the following equations (Tekinay and Davies, 2001): Introduction *Aquaculture is the most important sector in the world. Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Digestive Enzymes The digestive enzyme activity levels for each experimental group are shown in Table 2. No significant differences in the activity of amylase, lipase or trypsin were observed for fish supplemented with 0.01%, 0.05%, or 0.1% T. tomentosa extract compared with the control group. Table 2. Digestive enzyme activity in the intestines of Cyprinus carpio supplemented with a methanolic extract of T. tomentosa for 45 days. Groups Enzymes Amylase Lipase Trypsin Control 1.84 ± 0.54 0.009a ± 0.003 0.11 ± 0.01 0.01% 1.58 ± 0.51 0.009 ± 0.001 0.08 ± 0.01 0.05% 3.95 ± 1.26 0.004 ± 0.002 0.12 ± 0.01 0.10% 3.96 ± 1.58 0.006 ± 0.015 0.08 ± 0.05 Note: Values represent the mean ± SE. Haematological Profiles White blood cell counts (WBC × 107 mm−3), red blood cell counts (RBC × 106 mm−3), haemoglobin levels (Hb, g/dL) and haematocrit measurements (Hct, %) were measured according to the methods described by Blaxhall and Daisley (1973). Blood indices included the mean cell volume (MCV, fL), mean cell Hb (MCH, pg) and mean cell Hb concentration (MCHC, %), which were calculated according to the formulae of Lewis et al. (2001). Note: Values represent the mean ± SE; different superscript letters in a column indicate significant differences between groups (P < .05). IW: initial weight, FW: final weight, WG: weight gain, FCR: feed conversion ratio, SGR: specific growth rate, SR: survival rate. Nonspecific Immune Parameters Significant increases (P < 0.05) in FW, WG and SGR were observed for fish supplemented with 0.01% and 0.1% extract compared to the control. In addition, the FCR was significantly (P < 0.05) lower in the 0.05% and 0.1% supplemented groups compared with the control group. No significant differences were observed between the FW, FCR, SGR and SR of the other treated groups compared with the control group. Head kidney cells were isolated from euthanised C. carpio according to the method of Kono et al. (2012) with slight modifications as follows. Briefly, the head kidney tissue was carefully removed and gently pushed through a 100-µm nylon mesh (John Stanier & Co., Whitefield, Manchester, UK) into RPMI-1640 medium (Invitrogen) supplemented with 5% foetal bovine serum (Invitrogen) and a 1% solution of 10,000 g/mL streptomycin plus 10,000 U/mL penicillin (Invitrogen). This mixture was then pushed through a 40-µm nylon mesh cell strainer (Becton, Dickinson & Co., Franklin Lakes, NJ, USA). The final homogenate was placed in a 3-mL Falcon tube. Head kidney cell suspensions were pelleted at 1,800 rpm for 3 min at 4 °C. After centrifugation, the supernatant was collected to measure myeloperoxidase activity using 3,3,5,5- tetramethyl benzidine hydrochloride (Sigma-Aldrich) as the substrate (Sahoo et al., 2005). The lysozyme activity was measured using a lyophilised Micrococcus lysodeikticus bacterial cell solution (Sigma-Aldrich) as the substrate (Bilen et al., 2014). Each pellet was resuspended in 1 mL of the same medium to directly assay nitroblue tetrazolium (Sigma- Aldrich) reduction, according to the method described by Biswas et al. (2013). Statistical Analysis The results were analysed with SPSS software. One-way ANOVA and Duncan’s multiple range test were used to determine the significant differences between groups. All results were expressed as the mean ± SE and P < 0.05 was considered statistically significant. Results supernatant was removed and stored at −80 °C to test for digestive enzyme activity as follows. The amylase activity was determined using 2% starch (Sigma-Aldrich) as a substrate according to the Worthington (1991) method. The lipase activity was determined using hydrolysis of 4-nitrophenyl myristate (Sigma-Aldrich) according to the method described by Gawlicka et al. (2000). The trypsin activity was determined using the method of Erlanger et al. (1961) and benzoyl-DL-arginine p-nitroanilide (Sigma-Aldrich) as the substrate. The protein concentrations were evaluated with Bradford (1976) method. The final weight (FW), FCR, SGR, WG and SR of common carp fed on the experimental diets for 45 days were determined and presented in Table 1. Table 1. Growth indices of Cyprinus carpio supplemented with a methanolic extract of T. tomentosa for 45 days. Groups IW (g) FW (g) WG (%) FCR SGR (%/day) Control 4.09 ± 0.10a 6.88 ± 0.08a 168.07 ± 5.06a 1.68 ± 0.01a 1.15 ± 0.04a 0.01% 4.06 ± 0.05a 7.71 ± 0.09b 189.87 ± 6.28b 1.78 ± 0.02a 1.42 ± 0.02b 0.05% 4.23 ± 0.06a 7.25 ± 0.07a 171.23 ± 4.57c 1.24 ± 0.01b 1.20 ± 0.02a 0.10% 4.12 ± 0.14a 7.56 ± 0.21b 183.22 ± 6.19d 1.56 ± 0.01c 1.34 ± 0.03b Note: Values represent the mean ± SE; different superscript letters in a column indicate significant differences between groups (P < .05). IW: initial weight, FW: final weight, WG: weight gain, FCR: feed conversion ratio, SGR: specific growth rate, SR: survival rate. Groups IW (g) FW (g) WG (%) FCR SGR (%/day) Control 4.09 ± 0.10a 6.88 ± 0.08a 168.07 ± 5.06a 1.68 ± 0.01a 1.15 ± 0.04a 0.01% 4.06 ± 0.05a 7.71 ± 0.09b 189.87 ± 6.28b 1.78 ± 0.02a 1.42 ± 0.02b 0.05% 4.23 ± 0.06a 7.25 ± 0.07a 171.23 ± 4.57c 1.24 ± 0.01b 1.20 ± 0.02a 0.10% 4.12 ± 0.14a 7.56 ± 0.21b 183.22 ± 6.19d 1.56 ± 0.01c 1.34 ± 0.03b Note: Values represent the mean ± SE; different superscript letters in a column indicate significant differences between groups (P < .05). IW: initial weight, FW: final weight, WG: weight gain, FCR: feed conversion ratio, SGR: specific growth rate, SR: survival rate. Experimental Design The intestine samples were homogenised with a Potter Elvehjem homogeniser in cold double-distilled water (0.1 g/mL) and centrifuged at 9,000 rpm for 20 min at 4 °C. The Common carp (C. carpio) with an average body weight of 13 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Nonspecific Immune Parameters The immunostimulatory effects of the T. tomentosa extract are shown in Figures 1, 2 and 3. The lysozyme activity of fish supplemented with different concentrations of T. tomentosa extract was increased on day 30 of the experiment; this increase was found to be significant (P ˂ 0.05) only for the 0.1% supplementation compared with the control (Figure 1). On the other hand, the lysozyme activity decreased significantly on day 45 of the experiment for the 0.01% supplementation compared with the control (P ˂ 0.05). The lysozyme activity levels of the other supplementation groups did not change significantly over time. Figure 1. Lysozyme activity in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Different symbols above the bars indicate significant differences between groups (P ˂ 0.05). Figure 1. Lysozyme activity in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Different symbols above the bars indicate significant differences between groups (P ˂ 0.05). The myeloperoxidase activity of the treated fish did not significantly differ from that of the control fish during the g y g Table 3. Haematological profiles of Cyprinus carpio supplem Table 3. Haematological profiles of Cyprinus carpio supplemented with a methanolic extract of T. tomentosa for 45 days. Nonspecific Immune Parameters Groups WBC (×107cells mm−3) RBC (×106cells mm−3) Hb (g dl−1) Hct (%) MCV (fl) MCH (pg) MCHC (%) 15th day Control 24.47 ± 0.18 1.98 ± 0.04 8.22 ± 0.07 27.82 ± 0.84 141.11 ± 7.12 41.57 ± 1.05 296.47 ± 7.63 0.01% 25.63 ± 1.26 1.86 ± 0.09 7.77 ± 0.25 27.68 ± 0.55 148.92 ± 3.73 41.76 ± 0.78 281.13 ± 4.32 0.05% 22.57 ± 1.48 1.97 ± 0.06 8.07 ± 0.10 27.88 ± 0.53 142.44 ± 6.20 41.14 ± 0.89 289.85 ± 8.25 0.10% 26.03 ± 0.97 1.98 ± 0.05 8.27 ± 0.11 28.09 ± 0.87 143.29 ± 8.22 42.02 ± 1.08 295.31 ± 10.34 30th day Control 22.61 ± 1.67 1.97± 0.08 8.37 ± 0.17 25.99 ± 0.47 132.72 ± 2.71 42.69 ± 0.89 321.95 ± 2.56* 0.01% 26.84 ± 0.91 1.76 ± 0.09 7.72 ± 0.15 26.72 ± 0.33 153.71 ± 6.98* 44.23 ± 1.42 289.10 ± 4.66 0.05% 26.34 ± 1.00 1.81 ± 0.03 7.68 ± 0.36 25.59 ± 0.73 141.85 ± 2.26 42.51 ± 1.21 300.12 ± 7.38 0.01% 26.35 ± 0.45 1.93 ± 0.07 8.08 ± 0.10 26.84 ± 0.09 140.89 ± 5.95 42.1 2± 1.32 301.50 ± 4.48 45th day Control 23.76 ± 1.85 1.90 ± 0.09 7.31 ± 0.33 25.59 ± 0.62 134.97 ± 4.07 38.45 ± 0.55 285.78 ± 7.19 0.10% 24.16 ± 1.73 2.02 ± 0.05 7.81 ± 0.19 26.35 ± 0.64 131.08 ± 4.68 38.70 ± 1.29 269.27 ± 1.26 0.50% 26.74 ± 1.13 1.97 ± 0.07 7.80 ± 0.10 26.18 ± 0.27 133.49 ± 5.52 39.65 ± 1.08 298.42 ± 6.54 0.01% 27.41 ± 0.15 1.92 ± 0.06 7.70 ± 0.23 26.87 ± 0.48 140.42 ± 4.23 40.14 ± 0.30 286.51 ± 7.17 The myeloperoxidase activity of the treated fish did not significantly differ from that of the control fish during the study period (Figure 2). treated groups on day 15 compared to the control group. On day 30 of the experiment, significantly higher levels of respiratory burst were observed for the 0.05% and 0.1% groups, while a significantly lower level was observed for the 0.01% group compared with the control (P ˂ 0.05). The same pattern was exhibited on day 45 of the experiment (P ˂ 0.05). The respiratory burst level of the 1% group was significantly lower (P ˂ 0.05) than that of the control on day 15 of the experiment (Figure 3). Haematological Profiles Fish supplemented with T. tomentosa extract at different concentrations had the same haematological parameters as 14 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 study period (Figure 2). Figure 1. Lysozyme activity in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Different symbols above the bars indicate significant differences between groups (P ˂ 0.05). the control group throughout the experimental periods (Table 3), with the exception of the MCV of the 0.01% group on day 30 of the experiment (P ˂ 0.05) and the MCHC of the control on day 30 of the experiment (highest recorded result compared with the other groups; P ˂ 0.05). the control group throughout the experimental periods (Table 3), with the exception of the MCV of the 0.01% group on day 30 of the experiment (P ˂ 0.05) and the MCHC of the control on day 30 of the experiment (highest recorded result compared with the other groups; P ˂ 0.05). Nonspecific Immune Parameters No significant differences in the respiratory burst level were observed among the other 15 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Aloe vera did not promote the growth performance of rainbow trout (Oncorhynchus mykiss). Yılmaz et al. (2012) reported that the WG, FCR and SGR of sea bass (Dicentrarchus labrax) were unaffected by 1000 mg/kg dietary rosemary (Rosmarinus officinalis) and fenugreek. Also, the growth rate of koi carp (Cyprinus carpio) was unaffected by dietary supplementation with tetra (Cotinus coggygria) (Bilen et al., 2013). Figure 2. Myeloperoxidase activity in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Figure 2. Myeloperoxidase activity in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 Digestive enzymes play a significant role in the hydrolysis of proteins, lipids and carbohydrates, thereby assisting with the assimilation of nutrients. These nutrients are transported into the tissues and incorporated into cellular materials or used as an energy source for growth and reproduction (Furne et al., 2005). The digestion of foods begins with the digestive enzymes in the stomach and continues in the intestine with the digestive enzymes secreted by the pancreas, such as trypsin, chymotrypsin, amylase and lipase (Cockson and Bourne, 1972; Moriarty, 1973; Fang and Chiou, 1989). Figure 2. Myeloperoxidase activity in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Figure 2. Myeloperoxidase activity in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Figure 3. Respiratory burst level in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Different letters above the bars indicate significant differences between groups (P ˂ 0.05). Our study did not find any significant differences in the amylase, lipase and trypsin activity levels in any of the experimental groups compared with the control group. This result is in line with a study done by Iqbal et al. (2016), who found no significant differences in the digestive enzyme activity and haematology of juvenile Labeo rohita following supplementation with different plant and animal origin feeds (fishmeal). In contrast, a study by Kawai and Ikeda (1973) reported an increase in amylase activity in O. Nonspecific Immune Parameters mykiss when fed a diet containing increased amounts of plant protein. Many fish physiologists have concentrated on haematological studies as this has proved a valuable diagnostic approach for evaluating fish quality (Kori-Siakpre et al., 2005; Oluyemi et al, 2008; Patra et al., 2014). Variations in the haematological parameters of fish are due to environmental stress (Hickey, 1982), malnutrition (Casillas and Smith, 1977), gender (Siddique and Naseem, 1979; Collazos et al., 1998), fish size (Garcia et al., 1992), seasonal differences and breeding efficiency (Cech and Wohlschlang, 1981). The blood characteristics of fish are therefore an effective and sensitive index for monitoring physiological and pathological changes (Iwama et al., 1976; Chakrabarti and Banerjee, 1988; Orun et al., 2003; Patra et al., 2014). The present study indicated no significant effect on the blood parameters of C. carpio supplemented with 0.01%, 0.05%, or 0.1% T. tomentosa extract over the study period. This result suggested that the extract tested here did not stress the fish physiologically. Figure 3. Respiratory burst level in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Different letters above the bars indicate significant differences between groups (P ˂ 0.05). Figure 3. Respiratory burst level in kidney leucocytes of Cyprinus carpio fed with different experimental diets for 45 days. Values are expressed as the mean ± SE. Different letters above the bars indicate significant differences between groups (P ˂ 0.05). Discussion In the present study, T. tomentosa was shown to promote the growth of common carp based on observations of increased WG, SGR and efficiency of feed conversion. These results agree with those of previous studies demonstrating that medicinal plants promote the growth of various aquatic animals (Kour et al., 2004; Kaleeswaran et al., 2011; Ojha et al., 2014). The WG was significantly improved when Japanese flounder (Paralichthys olivaceus) were supplemented with an herbal mixture to 500 mg/kg (Seung-Cheol et al., 2007). Most probably fat was used for energy, and protein was used for growth in the herbal-supplemented diet (Yılmaz et al., 2012). Nile tilapia fingerlings fed with a basal diet containing 0, 0.5, 1 and 1.5 g/100 g fenugreek (Trigonella foenum-graecum) seed meal for three months (Mostafa et al. 2009) and they found that the use of 1 g/100 g fenugreek seed meal improved the fish growth performance. However, although some herbs have positive effects on the fish growth (Xie et al., 2008; Mahdavi et al., 2013), other herbal supplements have not been observed to have any effects. For example, Farahi et al. (2012) found that dietary supplementation with Lysozyme is an important enzyme in the humeral nonspecific defence mechanism that provides defence against microbial invasion (Evelyn, 2002). The bactericidal action of this enzyme involves the hydrolysation of the peptidoglycan layers of the bacterial cell wall, which lyses the cells and prevents colonisation by microorganisms (Saurabh and Sahoo, 2008). Lysozyme also induces antibacterial activity in the presence of a complement (Harikrishnan et al., 2011). The present study recorded significantly increased lysozyme activity in the 1% supplemented group compared with the control. Increasing lysozyme activity is in agreement with several reports on herbal immunostimulants (Rao et al., 2006; 16 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Choi et al., 2008; Bilen et al., 2011). Similar results were observed for common carp when fed a diet supplemented with methanolic extracts of Cotinus coggygria (Bilen et al., 2014) or various Chinese herbal extracts (Jian and Wu, 2004). enhanced the non-specific immune mechanisms and disease resistance in Oreochromis mossambicus. Fish & Shellfish Immunology, 29(5): 765-772. Alishahi, M., Ranjbar, M., Ghorbanpoor, M., Peyghan, R. & Mesbah, M. (2010). Effects of dietary Aloe vera on some specific and nonspecific immunity in the common carp (Cyprinus carpio). Iranian Journal of Veterinary Medicine, 4(3): 189–195. Discussion Neutrophils contain myeloperoxidase in their cytoplasmic granules (Rodriguez et al., 2003). Myeloperoxidase is an important enzyme with microbiocidal properties as it utilises a reactive oxygen species (H2O2) to produce hypochlorous acid (Dalmo et al., 1997). This process is believed to be key in killing microorganisms (Johnston, 1978). Although some studies have shown increased myeloperoxidase activity in fish following supplementation with, for example, quercetin or black cumin seed oil (Awad et al., 2013; Alexander et al., 2010; Bilen et al. 2013), our study showed no significant effect on myeloperoxidase activity following supplementation with T. tomentosa extract at different concentrations. Awad, E., Austin, D. & Lyndon, A.R. (2013). Effect of black cumin seed oil (Nigella sativa) and nettle extract (Quercetin) on enhancement of immunity in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture, 388: 193-197. Baser, K.H.C., Tümen, G., Malyer, H. & Kirimer,N. (2006). Plants used for common cold in Turkey. In Proceedings of the IVth International Congress of Ethnobotany (ICEB 2005), 133, p. 137. Bilen, S., Biswas, G., Otsuyama, S., Kono, T., Sakai, M. & Hikima, J.I. (2014). Inflammatory responses in the Japanese pufferfish (Takifugu rubripes) head kidney cells stimulated with an inflammasome-inducing agent, nigericin. Developmental & Comparative Immunology, 46(2): 222-230. Phagocytosis and the respiratory burst response by phagocytes in blood and tissues present a major antibacterial defense mechanism in fish (Secombes, 1996). Respiratory burst activity measured by nitroblue tetrazolium (NBT) is one of the most important bactericidal mechanisms in fish (Secombes and Fletcher, 1992). In this study, the respiratory burst levels of common carp were significantly decreased on days 15, 30 and 45 of supplementation with 0.1%, 0.05% and 0.01% T. tomentosa extract, respectively, compared with the control group. For the groups supplemented with 0.05% and 0.1% extract, on days 30 and 45 of the experiment we found a significant increase in this parameter compared with the control. This was similar to results observed by Bilen et al. (2011) when O. mykiss was supplemented with Cotinus coggygria leaves. Harikrishnan et al. (2010) reported a significant increase in respiratory bursts for olive flounder supplemented with three different Korean plants. Haghighi and Rohani (2013) reported a significantly higher respiratory burst level in rainbow trout fed a commercial diet containing Zingiber officinale. Our results are also in agreement with several studies on the use of dietary immunostimulants in various fish species (Yin et al., 2009; Bilen and Bulut, 2010). Bilen, S., Ünal, S. Acknowledgement Bilen, S., Yılmaz, S., Bilen, A.M. & Biswas, G. (2014). Effects of Dietary Incorporation of Tetra (Cotinus coggygria) Extract on Immune Response and Resistance to Aeromonas hydrophila in Koi Carp (Cyprinus carpio). The Israeli Journal of Aquaculture-Bamidgeh, 66: 1-6. An earlier version of this paper has been presented at International Congress on Engineering and Life Science ICELIS 2018, Kastamonu, Turkey. Biswas, G., Korenaga, H., Nagamine, R., Kawahara, S., Takeda, S., Kikuchi, Y., Dashnyam, B., Yoshida, T. & Sakai, M. (2013). Cytokine mediated immune responses in the Japanese pufferfish (Takifugu rubripes) administered with heat-killed Lactobacillus paracasei spp. paracasei (06TCa22) isolated from the Mongolian dairy product. International Immunopharmacology, 17(2): 358-365. Discussion & Güvensoy, H. (2016). Effects of oyster mushroom (Pleurotus ostreatus) and nettle (Urtica dioica) methanolic extracts on immune responses and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss). Aquaculture, 454: 90-94. Bilen, S. & Bulut, M. (2010). Effects of laurel (Laurus nobilis) on the non-specific immune responses of rainbow trout (Oncorhynchus mykiss, Walbaum). Journal of Animal and Veterinary Advances, 9(8): 1275-1279. Bilen, S., Bulut, M. & Bilen, A.M. (2011). Immunostimulant effects of Cotinus coggyria on rainbow trout (Oncorhynchus mykiss). Fish & Shellfish Immunology, 30(2): 451-455. Bilen, S., Yılmaz, S., & Bilen, A.M. (2013). Influence of tetra (Cotinus coggygria) extract against Vibrio anguillarum infection in koi carp, Cyprinus carpio with reference to haematological and immunological changes. Turkish Journal of Fisheries and Aquatic Sciences, 13(3): 517– 522. Conflict of Interest The authors declare that there is no conflict of interest. References The effects of powdered ginger (Zingiber officinale) on the haematological and immunological parameters of rainbow trout Oncorhynchus mykiss. Journal of medicinal Plant and Herbal Therapy Research, 1(1): 8-12. Citarasu, T. (2010). Herbal biomedicines: a new opportunity for aquaculture industry. Aquaculture International, 18(3): 403-414. Haghighi, M., Sharif Rohani, M., Samadi, M., Tavoli, M., Eslami, M. & Yusefi, R. (2014). Study of effects Aloe vera extract supplemented feed on hematological and immunological indices of rainbow trout (Oncorhynchus mykiss). International Journal of Advanced Biological and Biomedical Research, 2(6): 2143-2154. Cockson, A. & Bourne, D. (1972). Enzymes in the digestive tract of two species of euryhaline fish. Comparative Biochemistry and Physiology--Part A: Physiology, 41(4): 715-718. Collazos, M.E., Ortega, E., Barriga, C. & Rodrìguez, A.B. (1998). Seasonal variation in haematological parameters in male and female Tinca tinca. Molecular and Cellular Biochemistry, 183(1-2): 165-168. Harikrishnan, R., Balasundaram, C. & Heo, M.S. (2011). Impact of plant products on innate and adaptive immune system of cultured finfish and shellfish. Aquaculture, 317(1-4): 1-15. Dalmo, R.A., Ingebrigtsen, K. & Bøgwald, J. (1997). Non‐ specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). Journal of Fish Diseases, 20(4), 241-273. Harikrishnan, R., Jin, C.N., Kim, M.C., Kim, J.S., Balasundaram, C. & Heo, M.S. (2010). Effectiveness and immunomodulation of chemotherapeutants against scuticociliate Philasterides dicentrarchi in olive flounder. Experimental Parasitology, 124(3): 306-314. Erlanger, B.F., Kokowsky, N. & Cohen, W. (1961). The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics, 95(2): 271-278. Hickey, C.R. (1976). Fish hematology, its uses and significance. NY Fish Game J, 23(2): 170-175. Evelyn, T.P.T. (2002). Finfish immunology and its use in preventing infectious diseases in cultured finfish. Diseases in Asian Aquaculture IV, (CR Lavilla-Pitogo & ER Cruz-Lacierda ed.), 303-324. Ieri, F., Innocenti, M., Possieri, L., Gallori, S. & Mulinacci, N. (2015). Phenolic composition of “bud extracts” of Ribes nigrum L., Rosa canina L. and Tilia tomentosa M. Journal of Pharmaceutical and Biomedical Analysis, 115: 1-9. Farahi, A., Kasiri, M., Sudagar, M., Soleimani Iraei, M. & Zorriehzahra, S.M.J. (2012). Effect of dietary supplementation of Melissa officinalis and Aloe vera on hematological traits, lipid oxidation of carcass and performance in rainbow trout (Oncorhynchus mykiss). Online Journal of Animal and Feed Research, 1: 1-5. Iqbal, K.J., Ashraf, M., Javid, A., Khan, N., Abbas, F., Hafeez-ur-Rehman, M., Rafique, M.K., Rasool, F., Azmat, H. & Altaf, M. (2016). References Blaxhall, P.C. & Daisley, K.W. (1973). Routine haematological methods for use with fish blood. Journal of Fish Biology, 5(6): 771-781. Aklakur, M., Rather, M.A. & Kumar, N. (2015). Nano delivery: an emerging avenue for nutraceuticals and drug delivery. Critical reviews in Food Science and Nutrition, 56(14): 2352-2361. Aklakur, M., Rather, M.A. & Kumar, N. (2015). Nano delivery: an emerging avenue for nutraceuticals and drug delivery. Critical reviews in Food Science and Nutrition, 56(14): 2352-2361. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2): 248-254. Alexander, C.P., Kirubakaran, C.J.W. & Michael, R.D. (2010). Water soluble fraction of Tinospora cordifolia leaves 17 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 haematocrit value of two teleosts: rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax L.). Comparative biochemistry and physiology. Comparative Physiology, 101(4): 733- 736. Casillas, E. & Smith, L.S. (1977). Effect of stress on blood coagulation and haematology in rainbow trout (Salmo gairdneri). Journal of Fish Biology, 10(5): 481-491. Casillas, E. & Smith, L.S. (1977). Effect of stress on blood coagulation and haematology in rainbow trout (Salmo gairdneri). Journal of Fish Biology, 10(5): 481-491. Cech Jr, J.J. & Wohlschlag, D.E. (1982). Seasonal patterns of respiration, gill ventilation, and hematological characteristics in the striped mullet, Mugil cephalus L. Bulletin of Marine Science, 32(1): 130-138. Gawlicka, A., Parent, B., Horn, M.H., Ross, N., Opstad, I. & Torrissen, O.J. (2000). Activity of digestive enzymes in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus): indication of readiness for first feeding. Aquaculture, 184(3-4): 303-314. Chakrabarty, P. & Banerjee, V. (1988). Effect of organophosphorus pesticides on the peripheral hemogram of the fish Channa punctatus. Environment and Ecology, 6(2): 390-394. Gjedrem, T., Robinson, N. & Rye, M. (2012). The importance of selective breeding in aquaculture to meet future demands for animal protein: a review. Aquaculture, 350: 117-129. Chakrabarti, R. & Vasudeva, R.Y. (2006). Achyranthes aspera stimulates the immunity and enhances the antigen clearance in Catla catla. International Immunopharmacology, 6(5): 782-790. Gudding, R., Lillehaug, A. & Evensen, Ø. (1999). Recent developments in fish vaccinology. Veterinary Immunology and Immunopathology, 72(1-2): 203-212. Choi, S.H., Park, K.H., Yoon, T.J., Kim, J.B., Jang, Y.S. & Choe, C.H. (2008). Dietary Korean mistletoe enhances cellular non-specific immune responses and survival of Japanese eel (Anguilla japonica). Fish & Shellfish Immunology, 24(1): 67-73. Haghighi, M. & Rohani, M.S. (2013). References Effect of Different Plant and Animal Origin (Fishmeal) Feeds on Digestive Enzyme Activity and Haematology of Juvenile Labeo rohita. Pakistan Journal of Zoology, 48(1): 201-207. Fukumoto, L. R. & Mazza, G. (2000). Assessing antioxidant and prooxidant activities of phenolic compounds. Journal of Agricultural and Food Chemistry, 48(8): 3597-3604. Iwama, G.K., Greer, G.L. & Larkin, P.A. (1976). Changes in some hematological characteristics of coho salmon (Oncorhynchus kisutch) in response to acute exposure to dehydroabietic acid (DHAA) at different exercise levels. Journal of the Fisheries Board of Canada, 33(2): 285-289. Furne, M., Hidalgo, M.C., Lopez, A., Garcia-Gallego, M., Morales, A.E., Domezain, A., Domezaine, J. & Sanz, A. (2005). Digestive enzyme activities in Adriatic sturgeon Acipenser naccarii and rainbow trout Oncorhynchus mykiss. A comparative study. Aquaculture, 250(1-2): 391-398. Jian, J. & Wu, Z. (2004). Influences of traditional Chinese medicine on non-specific immunity of Jian carp (Cyprinus carpio var. Jian). Fish & Shellfish Immunology, 16(2): 185-191. Garcia, M.P., Echevarria, G., Martinez, F.J. & Zamora, S. (1992). Influence of blood sample collection on the 18 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Johnston, J.R. (1978). Oxygen metabolism and the microbicidal activity of macrophages. Federation Proceedings, 37(13): 2759-2764. Chandraprakash, S.P. (2014). Effect of ethanolic extract of Mucuna pruriens on growth, metabolism and immunity of Labeo rohita (Hamilton, 1822) fingerlings. International Journal of Fauna & Biological Studies, 1(5): 1-9. Johnston, J.R. (1978). Oxygen metabolism and the microbicidal activity of macrophages. Federation Proceedings, 37(13): 2759-2764. Kaleeswaran, B., Ilavenil, S. & Ravikumar, S. (2011). Dietary supplementation with Cynodon dactylon (L.) enhances innate immunity and disease resistance of Indian major carp, Catla catla (Ham.). Fish & Shellfish Immunology, 31(6): 953-962. Oluyemi, K.G., Adecprusi, E.A. & Olanrewage, J. (2008). Basic hematological parameters in African cat fish, Clarias gariepinus (Burchell, 1822) FED ascribe acid supplemented diets. Research Journal of Animal Sciences, 2(1): 17-21. Kawai, S. & Ikeda, S. (1973). Studies in digestive enzymes of fishes. III. Development of the digestive enzymes of rainbow trout after hatching and the effect of dietary change on the activities of digestive enzymes in the juvenile stage. Bulletin of the Japanese Society for the Science of Fish, 38: 265-270. Örün, I., Dörücü, M. & Yazlak, H. (2003). Haematological parameters of three cyprinid fish species from Karakaya Dam Lake, Turkey. OnLine Journal of Biological Sciences, 3: 320-328. Pakravan, S., Hajimoradloo, A. & Ghorbani, R. (2012). References Effect of dietary willow herb, Epilobium hirsutum extract on growth performance, body composition, haematological parameters and Aeromonas hydrophila challenge on common carp, Cyprinus carpio. Aquaculture Research, 43(6): 861-869. Khondoker, S., Hossain, M.M.M., Hasan-Uj-Jaman, M., Alam, M.E., Zaman, M.F.U. & Tabassum, N. (2016). Effect of Nigella sativa (Black Cumin Seed) to Enhance the Immunity of Common Carp (Cyprinus carpio) Against Pseudomonas fluorescens. American Journal of Life Sciences, 4(3): 87-92. Patra, B.B., Panda, R.P. & Patra, A.K. (2014). Seasonal Variations in Certain Hematological Factors of Catla catla (Hamilton 1822). Journal of Applied Physics, 6: 1-7. Kono, T., Hamasuna, S., Korenaga, H., Iizasa, T., Nagamine, R., Ida, T. & Sakai, M. (2012). The role of neuromedin U during inflammatory response in the common carp. Fish & Shellfish Immunology, 32(1): 151-160. Kori-Siakpere, O., Ake, J.E.G. & Idoge, E. (2005). Haematological characteristics of the African snakehead, Parachanna obscura. African Journal of Biotechnology, 4(6): 527-530. Rao, Y.V., Das, B.K., Jyotyrmayee, P. & Chakrabarti, R. (2006). Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Fish & Shellfish Immunology, 20(3):, 263- 273. Kour, D., Sharma, L.L. & Sharma, B.K. (2004). Use of herb, Bala (Sida cordifolia Linn.) as growth promoter in the supplementary feed of Cirrhinus mrigala (Ham.). Indian Journal of Fisheries, 51(4): 501-504. Rodríguez, A., Esteban, M. & Meseguer, J. (2003). Phagocytosis and peroxidase release by seabream (Sparus aurata L.) leucocytes in response to yeast cells. The Anatomical Record, 272(1): 415-423. Kumar, I.V., Chelladurai, G., Veni, T., Peeran, S.S.H. & Mohanraj, J. (2014). Medicinal plants as immunostimulants for health management in Indian cat fish. Journal of Coastal Life Medicine, 2(6): 426- 430. Sahoo, P.K., Kumari, J. & Mishra, B.K. (2005). Non‐specific immune responses in juveniles of Indian major carps. Journal of Applied Ichthyology, 21(2): 151-155. Saurabh, S. & Sahoo, P.K. (2008). Lysozyme: an important defence molecule of fish innate immune system. Aquaculture Research, 39(3): 223-239. Lee-Shing, F. & Shu-Fen, C. (1989). Effect of salinity on the activities of digestive proteases from the tilapia fish, Oreochromis niloticus in different culture environments. Comparative Biochemistry and Physiology Part A: Physiology, 93(2): 439-443. Secombes, C.J. (1996). The nonspecific immune system: cellular defenses. The Fish Immune System: Organism, Pathogen and Environment, 15: 63-103. Lewis, S.M., Bain, B.J., and Bates, I. (2001). Dacie and Lewis Practical Haematology, 9th ed. London: Churchill Livingstone; p 633. Secombes, C.J. & Fletcher, T.C. (1992). Xie, J., Liu, B., Zhou, Q., Su, Y., He, Y., Pan, L. Ge, X. & Xu, P., (2008). Effects of anthraquinone extract from rhubarb Rheum officinale Bail on the crowding stress response and growth of common carp Cyprinus carpio var. Jian. Aquaculture, 281: 5–11. Yin, G., Ardó, L., Thompson, K.D., Adams, A., Jeney, Z. & Jeney, G. (2009). Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of Yılmaz, S., Ergün, S. & Çelik, E.Ş. (2012). Effects of herbal supplements on growth performance of sea bass (Dicentrarchus labrax): Change in body composition and some blood parameters. Journal of BioScience and Biotechnology, 1(3): 217-222. References The role of phagocytes in the protective mechanisms of fish. Annual Review of Fish Diseases, 2: 53-71. Mahdavi, M., Hajimoradloo, A. & Ghorbani, R., (2013). Effect of Aloe vera extract on growth parameters of common carp (Cyprinus carpio). World Journal of Medical Sciences, 9: 55–60. Seung-Cheol, J.I., Jeong, G., Gwang-Soon, I.M., Lee, S., Yoo, J. & Takii, K. (2007). Dietary medicinal herbs improve growth performance, fatty acid utilization, and stress recovery of Japanese flounder. Fisheries Science, 73(1): 70-76. Moriarty, D.J.W. (1973). The physiology of digestion of blue‐ green algae in the cichlid fish, Tilapia nilotica. Journal of Zoology, 171(1): 25-39. Siddiqui, A.Q. & Naseem, S.M. (1979). The haematology of Rohu, Labeo rohita. Journal of Fish Biology, 14(1): 67- 72. Mostafa, A.A.Z.M., Ahmad, M.H., Mousallamy, A. & Samir, A. (2009). Effect of using dried Fenugreek seeds as natural feed additives on growth performance, feed utilization, whole-body coMyeloperoxidase activity sition and entropathogenic Aeromonas hydrophila- challinge of monsex Nile tilapia O. niloticus (L) fingerlings. Australian Journal of Basic and Applied Sciences, 3(2): 1234-1245. Tekinay, A.A. & Davies, S.J. (2001). Dietary carbohydrate level influencing feed intake, nutrient utilisation and plasma glucose concentration in the rainbow trout, Oncorhynchus mykiss. Journal of Veterinary and Animal Sciences, 25(5): 657-666. Worthington, C.S (1991). Worthington enzyme manual related Biochemical. Freehold, New Jersey, USA. Ojha, M.L., Chadha, N.K., Saini, V.P., Damroy, S. & Ojha, 19 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 carp, Cyprinus carpio, and protection against Aeromonas hydrophila. Fish & Shellfish Immunology, 26(1): 140-145. carp, Cyprinus carpio, and protection against Aeromonas hydrophila. Fish & Shellfish Immunology, 26(1): 140-145. Yılmaz, S., Ergün, S. & Çelik, E.Ş. (2012). Effects of herbal supplements on growth performance of sea bass (Dicentrarchus labrax): Change in body composition and some blood parameters. Journal of BioScience and Biotechnology, 1(3): 217-222. 20
https://openalex.org/W2119455012
https://europepmc.org/articles/pmc3224055?pdf=render
English
null
Snake bite on scrotum – a case report
˜The œPan African medical journal
2,011
cc-by
1,600
Abstract A 22-year old man was bitten by a snake on his scrotum. This interesting and unusual case occurred in the rural area of District Aligarh, India. The uniqueness of the case lies in the fact that scrotum is an extremely rare and unusual site for snake bite. Further, with negligible local signs of envenoming the patient presented with classical signs of neurotoxicity. Due to numerous superstitions associated with snake bite, the patient was treated with traditional home made ointment before coming to hospital. The authors realized that the case may be brought to the notice of the readers because the scrotal bite by the snake with no local signs of envenomation is the first reported case. Received: 21/08/2011 - Accepted: 07/09/2011 - Published: 20/10/2011 Received: 21/08/2011 - Accepted: 07/09/2011 - Published: 20/10/2011 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Anjum Arshad1,&, Mateen Azfar1, Husain Munawwar1, Usmani A Jawed1 1Department of Forensic medicine and Toxicology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India Department of Forensic medicine and Toxicology, Jawaharlal Nehru Medical College, Aligarh Muslim University, A &Corresponding author: Anjum Arshad, Department of Forensic medicine and Toxicology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India Key words: Snake bite, scrotum, India Case presentation The patient Mr ABC (patient details kept anonymous), 22 years old male, is a resident of Iglas, a small town which is about 25 km from the main city Aligarh. In the city, adequate facilities are available for treatment of all sorts of snake bite, including critical care management in the worst eventuality. However, due to ritual-based customs and dogma, the victim was treated by home-made ointment (Figure 1). The patient was bitten by the snake at about 12.30 am and hence precious six hours were lost while the patient was shunted from local quack to “tantrik” who generally claim to have cure for all sorts of illnesses. Finally, the patient was brought to the Emergency Section of Jawaharlal Nehru Medical College Hospital, Aligarh, at 06:30 am. According to the history given by the relatives of the patient, and which was substantiated and authenticated by the patient himself upon recovery, he was sleeping on the roof-top of his mud constructed house wearing a “lungi” (the loin cloth). At 12:30 am he felt something crawling up his thighs. By the time his reflexes worked the snake bit him on the scrotum and he felt immediate pain followed by little ooze of blood from the bite site. Immediately, driven by fright he caught the snake and threw it violently on the roof. It was killed by stamping over. No effort was made to identify it whether it was the poisonous or non poisonous variety. In India, cutting across all distinction between rich or poor, educated or illiterate, it is an ingrained belief that belligerent snake should be burnt after killing as the picture of its killer gets recorded in the eyes of it. Later, people believe the female snake particularly distinguish the killer by seeing the eyes of the killed snake and take revenge. Hence the snake was not available to identify its species. Pan African Medical Journal. 2011; 10:25 This article is available online at: http://www.panafrican-med-journal.com/content/article/10/25/full/ © Anjum Arshad et al. The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page number not for citation purposes 1 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes 1 Background Since the dawn of civilization, snakes have inspired a mystic feeling of good and evil in human mind. In India popular folklores and deep rooted superstitions have put the asp in some places at the height of God and somewhere at the depth of hell as the evil incarnate. In India, snakes are found everywhere from 12,000 feet altitude of the Himalayas down to Cape Comorin, but different areas have different species preponderance. India is inhabited by more than 60 species of venomous snakes out of which only four have been popularly known to be dangerously poisonous to man; cobra, common krait, Russell viper and Saw Scaled Viper [1]. In India each year approximately 200,000 number of cases of snake bite are reported, out of which 45,000 to 50,000 succumb to death [2]. The problem is so under-rated that it was only added to WHO´s list of neglected tropical diseases in April 2009 [2]. Many cases are non poisonous in nature, but emotional calamity and fright render them disastrous to the victims and their families. The most frequent site of bites is the lower extremity [3]. However, till date snake bite on the scrotum with negligible local signs has not been reported in scientific journals. Signs and symptoms The patient presented with a classic sings of neurotoxicity. He had ptosis, drooling of saliva, sluggishness, apathy, disorientation, slurring of speech, inability to hold neck and difficulty in respiration (Figure 2). On examination of bite site, there was slight redness on inferior aspect of scrotum without any features of swelling, bruising, blistering, local bleeding, etc. The patient was shifted to ICU for support of mechanical ventilation and was promptly administered the Anti Snake Venom (ASV). The adjunct therapy included atropine, neostigmine, antibiotic, i.v. fluids etc. This standard treatment continued, and he was weaned away from the ventilator after 36 hours and later shifted to ward for observation. The patient made a remarkable recovery and was discharged without any sequelae after ten days of hospital admission. He is asked to report after 1 week for further evaluation. Page number not for citation purposes 2 Conclusions In Indian subcontinent, people particularly in rural areas sleep on roof tops, which make them prone to Snake bites. Scrotum is a very rare and unusual site for snake bite. There are numerous superstitions associated with snakes and people still rely on traditional measures as first line of treatment, and thus valuable time is lost before patient is brought to hospital. Author’s contributions AA collected the data and did analysis and interpretation of literature and made the case report; AM have been involved in collecting pictures and review articles; MH drafted the manuscript and did proof reading and JAU have given final approval of the version to be published. All authors read and approved the final manuscript. Proposal The patient was a married man who tied the nuptial knot three months before the unfortunate incident. His wife was not pregnant at the time of the tragic occurrence. It is justifiably proposed that the patient would be followed up periodically to assess his virility and fertility. Regular examination of sperm count and normalcy would be done with the consent of the patient. A second reporting shall be done after adequate follow- up of the case. Figures Figure 1: Homemade ointment rubbed over the scrotum and on bitten part (arrow) as a measure of first line of treatment in a patient victim of snake bite on scrotum Figure 2: Patient victim of a snake bite on scrotum, admitted in ICU with typical neurotoxic symptoms (ptosis and inability to hold neck) Consent Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Author’s contributions 2. WHO SEARO (2010): Guidelines on management of snake-bites. New Delhi: WHO Regional Office for South-East Asia, Available at: http://www.searo.who.int/LinkFiles/BCT_snake_bite_guidelines.pdf. Accessed 1 September 2011 1. Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM, Rodriguez PS, Mishra K, Whitaker R, Jha P; Million Death Study Collaborators. Snake bite mortality in India: A nationally representative mortality survey. PLoS Negl Trop Dis. 2011 Apr 12;5(4):e1018. This article on PubMed 3. Hati AK, Mandal M, De MK, Mukherjee H, Hati RN. Epidemiology of snake bite in the district of Burdwan, West Bengal. J Indian Med Assoc. 1992 Jun;90(6):145-7. This article on PubMed Page number not for citation purposes 4 Page number not for citation purposes References References 1. Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM, Rodriguez PS, Mishra K, Whitaker R, Jha P; Million Death Study Collaborators. Snake bite mortality in India: A nationally representative mortality survey. PLoS Negl Trop Dis. 2011 Apr 12;5(4):e1018. This article on PubMed 1. Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM, Rodriguez PS, Mishra K, Whitaker R, Jha P; Million Death Study Collaborators. Snake bite mortality in India: A nationally representative mortality survey. PLoS Negl Trop Dis. 2011 Apr 12;5(4):e1018. This article on PubMed 2. WHO SEARO (2010): Guidelines on management of snake-bites. New Delhi: WHO Regional Office for South-East Asia, Available at: http://www.searo.who.int/LinkFiles/BCT_snake_bite_guidelines.pdf. Accessed 1 September 2011 2. WHO SEARO (2010): Guidelines on management of snake-bites. New Delhi: WHO Regional Office for South-East Asia, Available at: http://www.searo.who.int/LinkFiles/BCT_snake_bite_guidelines.pdf. Accessed 1 September 2011 Page number not for citation purposes 3. Hati AK, Mandal M, De MK, Mukherjee H, Hati RN. Epidemiology of snake bite in the district of Burdwan, West Bengal. J Indian Med Assoc. 1992 Jun;90(6):145-7. This article on PubMed 3. Hati AK, Mandal M, De MK, Mukherjee H, Hati RN. Epidemiology of snake bite in the district of Burdwan, West Bengal. J Indian Med Assoc. 1992 Jun;90(6):145-7. This article on PubMed Page number not for citation purposes 4
https://openalex.org/W4389485400
https://rmdopen.bmj.com/content/rmdopen/9/4/e003507.full.pdf
English
null
Rituximab to treat prolidase deficiency due to a novel pathogenic copy number variation in<i>PEPD</i>
RMD open
2,023
cc-by
4,689
on October 23, 2024 by guest. Protected by copyrig http://rmdopen.bmj.com/ RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Autoimmunity RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 Decem CLINICAL CASE WHAT THIS STUDY ADDS ⇒We report the second large scale deletion in PEPD gene, expanding the genetic spectrum of PD. ⇒We report the second large scale deletion in PEPD gene, expanding the genetic spectrum of PD.i ►Additional supplemental material is published online only. To view, please visit the journal online (http://​dx.​doi.​org/​10.​ 1136/​rmdopen-​2023-​003507). ⇒It suggests the therapeutic efficacy of rituximab in treating PD-­associated autoimmunity. To cite: Atschekzei F, Fedchenko M, Elsayed A, et al. Rituximab to treat prolidase deficiency due to a novel pathogenic copy number variation in PEPD. RMD Open 2023;9:e003507. doi:10.1136/ rmdopen-2023-003507 WHAT IS ALREADY KNOWN ON THIS TOPIC Prolidase deficiency (PD) is a rare autosomal recessive inborn error of immunity caused by biallelic homozygous or compound heterozygous loss-­of-­function mutations in PEPD, the gene that encodes prolidase. PD typically manifests with variable dysmorphic features, chronic cutaneous ulcers, recurrent infections and autoimmune features, including systemic lupus erythematosus. So far, there is no consensus regarding treatment of PD and its autoimmune manifestations. Here, we present a 28-­year-­ old female patient with PD due to a novel homozygous intragenic deletion in PEPD, diagnosed at the age of 6 years and 7 months with an undifferentiated connective tissue disease that, apart from its very early onset, would be consistent with the diagnosis of Sjögren’s syndrome. Steroids and diverse conventional synthetic disease-­ modifying antirheumatic drugs failed to control PD-­ associated vasculitis and mucocutaneous ulcerations and led to infectious complications, including cytomegalovirus colitis. Introduction of rituximab (RTX) treatment in this patient led to sustained recession of mucocutaneous ulceration, enabling tapering of steroids. High interleukin-­ 1β (IL-­1β) production by this patient’s monocytes, together with the detection of both IL-­1β and interleukin-­18 (IL-­18) in her serum, suggest enhanced inflammasome activation in PD, whereas the therapeutic efficacy of RTX implies a role for CD20 positive B cells in the complex immunopathogenesis of PD. ⇒Prolidase deficiency (PD) is an inborn error of immu- nity associated with autoimmunity, especially with systemic lupus erythematosus (SLE). ⇒Prolidase deficiency (PD) is an inborn error of immu- nity associated with autoimmunity, especially with systemic lupus erythematosus (SLE). ⇒Prolidase deficiency (PD) is an inborn error of immu- nity associated with autoimmunity, especially with systemic lupus erythematosus (SLE). HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒This study may raise awareness among rheumatol- ogists of PD and lead to its consideration in case of patients with dysmorphic features and early-­ onset autoimmunity mimicking SLE or Sjögren’s syndrome. ⇒This study may raise awareness among rheumatol- ogists of PD and lead to its consideration in case of patients with dysmorphic features and early-­ onset autoimmunity mimicking SLE or Sjögren’s syndrome. Received 17 July 2023 Accepted 23 November 2023 Received 17 July 2023 Accepted 23 November 2023 ⇒Early consideration of rituximab treatment in pa- tients with PD may spare infectious and autoim- mune complications. Biallelic homozygous or compound hetero- zygous loss-­of-­function mutations in PEPD, the gene encoding prolidase, cause an auto- somal recessive inborn error of immunity (IEI), prolidase deficiency (PD), falling under diseases of immune dysregulation and in particular, under the subgroup of auto- immunity.3 4 Typical manifestations of PD include dysmorphic features, chronic skin ulcers, recurrent infections and features of autoimmune connective tissue diseases. Asso- ciated laboratory findings include throm- bocytopaenia, hypergammaglobulinaemia, detection of diverse autoantibodies and hypo- complementaemia. So far, 92 cases and 35 pathogenic mutant alleles have been reported worldwide.3 5 Diagnosis of PD is based on the identification of high excretion of imidodi- peptides in urine or the reduced enzymatic activity of prolidase in erythrocytes and ►Additional supplemental material is published online only. To view, please visit the journal online (http://​dx.​doi.​org/​10.​ 1136/​rmdopen-​2023-​003507). Rituximab to treat prolidase deficiency due to a novel pathogenic copy number variation in PEPD on October 23, 2024 by guest. Protected by copyright. http://rmdopen.bmj.com/ st published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from http://rmdo blished as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Faranaz Atschekzei,1,2 Mykola Fedchenko,3 Abdulwahab Elsayed,1,2 Natalia Dubrowinskaja,1 Theresa Graalmann,1,4,5 Felix C Ringshausen,5,6,7 Torsten Witte,1,2 Georgios Sogkas1,2 Faranaz Atschekzei,1,2 Mykola Fedchenko,3 Abdulwahab Elsayed,1,2 Natalia Dubrowinskaja,1 Theresa Graalmann,1,4,5 Felix C Ringshausen,5,6,7 Torsten Witte,1,2 Georgios Sogkas1,2 To cite: Atschekzei F, Fedchenko M, Elsayed A, et al. Rituximab to treat prolidase deficiency due to a novel pathogenic copy number variation in PEPD. RMD Open 2023;9:e003507. doi:10.1136/ rmdopen-2023-003507 INTRODUCTION Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with rituximab (RTX). Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line highlights upper limit of reference range). on Octob http://rmdopen.bmj.com/ 36/rmdopen-2023-003507 on 7 December 2023. Downloaded from 36/rmdopen-2023-003507 on 7 December 20 tt 6/ dope 0 3 00350 o ece be 0 3 o oaded o Figure 1  Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency (PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis (B). Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with rituximab (RTX). Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line highlights upper limit of reference range). leukocytes in patients with characteristic clinical features, which leads to genetic testing for PEPD mutations. parents of Turkish descent. She displayed dysmorphic features, including a low hairline, a depressed nasal root and micrognathia and telangiectasia predomi- nantly at her hands and feet. Since the age of 3 years and 7 months, she displayed painful mucocutaneous ulcers in her mouth, nose and feet. In addition, she displayed focal painful parchment-­like skin lesions at both her feet. She had a history of recurrent bron- chitis since the age of 2 years. At the age of 6.7 years, she was diagnosed with an undifferentiated connec- tive tissue disease, whose diagnostic workup revealed Here, we report a 28-­year-­old female with PD, displaying features of early-­onset Sjögren’s syndrome (SjS) and vasculitis, due to a novel homozygous large deletion in PEPD. Rituximab (RTX) treatment in this patient was successful in controlling vasculitis and cutaneous ulcerations. uest. Protected by copyright. INTRODUCTION Prolidase or peptidase D (PEPD) is a cytosolic metalloproteinase hydrolysing dipeptides with a C-­terminal proline or hydroxyproline.1 Proline and hydroxyproline are ubiquitous collagen amino acids, constituting more than 10% of the residues of collagen proteins.2 Hence, dipeptides produced during the catabolism of collagen are major prolidase substrates.1 Given its involvement in collagen catabolism, prolidase plays an essential role in extracellular matrix remodelling and conse- quently in wound healing and inflammation. Correspondence to Dr Georgios Sogkas; ​sogkas.​georgios@​mh-​hannover.​ de Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 1 RMD Open RMD Open RMD Open Figure 1  Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency (PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis (B). Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with rituximab (RTX) Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line on Oc http://rmdopen.bmj.com/ RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from on October 23, http://rmdopen.bmj.com/ RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from RMD Open RMD Open RMD Open Figure 1  Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency (PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis (B). Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with rituximab (RTX). Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line highlights upper limit of reference range). Figure 1  Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency (PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis (B). Case presentation The index patient (figure 1A) is the older one of two female siblings born to healthy non-­consanguineous Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 on October 23, 2024 by guest. Protected by c http://rmdopen.bmj.com/ RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Autoimmunity Autoimmunity Autoimmunity Figure 2  Histopathological findings from a patient with prolidase deficiency. Biopsy of nasal septal mucosa revealing vascular occlusion with leukocytoclasia and fibrinoid vessel wall necrosis (marked with *) (A) and immunohistochemistry of same biopsy revealing substantial C1q, C3 and IgM deposition at the wall of inflamed arterioles (B–D, respectively). Rectum biopsy (E), revealing chronic active ulcerating colitis. Splenic section after splenectomy, revealing an infarction of unknown aetiology (marked with *) (F). Liver biopsy (G), showing ectasia of small portal vein branches and adjoining sinusoids, and minimal lobular and portal hepatitis. on October 23, 2 http://rmdopen.bmj.com/ RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded f Figure 2  Histopathological findings from a patient with prolidase deficiency. Biopsy of nasal septal mucosa revealing vascular occlusion with leukocytoclasia and fibrinoid vessel wall necrosis (marked with *) (A) and immunohistochemistry of same biopsy revealing substantial C1q, C3 and IgM deposition at the wall of inflamed arterioles (B–D, respectively). Rectum biopsy (E), revealing chronic active ulcerating colitis. Splenic section after splenectomy, revealing an infarction of unknown aetiology (marked with *) (F). Liver biopsy (G), showing ectasia of small portal vein branches and adjoining sinusoids, and minimal lobular and portal hepatitis. the lungs revealed bilateral basilar bronchiectasis, when she was 20 years old (figure 1B). Same CT scan showed a thymus hyperplasia. An infection history and the latter finding led to immunological investi- gations that revealed reduced T-­cells and especially CD4+-­T cell counts (online supplemental table S1), a finding that led to cotrimoxazole prophylaxis. At the age of 23 years, she displayed ascites and sple- nomegaly and was diagnosed with portal hyperten- sion, whose aetiology remains unknown. Shortly thereafter, she developed pancytopenia attributed to hypersplenism, which led to splenectomy (figure 2F) and consequently improvement of pancytopenia. Given the refractory course of cutaneous ulcerations and pathological evidence suggesting their vasculitic aetiology, an RTX treatment was initiated at the age of 25 years. This included initially two intravenous 1 g infusions separated by 2 weeks, followed by single 1 g infusions every 6 months. Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 Case presentation Since the introduction of RTX, chronic mucocutaneous ulcerations resolved completely and C3 as well as C4 complement values remained normal. These findings associated with a sustained reduction in the European Alliance of Asso- ciations for Rheumatology SjS Disease Activity Index (figure 1D).8 Within the 44-­month follow-­up period since the introduction of RTX no new mucocuta- neous ulcerations appeared. She further displayed no additional opportunistic or other severe infections. During the aforementioned follow-­up period, only three upper respiratory tract infections (detection of Haemophilus influenzae in sputum tests in two of those) have been documented (figure 1E). A chronological fulfilled classification criteria of SjS (SjS), though very early disease onset was not typical for primary SjS.6 7 This diagnosis was made on the basis of clin- ical findings, including bilateral keratoconjuncti- vitis sicca with positive Schirmer’s test, Raynaud’s phenomenon and cutaneous vasculitis, histopatho- logical findings confirming vasculitic aetiology of mucosal ulcerations (figure 2A–D) as well as labora- tory findings and in particular, polyclonal hypergam- maglobulinaemia, hypocomplementeamia and anti- nuclear antibodies with positive Ro (SS-­A) and La (SS-­B) antibodies. Due to aforementioned diagnosis, prednisolone treatment was started, initially as intra- venous pulse treatment with a dose of 10 mg/kg and combined with a variety of conventional synthetic disease-­modifying antirheumatic drugs (csDMARDs) (methotrexate, azathioprine, ciclosporin). Aggrava- tion of ulcerations, especially at patient’s feet, led also to high-­dose immunoglobulin treatment. All aforementioned treatments failed to control chronic ulcers, which led to a bilateral Syme’s amputation at the age of 11 years and 6 months, a transtibial ampu- tation at the age of 14 years and repetitive wound debridement thereafter. At the age of 13 years, the patient presented with chronic diarrhoea and was diagnosed with colitis ulcerosa. Besides infected skin ulcers and surgical wounds, the patient displayed no discernible infections during her middle and late childhood. However, at the age of 19 years she was diagnosed with cytomegalovirus colitis that was attributed to the immunosuppressive effect of ciclo- sporin and prednisolone treatment. A CT scan of on October 23, 2024 by guest. Protected by copyright. mdopen.bmj.com/ ober 23, 2024 by guest. Protected by copyright. 3 Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 on October 23, 2 http://rmdopen.bmj.com/ RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Case presentation Downloaded from p Figure 3  Integrative genomics viewer (IGV) screenshot from whole genome sequencing (WGS) analysis of the studied patient, showing the large 3 kb deletion in PEPD, spanning exon 4 (A). Western blotting performed with peripheral blood mononuclear cell (PBMC)-­derived protein showing the loss of prolidase expression (B). Urine proline and hydroxyproline levels measured in a 24-­hour urine collection (C; red line highlights upper limit of reference range). High serum levels of interleukin-­1β (IL-­1β) and interleukin-­18 (IL-­18) in studied PD patient compared with a healthy blood donor (HD). IL-­1β and IL-­18 were measured with standard ELISAs (D). Higher IL-­1β secretion by monocytes from studied PD-­patient, compared with an HD, stimulated with lipopolysaccharide (LPS; 500 ng/mL) and (ATP; 1 mM) (E). Finally, enhanced production of tumour necrosis factor α (TNF-α) by monocytes from studied PD-­patient, compared with an HD, stimulated with ultrapure lipopolysaccharide (LPS; 500 ng/mL) (F). Figure 3  Integrative genomics viewer (IGV) screenshot from whole genome sequencing (WGS) analysis of the studied patient, showing the large 3 kb deletion in PEPD, spanning exon 4 (A). Western blotting performed with peripheral blood mononuclear cell (PBMC)-­derived protein showing the loss of prolidase expression (B). Urine proline and hydroxyproline levels measured in a 24-­hour urine collection (C; red line highlights upper limit of reference range). High serum levels of interleukin-­1β (IL-­1β) and interleukin-­18 (IL-­18) in studied PD patient compared with a healthy blood donor (HD). IL-­1β and IL-­18 were measured with standard ELISAs (D). Higher IL-­1β secretion by monocytes from studied PD-­patient, compared with an HD, stimulated with lipopolysaccharide (LPS; 500 ng/mL) and (ATP; 1 mM) (E). Finally, enhanced production of tumour necrosis factor α (TNF-α) by monocytes from studied PD-­patient, compared with an HD, stimulated with ultrapure lipopolysaccharide (LPS; 500 ng/mL) (F). summary of the disease course and immunomodula- tory medication of the patient is shown in figure 1C. patient-­derived monocytes displayed higher IL-­1β secretion as measured by ELISA (figure 3E), that was already present after stimulation with LPS only. tory medication of the patient is shown in figure 1C. Dysmorphic features, the early onset of autoimmu- nity and the multiple affected organs suggested an underlying IEI. Therefore, we initiated genetic testing, by means of targeted next-­generation sequencing, aiming at evaluating the diagnosis of an autoinflam- matory disorder and in particular, a type I interfer- onopathy, as performed previously,9 which yielded no pathogenic variant. Case presentation Thereafter, we performed whole genome sequencing (WGS), which revealed a homo- zygous intragenic deletion of approximately 3 kb in PEPD gene, spanning exon 4 (NC_000019.10:g. (33989982_33992982del); (33989982_33992982del)) (figure 3). To confirm the diagnosis of PD we initiated a 24-­hour urine collection, which led to detection of high urinary excretion of proline and hydroxy- proline, consistent with a PD-­associated imidopepti- duria. Western blotting of protein from the patient’s peripheral blood mononuclear cell (PBMC) revealed the absence of prolidase expression. Consistent with previous reports, the present patient displayed elevated serum levels of IL-­18. In addition, we detected elevated serum levels of IL-­1β. Evaluation of IL-­1β- and IL-­18 in serum has been only performed at last follow-­up visit, after introduction of RTX treat- ment and was suggestive of enhanced inflammasome activation, that however, did not correlate with clin- ically evident disease activity (figure  1D). To eval- uate the latter, monocytes were isolated from the patient’s PBMC and stimulated with LPS-­ATP. Indeed, DISCUSSION Here, we report a case of PD due to a novel large copy number variation (CNV), spanning exon 4 of PEPD, iden- tified through WGS. With the exception of a previously reported large deletion in PEPD,10 all so far reported pathogenic variants in PEPD were small scale ones.1 Considering aforementioned novel variant, 36 patho- genic variants have been reported to cause PD. Those include 16 missense/nonsense variants, 9 indels, 9 splice variants and 2 CNVs. Autoimmunity in PD can manifest as systemic lupus erythematosus (SLE)-­like disease in approximately 10% of patients ranging from typical serological evidence of SLE to severe manifestations such as nephritis and vasculitis.1 3 5 Additional immune-­related manifesta- tions include vasculitis, AIHA, dermatitis and arthritis. Very early SjS or fulfilment of the relevant diagnostic criteria6 in the present patient, expands the pheno- typic spectrum of PD. The exact mechanism of auto- immunity in PD remains unclear.1 Thymic hyperplasia in the present patient may indicate a defect in central tolerance.11 Consistent with previous reports,3 5 the present patient displayed elevated serum levels of IL-­18, suggesting increased inflammasome activation, which was confirmed by the higher LPS and ATP-­induced IL-­1β secretion by patient-­derived monocytes. The efficacy of ober 23, 2024 by guest. Protected by copyright. est. Protected by copyright. Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 4 Autoimmunity Autoimmunity Autoimmunity RTX in controlling PD-­associated autoimmunity suggests the pathogenic relevance of autoantibodies and new plasma cell differentiation (as targeted CD20+ B cells are required intermediary cells) or the pathogenicity of alternative B cell functions other than the production of autoantibodies, such as their antigen-­presenting role.12 13 6Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School, Hannover, Germany 7European Reference Network on Rare and Complex Respiratory Diseases (ERN-­ LUNG), Frankfurt, Germany Acknowledgements  We thank all nurses and physicians of the outpatient clinics of the department of Rheumatology and Immunology of the Hannover Medical School for collecting blood samples and documenting patient’s course and medications. DISCUSSION g p g Efforts to treat PD with replacement of prolidase activity included blood transfusions, gene therapy with an adenoviral vector and enzyme replacement with liposome-­coated prolidase.3 5 All those approaches were of limited efficacy.3 Allogenic hematopoietic stem cell transplantation (HSCT) has been tried in a single patient, who despite reconstitution of prolidase activity died 3 months after HSCT of an invasive fungal infection.14 In this case, steroids, diverse csDMARDs and high-­dose intravenous immunologlobulin treatment were unsuc- cessful in treating PD and associated immune dysregu- lation. Remission of vasculitis and consequently steroid tapering were only possible after the introduction of RTX treatment. Consistent with a previous report by Sato et al, reporting the efficacy of RTX as an induction treat- ment for lupus nephritis and skin ulcers in a 16-­year-­old male with PD,15 here we report sustained regression of vasculitis and mucocutaneous ulcers in a patient with PD. In case of presented patients, csDMARDs were prior- itised and repetitive surgical interventions and infectious complications led to a relatively late introduction of RTX. This case together with the report by Sato et al suggest the early consideration of RTX treatment in patients with PD displaying autoimmunity. http://rmdope hed as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Contributors  GS and FA conceived and planned the study. GS took the lead in writing the manuscript. FA, MF, TG, FCR and TW significantly contributed to drafting and revision of the paper. FA and ND performed GS. AE performed western blotting and ELISAs. GS, TG, FCR and TW were main treating physicians of studied patient. All authors approved the final version. Funding  This project was primarily funded by the Rosemarie-­Germscheid foundation. It was additionally supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC 2155 'RESIST'—Project ID 39087428 and the German network for multiorgan autoimmune diseases (GAIN_01GM2206B) and the German Center for Infection Research (DZIF TTU 01.801). Competing interests  None declared. Patient consent for publication  Consent obtained directly from patient(s). Ethics approval  This study involves human participants and this study was conducted in accordance with the Declaration of Helsinki and was also approved from the Ethical committee of the Hannover Medical School (approval number: 5582; 8875_BO_K_2020). All patients signed an informed consent form. Participants gave informed consent to participate in the study before taking part. Provenance and peer review  Not commissioned; externally peer reviewed. DISCUSSION Supplemental material  This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-­reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. on October 23, 2024 by guest. Protected by http://rmdopen.bmj.com/ 23. Downloaded from p y g y Identification of IEIs among patients with well-­ classifiable rheumatic disorders can be a clinically rele- vant diagnostic challenge for rheumatologists.16 Red flags suggesting an underlying IEI in rheumatic patients include infectious complications, persistent secondary hypogammaglobulinaemia and secondary haemophago- cytic lymphohistiocytosis or macrophage activation syndrome.16–19 In the present case and overall in PD, early-­onset treatment-­refractory connective tissue disease associating with dysmorphic features should lead to the diagnostic consideration of PD. In addition, chronic typi- cally very painful ulcers and atrophic stellate scars, espe- cially on the feet (atrophie blanche) are very suggestive for this rare disorder.5 20 Raising awareness of PD and other rare IEIs, especially disorders of immune dysreg- ulation whose clinical spectrum overlaps with rheumatic conditions, may lead to timely genetic diagnosis and improved clinical outcomes. Open access  This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/​ licenses/by/4.0/. REFERENCES 1 Eni-­Aganga I, Lanaghan ZM, Balasubramaniam M, et al. PROLIDASE: a review from discovery to its role in health and disease. Front Mol Biosci 2021;8:723003. 1 Eni-­Aganga I, Lanaghan ZM, Balasubramaniam M, et al. PROLIDASE: a review from discovery to its role in health and disease. Front Mol Biosci 2021;8:723003. 2 Krane SM. The importance of proline residues in the structure, stability and susceptibility to proteolytic degradation of collagens Amino Acids 2008;35:703–10. 2 Krane SM. The importance of proline residues in the structure, stability and susceptibility to proteolytic degradation of collagens. Amino Acids 2008;35:703–10. 3 Spodenkiewicz M, Spodenkiewicz M, Cleary M, et al. Clinical genetics of prolidase deficiency: an updated review. Biology (Basel) 2020;9:108. i 4 Tangye SG, Al-­Herz W, Bousfiha A, et al. Human inborn errors of immunity: 2022 update on the classification from the International Union of immunological societies expert committee. J Clin Immunol 2022;42:1473–507. 5 Alrumayyan N, Slauenwhite D, McAlpine SM, et al. Prolidase deficiency, a rare inborn error of immunity, clinical phenotypes, immunological features, and proposed treatments in twins. Allergy Asthma Clin Immunol 2022;18:17. ; 6 Shiboski CH, Shiboski SC, Seror R, et al. American college of rheumatology/European league against rheumatism classification criteria for primary Sjögren’s syndrome: a consensus and data-­ driven methodology involving three international patient cohorts. Arthritis Rheumatol 2017;69:35–45. Author affiliations 1 1Rheumatology and Immunology, Hannover Medical School, Hannover, Germany 2Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany 3 1Rheumatology and Immunology, Hannover Medical School, Hannover, Germany 2Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany 7 Yayla ME, Karaman Z, Torgutalp M, et al. Early onset primary Sjögren syndrome, clinical and laboratory characteristics. Clin Rheumatol 2020;39:2689–96. 3Institute of Pathology, Hannover Medical School, Hannover, Germany 4Junior Research Group for Translational Immunology, TWINCORE, Center for Infection Research and the Hannover Medical School, Hannover, Germany 5Biomedical Research in End-­Stage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), Hannover, Germany 8 Seror R, Bowman SJ, Brito-­Zeron P, et al. EULAR Sjögren’s syndrome disease activity index (ESSDAI): a user guide. RMD Open 2015;1:e000022. Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 5 on October 23, 2024 by guest. Protected by c http://rmdopen.bmj.com/ RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from RMD Open RMD Open RMD Open 20 Dunn R, Varigos G, Winship I. A photographic essay of prolidase deficiency. Clin Dysmorphol 2011;20:194–9. RMD Open RMD Open RMD Open 15 Sato S, Ohnishi T, Uejima Y, et al. Induction therapy with rituximab for lupus nephritis due to prolidase deficiency. Rheumatology (Oxford) 2020;59:e57–9. 9 Atschekzei F, Dubrowinskaja N, Anim M, et al. Identification of variants in genes associated with autoinflammatory disorders in a cohort of patients with psoriatic arthritis. RMD Open 2022;8:e002561. 15 Sato S, Ohnishi T, Uejima Y, et al. Induction therapy with rituximab for lupus nephritis due to prolidase deficiency. Rheumatology (Oxford) 2020;59:e57–9. ( ) 16 Sogkas G, Witte T. The link between rheumatic disorders and inborn errors of immunity. EBioMedicine 2023;90:104501. 10 Hintze JP, Kirby A, Torti E, et al. Prolidase deficiency in a Mexican-­ American patient identified by array CGH reveals a novel and the largest PEPD gene deletion. Mol Syndromol 2016;7:80–6. 17 Sogkas G, Adriawan IR, Ringshausen FC, et al. A novel NFKBIA variant substituting serine 36 of IκBα causes immunodeficiency with warts, bronchiectasis and juvenile rheumatoid arthritis in the absence of ectodermal dysplasia. Clin Immunol 2020;210:108269. 11 Sogkas G, Atschekzei F, Adriawan IR, et al. Cellular and molecular mechanisms breaking immune tolerance in inborn errors of immunity. Cell Mol Immunol 2021;18:1122–40. http://rmdopen shed as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from y p 18 Zhang M, Behrens EM, Atkinson TP, et al. Genetic defects in cytolysis in macrophage activation syndrome. Curr Rheumatol Rep 2014;16:439. 12 Randall KL. Rituximab in autoimmune diseases. Aust Prescr 2016;39:131–4. i 19 Sogkas G, Dubrowinskaja N, Adriawan IR, et al. High frequency of variants in genes associated with primary immunodeficiencies in patients with rheumatic diseases with secondary hypogammaglobulinaemia. Ann Rheum Dis 2021;80:392–9. 13 Kurien BT, D’Sousa A, Bruner BF, et al. Prolidase deficiency breaks tolerance to lupus-­associated antigens. Int J Rheum Dis 2013;16:674–80. 19 Sogkas G, Dubrowinskaja N, Adriawan IR, et al. High frequency of variants in genes associated with primary immunodeficiencies in patients with rheumatic diseases with secondary hypogammaglobulinaemia. Ann Rheum Dis 2021;80:392–9. 14 Caselli D, Cimaz R, Besio R, et al. Partial rescue of biochemical parameters after hematopoietic stem cell transplantation in a patient with prolidase deficiency due to two novel PEPD mutations. JIMD Rep 2012;3:71–7. 14 Caselli D, Cimaz R, Besio R, et al. Partial rescue of biochemical parameters after hematopoietic stem cell transplantation in a patient with prolidase deficiency due to two novel PEPD mutations. JIMD Rep 2012;3:71–7. 20 Dunn R, Varigos G, Winship I. A photographic essay of prolidase deficiency. Clin Dysmorphol 2011;20:194–9. RMD Open RMD Open RMD Open 6 Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507
https://openalex.org/W4306145593
https://zenodo.org/records/7192713/files/ZDIT2488.pdf
Latin
null
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Zenodo (CERN European Organization for Nuclear Research)
2,022
cc-by
2,564
«Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS Meliyev Noʻmonxon Ibodulla oʻgʻli Toshkent state university of oriental studies Assistant teacher https://doi.org/10.5281/zenodo.7192713 «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya “In fact, the "fire of war" was imposed on the Afghan people from the outside, this is not their choice. Over the years, hundreds of thousands of civilians have become victims of the confrontation, and millions of people were forced to leave their homes and seek refuge in other countries. The involvement of more and more forces in the conflict led to its unprecedented escalation. It has ceased to be purely Afghan, becoming more and more of a complex international problem. The expansion of the presence of international terrorist groups in Afghanistan, the ongoing violence and bloodshed, the drug business - all this indicate the inadmissibility of the world community ignoring the situation in this country. Most importantly, an entire generation has grown up in Afghanistan in conditions of armed confrontation and violence. But, nevertheless, this is not the "lost generation", as some experts cynically claim. These are just people who are tired of war, deprivation and hardships, who want and strive to put an end to hostility, return to peaceful life and creation for the benefit of their country. I am firmly convinced that the Afghan people have the strength, wisdom, courage and resilience to start a new, peaceful life, build it for the well-being of their children and future generations...” [1] These words were voiced by the President of Uzbekistan during his speech at the Tashkent conference. Where in this way he opened a new page in the relationship between Afghanistan and Uzbekistan, and not only. Now the Republic of Uzbekistan has determined for itself new priorities in foreign policy: economic openness and attractiveness of the country for investment and business; focus on solving all problematic issues on the basis of consensus, mutual respect and understanding; balanced and mutually beneficial relations with world powers and other states; the desire for interaction on the basis of political trust and respect for international law - this is how the country's current foreign policy is trying to be built. Before turning to specific areas of foreign policy, I would like to say a few words about how the country's foreign policy priorities are formed in principle. It is known that, speaking of priorities, many people have in mind bilateral relations with certain countries, especially singling out the world's leading states with significant political, economic, military and other potential. ` «Zamon va ama ` «Zamon va ama «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS Meliyev Noʻmonxon Ibodulla oʻgʻli Toshkent state university of oriental studies Assistant teacher https://doi.org/10.5281/zenodo.7192713 This is of course obvious and even necessary. 354 354 «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya However, it is also necessary to proceed from what the main tasks in general hould be solved by foreign policy. The most important of them is the creation of avorable external conditions for the successful internal development of the state and society, the implementation of reforms, ensuring the growth of the welfare of he population, and improving the quality of life. The second task is to form a belt of peace stability and security around ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariy va amaliyot» nomli ilmiy, masofaviy, onlayn konferensi However, it is also necessary to proceed from what the main tasks in general should be solved by foreign policy. The most important of them is the creation of favorable external conditions for the successful internal development of the state and society, the implementation of reforms, ensuring the growth of the welfare of the population, and improving the quality of life. The second task is to form a belt of peace, stability and security around Uzbekistan. It is security in the region where the Republic of Uzbekistan is located that is the main condition for sustainable development. From which we can see that Afghanistan occupies a special place in the foreign policy of the Republic of Uzbekistan. Today, Uzbekistan and Afghanistan are developing close political, trade, economic, cultural and humanitarian relations. The security of Afghanistan is perceived as the security of Uzbekistan, a guarantee of stability and prosperity for the entire vast region. Uzbekistan's policy towards Afghanistan is based on the understanding that the prospects for stable development in Central Asia are closely linked to the achievement of peace in neighboring Afghanistan. ` y y q q y va amaliyot» nomli ilmiy, masofaviy, onlayn konferens However, it is also necessary to proceed from what the main tasks in general should be solved by foreign policy. The most important of them is the creation of favorable external conditions for the successful internal development of the state and society, the implementation of reforms, ensuring the growth of the welfare of the population, and improving the quality of life. However, it is also necessary to proceed from what the main tasks in general should be solved by foreign policy. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS Meliyev Noʻmonxon Ibodulla oʻgʻli Toshkent state university of oriental studies Assistant teacher https://doi.org/10.5281/zenodo.7192713 On the other hand, Uzbekistan is trying to pursue its own economic policy in relation to Afghanistan, and the northern regions of the country, in particular.[2] 355 355 ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariy va amaliyot» nomli ilmiy, masofaviy, onlayn konferens It should be noted that the participation of Uzbekistan in the Afghan processes remains hardly noticeable in the international information environment. This is largely due to the weak information policy of Tashkent and the lack of a PR component of foreign policy. At the same time, Uzbekistan remains one of the most active actors in Afghanistan. After declaring independence in 1991, Uzbekistan found itself in a rather difficult situation. Internal problems associated with the need to reform the public administration system, the transition to a market economy, the implementation of social reforms, and the prevention of interethnic conflicts put serious pressure on the stability of the state. At the same time, external factors related to the destabilization of neighboring Afghanistan and Tajikistan also raised concerns. In this regard, the Afghan issue has firmly taken its place as one of the highest priorities on the foreign policy agenda of Uzbekistan. Tashkent tried to make the most of the stands of international organizations to draw the attention of the international community to the aggravation of the situation in the neighboring country. In the 90s. the process of forming the position of Uzbekistan on the events in Afghanistan took place. Tashkent identified the destabilization of Afghanistan as the main threat to the security of all of Central Asia. At the same time, Uzbekistan advocated a peaceful settlement of the conflict through negotiations. In addition, great importance, in the opinion of the Uzbek side, should have been given to ensuring conditions for disarming the parties and preventing the use of force. In particular, during the 50th session of the UN General Assembly in 1995, Tashkent put forward an initiative to impose an embargo on the supply of weapons to Afghanistan.[3] ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen It should be noted that the participation of Uzbekistan in the Afghan processes remains hardly noticeable in the international information environment. This is largely due to the weak information policy of Tashkent and the lack of a PR component of foreign policy. At the same time, Uzbekistan remains one of the most active actors in Afghanistan. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS Meliyev Noʻmonxon Ibodulla oʻgʻli Toshkent state university of oriental studies Assistant teacher https://doi.org/10.5281/zenodo.7192713 The most important of them is the creation of favorable external conditions for the successful internal development of the state and society, the implementation of reforms, ensuring the growth of the welfare of the population, and improving the quality of life. From which we can see that Afghanistan occupies a special place in the foreign policy of the Republic of Uzbekistan. Today, Uzbekistan and Afghanistan are developing close political, trade, economic, cultural and humanitarian relations. The security of Afghanistan is perceived as the security of Uzbekistan, a guarantee of stability and prosperity for the entire vast region. Uzbekistan's policy towards Afghanistan is based on the understanding that the prospects for stable development in Central Asia are closely linked to the achievement of peace in neighboring Afghanistan. According to the Concept of Foreign Policy of Uzbekistan, adopted in 2012, ensuring a peaceful settlement of the conflict, stabilizing Afghanistan and building pragmatic relations with the southern neighbor are among the main priorities of the Uzbek foreign policy. The Uzbek strategy towards Afghanistan began to take shape as early as AD. 90s The position of Uzbekistan on the Afghan crisis, announced during the 48th and 50th sessions of the UN General Assembly in 1993 and 1995 hasn't changed much in the last 20 years. Tashkent believes that the settlement of the conflict is possible only through diplomatic means, without the use of force. In addition, special attention is expected to be paid to building a strong vertical of power, economic recovery, and creating conditions for the observance of the rights of all ethnic and national minorities in Afghanistan. The Uzbek strategy is being implemented in two directions. On the one hand, Tashkent participates in the process of resolving the Afghan crisis at the political and diplomatic level: the initiative to create the Contact Group "6 + 2" (1997), proposals to impose an embargo on arms supplies to Afghanistan (1995) and the demilitarization of the country (2001). .), calls to resume the activities of the Contact Group (2008), the provision of territory for the transportation of goods of the international coalition. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS Meliyev Noʻmonxon Ibodulla oʻgʻli Toshkent state university of oriental studies Assistant teacher https://doi.org/10.5281/zenodo.7192713 The achievements of Uzbekistan in this matter is that on March 26-27, 2018, Tashkent hosted a High-Level International Conference on Afghanistan, which was attended by the President of the IRA A. Ghani, the High Representative of the European Union for Foreign Affairs and Security Policy F. Mogherini and representatives member states of the UN Security Council. It was the first time that such a large-scale forum dedicated to solving the problem of Afghanistan was held at such a high level. The main result and success of the Tashkent conference is that it was possible to develop and approve a consolidated international approach to the issue of settling the Afghan crisis. All this is reflected in the Tashkent Declaration, which was recognized by the absolute majority of the states involved in the Afghan dialogue. The Tashkent conference and, in general, Uzbekistan's policy towards Afghanistan led to the intensification of international efforts to resolve the Afghan 356 356 ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariy va amaliyot» nomli ilmiy, masofaviy, onlayn konferens conflict and involve this country in regional trade, economic and infrastructure projects that should help advance the peace process. Uzbekistan began economic cooperation with Afghanistan in 2002. This cooperation affects several aspects: infrastructure construction, energy and trade. The opening of the Hairatan bridge on the Uzbek-Afghan border in 2002 was the first step towards the implementation of the strategy. Already the following year, the Airitom customs post began to function in Termez. These steps were aimed at ensuring the delivery of humanitarian aid to Afghanistan.[4] In addition, at the request of the Afghan government, Uzbekistan restored 11 bridges between Mazar-i-Sharif and Kabul that were destroyed during the civil war. This was of strategic importance, because. these bridges link the northern and eastern regions of Afghanistan and are an important component for economic recovery in the region.[5] Much attention is paid to the development of transport communications. In 2008, the Asian Development Bank (ADB), Uzbekistan and Afghanistan signed a protocol of understanding to expand cooperation in the field of railway construction. In the period 2008-2010. Uzbekistan Railways (Uzbek Railways) implemented the ADB-financed Hairatan-Mazari-Sharif railway construction project with a total cost of $170 million. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS Meliyev Noʻmonxon Ibodulla oʻgʻli Toshkent state university of oriental studies Assistant teacher https://doi.org/10.5281/zenodo.7192713 Источник: Пресс-служба Президента Республики Узбекистанhttp://isrs.uz/ru/page/pdf/o-mezdunarodnoj-konferenci-po- afganistanu afganistanu [2] Скромный актор в афганском урегулировании: стратегия Узбекистана в Афганистане 21 августа, 2015 Юрий Саруханян [3] http://www.press-service.uz/ru/document/4825/ [4] Akmalov, Shoislam, 2009 « Uzbekistan’s Role in Stability and Development of Afghanistan », in Policy Perspectives http://www.ips.org.pk/pakistan-and-its- neighbours/1048-uzbekistans-role-in-stability-and-development-of- afghanistan.html [5] Эргашев, Бахтиер, 2012 «Узбекистан и Афганистан: на пути к региональной безопасности в Центральной Азии» http://www.12news.uz/news/2012/12/uzbekistan-i-afganistan-na-puti-k- regi/ [6] http://www.railway-technology.com/projects/hairatanuzbekistanra/ [7] «Узбекистан в 2013 году приступит к строительству ж/д Мазари- Шариф-Андхой», 2013 http://www.12news.uz/news/2013/02/uzbekistan-v- 2013-godu-pristupit-k-stroit/ [7] «Узбекистан в 2013 году приступит к строительству ж/д Мазари- Шариф-Андхой», 2013 http://www.12news.uz/news/2013/02/uzbekistan-v- 2013-godu-pristupit-k-stroit/ «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari va amaliyot» nomli ilmiy, masofaviy, onlayn konferen FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS Meliyev Noʻmonxon Ibodulla oʻgʻli Toshkent state university of oriental studies Assistant teacher https://doi.org/10.5281/zenodo.7192713 Its length is 75 km, the volume of cargo transportation is 7 million tons with a possible increase to 20 million tons.[6] According to official data, in 2013 Tashkent began construction of a 230- kilometer section of the railway between Mazar-i-Sharif and Andkhoy, which is part of the Sherkhan-Bandar-Kunduz-Khulm-Naybabad-Andkhoy-Herat railway project, implemented under the program of the Central -Asian Regional Economic Cooperation (CAREC).[7] It should be noted that such projects are important for strengthening the Afghan economy by uniting the Afghan provinces through the transport network. At the same time, the effective functioning of transport corridors is an opportunity for Uzbekistan to reduce the negative impact of the country's isolation and gain access to South Asian markets and the Persian Gulf. Despite the difficult situation, the long-term systemic crisis in Afghanistan, the foreign policy initiative of Uzbekistan calls on the countries of the region to consider Afghanistan as a friendly partner and neighbor. Analyzing the priority directions of the foreign policy of the Republic of Uzbekistan, which pursue different goals, one can come to the conclusion that this state is taking all measures to resolve the Afghan conflict, namely political and economic measures. The foreign policy strategy of Uzbekistan is capable of ensuring a sufficiently effective participation in the processes of settling the Afghan conflict, both at the regional level and within the framework of bilateral relations. Despite the 357 ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya scenario of further development of the situation in the country, Uzbekistan remains interested in minimizing the negative impact of Afghanistan on the ` «Zamon va amal «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya scenario of further development of the situation in the country, Uzbekistan remains interested in minimizing the negative impact of Afghanistan on the countries of the region and continues to play an important role in resolving the Afghan military-political crisis. [1] Выступление Президента Республики Узбекистан Шавката Мирзиёева на международной конференции по Афганистану: "Мирный процесс, сотрудничество в сфере безопасности и региональное взаимодействие" 27 мар. 2018 г.
https://openalex.org/W2131746445
https://zenodo.org/record/2312946/files/article.pdf
English
null
LXXVII.—Steric influence: static and dynamic. Part II
Journal of the Chemical Society. Transactions
1,915
public-domain
2,628
View Article Online / Journal Homepage / Table of Contents for this issue View Article Online / Journal Homepage / Table of Contents for this issue 728 DAVIS AND RIXON : STERIC lNFLUENCE : By OLIVER CHARLES MINTY DAVIS arid FREDERIC WILLIAM RIXON. IN a previous communication (Davis, ZeitscA. physikal. Chern., 1912, 78, 353) the results of an investigation on the interaction of formic acid with aniline and its congeners were described, the reaction being expressed by the general equation : H*CO,H + R*NH, R*NH*COH + H,O. View Article Online View Article Online 729 STATIC AND DYNAMIC. rmr 11. I n such a system it is possible to measure under suitable con- ditions : d on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 915. Downloaded by University of Chicago on 29/10/2014 17:47:52. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. (1) The velocity of formation of the anilide. (1) The velocity of formation of the anilide. ( ) y (2) The velocity of decomposition of the anilide. y (2) The velocity of decomposition of the anilide. ( ) y p (3) The position of the point of equilibrium obtained by a direct method. (3) The position of the point of equilibrium obtained by a direct method. The research described dealt almost exclusively with the third possibility above mentioned, and numerous equilibrium points were determined by a static method. The main object of the present iiivestigatioii was to obtain three independent sets of data by a study of the measurements enumerated above, with a view of Published on 01 January 1915. Downloaded by University of Chicago on 29/10 Published on 01 January 1915. Downloaded by University of Chicag Frc. 1. Decomposition of for Inn 11 ilicle. Published on 01 January 1915. Downloaded by Univ l6 I .4 P 12 5 8 4 / / / / /* / / / / 1 / / # / # &#* AM’ 4 8 12 16 20 / / / / /* / / / / 1 / / # / # &#* AM’ 4 8 12 16 20 Hozrrs. cornparing their relative value, as applied particularly to steric influence. I n order to enable such a comparison to be made, the three setis of data have been determined, working under conditions as similar as possible in all cases. By OLIVER CHARLES MINTY DAVIS arid FREDERIC WILLIAM RIXON. l6 I .4 P 12 5 8 4 / / / / /* / / / / 1 / / # / # &#* AM’ 4 8 12 16 20 / / / / /* / / / / 1 / / # / # &#* AM’ 4 8 12 16 20 Hozrrs. .4 P 5 8 Hozrrs. cornparing their relative value, as applied particularly to steric influence. I n order to enable such a comparison to be made, the three setis of data have been determined, working under conditions as similar as possible in all cases. Experimental Conditio?ts.-A solvent was employed consisting of two volumes of pyridine and one of water, this solvent giving homogeneous solutdons. The temperatlure of reaction was that of boiling water, variations due to barometric changes being of 110 importance. Preliminary experiments were made with solutions of the follow- iiig concentrations : View Article Online 730 DAVIS AND RIXON : STERIC INFLUENCE : Rate of Forrnafion .-One gram-molecule of formic acid and 1 gram-molecule of aniline per litre. 915. Downloaded by University of Chicago on 29/10/2014 17:47:52. shed on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52 Rate of Decomposition.-One gram-molecule of the anilide and 1 gram-molecule of water per litre." The measurement of rates of formation and equilibria in such solutions could be carried out satisfactorily, but the determination of the rates of decomposition were not completely successful. The following table and curve show the results obtained for the decom- position of formanilide. Published on 01 January 1915. Downloaded by University of Chicago on Time in hours. Per cent. decomposed. 2 0.26 3 0.51 5 1-09 6 1-34 16 9.1 20 14.83 Time in hours. Per cent. decomposed. 2 0.26 3 0.51 5 1-09 6 1-34 16 9.1 20 14.83 The reaction appears to- be catalysed by one of the reaction products, and the addition of formic acid produces a considerable increase in t,he initial rate of decomposition, thus: Per cent. decomposed. A r 7 After the addition Molecular of 0.1 gram-mol. Substance. Hours. solution. of formic acid. Formanilide ... ... 1 ; Formo-o-toIuidide ... ... * This water is in addition to that already present in the solvent. By OLIVER CHARLES MINTY DAVIS arid FREDERIC WILLIAM RIXON. { 0.26 0.51 0-53 1-14 3.45 4.77 3-8 6.2 1 This effect is confined to the decomposition measurements, a similar excess of acid in the other determinations pro'ducing altera- tions which could be attributed normally to the increase in the mass of the formic acid; thus in the reaction between aniline and formic acid 59.2 per cent. of the anilide was formed in molecular solution, whilst with an excess of the acid as stated, the formation was 62.38 per cent. The solutions used throughout the following experiments were made up as follows: i ili d i i 1 l li Rate of Formation-Aniline or derivative, 1 gram-mol. per litre, and formic acid, 1.1 gram-mol. per litre. Rate of D1ecomposition.-Formanilide or derivative, 1 gram-mol. per litre; formic acid, 0.1 gram-mol. per litre; and water, 1 gram- mol. per litre (in addition to water present in the solvent). View Article Online STATIC AND DYNAMIC. PART 11. 731 t A slight variation was introduced, the formic acid solution and the water being measured into the flask instead of being weighed. * The loss due to volatilisation was found to be negligible. t Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 15. Downloaded by University of Chicago on 29/10/2014 17:47:52. Eate of Fornintion.-The aniline or derivative was weighed into a stoppered measuring flask, a weighed quantity of standardised formic acid solution in the pyridine-water solvent was added, the required volume made up with the solvent, and the flask weighed. Of this solution one portion was weighed in a stoppered flask and titrated with N / 10-sodium hydroxide, and six other portions were placed in tubes of Jena-glass, previously steamed. These tubes, closed by corks having narrow grooves cut in them,%- were lieatecl on a water-bath, being supported in the lid of the bath by enlarge- ments on the tubes. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 915. Downloaded by University of Chicago on 29/10/2014 17:47:52. shed on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52 E'quili 6 riuin : a Kw=- b+c' where a, 6, c are the concentrations of the anilide or derivative, aniline or derivative, and formic acid respectively. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 Published on 01 January 1915. Downloaded by University of Chicag At intervals of iift’eer. minutes a tube was taken out and rapidly cooled, and the contents were poured into a stoppered flask, weighed, and titrated. Rate of Decomposition.-The method adopted for making the solution was that described above. A portion of the solution having been titrated, the remainder was heated in a Jena flask fitted with a ground-in reflux condenser. Portions of the solution were removed by a pipette every hour, rapidly cooled, weighed, and i5trated.t Equilibrium.-Portions of the solutions prepared for the above determinations were sealed in glass tubes, the method used being that described by Davis (Zoc. cit.). Treatment of Data.-Empirical formula: have been used to cal- culate the constants, giving fairly consistent results, as will be seen from the table which follows. Water being present in com- paratively large quantity, a practical constancy been assumed in calculating the decomposition equilibrium constant. in its amount has constant and the The formulae used are: Formation : where t =time in minutes, a= concentration of the aniline or deriv- ative = 1, b =concentration of the formic acid = 1.1, and x = amount of the anilide formed. where t =time in minutes, a= concentration of the aniline or deriv- ative = 1, b =concentration of the formic acid = 1.1, and x = amount of the anilide formed. D e co m Position : 1 a(b+x) k - -log- ’ - t(u - 6) 6(a - x)’ where a =concentration of the anilide or derivative = 1, b = concen- * * The loss due to volatilisation was found to be negligible. t t A slight variation was introduced, the formic acid solution and the water being measured into the flask instead of being weighed. View Article Online View Article Online 732 DAVIS AKD RIXON : STERIC INFLUENCE : tratioii of the formic acid = 0.1, ~t: =ainouiit of the anilide decoiii- posed in t minutes. tratioii of the formic acid = 0.1, ~t: =ainouiit of the anilide decoiii- posed in t minutes. Discussion of R eszdts. Published on 01 January 1915. Downloaded by University of Chica It is possible1 t o compare the results in two distinct ways; either the effect of the position of a single subst.ituent group may be FIG. 2. FIG. 2. 11 10 4 0 g 9 -J 0 000 considered, or the result of substituting different groups in the same position may be taken as a basis of comparison. FIG. 2. 11 10 4 0 g 9 -J 0 000 4 0 g 4 0 g considered, or the result of substituting different groups in the same position may be taken as a basis of comparison. It will be seen by consulting the table already given or the one following that the rates of formation and the percentage of substance formed run approximately parallel, and that the values in the case of ortho-, meta-, and para-isomerides increase in the order mentioned, the increase being particularly pronounced for 0- and m-halogen-substituted derivatives. g No such regularity is found in thel rates of decomposition. Refelrring to the effects of the various groups when substituted in similar pwitions, it may be said that it is difficult to arrange d on 01 January 1915. D de .............................. oluidide ..................... toluidide ..................... oluidide ........................ nisidide ..................... isidide ..................... ormanilide ..................... ormanilide ..................... rmanilide ..................... ormanilide ..................... rmanilide ..................... naphthalide .................. naphthalide .................. ormanilide ...................... Average 4 0.0045 0.00108 0.0053 0.0067 0.0018 0.0099 0.00028 0.00 14 0.0021 0.00013 0.00154 0.0025 0.00072 0-0036 Maximum difference of any constant from average. 0.0004 0.00005 0.0007 0.0003 0*0001 0*0004 0-0001 0.00007 0~0001 0.00008 0.00004 0~0002 0~00002 0.0005 rather large maximum difference from the average i the catalytic action whic View Article Online View Article Online DAVIS AND RIXON : STEHIC INFLUENCE : 734 them in order of influence. I n the orthepositions they may be ordered thus: ,@-ring, O*CH,, CH,, a-ring, Br, Cl.* them in order of influence. I n the orthepositions they may be ordered thus: ,@-ring, O*CH,, CH,, a-ring, Br, Cl.* 5. Downloaded by University of Chicago on 29/10/2014 17:47:52. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. I n the meta- and para-positions the effect of the halogen atoms is in tho reverse order. Stress cannot be laid on this owing to the slight differences in the numbers obt'ained for these particular substances, especially in the case of the ortho- and meta- isonierides. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 The rates of decomposition do not lend themselves to any ordered comparison. Furtlier comparisons may be readily made by the aid of the table on p. 735. Conclusion. Equilibrium measurements applied to a reaction or series of reactions give more reliable results than do the determinations of either the rates of formation or decomposition, and should be, if possible, selected for the investigation of a reaction provided it is only intended to make one type of observation. On the other hand, the comparison of velocity measurements with equilibrium data gives, with regard t o the mechanism of a reaction, much more complete and valuable information, which may probably be used to discriminate between the various factors in the reaction. Published on 01 January 1915. Downloaded by Unive Published on 01 January 1915. Down Factors of the Reactions. I n the formation and the decomposition of the anilides the steric influence is one factor, the chemical effect of the substituent may be regarded as a second, and the action of the formic acid as a third; there may be other factors, but these three are9 obvious. y I f H represents a factor compounded of the firs6 and second, whilst F represents the third, the formation of a substance may be expressed thus : klaF/li, klaF/li, and its decomposition by: kaaFH. * The a- and &rings are included, although the inclusion can hardly be justified; this remark applies equally to other comparisons that are made in which the a- and 8-rings are involved. kaaFH. By combining these two relations, the factor F, which would be supposed to have equal accelerative effect on both reactions, can be eliminated, and 1 H2k,aL2 or H2a k, 1 k,. * The a- and &rings are included, although the inclusion can hardly be justified; this remark applies equally to other comparisons that are made in which the a- and 8-rings are involved. View Article Online STATIC AND DYNAMIC. PART 11. 735 s I 2 t- o 4 \O 0 . J‘ bil Fr; .r( CD rl 0 to H 0 M z In rl rl c.1 z m 8 s t- d 0 0 z c? d t- rl o c3 8 s 0 3 L cvn. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. [Received, February 20th, 19 15.1 View Article Online View Article Online LE SUEUR AND WI'I'HELiS : HENEICOSOIC ACID. 736 Making this assumption, the values of 15 may be expressed propor- tionally in round numbers as follows: Making this assumption, the values of 15 may be expressed propor- tionally in round numbers as follows: Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 15. Downloaded by University of Chicago on 29/10/2014 17:47:52. hed on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52 Group. 0. m. p. Ring. CH, ............... 15 9 7 a 19 6 O'CH, ............ 15 Br .................. 53 19 17 8 9 c1 ..................... 53 18 13 - - - These numbers are merely proportional, and are derived, in the manner indicated, from the table in which the parent substance formanilide is the unit,. Published on 01 January 1915. Downloaded by University of Chicago on The similar results obtained from the halogens point to the conclusion that the extra weight of the bromine atom is counter- balanced by the, chemical activity of the chlorine, the two atoms exercising an equal hindering effect. Published on 01 January 1915. Downloaded by University I f steric influence is proportional to the weight of the substituent group, the factor H might be divided by the weight of t,he group in question ; the quotient would be a proportional representation of what might be termed "chemical hindrance." The above method of treatment is purely tentative, and is sug- gested as possibly allowing a direct comparison t o be drawn between the effects produced by substituting different groups in a reacting system, and a discrimination to be made between certain of the reaction factors. The' expenses in connexion with the work have been partly met by a grant from the Royal Society, for which the authors express their thanks, CHEMICAL DEPARTMEN BRISTOL UNIVERSITY [Received, February 20th, 19 15.1
https://openalex.org/W4317852656
https://figshare.com/articles/preprint/3D_Quantum-inspired_Self-supervised_Tensor_Network_for_Volumetric_Segmentation_of_Brain_MR_Images/12909860/4/files/38883345.pdf
English
null
3D Quantum-inspired Self-supervised Tensor Network for Volumetric Segmentation of Medical Images
null
2,023
cc-by
13,302
3D Quantum-inspired Self-supervised Tensor Network for Volumetric Segmentation of Medical Images This paper was downloaded from TechRxiv (https://www.techrxiv.org). LICENSE SUBMISSION DATE / POSTED DATE 02-09-2020 / 24-01-2023 SUBMISSION DATE / POSTED DATE 02-09-2020 / 24-01-2023 g Debanjan Konar, SMIEEE, Siddhartha Bhattacharyya, SMIEEE, Tapan K. Gandhi, SMIEEE, Bijaya K. Panigrahi, SMIEEE, and Richard Jiang, SMIEEE Debanjan Konar, SMIEEE, Siddhartha Bhattacharyya, SMIEEE, Tapan K. Gandhi, SM Bijaya K. Panigrahi, SMIEEE, and Richard Jiang, SMIEEE for accurate and fast segmentation of volumetric medical images with different modalities.i Abstract—This paper introduces a novel shallow 3D self- supervised tensor neural network for volumetric segmentation of medical images with merits of obviating training and supervision. The proposed network is referred to as the 3D Quantum-inspired Self-supervised Tensor Neural Network (3D-QNet). The underly- ing architecture of 3D-QNet is composed of a trinity of volumetric layers viz. input, intermediate, and output layers inter-connected using an S-connected third-order neighborhood-based topology for voxel-wise processing of 3D medical image data, suitable for semantic segmentation. Each of the volumetric layers contains quantum neurons designated by qubits or quantum bits. The incorporation of tensor decomposition in quantum formalism leads to faster convergence of the network operations to preclude the inherent slow convergence problems faced by the classical supervised and self-supervised networks. The segmented volumes are obtained once the network converges. The suggested 3D-QNet is tailored and tested on the BRATS 2019 Brain MR image data set and Liver Tumor Segmentation Challenge (LiTS17) data set extensively in our experiments. 3D-QNet has achieved promising dice similarity as compared to the time intensive supervised convolutional neural network-based models like 3D-UNet, Vox- ResNet, DRINet, and 3D-ESPNet, thereby showing a potential advantage of our self-supervised shallow network on facilitating semantic segmentation. The unified concept of quantum-inspired neural networks (QINN) enhances the approximation and generalization capa- bilities of classical neural networks and has emerged to process information faster in the field of computer science [8], [9]. Of late, quantum-inspired neural networks are becoming popular in solving problems in the domain of pattern recognition and classification [10], [11], [14], [15], employing the inherent characteristics of quantum computation. However, the complex and time-intensive quantum back-propagation algorithm in the aforementioned QINN models suffers from slow convergence problems. In addition, the fixed activation schemes adopted in the QINN models restrict their application to gray-scale image segmentation. Given 3D medical image data, the primary aim of our proposed 3D-QNet architecture is to perform volumetric organ and lesions segmentation with expert-level accuracy for tumor identification alleviating supervision or training. Our pro- posed 3D-QNet architecture centres on the self-supervised bi- directional counter propagation of the quantum states obviating the time-intensive quantum back-propagation algorithm for faster convergence. D. Konar is with the CASUS - Center for Advanced Systems Understand- ing, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Görlitz, Germany, Email: d.konar@hzdr.de g Debanjan Konar, SMIEEE, Siddhartha Bhattacharyya, SMIEEE, Tapan K. Gandhi, SMIEEE, Bijaya K. Panigrahi, SMIEEE, and Richard Jiang, SMIEEE The network hyper-parameters associated with the gray-level thresholding process are adaptive, and voxel-wise context-sensitive information is exhibited in quan- tum formalism, as reported in this article. Index Terms—Quantum Computing, Volumetric Medical Im- age Segmentation, QIS-Net, Tensor Network CITATION Konar, Debanjan; Bhattacharyya, Siddhartha; Gandhi, Tapan Kumar; Panigrahi, Bijaya Ketan; Jiang, Richard (2020): 3D Quantum-inspired Self-supervised Tensor Network for Volumetric Segmentation of Medical Images. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.12909860.v4 10.36227/techrxiv.12909860.v4 10.36227/techrxiv.12909860.v4 1 T. K. Gandhi, and B. K. Panigrahi are with the Department of Electrical Engineering, Indian Institute of Technology Delhi, New Delhi, India, Email: tgandhi@ee.iitd.ac.in and bkpanigrahi@ee.iitd.ac.in S. Bhattacharyya is with Rajnagar Mahavidyalaya, Birbhum, West Bengal, India, Email: dr.siddhartha.bhattacharyya@gmail.com D. Konar is with the CASUS - Center for Advanced Systems Understand- ing, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Görlitz, Germany, Email: d.konar@hzdr.de T. K. Gandhi, and B. K. Panigrahi are with the Department of Electrical Engineering, Indian Institute of Technology Delhi, New Delhi, India, Email: tgandhi@ee.iitd.ac.in and bkpanigrahi@ee.iitd.ac.in S. Bhattacharyya is with Rajnagar Mahavidyalaya, Birbhum, West Bengal, India, Email: dr.siddhartha.bhattacharyya@gmail.com R. Jiang is with the School of Computing and Communications, Lancaster University, Lancaster, UK, Email: r.jiang2@lancaster.ac.uk R. Jiang is with the School of Computing and Communications, Lancaster University, Lancaster, UK, Email: r.jiang2@lancaster.ac.uk I. INTRODUCTION Despite popu- larity among the medical and computer vision researchers, U-Net architectures [24] fall short in scalability and cannot distinguish the distinctive features (shape, size, intensity, lo- cation etc.) learned at the convolutional layers. Moreover, they suffer from the vanishing gradient problem when the number of feature layers is increased to better represent the features. Various deeper network architectures obviating the vanishing gradient problem have been proposed concurrently for voxel- wise medical image segmentation including VoxResNet [26], DRINet [27], and 3D-ESPNet [28]. The current voxel-wise segmentation work has significant contributions over 2D medical image segmentation [1]–[3], [14] as given below. 1) We propose a novel quantum-inspired self-supervised shallow voxel-wise neural network referred to as 3D- QNet, which has relevance on volumetric medical image segmentation. 2) In this work, an S-connected quantum fuzzy context- sensitive voxel information is processed to integrate the appearance of low-level and high-level local image features with wide intensity variations and implicit shape of the VOIs, thereby enabling accurate volumetric seg- mentation of 3D medical images. 3) A novel generalized quantum-inspired self-supervised learning is proposed using a tensor representation of the weight vectors for high dimensional data employed in our suggested 3D-QNet for 3D medical image segmen- tation. The non-tensorized implementation of 3D-QNet, referred to as 3D-QNet-NonTensor, is also demonstrated in our experiments. 4) The convergence analysis of the proposed 3D-QNet is also demonstrated with super-linearity. The primary aim of incorporating quantum computing in our proposed 3D network architecture is to exploit the features of quantum correlation and accelerate the speed of convergence of the network operation, thereby simultaneously improv- ing the discrimination ability to yield fast and accurate segmentation. However, these deeply supervised network architectures suffer in computational complexity and slow-convergence with an increase in the number of feature layers in the network ar- chitecture. Currently, self-supervised/semi-supervised/weakly supervised networks have gained significant attention among the computer vision and medical imaging research community due to lack of annotated images for deep supervision [29]– [32]. Nevertheless, these self-supervised networks [29]–[32] for volumetric medical image segmentation rely on pre-trained 3D CNN models, and hence these are not fully self-supervised networks. Moreover, these networks are characterized by significant memory footprints which often poses a serious obstacle in employing them in various medical imaging ap- plication settings. It inspires us to develop 3D self-supervised neural network architectures for volumetric medical image segmentation. I. INTRODUCTION A A utomatic volumetric medical image segmentation as- sisted by contextual information yields Volumes of In- terest (VOIs), which are critical to cancer patients. Deeply su- pervised Convolutional Neural Networks (CNN) have achieved respectable accuracy in 2D medical image segmentation [1]– [4]. However, in automatic 3D medical image data segmen- tation, deeply supervised 3D-CNNs suffer from manually affected challenges viz. acquiring sufficient 3D annotated data for suitable training, high heterogeneity and dimensionality of 3D medical images, complex anatomical environments, and the need for optimizing the 3D neural networks [5]–[7]. Hence, 3D medical imaging research calls for self-supervised learning Translational medicine (TM) is an emerging concept and practice that facilitates the rapid transfer of medical break- throughs from scientists to clinicians. Recently, there have been a number of instructive instances in which the trans- lation of research has resulted in undesirable effects need- ing prompt intervention [12], [13]. A greater emphasis on three-dimensional (3D) simulation, biomarkers, and artificial intelligence may enable orthopaedic surgeons to forecast the optimal surgical techniques prior to surgery. Utilizing the most advanced imaging methods may enhance the precision and accuracy of tumor resections. This article is aimed at young surgeon scientists, specifically orthopaedic residents, to help them better understand how 3D quantum-inspired models can be used to process high mega pixel volumetric medical images in a faster, self-supervised manner, with merits of obviating training or very limited training. Heuristically, the suggested 3D-QNet has the ability to investigate the inherent features of quantum parallelism in order to concurrently compute high-resolution image voxels. This expedites the transfer of 2 information from the laboratory to the patient’s bedside. The current voxel-wise segmentation work has significant contributions over 2D medical image segmentation [1]–[3], [14] as given below. volumetric medical images [22]–[24]. Kamnitas et al. [22] suggested a dual path 3D CNN incorporating local and larger contextual feature information to obviate the computationally complex 3D medical image processing and exhibit dense inference on medical image segmentation. A flexible network, 3D-UNet architecture [24] achieved remarkable success on brain MR image semantic segmentation. Of late, to exploit the 3D contextual information, Brebisson et al. [25] employed 2D CNNs on three orthogonal 2D patches and formed 3D patches in combination to reduce the memory requirements. However, 3D CNN networks suffer from slow convergence problems owing to computationally exhaustive 3D convolution operations and extensive training procedures. I. INTRODUCTION The organization of the remaining sections of the manuscript is as follows: a comprehensive literature review about various deep learning-based volumetric segmentation of medical images and the challenges are presented in Section II. Section III illustrates the fundamental concepts of quantum computing. The novel self-supervised 3D-QNet architecture with a quantum-inspired tensor network model is introduced in Section IV. Section V elucidates voxel-wise segmentation of 3D medical images using the proposed 3D-QNet architecture. The experimental datasets, experimental setup, and outcome are provided in Section VI. The advantages and limitations of the proposed work have been discussed in Section VII. Section VIII states the concluding remarks of the proposed work and sheds light on the future directions of research. The main problem with the classical self-supervised neural network models lies in the fact that they do not converge fast and hence the segmented outcome is distorted due to the slower convergence problems [33], [34]. Numerous quan- tum neural networks have been evolved in the last few decades replicating classical neural networks and offering faster processing while compared with the classical counter- parts [9], [35]–[38]. The quantum versions of the classical self-supervised neural network architectures [10], [11], [39], [40] offer a potential candidate for faster and efficient image segmentation and surpasses the classical counterparts. Konar et al. recently developed quantum-inspired neural network mod- els referred to as QIS-Net [14], QFS-Net [15], and QIBDS- Net [41] suitable for brain MR image segmentation. These networks have been found to attain a promising outcome in complete brain tumor segmentation and serve as the motivation behind the assimilation of quantum-inspired computing in the current 3D-QNet architecture. A. Quantum Bits and Tensor Products The basic element equivalent to classical bits in quantum computing is known as quantum bit or qubit and is represented using Dirac notations |0⟩and |1⟩. However, unlike classical computing, quantum bits are expressed as a linear combination of probability amplitudes often known as superposition as follows [9]. (7) III. FUNDAMENTALS OF QUANTUM COMPUTING To enhance and extract the contextual information from high dimensional data, Tucker tensor decomposition is suitable for neural network layer decomposition [43]. The inner product of the two equal sized tensors V, Ψ ∈Rm×n×p is defined as follows. The basic concept of quantum computing deals with the principles of quantum mechanics and offers to demonstrate the quantum computing algorithms which rely on quantum bits having quantum operations on qubits [42]. |ξ⟩= ⟨V|Ψ⟩= X l ⟨V(l), Ψ(l)⟩= M X i=1 θi|i⟩· N X j=1 ϕj|j⟩ =      θ1 θ2 ... θM     ·      ϕ1 ϕ2 ... ϕN     , θi ∈V, ϕj ∈Ψ (7) IV. 3D QUANTUM-INSPIRED SELF-SUPERVISED TENSOR NEURAL NETWORK (3D-QNET) ARCHITECTURE In quantum formalism, the tensor products of the subspace form the full Hilbert space, H as (2) H = ⊗n t=1Ht (2) H = ⊗n t=1Ht (2) A set of n basis states (designated as |ϕj⟩) comprising 0 −1 can form a qubit system |ψ⟩, of size log n in the Hilbert space, H as follows. n A set of n basis states (designated as |ϕj⟩) comprising 0 −1 can form a qubit system |ψ⟩, of size log n in the Hilbert space, H as follows. n |ψ⟩= n X j pj|ϕj⟩ (3) (3) where, pj is the probability amplitude and |ϕj⟩= |ϕ1⟩⊗ |ϕ2⟩⊗. . . |ϕn⟩. For example, using two qubits, four distinct tensor sub-spaces can be created as basis |0⟩⊗|0⟩, |0⟩⊗|1⟩, |1⟩⊗|0⟩and |1⟩⊗|1⟩often represented as |00⟩, |01⟩, |10⟩, and |11⟩, respectively. II. RELATED WORKS Recent years have witnessed a surge in the application of deep learning networks in various tumor segmentation [16]– [20] tasks with respectable accuracy in 2D medical image segmentation [2], [21]. However, in contrast to automated volumetric segmentation of medical images, 2D convolutional neural network architectures (CNNs) [16], [18], [19] process the medical images in a 2D independent slice-wise fashion which leads to non-optimal use of the 3D contextual feature information of volumetric medical image data (3D Computed Tomography (CT) and Magnetic Resonance Imaging (MRI)). In turn, 3D CNN based architectures extract rich spatial and contextual features and perform voxel-wise segmentation of This manuscript presents a novel fully 3D self-supervised 3 functions defined over α ∈[0, 1]N. Considering the dimen- sions of the input feature vector restricted to N = 2, ϕ(α) is defined as quantum-inspired shallow neural network architecture for vol- umetric medical image segmentation to obviate the compre- hensive challenges faced by deep supervision of complex 3D CNNs. ϕ(0) = [0, 1], ϕ(1) = [1, 0] (6) (6) IV. 3D QUANTUM-INSPIRED SELF-SUPERVISED TENSOR NEURAL NETWORK (3D-QNET) ARCHITECTURE |ϕ⟩= cos α 2 |0⟩+ ei θ 2 sin α 2 |1⟩ (1) (1) In this article, a 3D Quantum-inspired Self-supervised Tensor Network (3D-QNet) architecture with self-supervised tensor learning is proposed for automatic voxel-wise segmen- tation of medical images. The 3D-QNet architecture comprises a trinity of volumetric layers of quantum neurons arranged as input, intermediate and output layers. A schematic outline of the proposed 3D-QNet architecture is shown in Figure 1. The input volume (M ×N ×P) is normalized and propagated from the 3D input layer to the successive 3D hidden and output layers of the 3D-QNet architecture for processing through S- connected voxels. Each of the three volumetric layers of the 3D-QNet architecture is fully intra-linked with qubits using a 3D-matrix representation. Each 3D layer of the proposed architecture is intra-connected through quantum neurons with intra-connection strengths set to π 2 (quantum 1 logic). The basic processing unit of each volumetric layer of the 3D-QNet architecture is the S-connected neighborhood-based voxel- wise orientation of each candidate neuron as illustrated in Figure 2. The inter-layer connection between the 3D input to 3D intermediate and the 3D intermediate to 3D output layer is formed using the S-connected voxel-wise neighborhood orientation. The contribution of the S number of neighborhood quantum neurons (pixels) of a candidate neuron at one 3D layer is propagated in the forward direction and accumulated at the corresponding candidate neuron of the subsequent 3D layer. Consequently, the voxel-wise information from the 3D output layer to the 3D intermediate layer is counter-propagated for further processing. The voxel-wise processing of each 3D layer is performed along with the depth of the 3D layer for semantic segmentation. The inter-linked connections between two successive 3D layers are represented using 3D weight matrices of qubits and each inter-connection weight is updated using rotation gate for faster processing. The relevant details about the principle of operation of the proposed 3D-QNet architecture for volumetric segmentation are provided in the following subsections using a self-supervised tensor learning model in quantum formalism. where, 0 ≤α ≤π and 0 ≤θ ≤2π. where, 0 ≤α ≤π and 0 ≤θ ≤2π. Hence, qubits reside in the Hilbert space parametrized by the continuous variables θ and α. B. Input Data Encoding and Tensor Decomposition A tensor product basis relies on the local input feature map {Φdj(αj)} in the Hilbert space of functions over αj ∈[0, 1] as [38] |Φd1,d2,...dN (α)⟩= |ϕd1(α1)⟩⊗|ϕd2(α2)⟩⊗. . . ⊗|ϕdN (αN)⟩ (4) where, dj varies from 1 . . . N (N-dimensional vector). A function, f l(α) can be realized using the inner product of the input local feature map ϕdj(αj) and the network weight decomposition Ψ, as follows. f l(α) = ⟨Ψl|Φ(α)⟩ = ⟨Ψd1d2...dN |ϕ(αd1 1 )⟩⊗|ϕ(αd2 2 )⟩. . . ⊗|ϕ(αdN N )⟩ (5) (5) Hence, the local feature map ϕdj(αj) forms a basis for a Hilbert space of functions defined over α ∈[0, 1] and the tensor product basis Φd1,d2,...dN (α) forms a Hilbert space of Fig. 1: 3D Quantum-inspired Self-supervised Tensor Neural Network (3D-QNet) architecture. For every candidate neuron as marked in red color, it forms a S-connected neighborhood oriented Inter-layer connections and here only three Inter- layer connection is illustrated for better visibility. A classical information, α is mapped to quantum bits or qubits as, ϕ(α) =  cos( π 2 α) sin( π 2 α)  . The inter-connection weights between the input and hidden layers of the 3D-QNet architecture are denoted by |φl,d k,j⟩, for the hidden layers to the output layers are indicated by |φl,d j,i⟩and for the output to the hidden layers are |φl,d i,j⟩ at a layer l with depth d. The classical interconnection weight [0, 1] is transformed in quantum formalism as |φ(ω)⟩=  cos( π 2 ω) sin( π 2 ω)  , where the angle of rotation (ω) is measured using the relative difference of fuzzy intensities of the candidate pixel and the neighborhood pixels in S-connected (here, S = 26) neighborhood oriented quantum neurons in a 3 × 3 × 3 voxel (v) as ωi,j = 1 −(αi −αi,j); j ∈{1, 2, 3, . . . S}. 3D-volumes of dimensions 3 × 3 × 3 are processed along the depth of the 3D-layers as shown in the Figure. Fig. 1: 3D Quantum-inspired Self-supervised Tensor Neural Network (3D-QNet) architecture. For every candidate neuron as marked in red color, it forms a S-connected neighborhood oriented Inter-layer connections and here only three Inter- layer connection is illustrated for better visibility. A classical information, α is mapped to quantum bits or qubits as, ϕ(α) =  cos( π 2 α) sin( π 2 α)  . B. Input Data Encoding and Tensor Decomposition The inter-connection weights between the input and hidden layers of the 3D-QNet architecture are denoted by |φl,d k,j⟩, for the hidden layers to the output layers are indicated by |φl,d j,i⟩and for the output to the hidden layers are |φl,d i,j⟩ at a layer l with depth d. The classical interconnection weight [0, 1] is transformed in quantum formalism as |φ(ω)⟩=  cos( π 2 ω) sin( π 2 ω)  , where the angle of rotation (ω) is measured using the relative difference of fuzzy intensities of the candidate pixel and the neighborhood pixels in S-connected (here, S = 26) neighborhood oriented quantum neurons in a 3 × 3 × 3 voxel (v) as ωi,j = 1 −(αi −αi,j); j ∈{1, 2, 3, . . . S}. 3D-volumes of dimensions 3 × 3 × 3 are processed along the depth of the 3D-layers as shown in the Figure. Fig. 2: S-connected neighborhood oriented quantum neurons form a voxel (The red pixel is the candidate neuron and the black pixels represent the corresponding neighborhood neurons). function ϕ(αi) as follows. ϕ(αi) = h cos(π 2 αi) sin(π 2 αi) i ∀i = 1, . . . M, j = 1, . . . N (8) (8) The classical interconnection weight [0, 1] is transformed into quantum formalism as |φ(ωi,j)⟩= h cos(π 2 ωi,j) sin(π 2 ωi,j) i (9) (9) Fig. 2: S-connected neighborhood oriented quantum neurons form a voxel (The red pixel is the candidate neuron and the black pixels represent the corresponding neighborhood neurons). Hence, the strength of inter-connection between neuron j (neighborhood of the candidate neuron i) of a layer to the cor- responding candidate neuron of the adjacent layer is mapped using φ. Also, ωi,j is designated as the angle of rotation and measured as the relative intensity difference between the candidate pixel (αi) and one of its neighborhood pixels αi,j as follows. Hence, the strength of inter-connection between neuron j (neighborhood of the candidate neuron i) of a layer to the cor- responding candidate neuron of the adjacent layer is mapped using φ. Also, ωi,j is designated as the angle of rotation and measured as the relative intensity difference between the candidate pixel (αi) and one of its neighborhood pixels αi,j as follows. A. 3D-Quantum-Inspired Self-supervised Tensor Network Model A. 3D-Quantum-Inspired Self-supervised Tensor Network Model V, Ψ are third-order tensors (1 ≤m ≤M, 1 ≤n ≤N, 1 ≤p ≤P)). According to Tucker Tensor decomposition [43] X = V ×1 Ψ1 ×2 Ψ2 ×3 Ψ3 (11) (11) where, X ∈Rm×n×p is the tensor outcome, Ψn is the weight matrix in terms of n factor matrix and ×n is the mod −n product of a tensor with a matrix. Each layer of the proposed 3D-QNet architecture is transformed to lower- dimensional tensors. Such types of M×N×P tensors in voxel form each layer in the underlying network architecture. Each volumetric layer of the 3D-QNet architecture forms M×N×P volumetric patches (voxels) of size S corresponding to the candidate pixels as where, X ∈Rm×n×p is the tensor outcome, Ψn is the weight matrix in terms of n factor matrix and ×n is the mod −n product of a tensor with a matrix. Each layer of the proposed 3D-QNet architecture is transformed to lower- dimensional tensors. Such types of M×N×P tensors in voxel form each layer in the underlying network architecture. Each volumetric layer of the 3D-QNet architecture forms M×N×P volumetric patches (voxels) of size S corresponding to the candidate pixels as Quantum fuzzy context-sensitive thresholding determines the bi-directional propagation of quantum information between the layers of the 3D-QNet architecture by means of self- organization of the inter-linked weight matrices. Reduction of feature dimensions using tensor decomposition followed by voxel-wise information processing of the proposed 3D-QNet architecture is inspired by the basic quantum neural network input-output model [14] as follows. v = vox(V) (12) (12) Here, V comprises all the 3D-patches (voxels), v ∈Rm×n×p for a network layer in the proposed 3D-QNet architecture. The spatial features in terms of the neighborhood pixels of every seed pixel at the network layer, are extracted and propagated to the next subsequent layers as inputs guided by a Quantum-inspired voxel-wise multi-level Sigmoidal (Vox- QSig) activation function, σ3D−QNet as follows. Here, V comprises all the 3D-patches (voxels), v ∈Rm×n×p for a network layer in the proposed 3D-QNet architecture. The spatial features in terms of the neighborhood pixels of every seed pixel at the network layer, are extracted and propagated to the next subsequent layers as inputs guided by a Quantum-inspired voxel-wise multi-level Sigmoidal (Vox- QSig) activation function, σ3D−QNet as follows. A. 3D-Quantum-Inspired Self-supervised Tensor Network Model In the suggested 3D-QNet architecture, the high dimensional weight vector Ψ is represented using a tensor to optimize the network operations and to facilitate the extraction of significant semantic feature information in the quantum-inspired self- supervised model. The internal kernels associated with the network operate in parallel, thereby accelerating convergence of the 3D-QNet architecture. The input quantum neurons containing the pixel intensity are expressed as qubits and the inter-connection weights are represented using quantum rotation gates. The classical intensity of any ith normalized gray-scale image pixel of MR or CT volume (denoted as αi ∈[0, 1]) is transformed into quantum state using a mapping ωi,j = 1 −(αi −αi,j); j ∈{1, 2, 3, . . . S} (10) (10) The angle of rotation is measured using the relative difference of fuzzy intensities of the candidate pixel and the neighbor- hood pixels in quantum formalism. This relative measure en- coding scheme [14], [15], [39] helps to segment the foreground and background regions of an image. However, there are other encoding schemes like variational and amplitude encoding, which are often used for variational quantum circuits. The angle of rotation is measured using the relative difference of fuzzy intensities of the candidate pixel and the neighbor- hood pixels in quantum formalism. This relative measure en- coding scheme [14], [15], [39] helps to segment the foreground and background regions of an image. However, there are other encoding schemes like variational and amplitude encoding, which are often used for variational quantum circuits. In this proposed tensor network model, the 3D-QNet layer is decomposed as voxel (core tensor) using Tucker Tensor decomposition [43] to reduce the input dimensions and the 5 TABLE I: Variational parameters used in the counter- propagation algorithm of the proposed 3D-QNet Symbol Description αl,d i The intermediate output at pixel i at layer l and depth d ωl,d The rotation angle for inter-connection weight at layer l with depth d χS It corresponds the contribution of S-connected third- order neighborhood pixels ϑl i The fuzzy context sensitive activation in quantum for- malism at layer l δl,d i The phase transformation parameters at layer l−1 with depth d interconnection wights as factor matrices. Let us consider tensor V, Ψ ∈Rm×n×p, where V is the voxel-wise input of 3D medical images and the corresponding inter-connection 3D weight matrix is evaluated in Eq. 9, respectively (m, n, p denote the row, column, and slice number. A. 3D-Quantum-Inspired Self-supervised Tensor Network Model |ϕl(αd i )⟩= σ3D−QNet( m×n×p X j f l−1(αd i )⟨φl j|χl,d i ⟩) (18) (18) yl = σ3D−QNet(vl−1 · Ψl(ω)) (13) (13) where, vl−1 ∈RM×N×P , Ψl(ω) ∈RM×N×P ×S at the network layer l = 2, 3, yl ∈RS, and “·” is the inner product operator. The Vox-QSig activation function, σ3D−QNet with slope λ and activation ϑ, is defined as where, |ϕl(αd i )⟩denotes the intermediate output of the ith seed quantum neuron at the 3D network layer in the lth sample with depth (slice#) d = 1, 2, . . . P. σ3D−QNet is the Quantum-inspired voxel-wise multi-level Sigmoidal activation (Vox-QSig) function with activation as |χl,d i ⟩. The output |ϕl(αd i )⟩can be written as σ3D−QNet(x) = 1 βτ + e−λ(x−ϑ) , 0 ≤βτ ≤π 2 (14) (14) |ϕl(αd i )⟩= f(π 2 δl,d i −arg{ m×n×p X j f l(ωd j,i)f l−1(αd i ) −f l(χd i )}) = σ3D−QNet( m×n×p X j f l−1(αd i )(cos(ωl,d j,i −ϑl i)+ γ sin(ωl,d j,i −ϑl i))) (19) where, βτ describes the multi-level class responses exhibited by the S-connected third-order neighborhood pixels expressed βτ = χS ρτ −ρτ−1 (15) (15) (19) where, ρτ and ρτ are the τ th and (τ −1)th class outcome, respectively and the contribution of the S-connected neighbor- hood gray-level pixels is χS. The fuzzy context-sensitive activation (designated as χi) for semantic segmentation in quantum formalism is defined as follows.   The fuzzy context-sensitive activation (designated as χi) for semantic segmentation in quantum formalism is defined as follows.   Here, the designated rotation angles associated with the inter- connection weights between input neuron j to output neuron i are represented by ωl,d j,i and δl,d i is the phase transfer parameter. Here, the designated rotation angles associated with the inter- connection weights between input neuron j to output neuron i are represented by ωl,d j,i and δl,d i is the phase transfer parameter. The variational parameters used in the counter-propagation algorithm are provided in Table I. The true classical output state (|1⟩) from the ith quantum neuron is obtained by consid- ering the imaginary part (sin) of the above expression where, γ is an imaginary unit. V. VOLUMETRIC MEDICAL IMAGE SEGMENTATION USING 3D-QNET where, L corresponds to the number of class levels. The multi- class responses for various hyper-parameters employed in the Vox-QSig activation functions are provided in Figure 3. where, L corresponds to the number of class levels. The multi- class responses for various hyper-parameters employed in the Vox-QSig activation functions are provided in Figure 3. Minimum pre-processing is performed before the medical image slices are fed as inputs to 3D-QNet. The input medical image volume is normalized as a fuzziness measure ([0, 1]) before transforming it to qubits as follows. A. 3D-Quantum-Inspired Self-supervised Tensor Network Model (a) L = 3 (b) L = 5 |ϕl(αd i )⟩= σ3D−QNet( m×n×p X j f(π 2 yl,d j )⟨φl,d ji |ϑl,d j ⟩) = σ3D−QNet( m×n×p X j f(π 2 × σ3D−QNet( m×n×p X k f(π 2 yl,d j ) ⟨φl,d kj |ϑl,d k ⟩)⟨φl,d ji |ϑl,d j ⟩) (20) |ϕl(αd i )⟩= σ3D−QNet( m×n×p X j f(π 2 yl,d j )⟨φl,d ji |ϑl,d j ⟩) = σ3D−QNet( m×n×p X j f(π 2 × σ3D−QNet( m×n×p X k f(π 2 yl,d j ) ⟨φl,d kj |ϑl,d k ⟩)⟨φl,d ji |ϑl,d j ⟩) (20) (a) L = 3 (b) L = 5 (c) L = 6 (d) L = 8 i.e., |ϕl(αd i )⟩= σ3D−QNet( m×n×p X j Net( m×n×p X k f(π 2 yl,d j ) cos(ωl,d k,j −ϑl,d j ) γ sin(ωl,d k,j −ϑl,d j ) sin(ωl,d j,i −ϑl,d i )))) (21) |ϕl(αd i )⟩= σ3D−QNet( m×n×p X j f(π 2 × σ3D−QNet( m×n×p X k f(π 2 yl,d j ) cos(ωl,d k,j −ϑl,d j ) cos(ωl,d j,i −ϑl,d i ) + γ sin(ωl,d k,j −ϑl,d j ) sin(ωl,d j,i −ϑl,d i )))) (21) (d) L = 8 (c) L = 6 f(π 2 × σ3D−QNet( m×n×p X k f(π 2 yl,d j ) cos(ωl,d k,j −ϑl,d j ) cos(ωl,d j,i −ϑl,d i ) + γ sin(ωl,d k,j −ϑl,d j ) sin(ωl,d j,i −ϑl,d i )))) (21) Fig. 3: Multi-level class response of Vox-QSig activation function for λ = 15, 20, 25. )) (21) and and where, γ is an imaginary number. where, γ is an imaginary number. ϑι+1,d = ϑι,d + △ϑι,d (26) (26) Equations 25 and 26 refer to updating the angles of rota- tion and activation, respectively. The error or loss function, ζ(ωι,d, ϑι,d) in the suggested 3D-QNet architecture is eval- uated in terms of Root Mean Square Error (RMSE) of the 3D-weight matrices at depth d (or the slice #d) in the lth epoch and is defined on the phase angles ωι,d, ϑι,d as Equations 25 and 26 refer to updating the angles of rota- tion and activation, respectively. The error or loss function, ζ(ωι,d, ϑι,d) in the suggested 3D-QNet architecture is eval- uated in terms of Root Mean Square Error (RMSE) of the 3D-weight matrices at depth d (or the slice #d) in the lth epoch and is defined on the phase angles ωι,d, ϑι,d as B. A. 3D-Quantum-Inspired Self-supervised Tensor Network Model Quantum-inspired Voxel-wise multi-level Sigmoidal (Vox- QSig) activation function Equations 25 and 26 refer to updating the angles of rota- tion and activation, respectively. The error or loss function, ζ(ωι,d, ϑι,d) in the suggested 3D-QNet architecture is eval- uated in terms of Root Mean Square Error (RMSE) of the 3D-weight matrices at depth d (or the slice #d) in the lth epoch and is defined on the phase angles ωι,d, ϑι,d as In this 3D-QNet architecture, a Quantum-inspired Voxel- wise multi-level Sigmoidal (Vox-QSig) activation function is proposed for voxel-wise processing of S-connected spatially oriented neighborhood-based pixels. The generalized form of the Vox-QSig activation function is obtained by leveraging the activation function hyper-parameters employed in Equation 14 as ζ(ωι,d, ϑι,d) = 1 N N X i=1 S X j=1 h φij(ωι+1,d ij , ϑι+1,d i ) −φij(ωι,d ij , ϑι,d i ) i2 (27) ζ(ωι,d, ϑι,d) = (27) σV ox−QSig(x; βτ, ρτ) = L X τ=1 1 βτ + e−λ(x−(τ−1)ρτ−1−ϑ) (22) A. 3D-Quantum-Inspired Self-supervised Tensor Network Model Assume that the inter-connection weights between the input and hidden layers of the 3D-QNet architecture are denoted by |φl,d k,j⟩and for the hidden layer to the output layer, are indicated by |φl,d j,i⟩in the lth sample sets. The activation at the hidden and output layers are designated using |χl,d j ⟩and |χl,d i ⟩, respectively. Considering any quantum |χi⟩=  cos ϑi sin ϑi  (16) (16) where, the angle of rotation, ϑi is evaluated using the sum- mation of the intensities of the third-order S-connected neigh- borhood pixels (denoted as αi,j, j = 1, 2, . . . S) of a candidate pixel i (neuron) in quantum formalism using the following equation. ϑi = 2π × ( S X j=1 αi,j) (17) (17) 6 seed neuron k from the sample of input neurons at the input layer, the corresponding seed neuron at the hidden layer be j and the output seed neuron be i, the response at the ith neuron with depth d in the lth sample sets is expressed as (a) L = 3 (b) L = 5 (c) L = 6 (d) L = 8 Fig. 3: Multi-level class response of Vox-QSig activation function for λ = 15, 20, 25. C. Experimental Results Experiments have been carried out in the current setup, and the results have been reported together with numerical and statistical analysis using the proposed 3D-QNet, 3D-QNet- NonTensor, 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-ESPNet [28] on BRATS 2019 data set [47] and LiTS17 data set [48]. Table II presents the complete tumor (WT) segmentation on BraTS19-CBICA-AAG-1-flair-slice#69. It is evident from the experimental data reported in Table II that the proposed 3D-QNet performs optimally for com- plete brain tumor segmentation of four different modalities of MR volumes (viz. T1, T1 −CE, FLAIR, and T2) using the activation guided by 26-connected heterogeneous voxel intensities (υξ) with L = 8 in comparison with other thresholding schemes under the four evaluation parameters (AC, DS, PV, SS) [18]. The 3D-QNet segmented brain MR slices collected from two different volumes BRATS19-CBICA- AAG and BRATS19-CBICA-AAB using class level L = 8 with activation scheme υξ, are shown in Figures 4 and A. Data Set The proposed 3D-QNet and its non-tensorized implementa- tion (3D-QNet-NonTensor) are validated extensively using the BRATS 2019 data set [47] and the Liver Tumor Segmentation Benchmark (LiTS17) data set [48]. The BRATS 2019 data set is composed of 315 (239 HGG and 76 LGG) 3D MRI volumes. Each MRI volume comprises 155 slices of resolution 240×240 with the ground truth segmented labels and includes four different modalities of 3D MR images viz. T1, T1 with Contrast-Enhanced (T1 −CE), T2, and FLAIR. The segmented labels are annotated with three distinct tumor sub- regions, viz. tumor core (TC), tumor enhancing (TE), necrosis and non-enhancing core region. These three annotations form a complete tumor (WT). The BRATS 2019 data set is divided into 8 : 2 ratio for training (252) and testing (63) due to GPU limitations. The Liver Tumor Segmentation Benchmark (LiTS17) data set [48] consists of 131 CT scans with various types of tumors. The LiTS17 data set is also divided into 8 : 2 ratio for training and testing in the study. Each CT volume consists of variable number of slices of resolution 512 × 512. C. Adjustment of Inter-connection Weights of 3D-QNet and Loss Function We have trained 3D-UNet [24] and VoxResNet [26] rigorously with the Stochastic Gradient Descent (SGD) algorithm on Caffe library1 using an Nvidia Tesla V 100 −SXM2 GPU Cluster with 32 GB of memory and 640 Tensor cores with 8 cores of Intel(R) Xeon(R) CPU E5-2683 v4@2.1GHz. The 3D-ESPNet is implemented using Pytorch from the code available in Github2 with 100 epochs using adam optimizer with an initial learning rate of 0.01. The DRINet is also implemented using adam optimizer with an initial learning rate of 0.01 and kernel size of 3 × 3. In order to detect a complete tumor, the segmented output images are resized to match the binary mask’s dimensions, with the result 1 representing the tumor region and 0 representing the backdrop. The dice similarity (DS) [18], which is regarded as a standard assessment technique in automatic medical image segmentation, can be evaluated by pixel to pixel comparison with manually segmented regions of interest or lesion mask. The ground truth for the evaluation is a manually segmented lesion mask, and each 2D pixel is predicted as True Positive (TRP ) or True Negative (TRN) or False Positive (TRN) or False Negative (FLN). The empirical goodness measures [Positive Predictive Value (PV ), Sensitivity (SS), Accuracy (AC) and Dice Similarity (DS) [18]] are assessed to evaluate the results. ξ (4) Activation guided by S-connected fuzzy voxel cardinality estimates (υκ). In addition, to investigate a number of optimal thresh- olds {T1, T2, · · · , TCl−1} in multi-class settings, Otsu’s method [45] is explored. The optimal thresholds maximize the class variance as follows [45]. O = hn{T1, T2, · · · , TCl−1}θi(µi −ω) (29) (29) where, Cl represents the number of defined classes in C ={C1, C2, . . . , CCl} and θi = X i∈Cl pi , µi = X i∈Cl ipi/θl (30) (30) where, the ith pixel is defined as pi. The probability of class Ci is represented as µi and its mean value is given by µi. ω is known to be the mean of class C.i To refine the segmentation accuracy and dice score for false- positive reduction in brain tumor detection, 3D-QNet seg- mented highly representative volumetric intensity features are post processed using the k-Means algorithm [46] for initial label segmentation and segmented into pre-defined number of clusters. C. Adjustment of Inter-connection Weights of 3D-QNet and Loss Function Moreover, the Vox-QSig is guided by four distinct activation schemes (υβ, υξ, υζ, υκ) [14], [34], [44]. Experiments have also been performed using the 3D- UNet [24] architecture, Deep Voxel-wise Residual Network (VoxResNet) [26], Dense-Res-Inception Net (DRINet) [27], and 3D-ESPNet [28] on the BRATS 2019 data set [47] and on the LiTS17 data set [48]. We have trained 3D-UNet [24] and VoxResNet [26] rigorously with the Stochastic Gradient Descent (SGD) algorithm on Caffe library1 using an Nvidia Tesla V 100 −SXM2 GPU Cluster with 32 GB of memory and 640 Tensor cores with 8 cores of Intel(R) Xeon(R) CPU E5-2683 v4@2.1GHz. The 3D-ESPNet is implemented using Pytorch from the code available in Github2 with 100 epochs using adam optimizer with an initial learning rate of 0.01. The DRINet is also implemented using adam optimizer with an initial learning rate of 0.01 and kernel size of 3 × 3. In order to detect a complete tumor, the segmented output images are resized to match the binary mask’s dimensions, with the result 1 representing the tumor region and 0 representing the backdrop. The dice similarity (DS) [18], which is regarded as a standard assessment technique in automatic medical image segmentation, can be evaluated by pixel to pixel comparison with manually segmented regions of interest or lesion mask. The ground truth for the evaluation is a manually segmented lesion mask, and each 2D pixel is predicted as True Positive (TRP ) or True Negative (TRN) or False Positive (TRN) or False Negative (FLN). The empirical goodness measures [Positive Predictive Value (PV ), Sensitivity (SS), Accuracy (AC) and Dice Similarity (DS) [18]] are assessed to evaluate the results. been observed that in majority of cases, λ = 0.232 and S = 26 (3 × 3 × 3 volume) yield optimal performance. For the other two modalities T1 and T1-CE, λ = 0.238 and S = 26 (3×3×3 volume) yield optimal performance. On contrary, the LiTS17 CT volume data has performed optimally for λ = 0.239 and S = 26 (3×3×3 volume). Moreover, the Vox-QSig is guided by four distinct activation schemes (υβ, υξ, υζ, υκ) [14], [34], [44]. Experiments have also been performed using the 3D- UNet [24] architecture, Deep Voxel-wise Residual Network (VoxResNet) [26], Dense-Res-Inception Net (DRINet) [27], and 3D-ESPNet [28] on the BRATS 2019 data set [47] and on the LiTS17 data set [48]. C. Adjustment of Inter-connection Weights of 3D-QNet and Loss Function αd i,j = αd i,j −min(αd i,j) max(αd i,j) −min(αd i,j) (28) (28) Each inter-connection link for each candidate pixel of the S-connected medical image volume and its corresponding activation are updated using quantum rotation gates, thereby enabling faster convergence of the proposed 3D-QNet archi- tecture. The inter-connection weight, φl,d and its activation, χl,d are updated as follows. Medical image volumes exhibit heterogeneous responses over the local intensities in the S-connected neighborhood regions, Medical image volumes exhibit heterogeneous responses over the local intensities in the S-connected neighborhood regions, owing to the wide variations of gray-levels. Inspired by the authors’ previous works [14], [34], [44], the proposed Vox-QSig activation function employs four different adaptive thresholding schemes suitable for efficient gray-scale segmen- tation in the 3D-QNet architecture. Medical image volumes exhibit heterogeneous responses over the local intensities in the S-connected neighborhood regions, owing to the wide variations of gray-levels. Inspired by the authors’ previous works [14], [34], [44], the proposed Vox-QSig activation function employs four different adaptive thresholding schemes suitable for efficient gray-scale segmen- tation in the 3D-QNet architecture. |φι+1,d⟩=  cos △ωι+1,d −sin △ωι+1,d sin △ωι+1,d cos △ωι+1,d  |φι,d⟩ (23) |χι+1,d⟩=  cos △ϑι+1,d −sin △ϑι+1,d sin △ϑι+1,d cos△ϑι+1,d  |χι,d⟩ (24) where, ωι+1,d ωι,d + △ωι,d (25) |φι+1,d⟩=  cos △ωι+1,d −sin △ωι+1,d sin △ωι+1,d cos △ωι+1,d  |φι,d⟩ (23) |χι+1,d⟩=  cos △ϑι+1,d −sin △ϑι+1,d sin △ϑι+1,d cos△ϑι+1,d  |χι,d⟩ (24) where, ωι+1,d = ωι,d + △ωι,d (25) |φι+1,d⟩=  cos △ωι+1,d −sin △ωι+1,d sin △ωι+1,d cos △ωι+1,d  |φι,d⟩ (23) |χι+1,d⟩=  cos △ϑι+1,d −sin △ϑι+1,d sin △ϑι+1,d cos△ϑι+1,d  |χι,d⟩ (24) (1) Activation guided by β-distribution of the intensity of S- connected neighborhood voxels (υβ). (2) Activation guided by S-connected voxels based on Skew- ness (υχ). (2) Activation guided by S-connected voxels based on Skew- ness (υχ). ωι+1,d = ωι,d + △ωι,d (25) ωι+1,d = ωι,d + △ωι,d (25) (3) Activation guided by S-connected heterogeneous voxel (3) Activation guided by S-connected heterogeneous voxel 7 been observed that in majority of cases, λ = 0.232 and S = 26 (3 × 3 × 3 volume) yield optimal performance. For the other two modalities T1 and T1-CE, λ = 0.238 and S = 26 (3×3×3 volume) yield optimal performance. On contrary, the LiTS17 CT volume data has performed optimally for λ = 0.239 and S = 26 (3×3×3 volume). 1https://doi.org/10.1145/2647868.2654889 2https://github.com/sacmehta/3D-ESPNet B. Experimental Setup Table IV presents the results reported using the proposed 3D-QNet, 3D-UNet [24], VoxResNet [26], 3D-QNet-NonTensor, DRINet [27], and 3D- ESPNet [28] on LiTS17 data set [48] in detecting complete Liver tumor regions. A sample of segmented Liver tumor using 3D-QNet with manually segmented tumors is shown in Fig- ure 7. It is observed from Table IV that 3D-QNet has reported with an average Dice Score (DS) of 0.958. Furthermore, a one-sided two-sample Kolmogorov-Smirnov (KS) [49] test is performed with a significance level of α = 0.05, and the experimental data given in Table III and Table IV show that 3D-QNet is capable of segmenting 3D medical image data. Despite being characterized by fully self-supervised quantum- inspired learning, the 3D-QNet has demonstrated compara- 5, respectively. The human expert annotated ground truth slices for all the four different modalities are illustrated in Figure 6. 3D-UNet [24], VoxResNet [26], DRINet [27], 3D- ESPNet [28], and 3D-QNet-NonTensor segmented brain MR volumes from BRATS19-CBICA-AAG are also demonstrated in the Supplementary Materials. It has been observed from the segmented MR slices that the proposed 3D-QNet is suitable in segmenting the correct position and size of the complete tumor while compared with the ground truth segmentation. However, it is not efficient in mapping the sharp contour of the core and enhanced tumor sub-regions outlined in the annotated slices. The convergence analysis of the proposed 3D-QNet architec- ture is discussed in the Appendix and experimentally demon- strated with the non-tensorized implementation of the network (3D-QNet-NonTensor). The convergence analysis of the pro- posed 3D-QNet and 3D-QNet-NonTensor architectures for all types of brain tumor images (T1, T1 −CE, Flair, and T2) from the BRATS 2019 datasets [47] and for LiTS17 liver volumes [48] are provided in the Supplementary Materials. Table V reports the average number of iterations required to converge the proposed 3D-QNet architecture and its non- tensorized implementation, 3D-QNet-NonTensor. It is evident from Table V that an optimal convergence of the proposed 3D-QNet architecture is observed for Flair with υξ. Hence, the non-tensorized implementation (3D-QNet without tensor decomposition) underperforms in terms of average iterations in comparison to 3D-QNet. It serves as the inspiration behind the incorporation of tensor-decomposition in 3D-QNet imple- mentation. it is not efficient in mapping the sharp contour of the core and enhanced tumor sub-regions outlined in the annotated slices. B. Experimental Setup Table III presents the quantitative results reported using the proposed 3D-QNet, 3D-QNet-NonTensor, 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-ESPNet [28] on evalu- ating the average accuracy (AC), dice similarity score (DS), positive prediction value (PV ), and sensitivity (SS) [18]. It has been observed from the 3D-QNet segmented brain MR slices and the results reported in Table III, that optimal segmentation is achieved for FLAIR reported with an average of 0.821 dice score (DS). The proposed 3D-QNet marginally outperforms the convolutional architectures (3D-UNet [24], VoxResNet [26], DRINet [27], 3D-ESPNet [28]), and 3D- QNet-NonTensor in predicting complete brain tumor detection. However, it may be noted that the proposed 3D-QNet does not intend to predict the core, enhanced tumor and necrosis sub regions owing to lack of optimization of the parameters in the suggested 3D-QNet architecture. The box plots are also demonstrated in the Supplementary Materials citing the out- come reported in Table III. Moreover, to show the effectiveness of the proposed 3D-QNet architecture over 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-ESPNet [28], we have also conducted experiments on the Liver Tumor Segmentation Benchmark (LiTS17) data set [48]. Table IV presents the results reported using the proposed 3D-QNet, 3D-UNet [24], VoxResNet [26], 3D-QNet-NonTensor, DRINet [27], and 3D- ESPNet [28] on LiTS17 data set [48] in detecting complete Liver tumor regions. A sample of segmented Liver tumor using 3D-QNet with manually segmented tumors is shown in Fig- ure 7. It is observed from Table IV that 3D-QNet has reported with an average Dice Score (DS) of 0.958. Furthermore, a one-sided two-sample Kolmogorov-Smirnov (KS) [49] test is performed with a significance level of α = 0.05, and the experimental data given in Table III and Table IV show that 3D-QNet is capable of segmenting 3D medical image data. Despite being characterized by fully self-supervised quantum- inspired learning, the 3D-QNet has demonstrated compara- ble accuracy (AC) and dice similarity (DS) in comparison to 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D- ESPNet [28]. Hence, the performance of the 3D-QNet model on the BRATS 2019 and LiTS17 data sets are statistically significant and offers a promising alternative to self-supervised deep learning for 3D-medical image segmentation. B. Experimental Setup Further- more, in case of brain MR image segmentation, the num- ber of parameters required in the 3D-UNet [24] architecture is 19, 069, 955, whereas the proposed 3D-QNet architecture employs maximum 2, 995, 200 parameters (considering each voxel as a candidate in a 240 × 240 dimensional slice, there are total 240 × 240 × 26 × 2 connections) in bi-directional VII. DISCUSSIONS The proposed 3D-QNet is computed and tested on a clas- sical system. Hence, the proposed model architecture is not quantum in the real sense. Instead, it is quantum-inspired. The pixel intensities and interconnection weight matrices are expressed in quantum formalism in classical simulations using real vectors. Here, each pixel information and weight term are presented as a vector with better expressibility and non-linearity than classical neurons. The 3D-QNet network architecture and the use of a unitary matrix representation in network weights ensure the authenticity of quantum analogies without sacrificing efficacy or efficiency, as shown by a full comparison with state-of-the-art models on two benchmark datasets. The incorporation of quantum-inspired computing and tensor-based learning in the suggested network model aims to provide faster convergence of the 3D-QNet architecture over its non-tensorized implementation, thereby enabling accurate segmentation results. g However, the proposed 3D-QNet architecture may be im- plemented utilizing a quantum-classical hybrid paradigm for NISQ devices on the real quantum processor. In this hybrid quantum classical framework, the N-connected third-order neighborhood-based interconnection needed N (here N = 26) input qubits, Hadamard gates, and Rotation gates, as well as N(N −1) numbers of CNOT gates [15], which resulted in quantum superposition and entanglement of the input states. This quantum-classical model lays the way for the imple- mentation of quantum machine learning on near-term NISQ devices using variational quantum circuits (VQCs) — a kind of Quantum circuits with improved gate settings. It is worth noting that 3D-QNet has the potential to be significantly more computationally efficient than the models presented in the experiments, especially in multi-level seg- mentation of BRATS 2019 MR images. The performance of many quantum-inspired algorithms derives directly from quantum parallelism, which is a fundamental characteristic of many quantum systems. Heuristically, the proposed 3D-QNet However, the proposed 3D-QNet architecture may be im- plemented utilizing a quantum-classical hybrid paradigm for NISQ devices on the real quantum processor. In this hybrid quantum classical framework, the N-connected third-order neighborhood-based interconnection needed N (here N = 26) input qubits, Hadamard gates, and Rotation gates, as well as N(N −1) numbers of CNOT gates [15], which resulted in quantum superposition and entanglement of the input states. This quantum-classical model lays the way for the imple- mentation of quantum machine learning on near-term NISQ devices using variational quantum circuits (VQCs) — a kind of Quantum circuits with improved gate settings. B. Experimental Setup Experiments have been carried out using 3D-QNet and 3D- QNet-NonTensor on 3D brain MR volumes collected from the BRATS 2019 dataset of size 240×240 and on the LiTS17 data set of size 512×512 with MATLAB 2020a. The proposed 3D- QNet and 3D-QNet-NonTensor architectures are implemented with the multi-level gray-scale images using distinct multi- class levels L = 4, 6, and 8 characterized by the Vox-QSig activation function. The steepness λ is varied in the range 0.23 to 0.24 with step size of 0.001. For FLAIR and T2, it has 8 5, respectively. The human expert annotated ground truth slices for all the four different modalities are illustrated in Figure 6. 3D-UNet [24], VoxResNet [26], DRINet [27], 3D- ESPNet [28], and 3D-QNet-NonTensor segmented brain MR volumes from BRATS19-CBICA-AAG are also demonstrated in the Supplementary Materials. It has been observed from the segmented MR slices that the proposed 3D-QNet is suitable in segmenting the correct position and size of the complete tumor while compared with the ground truth segmentation. However, it is not efficient in mapping the sharp contour of the core and enhanced tumor sub-regions outlined in the annotated slices. Table III presents the quantitative results reported using the proposed 3D-QNet, 3D-QNet-NonTensor, 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-ESPNet [28] on evalu- ating the average accuracy (AC), dice similarity score (DS), positive prediction value (PV ), and sensitivity (SS) [18]. It has been observed from the 3D-QNet segmented brain MR slices and the results reported in Table III, that optimal segmentation is achieved for FLAIR reported with an average of 0.821 dice score (DS). The proposed 3D-QNet marginally outperforms the convolutional architectures (3D-UNet [24], VoxResNet [26], DRINet [27], 3D-ESPNet [28]), and 3D- QNet-NonTensor in predicting complete brain tumor detection. However, it may be noted that the proposed 3D-QNet does not intend to predict the core, enhanced tumor and necrosis sub regions owing to lack of optimization of the parameters in the suggested 3D-QNet architecture. The box plots are also demonstrated in the Supplementary Materials citing the out- come reported in Table III. Moreover, to show the effectiveness of the proposed 3D-QNet architecture over 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-ESPNet [28], we have also conducted experiments on the Liver Tumor Segmentation Benchmark (LiTS17) data set [48]. VII. DISCUSSIONS Heuristically, the proposed 3D-QNet 9 TABLE II: Results obtained using proposed 3D-QNet for complete tumor (WT) segmentation on BraTS19-CBICA-AAG-1- flair-slice#69 TABLE II: Results obtained using proposed 3D-QNet for complete tumor (WT) segmentation on BraTS19-CBICA-AAG-1- flair-slice#69 flair-slice#69 Level Modality AC = TRP +TRN TRP +FLP +TRN +FLN DS = 2TRP 2TRP +FLP +FLN PV = TRP TRP +FLP SS = TRP TRP +FLN υβ υχ υξ υκ υβ υχ υξ υκ υβ υχ υξ υκ υβ υχ υξ υκ L = 4 T 1 0.99 0.99 0.99 0.99 0.62 0.80 0.80 0.79 0.44 0.66 0.65 0.65 0.99 0.99 0.99 0.99 T 1 −CE 0.99 0.99 0.99 0.99 0.79 0.80 0.79 0.80 0.66 0.66 0.66 0.66 0.99 0.99 0.99 0.99 F LAIR 0.99 0.99 0.99 0.99 0.62 0.80 0.80 0.79 0.44 0.66 0.66 0.65 0.99 0.99 0.99 0.99 T 2 0.99 0.99 0.99 0.99 0.62 0.80 0.80 0.79 0.44 0.66 0.66 0.65 0.99 0.99 0.99 0.99 L = 6 T 1 0.99 0.99 0.99 0.99 0.62 0.80 0.80 0.79 0.44 0.66 0.66 0.65 0.99 0.99 0.99 0.99 T 1 −CE 0.99 0.99 0.99 0.99 0.62 0.80 0.80 0.79 0.44 0.66 0.66 0.65 0.99 0.99 0.99 0.99 F LAIR 0.99 0.99 0.99 0.99 0.62 0.80 0.80 0.79 0.44 0.66 0.66 0.65 0.99 0.99 0.99 0.99 T 2 0.99 0.99 0.99 0.99 0.62 0.80 0.80 0.79 0.44 0.66 0.66 0.65 0.99 0.99 0.99 0.99 L = 8 T 1 0.99 0.99 0.99 0.99 0.82 0.82 0.82 0.81 0.70 0.69 0.69 0.68 0.99 0.99 0.99 0.99 T 1 −CE 0.99 0.99 0.99 0.99 0.81 0.81 0.81 0.81 0.68 0.68 0.68 0.68 0.99 0.99 0.99 0.99 F LAIR 0.99 0.99 0.99 0.99 0.84 0.84 0.84 0.84 0.73 0.73 0.73 0.72 0.99 0.99 0.98 0.98 T 2 0.99 0.99 0.99 0.99 0.82 0.82 0.82 0.82 0.69 0.69 0.70 0.70 0.99 0.99 0.99 0.99 TABLE III: Comparative analysis of proposed 3D-QNet with 3D-QNet-NonTensor, 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-ESPNet [28] [The bold numbers rep- resent evaluation metrics completed with a one-sided two- sample KS test with a significance threshold of α = 0.05 [49]] TABLE III: Comparative analysis of proposed 3D-QNet with 3D-QNet-NonTensor, 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-ESPNet [28] [The bold numbers rep- resent evaluation metrics completed with a one-sided two- sample KS test with a significance threshold of α = 0.05 [49]] TABLE IV: Comparative Results on Liver Segmentation using proposed 3D-QNet, 3D-UNet [24], VoxResNet [26], 3D-QNet- NonTensor, DRINet [27], and 3D-ESPNet [28] [The bold numbers represent evaluation metrics completed with a one- sided two-sample KS test with a significance threshold of α = 0.05 [49]] Methods Modality AC DS PV SS 3D-UNet [24] T 1 0.990 0.811 0.736 0.941 T 1 −CE 0.990 0.807 0.732 0.938 F LAIR 0.992 0.823 0.737 0.943 T 2 0.989 0.812 0.735 0.944 VoxResNet [26] T 1 0.990 0.810 0.737 0.937 T 1 −CE 0.989 0.813 0.732 0.943 F LAIR 0.991 0.822 0.751 0.942 T 2 0.990 0.807 0.729 0.944 DRINet [27] T 1 0.989 0.793 0.701 0.958 T 1 −CE 0.988 0.800 0.711 0.959 F LAIR 0.989 0.805 0.708 0.969 T 2 0.987 0.789 0.700 0.958 3D-ESPNet [28] T 1 0.989 0.801 0.709 0.961 T 1 −CE 0.989 0.813 0.721 0.966 F LAIR 0.989 0.800 0.715 0.959 T 2 0.988 0.802 0.714 0.957 3D-QNet T 1 0.989 0.801 0.736 0.965 T 1 −CE 0.989 0.811 0.740 0.957 F LAIR 0.991 0.821 0.751 0.957 T 2 0.990 0.814 0.736 0.960 3D-QNet-NonTensor T 1 0.987 0.776 0.678 0.959 T 1 −CE 0.987 0.772 0.678 0.958 F LAIR 0.989 0.786 0.697 0.956 T 2 0.988 0.788 0.696 0.957 Methods AC DS PV SS VoxResNet [26] 0.991 0.961 0.798 0.973 3D-QNet 0.989 0.958 0.801 0.965 3D-UNet [24] 0.991 0.959 0.830 0.964 3D-QNet-NonTensor 0.982 0.953 0.799 0.879 DRINet [27] 0.980 0.951 0.802 0.988 3D-ESPNet [28] 0.987 0.943 0.798 0.961 TABLE V: Average number of iterations required for conver- gence of the proposed 3D-QNet and 3D-QNet-NonTensor TABLE V: Average number of iterations required for conver- gence of the proposed 3D-QNet and 3D-QNet-NonTensor Methods Modality Number of iterations υβ υχ υξ υκ 3D-QNet T 1 30.89 30.64 30.38 30.91 T 1−CE 31.60 31.46 31.43 31.68 F lair 30.78 30.23 30.18 30.57 T 2 32.25 32.66 32.14 32.73 Liver 27.11 27.89 26.99 27.08 3D-QNet-NonTensor T 1 31.46 31.59 31.43 31.69 T 1−CE 32.98 32.33 32.39 32.56 F lair 31.69 31.12 31.19 31.26 T 2 32.88 32.34 32.51 32.79 Liver 28.52 28.16 28.12 28.17 architecture has the potential to explore the intrinsic properties of quantum parallelism to simultaneously compute the image pixels. VII. DISCUSSIONS However, 3D-QNet is computed and experimented on a classical system, and hence the quantum parallelism has not been fully explored in the proposed quantum-inspired framework. Despite the fact that quantum simulation requires a great deal of resources in general, the proposed quantum- inspired model, 3D-QNet, requires a lesser number of param- eters compared to the 3D classical CNN models. 3D-QNet architecture to promote automatic semantic segmen- tation of 3D medical images in real-time with minimum human intervention, still being considered as an uphill task in the field of volumetric medical image segmentation. Despite being a 3D self-supervised network model, 3D-QNet achieved similar dice similarity score on complete tumor detection as deeply supervised 3D-UNet, Vox-ResNet, DRINet and ESPNet, thus promoting self-supervised network learning for volumetric segmentation of medical images. In principle, the proposed 3D-QNet is a general self-supervised network architecture which can be extended to many other 3D medical image segmentation avenues, where the segmented annotations are limited. Furthermore, the proposed 3D self-supervised model can be used immediately in any application (e.g., medical IoT devices) where 3D deep learning models face VII. DISCUSSIONS It is worth noting that 3D-QNet has the potential to be significantly more computationally efficient than the models presented in the experiments, especially in multi-level seg- mentation of BRATS 2019 MR images. The performance of many quantum-inspired algorithms derives directly from quantum parallelism, which is a fundamental characteristic of many quantum systems. VIII. CONCLUSION (a) Slice#44 (b) Slice#59 (c) Slice#64 (d) Slice#69 (d) Slice#69 (e) Slice#44 (f) Slice#59 (g) Slice#64 (h) Slice#69 (f) Slice#59 (g) Slice#71 (h) Slice#95 (h) Slice#95 Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA- AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the BRATS 2019 data set [47] (Complete tumor (WT) region comprises a union of brown, light green and green yellow, core tumor (TC) is the union of light green and green yellow, and green yellow corresponds to the tumor enhancing (TE)). Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA- AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the BRATS 2019 data set [47] (Complete tumor (WT) region comprises a union of brown, light green and green yellow, core tumor (TC) is the union of light green and green yellow, and green yellow corresponds to the tumor enhancing (TE)). (m) Slice#44 (a) Slice#172 (b) Slice#181 (c) Slice#198 (d) Slice#206 (e) Slice#172 (f) Slice#181 (g) Slice#198 (h) Slice#206 Fig. 7: (a −d) 3D-QNet segmented volumetric Liver image slices (scan#64), (e −h) manually segmented image slices (scan#64) from data set [48] (Union of overlapped brown and red corresponds to a complete tumor (WT) region). Fig. 4: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG- 1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19- CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union of overlapped brown/yellow and green corresponds to a com- plete tumor (WT) region). Fig. 4: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG- 1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19- CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union of overlapped brown/yellow and green corresponds to a com- plete tumor (WT) region). ( ) Sli #172 (f) Sli #181 ( ) Sli #198 (h) Sli #206 plete tumor (WT) region). (a) Slice#52 (b) Slice#60 (c) Slice#71 (d) Slice#95 (e) Slice#52 (f) Slice#60 (g) Slice#71 (h) Slice#95 (i) Slice#52 (j) Slice#60 (k) Slice#71 (l) Slice#95 (m) Slice#52 (n) Slice#60 (o) Slice#71 (p) Slice#95 Fig. 5: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAB-1-flair, (e−h) BraTS19-CBICA-AAB- 1-t2, (i −l) BraTS19-CBICA-AAB-1-t1ce, (m −p) BraTS19- CBICA-AAB-1-t1 from the BRATS 2019 data set [47] (Union of overlapped brown/yellow and green corresponds to a com- plete tumor (WT) region). p ( ) g ) (e) Slice#172 (f) Slice#181 (e) Slice#172 (f) Slice#181 (g) Slice#198 (h) Slice#206 (g) Slice#198 (h) Slice#206 Fig. VIII. CONCLUSION 7: (a −d) 3D-QNet segmented volumetric Liver image slices (scan#64), (e −h) manually segmented image slices (scan#64) from data set [48] (Union of overlapped brown and red corresponds to a complete tumor (WT) region). (a) Slice#52 (b) Slice#60 (c) Slice#71 (d) Slice#95 (b) Slice#60 significant challenges. However, the 3D-QNet fails to yield optimal outcome for multi-level segmentation on the BRATS 2019 data set. The authors are currently engaged in extend- ing the 3D-QNet architecture by up-scaling the intermediate volumetric features in the network and optimizing its hyper- parameters to yield optimal segmentation outcome. (e) Slice#52 (f) Slice#60 (g) Slice#71 (h) Slice#95 VIII. CONCLUSION A 3D Quantum-inspired Self-supervised Tensor Network (3D-QNet) architecture characterized by S-connected voxel- wise processing for fully automated semantic segmentation of Brain MR volumes and 3D Liver CT scans, is presented in this work. Intensive validation using the BRATS 2019 and LiTS17 data sets shows the efficacy of the proposed self-supervised 10 10 (a) Slice#44 (b) Slice#59 (c) Slice#64 (d) Slice#69 (e) Slice#44 (f) Slice#59 (g) Slice#64 (h) Slice#69 (i) Slice#44 (j) Slice#59 (k) Slice#64 (l) Slice#69 (m) Slice#44 (n) Slice#59 (o) Slice#64 (p) Slice#69 Fig. 4: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG- 1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19- CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union of overlapped brown/yellow and green corresponds to a com- plete tumor (WT) region). (a) Slice#52 (b) Slice#60 (c) Slice#71 (d) Slice#95 (e) Slice#52 (f) Slice#60 (g) Slice#71 (h) Slice#95 (i) Slice#52 (j) Slice#60 (k) Slice#71 (l) Slice#95 (m) Slice#52 (n) Slice#60 (o) Slice#71 (p) Slice#95 Fig. 5: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAB-1-flair, (e−h) BraTS19-CBICA-AAB- 1-t2, (i −l) BraTS19-CBICA-AAB-1-t1ce, (m −p) BraTS19- CBICA-AAB-1-t1 from the BRATS 2019 data set [47] (Union (a) Slice#44 (b) Slice#59 (c) Slice#64 (d) Slice#69 (e) Slice#52 (f) Slice#60 (g) Slice#71 (h) Slice#95 Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA- AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the BRATS 2019 data set [47] (Complete tumor (WT) region comprises a union of brown, light green and green yellow, core tumor (TC) is the union of light green and green yellow, and green yellow corresponds to the tumor enhancing (TE)). (a) Slice#172 (b) Slice#181 (c) Slice#198 (d) Slice#206 (e) Slice#172 (f) Slice#181 (g) Slice#198 (h) Slice#206 Fig. 7: (a −d) 3D-QNet segmented volumetric Liver image slices (scan#64), (e −h) manually segmented image slices (scan#64) from data set [48] (Union of overlapped brown and red corresponds to a complete tumor (WT) region). significant challenges. However, the 3D-QNet fails to yield optimal outcome for multi-level segmentation on the BRATS 2019 data set. The authors are currently engaged in extend- ing the 3D-QNet architecture by up-scaling the intermediate volumetric features in the network and optimizing its hyper- parameters to yield optimal segmentation outcome. APPENDIX A. Convergence Analysis of 3D-QNet Le us assume the optimal phase angles at depth d for the weighted matrix and the activation are denoted as ωd and ϑd, respectively. VIII. CONCLUSION Now, consider Wι,d = ωι,d −ωd (31) Vι,d = ϑι,d −ϑd (32) and Dι,d = ωι+1,d −ωι,d = Wι+1,d −Wι,d (33) Pι,d = ϑι+1,d −ϑι,d = Vι+1,d −Vι,d (34) The loss function ζ(ωι,d, ϑι,d) is differentiable with respect to ωι,d and ϑι,d as ∂ζ(ωι,d, ϑι,d) ∂ωι,d ij = 2 N N X i=1 S X j=1 △φι,d ij (ωι,d ij , ϑι,d j ) " ∂φι+1,d ij (ωι+1,d ij , ϑι+1,d j ) − ∂φι,d ij (ωι,d ij , ϑι,d j ) # (35) (a) Slice#44 (b) Slice#59 (c) Slice#64 (d) Slice#69 (e) Slice#44 (f) Slice#59 (g) Slice#64 (h) Slice#69 (i) Slice#44 (j) Slice#59 (k) Slice#64 (l) Slice#69 (m) Slice#44 (n) Slice#59 (o) Slice#64 (p) Slice#69 Fig. 4: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG- 1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19- CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union of overlapped brown/yellow and green corresponds to a com- plete tumor (WT) region). (a) Slice#44 (b) Slice#59 (c) Slice#64 (d) Slice#69 (e) Slice#52 (f) Slice#60 (g) Slice#71 (h) Slice#95 Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA- AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the BRATS 2019 data set [47] (Complete tumor (WT) region comprises a union of brown, light green and green yellow, core tumor (TC) is the union of light green and green yellow, and green yellow corresponds to the tumor enhancing (TE)). (a) Slice#172 (b) Slice#181 (c) Slice#198 (d) Slice#206 (a) Slice#44 (b) Slice#59 (c) Slice#64 (d) Slice#69 (e) Slice#52 (f) Slice#60 (g) Slice#71 (h) Slice#95 Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA- AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the BRATS 2019 data set [47] (Complete tumor (WT) region comprises a union of brown, light green and green yellow, core tumor (TC) is the union of light green and green yellow, and green yellow corresponds to the tumor enhancing (TE)). (a) Slice#44 (b) Slice#59 (c) Slice#64 (d) Slice#69 (e) Slice#44 (f) Slice#59 (g) Slice#64 (h) Slice#69 (i) Slice#44 (j) Slice#59 (k) Slice#64 (l) Slice#69 (m) Slice#44 (n) Slice#59 (o) Slice#64 (p) Slice#69 Fig. 4: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG- 1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19- CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union of overlapped brown/yellow and green corresponds to a com- plete tumor (WT) region). A. Convergence Analysis of 3D-QNet Le us assume the optimal phase angles at depth d for the weighted matrix and the activation are denoted as ωd and ϑd, respectively. Now, consider (j) Slice#60 (k) Slice#71 (k) Slice#71 (l) Sli (k) Slice#71 Wι,d = ωι,d −ωd (31) Vι,d = ϑι,d −ϑd (32) and Dι,d = ωι+1,d −ωι,d = Wι+1,d −Wι,d (33) Pι,d = ϑι+1,d −ϑι,d = Vι+1,d −Vι,d (34) (o) Slice#71 (m) Slice#52 (m) Slice#52 (p) Slice#95 (n) Slice#60 The loss function ζ(ωι,d, ϑι,d) is differentiable with respect to ωι,d and ϑι,d as n ζ(ωι,d, ϑι,d) is differentiable with respect to ωι,d and ϑι,d as Fig. 5: 3D-QNet segmented Brain MR volume (a −d) BraTS19-CBICA-AAB-1-flair, (e−h) BraTS19-CBICA-AAB- 1-t2, (i −l) BraTS19-CBICA-AAB-1-t1ce, (m −p) BraTS19- CBICA-AAB-1-t1 from the BRATS 2019 data set [47] (Union of overlapped brown/yellow and green corresponds to a com- plete tumor (WT) region). REFERENCES ζ(ωι+1,d) ≤ζ(ωι,d) + ⟨∇ωζ(ωι,d), ωι+1,d −ωι,d⟩+ ι 2 ||ωι+1,d −ωι,d||2 = ζ(ωι,d) + ⟨∇ωζ(ωι,d) −ρ∇ωζ(ωι,d)⟩+ L 2 || −ρ∇ωζ(ωι,d)||2 = ζ(ωι,d) −ρ||∇ωζ(ωι,d)||2 + ρ2 L 2 ||∇ωζ(ωι,d)||2 = ζ(ωι,d) −ρ(1 −ρ L 2 )||∇ωζ(ωι,d)||2 ≤ζ(ωι,d) −ρ 2 ||∇ωζ(ωι,d)||2 (Assuming, ρ ∈(0, 1 L ]) ≤ζ(ωd) + ⟨∇ωζ(ωι,d), ωι,d −ωd⟩−ρ 2 ||∇ωζ(ωι,d)||2, (ζ is convex) = ζ(ωd) + ⟨∇ωζ(ωι,d), ωι,d −ωd⟩−ρ 2 ||∇ωζ(ωι,d)||2+ 1 2ρ (||ωι,d −ωd||2 −||ωι,d −ωd||2) = ζ(ωd) + 1 2ρ (||ωι,d −ωd||2 −(||ωι,d||2 −2⟨ωι,d, ωd⟩+ ||ωd||2 −2ρ⟨∇ωζ(ωι,d), ωι,d −ωd⟩+ ρ2||∇ωζ(ωι,d)||2)) = ζ(ωd) + 1 2ρ (||ωι,d|| −ωd||2 −(||ωι,d −ρ∇ωζ(ωι,d)||2− 2⟨ωι,d −∇ωζ(ωι,d), ωd⟩+ ||ωd||2)) = ζ(ωd) + 1 2ρ (||ωι,d −ωd||2 −||ωι+1,d −ωd||2) ∴, ζ(ωι+1,d) −ζ(ωd) ≤1 2ρ (||ωι,d −ωd||2 −||ωι+1,d −ωd||2) [7] Q. Doua, L. Yua, H. Chena, Y.Jina, X. Yanga, J. Q. Pheng, and A. Heng, “3D deeply supervised network for automated segmentation of volumetric medical images," Medical Image Analysis, vol. 41, pp. 40–54, 2017, doi: https://doi.org/10.1016/j.media.2017.05.001. [8] V. Gandhi, G. Prasad, D. Coyle, L. Behera, and T. M. McGinnity, “Quantum neural network-based EEG filtering for a brain-computer interface,” IEEE Transaction on Neural Network and Learning Systems, vol. 25, no. 2, pp. 278-–288, 2014, doi: 10.1109/TNNLS.2013.2274436. [9] P. Li, H. Xiao, F. Shang, X. Tong, X. Li, and M. Cao, “A hybrid quantum- inspired neural networks with sequence inputs,” Neurocomputing, vol. 117, pp. 81-–90, 2013, doi: https://doi.org/10.1016/j.neucom.2013.01.029. [10] N. Masuyama, C. K. Loo, M. Seera, and N. Kubota, “Quantum- Inspired Multidirectional Associative Memory With a Self-Convergent Iterative Learning,” IEEE Transaction on Neural Network and Learning Systems, vol. 29, no. 4, pp. 1058—1068, 2018, doi: 10.1109/TNNLS. 2017.2653114. [11] S. Bhattacharyya, P. Pal and S. Bhowmick, “Binary Image Denoising Using a Quantum Multilayer Self Organizing Neural Network," Applied Soft Computing, vol. 24, pp. 717–729, 2014, doi: https://doi.org/10.1016/ j.asoc.2014.08.027. [12] M. Mediouni, D. R. Schlatterer, H. Madry, M. Cucchiarini and B. Rai, “A review of translational medicine. The future paradigm: how can we connect the orthopedic dots better?," Current Medical Research and Opinion, vol. 34, no. 7, pp. 1217–1229, 2018, doi: 10.1080/03007995. 2017.1385450. Similarly, it can also be shown that Similarly, it can also be shown that ζ(ϑι+1,d) −ζ(ϑι,d) ≤1 2ρ (||ϑι+1,d −ϑd||2 −||ϑι+1,d −ϑd||2) (45) Now, according to Thaler formula ζ(ϑι+1,d) −ζ(ϑι,d) ≤1 2ρ (||ϑι+1,d −ϑd||2 −||ϑι+1,d −ϑd||2) (45) [ ζ(ϑι+1,d) −ζ(ϑι,d) ≤1 2ρ (||ϑι+1,d −ϑd||2 −||ϑι+1,d −ϑd||2) (45) Now, according to Thaler formula [13] M. Mediouni, R. Madiouni, M. Gardner, N. A. Convergence Analysis of 3D-QNet ∂ζ(ωι,d, ϑι,d) ∂ωι,d ij = 2 N N X i=1 S X j=1 △φι,d ij (ωι,d ij , ϑι,d j ) " ∂φι+1,d ij (ωι+1,d ij , ϑι+1,d j ) ∂ωι+1,d ij − ∂φι,d ij (ωι,d ij , ϑι,d j ) ∂ωι,d ij # (35) (35) 11 ∂ζ(ωι,d, ϑι,d) ∂ϑι,d j = 2 N N X i=1 S X j=1 △φι,d ij (ωι,d ij , ϑι,d j ) " ∂φι+1,d ij (ωι+1,d ij , ϑι+1,d j ) ∂ϑι+1,d j − ∂φι,d ij (ωι,d ij , ϑι,d j ) ∂ϑι,d j # (36) ≈ " {−ρι,d ij ∂ζ(ωι,d, ϑι,d) ∂ωι,d ij }2 + {−κι,d j ∂ζ(ωι,d, ϑι,d) ∂ϑl,d j }2 # {ζ(ωι,d, ϑι,d)} 1 ι (47) It is obvious that (ζ(ωι+1,d, αι+1,d) −ζ(ωι,d, ϑι,d)) ≤0 and the sequences of {ωι,d} and {ϑι,d} are monotonically decreasing as ≈ " {−ρι,d ij ∂ζ(ωι,d, ϑι,d) ∂ωι,d ij }2 + {−κι,d j ∂ζ(ωι,d, ϑι,d) ∂ϑl,d j }2 # {ζ(ωι,d, ϑι,d)} 1 ι (47) (47) (47) It is obvious that (ζ(ωι+1,d, αι+1,d) −ζ(ωι,d, ϑι,d)) ≤0 and the sequences of {ωι,d} and {ϑι,d} are monotonically decreasing as where, where, lim ι→∞ζ(ωι,d, ϑι,d) = (ωd, ϑ d) (48) lim ι→∞ζ(ωι,d, ϑι,d) = (ωd, ϑ d) (48) △φι,d ij (ωι,d ij ϑj) = |φι+1,d ij (ωι+1,d ij , ϑι+1,d j ) −φι,d ij (ωι,d ij , ϑι,d j )| (37) (37) and The following equations evaluate the change in phase or angles (△ω and △α) of the rotation gates as lim ι→∞ ||ζ(ωι+1,d, ϑι+1,d) −(ωd, ϑ d)|| ||ζ(ωι,d, ϑι,d) −(ωd, ϑ d)|| ≤1 (49) (49) △ωι,d ij = −ρι,d ij { ∂φ(ωι,d, ϑι,d) ∂ωι,d ij φ(ωι,d, ϑι,d)} 1 τ (38) △ϑι,d j = −κι,d j { ∂φ(ωι,d, ϑι,d) ∂ϑι,d i φ(ωι,d, ϑι,d))} 1 τ (39) (38) B. Code Availability 3D-QNet implementation is made available in GitHub: https://github.com/konar1987/ 3D-QNet for brain volume image segmentation with few samples, tailored and tested for FLAIR and T2 from the BRATS 2019 data set [47]. where, ρij and κj refer to the learning rates for the adjustments of weights and activation, respectively and are evaluated as REFERENCES ρι,d ij = X ι,d i −X ι,d ij ∀j = 1, 2 . . . 8 κι,d j = ( X j X ι,d i,j )∀j = 1, 2 . . . 8 (40) [1] G. Litjens et al., “A survey on deep learning in medical image analysis,” Medical Image Analysis, vol. 42, pp. 60—88, 2017. doi: https://doi.org/ 10.1016/j.media.2017.07.005. (40) j [2] M. Huang, W. Yang, Y. Wu, J. Jiang, W. Chen, and Q. Feng, “Brain Tumor Segmentation Based on Local Independent Projection-Based Classifica- tion," IEEE Transactions on Biomedical Engineering, vol. 61, no. 10, pp. 2633–2645, 2014, doi: 10.1109/TBME.2014.2325410. The sequences of {ωι,d} and {ϑι,d} converge super-linearly subject to the following conditions [15]. || ι+1 d d|| lim ι→∞ ||ωι+1,d −ωd|| ||ωι,d −ωd|| ≤1 lim ι→∞ ||ωι+1,d −ωd|| ||ωι,d −ωd|| ≤1 (41) lim ι→∞ ||ωι+1,d −ωd|| ||ωι,d −ωd|| ≤1 (41) and ||Wι+1,d|| = O||Dι,d|| (42) Also, lim ι→∞ ||ϑι+1,d −ϑ d|| ||ϑι −ϑ d|| ≤1 (43) and ||Vι+1,d|| = O||Pι,d|| (44) (41) [3] Z. Wu, K. D. Paulsen, and J. M. Sullivan, “Adaptive model initialization and deformation for automatic segmentation of T1-weighted brain MRI data", IEEE Transactions on Biomedical Engineering, vol. 52, no. 6, pp. 1128–1131, 2005, doi: 10.1109/TBME.2005.846709. and ||Wι+1,d|| = O||Dι,d|| (42) Also, lim ι→∞ ||ϑι+1,d −ϑ d|| ||ϑι −ϑ d|| ≤1 [4] M. Chung, J. Lee, M. Lee, J. Lee, Y-G. Shin, “Deeply self-supervised contour embedded neural network applied to liver segmentation," Com- puter Methods and Programs in Biomedicine, vol. 192, pp. 105447, 2020, https://doi.org/10.1016/j.cmpb.2020.105447. (43) and ||Vι+1,d|| = O||Pι,d|| (44) [5] F. Milletari, N. Navab, and S. A. Ahmadi, “V-Net: Fully Convolutional Neural Networks for Volumetric Medical Image Segmentation" 2016 Fourth International Conference on 3D Vision (3DV), pp. 565-–571, 2016 doi:10.1109/3DV.2016.79. The convergence of the sequence {ωι,d} according to L-Lipschitz continuity is illustrated as [50] [6] O. Ronneberger, P. Fischer, T. Brox, “U-Net: convolutional networks for biomedical image segmentation. International Conference on Med- ical Image Computing and Computer-Assisted Intervention (MICCAI 2015), vol. 9351, pp. 234-–241. 2016. doi: https://doi.org/10.1007/ 978-3-319-46723-8_4. REFERENCES Maulik, “Multilevel image segmen- tation with adaptive image context based thresholding,” Applied Soft Computing, vol. 11, no.1, pp. 946–962, 2011, doi: https://doi.org/10.1016/ j.asoc.2010.01.015. [25] A. Brebisson, and G. Montana, “Deep Neural Networks for Anatomical Brain Segmentation,” In: Proceedings of the IEEE Conference on Com- puter Vision and Pattern Recognition Workshops, pp. 20-–28, 2015, doi: 10.1109/CVPRW.2015.7301312. [45] N. Otsu, “A threshold selection method from gray level histograms," IEEE Transactions on Systems, Man, and Cybernetics, vol. 9, no. 1, pp. 62–66, 1979, doi: 10.1109/TSMC.1979.4310076. [26] H. Chen, Q. Dou, L. Yu, J. Qin, and P. A. Heng, 2017. “VoxResNet: Deep voxelwise residual networks for brain segmentation from 3D MR images,” NeuroImage, vol. 170, pp. 446-455, 2017, doi: https://doi.org/ 10.1016/j.neuroimage.2017.04.041. [46] H. P. Ng, S. H. Ong, K. W. C. Foong, P. S. Goh, and W. L. Nowinski, “Medical Image Segmentation Using K-Means Clustering and Improved Watershed Algorithm," 2006 IEEE Southwest Symposium on Image Analysis and Interpretation, 2006, doi: 10.1109/SSIAI.2006.1633722. [27] L.Chen , P. Bentley, K. Mori, K. Misawa, M. Fujiwara, and D. Rueckert, “DRINet for Medical Image Segmentation,” IEEE Transaction on Medical Imaging, vol. 37, no. 11, 2018, doi: 10.1109/TMI.2018.2835303.i [47] B. H. Menze et al., “The multimodal brain tumor image segmentation benchmark (BRATS)," IEEE Transactions on Medical Imaging, vol. 34, no. 10, pp. 1993-–2024, 2015, doi: 10.1109/TMI.2014.2377694. [28] N. Nuechterlein and S. Mehta, “3D-ESPNet with Pyramidal Refinement for Volumetric Brain Tumor Image Segmentation,” International MICCAI Brainlesion Workshop (BrainLes 2018), pp. 245–253, 2018, doi: https: //doi.org/10.1007/978-3-030-11726-9_22. [48] P. Bilic et al., “The Liver Tumor Segmentation Benchmark (LiTS)," arXiv, 2019, arXiv:1901.04056. [29] G. Wang et al., “Interactive Medical Image Segmentation Using Deep Learning With Image-Specific Fine Tuning,” IEEE Transactions on Med- ical Imaging, vol. 37, no. 7, 2018, doi: 10.1109/TMI.2018.2791721. [49] M. H. Gail and S. B. Green, “Critical values for the one-sided two- sample Kolmogorov–Smirnov statistic," J. Am. Stat. Assoc., vol. 71, pp. 757–760, 1976. [30] N. Tajbakhsh et al., “Convolutional Neural Networks for Medical Image Analysis: Full Training or Fine Tuning?,” IEEE Transactions on Medical Imaging, vol. 35, no. 5, pp. 1299–1312, 2016, doi: 10.1109/TMI.2016. 2535302. [50] H. Kim, J. Kang, W. Park, S. Ko, Y. Cho, D. Yu, Y. Song, and J. Choi, “Convergence Analysis of Optimization Algorithms," 2017, arXiv:1707. 01647. [31] X. Zhuang, Y. Li, Y. Hu, K. Ma, Y. Yang, and Y. REFERENCES Vries, M. J. N. L. Benders and I. Išgum, “Automatic Segmentation of MR Brain Images With a Convolutional Neural Network,”IEEE Transactions on Medical Imaging, vol.35, no.5, 2016, doi: 10.1109/TMI.2016.2548501. [39] D. Konar, S. Bhattacharya, B. K. Panigrahi, K. Nakamatsu “A quantum bi-directional self-organizing neural network (QBDSONN) architecture for binary object extraction from a noisy perspective,” Applied Soft Computing, vol.46, pp. 731–752, 2016, doi: https://doi.org/10.1016/j.asoc. 2015.12.040. [20] G. Wang et al., “DeepIGeoS: A Deep Interactive Geodesic Framework for Medical Image Segmentation," IEEE Transactions on Pattern Analysis and Machine Intelligence, vol. 41, no. 7, pp. 1559–1572, 2019, doi: 10. 1109/TPAMI.2018.2840695. [40] D. Konar, S. Bhattacharya, U. Chakraborty, T. K.Gandhi, B. K. Pani- grahi, “A quantum parallel bi-directional self-organizing neural network (QPBDSONN) architecture for extraction of pure color objects from noisy background,” Proc. IEEE International Conference on Advances in Computing, Communications and Informatics (ICACCI), 2016, pp. 1912– 1918, 2016, doi: 10.1109/ICACCI.2016.7732330. [21] S. Banerjee, L. Magee, D. Wang et al., “Semantic segmentation of microscopic neuroanatomical data by combining topological priors with encoder–decoder deep networks," Nature Machine Intelligence, vol. 2, pp. 585—594, 2020, doi: https://doi.org/10.1038/s42256-020-0227-9.i [22] K. Kamnitsas, “Efficient multi-scale 3D CNN with fully connected CRF for accurate brain lesion segmentation,” Medical Image Analysis, vol. 36, pp. 61–78, 2017, doi: https://doi.org/10.1016/j.media.2016.10.004. [41] D. Konar, S. Bhattacharyya and B. K. Panigrahi, “QIBDS Net: A Quantum-Inspired Bi-Directional Self-supervised Neural Network Archi- tecture for Automatic Brain MR Image Segmentation,” Proc. 8th Inter- national Conference on Pattern Recognition and Machine Intelligence (PReMI 2019), vol. 11942, pp. 87–95, 2019, doi:https://doi.org/10.1007/ 978-3-030-34872-4_64. [23] Q. Dou, H. Chen, L. Yu, L. Zhao, J. Qin, D. Wang, V. C. Mok, L. Shi, and P. A. Heng. “Automatic Detection of Cerebral Microbleeds From MR Images via 3D Convolutional Neural Networks, IEEE Transactions on Medical Imaging, vol. 35, no. 5, pp. 1182-–1195, 2016, doi: 10.1109/ TMI.2016.2528129. [42] M. A. Nielson and I. L. Chung, Quantum computation and quantum information, Cambridge University press, 2002. [24] Ö. Çiçek, A. Abdulkadir, S. S. Lienkamp, T. Brox, and O. Ronneberger, “3D U-Net: Learning Dense Volumetric Segmentation from Sparse An- notation,” International Conference on Medical Image Computing and Computer-Assisted Intervention (MICCAI 2016), pp. 424-–432, 2016, doi: https://doi.org/10.1007/978-3-319-46723-8_49. [43] T. G. Kolda, B. W. Bader, “Tensor Decompositions and Applications,” SIAM Review, vol. 51, no. 3, pp. 455-–500, 2009, doi: https://doi.org/10. 1137/07070111X. [44] S. Bhattacharyya, P. Dutta and U. REFERENCES Vaughan, Neil, “Trans- lational medicine: Challenges and new orthopaedic vision (Mediouni- Model)," Current Orthopaedic Practice, vol. 31. no. 2, pp. 196-200, 2020, doi: 10.1097/BCO.0000000000000846. Now, according to Thaler formula Now, according to Thaler formula ζ(ωι+1,d, ϑι+1,d) −ζ(ωι,d, ϑι,d) = (46) d d ζ(ωι+1,d, ϑι+1,d) −ζ(ωι,d, ϑι,d) = (46) [14] D. Konar, S. Bhattacharyya, T. K. Gandhi and B. K. Panigrahi, “A quantum-inspired self-supervised Network model for automatic segmen- tation of brain MR images," Applied Soft Computing, vol. 93, 2020, doi: https://doi.org/10.1016/j.asoc.2020.106348. ζ( , ) ζ( , ) ( h △ωι,d ij △ϑι,d j i   ∂ζ(ωι,d,ϑι,d) ∂ωι,d ij ∂ζ(ωι,d,ϑι,d ∂ϑι,d j  + O h ||△ωι,d ij △ϑι,d i || i 12 [15] D. Konar, S. Bhattacharyya, B. K. Panigrahi and E. C. Behrman, “Qutrit- Inspired Fully Self-Supervised Shallow Quantum Learning Network for Brain Tumor Segmentation," IEEE Transactions on Neural Networks and Learning Systems, vol. 33, no. 11, pp. 6331–6345, Nov. 2022, doi: 10. 1109/TNNLS.2021.3077188. [34] S. Bhattacharyya, P. Dutta and U. Maulik, “A parallel bi-directional self-organizing neural network (PBDSONN) architecture for color image extraction and segmentation," Neurocomputing, vol. 86, pp. 1–23, 2012, doi: https://doi.org/10.1016/j.neucom.2011.11.025. [35] G. Purushothaman, N. B. Karayiannis, “Quantum neural networks (QNNs): inherently fuzzy feedforward neural networks,” IEEE Transac- tions on Neural Networks, vol. 8 , no. 3, 1997, doi: 10.1109/72.572106. [16] M. Havaei et al., “Brain tumor segmentation with deep neural networks,” Medical Image Analysis, vol. 35, pp. 18–31, 2017, doi: https://doi.org/10. 1016/j.media.2016. [36] T. C. Lu, G. R. Yu, and J. C. Juang, “Quantum-based algorithm for optimizing artificial neural networks,” IEEE Transaction on Neural Network and Learning Systems, vol. 24, no. 8, pp. 1266—1278, 2013, doi: 10.1109/TNNLS.2013.2249089. j [17] E. Shelhamer, J. Long, and T. Darrell, “Fully convolutional networks for semantic segmentation,” IEEE Transactions on Pattern Analysis and Machine Intelligence, vol. 39, no. 4, pp. 640–651, 2017, doi: 10.1109/ TPAMI.2016.2572683. [37] Nam-H. Nguyen ; E. C. Behrman, A. Moustafa, J. E. Steck, “Bench- marking Neural Networks For Quantum Computations,” IEEE Trans- actions on Neural Networks and Learning Systems, pp. 1-10, 2019, doi:10.1109/TNNLS.2019.2933394. [18] S. Pereira, A. Pinto, V. Alves, and C. A. Silva, “Brain Tumor Seg- mentation Using Convolutional Neural Networks in MRI Images,” IEEE Transactions on Medical Imaging, vol.35, no. 5, 2016, doi: 10.1109/TMI. 2016.2538465. [38] E. M. Stoudenmire, and D. J. Schwab, “Supervised Learning with Quantum-Inspired Tensor Networks,” https://www.arxiv-vanity.com/ papers/1605.05775/, 2020. [19] P. Moeskops, M. A. Viergever, A. M. Mendrik, L. S. REFERENCES Zheng, “Self-supervised Feature Learning for 3D Medical Images by Playing a Rubik’s Cube,” International Conference on Medical Image Computing and Computer- Assisted Intervention (MICCAI 2019), pp. 420–428, https://doi.org/10. 1007/978-3-030-32251-9_46. [32] M. Blendowski, H. Nickisch, M. P. Heinrich, “How to Learn from Unla- beled Volume Data: Self-supervised 3D Context Feature Learning," Proc. Medical Image Computing and Computer Assisted Intervention – MIC- CAI 2019, vol 11769, 2019, https://doi.org/10.1007/978-3-030-32226-7_ 72. [33] A. Ghosh, N. R. Pal, and S. K. Pal, “Self organization for object extraction using a multilayer neural network and fuzziness measures," IEEE Transactions on Fuzzy Systems, vol. 1, no.1, pp. 54–68, 1993, doi: 10.1109/TFUZZ.1993.390285.
https://openalex.org/W3011224673
https://periodicos.ufrn.br/bibliocanto/article/download/18864/12498
Portuguese
null
Um Olhar ao estudante com surdez da Universidade Federal do Rio Grande do Norte:
BiblioCanto
2,019
cc-by
8,284
2 Doutora em Educação - UFRGS; Mestra em Educação - UFRGS; Especialização em Museologia Patrimônio Cultural - UFRGS; Graduação em Artes Plásticas - UFRGS. Lattes: http://lattes.cnpq.br/1990500214696545 1 Especialização em andamento em LIBRAS - UFRN; Especialista em Gestão Documental - UFRN; Graduada em Biblioteconomia - UFRN. Lattes: http://lattes.cnpq.br/0834125840536553 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 1 Especialização em andamento em LIBRAS - UFRN; Especialista em Gestão Documental UM OLHAR AO ESTUDANTE COM SURDEZ DA UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE: ​ANÁLISE SOBRE A ACESSIBILIDADE À INFORMAÇÃO NA BIBLIOTECA CENTRAL ZILA MAMEDE A LOOK AT THE STUDENT WITH AMAZING FEDERAL UNIVERSITY OF RIO GRANDE DO NORTE​: ANALYSIS ON INFORMATION ACCESSIBILITY IN THE ZILA MAMEDE CENTRAL LIBRARY Michele Rodrigues Dias 1 michele-1011@hotmail.com Gabriela Bon 2 gabibon@gmail.com Resumo: Este estudo trata do atendimento ao aluno com surdez em bibliotecas universitárias, em especial, na Biblioteca Central Zila Mamede, integrante do Sistema de Bibliotecas da Universidade Federal do Rio Grande Norte. Aborda também a legislação vigente acerca do Acesso à informação e da Acessibilidade Universal nestes ambientes, bem como, apresenta a Política de Inclusão e Acessibilidade para os estudantes com deficiência na Universidade Federal do Rio Grande Norte. Discorre sobre as políticas e diretrizes criadas e disponibilizadas para que as bibliotecas se adequem em prol da inclusão informacional dos usuários com surdez nos ambientes biblioteconômicos. Utiliza como metodologia, pesquisa de caráter exploratório. Como instrumento de coleta de informações foi aplicada técnica de observação e entrevistas com docentes do curso de Letras/LIBRAS e bibliotecários da Biblioteca Central Zila Mamede. Em decorrência, é explicitado como é realizado o atendimento para este público no Laboratório de Acessibilidade da Biblioteca Central Zila Mamede. Sugere recomendações, se adotadas, podem oferecer ao aluno com surdez acesso aos serviços das bibliotecas de forma BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 83 igualitária, derrubando as barreiras informacionais e comunicacionais existentes, e por fim, prioriza a criação de coleções acessíveis para este público. Palavras- chave: ​Acessibilidade. Surdez. Desenvolvimento de coleções. Acervo acessível. Biblioteca Universitária. 3 ​Documento não paginado. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 1 INTRODUÇÃO Ao longo da história, as pessoas com deficiência sempre foram rotuladas em consequência de conceitos errôneos e representações construídas por sua condição, carregando com isso um estigma da deficiência e da incapacidade. De acordo com dados do Censo Demográfico do Instituto Brasileiro de Geografia e Estatística - IBGE (2010) , 23,9% da população brasileira, em torno de 46 milhões de 3 pessoas possui pelo menos uma deficiência. Deste total, uma grande parcela é excluída do acesso à informação, apesar dos avanços legislativos em nosso país nas últimas décadas. Dentre as deficiências existentes, enfatiza-se neste artigo a deficiência auditiva, apresentando a necessidade da aplicabilidade das políticas públicas a partir da evolução da legislação brasileira. Cabe ressaltar, que estas leis foram criadas a fim de garantir o acesso da pessoa surda à informação em todos os níveis sociais, dentre os quais, se incluem os acervos biblioteconômicos. Além disso, este acesso só pode ser assegurado através de um processo de gestão da informação nas bibliotecas e da formação e desenvolvimento de coleções adequadas a este público específico dado a complexidade de sua cultura própria. Some-se a isso, a relevância das bibliotecas como elemento essencial para o desenvolvimento da cidadania de forma incontestável. Apesar de o termo ‘​Acessibilidade’ ser um tema abordado em diversas áreas do conhecimento e em fontes teóricas, nota-se que no âmbito das bibliotecas, o trabalho com a pessoa com deficiência auditiva ainda é bastante insipiente. Somado BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 84 84 4 https://digitaispuccampinas.wordpress.com/2012/10/06/numero-de-alunos-com-deficiencia-no-ensino- superior-aumentou-9336-aponta-mec/​. Acesso em: 30 nov. 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Além disso, foi identificada uma pequena quantidade de publicações na área da ciência da informação, sobre acessibilidade à informação nas bibliotecas para este público específico. 2 METODOLOGIA E DISCUSSÃO DOS RESULTADOS Os dados abordados no trabalho foram obtidos a partir de pesquisa de caráter exploratório que, segundo Gil, tem “como principal finalidade desenvolver, esclarecer e modificar conceitos e ideias, tendo em vista a formulação de problemas mais precisos ou hipótese pesquisáveis para estudos posteriores” (GIL, 2008, p. 27). E, para a obtenção de uma análise mais profunda do assunto a ser estudado foi utilizado o método quanti-qualitativo, utilizando como técnica de pesquisa a coleta de dados e, como procedimentos para essa coleta, foram aplicadas 02 entrevistas com docentes do curso de Letras/LIBRAS e com 02 bibliotecários do LA, bem como, técnica de observação no atendimento fornecido pela BCZM. Depois de obtidos os dados, o passo seguinte foi a análise e discussão das questões referentes à pessoa surda e seus avanços legais, bem como a ​Inclusão de alunos com Necessidades Educacionais Especiais na UFRN e a necessidade de uma Gestão de Coleção Acessível ao Estudante com Surdez no SISBI da UFRN. a isso, percebe-se a crescente demanda de pessoas com surdez no ensino superior. Apontou o Ministério da Educação (MEC) que entre 2000 e 2010, a quantidade de 4 matrículas em ensino superior aumentou 933,6%. Por estes motivos, o enfoque deste artigo será o atendimento da pessoa com surdez em bibliotecas universitárias, em especial na Biblioteca Central Zila Mamede (BCZM), integrante do Sistema de Bibliotecas (SISBI) da Universidade Federal do Rio Grande do Norte (UFRN). Além disso, esta pesquisa pretende verificar em que medida a Política de Acessibilidade do SISBI atende às necessidades informacionais dos estudantes surdos, identificando também, quais parâmetros são adotados pela BCZM na acessibilidade à informação para este público, bem como, identificar quais as barreiras que impedem o acesso destes estudantes ao Laboratório de Acessibilidade da Biblioteca Central Zila Mamede (LA). O papel das bibliotecas em nossa sociedade é bastante claro: garantir o acesso à informação para toda a sua comunidade, consequentemente, o bibliotecário assume a postura de intermediário entre a informação e o usuário e, para isso, precisa estar habilitado a comunicar-se com ele. Da mesma maneira, quando o bibliotecário atende um usuário com surdez, deve estar minimamente habilitado a comunicar-se através da Língua Brasileira de Sinais (LIBRAS), que é a língua usada pela comunidade surda. Em consequência disso, a pesquisa se justifica também, quanto ao currículo do curso de graduação em Biblioteconomia da UFRN, que não oferece disciplina obrigatória que desperte nos futuros profissionais a sensibilização para a prestação de serviço às pessoas com deficiência. Existe a disciplina LIBRAS como optativa, com a carga horária de 60h, tendo sido inserida na matriz curricular do curso no período letivo 2011.1. Sendo que, nesta categoria não é possível adquirir todo conhecimento necessário para este atendimento específico, tendo que ser complementada com cursos básicos e/ou especializações na área. 85 85 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 5 ​Documento não paginado. 3 ACESSIBILIDADE UNIVERSAL E ACESSO À INFORMAÇÃO PARA AS PESSOAS COM DEFICIÊNCIA Quando uma instituição possui o intuito de promover um local onde todas as pessoas fazem parte, no qual é permitido o acesso de toda e qualquer pessoa, tenha ela alguma necessidade específica ou não, devemos colocar em prática os conceitos de Acessibilidade Universal, que segundo GelpI, KaliL e Becker: Pode ser entendida como o direito de ir e vir de todos os cidadãos, inclusive daquelas pessoas com deficiências permanentes ou ocasionais. Isto incluía BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 86 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. todos, em todos os momentos e períodos de sua vida, quer sejam usuários de cadeiras de rodas, pessoas com deficiências visuais ou auditivas, gestantes, obesos, idosos ou ainda, as crianças. Os espaços devem permitir o trânsito e acesso a todos os espaços da cidade, prédios públicos, institucionais, usar transporte, equipamentos públicos e mobiliários urbanos, como telefones, sanitários, rede bancária, cabinas, assentos, bebedouros, etc. O importante e prioritário, está na abordagem da arquitetura, do urbanismo e da mobilidade urbana através do desenho universal, acessível, criando uma cidade ao alcance de todos os cidadãos com mobilidade reduzida ou não, democratizando todos os espaços, entendendo o desenho universal como a capacidade de comunicar e integrar a todos. (GELPI; KALIL; BECKER, 2015, p. 5). todos, em todos os momentos e períodos de sua vida, quer sejam usuários de cadeiras de rodas, pessoas com deficiências visuais ou auditivas, gestantes, obesos, idosos ou ainda, as crianças. Os espaços devem permitir o trânsito e acesso a todos os espaços da cidade, prédios públicos, institucionais, usar transporte, equipamentos públicos e mobiliários urbanos, como telefones, sanitários, rede bancária, cabinas, assentos, bebedouros, etc. O importante e prioritário, está na abordagem da arquitetura, do urbanismo e da mobilidade urbana através do desenho universal, acessível, criando uma cidade ao alcance de todos os cidadãos com mobilidade reduzida ou não, democratizando todos os espaços, entendendo o desenho universal como a capacidade de comunicar e integrar a todos. (GELPI; KALIL; BECKER, 2015, p. 5). Com isso, promovesse também, a valorização da dignidade humana, que segundo Moraes é “Um valor espiritual e moral inerente da pessoa, que se manifesta singularmente na autodeterminação consciente e responsável da própria vida e que traz consigo a pretensão ao respeito por parte das demais pessoas” (MORAES, 2005, p. 41). Em 1988, surge a primeira lei que trata sobre o assunto na Constituição Federal Brasileira, a qual estabelece um título próprio aos Princípios Fundamentais da Dignidade Humana. p g 7 ​Documento não paginado. 6 ​Documento não paginado. Em seu artigo 1º, inciso I ao V, ela nos traz como fundamentos: A soberania, a cidadania, a dignidade da pessoa humana, os valores sociais do trabalho e da livre iniciativa e o pluralismo político [...] E, como objetivos fundamentais, se constitui em “construir uma sociedade livre, justa e solidária; garantir o desenvolvimento nacional; erradicar a pobreza e a marginalização e reduzir as desigualdades sociais e regionais; promover o bem de todos, sem preconceitos de origem, raça, sexo, cor, idade e quaisquer outras formas de discriminação. (BRASIL, 1988) . 5 A soberania, a cidadania, a dignidade da pessoa humana, os valores sociais do trabalho e da livre iniciativa e o pluralismo político [...] E, como objetivos fundamentais, se constitui em “construir uma sociedade livre, justa e solidária; garantir o desenvolvimento nacional; erradicar a pobreza e a marginalização e reduzir as desigualdades sociais e regionais; promover o bem de todos, sem preconceitos de origem, raça, sexo, cor, idade e quaisquer outras formas de discriminação. (BRASIL, 1988) . 5 Com isso, a lei deixa clara a importância de se respeitar, acima de qualquer coisa o outro, independente de suas manifestações culturais e sociais, sendo primordial a não exclusão. Sabe-se que não é uma tarefa fácil, mas este marco legal está avançando, ajudando na criação de políticas públicas mais ajustadas com os Direitos Humanos e com a Acessibilidade. Nesta mesma perspectiva e com o intuito de promover a Acessibilidade e de reduzir as desigualdades sociais (um dos princípios defendidos por nossa BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 87 87 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. [...] possibilidade e condição de alcance para utilização, com segurança e autonomia, de espaços, mobiliários, equipamentos urbanos, edificações, transportes, ​informação e comunicação​, inclusive seus sistemas e tecnologias, bem como de outros serviços e instalações abertos ao público, de uso público ou privados de uso coletivo, tanto na zona urbana como na rural, por pessoa com deficiência ou com mobilidade reduzida. (BRASIL, 2015, grifo nosso) . 6 autoridades nacionais devem encontrar meio para evitar efeitos possíveis de exclusão nas áreas de acesso à informação e ao conhecimento, de difusão de Novas Tecnologias de Informação e Comunicação (NTIC) e de desenvolvimento do plurilinguismo na internet. Serão abordadas em seguida, peculiaridades sobre uma deficiência específica, a surdez, onde serão apresentados alguns conceitos de estudiosos da área, bem como, avanços na legislação acerca desse público. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Artigo 2º - garante, por parte do poder público em geral e empresas concessionárias de serviços públicos, formas institucionalizadas de apoiar o uso e difusão da Língua Constituição), a gestão da informação é vista como uma ferramenta essencial no desenvolvimento das organizações, do ponto de vista de Miranda e Streit (2007, p. 4) “As organizações processam e gerem informações para diminuir a ambiguidade e a incerteza provindas do ambiente em que atuam, e/ou para elaborar produtos e serviços de informação”. Ou seja, a gestão da informação vem dar suporte ao trabalho de organizar a informação, de maneira que os serviços e produtos possam ser úteis aos seus clientes, sem distinção. Desde 1988, após amplos debates nacionais e internacionais sobre o tema, a legislação brasileira foi sendo aprimorada e complementada com novas leis. Dentre estes avanços, podemos citar a introdução do conceito de acessibilidade à informação e à comunicação presente na Lei 13.146/15, de 6 de julho de 2015, Lei Brasileira de Inclusão da Pessoa com Deficiência (Estatuto da Pessoa com Deficiência), que conceitua acessibilidade como sendo: [...] possibilidade e condição de alcance para utilização, com segurança e autonomia, de espaços, mobiliários, equipamentos urbanos, edificações, transportes, ​informação e comunicação​, inclusive seus sistemas e tecnologias, bem como de outros serviços e instalações abertos ao público, de uso público ou privados de uso coletivo, tanto na zona urbana como na rural, por pessoa com deficiência ou com mobilidade reduzida. (BRASIL, 2015, grifo nosso) . 6 Vale salientar que em outros artigos específicos, esta lei reafirma um conjunto de direitos das pessoas com deficiência, mas também define, de forma mais clara, sanções relativas ao seu não cumprimento. Com isso, esta lei desde a sua promulgação em 2015, vem influenciando e alavancando a criação de outras políticas, programas e projetos voltados para a temática do acesso à informação. É importante ressaltar que a informação deve estar acessível a toda e qualquer pessoa, sem distinção. Podemos evidenciar tal afirmação, de acordo com o manifesto da Declaração Universal dos Direitos Humanos da Organização das Nações Unidas para a Educação, a Ciência e a Cultura (UNESCO) (ASSEMBLÉIA GERAL DAS NAÇÕES UNIDAS, 1998) onde as instituições internacionais e as 7 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 88 8 ​Documento não paginado. 3.1 A Pessoa Surda e seus Avanços Legais Para as pessoas surdas, o aspecto cultural é que define sua necessidade especial, até porque, possuem sua própria língua e forma de se comunicar, a Língua Brasileira de Sinais (LIBRAS), a qual envolve muito mais do que a simples tradução da Língua Portuguesa sob forma de sinais, o que para a sociedade é difícil de entender, uma vez que alguns ainda veem a surdez exclusivamente como um fenômeno físico, conforme afirma Gesser (2008, p. 230) que “infelizmente, os surdos têm sido narrados e definidos exclusivamente a partir da realidade física da falta de audição e, portanto, aos olhos da sociedade majoritária ouvinte tem sido vistos exclusivamente a partir desse fato”. É importante abordar o marco principal de avanço da comunidade surda, que de fato oficializou a Língua Brasileira de Sinais como meio legítimo de comunicação da pessoa com surdez, que foi a criação da Lei nº 10.436, de 24 de abril de 2002, ​a qual ficou conhecida na comunidade surda como a “Lei de LIBRAS”. Os artigos 1º e 2º desta Lei, respectivamente, tratam: Artigo 1º - é reconhecido como meio legal de comunicação e expressão a Língua Brasileira de Sinais - LIBRAS e outros recursos de expressão a ela associados. Artigo 2º - garante, por parte do poder público em geral e empresas concessionárias de serviços públicos, formas institucionalizadas de apoiar o uso e difusão da Língua BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 89 Brasileira de Sinais - LIBRAS como meio de comunicação objetiva e de utilização corrente das comunidades surdas do Brasil (BRASIL, 2002) . 8 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. unicamente porque não ouve, mas porque desenvolve potencialidades psicoculturais diferentes das dos ouvintes” (BEHARES, 2000, p. 2). Desta forma, pode-se dizer que a pessoa com surdez tem sua própria cultura, possui sua própria identidade, com seus valores, regras, comportamentos e tradições inerentes a elas. Além disso, por várias questões sociais e políticas, costumam criar comunidades onde vivem e compartilham juntos anseios e metas, além de se ajudarem nas suas lutas e na diminuição dos preconceitos que os cercam. Dentre os avanços das pessoas com deficiência, a seguir, serão relatadas as políticas de inclusão dos estudantes com necessidades especiais no âmbito da UFRN. 9 ​Documento não paginado Brasileira de Sinais - LIBRAS como meio de comunicação objetiva e de utilização corrente das comunidades surdas do Brasil (BRASIL, 2002) . 8 Portanto, somente a partir desta data foi possível realizar, em âmbito nacional, discussões relacionadas às necessidades das pessoas surdas, deixando claro para as pessoas que a LIBRAS é uma língua formada por sinais, com significados específicos, possuidora de uma composição gramatical própria, podendo expressar conceitos dentro da realidade em que o surdo está inserido. Strobel, afirma que: Ela [LIBRAS] é uma das principais marcas da identidade de um povo surdo por ser uma das peculiaridades da cultura surda. É uma forma de comunicação que capta as experiências visuais dos sujeitos surdos, sendo que é esta língua que vai levar o surdo a transmitir e proporcionar-lhe a aquisição de conhecimento universal (STROBEL, 2008, p. 42-43). É notória a importância da LIBRAS na vida das pessoas surdas, sendo usada como um meio de garantia deste grupo menorizado na socialização e interação na sociedade. Abordasse o conceito de surdez, uma vez que já foi discorrido sobre a legislação que configura a LIBRAS como língua reconhecida no Brasil. Sá, ao definir o termo surdo, diz que: [...] é o termo com o qual as pessoas que não ouvem referem-se a si mesmos e a seus pares. Podemos definir uma pessoa surda como àquela que vivencia um ​déficit de audição que o impede de adquirir, de maneira natural, a língua oral/auditiva usada pela comunidade majoritária e que constrói sua identidade calcada principalmente nesta diferença, utilizando-se de estratégias cognitivas e de manifestações comportamentais e culturais diferentes da maioria das pessoas que ouvem. (SÁ, 2006, p. 2, grifo nosso). Para este grupo, o termo deficiente não é bem-aceito e o uso desta terminologia reforça a segregação e a exclusão que já perdura desde seus antepassados. Além disso, o termo pode ser associado ao fato de que muitas pessoas com deficiência sofriam torturas, abandono e discriminação, por parte, principalmente, de sua própria família. Behares (2000) nos mostra que nos estudos sobre os surdos, é enfatizado a diferença, e não a deficiência, por que: “Cremos que é nela que se baseia a essência psicossocial da surdez: ele (o surdo) não é diferente BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 90 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. especializados; V – oferta de capacitação que possa contribuir para o aperfeiçoamento do processo de ensino-aprendizagem (​Ibidem​). Este documento formal tem a missão de instituir a Política de Inclusão e Acessibilidade para as Pessoas com Deficiência na Universidade Federal do Rio Grande do Norte, para fim de sua aplicação. No entanto, é a Comissão de Apoio a Estudantes com Necessidades Educacionais Especiais (CAENE), a responsável pela institucionalização da Política de Inclusão dos alunos com Necessidades Educacionais Especiais (NEE), e afiança o direito da pessoa com deficiência à educação superior na UFRN. Esta Comissão atua sob demanda de solicitação espontânea do aluno com NEE, em seguida, a solicitação é enviada à coordenação do curso do aluno para identificação e formalização de acompanhamento e triagem com o intuito de investigar mais sobre sua realidade, bem como suas necessidades efetivas. A seguir, será discorrido sobre a acessibilidade à informação da BCZM para o atendimento ao NEE, focando no público surdo. 4 INCLUSÃO DE ALUNOS COM NECESSIDADES EDUCACIONAIS ESPECIAIS NA UFRN De acordo com a Resolução 193/2010 da Política de Inclusão e Acessibilidade para as Pessoas com Deficiência na UFRN em seu artigo 1º, entende-se por estudante com necessidade educacional especial aquele com: “I – deficiência nas áreas: auditiva, visual, física, intelectual ou múltipla; II – transtornos globais do desenvolvimento; III - Altas habilidades/superdotação; IV – transtornos específicos” (UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE, 2010) . 9 E, em seu artigo 2º, a mesma resolução determina: Que os dirigentes das unidades acadêmicas deverão prover iniciativas que contemplem o princípio da inclusão social nas propostas curriculares de seus cursos presenciais e à distância, garantindo ações voltadas para o atendimento às demandas dos estudantes com necessidades educacionais especiais. § 2º A inclusão mencionada no caput deste artigo refere-se às responsabilidades concernentes dos estudantes com necessidades educacionais especiais, como: I – recursos didático-pedagógicos adequados; II – acesso às dependências das unidades acadêmicas; III – pessoal docente e técnico especializado; IV – serviços de apoio BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 91 especializados; V – oferta de capacitação que possa contribuir para o aperfeiçoamento do processo de ensino-aprendizagem (​Ibidem​). BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Em relação aos usuários com deficiência, as suas necessidades informacionais em linhas gerais não diferem das necessidades dos demais usuários, o que diferencia é o suporte físico da informação e o acesso a esta. Nesse contexto, a problemática está no acesso à informação e tipos de suporte que obedeçam ao conceito de desenho universal. (MELO, 2015, p. 33). 4.1 Acessibilidade à informação ao estudante com surdez da ufrn e a gestão de coleções acessíveis da BCZM De acordo com dados fornecidos pela CAENE, existem, atualmente na UFRN, 395 alunos com deficiência, e desse total, 15 correspondem a alunos com surdez. Para complementar este quantitativo de pessoas com surdez na UFRN, em visita ao departamento de Letras, foi informado que atualmente existem 05 professores com surdez no curso de graduação Letras/LIBRAS – Departamento de Letras do ​Centro de Ciências Humanas, Letras e Artes (CCHLA) e 01 professor surdo no Curso de Letras - Departamento de Letras (DLC) do Centro de Ensino Superior do Seridó (CERES) no Campus de Currais Novos. Diante do quantitativo apresentado e da presença do curso de Letras/ LIBRAS, que visa a formar professores para atuarem no ensino de Língua Brasileira de Sinais e de Língua Portuguesa para surdos, é de suma importância que o SISBI e a BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 92 92 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. CAENE disponibilizem acervo informacional acessível que atenda às reais necessidades informacionais dos alunos e professores com surdez da UFRN, auxiliando-os no desenvolvimento social, científico e acadêmico. Nesse sentido, Miranda enfatiza o papel das bibliotecas no contexto da formação do ser humano, “[...] uma vez que oferece aos seus usuários o acesso à informação que irá contribuir para a formação de um cidadão mais consciente de seus direitos e deveres” (MIRANDA, 2015, p. 23). No entanto, as bibliotecas devem garantir um ambiente democrático e acessível a todos, contribuindo para o estabelecimento de produtos e serviços adequados as necessidades informacionais das pessoas com surdez, que também necessitam de informação para seu conhecimento. Diante desse contexto das bibliotecas, será enfatizada sua abordagem no âmbito das universidades. Leitão nos traz uma definição da biblioteca nas Universidades, uma vez que, tem o papel de “[...] estimular, apoiar, fomentar e desenvolver o saber em seus múltiplos aspectos por meio de seus acervos e das relações que nela se estabelecem” (LEITÃO, 2005, p. 25). Ou seja, a biblioteca além de garantir material bibliográfico para os estudantes, deve também promover a Acessibilidade Universal, que visa facilitar a ​aquisição à informação por todas as pessoas. Apesar de o termo acessibilidade ser tema de diversas áreas do conhecimento, nota-se que no âmbito das bibliotecas ainda existem limitações e necessitam de contribuições mais pontuais. Baseando-se nisso, Melo nos traz uma abordagem mais específica quanto às necessidades informacionais dos estudantes com surdez, uma vez que: Em relação aos usuários com deficiência, as suas necessidades informacionais em linhas gerais não diferem das necessidades dos demais usuários, o que diferencia é o suporte físico da informação e o acesso a esta. Nesse contexto, a problemática está no acesso à informação e tipos de suporte que obedeçam ao conceito de desenho universal. (MELO, 2015, p. 33). Vale salientar que, uma das atividades mais importantes de gestão nas bibliotecas é a formação e desenvolvimento de coleções, que nada mais é que a BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 93 93 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. seleção do material que irá compor o acervo da mesma. Miranda nos relata a relevância desta atividade: seleção do material que irá compor o acervo da mesma. Miranda nos relata a relevância desta atividade: A gestão da coleção é fator fundamental à plena consecução dos objetivos das bibliotecas. A formação, desenvolvimento e organização do acervo devem ser encarados como um processo permanente no qual as atividades de seleção, aquisição e avaliação de materiais devem permanecer em contínua sintonia com as necessidades de informação da comunidade de usuários. (MIRANDA, 2007, p. 87). Fica claro que a formação e desenvolvimento de coleções é um processo fundamental e de grande importância para as bibliotecas, principalmente, devido à explosão bibliográfica. Tal informação pode ser corroborada com as diretrizes da Federação Internacional de Associações e Instituições Bibliotecárias (IFLA) (2010, p. 59) onde relata que “uma grande coleção não significa uma boa coleção, especialmente no novo mundo digital. A relevância da coleção para as necessidades da comunidade local é mais importante do que o seu tamanho”. É evidente que uma biblioteca com um grande acervo é fundamental, mas deve-se garantir, principalmente, que estes materiais sejam utilizados, que serão encontrados por seus usuários, e vice-versa. Baseando-se nisso, o Manual Orientador de Fortalecimento de Bibliotecas Inclusivas e Acessíveis (2016) sugere que é imprescindível que se tenha acervos em diferentes formatos, em especial, nos formatos acessíveis. Este manual ainda define como seriam estes formatos de acervos: “[...] são aqueles que incluem livros e outros materiais com recursos de acessibilidade (livros em braille, em tinta e braille, audiolivros, livros digitais bilíngües Português/LIBRAS etc. Que possibilitam o acesso ao livro e à leitura para pessoas com deficiência” (BRASIL, 2016, p. 25). O acervo acessível é a adaptação da informação registrada nos diferentes materiais da biblioteca para os formatos que sejam acessíveis aos usuários com NEE. Melo (2015) relata que a formação do acervo acessível só ocorre após o levantamento das necessidades informacionais dos usuários, o qual se pode iniciar o processo de (re)formulação do acervo para torná-lo acessível aos usuários com deficiência. Com isso, faz-se necessário que os bibliotecários tenham conhecimento BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 94 94 10 ​Documento não paginado. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. De acordo com Melo, Costa e Soares, Tecnologias Assistivas são: [...] recursos e serviços que visam facilitar o desenvolvimento de atividades da vida diária por pessoas com deficiências. Procuram aumentar capacidades funcionais e assim promover a autonomia e a independência de quem as utiliza. (MELO; COSTA; SOARES, 2008, ​apud ​ARAÚJO, 2017, p. 4). sobre possíveis formatos que possam ser utilizados para que ocorra a acessibilidade à informação para os usuários com surdez. Visto isso, pode-se contar também com a parceria das editoras, que no ato da compra podem disponibilizar o arquivo digital das obras, onde é possível converter para o formato acessível desenvolvido para cada deficiência. Inclusive, de acordo com o disposto na Lei n. 13.146/2015, que em seu Art. 68, afirma que: O poder público deve adotar mecanismos de incentivo à produção, à edição, à difusão, à distribuição e à comercialização de livros em formatos acessíveis, inclusive em publicações da administração pública ou financiadas com recursos públicos, com vistas a garantir à pessoa com deficiência o direito de acesso à leitura, à informação e à comunicação. § 1o Nos editais de compras de livros, inclusive para o abastecimento ou a atualização de acervos de bibliotecas em todos os níveis e modalidades de educação e de bibliotecas públicas, o poder público deverá adotar cláusulas de impedimento à participação de editoras que não ofertem sua produção também em formatos acessíveis. § 2o Consideram-se formatos acessíveis os arquivos digitais que possam ser reconhecidos e acessados por softwares leitores de telas ou outras tecnologias assistivas que vierem a substituí-los, permitindo leitura com voz sintetizada, ampliação de caracteres, diferentes contrastes e impressão em Braille. (BRASIL, 2015) . 10 Diante do exposto, a BCZM tem a possibilidade de adequar sua Política de Formação e Desenvolvimento de Coleções de acordo com o disposto das exigências legais, para que assim, possa oferecer um atendimento adequado para esta demanda de usuários existentes. Para que isso ocorra, conta-se também com a CAENE, que segundo Ferreira, uma das suas missões é “propor ações através da consolidação de redes de apoio e serviço institucional que incidam na eliminação de barreiras arquitetônicas, atitudinais, pedagógicas e de comunicação na UFRN” (FERREIRA, 2016, p. 73). Em conformidade a isto, foi criado em 2011, o Laboratório de Acessibilidade (LA) localizado na BCZM. O LA tem o intuito de promover a inclusão informacional das pessoas com NEE com limitações e/ou dificuldades na leitura impressa, aliado aos BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 95 95 equipamentos e recursos da Tecnologia Assistiva. Dentre suas atividades estão: digitalização de textos; produção de materiais em formatos acessíveis; empréstimo de tecnologias Assistivas; revisão Braille e Repositório de Informação Acessível (RIA). 96 Ter materiais referentes à surdez e a deficiência auditiva é importante não apenas para os próprios usuários com essa deficiência, mas para que todos tenham acesso a informações que transmitam mais conhecimentos sobre essa temática contribuindo para mudanças no comportamento, nas ações das pessoas em geral em relação aos surdos e pessoas com deficiência auditiva (MIRANDA, 2015, p. 127). BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. equipamentos e recursos da Tecnologia Assistiva. Dentre suas atividades estão: digitalização de textos; produção de materiais em formatos acessíveis; empréstimo de tecnologias Assistivas; revisão Braille e Repositório de Informação Acessível (RIA). De acordo com Melo, Costa e Soares, Tecnologias Assistivas são: [...] recursos e serviços que visam facilitar o desenvolvimento de atividades da vida diária por pessoas com deficiências. Procuram aumentar capacidades funcionais e assim promover a autonomia e a independência de quem as utiliza. (MELO; COSTA; SOARES, 2008, ​apud ​ARAÚJO, 2017, p. 4). Como uma das missões da BCZM é tornar a informação acessível ao usuário, foi criado em 2012, o Repositório de Informações Acessíveis (RIA) que tem o objetivo de reunir, integrar e disponibilizar os textos adaptados pelo LA. Em consulta ao site da BCZM, o RIA atualmente só tem acessibilidade ao estudante com deficiência visual. Pode-se constatar tal informação abaixo: Seu principal objetivo é armazenar, preservar, divulgar e permitir acesso ao estudante com deficiência visual dos cursos de Graduação e Pós-Graduação ao material de estudo e pesquisa necessários à sua formação acadêmica. O material disponibilizado no RIA é destinado à pessoa com deficiência visual e representa uma tentativa de promoção à igualdade de condições no acesso ao conhecimento (BIBLIOTECA CENTRAL ZILA MAMEDE, [201-]). Em resultante, é possível notar a inexistência de suporte informacional na biblioteca ao usuário surdo da UFRN. Uma vez que vai de encontro ao compromisso formal da Instituição em gerar acessibilidade à informação para todos os estudantes com NEE. Pode-se notar também, a não inclusão das pessoas com surdez nos objetivos do Laboratório de Acessibilidade, que é amparado pelo Decreto 5.296, de 02 de dezembro de 2004, que determina a garantia da acessibilidade e utilização de serviços e atendimentos das pessoas portadoras de deficiência, ou com mobilidade reduzida. Quanto ao termo “portador”, recomenda-se a não utilização do termo, sendo aconselhado utilizar “Pessoa com Deficiência (PCD)”, sendo empregado para demonstrar a existência de alguma deficiência, sem indicar qual (BRASIL, 2004). Retomando ao suporte informacional ao usuário surdo, concordasse com Miranda ao dizer que: BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Ter materiais referentes à surdez e a deficiência auditiva é importante não apenas para os próprios usuários com essa deficiência, mas para que todos tenham acesso a informações que transmitam mais conhecimentos sobre essa temática contribuindo para mudanças no comportamento, nas ações das pessoas em geral em relação aos surdos e pessoas com deficiência auditiva (MIRANDA, 2015, p. 127). As bibliotecas e os bibliotecários não podem mais se comportar como se as pessoas surdas não existissem, como se fossem invisíveis, principalmente, porque seus usuários estão cada vez mais conscientes de seus direitos a produtos e serviços com qualidade​. Miranda (2015, p. 61-63) relata que a IFLA preocupada com este público pouco assistido nas bibliotecas, elaborou um documento chamado “Diretrizes para Serviços de Bibliotecas para Surdos”. Enfatiza ainda, que estas diretrizes foram idealizadas em 1988, durante um simpósio sobre serviços de bibliotecas para surdos que aconteceu na Austrália. As diretrizes servem para nortear as bibliotecas nas adaptações necessárias para que possam oferecer um atendimento adequado para as pessoas com surdez. Essas recomendações são divididas em 5 categorias: pessoal, comunicação, acervo, serviços e divulgação dos programas das bibliotecas. Destaca ainda, que é de responsabilidades das bibliotecas “garantir que suas coleções e serviços sejam acessíveis aos surdos e que os surdos estejam cientes dos serviços que as bibliotecas podem lhes prover” (IFLA, 2000, p. 6, ​apud MIRANDA, 2015, p. 20). Vale ressaltar que a biblioteca e seus profissionais não precisam esperar essa demanda vir até a biblioteca para que se planejem, para que se inicie um projeto de acessibilidade, visto que, podem se antecipar iniciando uma gestão informacional para esse público, criando um fluxo que atenda às suas especificidades, fazendo uso das recomendações sugeridas pelas diretrizes da IFLA. Atualmente, o SISBI realiza o atendimento por demanda direcionada pela CAENE. À frente, além de expor os resultados das observações realizadas sobre o atendimento ao estudante com surdez da UFRN na BCZM, serão apresentadas algumas recomendações como sugestão. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 97 97 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 5 CONSIDERAÇÕES FINAIS 5 CONSIDERAÇÕES FINAIS Diante das investigações que embasaram esta pesquisa e considerando a eficácia da Política de Acessibilidade do SISBI em atender as necessidades informacionais dos estudantes surdos, os resultados demonstraram que o Sistema de Bibliotecas da UFRN: (a) atende, somente parcialmente, as necessidades desse grupo, e (b) não dispõe de pessoal habilitado em LIBRAS para facilitar a comunicação com os usuários surdos. Além disso, os resultados também apontaram a existência de um desejo por parte desses alunos de frequentar as bibliotecas do SISBI. Esta disposição, porém, só não se realiza em razão de barreiras comunicacionais e humanas que acabam por desmotivar o público surdo. Como já mencionado, a BCZM possui um Laboratório de Acessibilidade destinado, a princípio, a atender todas as pessoas com deficiência. No entanto, este Laboratório se restringe atualmente a atender apenas o público com deficiência visual. Apesar de o Laboratório estar aberto às demandas das pessoas surdas, identifica-se também que os bibliotecários que lá atuam não têm habilidade em LIBRAS, mas que existe o anseio por aprender. Além disso, verificamos que o local não dispõe de tecnologias específicas, como, por exemplo: ​o software Vlibras, que possui uma série de ferramentas para tradução de conteúdos de sites, áudio e textos para a Língua Brasileira de Sinais – LIBRAS. Outra ferramenta é a WIKILIBRAS, que consiste em um sistema de correção e inclusão de novos sinais. Outro bastante utilizado é o Hand Talk, esse aplicativo transforma as imagens e textos em linguagens de sinais. Existe também o Prodeaf Móvel, onde seu principal objetivo é a comunicação entre as pessoas com necessidades especiais (OLIVEIRA, 2016). Estas ferramentas favorecem a autonomia das pessoas com deficiência auditiva na busca de informações. A partir destas corroborações, conclui-se que a BCZM não atende aos anseios da comunidade surda da UFRN. E, como forma de aprimorar os serviços oferecidos pelo Laboratório de Acessibilidade e o Sistema de Bibliotecas da UFRN como um todo, foram produzidas algumas recomendações, baseadas no Manual BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 98 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. orientador para fortalecimento de bibliotecas acessíveis e inclusivas, como também, recomendações da IFLA, e de autores referenciados no trabalho. Acredita-se que assim, possam ser minimizadas algumas das barreiras informacionais que impedem o acesso à informação aos usuários com surdez na BCZM. São elas: 1. Realizar coletas de materiais informacionais digitais, como trabalhos de conclusão de cursos de outras universidades, artigos, publicações em revistas, nacionais e internacionais, que estejam relacionados especificamente à surdez, com a finalidade de criar um repositório sobre a temática; 2. Verificar se existe banco de dados com materiais acessíveis às pessoas com surdez, almejando possíveis parcerias; 3. Explicitar de forma mais objetiva e transparente a Política de Desenvolvimento de Coleções voltadas para as pessoas com NEE atendidas pelo LA; 4. Solicitar às editoras, na etapa de compra, arquivo digital compatível para que seja convertido pelas tecnologias assistivas e adaptado no formato acessível para o usuário com surdez; 5. Implantar e disponibilizar tecnologia assistiva para o usuário surdo, tais como tradutores automáticos da Língua Portuguesa para a LIBRAS; 6. Organizar visualmente as informações nas estantes e demais espaços da biblioteca, utilizando a datilologia ou imagens dos sinais em LIBRAS, de maneira que a escrita em Língua Portuguesa não seja a única forma de sinalização; 7. Disponibilizar cursos de capacitação em LIBRAS para bibliotecários, técnicos administrativos e demais profissionais do SISBI, visando uma melhoria da comunicação no atendimento ao usuário surdo da BCZM. 7. Disponibilizar cursos de capacitação em LIBRAS para bibliotecários, técnicos administrativos e demais profissionais do SISBI, visando uma melhoria da comunicação no atendimento ao usuário surdo da BCZM. 8. Divulgar os serviços oferecidos pela BCZM de forma que atraia os estudantes e docentes surdos para este ambiente informacional. Todas estas recomendações e estratégias se coadunam com Aranha (2000) que nos afirma que os profissionais da informação não podem ser meros organizadores e controladores do acesso às estantes, mas sim, se adequarem às BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 99 99 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. and communication barriers, and ultimately prioritizing the creation of collections accessible to this audience. Keywords: ​Acessibility. Deafness. Collection development. Accessible colllection. University library Keywords: ​Acessibility. Deafness. Collection development. Accessible colllection. Keywords: ​Acessibility. Deafness. Collection development. Accessible colllection. University library. University library. necessidades informacionais de interesse dos seus clientes, cumprindo seu papel de intermediador entre o leitor e a informação. O bibliotecário tem que estar atento às necessidades de seus usuários, buscando se qualificar e ter uma postura em prol da inclusão das pessoas com deficiência, independente de qual seja, passando da simples tarefa de organizar a informação que se encontra dentro da biblioteca e se adaptar à comunidade usuária da instituição que nela está inserida. Deve também acompanhar os avanços das tecnologias, estando atento sobre novas soluções que estejam disponíveis que facilite o acesso à informação para as pessoas com surdez. Desta forma, para que as bibliotecas universitárias em geral se tornem espaços realmente profícuos para as pessoas com deficiência auditiva, é imprescindível que sejam tomadas medidas que viabilizem seu acesso. Ademais, para que o aluno surdo possa usufruir igualitariamente de todos os serviços do SISBI, as adaptações sugeridas neste artigo, dentre outras, promovem a autonomia para a pessoa com surdez inserida na UFRN e que anseiam por fazer uso efetivo de suas bibliotecas. Abstract: ​This study deals with the care of students with deafness in university libraries, especially in the Central Library Zila Mamede, member of the Library System of the Federal University of Rio Grande Norte. It also discusses the current legislation on Access to Information and Universal Accessibility in these environments, as well as presents the Inclusion and Accessibility Policy for students with disabilities at the Federal University of Rio Grande Norte. It discusses the policies and guidelines created and made available for libraries to adapt to the informational inclusion of deaf users in library environments. It uses as methodology, exploratory research. As an instrument for collecting information, observation technique and interviews with teachers of the course of Letters / LIBRAS and librarians of the Central Library Zila Mamede were applied. As a result, it is explained how the attendance for this public is performed in the Accessibility Laboratory of the Central Library Zila Mamede. It suggests recommendations, if adopted, may offer the deaf student equal access to library services, breaking down existing informational BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 100 REFERÊNCIAS ARANHA, Francisco. E- Service em Bibliotecas: geração de valor para pesquisadores por meio de cooperação indireta. ​Revista de Administração de Empresas​, São Paulo, n. 4, v. 49, out/dez. 2000. p. 84-93. Disponível em: http://www.scielo.br/pdf/rae/v40n4/v40n4a08.pdf. Acesso em: 08 ago. 2019. ARANHA, Francisco. E- Service em Bibliotecas: geração de valor para pesquisadores por meio de cooperação indireta. ​Revista de Administração de Empresas​, São Paulo, n. 4, v. 49, out/dez. 2000. p. 84-93. Disponível em: http://www.scielo.br/pdf/rae/v40n4/v40n4a08.pdf. Acesso em: 08 ago. 2019. ARAÚJO, A. K. DA S. O livro acessível: um auxiliar no acesso à informação para deficientes visuais. ​Revista Informação na Sociedade Contemporânea​, v. 1, n. 2, p. 1-25, 5 abr. 2017. Disponível em: https://periodicos.ufrn.br/informacao/article/view/11353/7996. Acesso em: 08 ago. 2019. ARAÚJO, A. K. DA S. O livro acessível: um auxiliar no acesso à informação para deficientes visuais. ​Revista Informação na Sociedade Contemporânea​, v. 1, n. 2, p. 1-25, 5 abr. 2017. Disponível em: https://periodicos.ufrn.br/informacao/article/view/11353/7996. Acesso em: 08 ago. 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BRASIL. Ministério da Cultura. Fortalecimento de bibliotecas acessíveis e inclusivas: manual orientador​. São Paulo: Diferenças, 2016. FERREIRA, Érika Luzia Lopes da Silva. ​A Política de Inclusão de estudantes cegos na educação superior na UFRN​: do acesso a permanência. 2016. Dissertação (Mestrado em Serviço social) - Centro de Ciências Sociais Aplicadas, Programa de Pós- Graduação em Serviço Social, Universidade Federal do Estado do Rio Grande do Norte, Natal, 2016. FERREIRA, Érika Luzia Lopes da Silva. ​A Política de Inclusão de estudantes cegos na educação superior na UFRN​: do acesso a permanência. 2016. Dissertação (Mestrado em Serviço social) - Centro de Ciências Sociais Aplicadas, Programa de Pós- Graduação em Serviço Social, Universidade Federal do Estado do Rio Grande do Norte, Natal, 2016. FERREIRA, Érika Luzia Lopes da Silva. ​A Política de Inclusão de estudante cegos na educação superior na UFRN​: do acesso a permanência. 2016. GELPI, Adriana; KALIL, Rosa Maria Locatelli; BECKER, Alcione. Acessibilidade universal em instituições de ensino. ​In​: SEMINÁRIO NACIONAL DE CONSTRUÇÕES SUSTENTÁVEIS, 4.; FÓRUM DESEMPENHO DAS EDIFICAÇÕES, 1., 2015, Rio Grande do Sul. ​Anais​ [...]. 2015. Disponível em: file:///C:/Users/01176410490/Desktop/Acessibilidade%20Universal%20em%20Institui ções%20de%20Ensino.pdf. Acesso em: 01 dez. 2019. GELPI, Adriana; KALIL, Rosa Maria Locatelli; BECKER, Alcione. Acessibilidade universal em instituições de ensino. ​In​: SEMINÁRIO NACIONAL DE CONSTRUÇÕES SUSTENTÁVEIS, 4.; FÓRUM DESEMPENHO DAS EDIFICAÇÕES, 1., 2015, Rio Grande do Sul. ​Anais​ [...]. 2015. Disponível em: file:///C:/Users/01176410490/Desktop/Acessibilidade%20Universal%20em%20Institui ções%20de%20Ensino.pdf. Acesso em: 01 dez. 2019. GESSER, Audrei. Do patológico ao cultural na surdez: para além de um e de outro ou para uma reflexão crítica dos paradigmas. ​Trabalhos em lingüística aplicada, Campinas, v. 47, n. 1, p. 223 – 239, 2008. Disponível em: http://www.scielo.br/pdf/tla/v47n1/v47n1a12.pdf​. Acesso em: 30 nov. 2019. GESSER, Audrei. Do patológico ao cultural na surdez: para além de um e de outro ou para uma reflexão crítica dos paradigmas. ​Trabalhos em lingüística aplicada, Campinas, v. 47, n. 1, p. 223 – 239, 2008. Disponível em: http://www.scielo.br/pdf/tla/v47n1/v47n1a12.pdf​. Acesso em: 30 nov. 2019. GIL, Antonio Carlos. ​Como elaborar projetos de pesquisa​. 6. ed. São Paulo: Atlas, 2008. ASSEMBLÉIA GERAL DAS NAÇÕES UNIDAS. ​Declaração Universal dos Direitos Humanos​. 1998. Disponível em: ASSEMBLÉIA GERAL DAS NAÇÕES UNIDAS. ​Declaração Universal dos Direitos Humanos​. 1998. Disponível em: p https://unesdoc.unesco.org/ark:/48223/pf0000139423. Acesso em: 22 jul. 2019. BEHARES, Luis Ernesto. ​Novas correntes na educação do surdo​: dos enfoques clínicos aos culturais. Santa Maria: UFSM, 2000. BIBLIOTECA CENTRAL ZILA MAMEDE​. Repositório de informação acessível da UFRN – RIA. [201-]. Disponível em: http://www.sisbi.ufrn.br/bczm/pagina.php?a=ria#.Xe-I8pNKgdU​. Acesso em: 05 ago. 2019. BIBLIOTECA CENTRAL ZILA MAMEDE​. Repositório de informação acessível da UFRN – RIA. [201-]. Disponível em: http://www.sisbi.ufrn.br/bczm/pagina.php?a=ria#.Xe-I8pNKgdU​. Acesso em: 05 ago. UFRN – RIA. [201-]. Disponível em: http://www.sisbi.ufrn.br/bczm/pagina.php?a=ria#.Xe-I8pNKgdU​. Acesso em: 05 ago. 2019. BRASIL. Constituição (1988). Constituição da República Federativa do Brasil. ​Diário Oficial da União​, Brasília, DF. Disponível em: http://www.planalto.gov.br/ccivil_03/constituicao/constituicao.htm. Acesso em: 30 jul. 2019. BRASIL. ​Lei nº 10.436 de 24 de abril de 2002​. Dispõe sobre a Língua Brasileira de Sinais - Libras e dá outras providências. Brasília, DF. 2002. Disponível em: http://www.planalto.gov.br/ccivil_03/LEIS/2002/L10436.htm​. Acesso em: 30 jul. 2019. BRASIL. ​Lei nº 10.436 de 24 de abril de 2002​. Dispõe sobre a Língua Brasileira de Sinais - Libras e dá outras providências. Brasília, DF. 2002. Disponível em: http://www planalto gov br/ccivil 03/LEIS/2002/L10436 htm Acesso em: 30 jul 2019 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 101 BRASIL. ​Decreto 5.296, de 02 de dezembro de 2004. ​Regulamenta as Leis n​o​s​ 10.048, de 8 de novembro de 2000, que dá prioridade de atendimento às pessoas que especifica. 2004. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2004-2006/2004/decreto/d5296.htm​. Acesso em: 30 jul. 2019. BRASIL. ​Lei nº 13.146 de 06 de julho de 2015​. Institui a Lei Brasileira de Inclusão da Pessoa com Deficiência (Estatuto da Pessoa com Deficiência). Brasília, DF. Disponível em: BRASIL. ​Lei nº 13.146 de 06 de julho de 2015​. Institui a Lei Brasileira de Inclusão da Pessoa com Deficiência (Estatuto da Pessoa com Deficiência). Brasília, DF. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2015-2018/2015/lei/l13146.htm. Acesso em: 30 jul. 2019. BRASIL. ​Lei nº 13.146 de 06 de julho de 2015​. Institui a Lei Brasileira de Inclusão da Pessoa com Deficiência (Estatuto da Pessoa com Deficiência). Brasília, DF. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2015-2018/2015/lei/l13146.htm. Acesso em: 30 jul. 2019. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2015-2018/2015/lei/l13146.htm. Acesso em: 30 jul. 2019. BRASIL. Ministério da Cultura. Fortalecimento de bibliotecas acessíveis e inclusivas: manual orientador​. São Paulo: Diferenças, 2016. BRASIL. Ministério da Cultura. Fortalecimento de bibliotecas acessíveis e inclusivas: manual orientador​. São Paulo: Diferenças, 2016. https://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf​. Acesso em: 14 set. 2019. LEITÃO, Bárbara Júlia Menezello. ​Avaliação qualitativa e quantitativa numa biblioteca universitária​: grupos de foco. Rio de Janeiro: Intertexto; Interciência, 2005. MELO, Érika Simony Fernandes de. ​Formação de acervos acessíveis em bibliotecas universitárias​: o caso da Biblioteca Central Zila Mamede. BiblioCanto, Natal, v. 1, n. 1, p. 31-44, 2015. MIRANDA, Ana Claudia Carvalho de. Desenvolvimento de coleções em bibliotecas universitárias. ​Revista Digital de Biblioteconomia e Ciência da Informação​, Campinas, v. 4, n. 2, p. 01-19, 2007. Disponível em: http://www.brapci.inf.br/_repositorio/2010/05/pdf_8d3e5e1ab3_0010629.pdf. Acesso em: 09 ago. 2019. MIRANDA, Silvânia Vieira de; STREIT, Rosalvo Ermes. O processo de gestão da informação em organizações públicas. ​In​: ENCONTRO DE ADMINISTRAÇÃO DA INFORMAÇÃo, 1., 2007, Florianopolis. ​Anais​ [...]. Florianopolis, 2007. Disponível em: file:///C:/Users/01176410490/Documents/BIBLIOCANTO/enadi309.pdf. Acesso em: 01 dez. 2019. MIRANDA, Sulamita Nicolau de. ​Acessibilidade ao usuário surdo e com deficiência auditiva em bibliotecas universitárias:​ o caso da UNIRIO. 2015. Dissertação (Mestrado Profissional em Biblioteconomia) - Programa de Pós- Graduação em Biblioteconomia, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, 2015. Disponível em: http://www.unirio.br/ppgb/arquivo/Dissertacao%20Sulamita%20Miranda.pdf. Acesso em: 12 jun. 2019. INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. ​Censo Demográfico. 2010. Disponível em: https://www.ibge.gov.br/estatisticas/sociais/populacao/9662-censo-demografico-2010 .html?edicao=9748&t=destaques. Acesso em: 04 ago. 2019. https://www.ibge.gov.br/estatisticas/sociais/populacao/9662-censo-demografico-2010 .html?edicao=9748&t=destaques. Acesso em: 04 ago. 2019. INTERNATIONAL FEDERATION OF LIBRARY ASSOCIATION (IFLA). UNITED NATIONS EDUCATIONAL, SCIENTIFIC AND CULTURAL ORGANIZATION (UNESCO). ​Diretrizes da IFLA para biblioteca escolar​. 2010. Disponível em: BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 102 https://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf​. Acesso em: 14 set. 2019 ttps://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf​. Acesso em: 14 https://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf​. Acesso em: 14 set. 2019 MORAES, ​Alexandre de. ​Constituição do Brasil Interpretada​. 5. ed. São Paulo: Atlas, 2005. Disponível em: // / / / / / O CO S C O MORAES, ​Alexandre de. ​Constituição do Brasil Interpretada​. 5. ed. São Paulo: Atlas, 2005. Disponível em: https://jornalistaslivres org/wp-ontent/uploads/2017/02/DIREITO CONSTITUCIONAL , p https://jornalistaslivres.org/wp-ontent/uploads/2017/02/DIREITO_CONSTITUCIONAL -1.pdf. Acesso em: 20 jun. 2019. OLIVEIRA, Nazareth Ana Lúcia de. ​As​ ​Tecnologias e a Educação de Alunos Surdos​. 2016. Disponívem em: https://monografias.brasilescola.uol.com.br/imprimir/15432​. Acesso em: 01 dez. 2019. OLIVEIRA, Nazareth Ana Lúcia de. ​As​ ​Tecnologias e a Educação de Alunos Surdos​. 2016. Disponívem em: https://monografias brasilescola uol com br/imprimir/15432 Acesso em: 01 dez Surdos​. 2016. Disponívem em: https://monografias.brasilescola.uol.com.br/imprimir/15432​. Acesso em: 01 dez. 2019. SÁ, Nídia Limeira de. ​Cultura, poder e educação de surdos​. São Paulo: Paulinas, 2006. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. SÁ, Nídia Limeira de. ​Cultura, poder e educação de surdos​. São Paulo: Paulinas, 2006. STROBEL, K. ​As imagens do outro sobre a cultura surda​. Florianópolis: Editora da UFSC, 2008. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 103 UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. ​Resolução nº 193/2010​, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a estudantes com necessidades educacionais especiais na Universidade Federal do Rio Grande do Norte. 2010. Disponível em: file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10 ago. 2019. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. ​Resolução nº 193/2010​, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a estudantes com necessidades educacionais especiais na Universidade Federal do Rio Grande do Norte. 2010. Disponível em: file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10 ago. 2019. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. ​Resolução nº 193/2010​, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a estudantes com necessidades educacionais especiais na Universidade Federal do Rio Grande do Norte. 2010. Disponível em: file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10 ago. 2019. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. ​Resolução nº 193/2010​, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a estudantes com necessidades educacionais especiais na Universidade Federal do Rio Grande do Norte. 2010. Disponível em: file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10 ago. 2019. p file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10 ago. 2019. p file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10 ago. 2019. 104
https://openalex.org/W1841493807
http://diposit.ub.edu/dspace/bitstream/2445/132909/1/656770.pdf
English
null
First Observation of Top Quark Production in the Forward Region
Physical review letters
2,015
cc-by
8,525
DOI: 10.1103/PhysRevLett.115.112001 to integrated luminosities of 1.0 and 2.0 fb−1 collected at center-of-mass energies of ffiffiffis p ¼ 7 and 8 TeV in pp collisions with the LHCb detector. The W bosons are reconstructed using the W →μν decay with muons having a transverse momentum, pT, larger than 25 GeV (c ¼ 1 throughout this Letter) in the pseudorapidity range, 2.0 < η < 4.5. The analysis is performed using jets clus- tered with the anti-kT algorithm [5] using a distance parameter R ¼ 0.5. The jets are required to have 50 < pT < 100 GeV and 2.2 < η < 4.2. The muon and jet (j) must be separated by ΔRðμ; jÞ > 0.5, with ΔR ≡ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Δη2 þ Δϕ2 p . Here ΔηðΔϕÞ is the difference in pseudor- apidity (azimuthal angle) between the muon and jet momenta. The transverse component of the sum of the muon and jet momenta must satisfy pTðμ þ jÞ ≡ ½~pðμÞ þ ~pðjÞT > 20 GeV. The production of top quarks (t) from proton-proton (pp) collisions in the forward region is of considerable experimental and theoretical interest. In the standard model (SM), four processes make significant contributions to top quark production: t¯t pair production, single-top production via processes mediated by a W boson in the t channel (qb →q0t) or in the s channel (q¯q0 →t¯b), and single top produced in association with a W boson (gb →tW). The initial-state b quarks arise from gluon splitting to b¯b pairs or from the intrinsic b quark content in the proton. Top quarks decay almost entirely via t →Wb. The SM predicts that about 75% of t →Wb decays in the forward region are due to t¯t pair production. The remaining 25% are mostly due to t-channel single-top production, with s-channel and associated single-top production making percent-level contributions. The enhancement at forward rapidities of t¯t production via q¯q and qg scattering, relative to gg fusion, can result in larger charge asymmetries, which may be sensitive to physics beyond the SM [1,2]. Forward t¯t events can be used to constrain the gluon parton distribution function (PDF) at a large momentum fraction, resulting in reduced theoretical uncertainty for many SM predictions [3]. Furthermore, both single-top and t¯t cross-section measure- ments in the forward region will provide important exper- imental tests of differential next-to-next-to-leading order theoretical calculations as they become available [4]. *Full author list given at the end of the article. Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distri- bution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. First Observation of Top Quark Production in the Forward Region R. Aaij et al.* (LHCb Collaboration) j (LHCb Collaboration) (Received 3 June 2015; revised manuscript received 8 July 2015; published 8 September 2 Top quark production in the forward region in proton-proton collisions is observed for the first time. The W þ b final state with W →μν is reconstructed using muons with a transverse momentum, pT, larger than 25 GeV in the pseudorapidity range 2.0 < η < 4.5. The b jets are required to have 50 < pT < 100 GeV and 2.2 < η < 4.2, while the transverse component of the sum of the muon and b-jet momenta must satisfy pT > 20 GeV. The results are based on data corresponding to integrated luminosities of 1.0 and 2.0 fb−1 collected at center-of-mass energies of 7 and 8 TeV by LHCb. The inclusive top quark production cross sections in the fiducial region are σðtopÞ½7 TeV ¼ 239  53ðstatÞ  33ðsystÞ  24ðtheoryÞ fb; σðtopÞ½8 TeV ¼ 289  43ðstatÞ  40ðsystÞ  29ðtheoryÞ fb:These results, along with the observed differ- ential yields and charge asymmetries, are in agreement with next-to-leading order standard model predictions. PACS numbers: 14.65.Ha, 13.87.-a, 14.70.Fm DOI: 10.1103/PhysRevLett.115.112001 Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distri- bution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. *Full author list given at the end of the article. week ending 11 SEPTEMBER 2015 week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) DOI: 10.1103/PhysRevLett.115.112001 The signature for W þ jet events is an isolated high-pT muon and a well-separated jet originating from the same pp interaction. Signal events are selected by requiring a high-pT muon candidate and at least one jet with ΔRðμ; jÞ > 0.5. For each event, the highest-pT muon candidate that satisfies the trigger requirements is selected, along with the highest-pT jet from the same pp collision. The primary background to top quark production is direct W þ b production; however, Z þ b events, with one muon undetected in the decay Z →μμ, and di-b-jet events also contribute to the μ þ b-jet final state. Inclusive W þ jet production, i.e., where no SV-tag requirement is made on the jet, is only contaminated at the percent level by processes other than direct W þ jet production. Therefore, W þ jet production is used to validate both the theory predictions and the modeling of the detector response. Furthermore, the SM prediction for σðWbÞ=σðWjÞ has a smaller relative uncertainty than σðWbÞ alone, since the theory uncertainties partially cancel in the ratio. The analysis strategy is to first measure the W þ jet yields, and then to obtain predictions for the yields of direct W þ b production using the prediction for σðWbÞ=σðWjÞ. To an excellent approximation, many experimental effects, e.g., the muon reconstruction effi- ciency, are expected to be the same for both samples and do not need to be considered in the direct W þ b yield prediction. The anti-kT clustering algorithm is used as implemented in FastJet [20]. Information from all the detector subsystems is used to create charged and neutral particle inputs to the jet-clustering algorithm using a particle flow approach [21]. The reconstructed jets must fall within the pseudorapidity range 2.2 < ηðjÞ < 4.2. The reduced ηðjÞ acceptance ensures nearly uniform jet-reconstruction and heavy-flavor tagging efficiencies. The momentum of a reconstructed jet is corrected to obtain an unbiased estimate of the true jet momentum. The correction factor, typically between 0.9 and 1.1, is determined from simulation and depends on the jet pT and η, the fraction of the jet pT measured with the tracking system, and the number of pp interactions in the event. The W þ jet yield is determined by performing a fit to the pTðμÞ=pTðjμÞ distribution with templates, histograms obtained from data, as described in Ref. [19]. DOI: 10.1103/PhysRevLett.115.112001 However, when the dimuon invariant mass is in the range 60 < Mðμþμ−Þ < 120 GeV, such events are selected as ZðμμÞ þ jet candidates, which are used to determine the Z þ jet background. The jets are identified (tagged) as originating from the hadronization of a b or c quark by the presence of a secondary vertex (SV) with ΔR < 0.5 between the jet axis and the SV direction of flight, defined by the vector from the pp interaction point to the SV position. Two boosted decision trees (BDTs) [22,23], trained on the characteristics of the SV and the jet, are used to separate heavy-flavor jets from light-parton jets, and to separate b jets from c jets. The two-dimensional distribution of the BDT responses observed in data is fitted to obtain the SV-tagged b, c, and light-parton jet yields. The SV-tagger algorithm is described in Ref. [24], where the heavy-flavor tagging efficiencies and light-parton mistag probabilities are mea- sured in data. The data samples used in Ref. [24] are too small to validate the performance of the SV-tagger algo- rithm in the pTðjÞ > 100 GeV region. Furthermore, the mistag probability of light-parton jets increases with jet pT. Therefore, only jets with pT < 100 GeV are considered in the fiducial region, which, according to simulation, retains about 80% of all top quark events. FIG. 1 (color online). Distribution of pTðμÞ=pTðjμÞ with fit overlaid for all W þ jet candidates. next-to-leading order (NLO) with the MCFM package [15] and the CT10 PDF set [16], and are cross-checked using PowhegBox [17] with hadronization simulated by Pythia. next-to-leading order (NLO) with the MCFM package [15] and the CT10 PDF set [16], and are cross-checked using PowhegBox [17] with hadronization simulated by Pythia. The theoretical uncertainty on the cross-section predictions is a combination of PDF, scale, and strong-coupling (αs) uncertainties. The PDF and scale uncertainties are evalu- ated following Refs. [16] and [18], respectively. The αs uncertainty is evaluated as the envelope obtained using αsðMZÞ ∈½0.117; 0.118; 0.119 in the theory calculations. sð ZÞ ½ ; ;  y The event selection is the same as that in Ref. [19] but a reduced fiducial region is used to enhance the top quark contribution relative to direct W þ b production. DOI: 10.1103/PhysRevLett.115.112001 The LHCb detector is a single-arm forward spectrometer covering the pseudorapidity range 2 < η < 5, designed for the study of particles containing b or c quarks. It is described in detail in Refs. [6,7]. The trigger [8] consists of a hardware stage, based on information from the calorimeter and muon systems, followed by a software stage, which applies a full event reconstruction. This analysis requires at least one muon candidate that satisfies the trigger requirement of pT > 10 GeV. Global event cuts (GECs), which prevent high-occupancy events from domi- nating the processing time of the software trigger, have an efficiency of about 90% for W þ jet and top quark events. This Letter reports the first observation of top quark production in the forward region. The data used correspond Simulated pp collisions are generated using Pythia [9] with an LHCb configuration [10]. Decays of hadronic particles are described by EvtGen [11] in which final-state radiation is generated using Photos [12]. The interaction of the generated particles with the detector, and its response, are implemented using the Geant4 toolkit [13] as described in Ref. [14]. Further theory calculations are performed at 112001-1 © 2015 CERN, for the LHCb Collaboration 0031-9007=15=115(11)=112001(10) week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) ) μ j( T p )/ μ ( T p 0.5 0.6 0.7 0.8 0.9 1 Candidates / 0.05 5000 10000 Data W Z Jets LHCb +jet μ ) μ j( T p )/ μ ( T p 0.5 0.6 0.7 0.8 0.9 1 Candidates / 0.05 5000 10000 Data W Z Jets LHCb +jet μ FIG. 1 (color online). Distribution of pTðμÞ=pTðjμÞ with fit overlaid for all W þ jet candidates. to as the muon jet and denoted as jμ, is used to discriminate between W þ jet and dijet events [19]. No correction is applied to the momentum of the muon jet. The requirement pTðjμ þ jÞ > 20 GeV is made to suppress dijet back- grounds, which are well balanced in pT, unlike W þ jet events, where there is undetected energy from the neutrino. Events with a second, oppositely charged, high-pT muon candidate from the same pp collision are vetoed. DOI: 10.1103/PhysRevLett.115.112001 The muon trigger, reconstruction, and selection efficiencies are determined using Z →μμ events [21,25]. The GEC efficiency is obtained following Ref. [21]: an alternative dimuon trigger requirement, which requires a looser GEC, is used to determine the fraction of events that are rejected. Contamination from W →τ →μ decays are estimated to be 2.5% using both simulated W þ jet events and inclusive W data samples [26]. The fraction of muons that migrate out of the fiducial region due to final-state radiation is about 1.5% [26]. Figure 4 shows a fit to the pTðμÞ=pTðjμÞ distribution built from the b-tagged jets from the full data sample. For pTðμÞ=pTðjμÞ > 0.9 the data are dominantly from W decays. Figure 5 shows the yield and charge asymmetry distributions obtained as a function of pTðμ þ bÞ. The direct W þ b prediction is determined by scaling the inclusive W þ jet distribution observed in data by the SM prediction for σðWbÞ=σðWjÞ and by the b-tagging efficiency measured in data [24]. As can be seen, the data cannot be described by the expected direct W þ b con- tribution alone. The observed yield is about 3 times larger than the SM prediction without a top quark contribution, Migration of events in jet pT due to the detector response is studied with a data sample enriched in b jets using SV tagging. The pTðSVÞ=pTðjÞ distribution observed in data is compared to templates obtained from simulation in bins of jet pT. The resolution and scale for each jet pT bin are varied in simulation to find the best description of the data and to construct a detector response matrix. Figure 2 shows that the SM predictions, obtained with all detector response effects applied, agree with the inclusive W þ jet data. ) [GeV] c + μ ( T p ) c + W ( N 0 50 100 150 200 Data SM LHCb 20 45 70 95 ∞ FIG. 3 (color online). Results for W þ c compared to SM predictions at NLO obtained using MCFM. ) [GeV] c + μ ( T p ) c + W ( N 0 50 100 150 200 Data SM LHCb 20 45 70 95 ∞ The yields of W þ c and W þ b, which includes t →Wb decays, are determined using the subset of candidates with a SV-tagged jet and binned according to pTðμÞ=pTðjμÞ. DOI: 10.1103/PhysRevLett.115.112001 The Z þ jet contribution is fixed from the fully reconstructed ZðμμÞ þ jet yield, where the probability for one of the muons to escape detection is obtained using simulation. The con- tributions of b, c, and light-parton jets are each free to vary The high-pT muon candidate is not removed from the anti-kT inputs and so is clustered into a jet. This jet, referred 112001-2 P H Y S I C A L R E V I E W L E T T E R S week ending 11 SEPTEMBER 2015 PRL 115, 112001 (2015) ) [GeV] j + μ ( T p +jet) W ( N 0 2000 4000 6000 8000 LHCb Data SM 20 45 70 95 ∞ ) [GeV] j + μ ( T p Charge Asymmetry -0.4 -0.2 0 0.2 0.4 20 45 70 95 ∞ LHCb Data SM FIG. 2 (color online). Results for the inclusive W þ jet yield (left) and charge asymmetry (right) versus pTðμ þ jÞ compared to SM predictions at NLO obtained using MCFM. The data error bars are smaller than the marker size; the SM uncertainties are highly correlated across pTðμ þ jÞ bins. Charge Asymmetry FIG. 2 (color online). Results for the inclusive W þ jet yield (left) and charge asymmetry (right) versus pTðμ þ jÞ compared to SM predictions at NLO obtained using MCFM. The data error bars are smaller than the marker size; the SM uncertainties are highly correlated across pTðμ þ jÞ bins. A fit to the pTðμÞ=pTðjμÞ distribution built from the c- tagged jets from the full data sample is provided as Supplemental Material to this Letter [27]. Figure 3 shows that the W þ c yield versus pTðμ þ cÞ agrees with the SM prediction. Since the W þ c final state does not have any significant contributions from diboson or top quark pro- duction in the SM, this comparison validates the analysis procedures. in the fit. Figure 1 shows the fit for all candidates in the data sample. Such a fit is performed for each muon charge separately in bins of pTðμ þ jÞ; the differential W þ jet yield and charge asymmetry, defined as ½σðWþjÞ −σðW−jÞ=½σðWþjÞ þ σðW−jÞ, are given in Fig. 2. To compare the data to theory predictions, the detector response must be taken into account. All significant aspects of the detector response are determined using data-driven techniques. DOI: 10.1103/PhysRevLett.115.112001 4 (color online). Distribution of pTðμÞ=pTðjμÞ with fit overlaid for all W þ b candidates. while the SM prediction including both t¯t and single-top production does describe the data well. In Ref. [19], W þ b is studied in a larger fiducial region [pTðμÞ > 20 GeV; pTðjÞ > 20 GeV], where the top quark contribution is expected to be about half as large as that of direct Wþb production. The ratio ½σðWbÞþσðtopÞ=σðWjÞ is measured in the larger fiducial region to be 1.17  0.13 ðstatÞ  0.18 ðsystÞ% at ffiffiffis p ¼ 7 TeV and 1.29  0.08 ðstatÞ  0.19 ðsystÞ% at ffiffiffis p ¼ 8 TeV. These results agree with SM predictions, which include top quark production, of 1.23  0.24% and 1.38  0.26%, respec- tively. This validates the direct W þ b prediction, since direct W þ b production is the dominant contribution to the larger fiducial region. To determine the statistical significance of the top quark contribution, a binned profile likelihood test is performed. The top quark distribution and charge asymmetry versus pTðμ þ bÞ are obtained from the SM predictions. The total top quark yield is allowed to vary freely. Systematic uncertainties, both theoretical and experimental, are handled as Gaussian constraints. The profile likelihood technique is used to compare the SM hypotheses with and without a top quark contribution. The significance obtained using Wilks theorem [28] is 5.4σ, confirming the obser- vation of top quark production in the forward region. Various sources of systematic uncertainties are consid- ered and summarized in Table I. The direct W þ b prediction is normalized using the observed inclusive W þ jet data yields. Therefore, most experimental system- atic uncertainties cancel to a good approximation. The yield and charge asymmetry distributions versus pTðμ þ bÞ observed at ffiffiffis p ¼ 7 and 8 TeV are each consistent with the SM predictions. The excess of the observed yield relative to the direct W þ b prediction at each ffiffiffis p is attributed to top quark production, and used to Since the muon kinematic distributions in W þ jet and W þ b are similar, all muon-based uncertainties are neg- ligible with the exception of the trigger GEC efficiency. DOI: 10.1103/PhysRevLett.115.112001 In each pTðμÞ=pTðjμÞ bin, the two-dimensional SV-tagger BDT-response distributions are fitted to determine the yields of c-tagged and b-tagged jets, which are used to form the pTðμÞ=pTðjμÞ distributions for candidates with c-tagged and b-tagged jets. These pTðμÞ=pTðjμÞ distribu- tions are fitted to determine the SV-tagged W þ c and W þ b yields. FIG. 3 (color online). Results for W þ c compared to SM predictions at NLO obtained using MCFM. 112001-3 week ending 11 SEPTEMBER 2015 week ending 11 SEPTEMBER 2015 week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) ) μ j( T p )/ μ ( T p 0.5 0.6 0.7 0.8 0.9 1 Candidates / 0.1 100 200 300 Data W Z Jets LHCb -tag +b μ ) μ j( T p )/ μ ( T p 0.5 0.6 0.7 0.8 0.9 1 Candidates / 0.1 100 200 300 Data W Z Jets LHCb -tag +b μ FIG. 4 (color online). Distribution of pTðμÞ=pTðjμÞ with fit overlaid for all W þ b candidates. The data-driven GEC study discussed above shows that the efficiencies are consistent for W þ jet and W þ b, with the statistical precision of this study assigned as the systematic uncertainty. Mismodeling of the pTðμÞ=pTðjμÞ distribu- tions largely cancels, since this shifts the inclusive W þ jet and W þ b final-state yields by the same amount, leaving the observed excess over the expected direct W þ b yield unaffected. The one exception is possible mismodeling of the dijet templates, since the flavor content of the dijet background is not the same in the two samples. Variations of these templates are considered and a relative uncertainty of 5% is assigned on the W boson yields. The jet-reconstruction efficiencies for heavy-flavor and light-parton jets in simulation are found to be consistent within 2%, which is assigned as the systematic uncertainty for flavor dependencies in the jet-reconstruction efficiency. The SV-tagger BDT templates used in this analysis are two- dimensional histograms obtained from the data samples enriched in b and c jets used in Ref. [24]. Following Refs. [19,24], a 5% uncertainty on the b-tagged yields is assigned due to uncertainty in these templates. The pre- cision of the b-tagging efficiency measurement (10%) in data [24] is assigned as an additional uncertainty. FIG. DOI: 10.1103/PhysRevLett.115.112001 ) [GeV] b + μ ( T p ) W+b ( N 0 100 200 Data +top Wb Wb LHCb 20 45 70 95 ∞ ) [GeV] b + μ ( T p Charge Asymmetry -0.4 -0.2 0 0.2 0.4 Data +top Wb Wb LHCb 20 45 70 95 ∞ FIG. 5 (color online). Results for the W þ b yield (left) and charge asymmetry (right) versus pTðμ þ bÞ compared to SM predictions obtained at NLO using MCFM. ) [GeV] b + μ ( T p ) W+b ( N 0 100 200 Data +top Wb Wb LHCb 20 45 70 95 ∞ ) [GeV] b + μ ( T p Charge Asymmetry -0.4 -0.2 0 0.2 0.4 Data +top Wb Wb LHCb 20 45 70 95 ∞ Charge Asymmetry FIG. 5 (color online). Results for the W þ b yield (left) and charge asymmetry (right) versus pTðμ þ bÞ compared to SM predictions obtained at NLO using MCFM. 112001-4 week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) TABLE I. Relative systematic uncertainties. Source Uncertainty GEC 2% pTðμÞ=pTðjμÞ templates 5% Jet reconstruction 2% SV-tag BDT templates 5% b-tag efficiency 10% Trigger and μ selection 2%a Jet energy 5%a W →τ →μ 1%a Luminosity 1%–2%a Total 14% Theory 10% aAn uncertainty that only applies to the cross-section measurement and not the significance determination. Only the luminosity uncertainty depends on ffiffiffis p : 2% at 7 TeV and 1% at 8 TeV. TABLE I. Relative systematic uncertainties. Source Uncertainty GEC 2% pTðμÞ=pTðjμÞ templates 5% Jet reconstruction 2% SV-tag BDT templates 5% b-tag efficiency 10% Trigger and μ selection 2%a Jet energy 5%a W →τ →μ 1%a Luminosity 1%–2%a Total 14% Theory 10% aAn uncertainty that only applies to the cross-section measurement and not the significance determination. Only the luminosity uncertainty depends on ffiffiffis p : 2% at 7 TeV and 1% at 8 TeV. (France); BMBF, DFG, HGF, and MPG (Germany); INFN (Italy); FOM and NWO (The Netherlands); MNiSW and NCN (Poland); MEN/IFA (Romania); MinES and FANO (Russia); MinECo (Spain); SNSF and SER (Switzerland); NASU (Ukraine); STFC (United Kingdom); and NSF (U.S.). The Tier1 computing centers are supported by IN2P3 (France), KIT and BMBF (Germany), INFN (Italy), NWO and SURF (The Netherlands), PIC (Spain), and GridPP (United Kingdom). DOI: 10.1103/PhysRevLett.115.112001 We are indebted to the communities behind the multiple open source software packages on which we depend. We are also thankful for the computing resources and the access to software research and development tools provided by Yandex LLC (Russia). Individual groups or members have received support from EPLANET, Marie Skłodowska- Curie Actions and ERC (European Union), Conseil général de Haute-Savoie, Labex ENIGMASS and OCEVU, Région Auvergne (France), RFBR (Russia), XuntaGal and GENCAT (Spain), and the Royal Society and Royal Commission for the Exhibition of 1851 (United Kingdom). aAn uncertainty that only applies to the cross-section measurement and not the significance determination. Only the luminosity uncertainty depends on ffiffiffis p : 2% at 7 TeV and 1% at 8 TeV. measure the cross sections. Some additional systematic uncertainties that apply to the cross-section measurements do not factor into the significance determination. The uncertainties due to the muon trigger, reconstruction, and selection efficiencies are taken from the data-driven studies of Refs. [21,25]. The uncertainty due to the jet energy determination is obtained from the data-driven study used to obtain the detector response matrix. The uncertainty due to W →τ →μ contamination is taken as the difference between the contamination in simulation versus that of a data-driven study of inclusive W →μν production [26]. The luminosity uncertainty is described in detail in Ref. [29]. The total systematic uncertainty is obtained by adding the individual contributions in quadrature. [1] A. L. Kagan, J. F. Kamenik, G. Perez, and S. Stone, Probing New Top Physics at the LHCb Experiment, Phys. Rev. Lett. 107, 082003 (2011). [2] R. Gauld, Leptonic Top quark asymmetry predictions at LHCb, Phys. Rev. D 91, 054029 (2015). [3] R. Gauld, Feasibility of top quark measurements at LHCb and constraints on the large-x gluon PDF, J. High Energy Phys. 02 (2014) 126. [4] M. Czakon, P. Fiedler, and A. Mitov, Resolving the Tevatron Top Quark Forward-Backward Asymmetry Puzzle: Fully Differential Next-to-Next-to-Leading-Order Calculation, Phys. Rev. Lett. 115, 052001 (2015). The resulting inclusive top production cross sections in the fiducial region defined by pTðμÞ > 25 GeV, 2.0 < ηðμÞ < 4.5, 50 < pTðbÞ < 100 GeV, 2.2 < ηðbÞ < 4.2, ΔRðμ; bÞ > 0.5, and pTðμ þ bÞ > 20 GeV, are [5] M. Cacciari, G. P. Salam, and G. Soyez, The anti-kt jet clustering algorithm, J. High Energy Phys. 04 (2008) 063. DOI: 10.1103/PhysRevLett.115.112001 σðtopÞ½7 TeV ¼ 23953ðstatÞ33ðsystÞ24ðtheoryÞ fb; σðtopÞ½8 TeV ¼ 28943ðstatÞ40ðsystÞ29ðtheoryÞ fb: [6] A. A. Alves Jr. et al. (LHCb Collaboration), The LHCb detector at the LHC, JINST 3, S08005 (2008). [7] R. Aaij et al. (LHCb Collaboration), LHCb detector performance, Int. J. Mod. Phys. A 30, 1530022 (2015). The systematic uncertainties are nearly 100% correlated between the two measurements. [8] R. Aaij et al., The LHCb trigger and its performance in 2011, JINST 8, P04022 (2013). In summary, top quark production is observed for the first time in the forward region. The cross-section results are in agreement with the SM predictions of 180þ51 −41ð312þ83 −68Þ fb at 7(8) TeV obtained at NLO using MCFM. The differential distributions of the yield and charge asymmetry are also consistent with SM predictions. [9] T. Sjöstrand, S. Mrenna, and P. Skands, PYTHIA 6.4 physics and manual, J. High Energy Phys. 05 (2006) 026; , A brief introduction to PYTHIA 8.1, Comput. Phys. Commun. 178, 852 (2008). [10] I. Belyaev et al., Handling of the generation of primary events in Gauss, the LHCb simulation framework, J. Phys. Conf. Ser. 331, 032047 (2011). We express our gratitude to our colleagues in the CERN accelerator departments for the excellent performance of the LHC. We thank the technical and administrative staff at the LHCb institutes. We acknowledge support from CERN and from the national agencies: CAPES, CNPq, FAPERJ, and FINEP (Brazil); NSFC (China); CNRS/IN2P3 [11] D. J. Lange, The EvtGen particle decay simulation package, Nucl. Instrum. Methods Phys. Res., Sect. A 462, 152 (2001). [12] P. Golonka and Z. Was, PHOTOS Monte Carlo: A precision tool for QED corrections in Z and W decays, Eur. Phys. J. C 45, 97 (2006). 112001-5 week ending 11 SEPTEMBER 2015 week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) [21] R. Aaij et al. (LHCb Collaboration), Study of forward Z þ jet production in pp collisions at ffiffiffis p ¼ 7 TeV, J. High Energy Phys. 01 (2014) 033. [13] J. Allison et al. (Geant4 Collaboration), Geant4 develop- ments and applications, IEEE Trans. Nucl. Sci. 53, 270 (2006); S. Agostinelli et al. (Geant4 Collaboration), Geant4: A simulation toolkit, Nucl. Instrum. Methods Phys. Res., Sect. A 506, 250 (2003). [22] L. Breiman, J. H. Friedman, R. A. Olshen, and C. J. R. Aaij,38 B. Adeva,37 M. Adinolfi,46 A. Affolder,52 Z. Ajaltouni,5 S. Akar,6 J. Albrecht,9 F. Alessio,38 M. Alexander,51 S. Ali,41 G. Alkhazov,30 P. Alvarez Cartelle,53 A. A. Alves Jr.,57 S. Amato,2 S. Amerio,22 Y. Amhis,7 L. An,3 L. Anderlini,17,a J. Anderson,40 M. Andreotti,16,b J. E. Andrews,58 R. B. Appleby,54 O. Aquines Gutierrez,10 F. Archilli,38 P. d’Argent,11 A. Artamonov,35 M. Artuso,59 E. Aslanides,6 G. Auriemma,25,c M. Baalouch,5 S. Bachmann,11 J. J. Back,48 A. Badalov,36 C. Baesso,60 W. Baldini,16,38 R. J. Barlow,54 C. Barschel,38 S. Barsuk,7 W. Barter,38 V. Batozskaya,28 V. Battista,39 A. Bay,39 L. Beaucourt,4 J. Beddow,51 F. Bedeschi,23 I. Bediaga,1 L. J. Bel,41 I. Belyaev,31 E. Ben-Haim,8 G. Bencivenni,18 S. Benson,38 J. Benton,46 A. Berezhnoy,32 R. Bernet,40 A. Bertolin,22 M.-O. Bettler,38 M. van Beuzekom,41 A. Bien,11 S. Bifani,45 T. Bird,54 A. Birnkraut,9 A. Bizzeti,17,d T. Blake,48 F. Blanc,39 J. Blouw,10 S. Blusk,59 V. Bocci,25 A. Bondar,34 N. Bondar,30,38 W. Bonivento,15 S. Borghi,54 M. Borsato,7 T. J. V. Bowcock,52 E. Bowen,40 C. Bozzi,16 S. Braun,11 D. Brett,54 M. Britsch,10 T. Britton,59 J. Brodzicka,54 N. H. Brook,46 A. Bursche,40 J. Buytaert,38 S. Cadeddu,15 R. Calabrese,16,b M. Calvi,20,e M. Calvo Gomez,36,f P. Campana,18 D. Campora Perez,38 L. Capriotti,54 A. Carbone,14,g G. Carboni,24,h R. Cardinale,19,i A. Cardini,15 P. Carniti,20 L. Carson,50 K. Carvalho Akiba,2,38 G. Casse,52 L. Cassina,20,e L. Castillo Garcia,38 M. Cattaneo,38 Ch. Cauet,9 G. Cavallero,19 R. Cenci,23,j M. Charles,8 Ph. Charpentier,38 M. Chefdeville,4 S. Chen,54 S.-F. Cheung,55 N. Chiapolini,40 M. Chrzaszcz,40 X. Cid Vidal,38 G. Ciezarek,41 P. E. L. Clarke,50 M. Clemencic,38 H. V. Cliff,47 J. Closier,38 V. Coco,38 J. Cogan,6 E. Cogneras,5 V. Cogoni,15,k L. Cojocariu,29 G. Collazuol,22 P. Collins,38 A. Comerma-Montells,11 A. Contu,15,38 A. Cook,46 M. Coombes,46 S. Coquereau,8 G. Corti,38 M. Corvo,16,b B. Couturier,38 G. A. Cowan,50 D. C. Craik,48 A. Crocombe,48 M. Cruz Torres,60 S. Cunliffe,53 R. Currie,53 C. D’Ambrosio,38 J. Dalseno,46 P. N. Y. David,41 A. Davis,57 K. De Bruyn,41 S. De Capua,54 M. De Cian,11 J. M. De Miranda,1 L. De Paula,2 W. De Silva,57 P. De Simone,18 C.-T. Dean,51 D. Decamp,4 M. Deckenhoff,9 L. Del Buono,8 N. Déléage,4 M. Demmer,9 D. Derkach,55 O. Deschamps,5 F. Dettori,38 A. Di Canto,38 F. Di Ruscio,24 H. Dijkstra,38 S. Donleavy,52 F. Dordei,11 M. Dorigo,39 A. Dosil Suárez,37 D. Dossett,48 A. Dovbnya,43 K. Dreimanis,52 L. Dufour,41 G. Dujany,54 F. Dupertuis,39 P. Durante,38 R. Dzhelyadin,35 A. Dziurda,26 A. Dzyuba,30 S. Easo,49,38 U. Egede,53 V. Egorychev,31 S. Eidelman,34 S. Eisenhardt,50 U. Eitschberger,9 R. Ekelhof,9 L. Eklund,51 I. El Rifai,5 Ch. Elsasser,40 S. Ely,59 S. Esen,11 H. M. Evans,47 T. Evans,55 A. Falabella,14 C. Färber,11 C. Farinelli,41 N. Farley,45 DOI: 10.1103/PhysRevLett.115.112001 Britton,59 J. Brodzicka,54 N. H. Brook,46 A. Bursche,40 J. Buytaert,38 S. Cadeddu,15 R. Calabrese,16,b M. Calvi,20,e M. Calvo Gomez,36,f P. Campana,18 D. Campora Perez,38 L. Capriotti,54 A. Carbone,14,g 24 h 19 i 15 20 50 2 38 52 20 G. Carboni,24,h R. Cardinale,19,i A. Cardini,15 P. Carniti,20 L. Carson,50 K. Carvalho Akiba,2,38 G. Casse,52 L. Cassina,20,e L. Castillo Garcia,38 M. Cattaneo,38 Ch. Cauet,9 G. Cavallero,19 R. Cenci,23,j M. Charles,8 Ph. Charpentier,38 M. Chefdeville,4 S. Chen,54 S.-F. Cheung,55 N. Chiapolini,40 M. Chrzaszcz,40 X. Cid Vidal,38 G. Ciezarek,41 P. E. L. Clarke,50 M. Clemencic,38 H. V. Cliff,47 J. Closier,38 V. Coco,38 J. Cogan,6 E. Cogneras,5 V. Cogoni,15,k L. Cojocariu,29 G. Collazuol,22 P. Collins,38 A. Comerma-Montells,11 A. Contu,15,38 A. Cook,46 M. Coombes,46 S. Coquereau,8 G. Corti,38 M. Corvo,16,b B. Couturier,38 G. A. Cowan,50 D. C. Craik,48 A. Crocombe,48 M. Cruz Torres,60 M. De Cian,11 J. M. De Miranda,1 L. De Paula,2 W. De Silva,57 P. De Simone,18 C.-T. Dean,51 D. Decamp,4 M. Deckenhoff,9 8 4 9 55 5 38 38 24 112001-6 DOI: 10.1103/PhysRevLett.115.112001 Stone, Classification and Regression Trees (Wadsworth International Group, Belmont, CA, 1984). [14] M. Clemencic, G. Corti, S. Easo, C. R. Jones, S. Miglioranzi, M. Pappagallo, and P. Robbe, The LHCb simulation application, Gauss: Design, evolution and expe- rience, J. Phys. Conf. Ser. 331, 032023 (2011). ¯ [23] R. E. Schapire and Y. Freund, A decision-theoretic gener- alization of on-line learning and an application to boosting, J. Comput. Syst. Sci. 55, 119 (1997). [24] R. Aaij et al. (LHCb Collaboration), Identification of beauty and charm quark jets at LHCb, JINST 10, P06013 (2015). [15] J. M. Campbell and R. K. Ellis, Radiative corrections to Zb¯b production, Phys. Rev. D 62, 114012 (2000). [16] H.-L. Lai, M. Guzzi, J. Huston, Z. Li, P. M. Nadolsky, J. Pumplin, and C.-P. Yuan, New parton distributions for collider physics, Phys. Rev. D 82, 074024 (2010). [25] R. Aaij et al. (LHCb Collaboration), Measurement of the forward Z boson cross-section in pp collisions at ffiffiffis p ¼ 7 TeV, J. High Energy Phys. 08 (2015) 039. [17] S. Alioli, P. Nason, C. Oleari, and E. Re, A general framework for implementing NLO calculations in shower Monte Carlo programs: The POWHEG BOX, J. High Energy Phys. 06 (2010) 043. [26] R. Aaij et al. (LHCb Collaboration), Measurement of the forward W boson production cross-section in pp collisions at ffiffiffis p ¼ 7 TeV, J. High Energy Phys. 12 (2014) 079. [27] See Supplemental Material at http://link.aps.org/ supplemental/10.1103/PhysRevLett.115.112001 for distri- bution of pTðμÞ=pTðjμÞ with fit overlaid for all W þ c candidates. [18] K. Hamilton, P. Nason, E. Re, and G. Zanderighi, NNLOPS simulation of Higgs boson production, J. High Energy Phys. 10 (2013) 222. [19] R. Aaij et al. (LHCb Collaboration), Study of W boson production in association with beauty and charm, Phys. Rev. D 92, 052001 (2015). [28] S. S. Wilks, The large-sample distribution of the likelihood ratio for testing composite hypotheses, Ann. Math. Stat. 9, 60 (1938). [20] M. Cacciari, G. P. Salam, and G. Soyez, FastJet user manual, Eur. Phys. J. C 72, 1896 (2012). [29] R. Aaij et al. (LHCb Collaboration), Precision luminosity measurements at LHCb, JINST 9, P12005 (2014). S. Bifani,45 T. Bird,54 A. Birnkraut,9 A. Bizzeti,17,d T. Blake,48 F. Blanc,39 J. Blouw,10 S. Blusk,59 V. Bocci,25 A. Bondar,34 N. Bondar,30,38 W. Bonivento,15 S. Borghi,54 M. Borsato,7 T. J. V. Bowcock,52 E. Bowen,40 C. Bozzi,16 S. Braun,11 D. Brett,54 M. Britsch,10 T. 112001-6 week ending 11 SEPTEMBER 2015 week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) M. Grabalosa Gándara,5 R. Graciani Diaz,36 L. A. Granado Cardoso,38 E. Graugés,36 E. Graverini,40 G. Graziani,17 A. Grecu,29 E. Greening,55 S. Gregson,47 P. Griffith,45 L. Grillo,11 O. Grünberg,63 B. Gui,59 E. Gushchin,33 Yu. Guz,35,38 T. Gys,38 T. Hadavizadeh,55 C. Hadjivasiliou,59 G. Haefeli,39 C. Haen,38 S. C. Haines,47 S. Hall,53 B. Hamilton,58 T. Hampson,46 X. Han,11 S. Hansmann-Menzemer,11 N. Harnew,55 S. T. Harnew,46 J. Harrison,54 J. He,38 T. Head,39 V. Heijne,41 K. Hennessy,52 P. Henrard,5 L. Henry,8 J. A. Hernando Morata,37 E. van Herwijnen,38 M. Heß,63 A. Hicheur,2 D. Hill,55 M. Hoballah,5 C. Hombach,54 W. Hulsbergen,41 T. Humair,53 N. Hussain,55 D. Hutchcroft,52 D. Hynds,51 M. Idzik,27 P. Ilten,56 R. Jacobsson,38 A. Jaeger,11 J. Jalocha,55 E. Jans,41 A. Jawahery,58 F. Jing C. R. Jones,47 C. Joram,38 B. Jost,38 N. Jurik,59 S. Kandybei,43 W. Kanso,6 M. Karacson,38 T. M. Karbach,38 S. Karodia,51 M. Kelsey,59 I. R. Kenyon,45 M. Kenzie,38 T. Ketel,42 B. Khanji,20,38,e C. Khurewathanakul,39 S. Klaver,54 K. Klimaszewski,28 O. Kochebina,7 M. Kolpin,11 I. Komarov,39 R. F. Koopman,42 P. Koppenburg,41,38 M. Korolev,32 M. Kozeiha,5 L. Kravchuk,33 K. Kreplin,11 M. Kreps,48 G. Krocker,11 P. Krokovny,34 F. Kruse,9 W. Kucewicz,26,n M. Kucharczyk,26 V. Kudryavtsev,34 A. K. Kuonen,39 K. Kurek,28 T. Kvaratskheliya,31 V. N. La Thi,39 D. Lacarrere,38 P. Manning,59 A. Mapelli,38 J. Maratas,5 J. F. Marchand,4 U. Marconi,14 C. Marin Benito,36 P. Marino,23,38,j R. Märki,39 11 25 6 39 42 66 2 J. Marks,11 G. Martellotti,25 M. Martin,6 M. Martinelli,39 D. Martinez Santos,42 F. Martinez Vidal,66 D. Martins Tostes,2 A. Massafferri,1 R. Matev,38 A. Mathad,48 Z. Mathe,38 C. Matteuzzi,20 K. Matthieu,11 A. Mauri,40 B. Maurin,39 A. Mazurov,45 M. McCann,53 J. McCarthy,45 A. McNab,54 R. McNulty,12 B. Meadows,57 F. Meier,9 M. Meissner,11 D. Melnychuk,28 M. Merk,41 D. A. Milanes,62 M.-N. Minard,4 D. S. Mitzel,11 J. Molina Rodriguez,60 S. Monteil,5 22 27 6 23 j 27 50 59 50 T. Pilař,48 D. Pinci,25 A. Pistone,19 A. Piucci,11 S. Playfer,50 M. Plo Casasus,37 T. Poikela,38 F. Polci,8 A. Poluektov,48,34 I. Polyakov,31 E. Polycarpo,2 A. Popov,35 D. Popov,10,38 B. Popovici,29 C. Potterat,2 E. Price,46 J. D. Price,52 g g g B. Rachwal,26 J. H. Rademacker,46 B. Rakotomiaramanana,39 M. Rama,23 M. S. Rangel,2 I. Raniuk,43 N. Rauschmayr,38 112001-7 S. Farry,52 R. Fay,52 D. Ferguson,50 V. Fernandez Albor,37 F. Ferrari,14 F. Ferreira Rodrigues,1 M. Ferro-Luzzi,38 S. Filippov,33 M. Fiore,16,38,b M. Fiorini,16,b M. Firlej,27 C. Fitzpatrick,39 T. Fiutowski,27 K. Fohl,38 P. Fol,53 M. Fontana,10 F. Fontanelli,19,i R. Forty,38 O. Francisco,2 M. Frank,38 C. Frei,38 M. Frosini,17 J. Fu,21 E. Furfaro,24,h A. Gallas Torreira,37 D. Galli,14,g S. Gallorini,22,38 S. Gambetta,50 M. Gandelman,2 P. Gandini,55 Y. Gao,3 J. García Pardiñas,37 J. Garofoli,59 J. Garra Tico,47 L. Garrido,36 D. Gascon,36 C. Gaspar,38 U. Gastaldi,16 R. Gauld,55 L. Gavardi,9 G. Gazzoni,5 A. Geraci,21,l D. Gerick,11 E. Gersabeck,11 M. Gersabeck,54 T. Gershon,48 Ph. Ghez,4 A. Gianelle,22 S. Gianì,39 V. Gibson,47 O. G. Girard,39 L. Giubega,29 V. V. Gligorov,38 C. Göbel,60 D. Golubkov,31 A. Golutvin,53,31,38 A. Gomes,1,m C. Gotti,20,e M. Grabalosa Gándara,5 R. Graciani Diaz,36 L. A. Granado Cardoso,38 E. Graugés,36 E. Graverini,40 G. Graziani,17 A. Grecu,29 E. Greening,55 S. Gregson,47 P. Griffith,45 L. Grillo,11 O. Grünberg,63 B. Gui,59 E. Gushchin,33 Yu. Guz,35,38 T. Gys,38 T. Hadavizadeh,55 C. Hadjivasiliou,59 G. Haefeli,39 C. Haen,38 S. C. Haines,47 S. Hall,53 B. Hamilton,58 T. Hampson,46 X. Han,11 S. Hansmann-Menzemer,11 N. Harnew,55 S. T. Harnew,46 J. Harrison,54 J. He,38 T. Head,39 V. Heijne,41 K. Hennessy,52 P. Henrard,5 L. Henry,8 J. A. Hernando Morata,37 E. van Herwijnen,38 M. Heß,63 A. Hicheur,2 D. Hill,55 M. Hoballah,5 C. Hombach,54 W. Hulsbergen,41 T. Humair,53 N. Hussain,55 D. Hutchcroft,52 D. Hynds,51 M. Idzik,27 P. Ilten,56 R. Jacobsson,38 A. Jaeger,11 J. Jalocha,55 E. Jans,41 A. Jawahery,58 F. Jing,3 M. John,55 D. Johnson,38 C. R. Jones,47 C. Joram,38 B. Jost,38 N. Jurik,59 S. Kandybei,43 W. Kanso,6 M. Karacson,38 T. M. Karbach,38 S. Karodia,51 M. Kelsey,59 I. R. Kenyon,45 M. Kenzie,38 T. Ketel,42 B. Khanji,20,38,e C. Khurewathanakul,39 S. Klaver,54 K. Klimaszewski,28 O. Kochebina,7 M. Kolpin,11 I. Komarov,39 R. F. Koopman,42 P. Koppenburg,41,38 M. Korolev,32 M. Kozeiha,5 L. Kravchuk,33 K. Kreplin,11 M. Kreps,48 G. Krocker,11 P. Krokovny,34 F. Kruse,9 W. Kucewicz,26,n M. Kucharczyk,26 V. Kudryavtsev,34 A. K. Kuonen,39 K. Kurek,28 T. Kvaratskheliya,31 V. N. La Thi,39 D. Lacarrere,38 G. Lafferty,54 A. Lai,15 D. Lambert,50 R. W. Lambert,42 G. Lanfranchi,18 C. Langenbruch,48 B. Langhans,38 T. Latham,48 C. Lazzeroni,45 R. Le Gac,6 J. van Leerdam,41 J.-P. Lees,4 R. Lefèvre,5 A. Leflat,32,38 J. Lefrançois,7 O. Leroy,6 T. Lesiak,26 B. Leverington,11 Y. Li,7 T. Likhomanenko,65,64 M. Liles,52 R. Lindner,38 C. Linn,38 F. Lionetto,40 B. Liu,15 X. Liu,3 D. Loh,48 S. Lohn,38 I. Longstaff,51 J. H. Lopes,2 D. Lucchesi,22,o M. Lucio Martinez,37 H. Luo,50 A. Lupato,22 E. Luppi,16,b O. Lupton,55 F. Machefert,7 F. Maciuc,29 O. Maev,30 K. Maguire,54 S. Malde,55 A. Malinin,64 G. Manca,7 G. Mancinelli,6 P. Manning,59 A. Mapelli,38 J. Maratas,5 J. F. Marchand,4 U. Marconi,14 C. Marin Benito,36 P. Marino,23,38,j R. Märki,39 J. Marks,11 G. Martellotti,25 M. Martin,6 M. Martinelli,39 D. Martinez Santos,42 F. Martinez Vidal,66 D. Martins Tostes,2 A. Massafferri,1 R. Matev,38 A. Mathad,48 Z. Mathe,38 C. Matteuzzi,20 K. Matthieu,11 A. Mauri,40 B. Maurin,39 A. Mazurov,45 M. McCann,53 J. McCarthy,45 A. McNab,54 R. McNulty,12 B. Meadows,57 F. Meier,9 M. Meissner,11 D. Melnychuk,28 M. Merk,41 D. A. Milanes,62 M.-N. Minard,4 D. S. Mitzel,11 J. Molina Rodriguez,60 S. Monteil,5 M. Morandin,22 P. Morawski,27 A. Mordà,6 M. J. Morello,23,j J. Moron,27 A. B. Morris,50 R. Mountain,59 F. Muheim,50 J. Müller,9 K. Müller,40 V. Müller,9 M. Mussini,14 B. Muster,39 P. Naik,46 T. Nakada,39 R. Nandakumar,49 A. Nandi,55 I. Nasteva,2 M. Needham,50 N. Neri,21 S. Neubert,11 N. Neufeld,38 M. Neuner,11 A. D. Nguyen,39 T. D. Nguyen,39 C. Nguyen-Mau,39,p V. Niess,5 R. Niet,9 N. Nikitin,32 T. Nikodem,11 D. Ninci,23 A. Novoselov,35 D. P. O’Hanlon,48 A. Oblakowska-Mucha,27 V. Obraztsov,35 S. Ogilvy,51 O. Okhrimenko,44 R. Oldeman,15,k C. J. G. Onderwater,67 B. Osorio Rodrigues,1 J. M. Otalora Goicochea,2 A. Otto,38 P. Owen,53 A. Oyanguren,66 A. Palano,13,q F. Palombo,21,r M. Palutan,18 J. Panman,38 A. Papanestis,49 M. Pappagallo,51 L. L. Pappalardo,16,b C. Pappenheimer,57 C. Parkes,54 G. Passaleva,17 G. D. Patel,52 M. Patel,53 C. Patrignani,19,i A. Pearce,54,49 A. Pellegrino,41 G. Penso,25,s M. Pepe Altarelli,38 S. Perazzini,14,g P. Perret,5 L. Pescatore,45 K. Petridis,46 A. Petrolini,19,i M. Petruzzo,21 E. Picatoste Olloqui,36 B. Pietrzyk,4 112001-7 Vollhardt,40 10 46 30 34 63 41 63 48 q q g q G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. Vollhardt,40 10 46 30 34 63 41 63 48 G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardon R. Wallace,12 J. Walsh,23 S. Wandernoth,11 J. Wang,59 D. R. Ward,47 N. K. Watson,45 D. Web T. Williams,45 F. F. Wilson,49 J. Wimberley,58 J. Wishahi,9 W. Wislicki,28 M. Witek,26 G. Wormser,7 S. A. Wotton,47 S. Wright,47 K. Wyllie,38 Y. Xie,61 Z. Xu,39 Z. Yang,3 J. Yu,61 X. Yuan,34 O. Yushchenko,35 M. Zangoli,14 M. Zavertyaev,10,v L. Zhang,3 Y. Zhang,3 A. Zhelezov,11 A. Zhokhov,31 and L. Zhong3 (LHCb Collaboration) 1Centro Brasileiro de Pesquisas Físicas (CBPF), Rio de Janeiro, Brazil 2 2Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3 3Center for High Energy Physics, Tsinghua University, Beijing, China 4LAPP, Université Savoie Mont-Blanc, CNRS/IN2P3, Annecy-Le-Vieux, Fran 5 y t Université, Université Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 7LAL, Université Paris-Sud, CNRS/IN2P3, Orsay, France NHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France 9 et Marie Curie, Université Paris Diderot, CNRS/IN 9Fakultät Physik, Technische Universität Dortmund, Dortmund, Germany nstitut für Kernphysik (MPIK), Heidelberg, Germany Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Ger 12 12School of Physics, University College Dublin, Dublin, Ireland 13Sezione INFN di Bari, Bari, Italy 14Sezione INFN di Bologna, Bologna, Italy 15 15Sezione INFN di Cagliari, Cagliari, Italy 16 16Sezione INFN di Ferrara, Ferrara, Italy 17 17Sezione INFN di Firenze, Firenze, Italy 18Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy 19 19Sezione INFN di Genova, Genova, Italy 20Sezione INFN di Milano Bicocca, Milano, Italy 21 21Sezione INFN di Milano, Milano, Italy 22 22Sezione INFN di Padova, Padova, Italy 23 23Sezione INFN di Pisa, Pisa, Italy 24Sezione INFN di Roma Tor Vergata, Roma, Italy 25 25Sezione INFN di Roma La Sapienza, Roma, Italy k Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland ty of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, Poland 28National Center for Nuclear Research (NCBJ), Warsaw, Poland 29 ei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania 30 30Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 31Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 2 f p y 32Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia 33 33Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow, Russia 112001-8 M.-H. Schune,7 R. Schwemmer,38 B. Sciascia,18 A. Sciubba,25,s A. Semennikov,31 I. Sepp,53 N. Serra,40 J. Serrano,6 L. Sestini,22 P. Seyfert,20 M. Shapkin,35 I. Shapoval,16,43,b Y. Shcheglov,30 T. Shears,52 L. Shekhtman,34 V. Shevchenko,64 A. Shires,9 R. Silva Coutinho,48 G. Simi,22 M. Sirendi,47 N. Skidmore,46 I. Skillicorn,51 T. Skwarnicki,59 E. Smith,55,49 E. Smith,53 I. T. Smith,50 J. Smith,47 M. Smith,54 H. Snoek,41 M. D. Sokoloff,57,38 F. J. P. Soler,51 D. Souza,46 B. Souza De Paula,2 B. Spaan,9 P. Spradlin,51 S. Sridharan,38 F. Stagni,38 M. Stahl,11 S. Stahl,38 O. Steinkamp,40 O. Stenyakin,35 F. Sterpka,59 S. Stevenson,55 S. Stoica,29 S. Stone,59 B. Storaci,40 S. Stracka,23,j M. Straticiuc,29 U. Straumann,40 L. Sun,57 W. Sutcliffe,53 K. Swientek,27 S. Swientek,9 V. Syropoulos,42 M. Szczekowski,28 P. Szczypka,39,38 T. Szumlak,27 S. T’Jampens,4 T. Tekampe,9 M. Teklishyn,7 G. Tellarini,16,b F. Teubert,38 C. Thomas,55 E. Thomas,38 J. van Tilburg,41 V. Tisserand,4 M. Tobin,39 J. Todd,57 S. Tolk,42 L. Tomassetti,16,b D. Tonelli,38 S. Topp-Joergensen,55 N. Torr,55 E. Tournefier,4 S. Tourneur,39 K. Trabelsi,39 M. T. Tran,39 M. Tresch,40 A. Trisovic,38 A. Tsaregorodtsev,6 P. Tsopelas,41 N. Tuning,41,38 A. Ukleja,28 A. Ustyuzhanin,65,64 U. Uwer,11 C. Vacca,15,k V. Vagnoni,14 G. Valenti,14 A. Vallier,7 R. Vazquez Gomez,18 P. Vazquez Regueiro,37 C. Vázquez Sierra,37 S. Vecchi,16 J. J. Velthuis,46 M. Veltri,17,u G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. Vollhardt,40 D. Volyanskyy,10 D. Voong,46 A. Vorobyev,30 V. Vorobyev,34 C. Voß,63 J. A. de Vries,41 R. Waldi,63 C. Wallace,48 R. Wallace,12 J. Walsh,23 S. Wandernoth,11 J. Wang,59 D. R. Ward,47 N. K. Watson,45 D. Websdale,53 A. Weiden,40 M. Whitehead,48 D. Wiedner,11 G. Wilkinson,55,38 M. Wilkinson,59 M. Williams,38 M. P. Williams,45 M. Williams,56 T. Williams,45 F. F. Wilson,49 J. Wimberley,58 J. Wishahi,9 W. Wislicki,28 M. Witek,26 G. Wormser,7 S. A. Wotton,47 S. Wright,47 K. Wyllie,38 Y. Xie,61 Z. Xu,39 Z. Yang,3 J. Yu,61 X. Yuan,34 O. Yushchenko,35 M. Zangoli,14 M. Zavertyaev,10,v L Zh 3 Y Zh 3 A Zh l 11 A Zh kh 31 d L Zh 3 112001-7 week ending 11 SEPTEMBER 2015 week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) M.-H. Schune,7 R. Schwemmer,38 B. Sciascia,18 A. Sciubba,25,s A. Semennikov,31 I. Sepp,53 N. Serra,40 J. Serrano,6 L. Sestini,22 P. Seyfert,20 M. Shapkin,35 I. Shapoval,16,43,b Y. Shcheglov,30 T. Shears,52 L. Shekhtman,34 V. Shevchenko,64 A. Shires,9 R. Silva Coutinho,48 G. Simi,22 M. Sirendi,47 N. Skidmore,46 I. Skillicorn,51 T. Skwarnicki,59 E. Smith,55,49 E. Smith,53 I. T. Smith,50 J. Smith,47 M. Smith,54 H. Snoek,41 M. D. Sokoloff,57,38 F. J. P. Soler,51 D. Souza,46 B. Souza De Paula,2 B. Spaan,9 P. Spradlin,51 S. Sridharan,38 F. Stagni,38 M. Stahl,11 S. Stahl,38 O. Steinkamp,40 O. Stenyakin,35 F. Sterpka,59 S. Stevenson,55 S. Stoica,29 S. Stone,59 B. Storaci,40 S. Stracka,23,j M. Straticiuc,29 U. Straumann,40 L. Sun,57 W. Sutcliffe,53 K. Swientek,27 S. Swientek,9 V. Syropoulos,42 M. Szczekowski,28 P. Szczypka,39,38 T. Szumlak,27 S. T’Jampens,4 T. Tekampe,9 M. Teklishyn,7 G. Tellarini,16,b F. Teubert,38 C. Thomas,55 E. Thomas,38 J. van Tilburg,41 V. Tisserand,4 M. Tobin,39 J. Todd,57 S. Tolk,42 L. Tomassetti,16,b D. Tonelli,38 S. Topp-Joergensen,55 N. Torr,55 E. Tournefier,4 S. Tourneur,39 K. Trabelsi,39 M. T. Tran,39 M. Tresch,40 A. Trisovic,38 A. Tsaregorodtsev,6 P. Tsopelas,41 N. Tuning,41,38 A. Ukleja,28 A. Ustyuzhanin,65,64 U. Uwer,11 C. Vacca,15,k V. Vagnoni,14 G. Valenti,14 A. Vallier,7 R. Vazquez Gomez,18 P. Vazquez Regueiro,37 C. Vázquez Sierra,37 S. Vecchi,16 J. J. Velthuis,46 M. Veltri,17,u G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. Vollhardt,40 D. Volyanskyy,10 D. Voong,46 A. Vorobyev,30 V. Vorobyev,34 C. Voß,63 J. A. de Vries,41 R. Waldi,63 C. Wallace,48 R. Wallace,12 J. Walsh,23 S. Wandernoth,11 J. Wang,59 D. R. Ward,47 N. K. Watson,45 D. Websdale,53 A. Weiden,40 M. Whitehead,48 D. Wiedner,11 G. Wilkinson,55,38 M. Wilkinson,59 M. Williams,38 M. P. Williams,45 M. Williams,56 T. Williams,45 F. F. Wilson,49 J. Wimberley,58 J. Wishahi,9 W. Wislicki,28 M. Witek,26 G. Wormser,7 S. A. Wotton,47 S. Wright,47 K. Wyllie,38 Y. Xie,61 Z. Xu,39 Z. Yang,3 J. Yu,61 X. Yuan,34 O. Yushchenko,35 M. Zangoli,14 M. Zavertyaev,10,v L Zhang 3 Y Zhang 3 A Zhelezov 11 A Zhokhov 31 and L Zhong3 A. Vallier,7 R. Vazquez Gomez,18 P. Vazquez Regueiro,37 C. Vázquez Sierra,37 S. Vecchi,16 J. J. Velthuis,46 M. Veltri,17,u G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. 112001-8 f eAlso at Università di Milano Bicocca, Milano, Italy. f fAlso at LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. fAlso at LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. gAlso at Università di Bologna, Bologna, Italy. h hAlso at Università di Roma Tor Vergata, Roma, i iAlso at Università di Genova, Genova, Italy. j iAlso at Università di Genova, Genova, Italy. j jAlso at Scuola Normale Superiore, Pisa, Italy. jAlso at Scuola Normale Superiore, Pisa, Italy. k kAlso at Università di Cagliari, Cagliari, Italy. l kAlso at Università di Cagliari, Cagliari, Italy. l lAlso at Politecnico di Milano, Milano, Italy. lAlso at Politecnico di Milano, Milano, Italy. mAlso at Universidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. nAlso at AGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. o oAlso at Università di Padova, Padova, Italy. oAlso at Università di Padova, Padova, Italy. pAlso at Hanoi University of Science, Hanoi, Viet Nam. pAlso at Hanoi University of Science, Hanoi, Viet Nam. pAlso at Hanoi University of Science, Hanoi, V g nAlso at AGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. o 112001-8 week ending 11 SEPTEMBER 2015 week ending 11 SEPTEMBER 2015 P H Y S I C A L R E V I E W L E T T E R S PRL 115, 112001 (2015) 34Budker Institute of Nuclear Physics (SB RAS) and Novosibirsk State University, Novosibirsk, Russia 35Institute for High Energy Physics (IHEP), Protvino, Russia Imperial College London, London, United Kingdom ool of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 55 55Department of Physics, University of Oxford, Oxford, United Kingdom 56 56Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 57 57University of Cincinnati, Cincinnati, Ohio, USA 8 58University of Maryland, College Park, Maryland, USA 59 59Syracuse University, Syracuse, New York, USA 60Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil (associated with Institution Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil) 61Institute of Particle Physics Central China Normal University Wuhan Hubei China 60Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil (associated with Institution Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 61 61Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China (associated with Institution Center for High Energy Physics, Tsinghua University, Beijing, China) 62 Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia 62Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia 63Institut für Physik, Universität Rostock, Rostock, Germany associated with Institution Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, He 64 stitution Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany) 64 64National Research Centre Kurchatov Institute, Moscow, Russia (associated with Institution Institute of Theoretical and Experimental Physics (ITEP), Moscow, 65 tion Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia) 65 65Yandex School of Data Analysis, Moscow, Russia (associated with Institution Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russi 66 66Instituto de Fisica Corpuscular (IFIC), Universitat de Valencia-CSIC, Valencia, Spain (associated with Institution Universitat de Barcelona, Barcelona, Spain) 67Van Swinderen Institute, University of Groningen, Groningen, The Netherlands (associated with Institution Nikhef National Institute for Subatomic Physics, Amsterdam, The Netherlands) aAlso at Università di Firenze, Firenze, Italy. b bAlso at Università di Ferrara, Ferrara, Italy. c bAlso at Università di Ferrara, Ferrara, Italy. cAlso at Università della Basilicata, Potenza, Italy. d cAlso at Università della Basilicata, Potenza, Italy. d dAlso at Università di Modena e Reggio Emilia, Modena, Italy. dAlso at Università di Modena e Reggio Emilia, Modena, Italy. eAlso at Università di Milano Bicocca, Milano, Italy. 112001-9 week ending 11 SEPTEMBER 2015 vAlso at P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia. PRL 115, 112001 (2015) qAlso at Università di Bari, Bari, Italy. so at Università degli Studi di Milano, Milano, Ital sAlso at Università di Roma La Sapienza, Roma, Italy. tAlso at Università di Pisa, Pisa, Italy. uAlso at Università di Urbino, Urbino, Italy. uAlso at Università di Urbino, Urbino, Italy. vAlso at P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russ 112001-10
https://openalex.org/W2767015251
https://europepmc.org/articles/pmc5663095?pdf=render
English
null
Isolation and identification of halotolerant soil bacteria from coastal Patenga area
BMC research notes
2,017
cc-by
4,377
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Objective:  Halotolerant bacteria have multiple uses viz. fermentation with lesser sterility control and industrial pro‑ duction of bioplastics. Moreover, it may increase the crop productivity of coastal saline lands in Bangladesh by trans‑ ferring the salt tolerant genes into the plants. The study focused on the isolation and identification of the halotolerant bacteria from three soil samples, collected from coastal Patenga area. The samples were inoculated in nutrient media containing a wide range of salt concentrations. Results:  All the samples showed 2, 4 and 6% (w/v) salt tolerance. The isolates from Patenga soil (4, 6%) and beach soil (2%) showed catalase activity and all the isolates showed negative results for oxidase activity, indole produc‑ tion, lactose and motility. All the samples provided positive results for dextrose fermentation. Other tests provided mixed results. Based on the morphological characteristics, biochemical tests and ABIS software analysis the isolates fall within the Enterobacteriaceae, Clostridium and Corynebacterium, with a predominance of Vibrios. Overall the isolates can be considered as mild halotolerant, with the best growth observed at lower salinities and no halophilism detected. Among many possibilities, the genes responsible for the salt tolerant trait in these species can be identified, extracted and inserted into the crop plants to form a transgenic plant to result in higher yield for the rest of the year. Keywords: Halotolerant, Enterobacteriaceae, Clostridium, Corynebacterium, Salinity Keywords:  Halotolerant, Enterobacteriaceae, Clostridium, Corynebacterium, Salinity halotolerant. Non-halotolerant which can grow in low salt concentration about 1% w/v. Slightly tolerant as pseu- domonads, enterobacteria, and vibrios, can survive in up to 2–8%, moderately tolerant 18–20% and extremely tolerant microbes can grow over the whole range of salt concentrations from zero to saturation. The halotolerant organisms maintain a low level of ionic concentrations to synthesize compatible solutes to balance the osmotic level inside the cytoplasm with the outer medium. These maintenance mechanisms of the internal environment and the properties of the cytoplasmic membrane help them to adapt to changes in the saline environment as salt lakes, saline soils, and salted food products [4]. Isolation and identification of halotolerant soil bacteria from coastal Patenga area Shafkat Shamim Rahman1,2*  , Romana Siddique1 and Nafisa Tabassum1 *Correspondence: shafkatshamimrahman@gmail.com 1 Biotechnology Program, Department of Mathematics and Natural Sciences, BRAC University, 66, Mohakhali, Dhaka 1212, Bangladesh Full list of author information is available at the end of the article Rahman et al. BMC Res Notes (2017) 10:531 DOI 10.1186/s13104-017-2855-7 Rahman et al. BMC Res Notes (2017) 10:531 DOI 10.1186/s13104-017-2855-7 BMC Research Notes Biochemical tests Total 18 primarily screened isolates (2 each from 2, 4 and 6% plates) were sub-cultured on NA plates without salt with the same dilution factor and nine inocula were tested further. After 24-h of incubation at 37  °C, the plates were observed [19]. p Standard gram staining protocols were followed and then slide observed under a microscope. The presence or absence of bubbles or foam was observed to determine the capability of catalase activity [20]. Two drops of oxi- dase reagent p-aminodimethylaniline oxalate were added to the surface of test organisms’ growth in oxidase test [21]. Nitrate broth (beef extract 3  g/L, peptone 5  g/L, potassium nitrate 5  g/L) [22] and MR-VP broth (pep- tone, dextrose and potassium phosphate) was prepared and incubated at 24-h at 37 °C for Methyl Red test and VP test [23]. The triple sugar iodine agar was prepared and after inoculation, incubated for about 24-h at 35 °C for TSI test [24]. After inoculation, MIU media (MIU test [25]) and Simmon’s agar slants were also incubated for 24-h at 37 °C [22, 23]. Trypticase 10 g/L, NaCl 5 g/L, phe- nol red 0.018/L, sugar (glucose, lactose or sucrose) 5 g/L contained broth incubated at 37 °C for 24-h to determine the capability of fermenting carbohydrate substrate with acid and gas production [26]. Autoclaved starch agar (beef extract 3 g/L, soluble starch 10 g/L and agar 15 g/L) plates were incubated for 24-h to indicate the presence of α-amylase [27]. Sterile tubes containing inoculum was transferred in 6.5% NaCl solution and kept in 24-h incu- bation to assess salt hindrance. Halophilic and halotolerant bacteria are essential for salty foods production as Thai fish sauce, pickling brines and salt-cured bacon [10]. Isolates from effluents of tex- tile industries also showed the ability to decolorize the utilized azo dyes [11]. Halotolerant bacteria, recovered from the composting process, were able to produce hydrolases, lipases, proteases, amylases, cellulases and biopolymers [12]. Four halotolerant species (Bacillus atrophaeus, Halomonas shengliensis, Halomonas kore- ensis and Virgibacillus salarius) showed the ability to metabolize hydrocarbons and isolates as V. salarius and Brevibacillus sp. KUMAs1 has the potential to be used for bioremediation [13, 14]. Nevertheless, Corynebacte- rium xerosis was the potent degraders of hydrocarbons (petrol and diesel) [15]. Isolation and screening containing aminocyclopropane-1-carboxylate (ACC) deaminase, [6] examined their effect on salinity stress tol- erance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated plants exhibited higher germination percentage, growth parameters, and chloro- phyll content than control. 5 g of each of the soil samples were taken to prepare a suspension. Then serially diluted ­(10−3, ­10−5 and ­10−7, 100 μL) samples were taken and incubated on the nutri- ent agar plates containing 2% (w/v), 4% (w/v), 6% (w/v), 8% (w/v) and 10% (w/v) NaCl for 24-h at 37 °C. No bac- terial growth observed for 8% (w/v) and 10% (w/v) NaCl plates. In a salt stress improvement research for red pepper plants by 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase producing halotolerant bacteria was studied [7, 8]. The result showed salt stress ethylene production by increasing enzyme activities of a biosynthetic pathway. It was also reported that the growth promotion in inoc- ulated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria [9]. Biochemical tests Halophilic and halotolerant bac- teria can be used for the production of enzymes with dif- ferent immunological properties [16] and also essential for nutrient recycling and for maintaining the soil health in a salty environment [17].h The goal of the research was to isolate and identify halotolerant bacteria from natural sources [18]. Multiple uses of these species viz. fermentation with lesser control on sterility and industrial production of bioplastics, can be beneficial for different sectors in Bangladesh. Besides that, it may provide the salt tolerant genes for plants in future. Therefore, plants can uptake and store salt as a nutrient and result in a good yield throughout the year. Introduction Salinization is one of the root reasons for the crop destruction in Bangladesh [1, 2]. Almost 20% of the coun- try is covered as the coastal area from which about 53% of lands are affected by very slight to very strong salinity [3]. The upward movement of the saline ground water in the dry season (November–May) is the factor that initi- ates the development of saline soil. Nevertheless, the intrusion of the seawater also increases the degree of salinity of the coastal drinking water and cause severe health problems viz. hypertension or high blood pres- sure, stroke, heart diseases, pre-eclampsia. Bacteria that grow in the absence of salt and in the presence of high salt concentrations are known as It was reported that amelioration of salt stress inhibi- tory effect on the canola seed germination was attrib- uted to the inoculation of ACC deaminase-producing halotolerant bacteria modulating ethylene emission and inducing hydrolytic enzymes [5]. A research was exe- cuted on plant growth-promoting rhizobacteria (PGPR) Rahman et al. BMC Res Notes (2017) 10:531 Page 2 of 6 Results All the samples showed growth in NA media contain- ing 2% (w/v), 4% (w/v) and 6% (w/v) NaCl. But failed to grow in media containing 8% (w/v) and 10% (w/v) NaCl (Table 1). Main text Methods Sample collectionhf The isolates were all gram positive (Fig. 1; Table 2). Pat- enga 4, 6% were cocci shaped and rest were rod shaped. Sample collectionhf Patenga area 4% and Beach soil 2, 4, 6% hydrolyzed starch. Patenga 2, 4%, Beach soil 2, 6%, Land soil 2, 4 and 6% showed the ability to grow at 6.5% NaCl solution. Sample collectionhf soil 2%—Vibrio metschnikovii; Beach soil 4%—Vibrio metschnikovii; Beach soil 6%—Volucribacter psittaci- cida; Land soil 2%—Aggregatibacter (Haemophilus) segnis; Land soil 4%—Aggregatibacter (Haemophilus) seg- nis; Land soil 6%—Clostridium innocuum/Clostridium spiroforme. soil 2%—Vibrio metschnikovii; Beach soil 4%—Vibrio metschnikovii; Beach soil 6%—Volucribacter psittaci- cida; Land soil 2%—Aggregatibacter (Haemophilus) segnis; Land soil 4%—Aggregatibacter (Haemophilus) seg- nis; Land soil 6%—Clostridium innocuum/Clostridium spiroforme. became yellow for Patenga 2%, Beach soil 2, 4 and 6%. The only slant was acidic for Land soil 2 and 4% and Land soil 6% for the butt. Patenga 2%, Beach soil 2, 4 and 6% recorded as MR test positive. Patenga 4, 6%, Beach soil 2, 4%, Land soil 2, 4, and 6% found VP test positive because the color did not change to pink. All negative results recorded at MIU test, except non-motile Patenga 6% sample was urease positive. Simmon’s citrate test showed a positive result for Beach soil 2, 4% and land soil 4, 6%. Land soil 4, 6%, Patenga area 2, 4, 6%, Beach soil 2, 4, and 6% found positive for sucrose metabolism. No positive result observed for lactose fermentation. All the sam- ple result was positive for dextrose fermentation, except Land soil 2%. Patenga area 2% and Beach soil 4% pro- duced gas in fermentation. Patenga area 4% and Beach soil 2, 4, 6% hydrolyzed starch. Patenga 2, 4%, Beach soil 2, 6%, Land soil 2, 4 and 6% showed the ability to grow at 6.5% NaCl solution. became yellow for Patenga 2%, Beach soil 2, 4 and 6%. The only slant was acidic for Land soil 2 and 4% and Land soil 6% for the butt. Patenga 2%, Beach soil 2, 4 and 6% recorded as MR test positive. Patenga 4, 6%, Beach soil 2, 4%, Land soil 2, 4, and 6% found VP test positive because the color did not change to pink. All negative results recorded at MIU test, except non-motile Patenga 6% sample was urease positive. Simmon’s citrate test showed a positive result for Beach soil 2, 4% and land soil 4, 6%. Land soil 4, 6%, Patenga area 2, 4, 6%, Beach soil 2, 4, and 6% found positive for sucrose metabolism. No positive result observed for lactose fermentation. All the sam- ple result was positive for dextrose fermentation, except Land soil 2%. Patenga area 2% and Beach soil 4% pro- duced gas in fermentation. Sample collectionhf Three different soil samples (Patenga Beach soil 22°15′06.8″N, 91°45′29.5″E; Land soil 22°14′31.6″N, 91°47′15.9″E and Patenga area soil 22°14′25.4″N, 91°48′59.7″E) were collected from 10 to 12-in. depth at different locations of the coastal Chittagong area; these were kept in a sterile polythene packet at room temperature. In catalase test, hydrogen peroxide was used and bro- ken down to water and oxygen. Patenga 2, 6% and Beach soil 2% were positive. Oxidase test resulted as negative for all (Table 2). Only Land soil 2 and 4% was reduced nitrate by the change of color (red) (Table 2). Most of the samples were responsive to TSI test. Both slant and butt Page 3 of 6 Rahman et al. BMC Res Notes (2017) 10:531 Table 1  Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl Conc. Patenga soil Beach soil Land soil 2% 4% 6% 8% 10% 2% 4% 6% 8% 10% 2% 4% 6% 8% 10% Growth ✓ ✓ ✓ x x ✓ ✓ ✓ x x ✓ ✓ ✓ x x Fig. 1  Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Rahman et al. BMC Res Notes (2017) 10:531 Page 3 of 6 Table 1  Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl Conc. Patenga soil Beach soil Land soil 2% 4% 6% 8% 10% 2% 4% 6% 8% 10% 2% 4% 6% 8% 10% Growth ✓ ✓ ✓ x x ✓ ✓ ✓ x x ✓ ✓ ✓ x x Table 1  Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl Table 1  Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl Table 1  Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl Conc. Patenga soil Beach soil Land soil 2% 4% 6% 8% 10% 2% 4% 6% 8% 10% 2% 4% 6% 8% 10% Growth ✓ ✓ ✓ x x ✓ ✓ ✓ x x ✓ ✓ ✓ x x Fig. 1  Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Fig. 1  Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Fig. 1  Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Fig. Discussion In this study, three soil samples were collected from the coastal area of Chittagong [29–31]. The isolated bacteria from the samples had successfully survived in a limited range of salinities and no halophilism detected. All the iso- lates showed tolerance to 2% (w/v), 4% (w/v) and 6% (w/v) of NaCl (Table 1). The dilution factor was inversely pro- portional to the number of colonies. The study divulged the abundance of gram positive bacteria (Table  2). The isolates from Patenga area (2, 6%) and Beach (2%) showed catalase activity and all the isolates showed negative results for oxidase activity, indole production, lactose and motility. Aerobic and facultative aerobes exhibit oxidase activity whereas Enterobacteriaceae are oxidase nega- tive. This provided the evidence of Enterobacteriaceae in the samples. In the MIU test, only the isolates extracted from Patenga area that is 6% (w/v) NaCl tolerant showed urease positive. In addition to this, all the isolates pro- vided positive results for dextrose fermentation. Vibrios Finally, the test results were analyzed in ABIS online software [28], which is used to analyze the genus of the organism. Based on the morphology characteris- tics, Biochemical test and ABIS software the follow- ing results were predicted as Patenga 2%—Brevibacillus agri; Patenga 4%—Pantoea stewartii subsp. stewartii; Patenga 6%—Corynebacterium xerosis/Corynebacte- rium minutissimum/Corynebacterium kutscheri; Beach Rahman et al. BMC Res Notes (2017) 10:531 Page 4 of 6 Table 2  Results of biochemical and sugar tests of the isolates collected from nutrient agar K alkaline reaction, A acidic reaction, + positive reaction, − negative reaction Isolate no Sample isolate name Gram stain TSI MIU Catalase Oxidase Sim- mon’s citrate MR VP Nitrate reduc- tion Starch hydroly- sis Carbohydrate 6.5% NaCl solution Presumptive organism ± Shape BUTT SLANT H2S Gas Motility Indole Urease Sucrose Lactose Dextrose 1. Patenga area soil 2% + Rod A A − − − − − + − − + − − − + − + + Brevibacillus agri 2. Patenga area soil 4% + Cocci A K − − − − − − − − − + − + + − + + Pantoea stew- artii subsp. stewartii 3. Patenga area soil 6% + Cocci K A − − − − + + − − − + − − + − + − Corynebacte- rium xerosis/ Corynebacte- rium minutis- simum/ Corynebacte- rium kutscheri 4. Discussion Beach soil 2% + Rod A A − − − − − + − + + + − + + − + + Vibrio metschnikovii 5. Beach soil 4% + Rod A A − − − − − − − + + + − + + − + − Vibrio metschnikovii 6. Beach soil 6% + Rod A A − − − − − − − − + − − + + − + + Volucribacter psittacicida 7. Land soil 2% + Rod K A − − − − − − − − − + + − − − − + Aggregatibacter (Haemophi- lus) segnis 8. Land soil 4% + Rod K A − − − − − − − + − + + − + − + + Aggregatibacter (Haemophi- lus) segnis 9. Land soil 6% + Rod A K − − − − − − − + − + − − + − + + Clostridium innocuum/ Clostridium spiroforme Rahman et al. BMC Res Notes (2017) 10:531 Page 5 of 6 were predominating in the investigated soil along with Corynebacterium, Clostridium and Enterobacteriaceae. were predominating in the investigated soil along with Corynebacterium, Clostridium and Enterobacteriaceae. References 1. Elaine Colligan. Salinity issues in Bangladesh. Georget J Int Aff. 2011. http://journal.georgetown.edu/guide-to-salinity-issues-in-bangladesh/. Accessed 26 June 2017. Abbreviations MIU tilit i d MIU: motility indole urease test; et al.: and others; NA: nutrient agar; Mg: mil‑ ligram; sp.: species; mL: milliliter; TSI: triple sugar iodine; MR: methyl red; VP: Voges Proskauer; w/v: weight by volume; g/L: gram per liter. y 8. Glick BR. Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res. 2014;169(1):30–9. 8. Glick BR. Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res. 2014;169(1):30–9. 9. Nabti E, Schmid M, Hartmann A. Application of halotolerant bacteria to restore plant growth under salt stress. In: Halophiles. Berlin: Springer International Publishing; 2015. p. 235–59. 9. Nabti E, Schmid M, Hartmann A. Application of halotolerant bacteria to restore plant growth under salt stress. In: Halophiles. Berlin: Springer International Publishing; 2015. p. 235–59. Limitations 2. Rasel HM, Hasan MR, Ahmed B, Miah MS. Investigation of soil and water salinity, its effect on crop production and adaptation strategy. Int J Water Res Environ Eng. 2013;5(8):475–81. Extreme halotolerant species as Halomonas elongata CHR63, Thioalkalivibrio versutus or Sporosarcina pas- teurii wasn’t detected in the study [32]. The future pros- pect can be the plantation of transgenic plants in the coastal area for better agricultural use. The research also provides insights for adaption and use of soil microor- ganisms as natural fertilizers after natural calamity struck the coastal region. Before that, the success of this investi- gation is not properly accomplished. 3. Haque SA. Salinity problems and crop production in coastal regions of Bangladesh. Pak J Bot. 2006;38(5):1359–65. 4. Roberts MF. Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Syst. 2005;1(1):5. 5. Siddikee MA, Sundaram S, Chandrasekaran M, Kim K, Selvakumar G, Sa T. Halotolerant bacteria with ACC deaminase activity alleviate salt stress effect in canola seed germination. Appl Biol Chem. 2015;2(58):237–41. f 6. Habib SH, Kausar H, Saud HM. Plant growth-promoting rhizobacteria enhance salinity stress tolerance in okra through ROS-scavenging enzymes. Biomed Res Int. 2016;2016:1–10. 7. Siddikee MA, Glick BR, Chauhan PS, Jong Yim W, Sa T. Enhancement of growth and salt tolerance of red pepper seedlings (Capsicum annuum L.) by regulating stress ethylene synthesis with halotolerant bacteria containing 1-aminocyclopropane-1-carboxylic acid deaminase activity. Plant Physiol Biochem. 2011;49(4):427–34. Author details 1 12. Oliveira LC, Ramos PL, Marem A, Kondo MY, Rocha R, Bertolini T, Silveira MA, Cruz JB, Vasconcellos SP, Juliano L, Okamoto DN. Halotolerant bac‑ teria in the São Paulo Zoo composting process and their hydrolases and bioproducts. Braz J Microbiol. 2015;46(2):347–54. 1 Biotechnology Program, Department of Mathematics and Natural Sciences, BRAC University, 66, Mohakhali, Dhaka 1212, Bangladesh. 2 Present Address: United Surgical (BD) Ltd, Plot# 659‑661, Islampur, Kadda, Gazipur 1702, Bangladesh. 13. Kothari V, Panchal M, Srivastava N. Hydrocarbon degradation potential of halotolerant bacteria. In: Scientific study. 2014. http://www.grin.com/ en/e-book/268982/hydrocarbon-degradation-potential-of-halotolerant- bacteria. Accessed 27 June 2017. Authors’ contributions NT carried out experimental studies; analyzed, interpreted data and drafted the dissertation. SSR carried out the experimental studies partially and drafted, edited, finalized and revised the article and coordinated to the publisher. RS participated in designing, supervising and coordinating the study. All authors read and approved the final manuscript. 10. Margesin R, Schinner F. Potential of halotolerant and halophilic microor‑ ganisms for biotechnology. Extremophiles. 2001;5(2):73–83. 11. Asad S, Amoozegar MA, Pourbabaee A, Sarbolouki MN, Dastgheib SM. Decolorization of textile azo dyes by newly isolated halophilic and halo‑ tolerant bacteria. Bioresour Technol. 2007;98(11):2082–8. Competing interests The authors declare that they have no competing interests. Availability of data and materials All data presented within the manuscript. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Funding This study was self-funded by authors and their institution Department of Mathematics and Natural Sciences, BRAC University. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Received: 13 July 2017 Accepted: 23 October 2017 Competing interests The authors declare that they have no competing interests. Availability of data and materials All data presented within the manuscript. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Funding This study was self-funded by authors and their institution Department of Mathematics and Natural Sciences, BRAC University. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Received: 13 July 2017 Accepted: 23 October 2017 Ethics approval and consent to participate Not applicable. 19. Aditi FY, Rahman SS, Hossain MM. A study on the microbiological status of mineral drinking water. Open Microbiol J. 2017;11:31–44. https://doi. org/10.2174/1874285801711010031. Funding This study was self-funded by authors and their institution Department of Mathematics and Natural Sciences, BRAC University. 20. Reiner K. Catalase test protocol. Washington: American Society for Microbiology; 2010. http://www.microbelibrary.org/library/ laboratorytest/3226-catalase-test-protocol.htm. Accessed 5 July 2017. Acknowledgements Authors are grateful to Prof. Dr. AAZ Ahmad (Chairperson) and Prof. Naiyyum Choudhury of Department of Mathematics and Natural Sciences, BRAC University, Dhaka, for motivation. Their sincere appreciation goes to Dr. M. Mahboob Hossain and Jebunnesa Chowdhury for drawing novel thoughts. And, grateful to Asma Binte Afzal, Nahreen Mirza and Salman Khan Promon for their unremitting help. 14. Mallick I, Hossain ST, Sinha S, Mukherjee SK. Use of indigenous bacteria from arsenic contaminated soil for arsenic bioremediation. In: Manage‑ ment of natural resources in a changing environment. Berlin: Springer International Publishing; 2015. p. 155–65. 15. Jyothi K, Babu KS, Clara NK, Kumar A. Identification and isolation of hydrocarbon degrading bacteria by molecular characterization. Helix. 2012;2:105–11. Consent for publication 18. Grattieri M, Suvira M, Hasan K, Minteer SD. Halotolerant extremophile bacteria from the Great Salt Lake for recycling pollutants in microbial fuel cells. J Power Sources. 2017;356:310–8. Competing interests g The authors declare that they have no competing interests. 16. Shirazian P, Asad S, Amoozegar MA. The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: l-asparaginase and l-glutaminase. EXCLI J. 2016;15:268. 17. Lavik G, Stührmann T, Brüchert V, Van der Plas A, Mohrholz V, Lam P, Mußmann M, Fuchs BM, Amann R, Lass U, Kuypers MM. Detoxification of sulphidic African shelf waters by blooming chemolithotrophs. Nature. 2009;457(7229):581–4. Publisher’s Note Physi‑ ological characterization of a halotolerant anoxygenic phototrophic Fe(II)-oxidizing green-sulfur bacterium isolated from a marine sediment. FEMS Microbiol Ecol. 2017;93(5):fix054. i 32. Aljohny BO. Halophilic bacterium—a review of new studies. Biosci Bio‑ technol Res Asia. 2015;12(3):2061–9. https://doi.org/10.13005/bbra/1874. i 32. Aljohny BO. Halophilic bacterium—a review of new studies. Biosci Bio‑ technol Res Asia. 2015;12(3):2061–9. https://doi.org/10.13005/bbra/1874. 27. Starch hydrolysis test. http://www.vumicro.com/vumie/help/VUMICRO/ Starch_Hydrolysis_Test.htm. Accessed 5 July 2017. 27. Starch hydrolysis test. http://www.vumicro.com/vumie/help/VUMICRO/ Starch_Hydrolysis_Test.htm. Accessed 5 July 2017. Publisher’s Note 21. Shields P, Cathcart L. Oxidase test protocol. Washington: American Society for Microbiology; 2010. http://www.microbelibrary.org/library/ laboratory-test/3229-oxidase-test-protocol.htm. Accessed 5 July 2017. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Received: 13 July 2017 Accepted: 23 October 2017 Received: 13 July 2017 Accepted: 23 October 2017 22. Ferdous TA, Kabir SML, Amin MM, Hossain KMM. Identification and anti‑ microbial susceptibility of salmonella species isolated from washing and rinsed water of broilers in pluck shops. Int J Anim Vet Adv. 2013;5(1):1–8. Rahman et al. BMC Res Notes (2017) 10:531 Page 6 of 6 23. McDevitt S. Methyl red and Voges–Proskauer test protocols 2009 ASM microbe library. 2013. http://www.microbelibrary.org/component/ resource/laboratory-test/3204-methyl-red-andvoges-proskauer-test- protocols.htm. Accessed 5 July 2017. 24. Cappuccino JG, Sherman N. Microbiology a laboratory manual. Benjamin Cummings: New York; 2005. p. 125–79. 25. Acharya T. Tests for bacterial motility: procedure and results 2015. http:// microbeonline.com/tests-bacterial-motilityprocedure-results/. Accessed 5 July 2017. 26. Rahman SS, Hossain MM, Choudhury N. Effect of various parameters on the growth and ethanol production by yeasts isolated from natural sources. Bangladesh J Microbiol. 2013;30(1–2):49–54. https://doi. org/10.3329/bjm.v30i1-2.28453. 27. Starch hydrolysis test. http://www.vumicro.com/vumie/help/VUMICRO/ Starch_Hydrolysis_Test.htm. Accessed 5 July 2017. 28. Analysis of bacteria identification software. http://www.tgw1916.net/ bacteria_logare_desktop.html. Accessed 1 July 2017. 23. McDevitt S. Methyl red and Voges–Proskauer test protocols 2009 ASM microbe library. 2013. http://www.microbelibrary.org/component/ resource/laboratory-test/3204-methyl-red-andvoges-proskauer-test- protocols.htm. Accessed 5 July 2017. 29. Arias D, Cisternas LA, Rivas M. Biomineralization of calcium and mag‑ nesium crystals from seawater by halotolerant bacteria isolated from Atacama Salar (Chile). Desalination. 2017;405:1–9. 24. Cappuccino JG, Sherman N. Microbiology a laboratory manual. Benjamin Cummings: New York; 2005. p. 125–79. 30. Khansha J, Ranjbaran M, Amoozegar MA. Isolation and identification of halophilic and halotolerant bacteria from Badab-e Surt Travertine Spring, Kiasar, Iran, and investigation of calcite biomineralization induction. Geomicrobiol J. 2017:1. 25. Acharya T. Tests for bacterial motility: procedure and results 2015. http:// microbeonline.com/tests-bacterial-motilityprocedure-results/. Accessed 5 July 2017. 26. Rahman SS, Hossain MM, Choudhury N. Effect of various parameters on the growth and ethanol production by yeasts isolated from natural sources. Bangladesh J Microbiol. 2013;30(1–2):49–54. https://doi. org/10.3329/bjm.v30i1-2.28453. 31. Laufer K, Niemeyer A, Nikeleit V, Halama M, Byrne JM, Kappler A. Physi‑ ological characterization of a halotolerant anoxygenic phototrophic Fe(II)-oxidizing green-sulfur bacterium isolated from a marine sediment. FEMS Microbiol Ecol. 2017;93(5):fix054. 31. Laufer K, Niemeyer A, Nikeleit V, Halama M, Byrne JM, Kappler A. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
W4226119058.txt
https://indieskriflig.org.za/index.php/skriflig/article/download/2827/7360
fr
Table of Contents Vol 55 (2021)
In die skriflig/In die Skriflig
2,021
cc-by
2,752
Page i of iv Table of Contents Table of Contents In die Skriflig / In Luce Verbi ISSN: 1018-6441 (print) | ISSN: 2305-0853 (online) Vol 55, No 1 (2021) Book Review 1 A new commentary in the vernacular: Zephaniah: A review with a translation in isiZulu by Riens de Haan and Thulani Hlela Bob Wielenga Original Research 51 Science in service of theology: Gender and sexual orientation Pieter H. Labuschagne In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2665 | 22 February 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2643 | 22 April 2021 Book Review 3 Original Research With Calvin, beyond Calvin Robert Vosloo A two-tier protestant evaluative framework for cults applied to KwaSizabantu Mission Elfrieda Fleischmann, Ignatius W. Ferreira, Francois Muller In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2745 | 29 April 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2686 | 29 March 2021 Book Review 5 Original Research Leadership according to God’s model: Is it possible to use the Bible to define God’s leadership? Marius Nel Shifting negative migrant categories to encourage embrace and inclusivity: Perspective from Matthew 22:34–40 Christopher Magezi In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2757 | 26 May 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2664 | 30 March 2021 Book Review 7 Original Research 61 70 81 Nudged by Nathan Following a Lessing line in interfaith theology? Petrus P. Kruger Partnering for development: German-South African perspectives on religion and state cooperation Alfred R. Brunsdon In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2685 | 22 April 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2768 | 28 May 2021 Book Review 8 Original Research Listen to these voices! Elma M. Cornelius Marriage as a choice or duty: Considering Nigerian Christians’ attitude to singlehood from the biblical perspective Solomon O. Ademiluka In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2793 | 10 November 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2674 | 22 April 2021 Book Review 10 Original Research The phenomenon of multilingualism between ancient perspectives and contemporary developments Isabella Bonati The Christian scholar today and Bonhoeffer’s legacy of the transformative gospel Aleksandar S. Santrac In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2799 | 10 November 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2678 | 26 April 2021 Original Research 12 Original Research Church decline: A comparative investigation assessing more than numbers Ignatius W. Ferreira, Wilbert Chipenyu In the same boat? Jonah and Jesus as wave-beaten heralds Alistair I. Wilson In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2645 | 18 January 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2679 | 28 April 2021 Original Research 22 Original Research White fragility, white supremacy and white normativity make theological dialogue on race difficult Kelebogile T. Resane Introducing a re-reading of Lamentations through the lens of trauma studies: The challenge of the COVID-19 pandemic Johan L. Serfontein In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2661 | 25 January 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2688 | 28 April 2021 Original Research 32 Original Research ‘For the husband is the head of the wife’: A contextual re-reading of Ephesians 5:22–33 among Nigerian Yoruba Christians Solomon O. Ademiluka Die Algemene Sinode van die Nederduitse Gereformeerde Kerk van 1990 en die Gelofte van 1838 Piet J. Strauss In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2613 | 04 February 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2717 | 18 May 2021 Original Research 42 The role of spirituality in facilitating personal development according to the Pauline corpus Frederick J. de Beer, Jan A. du Rand Original Research Rethinking violence through the narrative of Genesis 4:1–16 Blessing O. Boloje In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2715 | 19 May 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2677 | 17 February 2021 http://www.indieskriflig.org.za Open Access 90 99 105 113 121 130 Page ii of iv Original Research 138 Table of Contents Original Research Ἐπιφάνεια as wederkomswoord in die Nuwe Testament H.P. Malan van Rhyn Critical evaluation of Theonomist eschatology Morne Diedericks In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2705 | 20 May 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2753 | 01 September 2021 Original Research 148 Original Research 236 243 Living as a diakonos of Christ and pastoral care to the narcissistically entitled person Gert Breed Guidelines towards plausible interpretation of gospel parables Aniedi M. Akpan, Francois P. Viljoen In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2701 | 27 May 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2742 | 13 September 2021 Original Research 157 ‘I Am the LORD your Healer’ Exodus 15:26 (‫)אנייהוהרפאך‬: Healing in the Old Testament and the African (Yoruba) context David T. Adamo Original Research 253 Theology and culture in dialogue towards harmonious multireligious and multi-cultural South Africa Kelebogile T. Resane In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2689 | 27 May 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2764 | 14 September 2021 Original Research 165 Hannah’s prayer for a male child: Interpreting 1 Samuel 1:11 in the Nigerian context Solomon O. Ademiluka Original Research 262 Exploring possibilities of the United Nations High Commissioner for Refugees’ integration with churches in refugee response Christopher Magezi In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2719 | 15 June 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2750 | 29 September 2021 Original Research 173 Practical theological perspectives on preaching to listeners experiencing angst or nothingness within the present reality of a post-pandemic world Ferdi P. Kruger Original Research 274 The insignificant impact of the historical Jesus Andre van Oudtshoorn In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2756 | 30 September 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2736 | 17 June 2021 Original Research 182 Homiletical perspectives on preaching the truth to post-pandemic postmodernist listeners with reference to the emotional appeal of the text Ferdi P. Kruger, Ben J. de Klerk Original Research 192 Jy moet jou naaste asook jou vyand liefhê: Liefde in Lukas 6:27–38 en 10:25–37 Jan G. van der Watt Original Research 200 The lame man at the pool of Bethesda: Christological and doxological significance of characterisation in John 5 Nathan Hahn, Ernest van Eck In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2774 | 27 October 2021 Original Research Original Research Key questions on the outmoded Bernese policy on religion Matthias G. Inniger, Jacobus M. Vorster, Riaan Rheeder 209 Women in the Bible: What can they teach us about gender equality? Christin E. Bøsterud In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2754 | 24 August 2021 218 311 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2763 | 29 November 2021 Original Research Calvin, Bucer and missionary opportunities in times of crises Stéphan van der Watt 302 Ἀποκάλυψισ en ἀποκαλύπτω as wederkomswoorde in die Nuwe Testament H.P. Malan van Rhyn In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2713 | 23 July 2021 Original Research 292 Die tempel-metafoor in Efesiërs 2:11–22 as deel van identiteitsvorming van die gelowige in-groep Aletta Vrey In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2760 | 27 October 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2744 | 21 July 2021 Original Research 284 Contextualisation within context: A pedagogical spectrum of six methodologies Brian A. DeVries In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2751 | 26 October 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2737 | 23 June 2021 Original Research Original Research 322 Calvin’s exposition of the sixth commandment as a trajectory in his catechetical works Rudolph M. Britz In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2773 | 30 November 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2761 | 27 August 2021 Original Research 227 The Deuteronomic roots of postexilic prophetic eschatology in Malachi Bob Wielenga Original Research Die koninkryk van God in die Ou Testament: ’n Kort oorsig Daniel F. O’Kennedy In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2787 | 15 December 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2759 | 01 September 2021 http://www.indieskriflig.org.za Open Access 331 Page iii of iv Correction 341 Erratum: Auto-memorialisation: Augustus’ Res Gestae as slanted narrative Johanna Maria Claassen Table of Contents Correction 342 Erratum: Background perspectives on infinity and God Danie F.M. Strauss In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2789 | 16 November 2021 In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2788 | 16 November 2021 Vol 55, No 2 (2021) Special Collection: Gert Breed Festschrift Editorial 343 Original Research Gert Breed Festschrift Foreword Albert J. Coetsee, Francois P. Viljoen A call for peacemaking: A perspective from the Sermon on the Mount Francois P. Viljoen In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2806 | 30 November 2021 In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2708 | 17 May 2021 Original Research 346 Original Research Contextual pastoral counselling: Paradigm shifts in practical theological development since the middle 20th century Amanda L. du Plessis Breed se verstaan van ’n bybelse pastorale model: ’n Verkennende, beskrywende perspektief Rineé Pretorius In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2696 | 18 March 2021 In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2707 | 24 June 2021 Original Research 355 Die behoefte aan apologetiese toerusting reeds by voorskoolse kategese Henk G. Stoker, G. Jonker Venter In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2722 | 29 October 2021 Original Research 363 Anagnorisis (processing forgiveness): The mystical praxis-space of diaconal reaching out to the Other/others (the hopeful case of Joseph and his brothers) Daniël J. Louw Original Research 410 418 427 Reconstructing communities and individuals after conflict and violence: An avant-garde quest for a forgiveness process that includes koinonia and diakonia Rudy A. Denton In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2724 | 20 July 2021 Original Research 436 Bekentenisse van ’n YouTube-prediker: Voorlopige outoetnografiese refleksies oor die opname en lei van ’n digitale erediens Jan-Albert van den Berg In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2651 | 31 March 2021 In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2740 | 23 August 2021 Original Research 373 Breed se bybelse pastorale model Skriftuurlik begrond in 2 Petrus 1:3–11: ’n Eksegetiese toeligting Douw G. Breed 443 Liturgy as an anti-racist praxis for Reformed Churches in South Africa Eugene Baron, Rantoa Letšosa In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2673 | 31 March 2021 Original Research Original Research In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2709 | 27 August 2021 382 A more comprehensive comprehension and appropriate application: An answer to dwindling faith commitment from the book of Hebrews Albert J. Coetsee Original Research 453 Peace is not the absence of war but the presence of a relationship founded by God – ‫שׁלֹום‬ ָ (shalom) in Isaiah and Micah Chris van der Walt In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2728 | 02 September 2021 In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2704 | 26 April 2021 Original Research 392 Fight, flight or faith: A pastoral model for spiritual coping Steve le Roux, George Lotter In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2700 | 29 April 2021 Original Research 461 Verspreiding van persoonlikheidstipes by predikante in die gereformeerde Kerke van Suid-Afrika – om risikogroepe vir diensverlating te identifiseer Gerhardus J. Niemann In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2727 | 03 November 2021 Original Research 401 Mentoring fathers who grapple with fatherhood issues in a faith-based context: A pastoral-theological review Fazel E. Freeks In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2698 | 30 April 2021 Correction Erratum: Breed se bybelse pastorale model Skriftuurlik begrond in 2 Petrus 1:3–11: ’n Eksegetiese toeligting Douw G. Breed In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2795 | 23 November 2021 http://www.indieskriflig.org.za Open Access 472 Page iv of iv Table of Contents Vol 55, No 3 (2021) Special Collection: Bible 200 Editorial 473 Foreword Bible 200 Francois P. Viljoen, Albert J. Coetsee Original Research 538 Eksegese van 2 Petrus 3:1–2 en die hermeneuse daarvan met spesifieke verwysing na standpunte van die sogenaamde Nuwe Ateïste Douw G. Breed In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2808 | 15 December 2021 In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2720 | 17 June 2021 Original Research 476 The impact of a Biblical Fatherhood Programme for faith communities in the Christiana district: A reflective and community engagement strategy Fazel E. Freeks In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2680 | 30 March 2021 Original Research 485 By everyone and for everyone: The principles underlying ‘justice’ in Deuteronomy 16:18–20 Albert J. Coetsee Original Research 548 To what extent did the Bible translations into indigenous languages of Southern Africa produced since 1966 reflect the purpose of providing meaning-based translations? Jacobus A. van Rooy In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2747 | 24 June 2021 Original Research 555 Die 2020-direkte vertaling in Afrikaans: Waarom en hoe? Gert J.C. Jordaan In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2654 | 31 March 2021 In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2758 | 25 June 2021 Original Research 496 The (mis)interpretation of the Bible in South Africa: Towards a better hermeneutic Bradley M. Trout Original Research 561 ‘Ek is Kain’: ’n Hermeneutiek van weerloosheid as ’n antwoord op die dekoloniale diskoers Gerrie F. Snyman In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2748 | 05 August 2021 In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2738 | 13 July 2021 Original Research 504 Oral-based Bible translation: A contextualised model for the nomadic Himba people of southern Africa Karen J. Floor Original Research 571 Translation types and niche translations: Comparing five Afrikaans translations Sebastian J. Floor In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2752 | 23 September 2021 In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2743 | 24 November 2021 Original Research 510 Die betroubaarheid van die apostels en hulle getuienis volgens 2 Petrus 1:3-4 en die betekenis daarvan vir die geskrifte van die Nuwe Testament: ‘n Eksegetiese studie Douw G. Breed Original Research 582 Die invloed van die lojaliteite en onderliggende vertaalfilosofie van die vertaler op die vertaalkeuses, met verwysing na die Afr2020 Bybelvertaling Roelie van der Spuy In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2703 | 17 May 2021 In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2755 | 15 December 2021 Original Research 519 Skrifgebruik en Skrifgesag in die postmodernistiese konteks Johan Janse van Rensburg In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2687 | 18 May 2021 Original Research 529 Translating psalms for Africa today: Involving the community and transmitting through performance June F. Dickie In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2726 | 26 May 2021 http://www.indieskriflig.org.za Correction 594 Erratum: The impact of a Biblical Fatherhood Programme for faith communities in the Christiana district: A reflective and community engagement strategy Fazel E. Freeks In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2794 | 23 November 2021 Reviewer Acknowledgement In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2826 | 22 December 2021 Open Access 595
https://openalex.org/W4281707586
https://link.springer.com/content/pdf/10.1007/s10853-022-07325-2.pdf
English
null
Corrosion performance of a steel surface modified by a robust graphene-based superhydrophobic film with hierarchical roughness
Journal of materials science
2,022
cc-by
8,607
Corrosion performance of a steel surface modified by a robust graphene-based superhydrophobic film with hierarchical roughness M. E. Mohamed1,* and B. A. Abd-El-Nabey1 M. E. Mohamed1,* and B. A. Abd-El-Nabey1 1Faculty of Science, Chemistry Department, Alexandria University, P. O. Box 426, Alexandria 21321, Egypt Received: 31 January 2022 Accepted: 10 May 2022 Published online: 4 June 2022  The Author(s) 2022 Received: 31 January 2022 Accepted: 10 May 2022 Published online: 4 June 2022  The Author(s) 2022 METALS & CORROSION J Mater Sci (2022) 57:11376–11391 Metals & corrosion METALS & CORROSION J Mater Sci (2022) 57:11376–11391 Metals & corrosion J Mater Sci (2022) 57:11376–11391 Handling Editor: Maude Jimenez. ABSTRACT Potentiostatic deposition of cobalt film and cobalt-graphene, Co-G, composite, followed by modification with low surface energy stearic acid (SA), was used to fabricate superhydrophobic films on a steel substrate successfully. A scanning electron microscope was used to analyze the surface morphology of the pre- pared superhydrophobic cobalt film modified by stearic acid, Co-SA, and the cobalt-graphene film modified by stearic acid, Co-G-SA. The findings show that both the fabricated films have micro-nanostructures. The Co-G-SA film shows a higher roughness due to the network structures of graphene and so exhibits higher superhydrophobicity. The Fourier transform infrared spectrophotometer, FTIR, results confirm the formation of Co-SA and Co-G-SA films on the steel surface. The wettability of the prepared films shows that they exhibit super- hydrophobicity, where the Co-SA and Co-G-SA films have contact angles of 155 and 158, respectively. The Potentiodynamic polarization results show that the value of the corrosion current density for steel coated with Co-SA (0.7094 lA) is lower than that of bare steel (0.1457 mA), while the coated steel with Co-G-SA film has the lowest value (0.1732 lA). The electrochemical impedance spectroscopy, EIS, results show that the charge transfer resistance for steel coated with Co-SA is 38 times that of bare steel, while steel coated with Co- SA is 57 times that of bare steel. Potentiodynamic polarization and EIS results show that the prepared Co-G-SA film superhydrophobic films exhibit higher corrosion resistance. Co-G-SA film has higher mechanical stability (maintains superhydrophobicity until 900 abrasion cycles), chemical stability (has super- hydrophobicity in the pH range 1–13), and long-term stability (retains super- hydrophobicity after 30 days in a 0.5 M NaCl solution) in 0.5 M NaCl solution. Handling Editor: Maude Jimenez. https://doi.org/10.1007/s10853-022-07325-2 https://doi.org/10.1007/s10853-022-07325-2 Address correspondence to E-mail: elshahatchemist93@gmail.com Introduction films typically show low substrate bonding strength and low hydrophobic efficiency, which decrease their applications significantly. Fortunately, doping with metals or non-metals also enhanced substrate adhe- sion and uniformity; moreover, some novel charac- teristics can be found based on retaining the original excellent efficiency [29]. Because of their significance in fundamental science and commercial applications, superhydrophobic surfaces have sparked great interest [1–5]. Superhy- drophobic surfaces have applications in diverse fields, such as anti-icing [6], oil–water separation [7], corrosion resistance [8], self-cleaning [9], drag reduction [10], and antifouling technologies [11]. Superhydrophobic surfaces have been fabricated using various techniques, such as electrospinning [12], etching [6], sol–gel process [13], electrodeposi- tion [14], laser fabrication [15], and electrochemical anodization [16]. However, most of these approaches require extreme conditions that restrict their practical aspects, such as complicated chemical treatments, high-cost materials, and multi-step manufacturing. Due to its low cost, flexibility, ambient temperature operation, and ability to monitor electrodeposition parameters, electrodeposition is an excellent tech- nique for creating artificial superhydrophobic sur- faces. Thus far, the commercialization of superhydrophobic surfaces has been significantly reduced due to their low surface chemical and mechanical stabilities and the fragility of their microscopic nanostructures characteristics [17]. The mechanical strength and durability of superhy- drophobic coatings have been a major focus of recent research [17, 18]. It is essential to improve the mechanical abrasion resistance and chemical stability of superhydrophobic surfaces in order to use them in industrial applications. y Steel materials are used in a wide range of indus- tries because of their high mechanical strength. However, steel structures have poor corrosion resis- tance due to their thermodynamic instability when exposed to extreme temperature, humidity, and pH [30]. Many techniques have been used to protect the steel surfaces; an important of them is the fabrication of superhydrophobic films, which greatly improve the corrosion resistance of steel [31]. Superhy- drophobic coatings were fabricated on different substrates using different techniques [32–37]. Philip et al. studied the fabrication of robust superhy- drophobic coating on ferritic steel with the self- cleaning ability and superior corrosion resistance using a template-free one-step electrodeposition method [32]. Siddaiah et al. studied the steel coatings by nickel (Ni) as well as Ni–graphene (Ni–Gr) [36]. On a Si substrate, Yan et al. created carbon-based films with excellent self-cleaning and corrosion resistance [37]. Dong et al. J Mater Sci (2022) 57:11376–11391 11377 Chemical stability A water droplet with various pH values (pH = 1–13) was placed on the prepared superhydrophobic sur- faces, and the CAs and SAs at each pH were mea- sured. Sodium hydroxide and sulphuric acid have been used to control the pH value of the water droplet. After electrodeposition, the Co and Co-G films were washed with distilled water and dried for 24.0 h at room temperature, immersed in 0.01 M ethanolic solution of stearic acid for 30 min, and then removed from the solution and left to dry at room conditions. After that, the as-prepared Co film modified by stearic acid, Co-SA, and the Co-G film modified by Characterization Sodium hydroxide, anhydrous ethanol, sodium chloride, sulfuric acid, cobalt chloride, boric acid, sodium carbonate, sodium dodecyl sulfate and stea- ric acid were purchased from Sigma-Aldrich. The surface morphology of prepared films was studied using a scanning electron microscope, SEM (model JSM-IT 200). A Fourier transform infrared spectrophotometer, FTIR (FTIR LX 18–5255 Perkin Elmer), was used to investigate the chemical com- position of the prepared films. The spectra were recorded in the wave number range of 4000–500 cm-1. X-ray diffraction (XRD) was done with monochromatic Cu K radiation (k = 0.154056 nm) by an X-ray diffractometer (Bruker D2 phaser). An optical contact angle meter (OCALS plus) was used to calculate the water contact angle, CA, and sliding angle, SA, with 5 ll water droplets. The recorded CAs and SAs are the averages of three measurements taken at various locations on the sample’s surface. A coating thickness gauge (FN Type CT-100) measured the thickness of the as-pre- pared superhydrophobic films. The reported film thickness is the average of three measurements done at different positions of the prepared sample. Sample preparation A steel plate with dimensions of 2.0 9 2.0 9 0.1 cm and chemical composition of (wt%): S, 0.04; C, 0.21; Mn, 2.5; Si, 0.35; P, 0.04 and Fe, 96.86 was used as a working electrode. The substrate was rubbed with emery paper of various grades before electrodeposi- tion, beginning with coarse (150 grade) and pro- gressing in steps to the finest (1200 grade). Then it was degreased for 30.0 min in an aqueous solution containing 20 g L-1 Na2CO3, 15 g L-1 NaOH and 8 g L-1 sodium dodecyl sulfate, followed by pickling and oxide removal in 2.0 M H2SO4 for 1.0 min, and finally rinsing with distilled water and ethanol. The elec- trodeposition was done in an aqueous solution con- taining CoCl2 (200 g L-1) and H3BO3 (30 g L-1) using a potential of 3.0 V and deposition time equals 20.0 min; then, the potential was increased to 7.0 V for a time of 1.0 min. The two-step electrodeposition was used to fabricate various superhydrophobic films [39–41]. In the first deposition step (at low potential), the crystal growth rate of cobalt is faster than the nucleation rate, so a coarse deposit of Co is formed. In the second electrodeposition step (at high potential), the nucleation rate of cobalt is faster than the crystal growth rate, so a nano deposit of Co is formed. For the manufacture of Co film, a platinum rod was used as the anode, and for the manufacture of Co film doped with graphene, Co-G film, a gra- phite rod was used as the anode, where the graphite is electrochemically exfoliated, giving graphene, which incorporated into the deposited layer of Co [42, 43]. Experimental stearic acid, Co-G-SA, were exposed to different evaluations and characterization techniques. Introduction [4] studied the fabrication of Ni–B4C superhydrophobic composite coatings at Q235 steel by electrodeposition and investigated its corrosion performance. Zhang et al. [38] investigated the preparation and performance of a biomimetic flower-like superhydrophobic coating on X80 pipe- line steel using a static self-assembly method. Abd- El-Nabey et al. [14] studied the construction of robust superhydrophobic films on steel surfaces and studied their corrosion performance, mechanical and chemi- cal stability. Cobalt (Co) is commonly used in aerospace, ship- building wear-resistant, automotive, corrosion resis- tance, high-strength alloys, catalysis, solar energy absorption and magnetic recording [19, 20]. The material’s internal characteristics and morphology decide these different characteristics [21, 22]. As a result, controlling the development of unique cobalt nanostructures has become a critical issue in the materials fabrication industry. Because of its strength, single atomic layer thickness, chemical inertness, and impermeability to most gases, graphene is thought to be a good material for coatings [23–26]. Graphene can be processed as nanoplatelets, nanosheets, and functionalized graphene using various methodolo- gies, such as chemical or electrochemical exfoliation, chemical vapor deposition, and single-crystal SiC crystal cleavage and annealing [27, 28]. Carbon-based This study aims to fabricate Co film and Co film doped with in situ prepared graphene by electro- chemical exfoliation method on the steel surface. Then, the as-prepared films were modified with stearic acid to fabricate superhydrophobic surfaces. To the best of our knowledge, this is the first study on constructing a superhydrophobic Co film doped with in situ produced graphene. The effect of graphene doping on the wettability, mechanical and chemical stability, long-term stability in 0.5 M NaCl, and cor- rosion resistance properties of a superhydrophobic cobalt film modified by stearic acid were investigated. 11378 J Mater Sci (2022) 57:11376–11391 Mechanical abrasion The abrasion test has been used to analyze the mechanical stability of the as-prepared films. Sand- paper (1800 mesh and length of 30 mm) has been used as an abrasion surface. The prepared superhy- drophobic film is oriented to face the sand surface, and a pressure of 5.0 kPa was applied to the super- hydrophobic film. Corrosion tests All electrochemical experiments were carried out in a three-electrode cell containing a 0.5 M NaCl aqueous solution at room temperature using a frequency SEM and wettability results SEM and wettability results One of the most outstanding parameters for investi- gating superhydrophobic properties is the surface morphology, so the SEM technique has been used to study the topography of the prepared superhy- drophobic films on the steel substrate. Figure 1a shows the micrograph of steel coated by Co; it is clear that the electrodeposited cobalt has micro-nanos- tructures of papillae structure. Figure 1b shows the micrograph of the magnified papillae structure, which has a hierarchical roughness. Figure 1c shows the micrograph of steel coated by Co-SA; it shows stearic acid’s white structures covering the elec- trodeposited micro-nano-papillae structure. Results and discussion The thickness of the Co-SA and Co-G-SA films are 19 lm and 25 lm, respectively. These results demonstrate that doping of Co film with graphene increases the thickness of the prepared film and improves the roughness and so shows higher super- hydrophobicity after modification with the stearic acid as a low surface energy material. Based on the Cassie–Baxter state [46], much air can be easily stored in the micro-nanostructures of the Co-G-SA film. Furthermore, the wettability of the superhydrophobic coated steel by Co-G-SA is superior to several pre- viously recorded values [47, 48]. 11379 J Mater Sci (2022) 57:11376–11391 response analyzer potentiostat (PARSTAT, USA). The reference and counter electrodes were an Ag/AgCl electrode and a platinum rod. The bare steel and steel covered by superhydrophobic Co-SA and Co-G-SA films have been used as working electrodes. The operating procedure was given in previous work [44]. The working electrode was placed in a cell containing 0.5 M NaCl solution that was opened to the environment at room temperature and left for 30 min before electrochemical measurements were taken to establish the equilibrium potential. The fre- quency range of the electrochemical impedance spectroscopy (EIS) measurements was 0.01 B f B 1.0 9 105 with an applied potential signal amplitude of 10 mV around the equilibrium poten- tial. The polarization curves were measured starting from cathodic potential (-300 mV) to anodic potential (? 450 mV) around the equilibrium potential at a 0.5 mV/sec scan rate. respectively. The value of contact and sliding angles for Co-SA film are 155 and 3, respectively, while the contact and sliding angles for Co-G-SA are 158 and 2, respectively. The contact angle image of the water droplet on the prepared superhydrophobic surfaces is shown in Fig. 2. These results indicate that the grafting of the Co film at the steel surfaces leads to an enhancement of the hydrophilic character of the bare steel, and the contact angle is decreased. While, when the Co film is doped with graphene, Co-G film, the contact angle shows a greater decrease reflecting higher hydro- philic characteristics. When the prepared Co and Co- G films are modified with stearic acid, superhy- drophobic surfaces were obtained with greater superhydrophobic characteristics of Co-G-SA film due to the presence of graphene, which enhances the surface roughness. These results can be discussed on the basis that as the surface roughness increases, the hydrophilic surfaces become more hydrophilic, while the hydrophobic surfaces show higher hydrophobic- ity [45]. FTIR and XRD results Figure 3 and Table 1 show the FTIR spectra and band assignments of stearic acid powder and steel coated by Co, Co-SA, Co-G, and Co-G-SA. The spectrum for steel coated with cobalt shows a peak at 576 cm-1 characteristics of the Co–O stretching vibration. In addition, the weak absorptions peaks at 3342 and 1660 cm-1 correspond to the stretching and bending vibration modes of the –OH group; this will prove the absorption of water by the deposited nanostructure of cobalt at the steel surface[49, 50]. The spectrum for the powder of stearic acid shows the characteristic peaks of the stearic acid. The peaks at 2866 cm-1 and 2944 cm-1 are assigned to the stretching vibrations of Figure 1d shows the micrograph of steel coated by Co-G; it is clear that the graphene forms a network layer of micro-nano-papillae structure; the porosity of the network gives a higher roughness, hierarchical roughness, to the formed layer. Figure 1e depicts the micrograph of steel coated by Co-G-SA; it is clear that stearic acid, white structures, graft the prepared film. The values of the contact angles of bare steel and steel coated with Co and Co-G films are 58, 21 and 15, 11380 J Mater Sci (2022) 57:11376–11391 gure 1 SEM images of the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and Co-G-SA film. Figure 1 SEM images of the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and e C G SA fil the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and Figure 1 SEM images of the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and e Co-G-SA film. peak at 3363 cm-1 is attributed to the bond tension vibration of O–H; the peak at 1151 cm-1 corresponds to the hydroxyl groups of the graphene. The peak at 1651 cm-1 is characteristic of the double bond’s gra- phene ring. The peak at 1078 cm-1 is attributed to the C–O–C group [53]. –CH2 groups. The peak at 1719 cm-1 is due to the stretching vibration of the carbonyl group of stearic acid. FTIR and XRD results The peak at 1314 cm-1 is attributed to the bending vibration of -OH, while the peak at 1477 cm-1 is due to the bending vibration of C–H [51, 52]. The spectrum for steel coated by Co-G depicts the characteristic peaks of the graphene. The 11381 J Mater Sci (2022) 57:11376–11391 Figure 2 Contact angle graphs of steel coated with a Co-SA film, and b Co-G-SA film. Figure 2 Contact angle graphs of steel coated with a Co-SA film, and b Co-G-SA film. Co3O4, and its sharpness indicates that the deposited cobalt has good crystallinity [54, 55]. For Co-SA coating, there are six diffraction peaks at 2h values of 30.7, 36.4, 38.1, 44.5, 59.1 and 64.9, which cor- respond to faced cubic centered, fcc, of Co3O4 (JCPDS No. 00-042-1467)[56]. The (311) plane has the highest intensity of the three peaks, indicating the preferred crystal orientation, with higher periodicity than the other orientations [57]. The Co-G-SA film has the same six diffraction peaks as the Co-SA film, with one greater diffraction peak at 2h values of 24.2, corre- sponding to graphene [58]. The graphene peak is broad, showing that graphene has a small particle size. Co3O4, and its sharpness indicates that the deposited cobalt has good crystallinity [54, 55]. For Co-SA coating, there are six diffraction peaks at 2h values of 30.7, 36.4, 38.1, 44.5, 59.1 and 64.9, which cor- respond to faced cubic centered, fcc, of Co3O4 (JCPDS No. 00-042-1467)[56]. The (311) plane has the highest intensity of the three peaks, indicating the preferred crystal orientation, with higher periodicity than the other orientations [57]. The Co-G-SA film has the same six diffraction peaks as the Co-SA film, with one greater diffraction peak at 2h values of 24.2, corre- sponding to graphene [58]. The graphene peak is broad, showing that graphene has a small particle size. Figure 3 FTIR spectra of stearic acid powder and steel coated by Co, Co-G, Co-G-SA, and Co-SA. Mechanical abrasion resistance Figure 3 FTIR spectra of stearic acid powder and steel coated by Co, Co-G, Co-G-SA, and Co-SA. The superhydrophobic surfaces are susceptible to mechanical abrasion. Improvement of superhy- drophobic coatings’ abrasion resistance has become the primary concern for their industrial applications [59]. The steel coated by Co-SA spectrum shows two peaks at 2919 cm-1 and 2850 cm-1 attributed to asymmetry and symmetry vibration of –CH2– of the stearic acid. The stretching vibration of C=O is responsible for the peak at 1702 cm-1, while the bending vibration of C-H is responsible for the peak at 1468 cm-1. The bending vibration absorption peak of –OH is at 1330 cm-1. The stearic acid peaks at around 2922, 2852, and 1703 cm-1 in the spectrum for steel coated with Co-G-SA suggest that the deposited graphene is modified by stearic acid. Abrasion testing was used to assess the resistance of the prepared superhydrophobic films to mechan- ical abrasion. The variations in water contact and sliding angles of the prepared superhydrophobic films as a function of the number of abrasion cycles are shown in Fig. 5. Increased abrasion cycles result in a decrease in contact angle values and an increase in sliding angle values, as seen in the graph. The superhydrophobic Co-SA film exhibits superhydrophobicity until 500 abrasion cycles; however, the superhydrophobic Co- G-SA film maintains superhydrophobicity until 900 The composition and crystal orientation of steel coated with Co-SA and Co-G-SA films were deter- mined using the XRD technique. The XRD patterns of Co-SA and Co-G-SA films are depicted in Fig. 4. Mechanical abrasion resistance The diffraction peaks imply that the deposited film is J Mater Sci (2022) 57:11376–11391 Table 1 FTIR band assignments for stearic acid powder and steel coated by Co, Co-G, Co-SA, and Co-G –SA films Film type Band wave numbers, cm-1 Assignment Co film 3342 stretching vibration of-OH group 1660 bending vibration of-OH group 576 Co–O stretching vibration Stearic acid powder 2944 asymmetric stretching vibration of CH2 2866 symmetric stretching vibration of CH2 1719 stretching vibration of the carbonyl group of stearic acid 1477 bending vibration of C-H 1314 bending vibration of -OH Co-G film 3363 bond tension vibration of O–H 2925 asymmetric stretching vibration of CH2 2854 symmetric stretching vibration of CH2 1651 the double bond’s graphene ring 1460 Bending Vibrations of CH 1151 hydroxyl groups of the graphene 1078 C–O–C group of the graphene Co-SA film 2919 asymmetric stretching vibration of CH2 2850 symmetric stretching vibration of CH2 1702 stretching vibration of C = O 1468 bending vibration of C-H 1330 bending vibration absorption peak of -OH Co-G –SA 2922 asymmetric stretching vibration of CH2 2852 symmetric stretching vibration of CH2 1703 stretching vibration of C=O 1471 bending vibration of C–H 1336 bending vibration absorption peak of –OH Figure 4 XRD patterns of steel coated by a Co-SA film and b Co-G-SA film. Figure 6 shows the SEM micrographs of steel coated with Co-SA and Co-G-SA films after the abrasion test. The micro-nano-papillae structure was destroyed, and the density of stearic acid (white structures) on the surface was decreased, so the sur- face lost its superhydrophobic characteristics. The contact angle shape of the water droplet on the pre- pared superhydrophobic surfaces after the abrasion test is shown in Fig. 7. The abrasion resistance of the superhydrophobic coated steel by Co-G-SA is supe- rior to several previously recorded values [32–35, 47, 60, 61]. Chemical stability Figure 4 XRD patterns of steel coated by a Co-SA film and b Co-G-SA film. To demonstrate that the prepared superhydrophobic film can be used in the industrial sector, a chemical stability test must be conducted. Figure 8 shows the relationship between each contact and sliding angles and the pH of the water droplets. abrasion cycles, which may be due to the higher adhesion of the low surface energy stearic acid to the rough structure of the Co-G composite than that of the Co film alone. J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 The Co-SA film has superhydrophobicity only in the pH range 3–11, while the Co-G-SA film has superhydrophobicity in the pH range 1–13, where the CAs are always greater than 150, and the SAs are less than 10. The two essential factors necessary for the fabrication of superhydrophobic films are low surface energy and surface roughness. So, the aggressive acidic and basic liquids could reduce the density of hydrophobic groups on the surface and destroys the micro/nanostructures of the surface, and so the sur- face loses its superhydrophobic characteristics [9, 45, 62, 63]. The chemical stability of the superhy- drophobic coated steel by Co-G-SA is superior to several previously recorded values [47, 64]. Figure 5 CAs and SAs as a function of the number of abrasion cycles for coated steel by a Co-SA film, and b Co-G-SA film. 11383 Long term stability The manufacture of surfaces with long-term stable superhydrophobicity remains a major problem that restricts superhydrophobic surface industrial applications. By measuring the contact angle every 2 days for 30 days, the long-term stability of the prepared Co-SA and Co-G-SA superhydrophobic films on steel substrate in 0.5 M NaCl solution was investigated. Figure 9 shows that the Co-SA film exhibit superhydrophobicity and has a contact angle greater than 150; after immersion for 20 days, the contact angle becomes smaller than 150, and the surface loses the superhydrophobic property. The Figure demonstrates that the Co-G-SA film retains superhydrophobicity after 30 days in a 0.5 M NaCl solution, implying that doping the Co-SA film with graphene improves the long-term stability of the superhydrophobic film prepared. The prepared superhydrophobic films will lose their superhy- drophobic characteristics after a definite immersion time in a 0.5 M NaCl solution as the Cl- ions attack Figure 5 CAs and SAs as a function of the number of abrasion cycles for coated steel by a Co-SA film, and b Co-G-SA film. Figure 6 SEM images of the superhydrophobic films a Co-SA film and b Co-G-SA film after abrasion test. Figure 6 SEM images of the superhydrophobic films a Co-SA film and b Co-G-SA film after abrasion test. 11384 J Mater Sci (2022) 57:11376–11391 Figure 7 Contact angle images of steel coated with a Co-SA film and b Co-G-SA film after abrasion test. Figure 7 Contact angle images of steel coated with a Co-SA film and b Co-G-SA film after abrasion test. Figure 7 Contact angle images of steel coated with a Co-SA film and b Co-G-SA film after abrasion test. the films and could decrease the hydrophobic groups’ density on the surface and destroy the micro/nanostructures of the surface, and so the sur- face loses its superhydrophobicity. The enhanced mechanical, chemical, and long-term stability of Co- G-SA layer in a 0.5 M NaCl solution is due to the synergistic effect of superhydrophobicity and the high chemical and mechanical stability, imperme- ability, hydrophobicity, and chemical inertness of graphene [58, 65–69]. The long-term stability of the superhydrophobic coated steel by Co-G-SA is Figure 8 Variation of pH values of a water droplet with the CAs and SAs of the coated steel by a Co-SA film and b Co-G-SA film. Figure 9 CAs and SAs as a function of immersion time of coated steel by a Co-SA film, and b Co-G-SA film. Corrosion resistance behaviour It is clear that the icorr. value for steel coated with Co-SA (0.7094 lA) is lower than that of bare steel (0.1457 mA); this can be attributed to coated steel’s superhydrophobic behaviour. The trapped air around the microstructures can reduce the contact area between the coated steel and the solution, resulting in a significant reduction in the icorr [75]. The steel coated with Co-G-SA film has a greater reduction in both the contact area between the coated steel and the medium and the icorr. value (0.1732 lA) because the presence of graphene increases the superhydrophobicity of the prepared Co-G-SA film as well as the high mechanical and chemical stability, hydrophobicity, impermeability, and chemical inert- ness of graphene [58, 65–69]. So, the inhibition effi- ciency of steel coated by Co-G-SA is higher than that of Co-SA. The value of Ecorr for steel coated by Co-G- SA is nobler than Co-SA, which is extremely noble than bare steel. The corrosion resistance of bare steel and superhy- drophobic coated steel by Co-SA and Co-G-SA has been investigated using the potentiodynamic polar- ization technique. Figure 10 shows the potentiody- namic polarization curves of bare steel and superhydrophobic coated steel in a 0.5 M NaCl aqueous solution. It is obvious that the cathodic polarization plots show a limiting diffusion current, IL, due to the reduction of oxygen according to Eq. (1). O2 þ 2H2O þ 4e ! 4OH ð1Þ ð1Þ O2 þ 2H2O þ 4e ! 4OH ð1Þ Thus, the cathodic process is controlled by mass transport. The rapid formation of corrosion products, in the case of bare steel, or formation of passive layer, in the case of the prepared superhydrophobic coated steel, on the electrode surface hinders the develop- ment of an ideal anodic Tafel region [72, 73]. J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 superior to several previously recorded values [5, 18, 70, 71]. superior to several previously recorded values [5, 18, 70, 71]. %P ¼ io corr:  icorr:   nio corr:    100 ð2Þ %P ¼ io corr:  icorr:   nio corr:    100 ð2Þ io corr. and icorr. are the corrosion current density for bare steel and superhydrophobic coated steel. io corr. and icorr. are the corrosion current density for bare steel and superhydrophobic coated steel. Corrosion resistance behaviour Potentiodynamic polarization results Corrosion resistance behaviour Long term stability Figure 8 Variation of pH values of a water droplet with the CAs and SAs of the coated steel by a Co-SA film and b Co-G-SA film. Figure 9 CAs and SAs as a function of immersion time of coated steel by a Co-SA film, and b Co-G-SA film. the films and could decrease the hydrophobic groups’ density on the surface and destroy the micro/nanostructures of the surface, and so the sur- face loses its superhydrophobicity. The enhanced mechanical, chemical, and long-term stability of Co- G-SA layer in a 0.5 M NaCl solution is due to the synergistic effect of superhydrophobicity and the high chemical and mechanical stability, imperme- ability, hydrophobicity, and chemical inertness of graphene [58, 65–69]. The long-term stability of the superhydrophobic coated steel by Co-G-SA is synergistic effect of superhydrophobicity and the high chemical and mechanical stability, imperme- ability, hydrophobicity, and chemical inertness of graphene [58, 65–69]. The long-term stability of the superhydrophobic coated steel by Co-G-SA is 11385 Electrochemical impedance spectroscopy results Table 2 displays the result of the bare steel and superhydrophobic coated steel’s potentiodynamic polarization parameters, including corrosion current density, icorr., corrosion potential, Ecorr., and protec- tion efficiency, % P. Equation 2 is used to measure the protection efficiency [74] The Nyquist and Bode plots of bare steel and superhydrophobic coated steel in 0.5 M NaCl solu- tion are shown in Fig. 11. At high frequency, the Nyquist plots show a depressed capacitive semicir- cle, accompanied by a diffusion tail at low frequency. The interfacial charge transfer reaction is responsible for the depressed capacitive semicircle of the Nyquist plots at high frequencies [76]. The diffusion tail at low frequency is due to the mass transport process. These results indicate that steel coated by Co-SA, which shows high charge transfer resistance compared to bare steel, has larger charge transfer resistance due to the presence of a protective superhydrophobic layer. Steel coated by Co-G-SA shows the highest capacitive semicircle, so it has the highest protection efficiency. The superhydrophobic coated steel blocks the active corrosion sites and limits the diffusion of the corro- sive species, such as Cl- and H2O, into the surface of steel metal. Figure 10 Potentiodynamic polarization curves for bare steel and superhydrophobic coated steel in 0.5 M NaCl solution. According to Fig. 11b, the Bode plots for prepared superhydrophobic coated steel in 0.5 M NaCl solu- tion show higher impedance magnitudes at the low frequency than bare steel. This indicates the protec- tive action of the prepared superhydrophobic coats Figure 10 Potentiodynamic polarization curves for bare steel and superhydrophobic coated steel in 0.5 M NaCl solution. 11386 J Mater Sci (2022) 57:11376–11391 Deposit -Ecorr (mV) ba (mV/decade) -bc (mV/decade) icorr (mA cm-2) %P Bare steel 473.5 155.5 183.5 0.1457064 – Steel ? Co-SA film 259.1 62.9 138.9 0.0007094 99.5 Steel ? Co-G-SA film 222.5 61.4 132.7 0.0001732 99.6 Table 2 The potentiodynamic polarization parameters for the bare steel and the prepared superhydrophobic coated steel in 0.5 M NaCl solution polarization parameters for the bare steel and the prepared superhydrophobic coated steel in 0.5 M NaCl solution p cor Bare steel 473.5 Steel ? Co-SA film 259.1 Steel ? Co-G-SA film 222.5 Figure 11 Nyquist and Bode plots of bare steel and superhydrophobic coated steel in 0.5 M NaCl solution. Steel ? Co-G-SA film 222.5 on the steel substrate. The phase angle plot, Fig. Conclusion 1. superhydrophobic Co-SA and Co-G-SA films were fabricated on the steel substrate. 1. superhydrophobic Co-SA and Co-G-SA films were fabricated on the steel substrate. 2. The doping of the superhydrophobic Co film with graphene greatly enhances the superhy- drophobicity, and the contact angle increases from 155 to 158. On the contrary, the steel coated by superhy- drophobic films has a micro-nanostructure covered by adsorbed hydrophobic material. The roughness of the superhydrophobic coatings allows air to be trapped easily within the valleys between the peaks of the rough surface. Consequently, the aggressive ion species such as Cl- in the electrolyte or corrosive environments can rarely attack the underlying sur- face due to trapped air’s obstructive influence [32, 45]. In fact, the air trapped on the superhy- drophobic surface acts as a passivation layer between the substrate and the corrosive environment. The enhanced corrosion resistance for steel coated by Co- G-SA films is due to the synergistic effect of super- hydrophobicity and the high mechanical and chemi- cal stability, impermeability, hydrophobicity, and chemical inertness of graphene. The schematic 3. The doping of the superhydrophobic Co film with graphene also greatly improves steel’s chemical, mechanical, long-term stability and corrosion resistance behaviour in 0.5 M NaCl solution. J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 Table 3 The impedance parameters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution Deposit Rs (X cm2) CPEf 9 10–6 (sn X-1 cm2) n1 Rf (X cm2) CPEdl 9 10–6 (sn X-1 cm2) n2 Rct (X cm2) %P Bare steel 2.28 281 0.78 56 278 0.77 73.2 – Steel ? Co-SA 3.95 91 0.76 214 88 0.74 2795 97.38 Steel ? Co-G-SA 4.22 59 0.68 273 53 0.72 4165 98.24 Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. Table 3 The impedance parameters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution Deposit Rs (X cm2) CPEf 9 10–6 (sn X-1 cm2) n1 Rf (X cm2) CPEdl 9 10–6 (sn X-1 cm2) n2 Rct (X cm2) %P Bare steel 2.28 281 0.78 56 278 0.77 73.2 – Steel ? Co-SA 3.95 91 0.76 214 88 0.74 2795 97.38 Steel ? Co-G-SA 4.22 59 0.68 273 53 0.72 4165 98.24 Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. meters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution le 3 The impedance parameters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared Mechanism of anti-corrosion performance representation of the proposed mechanism for cor- rosion protection of the prepared superhydrophobic films is shown in Fig. 13. representation of the proposed mechanism for cor- rosion protection of the prepared superhydrophobic films is shown in Fig. 13. Bare steel freely interacts with surrounding water molecules; the water molecules can be adsorbed to the steel surface. Along with water molecules, chlo- ride ions can also get adsorbed to the steel surface and form [FeClOH]-, which will lead to severe cor- rosion of the uncoated steel. So, water and Cl- ions easily reach the metal surface and initiate corrosion [32]. Electrochemical impedance spectroscopy results 11c, shows two times constant at low and moderate fre- quencies. The time constant appearing in the low- frequency range was due to the protective superhy- drophobic film or the unprotective corrosion prod- ucts in the case of bare steel. The time constant appearing at the moderate frequency was attributed to the electrical double layer [77–79]. The impedance parameters were determined using the Zsimpwin software to fit the Nyquist plots to the equivalent circuit shown in Fig. 12. The equivalent circuit includes solution resistance, Rs, film resis- tance, Rf, film constant phase element, CPEf, charge transfer resistance, Rct, and double-layer constant phase element, CPEdl. Table 3 shows the EIS param- eters of bare steel and superhydrophobic coated steel. Equation (3) is used to calculate the protection effi- ciency [74] %P ¼ Rct  Ro ct   =Rct    100 ð3Þ ð3Þ Rct o and Rct are the charge transfer resistance for the bare steel and superhydrophobic coated steel. It is clear that each of Rct, and %P increase in the fol- lowing order, bare steel \ steel ? Co-SA \ steel ? Co-G-SA, and so increasing the corrosion resistance in the same order. The corrosion resistance of the superhydrophobic coated steel by Co-G-SA compos- ite is superior to several previously reported values [5, 71, 80]. Figure 12 The equivalent circuit model used to fit the experimental Nyquist plots for steel in 0.5 M NaCl solution. Figure 11 Nyquist and Bode plots of bare steel and superhydrophobic coated steel in 0.5 M NaCl solution. Figure 12 The equivalent circuit model used to fit the experimental Nyquist plots for steel in 0.5 M NaCl solution. 11387 Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research did not receive any specific 11388 J Mater Sci (2022) 57:11376–11391 grant from funding agencies in the public, commer- cial, or not-for-profit sectors. Appl Surf Sci 562:150192. https://doi.org/10.1016/j.apsusc. 2021.150192 Appl Surf Sci 562:150192. https://doi.org/10.1016/j.apsusc. 2021.150192 [6] Hou W, Shen Y, Tao J et al (2020) Anti-icing performance of the superhydrophobic surface with micro-cubic array struc- tures fabricated by plasma etching. Colloids Surfaces A Physicochem Eng Asp 586:124180. https://doi.org/10.1016/ j.colsurfa.2019.124180 Declarations Conflict of interest The authors declare that there is no conflict of interest. [7] Chu Z, Feng Y, Seeger S (2015) Oil/water separation with selective superantiwetting/superwetting surface materials. Angew Chemie-Int Ed 54:2328–2338. https://doi.org/10.10 02/anie.201405785 Open Access This article is licensed under a Crea- tive Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Crea- tive Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/. [8] Mohamed ME, Abd-El-Nabey BA (2022) Superhydrophobic cobalt-graphene composite for the corrosion protection of copper bipolar plates in proton exchange membrane fuel cells. J Electrochem Energy Convers Storage 19:031007. h ttps://doi.org/10.1115/1.4053782 [9] Xu S, Wang Q, Wang N, Zheng X (2019) Fabrication of superhydrophobic green surfaces with good self-cleaning, chemical stability and anti-corrosion properties. J Mater Sci 54:13006–13016. https://doi.org/10.1007/s10853-019-0378 9-x [10] Li Z, Marlena J, Pranantyo D et al (2019) A porous super- hydrophobic surface with active air plastron control for drag reduction and fluid impalement resistance. J Mater Chem A 7:16387–16396. https://doi.org/10.1039/c9ta02745a [11] Qin L, Hafezi M, Yang H et al (2019) Constructing a dual- function surface by microcasting and nanospraying for effi- cient drag reduction and potential antifouling capabilities. Micromachines 10:490. https://doi.org/10.3390/mi10070490 J Mater Sci (2022) 57:11376–11391 11389 J Mater Sci (2022) 57:11376–11391 blocks for carbon-based materials. Small 6:711–723. http s://doi.org/10.1002/smll.200901934 [17] Verho T, Bower C, Andrew P et al (2011) Mechanically durable superhydrophobic surfaces. Adv Mater 23:673–678. https://doi.org/10.1002/adma.201003129 [30] Musa AY, Kadhum AAH, Mohamad AB et al (2010) On the inhibition of mild steel corrosion by 4-amino-5-phenyl-4H- 1, 2, 4-trizole-3-thiol. Corros Sci 52:526–533. https://doi. org/10.1016/j.corsci.2009.10.009 [18] Liang T, Yuan H, Li C et al (2020) Corrosion inhibition effect of nano–SiO2 for galvanized steel superhydrophobic surface. Surf Coat Technol. https://doi.org/10.1016/j.surfcoa t.2020.126673 [31] Kumar CMP, Venkatesha TV, Chandrappa KG (2012) Effect of surfactants on co-deposition of B 4C nanoparticles in Zn matrix by electrodeposition and its corrosion behavior. Surf Coat Technol 206:2249–2257. https://doi.org/10.1016/j.surfc oat.2011.09.075 [19] Vazquez-Arenas J, Altamirano-Garcia L, Treeratanaphitak T et al (2012) Co-Ni alloy electrodeposition under different conditions of pH, current and composition. Electrochim Acta 65:234–243. https://doi.org/10.1016/j.electacta.2012.01.050 [32] Rasitha TP, Vanithakumari SC, George RP, Philip J (2019) Template-free one-step electrodeposition method for fabri- cation of robust superhydrophobic coating on ferritic steel with self-cleaning ability and superior corrosion resistance. Langmuir 35:12665–12679. https://doi.org/10.1021/acs.lang muir.9b02045 [20] Yang J, Bao C, Zhu K et al (2014) High catalytic activity and stability of nickel sulfide and cobalt sulfide hierarchical nanospheres on the counter electrodes for dye-sensitized solar cells. Chem Commun 50:4824–4826. https://doi.org/ 10.1039/c4cc00001c [21] An ZG, Zhang JJ, Pan SL (2010) Simple synthesis and characterization of highly ordered sisal-like cobalt super- structures. Mater Chem Phys 123:795–800. https://doi.org/ 10.1016/j.matchemphys.2010.05.062 [33] Rasitha TP, Vanithakumari SC, Nanda Gopala Krishna D et al (2022) Facile fabrication of robust superhydrophobic aluminum surfaces with enhanced corrosion protection and antifouling properties. Prog Org Coatings 162:106560. http s://doi.org/10.1016/j.porgcoat.2021.106560 [22] Cheng G (2014) Facile synthesis of leaf-like cobalt microstructures at low temperature. Micro Nano Lett 9:312–314. https://doi.org/10.1049/mnl.2014.0027 [34] Tp R, Philip J (2022) Optimal condition for fabricating mechanically durable superhydrophobic titanium surface by rapid breakdown anodization: self cleaning and bouncing characteristics. Appl Surf Sci 585:152628. https://doi.org/10. 1016/j.apsusc.2022.152628 [23] Montemor MF (2014) Functional and smart coatings for corrosion protection: a review of recent advances. Surf Coatings Technol 258:17–37. https://doi.org/10.1016/j.surfc oat.2014.06.031 [24] Chen-Yang YW, Yang HC, Li GJ, Li YK (2005) Thermal and anticorrosive properties of polyurethane/clay nanocom- posites. J Polym Res 11:275–283. https://doi.org/10.1007/ s10965-005-3982-8 [35] Manoj TP, Rasitha TP, Vanithakumari SC et al (2020) A simple, rapid and single step method for fabricating super- hydrophobic titanium surfaces with improved water bounc- ing and self cleaning properties. Appl Surf Sci 512:145636. References [1] Khadka A, Subeshan B, Asmatulu R (2021) Studies on de- icing and anti-icing of carbon fiber-reinforced composites for aircraft surfaces using commercial multifunctional perma- nent superhydrophobic coatings. J Mater Sci 56(4):3078–3094. https://doi.org/10.1007/s10853-02 [1] Khadka A, Subeshan B, Asmatulu R (2021) Studies on de- icing and anti-icing of carbon fiber-reinforced composites for aircraft surfaces using commercial multifunctional perma- nent superhydrophobic coatings. J Mater Sci 56(4):3078–3094. https://doi.org/10.1007/s10853-02 [12] Zhang G, Wang P, Zhang X et al (2019) Preparation of hierarchically structured PCL superhydrophobic membrane via alternate electrospinning/electrospraying techniques. J Polym Sci Part B Polym Phys 57:421–430. https://doi.org/ 10.1002/polb.24795 [2] Zhu G, Zhao Y, Liu L et al (2021) Facile fabrication and evaluation of self-healing Zn-Al layered double hydroxide superhydrophobic coating on aluminum alloy. J Mater Sci 56:14803–14820. https://doi.org/10.1007/s10853-021-0624 7-9 [2] Zhu G, Zhao Y, Liu L et al (2021) Facile fabrication and evaluation of self-healing Zn-Al layered double hydroxide superhydrophobic coating on aluminum alloy. J Mater Sci 56:14803–14820. https://doi.org/10.1007/s10853-021-0624 7-9 [13] Vidal K, Go´mez E, Goitandia AM et al (2019) The synthesis of a superhydrophobic and thermal. Coatings 9:1–33 [14] Abd-El-Nabey BA, Ashour M, Aly A, Mohamed M (2022) Fabrication of robust superhydrophobic nickel films on steel surface with high corrosion resistance mechanical and chemical stability. J Eng Mater Technol 144:021007. http s://doi.org/10.1115/1.4052768 [3] Mohamed ME, Mahgoub FM, Ragheb DM, Abdel-Gaber AM (2021) Novel and facile method for fabrication of robust superhydrophobic film on copper surface and its chemical, mechanical, and corrosion performance. Surf Eng. https://d oi.org/10.1080/02670844.2021.1951502 [3] Mohamed ME, Mahgoub FM, Ragheb DM, Abdel-Gaber AM (2021) Novel and facile method for fabrication of robust superhydrophobic film on copper surface and its chemical, mechanical, and corrosion performance. Surf Eng. https://d oi.org/10.1080/02670844.2021.1951502 [15] Yang Z, Tian Y, Zhao Y, Yang C (2019) Study on the fab- rication of superhydrophobic surface on Inconel Alloy via nanosecond laser ablation. Materials (Basel). https://doi.org/ 10.3390/ma12020278 [4] Dong S, Yang Y, Liang T et al (2021) Construction and corrosion resistance of Ni-B4C superhydrophobic composite coatings on Q235 steel. Surf Coat Technol. https://doi.org/ 10.1016/j.surfcoat.2021.127551 [16] Saji VS (2020) Superhydrophobic surfaces and coatings by electrochemical anodic oxidation and plasma electrolytic oxidation. Adv Colloid Interface Sci 283:102245. https://doi. org/10.1016/j.cis.2020.102245 [5] Lv XS, Qin Y, Liang H et al (2021) A facile method for constructing a superhydrophobic zinc coating on a steel surface with anti-corrosion and drag-reduction properties. J Mater Sci (2022) 57:11376–11391 https://doi.org/10.1016/j.apsusc.2020.145636 [25] Jena G, Anandkumar B, Vanithakumari SC et al (2020) Graphene oxide-chitosan-silver composite coating on Cu-Ni alloy with enhanced anticorrosive and antibacterial proper- ties suitable for marine applications. Prog Org Coatings 139:105444. https://doi.org/10.1016/j.porgcoat.2019.105444 [36] Siddaiah A, Kumar P, Henderson A et al (2019) Surface energy and tribology of electrodeposited Ni and Ni-graphene coatings on steel. Lubricants. https://doi.org/10.3390/lubrica nts7100087 [26] Jena G, George RP, Philip J (2021) Fabrication of a robust graphene oxide-nano SiO2-polydimethylsiloxane composite coating on carbon steel for marine applications. Prog Org Coat 161:106462. https://doi.org/10.1016/j.porgcoat.2021. 106462 [37] Yan Q, Zhou S, Ma L et al (2019) Approach to excellent superhydrophobicity and corrosion resistance of carbon- based films by graphene and cobalt synergism. Surf Interface Anal 51:152–163. https://doi.org/10.1002/sia.6546 [38] Zhang W, Fan S, Wang Y et al (2021) Preparation and per- formance of biomimetic superhydrophobic coating on X80 pipeline steel for inhibition of hydrate adhesion. Chem Eng J 419:129651. https://doi.org/10.1016/j.cej.2021.129651 [27] Mattevi C, Kim H, Chhowalla M (2011) A review of chemical vapour deposition of graphene on copper. J Mater Chem 21:3324–3334. https://doi.org/10.1039/c0jm02126a [39] Hang T, Hu A, Ling H et al (2010) Super-hydrophobic nickel films with micro-nano hierarchical structure prepared by electrodeposition. Appl Surf Sci 256:2400–2404. https://doi. org/10.1016/j.apsusc.2009.10.074 [28] Lee DS, Riedl C, Krauss B et al (2008) Raman spectra of epitaxial graphene on SiC and of epitaxial graphene trans- ferred to SiO2. Nano Lett 8:4320–4325. https://doi.org/10. 1021/nl802156w [29] Compton OC, Nguyen ST (2010) Graphene oxide, highly reduced graphene oxide, and graphene: versatile building [40] Esmailzadeh S, Khorsand S, Raeissi K, Ashrafizadeh F (2015) Microstructural evolution and corrosion resistance of 11390 J Mater Sci (2022) 57:11376–11391 organic solvents. J Nanopart Res. https://doi.org/10.1007/s1 1051-014-2561-5 organic solvents. J Nanopart Res. https://doi.org/10.1007/s1 1051-014-2561-5 superhydrophobic electrodeposited nickel films. Surf Coat Technol 283:337–346. https://doi.org/10.1016/j.surfcoat.201 5.11.005 [53] Valencia C, Valencia CH, Zuluaga F et al (2018) Synthesis and application of scaffolds of chitosan-graphene oxide by the freeze-drying method for tissue regeneration. Molecules 23:2651. https://doi.org/10.3390/molecules23102651 [41] Khorsand S, Raeissi K, Ashrafizadeh F (2014) Corrosion resistance and long-term durability of superhydrophobic nickel film prepared by electrodeposition process. Appl Surf Sci 305:498–505. https://doi.org/10.1016/j.apsusc.2014.03. 123 [54] Prabaharan DDM, Sadaiyandi K, Mahendran M, Sagadevan S (2017) Precipitation method and characterization of cobalt oxide nanoparticles. Appl Phys A Mater Sci Process. http s://doi.org/10.1007/s00339-017-0786-8 [42] Yu P, Lowe SE, Simon GP, Zhong YL (2015) Electro- chemical exfoliation of graphite and production of functional graphene. Curr Opin Colloid Interface Sci 20:329–338. J Mater Sci (2022) 57:11376–11391 h ttps://doi.org/10.1016/j.cocis.2015.10.007 [55] Jadhav CH, Pisal KB, Chavan AR et al (2020) Electro- chemical supercapacitive performance study of spray pyr- olyzed cobalt oxide film. Mater Today Proc 43:2742–2746. h ttps://doi.org/10.1016/j.matpr.2020.06.477 [43] Su CY, Lu AY, Xu Y et al (2011) High-quality thin graphene films from fast electrochemical exfoliation. ACS Nano 5:2332–2339. https://doi.org/10.1021/nn200025p [56] Ikhuoria EU, Omorogbe SO, Sone BT, Maaza M (2018) Bioinspired shape controlled antiferromagnetic Co3O4 with prism like-anchored octahedron morphology: a facile green synthesis using manihot esculenta crantz extract. Sci Technol Mater. https://doi.org/10.1016/j.stmat.2018.02.003 [44] Mohamed ME, Abd-El-Nabey BA (2020) Facile and eco- friendly method for fabrication of superhydrophobic surface on copper metal. ECS J Solid State Sci Technol. https://doi. org/10.1149/2162-8777/ab9dc7 [57] Zhou J, Wang H (2003) The physical meanings of 5 basic parameters for an X-ray diffraction peak and their applica- tion. Chin J Geochem 22:38–44. https://doi.org/10.1007/bf 02831544 [45] Barati Darband G, Aliofkhazraei M, Khorsand S et al (2020) Science and engineering of superhydrophobic surfaces: review of corrosion resistance, chemical and mechanical stability. Arab J Chem 13:1763–1802. https://doi.org/10.10 16/j.arabjc.2018.01.013 [58] Shang W, Li J, Rabiei Baboukani A et al (2020) Study on the relationship between graphene dispersion and corrosion resistance of graphene composite film. Appl Surf Sci 511:145518. https://doi.org/10.1016/j.apsusc.2020.145518 [46] Cassie ABD, Baxter S (1944) Wettability of porous surfaces. Trans Faraday Soc 40:546–551 [47] Ma L, Wang J, Zhang Z et al (2021) Preparation of a superhydrophobic TiN/PTFE composite film toward self- cleaning and corrosion protection applications. J Mater Sci 56:1413–1425. https://doi.org/10.1007/s10853-020-05364-1 [59] Nguyen-Tri P, Tran HN, Plamondon CO et al (2019) Recent progress in the preparation, properties and applications of superhydrophobic nano-based coatings and surfaces: a review. Prog Org Coat 132:235–256. https://doi.org/10.101 6/j.porgcoat.2019.03.042 [48] Forooshani HM, Aliofkhazraei M, Bagheri H (2019) Fabri- cation of hierarchical dual structured (HDS) nickel surfaces and their corrosion behavior. J Alloys Compd 784:556–573. https://doi.org/10.1016/j.jallcom.2019.01.079 [60] Wang H, Zhu Y, Hu Z et al (2016) A novel electrodeposition route for fabrication of the superhydrophobic surface with unique self-cleaning, mechanical abrasion and corrosion resistance properties. Chem Eng J 303:37–47. https://doi. org/10.1016/j.cej.2016.05.133 [49] Zhang F, Yuan C, Lu X et al (2012) Facile growth of mesoporous Co 3O 4 nanowire arrays on Ni foam for high performance electrochemical capacitors. J Power Sources 203:250–256. https://doi.org/10.1016/j.jpowsour.2011.12. 001 [61] Tan C, Li Q, Cai P et al (2015) Fabrication of color-con- trollable superhydrophobic copper compound coating with decoration performance. Appl Surf Sci 328:623–631. J Mater Sci (2022) 57:11376–11391 [64] Du C, He X, Tian F et al (2019) Preparation of superhy- drophobic steel surfaces with chemical stability and corro- sion. Coatings 9:398. https://doi.org/10.3390/coating s9060398 [73] McCafferty E (2005) Validation of corrosion rates measured by the Tafel extrapolation method. Corros Sci 47:3202–3215. https://doi.org/10.1016/j.corsci.2005.05.046 [74] Fetouh HA, Abd-El-Nabey B, Goher YM, Karam MS (2018) An Electrochemical investigation in the anticorrosive prop- erties of silver nanoparticles for the acidic corrosion of aluminium. J Electrochem 24:89–100 [65] Zhu H, Yue L, Zhuang C et al (2016) Fabrication and characterization of self-assembled graphene oxide/silane coatings for corrosion resistance. Surf Coat Technol 304:76–84. https://doi.org/10.1016/j.surfcoat.2016.07.002 [75] Ou J, Liu M, Li W et al (2012) Corrosion behavior of superhydrophobic surfaces of Ti alloys in NaCl solutions. Appl Surf Sci 258:4724–4728. https://doi.org/10.1016/j.aps usc.2012.01.066 [66] Zhong F, He Y, Wang P et al (2019) Self-assembled gra- phene oxide-graphene hybrids for enhancing the corrosion resistance of waterborne epoxy coating. Appl Surf Sci 488:801–812. https://doi.org/10.1016/j.apsusc.2019.05.321 [76] Ghiamati Yazdi E, Ghahfarokhi ZS, Bagherzadeh M (2017) Protection of carbon steel corrosion in 3.5% NaCl medium by aryldiazonium grafted graphene coatings. J Chem 41:12470–12480. https://doi.org/10.1039/c7nj01655g [67] Chu JH, Tong LB, Zhang JB et al (2019) Bio-inspired gra- phene-based coatings on Mg alloy surfaces and their inte- grations of anti-corrosive/wearable performances. Carbon N Y 141:154–168. https://doi.org/10.1016/j.carbon.2018.09. 047 [77] Nady H, El-Rabiei MM, Samy M (2017) Corrosion behavior and electrochemical properties of carbon steel, commercial pure titanium, copper and copper–aluminum–nickel alloy in 3.5% sodium chloride containing sulfide ions. Egypt J Pet 26:79–94. https://doi.org/10.1016/j.ejpe.2016.02.008 [68] Singh BP, Nayak S, Nanda KK et al (2013) The production of a corrosion resistant graphene reinforced composite coating on copper by electrophoretic deposition. Carbon N Y 61:47–56. https://doi.org/10.1016/j.carbon.2013.04.063 [78] Mahdavian M, Naderi R (2011) Corrosion inhibition of mild steel in sodium chloride solution by some zinc complexes. Corros Sci 53:1194–1200. https://doi.org/10.1016/j.corsci.2 010.12.013 [69] Ding S, Xiang T, Li C et al (2017) Fabrication of self- cleaning superhydrophobic nickel/graphene hybrid film with improved corrosion resistance on mild steel. Mater Des 117:280–288. https://doi.org/10.1016/j.matdes.2016.12.084 [79] Berrani A, Benassaoui H, Zouarhi M et al (2018) Analytical & bioanalytical electrochemistry. Anal Bioanal Electrochem 10:1299–1316 [70] Polyakov NA, Botryakova IG, Glukhov VG et al (2021) Formation and anticorrosion properties of superhydrophobic zinc coatings on steel. Chem Eng J 421:127775. https://doi. J Mater Sci (2022) 57:11376–11391 http s://doi.org/10.1016/j.apsusc.2014.12.025 [50] Allaedini G, Muhammad A (2013) Study of influential fac- tors in synthesis and characterization of cobalt oxide nanoparticles. J Nanostruct Chem. https://doi.org/10.1186/ 2193-8865-3-77 [62] Wang J, Wu Y, Cao Y et al (2020) Influence of surface roughness on contact angle hysteresis and spreading work. Colloid Polym Sci 298:1107–1112. https://doi.org/10.1007/ s00396-020-04680-x [51] Zeng YX, Zhong XW, Liu ZQ et al (2013) Preparation and enhancement of thermal conductivity of heat transfer oil- based MoS2 nanofluids. J Nanomater. https://doi.org/10.11 55/2013/270490 [63] Guo M, Kang Z, Li W, Zhang J (2014) A facile approach to fabricate a stable superhydrophobic film with switchable water adhesion on titanium surface. Surf Coat Technol 239:227–232. https://doi.org/10.1016/j.surfcoat.2013.11.047 [52] Luo S, Yang D, Feng J, Ng KM (2014) Synthesis and application of non-agglomerated ITO nanocrystals via pyrolysis of indium-tin stearate without using additional 11391 J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 org/10.1016/j.cej.2020.127775 [80] Qu JE, Yu C, Nie C et al (2021) A new environmentally friendly approach to prepare superhydrophobic colored stainless steel surface for decoration, anti-corrosion and self- cleaning. J Mater Sci 56:854–869. https://doi.org/10.1007/ s10853-020-05293-z [71] Varshney P, Mohapatra SS, Kumar A (2021) Durable and regenerable superhydrophobic coating on steel surface for corrosion protection. J Bio-Tribo-Corrosion 7:1–11. https://d oi.org/10.1007/s40735-021-00518-3 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. [72] Flitt HJ, Schweinsberg DP (2005) Evaluation of corrosion rate from polarisation curves not exhibiting a Tafel region. Corros Sci 47:3034–3052. https://doi.org/10.1016/j.corsci.2 005.06.014
https://openalex.org/W2804465752
https://online-journal.unja.ac.id/jseb/article/download/5102/8945
Indonesian
null
Hubungan Faktor-faktor Alih Fungsi Lahan Padi Sawah dan Perbedaan Tingkat Penerimaan Usahatani Petani Di Kecamatan Keliling Danau Kabupaten Kerinci
Jurnal ilmiah sosio ekonomika bisnis/Jurnal ilmiah sosio-ekonomika bisnis
2,018
cc-by
8,108
1) Alumni Jurusan Agribisnis Program Studi Agribisnis Fakultas Pertanian Universitas Jambi 2) Staf Pengajar Jurusan Agribisnis Universitas Jambi Email: peni.agri@yahoo.co.id ABSTRAK Tujuan penelitian ini adalah untuk mengetahui hubungan faktor-faktor yang mempengaruhi alih fungsi lahan sawah pada tingkat petani dan mengetahui perbedaan penerimaan usahatani petani yang mengalihkan lahan sebelum dan sesudah alih fungsi di Desa Koto Dian dan Koto Tuo. Faktor-faktor alih fungsi adalah tingkatusia, lama pendidikan, luaslahan, jumlah tanggungan, dan pengalaman bertani. Metode pengambilan data diambil dari data primer dan sekunder. Data diolah dan dianalisis menggunakan analisis deskriptif untuk menggambarkan hubungan faktor-faktor alih fungsi lahan sawah, kemudian digunakan analisis uji beda dua rata-rata untuk mengetahui perbedaan penerimaan usahatani petani yang mengalih fungsikan lahan sawah sebelum dan sesudah alih fungsi lahan. Dengan jumlah responden sebanyak 37 petani yang mengalihkan sebagian lahan dan 20 petani responden yang mengalihkan semua lahan. Hasil analisis deskriptif yang menggambarkan hubungan faktor-faktor alih fungsi lahan adalah semakin tinggi tingkat usia, maka semakin tinggi tingkat alih fungsi lahan; semakin rendah tingkat pendidikan seorang petani, maka semakin tinggi tingkat alih fungsi lahan; semakin luas kepemilikan lahan, maka peluang petani untuk mengalih fungsikan lahannya lebihkecil; semakin banyak jumlah tanggungan yang harus ditanggung, maka alih fungsilahan akan semakin tinggi; dan semakin lama pengalaman dalam berusahatani, maka akan semakin berat dalam pengambilan keputusan untuk alih fungsi lahan. Kata kunci: hubungan faktor-faktor, alih fungsi lahan sawah, penerimaan usahatani. Key words: relationship factors, diversion of paddy fields, farming acceptance. Peniarti1), Rosyani2), Elwamendri3) 1) Alumni Jurusan Agribisnis Program Studi Agribisnis Fakultas Pertanian Universitas Jambi 2) Staf Pengajar Jurusan Agribisnis Universitas Jambi Email: peni.agri@yahoo.co.id ABSTRAK JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 PENDAHULUAN Indonesia merupakan negara agraris dimana pertanian merupakan basis utama perekonomian nasional.Sebagian besar masyarakat Indonesia masih menggantungkan hidupnya pada sektor pertanian. Sektor pertanian telah memberikan sumbangan besar dalam pembangunan nasional, seperti peningkatan ketahanan nasional, penyerapan tenaga kerja, peningkatan pendapatan masyarakat, peningkatan Pendapatan Domestik Regional Bruto (PDRB) , perolehan devisa melalui ekspor-impor, dan penekanan inflasi. Sektor pertanian, peternakan, kehutanan, dan perikanan merupakan sektor kedua setelah sektor industri pengolahan yang memberikan kontribusi besar terhadap peningkatan PDRB Indonesia.PDRB merupakan salah satu indikator yang menggambarkan pertumbuhan ekonomi suatu wilayah atau negara.Dalam menghadapi pembangunan, sektor pertanian masih terdapat banyak persoalan besar yang harus diselesaikan, salah satu diantaranya adalah permasalahan alih fungsi lahan pertanian menjadi lahan non-pertanian yang saat ini terus mengalami peningkatan. Menurut Utomo (1992) Alih fungsi lahan atau konversi lahan adalah berubahnya satu penggunanaan lahan ke penggunanaan lahan lainnya.Banyak faktor baik internal maupun eksternal yang mempengaruhi terjadinya alih fungsi lahan.Alih fungsi lahan pertanian sebenarnya bukan masalah baru. Sejalan dengan adanya peningkatan jumlah penduduk serta meningkatnya kebutuhan infrastruktur seperti, perumahan, jalan, industri, perkantoran, dan bangunan lain menyebabkan kebutuhan akan lahan meningkat. Selain itu, pertumbuhan ekonomi yang tinggi menyebabkan pertumbuhan yang sangat cepat di beberapa sektor ekonomi. Pertumbuhan tersebut juga membutuhkan lahan yang lebih luas sehingga terjadi peningkatan kebutuhan lahan untuk pembangunan, sementara ketersediaan lahan relatif tetap menyebabkan persaingan dalam pemanfaatan lahan. Kebanyakan lahan yang dialih fungsikan umumnya adalah lahan- lahan pertanian karena land rent (sewa lahan). Sewa ekonomi lahan (land rent) mengandung pengertian nilai ekonomi yang diperoleh oleh satu bidang lahan bila lahan tersebut digunakan untuk kegiatan proses produksi. Land rent lahan pertanian relatif lebih tinggi penggunaannya untuk non-pertanian dibandingkan dengan lahan pertanian yang dikelola oleh petani. Kabupaten Kerinci merupakan wilayah di Provinsi Jambi yang penghasil komuditas padi tertinggi dibanding dengan kabupaten–kabupaten lain. Hampir sebagian luas wilayah Kabupaten Kerinci merupakan lahan sawah. Namun Kabupaten Kerinci merupakan wilayah yang rawan akan masalah lahan, terutama karena adanya pemukiman penduduk. Adanya pertambahan jumlah penduduk Kabupaten Kerinci setiap tahun serta pemekaran wilayah menyebabkan kebutuhan baik pemukiman maupun perumahan terus meningkat ( Badan Pusat Statistik Kabupaten Kerinci 2015). Fenomena alih fungsi lahan pertanian merupakan dampak dari transformasi sruktur ekonomi (pertanian ke industri), dan demografi (pedesaan ke perkotaan) yang pada akhirnya mendorong transformasi sumberdaya lahan dari pertanian ke non-pertanian. Persoalan ini harus dicarikan solusipemecahannya karena melihat juga dampak yang ditimbulkan dari alih fungsi lahan ini dapat merugikan petani khususnya dan masyarakat Indonesia pada umumnya. ABSTRACT The purpose of this study was to determine the relationship of the factors that affect the transfer of paddy fields function at the farm level and determine differences in farming acceptance of farmers who divert land before and after conversion in Koto Dian and Koto Tuo village. Diversion factors are age, length of education, land area, number of dependents, and farming experience. The method of collecting data taken from primary and secondary data. Data were processed and analyzed using descriptive analysis to describe the relationship factors over paddy field function, and then used the analysis of two different test average to determine differences in farming acceptance of farmers who divert paddy field before and after land diversion. The number of respondents as many as 37 farmers who divert some of the land and 20 farmers respondent divert all land.Descriptive analysis that describes the relationship of the factors of land diversion is the higher the age, the higher the rate of land diversion; the lower the level of education of a farmer, the higher the rate of land diversion; more extensive land holdings, then the smaller chances of farmers to divert their land; more the number of dependents must be assured, then land diversion will be higher; and the longer experience in farming, more difficult in the decision to land diversion. - 1 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 METODE PENELITIAN Penelitian dilaksanakan didua desa, yakni Desa Koto Dian dan Desa Koto Tuo Kecamatan Keliling Danau Kabupaten Kerinci. Objek penelitian ini adalah petani yang mengalihkan sebagian lahan sawah dan yang mengalihkan semua lahan sawahnya.Penelitian dilakukan dengan mengambil sampel Desa Koto Dian dan Desa Koto Tuo. Pemilihan lokasi dilakukan secara sengaja (purposive) atau disebut juga judgemental samplingkarena wilayah tersebut merupakan wilayah yang mengalami alih fungsi lahan tertinggi di Kecamatan Keliling Danau . Sumber data Jenis data yang digunakan dalam penelitian ini adalah data primer dan data sekunder.Data primer digunakan untuk mengetahui faktor-faktor yang mempengaruhi alih fungsi lahan di tingkat petani, serta dampak alih fungsi lahan pertanian terhadap pendapatan usahatani petani.Data primer diperoleh dari hasil wawancara langsung dari pemilik lahan baik melalui kusioner maupun melalui wawancara mendalam. Data sekunder digunakan untuk faktor-faktor yang mempengaruhi alih fungsi lahan di tingkat wilaayah. Pengumpulan Data yang dilakukan kepada petani pemilik lahan yang mengalami alih fungsi lahan dan tidak mengalami alih fungsi lahan dilakukan secara purposive sampling. Penelitian dilaksanakan menggunakan metode sampling non-probability disebabkan oleh jumlah masing-masing populasi yang akan diteliti tidak diketahui secara pasti. Sampel pada samplingtidak acak akan menyebabkan populasi yang akan diteliti tidak memiliki kesempatan yang sama untuk dipilih sebagai sampel.Populasi di desa Koto Dian sebanyak 385 kk sedangkan di desa Koto Tuo sebanyak 444 kk. Responden dalam penelitian ini adalah petani setempat yang lahan usaha taninya pernah mengalami alih fungsi lahan. Penelitian yang dilaksanakan mengambil responden berjumlah 70 responden untuk petani di Desa Koto Dian dan 90 Responden di Desa Koto Tuo.Penetapan sampel ini disasarkan pada pendapat Bailey dalam Hasan (2002) yang menyatakan bahwa ukuran sampel minimum yang menggunakan analisis data statistik ialah 30 responden dimana populasi menyebar normal. Sampel merupakan bagian dari populasi yang diambil melalui cara-cara tertentu yang juga mewakili karateristik tertentu, jelas, dan lengkap yang bisa dianggap bisa mewakili populasi. Pengambilan data primer dilakukan melalui teknik wawancara dengan bantuan kuisioner kepada responden.Responden merupakan pihak yang memberikan informasi dan dapat mewakili dalam menjawab permasalahan penelitian. Petani sampel pada Penelitian ini adalah petani yang mengalih fungsikan lahan sawahnya dengan kata lain petani yang pada awalnya menanam komuditi padi pada lahan sawahnya kemudian pada saat ini sudah melakukan alih fungsi atau mengubah sebagian atau keseluruhan dari lahannya. Kecamatan Keliling Danau dipilih secara sengaja (purposive). PENDAHULUAN Adanya alih fungsi lahan pertanian khususnya lahan sawah akan mempengaruhi produksi beras yang mana merupakan makanan pokok masyarakat Indonesia sehingga akan berpengaruh terhadap ketahanan pangan. Fenomena alih fungsi lahan pertanian ke penggunaan non-pertanian saat ini terjadi sangat pesat di beberapa wilayah di Indonesia terutama di Pulau Sumatra. Satu wilayah penyumbang beras tertinggi khususnya di Provinsi Jambi sampai saat ini tetap mengalami alih fungsi lahan pertanian khususnya lahan sawah.Salah satu Kabupaten yang mengalami alih fungsi lahan pertanian di Provinsi Jambi adalah kabupaten Kerinci.Wilayah ini juga terkenal sebagai lumbung padi Provinsi karena merupakan daerah dataran tinggi. Kabupaten Kerinci menjadi salah satu penghasil padi terbesar di Provinsi Jambi. Dalam lima tahun terakhir Kecamatan Keliling Danau mengalami tingkat penurunan luas lahan padi sawah yang cukup tinggi, Tahun 2011 Luas panen sebesar 3.209 Ha namun terjadi penurunan sehingga pada Tahun 2013 luas panen hanya mencapai 2.949 Ha. Sementara pada Tahun 2014 - 2 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 penurunan luas panen semakin menungkat yaitu 2.888 ha dan kembali menurun menjadi 2.859 ha di Tahun 2015. Desa Koto dian dan Koto Tuo bukan merupaakan desa dengan Luas lahan sawah tertinggi di Kecamatan Keliling Danau namun tingkat pengalihan fungsi lahan tertinggi terletek didua desa tersebut.Lahan yang dialih fungsikan berupa lahan sawah produktif yang berada dipinggir jalan raya utama Desa Koto Dian dan Koto Tuo. Pembangunan di wilayah ini lebih banyak untuk perumahan. Banyak Masyarakat yang memilih membangun Pemukiman tepat disisi Jalan utama meskipun lahan tersebut merupakan lahan produktif bagi pertanian khususnya padi sawah. Tujuan dari penelitian ini adalah untuk mengidentifikasi faktor-faktor yang mempengaruhi alih fungsi lahan sawah pada tingkat petani dikedua desa serta hubungannya terhadap tingkat alih fungsi lahan sawah dan mengetahui perbedaan penerimaan usahatani Petani yang mengalihkan lahan sebelum dan sesudah alih fungsi lahan sawah kedua desa tersebut. 1. Tingkat Usia Usia merupakan salah satu faktor yang berpengaruh dalam suatu tindakan alih fungsi lahan. Faktor usia dianggap faktor utama pendorong bagi petani untuk mengalihkan lahan terutama dalam hal ini lahan sawah. Rentang usia petani sampelpun menjadi bahasan dalam proses penelitian ini. Berikut tabel yang menunjukkan Tingkat usia petani sampel yang mengalihkan sebagian lahan sawah dan semua lahan sebelum dan sesudah alih fungsi lahan. Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian bel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian Lahan Sawah dan Semua Lahan Sawah. Tabel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian Lahan Sawah dan Semua Lahan Sawah. Tabel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian Lahan Sawah dan Semua Lahan Sawah. Rentang Usia Frekuensi Petani yang Mengalihkan Sebagian Lahan Sawah Persentase Frekuensi Petani yang Mengalihkan Semua Lahan Sawah Persentase 29 – 35 tahun 5 jiwa 13,51 % 2 jiwa 10 36 – 42 tahun 12 jiwa 32,43 % 6 jiwa 30 43 - 49 tahun 9 jiwa 24,32 % 1 jiwa 5 50 - 56 tahun 4 jiwa 10,81 % 4 jiwa 20 57 – 63 tahun 5 jiwa 13,51 % 2 jiwa 10 64 – 70 tahun 1 2,70 % 3 jiwa 15 71 – 77 tahun - - 2 jiwa 10 Jumlah 37 jiwa 100 % 20 jiwa 100 Pada petani sampel yang mengalihkan sebagian lahan sawahnya terlihat jelas bahwa rentangan usia yang mengalihkan sebagian lahan sawah tergolong masih dalam usia yang produktif yaitu pada rentang usia 29- 35 tahun dengan jumlah persentase sekitar 13,51 % dari 37 sampel yang ada, 36- 42 tahun 32,43 %, 43- 49 tahun 24,32%, 50- 56 tahun 10,81 % , 57- 63 tahun 13,51 % sedangkan pada rentang usia 64 - 70 tahun persentase jumlah petani yang mengalihkan sebagian lahan yakni 2,70 % dari 37 sampel. Sementara itu petani yang mengalihkan semua lahan sawahnya memiliki persentase rentang usia yang berbeda pula yaitu pada rentang usia 29- 35 tahun 10 %, 36- 42 tahun 30 %, 43- 49 tahun 5 %, 50- 56 tahun 20 %, 57- 63 tahun 10 %, 64- 70 tahun 15 %, dan pada rentang usia 71- 77 tahun 10 % dari 20 sampel petani yang mengalihkan semua lahan. 1. Tingkat Usia Petani sampel yang berada pada usia rentan/ rawan dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan sementara sebesar 60% pada tingkat petani yang mengalihkan semua lahan. Petani sampel yang berada pada usia rentan/ rawan dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan sementara sebesar 60% pada tingkat petani yang mengalihkan semua lahan. Hal ini mengindikasikan b h i k i b h d lih f i l h di K K lili D K b Tabel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian Lahan Sawah dan Semua Lahan Sawah. Rentang Usia Frekuensi Petani yang Mengalihkan Sebagian Lahan Sawah Persentase Frekuensi Petani yang Mengalihkan Semua Lahan Sawah Persentase 29 – 35 tahun 5 jiwa 13,51 % 2 jiwa 10 36 – 42 tahun 12 jiwa 32,43 % 6 jiwa 30 43 - 49 tahun 9 jiwa 24,32 % 1 jiwa 5 50 - 56 tahun 4 jiwa 10,81 % 4 jiwa 20 57 – 63 tahun 5 jiwa 13,51 % 2 jiwa 10 64 – 70 tahun 1 2,70 % 3 jiwa 15 71 – 77 tahun - - 2 jiwa 10 Jumlah 37 jiwa 100 % 20 jiwa 100 Pada petani sampel yang mengalihkan sebagian lahan sawahnya terlihat jelas bahwa rentangan Pada petani sampel yang mengalihkan sebagian lahan sawahnya terlihat jelas bahwa rentangan usia yang mengalihkan sebagian lahan sawah tergolong masih dalam usia yang produktif yaitu pada rentang usia 29- 35 tahun dengan jumlah persentase sekitar 13,51 % dari 37 sampel yang ada, 36- 42 tahun 32,43 %, 43- 49 tahun 24,32%, 50- 56 tahun 10,81 % , 57- 63 tahun 13,51 % sedangkan pada rentang usia 64 - 70 tahun persentase jumlah petani yang mengalihkan sebagian lahan yakni 2,70 % dari 37 sampel. Sementara itu petani yang mengalihkan semua lahan sawahnya memiliki persentase rentang usia yang berbeda pula yaitu pada rentang usia 29- 35 tahun 10 %, 36- 42 tahun 30 %, 43- 49 tahun 5 %, 50- 56 tahun 20 %, 57- 63 tahun 10 %, 64- 70 tahun 15 %, dan pada rentang usia 71- 77 tahun 10 % dari 20 sampel petani yang mengalihkan semua lahan. Petani sampel yang berada pada usia rentan/ rawan dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan sementara sebesar 60% pada tingkat petani yang mengalihkan semua lahan. JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS uji beda rata-rata bertujuan untuk mengetahui pengaruhnya terhadap tingkat penerimaan usahata petani. 2. Lama Pendidikan METODE PENELITIAN Dari sebanyak 24 desa di Kecamatan Keliling Danau ini dipilih secara Proposive Desa Koto Dian dan Desa Koto Tuo karena ke 2 desa tersebut merupakan salah satu Desa yang paling tinggi tingkat penurunan luas lahan (Keliling Danau Dalam Angka). Berdasarkan imformasi langsung yang peneliti dapatkan dilapangan, kedua desa di Kecamatan Keliling Danau Kabupaten Kerinci jumlah Petani yang melakukan alih fungsi dari lahan sawah menjadisebanyak 70 Kk di Desa Koto Dian (Kantor Kepala Desa Koto Dian tahun 2015), sedangkan di Desa Koto Tuo sebanyak 90 Kk (Kantor Kepala Desa Koto Tuo tahun 2015) yang mengalih fungsikan lahan sawahnya ke Pemukiman. Dalam hal ini Formula yang digunakan untuk menentukan besarnya ukuran sampel adalah sebagai berikut (Slovin,1964 dan Nazir,2005). Metode analisis deskriftif digunakan dengan tujuan untuk memberikan penjelasan dan interpretasi atas data dan informasi pada tabulasi data serta faktor-faktor yang mempengaruhi alih fungsi lahan sawah. Kemudian metode analisis - 3 - 1. Tingkat Usia Tingkat pendidikan merupakan hal yang paling penting dalam usaha meningkatkan produksi dan kesejahteraan petani, maka diharapkan pola pikir petani akan rasional dalam mengambil keputusan sehingga usahatani yang dikelola akan meningkat baik dalam hal jumlah dan mutu produksi yang akan dihasilkan serta dengan memiliki pendidikan yang tinggi petani juga dapat memajukan dan mengelola kelompok tani yang telah ada. Pada Petani yang mengalihkan sebagian lahan sebanyak 8,10% yang berada pada tingkat sekolah dasar. Sedangkan lulusan SMP dan SMA persentasenya masing-masing 29,72% dan 37,83%, sementara pada petani yang mengalihkan semua lahan sawahnya sebanyak 30% berada pada tingkat SD dan pada tingkat SMP sebesar 20% serta tingkat SMA sebanyak 25%. Dari angka tersebut dapat dinyatakan bahwa rata-rata tingkat pendidikan formal petani sampel tergolong masih rendah. Rendahnya tingkat pendididkan ini dapat mempengaruhi pola pemikiran dalam mengelola usahataninya, sehingga pada akhirnya petani akan cenderung mengalihkan lahan usahatani padi sawah. Semakin rendah tingkat pendidikan seorang petani maka semakin tinggi tingkat alih fungsi lahan, terutama dalam hal ini lahan padi sawah.Petani yang memiliki pendidikan rendah cenderung gampang mengalih fungsikan lahan sawah disebakan petani tersebut hanya memikirkan keuntungan jangka pendek tanpa memikirkan kerugian dikemudian hari. Sedangkan petani yang memiliki tingkat pendidikan tinggi akan memiliki pola pikir kedepan dengan mempertimbangkan lagi kerugian jangka panjang apabila ia melakukan pengalihan fungsi lahan sawah. 1. Tingkat Usia Petani sampel yang berada pada usia rentan/ rawan dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan sementara sebesar 60% pada tingkat petani yang mengalihkan semua lahan. Hal ini mengindikasikan bahwa tingkat usia berpengaruh pada pengalihan fungsi lahan di Kecamatan Keliling Danau Kabupaten Kerinci. Semakin tinggi tingkat usia maka semakin tinggi tingkat alih fungsi lahan. Ini terjadi disebabkan karena semakin tinggi tingkat usia seseorang maka kondisi fisik akan semakin lemah. Mereka sudah tidak kuat lagi bekerja di sektor pertanian yang membutuhkan tenaga yang kuat. Kondisi ini membatasi kemampuan responden untuk menghasilkan sesuatu sehingga akan cenderung mengalih fungsikan lahan yang dimilikinya. Apalagi dengan melihat kondisi saat ini dimana anak-anak mereka yang tidak lagi mengikuti jejak orang tua mereka untuk bekerja di sektor pertanian. Dengan mengalih fungsikan lahan, mereka dapat bekerja disektor lain yang tidak membutuhkan tenaga lebih. - 4 - - 4 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 Pendidikan adalah bagian yang dinilai paling penting dalam kehidupan social masyarakat.Pendidikan menunjukkan tingkat pengetahuan, wawasan, pola pikIr dan perilaku seseorang. Tingkat pendidikan formal yang dimiliki petani memberikan gambaran sumberdaya manusia petani pada aspek formal.Pada masa sekarang lama seseorang berpendidikan menjadi suatu indikator dalam menetukan sejahtera atau tidak sejahteranya suatu individu. Tabel 2 . Distribusi frekuensi dan Persentase Pendidkan Petani sampel yang mengalihkan sebagian dan semua lahan sawah. Tingkat Pendidikan Sebagian Lahan sawah Semua Lahan Sawah Frekuensi Persentase (%) Frekuensi Persentase (%) SD 3 8,10 6 30 SMP 11 29,72 4 20 SMA 14 37,83 5 25 Perguruan Tinggi 9 24,32 5 25 Jumlah 37 100 20 100 Tingkat pendidikan merupakan hal yang paling penting dalam usaha meningkatkan produksi dan kesejahteraan petani, maka diharapkan pola pikir petani akan rasional dalam mengambil keputusan sehingga usahatani yang dikelola akan meningkat baik dalam hal jumlah dan mutu produksi yang akan dihasilkan serta dengan memiliki pendidikan yang tinggi petani juga dapat memajukan dan mengelola kelompok tani yang telah ada. Pada Petani yang mengalihkan sebagian lahan sebanyak 8,10% yang berada pada tingkat sekolah dasar. Sedangkan lulusan SMP dan SMA persentasenya masing-masing 29,72% dan 37,83%, sementara pada petani yang mengalihkan semua lahan sawahnya sebanyak 30% berada pada tingkat SD dan pada tingkat SMP sebesar 20% serta tingkat SMA sebanyak 25%. Dari angka tersebut dapat dinyatakan bahwa rata-rata tingkat pendidikan formal petani sampel tergolong masih rendah. Luas lahan yang dimaksud dalam penelitian ini adalah luas lahan sawah yang digarap petani selama melakukan kegiatan usahataninya. Dari hasil penelitian luas lahan yang dimiliki petani yang mengalihkan sebagian lahan sawahnya sebanyak 43,24% ≤ 0,31 ha dan 56,76% petani yang memiliki 1. Tingkat Usia Rendahnya tingkat pendididkan ini dapat mempengaruhi pola pemikiran dalam mengelola usahataninya, sehingga pada akhirnya petani akan cenderung mengalihkan lahan usahatani padi sawah. Semakin rendah tingkat pendidikan seorang petani maka semakin tinggi tingkat alih fungsi lahan, terutama dalam hal ini lahan padi sawah.Petani yang memiliki pendidikan rendah cenderung gampang mengalih fungsikan lahan sawah disebakan petani tersebut hanya memikirkan keuntungan jangka pendek tanpa memikirkan kerugian dikemudian hari. Sedangkan petani yang memiliki tingkat pendidikan tinggi akan memiliki pola pikir kedepan dengan mempertimbangkan lagi kerugian jangka panjang apabila ia melakukan pengalihan fungsi lahan sawah. 3 h Tabel 2 . Distribusi frekuensi dan Persentase Pendidkan Petani sampel yang mengalihkan sebagian dan semua lahan sawah. Tingkat Pendidikan Sebagian Lahan sawah Semua Lahan Sawah Frekuensi Persentase (%) Frekuensi Persentase (%) SD 3 8,10 6 30 SMP 11 29,72 4 20 SMA 14 37,83 5 25 Perguruan Tinggi 9 24,32 5 25 Jumlah 37 100 20 100 bel 2 . Distribusi frekuensi dan Persentase Pendidkan Petani sampel yang mengalihkan sebagian dan semua lahan sawah. Jumlah 37 100 20 100 Tingkat pendidikan merupakan hal yang paling penting dalam usaha meningkatkan produksi dan kesejahteraan petani, maka diharapkan pola pikir petani akan rasional dalam mengambil keputusan sehingga usahatani yang dikelola akan meningkat baik dalam hal jumlah dan mutu produksi yang akan dihasilkan serta dengan memiliki pendidikan yang tinggi petani juga dapat memajukan dan mengelola kelompok tani yang telah ada. Pada Petani yang mengalihkan sebagian lahan sebanyak 8,10% yang berada pada tingkat sekolah dasar. Sedangkan lulusan SMP dan SMA persentasenya masing-masing 29,72% dan 37,83%, sementara pada petani yang mengalihkan semua lahan sawahnya sebanyak 30% berada pada tingkat SD dan pada tingkat SMP sebesar 20% serta tingkat SMA sebanyak 25%. Dari angka tersebut dapat dinyatakan bahwa rata-rata tingkat pendidikan formal petani sampel tergolong masih rendah. Rendahnya tingkat pendididkan ini dapat mempengaruhi pola pemikiran dalam mengelola usahataninya, sehingga pada akhirnya petani akan cenderung mengalihkan lahan usahatani padi sawah. Semakin rendah tingkat pendidikan seorang petani maka semakin tinggi tingkat alih fungsi lahan, terutama dalam hal ini lahan padi sawah.Petani yang memiliki pendidikan rendah cenderung gampang mengalih fungsikan lahan sawah disebakan petani tersebut hanya memikirkan keuntungan jangka pendek tanpa memikirkan kerugian dikemudian hari. Sedangkan petani yang memiliki tingkat pendidikan tinggi akan memiliki pola pikir kedepan dengan mempertimbangkan lagi kerugian jangka panjang apabila ia melakukan pengalihan fungsi lahan sawah. 3. Luas Lahan Dalam hal ini luas lahan yang dilihat adalah luas lahan yang dimiliki petani dan luas lahan yang dialih fungsikan baik pada petani yang mengalihkan sebagian lahan dan yang mengalihkan semua lahan sawahnya.Berikut tabel luas lahan petani yang mengalihkan sebagian lahan dan semua lahan sawah. Dalam hal ini luas lahan yang dilihat adalah luas lahan yang dimiliki petani dan luas lahan yang dialih fungsikan baik pada petani yang mengalihkan sebagian lahan dan yang mengalihkan semua lahan sawahnya.Berikut tabel luas lahan petani yang mengalihkan sebagian lahan dan semua lahan sawah. Tabel 3 . Distribusi dan Frekuensi Luas Lahan Petani yang Mengalihkan Sebagian dan Semua Lahan Luas lahan Sebagian Lahan sawah Semua Lahan Sawah Frekuensi Persentase (%) Frekuensi Persentase (%) ≤ 0,31 ha 16 43,24 11 55 ≥ 0,31 ha 21 56,76 9 45 Jumlah 37 100 20 100 L l h di k d d l liti i i d l h l l h h di t i sawahnya.Berikut tabel luas lahan petani yang mengalihkan sebagian lahan dan semua lahan sawah. Tabel 3 . Distribusi dan Frekuensi Luas Lahan Petani yang Mengalihkan Sebagian dan Semua Lahan Luas lahan Sebagian Lahan sawah Semua Lahan Sawah Frekuensi Persentase (%) Frekuensi Persentase (%) ≤ 0,31 ha 16 43,24 11 55 ≥ 0,31 ha 21 56,76 9 45 Jumlah 37 100 20 100 Luas lahan yang dimaksud dalam penelitian ini adalah luas lahan sawah yang digarap petani selama melakukan kegiatan usahataninya. Dari hasil penelitian luas lahan yang dimiliki petani yang mengalihkan sebagian lahan sawahnya sebanyak 43,24% ≤ 0,31 ha dan 56,76% petani yang memiliki Luas lahan yang dimaksud dalam penelitian ini adalah luas lahan sawah yang digarap petani selama melakukan kegiatan usahataninya. Dari hasil penelitian luas lahan yang dimiliki petani yang mengalihkan sebagian lahan sawahnya sebanyak 43,24% ≤ 0,31 ha dan 56,76% petani yang memiliki - 5 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 lahan ≥ 0,31 ha. Sementara petani yang mengalihkan semua lahan sawahnya sebesar 55% bagi petani yang memiliki luas lahan ≤ 0,31 dan 45% petani yang lahannya ≥ 0,31 ha.Jika dijumlahkan petani yang memiliki luas lahan sedikit akan cenderung mengalih fungsikan lahan sawahnya. 4. Jumlah Tanggungan Dalam hal ini jumlah tanggungan bukanlah jumlah anak secara keseluruhan melainkan jumlah anak yang masih menjadi tanggungan dikarenakan belum menikah dan masih dalam tahap menempuh pendidikan.Dibawah ini adalah tabel jumlah tanggungan petani yang mengalihkan sebagian dan semua lahan sawah. bel 4 . Distribusi Frekuensi Jumlah Tanggungan Petani yang Mengalihkan Sebagian dan Semua Tabel 4 . Distribusi Frekuensi Jumlah Tanggungan Petani yang Mengalihkan Sebagian dan Semua Lahan Sawah. Jumlah tanggungan Sebagian Lahan sawah Semua Lahan Sawah Frekuensi Persentase (%) Frekuensi Persentase (%) Tidak memiliki tanggungan 4 10,81 9 45 ≤ 2 orang 30 81,08 11 55 ≥ 2 orang 3 8,10 - - Jumlah 37 100 20 100 Jumlah tanggungan yang dimaksud dalam penelitian ini adalah jumlah anak yang belum menikah dan masih dalam tahap menempuh pendidikan. Dari hasil penelitian mengenai jumlah tanggungan yang dimiliki petani yang mengalihkan sebagian lahannya didapatkan sebesar 10,81% petani tidak memiliki tanggungan, 81,08% petani yang memiliki jumlah tanggungan ≤ 2 orang sedangkan jumlah tanggungan ≥ 2 orang sebanyak 8,10%, Hal ini berbeda dengan petani yang memutuskan mengalihkan semua lahan sawahnya yaitu sebesar 45% untuk petani yang tidak memiliki tanggungan lagi, 55% untuk petani yang memiliki ≤ 2 orang tanggungan sedangkan jumlah tanggungan petani yang ≥ 2 orang tidak terdapat sama sekali. Sejatinya jumlah tanggungan yang harus ditanggung petani mempengaruhi alih fungsi lahan dimana semakin banyak jumlah tanggungan yang harus ditanggung, maka alih fungsi lahan akan semakin tinggi. Semakin banyak tanggungan yang dimiliki maka biaya yang dibutuhkan dalam memenuhi kebutuhan sehari-hari semakin banyak sehingga petani akan cenderung untuk mengalih fungsikan lahannya. Pada penelitian ini jumlah tanggungan petani tidak berpengaruh secara nyata terhadap tingkat alih fungsi lahan sawah, hal ini dikarenakan jumlah tanggungan kurang dari 3 orang maka dikategorikan sedikit (Winoto, 2005). 3. Luas Lahan Semakin luas kepemilikan lahan maka peluang petani untuk mengalih fungsikan lahannya lebih kecil dibandingkan petani yang melakukan alih fungsi lahan.Dalam tingkat luas pemilikan lahan, petani yang memiliki lahan cukup luas cenderung untuk tetap mempertahankan lahannya sehingga peluang terjadinya alih fungsi lahan kecil. Sedangkan bagi petani yang memiliki lahan kecil cenderung untuk mengalih fungsikan lahan sawahnya ke sektor lain. Hal ini diduga disebabkan karena luas lahan sangat berhubungan dengan penerimaan. Petani yang memiliki lahan lebih luas memiliki perolehan hasil produksi lebih besar sehingga penerimaan yang dihasilkan lebih besar dibandingkan dengan petani yang memiliki luas lahan lebih sempit. Hasil panen dari pengolahan lahan yang lebih sempit tidak sebanding dengan modal yang dikeluarkan petani sehingga secara tidak langsung akan mempengaruhi penerimaan yang diperoleh dalam mencukupi kehidupan sehari-hari. 5. Pengalaman Berusahatani Pengalaman berusahatani merupakan lama petani dalam mengusahakan kegiatan berusahatani dihitung dalam tahun. Petani dalam mengambil keputusan dan bijaksana mengenai usahataninya selalu mempertimbangkan resiko yang akan diterimanya. Kemampuan petani dalam menerima resiko akan berbeda antara petani satu dengan yang lainnya. Perbedaan Kemampuan menerima resiko ini dipengaruhi dari beberapa faktor antara lain adalah pengalaman petani. Pengalaman petani dalam mengusahan usahatani padi sawah dapat berpengaruh terhadap keputusan petani dalam mengalih fungsikan lahan sawahnya. Berikut Tabel 5 pengalaman berusahatani petani yang mengalihkan sebagian dan semua lahan sawah. Tabel 5. Pengalaman Berusahatani Petani yang Mengalihkan Sebagian dan Semua Lahan Sawah. Tabel 5. Pengalaman Berusahatani Petani yang Mengalihkan Sebagian dan Semua Lahan Sawah. - 6 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS Rentang Usia (Pengalaman berusahatani) Frekuensi Petani yang Mengalihkan Sebagian Lahan Sawah Persentase Frekuensi Petani yang Mengalihkan Sebagian Semua Lahan Sawah Persentase 1- 6 tahun 4 jiwa 10,81 % 4 jiwa 20 % 7 - 13 tahun 21 jiwa 56,75 % 3 jiwa 15 % 14 - 20 tahun 4 jiwa 10,81 % 2 jiwa 10 % 21 - 27 tahun 2 jiwa 5,40 % 2 jiwa 10 % 28 - 34 tahun 4 jiwa 10,81 % 1 jiwa 5 % 35 – 41 tahun 2 jiwa 5,40 % 6 jiwa 30 % 42 – 49 tahun - - 2 jiwa 10 % Jumlah 37 jiwa 100 % 20 jiwa 100 % Pada petani yang mengalihkan sebagian lahan terlihat jelas bahwa rentang tahun pengalaman berusahatani yaitu dari 1- 6 tahun terdapat 4 orang petani de3ngan persentase 10,81%, 7- 13 tahun terdapat 21 orang petani dengan persentase 56,75%, 14-20 tahun terdapat 4 orang petani dengan persentase 10,81%, 21-27 tahun terdapat 2 orang petani dengan persentase 5,40%, 28-34 tahun sebanyak 4 orang petani dengan persentase 10,81% dan 35-41 tahun ada 2 orang petani yang mengalihkan lahan dengan persentase 5,40%. Sedangkan pada petani yang mengalihkan semua lahan dapat dilihat bahwa rentang tahun pengalaman berusahatani yaitu dari 1-6 tahun ada 4 orang petani dengan persentase 20%, 7-13 tahun 3 orang dengan persentase 15%, 14-20 tahun terdapat 2 orang dengan persentase 10%, 21-27 tahun 2 orang petani dengan persentase 10%, 28-34 tahun Cuma ada 1 orang petani dengan persentase 5%, 35-41 tahun terdapat 6 orang petani dengan persentase 30% dan 42-49 tahun terdapat 2 orang dengan jumlah perssentase 10%. Seorang petani dikatakan berpengalaman jika lama berusahatani > 20 tahun, sementara petani dianggap pemula apabila lama berusahatani < 20 tahun (Winoto, 2005). 5. Pengalaman Berusahatani Pada tabel lama berusahatani dapat dilihat bahwa jumlah petani yang memiliki pengalaman < 20 tahun sebanyak 42 petani dengan total persentase 73,68%. Sedangkan untuk petani yang memiliki pengalaman > 20 tahun sebanyak 19 petani dengan jumlah persentase 33,33%.Semakin lama pengalaman dalam berusahatani, maka akan semakin berat dalam pengambilan keputusan untuk alih fungsi lahan. Hal ini disebabkan karena semakin lama pengalaman bertani, maka keahlian dalam bertani akan semakin tinggi sehingga petani akan cenderung untuk terus mempertahankan lahannya. Hal ini menyebabkan mereka tidak akan mengalih fungsikan lahannya. Memang pada saat peneliti terjun ke lapangan rata-rata petani yang mengalih fungsi lahan sawahnya adalah petani yang dikategorikan petani pemula. Jumlah Ada juga nenerapa responden memilih untuk menjadi TKI di luar negeri dimana ini menjadi fenomena yang tidak bias dipungkiri lagi menjadi sumber penerimaan yang dianggap mampu memenuhi kebetuhan hidup serta mampu meningkatkan taraf hidup masyarakat Kerinci khususnya di Kecamatan Keliling Danau.Selain dari hanya bersumber padi gaji PNS dan TKI ada juga beberape petani memilih bekerja disektor perdagangan, sebgai tukang, supir, pegawai honorer bahkan ada 3 responden yang tidak bekerja sama sekali karena faktor usia sehingga kebutuhan hidupnya ditanggung oleh anaknya. Kejadian-kejadian tersebut menunjukkan gejala akan terjadinya transformasi kegiatan dari sektor pertanian ke sektor non pertanian. Hal ini dapat dilihat dari adanya perubahan mata pencaharian utama dari petani.Namun, akibat keterbatasan keterampilan yang dimiliki serta pendidikan yang rendah, hanya pekerjaan dengan upah rendah yang bisa mereka peroleh. Perubahan mata pencaharian utama yang terjadi, secara otomatis akan berpengaruh terhadap penerimaan yang diperoleh saat ini. B t k P lih L h S h Pengalihan sumber penerimaanbagi petani yang mengalihkan semua lahan sawahnya memang masih ada yang tetap memilih berusahatani namun dalam hal ini bukan sektor usahatani padi sawah melain sebagai petani gurem dan berternak yang hanya 30% saja.Sementara dari sumber penerimaan diluar usahatani ada yang hanya menjadi PNS sekitar 3 responden dengan persentase 15% dari total 20 sampel. Ada juga nenerapa responden memilih untuk menjadi TKI di luar negeri dimana ini menjadi fenomena yang tidak bias dipungkiri lagi menjadi sumber penerimaan yang dianggap mampu memenuhi kebetuhan hidup serta mampu meningkatkan taraf hidup masyarakat Kerinci khususnya di Kecamatan Keliling Danau.Selain dari hanya bersumber padi gaji PNS dan TKI ada juga beberape petani memilih bekerja disektor perdagangan, sebgai tukang, supir, pegawai honorer bahkan ada 3 responden yang tidak bekerja sama sekali karena faktor usia sehingga kebutuhan hidupnya ditanggung oleh anaknya. Kejadian-kejadian tersebut menunjukkan gejala akan terjadinya transformasi kegiatan dari sektor pertanian ke sektor non pertanian. Hal ini dapat dilihat dari adanya perubahan mata pencaharian utama dari petani.Namun, akibat keterbatasan keterampilan yang dimiliki serta pendidikan yang rendah, hanya pekerjaan dengan upah rendah yang bisa mereka peroleh. Perubahan mata pencaharian utama yang terjadi, secara otomatis akan berpengaruh terhadap penerimaan yang diperoleh saat ini. B t k P lih L h S h Alih fungsi lahan sawah mulai terjadi pada awal tahun 2000-an dan mengalami peningkatan tiap tahunnya dikarenakan tingginya permintaan lahan untuk pemukiman, sementara lahan di Kabupaten Kerinci merupakan lahan yang perbukitan sehinnga lahan sawahlah yang menjadi pilihan para masyarakat untuk membangun pemukiman. Sumber Penerimaan Petani Setelah Alih Fungsi Lahan Usahatani padi sawah di Kecamatan Keliling Danau Kabupaten Kerinci masih menggunakan sistem jaringan irigasi sederhana dan setengah teknis. Sebenarnya pada awal tahun 2000 kelompok tani mulai dibentuk di daerah penelitian, namun karena kurang adanya perhatian dari Dinas Pertanian Kabupaten Kerinci maupun PPL kelompok tani yang telah ada tidak aktif atau tidak dijalankan. Akibat dari kurang adanya perhatian pemerintah lahan sawah yang awalnya membentang sepanjang jalan raya mulai berkurang karena tingginya permintaan lahan untuk pemukiman. Berusahatani padi sawahpun dianggap kurang menjinjikan mengingat penerimaan yang diperoleh dari sektor tersebut lebih kecil disbanding sektor lain. Untuk mengetahui sumber penerimaan petani yang mengalihkan sebagian lahan sawahnya dapat dilihat pada Tabel 6 dibawah ini : Sumber Penerimaan Petani yang Mengalihkan Sebagian Lahan Sawahnya Sebelum dan Sesudah Alih fungsi Lahan. Sesudah Alih fungsi Lahan. Sumber Penerimaan Jumlah Sampel (orang) Persentase Usahatani Padi dan PNS 7 18,91% Usahatani Padi dan Berdagang 4 10,81% Usahatani Padi dan Petani Gurem 5 13,51% Usahatani Padi dan Nelayan 4 10,81% Usahatani Padi dan TKI 7 18,91% Usahatani Padi dan Tukang 4 10,81% Usahatani Padi dan Supir 2 5,40% Hanya Usahatani Padi Sawah 4 10,81% - 7 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 Jumlah 100% Pada tabel dapat dilihat sumber penerimaan yang diperoleh oleh petani sampel sesudah alih fungsi lahan terdiri dari beberapa sumber penerimaan. Petani yang mengalihkan sebagian lahan di lapangan tempat penelitian berlangsung tentunya masih mengusahakan padi bsawah dan memiliki sumber penerimaan dari sektor lain diantaranya, sebagai PNS sebanyak 7 responden dengan persentase 18,91 % dari jumlah sampel, disaektor dagang ada 4 responden dengan persentase 10,81%, disektor petani gurem 5 responden dengan persentase 13,51%, sebagai nelayan ada 4 responden dengan persentase 10,81%, sumber penerimaan sebagai TKI sebanyak 7 responden dengan persentase 18,91%, sebagai tukang ada 4 responden dengan jumlah persentase 10,81%, sementara responden yang menjadi supir hanya ada 2 responden dengan persentase 5,40% dan yang hanya berusahatani padi tanpa sumber penerimaan smpingan ada 4 responden dengan jumlah persentase 10,91%. Sumber penerimaan dari sektor lain ini tentunya dapat membantu petani dalam memenuhi kebutuhan keluarga sehari-hari. Dengan adanya sumber penerimaan dari sektor lain petani kiranya dapat terus berusahatni padi tanpa harus mengalihkan semua luas lahan sawah yang dimiliki. Sementara itu mengetahui sumber penerimaan bagi petani yang mengalihkan semua lahan sawahnya dapat dilihat pada Tabel 7 berikut ini: Tabel 7. Sumber Penerimaan Petani yang Mengalihkan Semua Lahan Sawahnya Sesudah Alih f i L h Sumber Penerimaan Petani yang Mengalihkan Semua Lahan Sawahnya Sesudah Alih fungsi Lahan. fungsi Lahan. Sumber Penerimaan Jumlah Sampel (orang) Persentase PNS 3 15% Berdagang 2 10% Petani Gurem 3 15% Peternak 3 15% TKI 2 10% Tukang 1 5% Supir 1 5% Honorer 2 10% Tidak bekerja 3 15% Jumlah 20 100% g Sumber Penerimaan Jumlah Sampel (orang) Persentase PNS 3 15% Berdagang 2 10% Petani Gurem 3 15% Peternak 3 15% TKI 2 10% Tukang 1 5% Supir 1 5% Honorer 2 10% Tidak bekerja 3 15% Jumlah 20 100% Pengalihan sumber penerimaanbagi petani yang mengalihkan semua lahan sawahnya memang masih ada yang tetap memilih berusahatani namun dalam hal ini bukan sektor usahatani padi sawah melain sebagai petani gurem dan berternak yang hanya 30% saja.Sementara dari sumber penerimaan diluar usahatani ada yang hanya menjadi PNS sekitar 3 responden dengan persentase 15% dari total 20 sampel. Jumlah Untuk mengetahui bentuk peralihan lahan sawah dapat di lihat pada Tabel 19 dibawah ini. Bentuk Peralihan Lahan Sawah Bagi Petani yang Mengalihkan Sebagian dan Semua Lahan Bentuk Peralihan Lahan Sawah Bagi Petani yang Mengalihkan Sebagian dan Semua Lahan Sawah. Bentuk Peralihan Lahan Sawah Bagi Petani yang Mengalihkan Sebagian dan Semua Laha Sawah. Petani yang Mengalihkan Sebagian Lahan Bentuk Alih fungsi lahan Petani yang Mengalihkan Semua Lahan Petani yang Mengalihkan Sebagian Lahan - 8 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS Responden Persentase Responden Persentase Lahan Petani Gurem 3 15% 5 13,51% Pemukiman 3 15% 32 86,48% Lahan Ternak 14 70% - - Jumlah 20 100% 37 100% Lahan yang dijadikan pemukiman sekitar 86,48% petani yang mengalihkan sebagian lahan sawahnya dengan jumlah responden sebanyak 32 orang hal ini tentu saja menunjukkan tinggi alih fungsi lahan ke pemukiman. Dijadikan lahan selain untuk pemukiman ada juga beberapa responden petani mengalihkan lahan sawahnya ke sektor usahatani lain yakni sebagai lahan ternak sebanyak 70% bagi petani yang mengalihkan sebagian lahan dan untuk yang mengalihkan semua lahan sawah tidak ada yang menjadikan lahan ternak melainkan 13,51% responden petani yang mengalihkan ke lahan petani gurem, sedangkan 15% reponden dari petani yang mengalihkan semua lahan menjadi lahan pertanian gurem dan hanya ada 15% petani yang mengalihkan semua lahan ke pemukiman. Lahan-lahan yang memiliki lokasi dekat dengan jalan raya maka akan memiliki nilai jual lebih tinggi dibandingkan dengan lahan yang letaknya jauh dari jalan raya. Keadaan ini sesuai dengan teori yang diungkapkan oleh Von Thunendimana lokasi merupakan faktor yang menentukan pengunaan lahan.Hal ini pula yang mengindikasikan mengapa petani cenderung mengalih fungsikan lahan sawahnya untuk pemukiman terutama yang berada di pinggir jalan raya. Responden Persentase Responden Persentase Lahan Petani Gurem 3 15% 5 13,51% Pemukiman 3 15% 32 86,48% Lahan Ternak 14 70% - - Jumlah 20 100% 37 100% Lahan yang dijadikan pemukiman sekitar 86 48% petani yang mengalihkan sebagian lahan Lahan yang dijadikan pemukiman sekitar 86,48% petani yang mengalihkan sebagian lahan sawahnya dengan jumlah responden sebanyak 32 orang hal ini tentu saja menunjukkan tinggi alih fungsi lahan ke pemukiman. Jumlah Dijadikan lahan selain untuk pemukiman ada juga beberapa responden petani mengalihkan lahan sawahnya ke sektor usahatani lain yakni sebagai lahan ternak sebanyak 70% bagi petani yang mengalihkan sebagian lahan dan untuk yang mengalihkan semua lahan sawah tidak ada yang menjadikan lahan ternak melainkan 13,51% responden petani yang mengalihkan ke lahan petani gurem, sedangkan 15% reponden dari petani yang mengalihkan semua lahan menjadi lahan pertanian gurem dan hanya ada 15% petani yang mengalihkan semua lahan ke pemukiman. Lahan-lahan yang memiliki lokasi dekat dengan jalan raya maka akan memiliki nilai jual lebih tinggi dibandingkan dengan lahan yang letaknya jauh dari jalan raya. Keadaan ini sesuai dengan teori yang diungkapkan oleh Von Thunendimana lokasi merupakan faktor yang menentukan pengunaan lahan.Hal ini pula yang mengindikasikan mengapa petani cenderung mengalih fungsikan lahan sawahnya untuk pemukiman terutama yang berada di pinggir jalan raya. Lahan yang dijadikan pemukiman sekitar 86,48% petani yang mengalihkan sebagian lahan sawahnya dengan jumlah responden sebanyak 32 orang hal ini tentu saja menunjukkan tinggi alih fungsi lahan ke pemukiman. Dijadikan lahan selain untuk pemukiman ada juga beberapa responden petani mengalihkan lahan sawahnya ke sektor usahatani lain yakni sebagai lahan ternak sebanyak 70% bagi petani yang mengalihkan sebagian lahan dan untuk yang mengalihkan semua lahan sawah tidak ada yang menjadikan lahan ternak melainkan 13,51% responden petani yang mengalihkan ke lahan petani gurem, sedangkan 15% reponden dari petani yang mengalihkan semua lahan menjadi lahan pertanian gurem dan hanya ada 15% petani yang mengalihkan semua lahan ke pemukiman. Lahan-lahan yang memiliki lokasi dekat dengan jalan raya maka akan memiliki nilai jual lebih tinggi dibandingkan dengan lahan yang letaknya jauh dari jalan raya. Keadaan ini sesuai dengan teori yang diungkapkan oleh Von Thunendimana lokasi merupakan faktor yang menentukan pengunaan lahan.Hal ini pula yang mengindikasikan mengapa petani cenderung mengalih fungsikan lahan sawahnya untuk pemukiman terutama yang berada di pinggir jalan raya. Penerimaan Usahatani Luas Lahan Rata-rata (Ha) Produksi Rata-rata (Ton) Produktivitas Rata-rata (Ton/Ha) Penerimaan Rata- ratausahatani (Rp) Sebelum Alih Funsi Lahan 0,33 1,08 3,22 1.500.000 Sesudah Alih Fungsi Lahan 0,28 0,34 2,21 720.541 Jumlah 0,6 1,42 5,43 2.220.541 Dari tabel sudah terlihat dengan jelas perbedaaan penerimaantan usahatani petani yang Penerimaan Usahatani Penerimaan usahatani dikatakan sebagai hasil dari suatu kegitan usahatani yang dapat meningkatkan pendapatan rumah tangga petani. Namun pada hakekatnya para petani yang dulunya sangat bergantung pada sektor pertanian terutama menggarap sawah sekarang mulai meninggalkan dan beralih pada usaha lain. Beberapa dari petani sampai harus mengalihkan sawahnya untuk dijadikan pemukiman. Para petani menilai penerimaan yang dihasilkan dari kegiatan berusahatani tak mampu lagi menghasilkan atau dalam kata lain penerrimaan yang didapatkan tak sesuai dengan usaha yang dirasa oleh para petani. Beberapa petani ada yang memilih membangun usaha lain, seperti menjadi pedagang, bertani ladang, menjadi TKI di Negara tetangga dan memilih bidang pekerjaan lainnya. Berikut ini Tabel 9 dapat dilihat perbedaan penerimaan usahatani petani yang mengalihkan sebagian lahan sawah dan yang mengalihkan semua lahan sawah di daerah penelian pada tahun 2015. Tabel 9. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya Pada Tahun 2015. Luas Lahan Rata-rata (Ha) Produksi Rata-rata (Ton) Produktivitas Rata-rata (Ton/Ha) Penerimaan Rata- ratausahatani (Rp) Sebelum Alih Funsi Lahan 0,33 1,08 3,22 1.500.000 Sesudah Alih Fungsi Lahan 0,28 0,34 2,21 720.541 Jumlah 0,6 1,42 5,43 2.220.541 Dari tabel sudah terlihat dengan jelas perbedaaan penerimaantan usahatani petani yang mengalihkan sebagian lahan sawah sebelum mengalih fungsikan lahan dan sesudah alih fungsi lahan sawanya. Jika sebelum alih fungsi lahan luas lahan sawah rata-rata 0,33 ha dapat menghasilkan 1,08 ton dan produktivitasnya sekitar 3,22 dengan jumlah penerimaan Rp 1.500.000,- setiap satu kali masa panen. Sementara yang terjadi sesudah alih fungsi lahan sawah terjadi pengurangan jumlah lahan rata- rata yang ada hanya 0,27 ha dengan produksi hanya 0,34 ton dan produktivitas hanya mampu menyentuh angka 2,20 ton/ha serta penerimaan Rp 720.541,- saja. Sedangkan pada Tabel 10 dibawah dapat dilihat perbedaan penerimaan usahatani petani yang mengalihkan semua lahan sawahnya sebagai berikut.Pada saat Peneliti ke lapangan penerimaan usahatani yang sedemikian tidak menjadi sumber utama bagi pendapatan mereka, petani yang mengalihkan sebagian lahan sawah beralasan hanya berusahatani seadanya saja demi tetap menjaga produksi padi lokal walau hanya bisa menggarap sedikit lahan sawahnya sementara sebagian dijadikan pemukiman. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya Pada Tahun 2015. Tabel 9. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya Tabel 9. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya Pada Tahun 2015. Jumlah Dari tabel sudah terlihat dengan jelas perbedaaan penerimaantan usahatani petani yang mengalihkan sebagian lahan sawah sebelum mengalih fungsikan lahan dan sesudah alih fungsi lahan sawanya. Jika sebelum alih fungsi lahan luas lahan sawah rata-rata 0,33 ha dapat menghasilkan 1,08 ton dan produktivitasnya sekitar 3,22 dengan jumlah penerimaan Rp 1.500.000,- setiap satu kali masa panen. Sementara yang terjadi sesudah alih fungsi lahan sawah terjadi pengurangan jumlah lahan rata- rata yang ada hanya 0,27 ha dengan produksi hanya 0,34 ton dan produktivitas hanya mampu menyentuh angka 2,20 ton/ha serta penerimaan Rp 720.541,- saja. Sedangkan pada Tabel 10 dibawah dapat dilihat perbedaan penerimaan usahatani petani yang mengalihkan semua lahan sawahnya sebagai berikut.Pada saat Peneliti ke lapangan penerimaan usahatani yang sedemikian tidak menjadi sumber utama bagi pendapatan mereka, petani yang mengalihkan sebagian lahan sawah beralasan hanya berusahatani seadanya saja demi tetap menjaga produksi padi lokal walau hanya bisa menggarap sedikit lahan sawahnya sementara sebagian dijadikan pemukiman. - 9 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS Tabel 10. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Semua Lahan Sawahnya Pada Tahun 2015. Luas Lahan Rata-rata (Ha) Produksi Rata-rata (Ton) Produktivitas Rata-rata (Ton/Ha) Penerimaan usahatani Rata-rata (Rp) Sebelum Alih Funsi Lahan 0,30 1,075 3,15 1.460.000 Sesudah Alih Fungsi Lahan 0,30 yang dialih fungsikan Produksi di sektor usahatani lain Produktivitas disektor usahatani lain 595.000 (selain usahatani padi sawah) bel 10. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Semua Lahan Sawahnya Pada Tahun 2015. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Semua Lahan Sawahnya Pada Tahun 2015 Sangat jelas bahwa sebelum alih fungsi lahan sawah petani dapat menghasilkan rata-rata Rp 1.460.000,- dengan luas lahan 0,30 ha dan produksi yang mencapai 1,075 ton serta Produktivitas yang berada pada angka 3,15 ton/ha. Sedangkan sesudah petani mengalihkan semua lahan sawahnya luas lahan, produksi, produktivitas maupun penerimaan usahataninya berada pada sektor usahatani lain dengan rata-rata penerimaan Rp. 595.000,- .Semakin rendah penerimaan yang diperoleh dari hasil usahatani, maka akan semakin tinggi peluang petani dalam melakukan alih fungsi lahan. Jika penerimaan yang diperoleh dari hasil usahatani rendah maka ada kecenderungan untuk memilih penerimaan diluar sektor pertanian dan lahan yang dimiliki dialih fungsikan karena penerimaan usahatani tidak dapat memenuhi kebutuhan sehari-hari. Jumlah p Berdasarkan hasil penelitian di lapangan faktor-faktor alih fungsi lahan sawah sangat berhubungan dengan perbedaan tingkat penerimaan usahatani dikedua desa tersebut, untuk petani yang mengalihkan sebagian luas lahan sawahnya sebelum alih fungsi penerimaan rata-rata usahataninya dari Rp 1.500.000,- dan sesudah alih fungsi sebagian lahan sawah menjadi Rp 720.541,- sementara untuk petani yang mengalihkan semua luas lahan sawahnya penerimaan usahatani sebelum alih fungsi lahan dengan rata-rata Rp 1.460.000,- dan sesudah alih fungsi semua luas lahan sawhnya menjadi Rp 595.000,-. Kemudian untuk melihat lebih jelas perbedaan pendapatan usahatani yang dihasilkan tersebut dilakukan uji dengan alat uji beda dua rata-rata maka diperoleh 𝑡"#$>𝑡$%& yaitu 3,35 > 1,665 terima 𝐻( tolak 𝐻) untuk petani yang mengalihkan sebagian luas lahan sawahnya, sementara untuk petani yang mengalihkan semua lahan sawahnya 𝑡"#$>𝑡$%& yaitu 4,02 > 3,086 terima 𝐻( tolak 𝐻) artinya terdapat derajad perbedaan yang nyata antara penerimaan usahatani sebelum dan sesudah alih fungsi lahan sawah, dimana penerimaan usahatani sebelum alih fungsi lahan lebih besar daripada penerimaan usahatani sesudah alih fungsi lahan. KESIMPULAN Dari hasil penelitian yang telah dilaksanakan, maka dapat disimpulkan bahwa: 1). Faktor-faktor yang mempengaruhi alih fungsi lahan sawah terdiri dari lama pendidikan, umur petani, luas lahan, pengalaman bertani, jumlah tanggungan. 2). Hasil analisis uji beda dua rata-rata, maka diperoleh thitung > ttabel yaitu 3,35 > 1,665 terima H1 tolak H0 untuk petani yang mengalihkan sebagian luas lahan sawahnya, sementara untuk petani yang mengalihkan semua lahan sawahnya thitung > ttabel yaitu 4,02 > 1,665 terima H1 tolak H0 artinya terdapat derajat perbedaan yang nyata antara penerimaan usahatani sebelum dan sesudah alih fungsi lahan sawah, dimana penerimaan usahatani sebelum alih fungsi lahan lebih besar daripada penerimaan usahatani sesudah alih fungsi lahan. Terdapat perbedaan penerimaan usahatani petani sebelum alih fungsi lahan dan sesudah alih fungsi lahan yaitu dari Rata-rata penerimaan usahatani yang awalnya Rp. 1.500.000,-/ rata- rata 0,33 ha luas lahan turun menjadi Rp. 720.541,-/ rata- rata 0,27 ha atau ± Rp. 4.500.000,- / 1 ha luas lahan pada satu kali musim tanam, ini terjadi pada petani yang mengalihkan sebagian lahan sementara yang mengalihkan semua lahan penerimaan usahatani sebelum alih fungsi lahan yakni Rp. 1.460.000,-/ rata-rata 0,30 ha lahan atau ± Rp. 5.600.000,-/ 1 lahan menjadi Rp. 595.000/ 0,30 ha atau ± Rp. 1.800.000 / 1 ha lahan yang telah dialih fungsi ke sektor usahatani lain pada satu kali musim tanam. UCAPAN TERIMAKASIH - 10 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018 DOI: 10.22437/jiseb.v21i1 Ucapan terimakasih disampaikan kepada Dekan Fakultas Pertanian dan ketua Program Studi Agribisnis Fakultas Pertanian Universitas Jambi yang telah memfasilitasi pelaksanaan penelitian ini. Terimakasih kepada dosen pembimbing akademik ibu Ir. Adlaida Malik, Ms. Selain itu saya ucapkan terimakasih kepada Bapak Camat Keliling Danau yang telah sudi memberikan segala informasi dalam membantu penelitian ini. Kemudian ucapan terimakasih kepada Bapak Mukhsin sebagai Kepala Desa Koto Dian dan Bapak Erizal selaku Kepala Desa Koto tuo yang telah member izin untuk melakukan penelitian di Desa Koto Dian dan Koto Tuo dengan judul Hubungan Faktor-faktor Alih fungsi Lahan Padi Sawah dan Perbedaan Tingkat Penerimaan Usahatani Petani di Kecamatan Keliling Danau Kabupaten Kerinci. DAFTAR PUSTAKA Badan Pusat Statistik Provinsi Jambi. 2013. Jambi Dalam Angka. Jambi Badan Pusat Statistik Provinsi Jambi. 2013. Keliling Danau Dalam angka.Jambi. Barlowe R. 1978. Land Resource economics. Third edition.Prentice. Hall inc, New jersey. Hasan, Iqbal. Analisi Data Penelitian dengan Statistik. PT Bumi Aksara. Jakarta Kacabdis Pertanian Dan Perkebunan Tanaman Pangan Kecamatan Keliling Danau. Kerinci.Jambi. Mosher, A.T. 1983.Menggerakkan dan Membangun Pertanian. CV Yasaguna. Jakarta Nazir M. 1988. Metode Penelitian. Ghalia Indonesia, Jakarta. Utomo. 1992. Konversi Lahan Sawah di Indonesia.ITB. Bandung. Winoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bogor. Winoto.2005. Konversi Lahan Sawah Di Indonesia. Penebar Swadaya Badan Pusat Statistik Provinsi Jambi. 2013. Jambi Dalam Angka. Jambi Badan Pusat Statistik Provinsi Jambi. 2013. Keliling Danau Dalam angka.Jambi. Barlowe R. 1978. Land Resource economics. Third edition.Prentice. Hall inc, New jersey. Hasan, Iqbal. Analisi Data Penelitian dengan Statistik. PT Bumi Aksara. Jakarta Kacabdis Pertanian Dan Perkebunan Tanaman Pangan Kecamatan Keliling Danau. Kerinci.Jambi. Mosher, A.T. 1983.Menggerakkan dan Membangun Pertanian. CV Yasaguna. Jakarta Nazir M. 1988. Metode Penelitian. Ghalia Indonesia, Jakarta. Utomo. 1992. Konversi Lahan Sawah di Indonesia.ITB. Bandung. Winoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bogor. Winoto.2005. Konversi Lahan Sawah Di Indonesia. Penebar Swadaya Badan Pusat Statistik Provinsi Jambi. 2013. Jambi Dalam Angka. Jambi Badan Pusat Statistik Provinsi Jambi. 2013. Keliling Danau Dalam angka.Jambi. Barlowe R. 1978. Land Resource economics. Third edition.Prentice. Hall inc, New jersey. Hasan, Iqbal. Analisi Data Penelitian dengan Statistik. PT Bumi Aksara. Jakarta Kacabdis Pertanian Dan Perkebunan Tanaman Pangan Kecamatan Keliling Danau. Kerinci.Jambi. Mosher, A.T. 1983.Menggerakkan dan Membangun Pertanian. CV Yasaguna. Jakarta Nazir M. 1988. Metode Penelitian. Ghalia Indonesia, Jakarta. Utomo. 1992. Konversi Lahan Sawah di Indonesia.ITB. Bandung. Winoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bog Winoto.2005. Konversi Lahan Sawah Di Indonesia. Penebar Swadaya inoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bogor. - 11 -
https://openalex.org/W2444513720
https://academiccommons.columbia.edu/doi/10.7916/D8SN0MZQ/download
English
null
Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji
Scientific reports
2,016
cc-by
9,156
Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji received: 28 July 2015 accepted: 07 December 2015 Published: 25 January 2016 Erin K. Eastwood1,†,*, Elora H. López1,‡,* & Joshua A. Drew1,2 Erin K. Eastwood1,†,*, Elora H. López1,‡,* & Joshua A. Drew1,2 Coral reef fish serve as food sources to coastal communities worldwide, yet are vulnerable to mounting anthropogenic pressures like overfishing and climate change. Marine reserve networks have become important tools for mitigating these pressures, and one of the most critical factors in determining their spatial design is the degree of connectivity among different populations of species prioritized for protection. To help inform the spatial design of an expanded reserve network in Fiji, we used rapidly evolving mitochondrial genes to investigate connectivity patterns of three coral reef species targeted by fisheries in Fiji: Epinephelus merra (Serranidae), Halichoeres trimaculatus (Labridae), and Holothuria atra (Holothuriidae). The two fish species, E. merra and Ha. trimaculatus, exhibited low genetic structuring and high amounts of gene flow, whereas the sea cucumber Ho. atra displayed high genetic partitioning and predominantly westward gene flow. The idiosyncratic patterns observed among these species indicate that patterns of connectivity in Fiji are likely determined by a combination of oceanographic and ecological characteristics. Our data indicate that in the cases of species with high connectivity, other factors such as representation or political availability may dictate where reserves are placed. In low connectivity species, ensuring upstream and downstream connections is critical. Coral reefs are some of the planet’s most complex and diverse ecosystems, providing nearly US$ 30 billion in goods and services to global economies annually, through tourism, fisheries, and coastal protection1. However, the vivid splendor of these ecosystems is subject to numerous anthropogenic stressors including overfishing, pol- lution, sedimentation, and climate change, and nearly every coral reef worldwide is currently affected by human activities2–4. One tool commonly used to mitigate global reef degradation is the creation of marine protected areas (MPAs) – spatially explicit areas of ocean where human activities are regulated or prohibited. One subset of MPAs are no-take marine reserves, wherein direct harvesting of marine resources is prohibited both spatially and tempo- rally. When effectively implemented, no-take marine reserves are important tools for conservation and fisheries management. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 28 July 2015 accepted: 07 December 2015 Published: 25 January 2016 Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji They provide a refuge for exploited species, allow for fish and invertebrate stocks to recover, repro- duce, and reseed adjacent unprotected areas through larval export and adult movement, while they protecting existing habitat from further degradation5–8.i g g Networks of reserves confer additional benefits, protecting a greater diversity of habitats and promoting the stability of meta-populations9,10. The optimal orientation for reserve networks requires information concerning the degree of demographic or larval exchange occurring between populations11,12. Reserve networks that explic- itly incorporate connectivity in their design are more resilient to threats like overfishing, disease, and climate change because neighboring reserves can help reseed reefs in the event of disturbances, thereby boosting the stability of the system as a whole11,13,14. 1Department of Ecology, Evolution, and Environmental Biology, Columbia University, NY. 2Department of Vertebrate Zoology, American Museum of Natural History, NY. †Present address: National Oceanic and Atmospheric Administration, Climate Program Office, 1315 East-West Highway, Silver Spring MD 20910. ‡Present address: Department of Biology, Stanford University, Hopkins Marine Station, Pacific Grove, CA. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.A.D. (email: j.drew@ columbia.edu) Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 1 www.nature.com/scientificreports/ Figure 1. Sampling locations within Fiji, with island names in bold and village names adjacent to points. Division boundaries and path of the Bligh Water Current (denoted by red arrows) are approximate. Map © d-maps.com available at http://dmaps.com/carte.php?num_car= 28136&lang= en Figure 1. Sampling locations within Fiji, with island names in bold and village names adjacent to points. Division boundaries and path of the Bligh Water Current (denoted by red arrows) are approximate. Map © d-maps.com available at http://dmaps.com/carte.php?num_car= 28136&lang= en Species’ behaviour, locomotion, life history, and habitat all play into how organisms interact with their phys- ical environment, and thus influence connectivity. The larvae of many reef fishes possess an array of sensory modalities and the physiological capacity for directional swimming that allows for non-random settlement15–18, and some species with prolonged pelagic larval stages display natal reef preference and genetic structuring across populations18,19. Despite the capabilities of some larvae, oceanic currents can limit genetic connectivity between populations if individuals are unable to traverse those currents, as this reduces gene flow20–23. Conversely, ocean currents can promote connectivity if they transport larvae from upstream to downstream sites, thus generating asymmetrical gene flow24. Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji y gl In 2005, the Fiji government set the goal of protecting 30% of its marine waters by 202025. As of 2010, local management had effectively protected a considerable proportion of Fiji’s priority ecosystems for management26. However, the amount of area that can be protected by ad-hoc community-based management is nearing satura- tion, and these efforts need to be scaled up significantly if the national goal is to be reached26. Because of this, a systematic conservation planning approach that includes consideration of the connectivity and complementarity of potential reserve sites across the entirety of Fiji has been recommended27. Additional scientific input, particu- larly information concerning population connectivity, will be necessary if an expanded network of MPAs is to effectively utilize limited conservation resources in the country.l f y y Previous work on connectivity among reefs in Fiji revealed asymmetrical gene flow along an east-west gradi- ent for three of five coral reef fish species studied, potentially indicating that the Bligh Waters – a fast-moving cur- rent that bisects the main islands – could be facilitating larval transport for multiple taxa within Fiji28. However, this study was based on a suite of species that were not fisheries targets. A more relevant framework for conser- vation planning might instead be crafted based on the connectivity dynamics of economically and nutrition- ally significant species. Small-scale inshore fisheries provide food for about 50% of rural households in Fiji and contribute over $48 million to the national economy every year29,30, so the sustainable management of species contributing to these fisheries is paramount for ensuring food security. Here, we expand upon previous work and investigate the role of the Bligh Waters in shaping the connectivity patterns of three coral reef species commonly targeted by inshore fisheries across the Fijian archipelago (Fig. 1) – the honeycomb grouper Epinephelus merra (Serranidae), the three-spot wrasse Halichoeres trimaculatus (Labridae), and the black sea cucumber Holothuria atra (Holothuriidae). Results G ti atra populations at the island- assemblage level in Fiji. Significant p-values are in bold. Vanua Levu Taveuni Naviti Vanuabalavu (A) E. merra Vanua Levu –   Taveuni 0.00000 –   Naviti 0.05327 0.01336 –   Vanuabalavu 0.00702 0.00000 0.00000 –   Viti Levu 0.00602 0.00000 0.00602 0.00000 (B) Ha. trimaculatus Vanua Levu –   Taveuni 0.00547 –   Naviti 0.00000 0.00000 –   Vanuabalavu 0.00000 0.00000 0.00000 –   Viti Levu 0.07232 0.00196 0.02021 0.01350 (C) Ho. atra Vanua Levu –   Taveuni 0.3287 –   Naviti 0.0865 0.00000 –   Vanuabalavu 0.5042 0.00000 0.1395 – Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. atra populations at the island- assemblage level in Fiji. Significant p-values are in bold. Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. atra populations at the island- assemblage level in Fiji. Significant p-values are in bold. Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. atra populations at the island- assemblage level in Fiji. Significant p-values are in bold. Population Structure and Power Analyses. E. merra was characterized by very low and non-significant measures of genetic differentiation at the island level (pairwise ΦST values in Table 3A), after an AMOVA grouping the two Vanuabalavu sites together (Naracivo and Donicali) resulted in non-significant F-statistics for among-group (FCT), within-group (FSC), and within-population (FST) genetic differentiation (Table 4A). Sampling locations were also assembled into 4 groups that reflect the political units of Fiji (e.g. grouping Nagigi and Naselesele together, as they both belong to the Northern Division), and this second AMOVA also revealed non-significant F-statistics for each hierarchical level (Table 4B). gi Ha. trimaculatus displayed slightly different patterns of genetic partitioning. This species’ pairwise ΦST values were similarly low but exhibited significant north-south genetic partitioning between the Vanua Levu village of Nagigi and the Viti Levu village of Nabukavese (ΦST =  0.07 p =  0.032, Table 3B). An AMOVA clustering Ha. trimaculatus populations into 4 groups reflecting political units within Fiji also revealed non-significant F-statistics for each hierarchical level (Table 4C). Analysis of Ho. atra populations returned a wide range of pairwise ΦST values (Table 3C). Very low values were observed between Taveuni and both the eastern island of Vanuabalavu and the western island and Naviti, and relatively high values between Vanua Levu and all other island sites. The highest pairwise ΦST value occurred between Vanua Levu and Vanuabalavu. Results G ti Genetic Diversity. We sequenced 475 bp of the mtDNA control region from 82 individuals of E. merra, 376 bp of the same locus from 91 individuals of Ha. trimaculatus, and 376 bp of the COI gene from 40 individuals of Ho. atra (Table 1). Both fish species displayed high haplotype diversity and had highly polymorphic control regions (87 and 71 polymorphic loci within E. merra and Ha. trimaculatus sequences, respectively), while Ho. atra COI sequences were considerably less genetically diverse (Table 2) and had only 15 polymorphic loci. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 2 www.nature.com/scientificreports/ Division Island (Village) Latitude, Longitude Epinephelus merra (N =  82) Halichoeres trimaculatus (N =  91) Holothuria atra (N =  40) Northern Vanua Levu (Nagigi) S 16° 80’ 44.18” W 179° 48'05.53” 20 23 10 Northern Taveuni (Naselesele) S 16° 41’ 41.19” W 179° 52’ 1.52” 12 21 5 Western Naviti (Malevu) S 17° 6’ 54.12” E 177° 16’ 51.18” 11 16 7 Central Viti Levu (Nabukavesi) S 18° 13’ 20.42” E 178° 15’ 58.08” 16 15 – Eastern Vanuabalavu (Naracivo, Donicali) S 17° 13’ 15.84” W 178° 57’ 53.57” 23 16 18 Table 1. Geographic locations of sampling sites, and sample sizes for each species. Table 1. Geographic locations of sampling sites, and sample sizes for each species. Table 1. Geographic locations of sampling sites, and sample sizes for each species. Table 1. Geographic locations of sampling sites, and sample sizes for each species. Species Individuals Haplotypes π Θs (SD) Sequence Length Larval Duration Epinephelus merra 82 66 0.03 16.67 (4.51) 475 39 days Halichoeres trimaculatus 91 75 0.03 13.97 (3.77) 376 27 days Holothuria atra 40 8 0.01 3.18 (1.61) 376 18–25 days Table 2. Results of sequencing the mitochondrial control region, and larval durations for each study species64,73,74. Table 2. Results of sequencing the mitochondrial control region, and larval durations for each study species64,73,74. Vanua Levu Taveuni Naviti Vanuabalavu (A) E. merra Vanua Levu –   Taveuni 0.00000 –   Naviti 0.05327 0.01336 –   Vanuabalavu 0.00702 0.00000 0.00000 –   Viti Levu 0.00602 0.00000 0.00602 0.00000 (B) Ha. trimaculatus Vanua Levu –   Taveuni 0.00547 –   Naviti 0.00000 0.00000 –   Vanuabalavu 0.00000 0.00000 0.00000 –   Viti Levu 0.07232 0.00196 0.02021 0.01350 (C) Ho. atra Vanua Levu –   Taveuni 0.3287 –   Naviti 0.0865 0.00000 –   Vanuabalavu 0.5042 0.00000 0.1395 – Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 Results G ti Analysis of molecular variance using the mitochondrial control region between (A) five groups of E. merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages, (B) four groups of E. merra (Northern Division, Eastern Division, Western Division, Central Division) reflecting political boundaries, and (C) four groups of Ha. trimaculatus (Northern Division, Eastern Division, Western Division, Central Division) reflecting political boundaries. Negative values are presented, but are effectively equal to zero. Figure 2. Haplotype network of Ho. atra samples. Each circle represents a haplotype. Size of the circle is scaled to the number of individuals that share that haplotype, Hash marks on connecting lines indicate the number of base pair differences between haplotypes. Shading represents the island where individuals were sampled. Figure 2. Haplotype network of Ho. atra samples. Each circle represents a haplotype. Size of the circle is scaled to the number of individuals that share that haplotype, Hash marks on connecting lines indicate the number of base pair differences between haplotypes. Shading represents the island where individuals were sampled. island of Naviti had the highest number of individuals with private alleles, and the eastern island of Vanuabalavu had the lowest measures of intra-population genetic diversity.i Results of the statistical power analyses revealed that in both fish species, the control region markers had sufficient power to detect genetic differentiation above a ΦST value of 0.01, which corresponds with low levels of genetic differentiation and high amounts of connectivity (Fig. 3)31. Migration Estimates. MIGRATE analyses resolved high levels of migration but with complex patterns of directionality among populations. For E. merra, the individuals from Nabukavesi, located on the southern large island of Viti Levu appear to be a major source population, providing high numbers of migrants per generation to other localities – in some cases, up to 10 times the amount migrants received (Table 5). These patterns are the opposite of those observed in Ha. trimaculatus – with the site on northern large island of Vanua Levu serving as a source and Viti Levu as a sink (Table 5). Additionally, in both species the western island of Naviti provided more migrants per generation to the far eastern island of Vanuabalavu than vice versa (3.5 times more in E. merra, 2.5 times more in Ha. trimaculatus).i MIGRATE analyses of Ho. Results G ti Because genetic partitioning was detected between the same-Division islands of Vanua Levu and Taveuni at this initial step, an AMOVA further grouping locations together was not performed. Further, the majority of Ho. atra samples (n =  35) fit into one of three haplotypes (Fig. 2). The most highly represented haplotype (n =  29) was found in individuals from all four sampling locations. The western Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 3 www.nature.com/scientificreports/ Source of variation d.f. Sum of squares Percentage of variation Statistics P (A) Among groups 4 22.069 − 2.02 FCT =  − 0.020 0.785 Among populations within groups 1 6.865 1.98 FSC =  0.019 0.218 Within populations 76 437.262 100.04 FST =  0.000 0.424 (B) Among groups 3 17.781 1.59 FCT =  0.016 0.298 Among populations within groups 1 4.288 − 1.71 FSC =  − 0.017 0.617 Within populations 77 444.127 100.13 FST =  − 0.001 0.490 (C) Among groups 3 14.387 − 0.10 FCT =  − 0.001 0.723 Among populations within groups 1 5.034 0.61 FSC =  0.006 0.295 Within populations 85 377.046 99.49 FST =  0.005 0.281 Table 4. Analysis of molecular variance using the mitochondrial control region between (A) five groups of E. merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages, (B) four groups of E. merra (Northern Division, Eastern Division, Western Division, Central Division) reflecting political boundaries, and (C) four groups of Ha. trimaculatus (Northern Division, Eastern Division, Western Division, Central Division) reflecting political boundaries. Negative values are presented, but are effectively equal to zero. Source of variation d.f. Sum of squares Percentage of variation Statistics P (A) Among groups 4 22.069 − 2.02 FCT =  − 0.020 0.785 Among populations within groups 1 6.865 1.98 FSC =  0.019 0.218 Within populations 76 437.262 100.04 FST =  0.000 0.424 (B) Among groups 3 17.781 1.59 FCT =  0.016 0.298 Among populations within groups 1 4.288 − 1.71 FSC =  − 0.017 0.617 Within populations 77 444.127 100.13 FST =  − 0.001 0.490 (C) Among groups 3 14.387 − 0.10 FCT =  − 0.001 0.723 Among populations within groups 1 5.034 0.61 FSC =  0.006 0.295 Within populations 85 377.046 99.49 FST =  0.005 0.281 Table 4. Analysis of molecular variance using the mitochondrial control region between (A) five groups of . merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages, Table 4. Table 4.  Analysis of molecular variance using the mitochondrial control region between (A) five groups of E. merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages, (B) four groups of E. merra (Northern Division, Eastern Division, Western Division, Central Division) reflecting political boundaries, and (C) four groups of Ha. trimaculatus (Northern Division, Eastern Division, Western Division, Central Division) reflecting political boundaries. Negative values are presented, but are effectively equal to zero. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. POWSIM analysis results showing power to detect structure among populations of Epinephelus merra and Halichoeres trimaculatus at different FST levels. Power is expressed as the proportion of significant outcomes after 1,000 replicates. Dashed line is at 0.80, the minimum acceptable power level put forth by72. Figure 3. POWSIM analysis results showing power to detect structure among populations of Epinephelus merra and Halichoeres trimaculatus at different FST levels. Power is expressed as the proportion of significant outcomes after 1,000 replicates. Dashed line is at 0.80, the minimum acceptable power level put forth by72. Species Vanua Levu Taveuni Naviti Vanuabalavu Viti Levu E. merra Vanua Levu – 5850.0 543.3 896.7 576.7 Taveuni 3183.3 – 276.7 1516.7 1256.7 Naviti 7490.0 2676.7 – 656.7 690.0 Vanuabalavu 6536.7 2436.7 190.0 – 2023.3 Viti Levu 5530.0 1403.3 116.7 1790.0 – Ha. trimaculatus Vanua Levu – 296.7 916.7 1683.3 1050.0 Taveuni 523.3 – 683.3 450.0 1330.0 Naviti 736.7 450.0 – 1636.7 3930.0 Vanuabalavu 316.7 430.0 623.3 – 1936.7 Viti Levu 550.0 516.7 1123.3 1250.0 – Ho. atra Vanua Levu – 522.3 479.7 555 – Taveuni 455 – 514.3 411.7 – Naviti 438.3 487 – 388.3 – Vanuabalavu 533 672.3 582.3 – – Table 5. Results of MIGRATE analysis for each species. Median number of recruits per generation is shown, with migration occurring from row to column Table 5. Results of MIGRATE analysis for each species. Median number of recruits per generation is shown, with migration occurring from row to column. Table 5. Results of MIGRATE analysis for each species. Median number of recruits per generation is shown, with migration occurring from row to column. Finally, the Mantel test revealed negative or flat levels of correlation between pairwise measures genetic differentiation and geographic distance in all three species (E. merra R2 =  0.001, Ha. trimaculatus R2 =  0.005, Ho. atra R2 =  0.010), indicating that none of these species’ patterns of genetic differentiation can be explained by geographic distance between populations. Results G ti atra sequences showed that three of the five highest migration values were from the eastern island of Vanuabalavu out to more western sites. The highest estimated median number of migrants per generation was from Vanuabalavu to Taveuni (672.3), while the lowest estimated number of migrants per generation was from Malevu to Vanuabalavu (388.3-Table 5). There were more westward moving migrants than eastward moving migrants for four of the six pairs of sites. (Table 3). Tajima’s D values were negative for both E. merra and Ha. trimaculatus (−1.15228 and −1.20336, respec- tively), and positive for Ho. atra (0.71194). All values were non-significant (p =  0.109, p =  0.094, p =  0.712, respectively). Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 4 Discussionf More oceanographic investigation is necessary to understand if larvae spawned at Taveuni are brought out to sea by a current that bypasses the reefs on the southern edge of Vanua Levu. Skillings et al. 2011 found Ho. atra to be genetically differentiated across distances comparable to that between Vanua Levu and Taveuni (74.8 km), suggesting that there are many other factors besides this species’ larval potential to drift great distances that determine larval dispersal and consequent connectivity.h g p q y The most frequent Ho. atra haplotype, accounting for almost 58% of samples, is found in all four sampled loca- tions, suggesting an overall trend of connectivity across the region despite observed differences in the degree of connectivity in each pair of sites. The boom-bust demographic cycle of Ho. atra38 may explain how all populations came to share the dominant haplotype, but still display levels of differentiation between populations. This shared haplotype may be the vestige of a time when there were large population sizes and high rates of migration, while the varying levels of genetic differentiation may be the result of subsequent population contraction and lesser gene flow across the archipelago. This is supported by Ho. atra’s positive and non-significant Tajima’s D, which indicates that the sea cucumber populations studied may have undergone recent demographic contraction39. p p y g g p Results of the MIGRATE analyses were complex and idiosyncratic among all three species studied (Table 5). Gene flow patterns support a scenario of high connectivity for both fish species, with the magnitude of migrants exchanged among populations estimated at hundreds to thousands per generation. Patterns of directionality in E. merra and Ha. trimaculatus were complex and occasionally contradictory – for instance, Vanua Levu exported more migrants than it received from other locations in E. merra, whereas in Ha. trimaculatus the opposite was true. Both fish species also exhibited higher numbers of migrants coming from the western locality of Naviti and settling in the eastern island of Vanuabalavu than vice versa, indicating that western populations could be seeding eastern localities in spite of the prevailing westward-moving oceanic currents. Ho. atra shows a different pattern, in which the larvae seem to follow the oceanic currents resulting in the eastern island of Vanuabalavu exporting higher numbers of larval migrants to the western localities than vice versa. Discussionf This concurs with previous studies showing unidirectional patterns of east-to-west gene flow in several taxa28. Ho. atra’s east to west pattern of gene flow is also supported by the haplotype diversities of each location, as the western population of Naviti, in the Yasawa islands, has the highest intra-population genetic diversity while Vanuabalavu, which lies farthest to the east in the Lau group, has the lowest. This suggests that larvae spawned in Nagigi, Taveuni, Vanuabalavu and other unsampled populations may potentially recruit more often to reefs in the western Yasawa Islands, promoting high genetic diversity. These contrasting patterns of gene flow observed between the fishes and the sea cucumber could be due to demographic stochasticity, differences in reproductive mode or timing, or seasonal variation in the flow of the Bligh Waters, which is common in other intra-pelagic currents40.h g p g The dearth of literature on the inter- and intra-annual physical oceanography of the Fijian archipelago makes it difficult to assess the role of variation in currents on the data presented here. Genetic connectivity studies conducted in regions with better-studied oceanography have shown both inter-annual and intra-annual ocean- ographic features to be responsible for population connectivity or structure. Galarza et al.41 found oceanic fronts in the Mediterranean Sea to be associated with genetic structuring for several fish species, presumably due to the inability of larvae spawned on one side of the front to disperse across the front and admix with populations on the other side. Similarly, the Kuroshio Current in the East China Sea limits dispersal and therefore generates population differentiation between mudskippers in China and those in Japan42. Mesoscale eddies, which occur on a length scale of less than 100 km and last less than one month, have been implicated in keeping newly spawned larvae close to their natal site, as opposed to dispersing out into the open ocean43. Interpretations of genetic structuring and connectivity patterns found across species studied in the Fijian archipelago, as well as the idiosyn- crasies in the data, almost certainly require a better understanding of the underlying oceanography. This clearly presents a priority for future research.hi p p y The Tajima’s D values for both E. merra and Ha. trimaculatus were strongly negative and while non-significant, they may indicate that both species may have undergone rapid population expansion in recent evolutionary his- tory. Discussionf Different patterns of connectivity were observed for each of the three species in this study, highlighting the com- plexity of larval transport and population demography in the marine environment. High amounts of gene flow and very low amounts of genetic structure were observed for both fish species, indicating that on the relatively small spatial scale of the Fijian archipelago, populations of E. merra and Ha. trimaculatus are interconnected. High gene flow is a common phenomenon in marine species, as the marine environment has relatively few phys- ical barriers and long pelagic larval durations often promote long-distance dispersal32–34.h t The high level of connectivity we observed among populations of E. merra is not surprising, and mirrors con- nectivity levels found in the Philippines35 and the Western Indian Ocean36. Ha. trimaculatus generally exhibited high levels of connectivity among populations as well, but this species did show a subtle yet significant amount of genetic partitioning between the northern island of Vanua Levu and southern island of Viti Levu. These findings support the hypothesis that the Bligh Waters may be a major oceanographic feature affecting connectivity in Fiji – a |pattern observed in a previous comparative phylogeographic study28.l Fast-flowing currents like the Bligh Waters can act as barriers to dispersal by advecting larvae out of the system before they are able to settle on reefs opposite of the current, thus preventing genetic exchange between popu- lations20–23. Ha. trimaculatus’ low amount of genetic partitioning may indicate that the Bligh Waters has been acting as an oceanographic barrier for a relatively short evolutionary time scale, or that historical factors could Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 5 www.nature.com/scientificreports/ also have contributed to this species’ subtle phylogeographic structure – for instance, sea level shifts influencing habitat availability37. y In contrast, the sea cucumber Ho. atra exhibited complex patterns of connectivity among the four populations sampled. There was little to no genetic differentiation between Taveuni and the islands of the Lau group, but high amounts of partitioning between Taveuni and Vanua Levu despite these two locations’ geographic proximity. The populations in the Lau group were also genetically divergent from those in Vanua Levu. This may be due to oceanographic characteristics of the Somosomo Strait, the narrow channel that passes between the islands of Taveuni and Vanua Levu. www.nature.com/scientificreports/ www.nature.com/scientificreports/ study did miss populations exhibiting partitioning at these levels, the conclusions drawn would be effectively the same – that both fish species exhibit high amounts of connectivity between their populations31. study did miss populations exhibiting partitioning at these levels, the conclusions drawn would be effectively the same – that both fish species exhibit high amounts of connectivity between their populations31. i p g y p p In Ho. atra, on the other hand, this study observed very low haplotype diversities across all locations, even in Vanuabalavu where sample sizes were high (n =  18). While low sample sizes understandably lead to low observed genetic diversity, it is also likely that this species’ reproductive ecology (e.g. fission, or budding) has contributed to higher local densities of genetically identical individuals than in exclusively sexually reproducing species. Thus, while results for this species should certainly be taken with caution, it is possible that low sample sizes in this study may be capturing an adequate amount of the gene pool at each location.t y y p g q g p Critiques for mtDNA-based phylogeographic studies often concern the variable rate of mutation observed between these markers and others – mtDNA often reach higher FST values than nuclear DNA45 – and the lack of power caused by high levels of diversity46. However, rapid mutation rates of mtDNA have been shown to provide high sensitivity for detecting genetic differentiation on short evolutionary time scales, rendering these markers highly useful for studies like this one investigating population-level connectivity47–49. In addition, a recent anal- ysis has shown that mtDNA markers consistently display higher power to detect population divergence than any single nuclear marker when sample sizes are sufficient50. As a result, mtDNA sequences have been the markers of choice for marine connectivity studies in the Indo-Pacific for the past 20 years51. While multi-locus analyses certainly allow for a more nuanced evaluation of connectivity, it is not necessarily the case that adding nuclear loci would reveal significantly more information52. gi y Additionally, use of markers that have been used for previous phylogeography studies allows for easier com- parison of results across taxa and locations51. The control region was used to analyse genetic connectivity in the only other study of fish biogeography in Fiji28, and thus we chose this ‘legacy marker’ for our analyses of the fish data. www.nature.com/scientificreports/ Similarly, the COI was used in a Hawai’ian biogeography study of Ho. atra53, so we chose to use the same marker in this study for the sake of comparison. Using relatively inexpensive markers and technologies for analy- ses in biogeography studies makes such studies more doable across underrepresented taxa and regions, and thus broadens the scope of known genetic connectivity patterns across the Pacific. This is especially critical from a management perspective. Conclusions and Management Implicationsh The high connectivity observed among populations of the fish species suggests that prioritizing one region over another for the placement of marine reserves in Fiji is not necessary for the conservation and persistence of E. merra and Ha. trimaculatus populations. Instead, each region sampled should be considered equally important for protection, as each node in these meta-populations contributes ecologically and economically significant amounts of migrants to the others. This finding provides managers with greater flexibility in conservation pri- oritization, as they can focus on areas that have strong local support for conservation, or areas like the Vatu-i-Ra seascape (between Viti Levu and Vanua Levu), which have been identified as having exceptional habitat quality54. Placing some portion of habitat within these regions under protection will promote the persistence and resilience of the entire system, while enabling fine-scale locations of protected areas to be determined by habitat representa- tion, social and economic factors, or other aspects of reserve network design. This may help alleviate the tensions that often exist during marine spatial planning, between scientific information and social or economic priorities55. C l i h H l i ll d h h d h i l d hi h ti Conversely, with Ho. atra, some populations are well connected to each other and others are isolated, which necessitates a more nuanced spatial management scenario. For instance, since the Vanua Levu and Taveuni pop- ulations are genetically differentiated, it is recommended that they be treated as separate management areas to preserve their genetic diversity, despite the fact that both lie within Fiji’s Northern Division. Additionally, since the Vanuabalavu population in the Lau archipelago to the east likely provides larval recruits and thereby genetic diversity to western reefs, this region should be a conservation priority for the management of the species. Upstream areas, such as Vanuabalavu for Ho. atra and Naviti for the fish, should be considered priorities for mon- itoring, as the health of downstream populations is largely reliant on the well-being of upstream areas. Ultimately, our data suggest management at the larger Division or Provincial scale may be appropriate for species like E. merra and Ha. trimaculatus that exhibit high amounts of connectivity within the country, but for those like Ho. atra with low connectivity and high genetic partitioning among populations, management at the provincial or local scale may be required to ensure the persistence of isolated populations. Discussionf Because the program MIGRATE assumes stable population dynamics, migration estimates can be strongly influenced by population expansion or contraction. Thus, the results of gene flow in the fish species should be taken with caution, particularly in the case of directionality. While the Tajima’s D value for Ho. atra was slightly positive, potentially indicating that this species has undergone a recent population bottleneck, the p-value for this estimate was much larger than those of the fishes and therefore likely does not have a significant effect on MIGRATE analyses39. y Another caveat of this study is its low sample sizes for E. merra at two locations (n =  10 and 12), and for Ho. atra at three locations (n =  10, 7, and 5). High amounts of nucleotide and haplotype diversity, like those found in E. merra, can sometimes be a relic of low sample sizes - however, high genetic diversity is a very common find- ing in marine fishes, and studies with very large sample sizes often observe high haplotype diversities as well33,44. Additionally, Ha. trimaculatus displayed almost identical measures of haplotype and nucleotide diversity, while having larger sample sizes than E. merra at nearly every location. Our power analyses show that with the sample sizes used for E. merra and Ha. trimaculatus, the diverse mtDNA control region confers low statistical power in detecting very weak levels of differentiation (ΦST of 0.01 or below), thus limiting our study’s ability to rule out a type II error within this range. However, the magnitude of differentiation below 0.01 is so low that even if our Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 6 Methods d Study species. Epinephelus merra and Halichoeres trimaculatus are both widespread Indo-Pacific coral reef fishes, and are commonly targeted by fishermen in Fiji56,57. E. merra is typically caught by spear or by hook and line, and is most commonly used for subsistence and occasionally sold in fish markets. Ha. trimaculatus is caught primarily via net, and is more often used for subsistence or fed to livestock, depending on their size at harvest. Both species are found in protected seaward reefs and lagoonal areas, and both have small home ranges, rarely moving more than 1 km away from their adult home reefs58,59.ii g y Holothuria atra, known as the black sea cucumber or the lollyfish, is a major commercial fisheries target throughout the Indo-Pacific60,61. Ho. atra can reproduce both sexually via broadcast spawning, and asexually by fissioning into anterior and posterior parts and then regenerating a full body from each part38,62,63. Ho. atra’s larval duration lasts approximately 18–25 days53,64, and its range extends from the Western Indian Ocean to the Eastern Pacific Ocean. Like the fishes, they tend to be relatively sedentary as adults with congeners traveling < 20 m per day65. Ethics Statement. The study protocol was approved by the Columbia University Animal Care Committee (protocol no. AC-AAAF6300) and followed the laws of the Republic of Fiji and with permission of the traditional marine resource owners. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 7 www.nature.com/scientificreports/ Sample collection. We sampled at six locations across Fiji (Fig. 1). Study sites were chosen to maximize regional coverage within the country, and sites were spread across the four Divisions of Fiji (Northern, Western, Central, and Eastern). Sample collection. We sampled at six locations across Fiji (Fig. 1). Study sites were chosen to maximize regional coverage within the country, and sites were spread across the four Divisions of Fiji (Northern, Western, Central, and Eastern). , ) Fish were collected by net, spear or through purchase from local fishermen, while Ho. atra were collected by hand (Table 1). After samples were collected, gill clips from fish, or dermal clips from Ho. atra were taken and stored in ethanol or frozen in liquid nitrogen to preserve DNA. The whole samples were then fixed in formalin for accession into the ichthyology collections at the American Museum of Natural History. Genetic Analyses. Genomic DNA was extracted from gill clips and dermal samples using a DNeasy Tissue Kit (Qiagen, Hilden, Germany). Methods d For fish samples, we amplified the mitochondrial control region by PCR using the primers CR-A and CR-E66. For Ho. atra we amplified part of the cytochrome c oxidase 1 (COI) gene using primers designed by53. PCR parameters can be found in Supplementary Note S1 online. Sequences were gen- erated on an ABI 3730 sequencer (Applied Biosystems, Inc., Foster City, CA, USA) and sequence quality was checked and sequences were aligned using Geneious 8.067. Sequences were deposited in GENBANK with the following accession numbers: E. merra KT353114-KT353195, Ha. trimaculatus KT329098-KT329188, Ho. atra KT378456-KT378495. Data Analysis. To investigate the amount of genetic differentiation occurring between sampled populations, pairwise ΦST measures (a modified F-statistic specific to mitochondrial DNA) were calculated for each species in ARLEQUIN 3.568, using 1000 replicates to estimate significance. Because of high haplotype diversities observed in the fish species and relatively low sample sizes, the statistical power of the control region marker was evaluated with POWSIM69, using a Ne of 5,000 and adjusting generation time (t) to assess power at multiple FST values (0.001, 0.0025, 0.005, 0.01, and 0.02). Power was expressed as the proportion of significant outcomes for 1,000 replicates. p To test whether regional-level genetic structuring was present, analyses of molecular variance (AMOVAs) were also performed in ARLEQUIN. For E. merra, each of the 6 sampling localities was initially treated as a separate entity, and when no significant differences were detected between the villages of Donicali and Naracivo (13 km apart on the island of Vanuabalavu), the data were pooled into island-level assemblages and reanalysed. Extremely low sample sizes for Ha. trimaculatus and Ho. atra in Donicali (n =  3 and n =  4, respectively) would have made individual locality results unreliable, so these two species were grouped at the island-level at the outset. Further, if no differentiation was detected between the island-level assemblages for each species, analyses were repeated with populations grouped by Division to investigate whether Fiji’s large-scale political boundaries are appropriate for the management of biological populations.l pp p g g p p We calculated magnitude and direction of gene flow at the island-level using MIGRATE-n70. Methods d Two runs of 1,000,000 generations were conducted for each species with an initial 25% burn-in, Because of this program’s underlying assumptions that population size and migration rates have not changed over time, Tajima’s D statistics were determined in ARLEQUIN, to assess the likelihood of recent demographic expansion or contraction for each species. p To test whether any observed genetic differences could be due to geographic distance versus phylogeogra barriers, a Mantel test was also performed for each species using Isolation By Distance 3.2371. References 1. Cesar, H., Burke, L. & Pet-Soede, L. The Economics of Worldwide Coral Reef Degradation. (2003). Available at: http://eprints.eriub org/48/. Date of access: 01/09/2013.h y f p fh f 3. Roberts, C. et al. Marine biodiversity hotspots and conservation priorities for tropical reefs. Science 295, 1280–84 (i y p p p , 4 Myers R & Worm B Rapid worldwide depletion of predatory fish communities Nature 423, 280–83 (2003) y p p p 4. Myers, R. & Worm, B. Rapid worldwide depletion of predatory fish communities. Nature 423, 280–83 (2003).f i 5. Selig, E. & Bruno, J. A global analysis of the effectiveness of marine protected areas in preventing coral loss. PLoS One 5, (2010).h ( ) 6. Halpern, B. The impact of marine reserves: do reserves work and does reserve size matter? Ecol. Appl. 13, 117–137 (2003).fi h pp 7. Roberts, C., Bohnsack, J., Gell, F., Hawkins, J. & Goodridge, R. Effects of marine reserves on adjacent fisheries. Science 294, 1920–2 (2001).i 8. Gaines, S., White, C., Carr, M. & Palumbi, S. Designing marine reserve networks for both conservation and fisheries management. Proc. Natl. Acad. Sciences 107, 18286–93 (2010).i , ( ) 9. Kritzer, J. & Sale, P. Metapopulation ecology in the sea: from Levins’ Model to marine ecology and fisheries science. Fish Fish 5, 131–40 (2004). ( ) 10. Kininmonth, S. et al. Dispersal connectivity and reserve selection for marine conservation. Ecol. Modelling 222, 1272–82 (201 10. Kininmonth, S. et al. Dispersal connectivity and reserve selection for marine conservation. Ecol. Modelling 222, 1272–82 (2011). 11. Almany, G. et al. Connectivity, biodiversity conservation and the design of marine reserve networks for coral reefs. Coral Reefs 28, 339–51 (2009). 2. Roberts, C. et al. Application of ecological criteria in selecting marine reserves and developing reserve networks. Ecol. Appl. 13 215–28 (2003). 13. McLeod, E., Salm, R., Green, A. & Almany, J. Designing marine protected area networks to address the impacts of climate change. Front. Ecol. Environ. 7, 362–70 (2008).i 14. Green, A. et al. Designing marine reserves for fisheries management, biodiversity conservation, and climate change adaptation Coast. Manage. 42, 143–59 (2014).ii g ( ) 15. Dixson, D. et al. Coral reef fish smell leaves to find island homes. Proc. Royal Soc. B: Biol. Sci. 275, 2831–39 (2008).fi ii 16. Simpson, S., Meekan, M., Jeffs, A., Montgomery, J. & McCauley, R. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 20. Barber, P., Palumbi, S., Erdmann, M. & Moosa, M. Sharp genetic breaks among populations of Haptosquilla pulchella (Stomatop indicate limits to larval transport: patterns, causes, and consequences. Mol. Ecol. 11, 659–74 (2002).i 21. Rocha, L. Patterns of distribution and processes of speciation in Brazilian reef fishes. Journ. Biogeog. 30, 1161–71 (2003).h p pi g g 22. Thornhill, D., Mahon, A., Norenburg, J. & Halanych, K. Open-ocean barriers to dispersal: a test case with the Antarctic Polar and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae). Mol. Ecol. 17, 5104–5117 (2008). g ( ) , ( ) 23. Treml, E., Halpin, P., Urban, D. & Pratson, L. Modeling population connectivity by ocean currents, a graph-theoretic approach for marine conservation Landscape Ecol 23 19–36 (2008) 23. Treml, E., Halpin, P., Urban, D. & Pratson, L. Modeling population connectivity by ocean currents, a graph-theoretic approac marine conservation. Landscape Ecol. 23, 19–36 (2008). p g p p marine conservation. Landscape Ecol. 23, 19–36 (2008). p , ( ) 24. Vuilleumier, S. & Possingham, H. Does colonization asymmetry matter in metapopulations? Proc. Royal Soc. London B: Biol. Sci. 273, 1637–42 (2006). 25. Jupiter, S., Mills, M., Compley, J., Batibasaga, A. & Jenkins, A. Fiji marine ecological gap assessment: interim progress report. (2010) Wildlife Conservation Society, Suva, Fiji. 26 pp. Available at: http://www.wcsfiji.com/Resources/Reports.aspx Date of access: 01/04/2015.f 26. Mills, M., Jupiter, S., Pressey, R., Ban, N. & Comley, J. Incorporating effectiveness of community-based management in a nat marine gap analysis for Fiji. Cons. Biol. 25, 1155–64 (2011). 27. Mills, M., Adams, V., Pressey, R., Ban, N. & Jupiter, S. Where do national and local conservation actions meet? Simulating the expansion of ad hoc and systematic approaches to conservation into the future in Fiji: marginal benefits of conservation planning. Cons. Letters 5, 387–98 (2012).i 28. Drew, J. & Barber, P. Comparative phylogeography in Fijian coral reef fishes: a multi-taxa approach towards marine reserve design. PLoS ONE 7, e47710 (2012). 29. Asian Development Bank. Fisheries Sector Review: Republic of the Fiji Islands. Technical Assistance Report TA4403, 1 (2005). Available at: http://www.adb.org/projects/documents/fisheries-sector-review-0. Date of access: 01/09/2013.i 29. Asian Development Bank. Fisheries Sector Review: Republic of the Fiji Islands. Technical Assistance Report TA4403, 1 (2005). Available at: http://www.adb.org/projects/documents/fisheries-sector-review-0. Date of access: 01/09/2013.i g ji 0. Food and Agriculture Organization of the United Nations (FAO). Fiji: Fishery and Aquaculture Country Profile. (2005). Available at: http://www.fao.org/fishery/countrysector/FI-CP_FJ/1 Date of access: 01/09/2013. www.nature.com/scientificreports/ Strong genetic but not spatial subdivision of two reef fish species targeted by fishers on the Great Barrier Reef. Fish. Res. 102, 16–25 (2010).hf fishers on the Great Barrier Reef. Fish. Res. 102, 16–25 (2010).h i 5. Nabholz, B., Glémin, S. & Galtier, N. The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals. BMC Evol. Biol. 9, 54 (2009). i 45. Nabholz, B., Glémin, S. & Galtier, N. The erratic mitochondrial h diversity across birds and mammals. BMC Evol. Biol. 9, 54 (200 y 6. Galtier, N., Nabholz, B., Glémin, S. & Hurst, G. Mitochondrial DNA as a marker of molecular diversity: a reappraisal. Mol. Ecol. 18 4541–4550 (2009).i 47. Avise, J. et al. Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics. Ann. Rev. Ecol. Syst. 18, 489–522 (1987).ffl 48. Waples, R. Separating the wheat from the chaff: patterns of genetic differentiation in high gene flow species. Journ. Hered 438–450 (1998). ( ) 49. Hedgecock, D., Barber, P. & Edmands, S. Genetic approaches to measuring connectivity. Oceanography 20, 70–79 (2007). 49. Hedgecock, D., Barber, P. & Edmands, S. Genetic approaches g g y g p y 50. Larsson, L., Charlier, J., Laikre, L. & Ryman, N. Statistical power for detecting genetic divergence – organelle versus nuclear markers. Cons. Gen. 10, 1255–1264 (2009).hi 1. Keyse, J. et al. The scope of published population genetic data for Indo-Pacific marine fauna and future research opportunities in the region. Bull. Marine Sci. 90, 47–78 (2014). g 52. Karl, S., Toonen, R., Grant, W. & Bowen, B. Common misconceptions in molecular ecology: echoes of the modern synthesis. Mol. Ecol. 21, 4171–4189 (2012). 53. Skillings, D., Bird, C. & Toonen, R. Gateways to Hawai’i: genetic population structure of the tropical sea cucumber Holothuria atra. Journ. Marine Biol. 2011, 1–16 (2011). 4. Jupiter, S., Mcclennen, C. & Matthews, E. “Vatu-i-Ra Seascape, Fiji”. In Climate and Conservation, pp. 155–169 (Island Press/Center for Resource Economics, 2012).fii 55. Gurney, G. et al. Efficient and equitable design of marine protected areas in Fiji through inclusion of stakeholder-specific objectives in conservation planning. Cons. Biol. 29, 1378–1389 (2015).fif 56. Jennings, S. & Polunin, N. Relationships between catch and effort in Fijian multispecies reef fisheries subject to different levels of exploitation. Fish. Manage. Ecol. 2, 89–101 (1995).i p g ( ) 7. Golden, A., Naisilsisili, W., Ligairi, I. & Drew, J. www.nature.com/scientificreports/ p gi y y 1. Wright, S. Evolution and the Genetics of Populations, Vol. 4: Variability within and among populations. Univ. Chicago Press, Chicago (1978).ii 32. Craig, M., Eble, T. & Bowen, B. High genetic connectivity across the Indian and Pacific Oceans in the reef fish Myripristis berndti (Holocentridae). Mar. Ecol. Prog. Ser 334, 245–54 (2007).i g 3. Horne, J., van Herwerden, L., Choat, J. & Robertson, D. High population connectivity across the Indo-Pacific: congruent lack o phylogeographic structure in three reef fish Congeners. Mol. Phylogenet. Evol. 49, 629–38 (2008). i 34. Teske, P., Forget, F., Cowley, P., von der Heyden, S. & Beheregaray, L. Connectivity between marine reserves and exploited areas in the philopatric reef fish Chrysoblephus laticeps (Teleostei: Sparidae). Mar. Biol. 157, 2029–42 (2010).li i 35. Matias, A., Anticamara, J. & Quilang, J. High gene flow in reef fishes and its implications for ad-hoc no-take marine reserves. MDN 24, 584–95 (2013). 6. Muths, D., Tessier, E. & Bourjea, J. Genetic structure of the reef grouper Epinephelus merra in the West Indian Ocean appear congruent with biogeographic and oceanographic boundaries. Mar. Ecol. 36, 447–461 (2014).i 37. Ludt, W., Bernal, M., Bowen, B. & Rocha, L. Living in the past: phylogeography and population histories of Indo-Pacific wrasses (genus Halichoeres) in shallow lagoons versus outer reef slopes. PLoS ONE 9, e38042 (2012). 8. Uthicke, S., Schaffelke, B. & Byrne, M. A boom-bust phylum? Ecological and evolutionary consequences of density variations in echinoderms. Ecol. Monographs 79, 3–24 (2009).i g p 39. Tajima, F. Evolutionary relationship of DNA sequences in finite populations. Genetics 105, 437–460 (1983). j y p qi p p 40. Hu, J., Kawamura, H., Hong, H. & Qi, Y. A review on the currents in the South China Sea: seasonal circulation, South China Sea Warm Current and Kuroshio Intrusion. Journ. Oceanog. 56, 607–24 (2000).hli 1. Galarza, J. et al. The influence of oceanographic fronts and early-life-history traits on connectivity among littoral fish species. PNAS 106, 1473–1478 (2009). 42. He, L., Mukai, T., Chu, K., Ma, Q. & Zhang, J. Biogeographical role of the Kuroshio Current in the amphibious mudskipper Periophthalmus modestus indicated by mitochondrial DNA data. Sci. Rep. 5, 15645 (2015).ii 3. Karnauskas, M., Chérubin, L. & Paris, C. Adaptive significance of the formation of multi-species fish spawning aggregations near submerged capes. PLoS ONE 6, e22067 (2011).i g 4. Evans, R., van Herwerden, L., Russ, G. & Frisch, A. References Settlement-stage coral reef fish prefer the higher-frequency invertebrate-generated audible component of reef noise. Animal Behav. 75, 1861–68 (2008).hi 17. Igulu, M. et al. The potential role of visual cues for microhabitat selection during the early life phase of a coral reef fish (Lu fulviflamma). Journ. Exper. Marine Biol. Ecol. 401, 118–25 (2011).hi 7. gu u, . et al.h e pote t a o e o v sua cues o c o ab tat se ect o du g t e ea y e p ase o a co a e fulviflamma). Journ. Exper. Marine Biol. Ecol. 401, 118–25 (2011). Thi f fl p 18. Jones, G., Planes, S. & Thorrold, S. Coral reef fish larvae settle close to home. Current Biol. 15, 1314–18 (2005).i l . Jones, G., Planes, S. & Thorrold, S. Coral reef fish larvae settle cloi J , , ,h ,i , ( ) 9. Robertson, D. Population maintenance among tropical reef fishes: inferences from small-island endemics. Proc. Natl. Acad. Sci. 98 5667–5670 (2001). Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 8 ( ) 0. Bruckner, A., Johnson, K. & Field, J. Conservation strategies for sea cucumbers: can a CITES Appendix II listing promote sustainable international trade? SPC Beche-de-mer Info. Bull. 18, 24–33 (2003). www.nature.com/scientificreports/ 72. Cohen, J. Statistical power analysis for the behavioral sciences. Second edition, Acad. Press (1988).l 3. Planes, S., Parroni, M. & Chauvet, C. Evidence of limited gene flow in three species of coral reef fishes in the lagoon of New Caledonia. Marine Biol. 130, 361–368 (1998).i 73. Planes, S., Parroni, M. & Chauvet, C. Evidence of limited gene flow in three species of coral reef fishes in the lagoon of New Caledonia. Marine Biol. 130, 361–368 (1998).i 4. Victor, B. Duration of the planktonic larval stage of on hundred species of Pacific and Atlantic wrasses (family Labridae). Marine Biol. 90, 317–326 (1986). 74. Victor, B. Duration of the planktonic larval stage of on hundred species of Pacific and Atlantic wrasses (family Labridae). Marine Biol. 90, 317–326 (1986). Author Contributions Designed Experiment J.A.D. Conducted Experiment J.A.D., E.K.E. and E.H.L. Wrote Manuscript J.A.D., E.K.E. and E.H.L. Prepared Figures E.K.E. and E.H.L. All authors reviewed the Manuscript. Acknowledgements g We would like to thank the Fijian Locally Managed Marine Area (FLMMA) Network for supporting this research, as well as Waisea Naisilisili, Apete Tamani, Liliana Rabuku, and the people of Nagigi, Naselesele, Malevu, Donicali, Naracivo, and Nabukavesi for welcoming us into their homes and communities. We would also like to thank the Sackler Institute for Comparative Genomics for sequencing facilities and assistance. Funding for EHL was provided by the Columbia University Earth Institute Student Travel Grant, The Explorers Club’s Youth Activity Fund, the Columbia College Class of 1939 Summer Research Fellowship, the Department of Ecology, Evolution and Environmental Biology Department Thesis Research Award, and the Columbia University Scholars Program (CUSP) Summer Enhancement Fellowship. Funding for EKE was provided by the Columbia University Department of Ecology, Evolution and Environmental Biology and 45 SciFund challenge contributors. Funding to JAD was provided by the US State Department Embassy in Suva, Fiji and the Mindlin Foundation. No funders had any input into the development or execution of this work. www.nature.com/scientificreports/ 61. Purcell, S. et al. Sea cucumber fisheries: global analysis of stocks, management measures and drivers of overfishing. Fish Fish 34–59 (2013).f 61. Purcell, S. et al. Sea cucumber fisheries: global analysis of stocks, management measures and drivers of overfishing. Fish Fish. 14, 34–59 (2013). 62 Ch S Ch C & Al d S Fi i d it ff t l ti t t f H l th i t (E hi d t H l th id ) 34–59 (2013). 62. Chao, S., Chen, C. & Alexander, S. Fission and its effect on population structure of Holothuria atra (Echinodermata: Holothuroidea) 2. Chao, S., Chen, C. & Alexander, S. Fission and its effect on population structure of Holothuria atra (Echinodermata: Holothuroidea in Taiwan. Marine Biol. 116, 109–115 (1993).i ( ) 63. Conand, C. Asexual reproduction by fission in Holothuria atra: variability of some parameters in populations from the tropical Indo-Pacific. Oceanologica Acta 19, 209–216 (1996). i Indo-Pacific. Oceanologica Acta 19, 209–216 (1996). i g 4. Laxminarayana, A. Induced spawning and larval rearing of the sea cucumbers, Bohadschia mamorata and Holothuria atra in Mauritius. SPC Beche-de-mer Info. Bull. 22, 48–52 (2005). f 65. Navarro, P., García-Sanz, S., Barrio, J. & Tuya, F. Feeding and movement patterns of the sea cucumber Holothuria sanctori. Marine Biol. 160, 2957–2966 (2013). ( ) 6. Lee, W., Conroy, J., Howell, W. & Kocher, T. Structure and evolution of teleost mitochondrial control regions. Journ. Mol. Evol. 41 54–66 (1995).t ( ) 7. Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis o sequence data. Bioinformatics, 28, 1647–1649 (2012).fi q f 68. Excoffier, L. & Lischer, H. Arlequin Suite Ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol. Ecol. Res. 10, 564–67 (2010).f 9. Ryman, N. & Palm, S. POWSIM: A computer program for assessing statistical power when testing for genetic differentiation. Mol Ecol. 6, 600–602 (2006).i 0. Beerli, P. & Palczewski, M. Unified framework to evaluate panmixia and migration direction among multiple sampling locations Genetics 185, 313–26 (2010). 71. Jensen, J., Bohonak, A. & Kelley, S. Isolation by distance, web service. BMC Genetics 6: 13. v.3.23 (2005). Available at: http://ibdws. sdsu.edu/Date of access: 01/04/2015. 1. Jensen, J., Bohonak, A. & Kelley, S. Isolation by distance, web service. BMC Genetics 6: 13. v.3.23 (2005). Available at: http://ibdws sdsu.edu/Date of access: 01/04/2015. www.nature.com/scientificreports/ Combining natural history collections with fisher knowledge for community-based conservation in Fiji. PLoS ONE 9, e98036 (2014).hh 58. Liu, M. & Yeeting, B. 2008. Epinephelus merra. The IUCN Red List of Threatened Species. Version 2014.3. < www.iucnredlist.org> . Date of access: 12/04/2015.i 59. Green, A. et al. Larval dispersal and movement patterns of coral reef fishes, and implications for marine reserve network design. Biol. Reviews 90, 1215–1247 (2014). Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 9 www.nature.com/scientificreports/ Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare How to cite this article: Eastwood, E. K. et al. Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji. Sci. Rep. 6, 19318; doi: 10.1038/srep19318 (2016) How to cite this article: Eastwood, E. K. et al. Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji. Sci. Rep. 6, 19318; doi: 10.1038/srep19318 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 10
https://openalex.org/W2839531123
https://nottingham-repository.worktribe.com/preview/946788/sty1870.pdf
English
null
Cosmic CARNage II: the evolution of the galaxy stellar mass function in observations and galaxy formation models
Monthly Notices of the Royal Astronomical Society
2,018
cc-by
14,398
Accepted XXX. Received YYY; in original form ZZZ Cosmic CARNage II 1 Cosmic CARNage II 1 Department of Physics & Astronomy, University of Sussex, Brighton, BN1 9QH, UK Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 Department of Astronomy and Yonsei University Observatory, Yonsei University, 03722 Seoul, Republic of Kore 23Centro de Astro-Ingenieria, Universidad Catolica de Chile, Santiago, Chile 24 24International Centre for Radio Astronomy Research, University of Western Australia, 35 Stirling Highway, C Western Australia 6009, Australia by U e s ty o on 17 July 2018 Cosmic CARNage II: the evolution of the galaxy stellar mass function in observations and galaxy formation models Rachel Asquith,1⋆Frazer R. Pearce,1 Omar Almaini,1 Alexander Knebe,2,3 Violeta Gonzalez-Perez,4,5 Andrew Benson,6 Jeremy Blaizot,7,8,9 Jorge Carretero,10,11 Francisco J. Castander,10 Andrea Cattaneo,12 Sof´ıa A. Cora,13,14 Darren J. Croton,15 Julien E. Devriendt,16 Fabio Fontanot,17 Ignacio D. Gargiulo,13,14 Will Hartley,18 Bruno Henriques,19 Jaehyun Lee,20 Gary A. Mamon,21 Julian Onions,1 Nelson D. Padilla,22,23 Chris Power,24 Chaichalit Srisawat,25 Adam R. H. Stevens,15,24 Peter A. Thomas,25 Cristian A. Vega-Mart´ınez,13 Sukyoung K. Yi26 1School of Physics & Astronomy, University of Nottingham, Nottingham NG7 2RD, UK f y y, y f g , g , 2Departamento de F´ısica Te´orica, M´odulo 15, Facultad de Ciencias, Universidad Aut´onoma de Madrid, 28049 Madrid, Spain 3Centro de Investigaci´on Avanzada en F´ısica Fundamental (CIAFF), Facultad de Ciencias, Universidad Aut´onoma de Madrid, 28049 Madrid, Spain 2Departamento de F´ısica Te´orica, M´odulo 15, Facultad de Ciencias, Universidad Aut´onoma de Madrid, 28049 Madrid, Spain 3Centro de Investigaci´on Avanzada en F´ısica Fundamental (CIAFF), Facultad de Ciencias, Universidad Aut´onoma de Madrid, 28049 Madrid, Spain 4Institute for Computational Cosmology, Department of Physics, University of Durham, South Road, Durham, DH1 3LE, UK 5Institute of Cosmology & Gravitation, University of Portsmouth, Dennis Sciama Building, Portsmouth, PO1 3FX, UK 6Carnegie Observatories, 813 Santa Barbara Street, Pasadena, CA 91101, USA 4Institute for Computational Cosmology, Department of Physics, University of Durham, South Road, Durham, DH1 3LE, UK 5Institute of Cosmology & Gravitation, University of Portsmouth, Dennis Sciama Building, Portsmouth, PO1 3FX, UK 6Carnegie Observatories, 813 Santa Barbara Street, Pasadena, CA 91101, USA 7Universit`e de Lyon, Lyon, F-69003, France y , y , , 8Universit`e Lyon 1, Observatoire de Lyon, 9 avenue Charles Andr`e, Saint-Genis Laval, F-69230, France 9 8Universit`e Lyon 1, Observatoire de Lyon, 9 avenue Charles Andr`e, Saint-Genis Laval, F-69230, France 9C S C A S 11Port d’Informaci´o Cient´ıfica (PIC) Edifici D, Universitat Aut`onoma de Barcelona (UAB), E-08193 Bellaterra (Barcelona), Spain. 12GEPI, Observatoire de Paris, CNRS, 61, Avenue de l’Observatoire 75014, Paris France Instituto de Astrofısica de La Plata (CCT La Plata, CONICET, UNLP), Paseo del Bosque s/n, B1900FWA, La Plata, Argentina. 1 INTRODUCTION galaxies throughout their lives, one approach is to link a population of galaxies at high redshift to a population at low redshift that could be their descendants. This can be done by selecting galaxies at a constant comoving number density when ranked by mass or luminosity (Mundy et al. 2015). This method was partly motivated by the need to overcome ‘progenitor bias’, where new young star-forming galaxies enter the sample at low redshift that are not present at high redshift (Shankar et al. 2015). Not accounting for this bias correctly can lead to a poor selection of the set of galaxies being connected as progenitor and descendent and therefore incorrect conclusions being drawn about their evolution. Locally, low-mass galaxies tend to be disky, blue and star-forming, whereas high-mass galaxies are more likely to be spheroidal, red and passive (e.g. Kennicutt 1998; Strateva et al. 2001; Kauffmann et al. 2003; Baldry et al. 2004). At high redshift (z > 1) we also observe this bimodality in the galaxy population (Kovaˇc et al. 2014; Cirasuolo et al. 2007), but do not definitively know the mechanisms by which these galaxies evolve into the popula- tions we observe locally. Various mechanisms have been sug- gested to move galaxies from the ‘blue cloud’ to the ‘red se- quence’ and shut offtheir star formation in a process known as ‘quenching’. Potential quenching mechanisms include en- vironmental effects and feedback from active galactic nuclei (AGN) at high masses (for a review see Benson 2010), but these processes are still not fully understood. A powerful method to trace galaxies through redshift is to use semi-analytic models (SAMs) (for a review see Benson 2010; Somerville & Dav´e 2015), a type of galaxy formation model in which simple analytic prescriptions (in connection with merger trees from either cosmological simulations or extended Press-Schechter formalisms) are used to model the physical processes occurring during galaxy formation and evolution. These models are able to evolve the same popula- tion of galaxies through redshift and connect them without the limitations of observational methods. These models are also computationally inexpensive, so can be used to simu- late large volumes and produce large catalogues of galaxies with which to compare observational data. By comparing the models to key observables, e.g. the evolution of the stel- lar mass function (SMF), we can learn about the physics of galaxy formation. ⋆E-mail: rachel.asquith@nottingham.ac.uk ABSTRACT We present a comparison of the observed evolving galaxy stellar mass functions with the predictions of eight semi-analytic models and one halo occupation distribution model. While most models are able to fit the data at low redshift, some of them struggle to simultaneously fit observations at high redshift. We separate the galaxies into ‘passive’ and ‘star-forming’ classes and find that several of the models produce too many low-mass star-forming galaxies at high redshift compared to observations, in some cases by nearly a factor of 10 in the redshift range 2.5 < z < 3.0. We also find important differences in the implied mass of the dark matter haloes the galaxies inhabit, by comparing with halo masses inferred from observations. Galaxies at high redshift in the models are in lower mass haloes than suggested by observations, and the star formation efficiency in low-mass haloes is higher than observed. We conclude that many of the models require a physical prescription that acts to dissociate the growth of low-mass galaxies from the growth of their dark matter haloes at high redshift. Key words: methods:numerical – galaxies:haloes – galaxies: evolution – cosmol- ogy:theory – dark matter Cosmic CARNage II: the evolution of the galaxy stellar mass function in observations and galaxy formation models 14Facultad de Ciencias Astron´omicas y Geof´ısicas, Universidad Nacional de La Plata, Paseo del Bosque s/n, B1900FWA, La Plata, Argentina 15C t f A t h i d S ti S i b U i it f T h l H th Vi t i 3122 A t li s y Geofısicas, Universidad Nacional de La Plata, Paseo del Bosque s/n, B1900FWA, La Plata, Argentina rcomputing, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia Facultad de Ciencias Astronomicas y Geofısicas, Universidad Nacional de La Plata, Paseo del Bosque s/n, B190 Centre for Astrophysics and Supercomputing, Swinburne University of Technology, Hawthorn, Victoria 3122, Au Astrophysics, University of Oxford, Denys Wilkinson Building, Keble Road, Oxford, OX1 3RH, UK 17INAF - Astronomical Observatory of Trieste, via Tiepolo 11, I-34143 Trieste, Italy 18 servatory of Trieste, via Tiepolo 11, I-34143 Trieste, Ita 17INAF - Astronomical Observatory of Trieste, via Tiepolo 11, I-34143 Trieste, Italy 18Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT 19Max-Planck-Institut f¨ur Astrophysik, Karl-Schwarzschild-Str. 1, 85741 Garching b. M¨unchen, Germany 20Korea Institute for Advanced Study, 85 Hoegiro Dongdaemun-gu, Seoul 02455 Korea 22Instituto de Astrofisica, Universidad Catolica de Chile, Santiago, Chile 23Centro de Astro-Ingenieria, Universidad Catolica de Chile, Santiago, Chile 24 24International Centre for Radio Astronomy Research, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia 25Department of Physics & Astronomy, University of Sussex, Brighton, BN1 9QH, UK 26 2 R. Asquith et al. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 1 INTRODUCTION (2013) had on their own model, but found that it did not make much difference to the ob- served number density of low-mass galaxies. They conclude that this is due to the sensitivity of the results to how the gas reservoirs are tracked and treated in the different codes. Croton et al. (2016) also had similar problems with this ap- proach and found that it did not solve the problems with fitting the stellar mass function. This presents difficulties to the modelling community, as it means that different mod- els may require different changes to get them to match the observed evolution. This excess of low-mass galaxies at high redshift was investigated by Fontanot et al. (2009), who found that in three different SAMs, galaxies in the mass range 9 < log(M∗/M⊙) < 11 form too early and have little ongoing star formation at late times. They concluded that the physical processes operating on these mass scales, such as supernova feedback, needed a re-think. Weinmann et al. (2012) later used two SAMs and two cosmological hydrodynamical sim- ulations and examined the evolution of the observed number density of galaxies. They found that although the models fit well at z = 0, the low-mass galaxies were formed at early times. They conclude that as the current form of feedback is mainly dependent on host halo mass and time, it is un- likely to be able to separate the growth of galaxies from the growth of their dark matter haloes. It is also possible to try and match the galaxy stellar mass function at all redshifts without changing the physics involved in the model. For example, Rodrigues et al. (2017) used Galform to identify a small region of parameter space where the model matched the observational data out to z = 1.5, without needing to adapt any of the physics involved. They found that the parameters controlling the feedback processes were most strongly constrained, suggesting that these processes are important when fitting the evolution of the galaxy stellar mass function. Halo occupation distribution (HOD) models, rather than modelling the physical processes that we think go into galaxy formation, use statistical methods to match galaxies to their corresponding dark matter haloes (e.g. Berlind & Weinberg 2002; Zheng et al. 2005). 1 INTRODUCTION However, they allow these functions to scale with halo mass and redshift in an arbitrary way which may not be physically motivated. Hirschmann et al. (2016) also investigated this problem with their model and found that they improved their agreement with observations by ei- ther reducing the gas ejection rate with cosmic time or vary- ing the reincorporation timescale with halo mass, classed as ‘ejective’ and ‘preventative’ feedback schemes respectively. Although their results improve from their fiducial model, they still find too many low-mass, red, old galaxies between 0.5 < z < 2.0. galaxies form earlier with their abundance changing little from z ∼1 to the present day, whereas there is a rapid evo- lution in the number of low-mass galaxies at late times (e.g. Fontana et al. 2004, 2006; Faber et al. 2007; Pozzetti et al. 2007; Marchesini et al. 2009, 2010; Pozzetti et al. 2010; Ilbert et al. 2010, 2013; Muzzin et al. 2013). This is some- times referred to as ‘mass assembly downsizing’ (Cowie et al. 1996; Cimatti et al. 2006; Lee & Yi 2013). ) After much work understanding both AGN feed- back and the mass assembly of high-mass galaxies (e.g. Benson et al. 2003; Di Matteo et al. 2005; Bower et al. 2006; Croton et al. 2006), models are now able to reproduce the high-mass end of the galaxy stellar mass function over a range of redshifts. However, models still typically overpro- duce the number of low-mass galaxies at high redshift. The main reason for this discrepancy appears to be that galaxies in the models follow the growth of their dark matter haloes too closely (Weinmann et al. 2012; Somerville & Dav´e 2015; Guo et al. 2016). Halo mass growth is the main driver of gas accretion rate in galaxies, which then in turn drives the star formation rate. The star formation history then traces the dark matter mass accretion history, which, in the favoured ΛCDM structure formation scenario, is approximately self- similar for haloes of different masses. However, in the real Universe it appears that there is not such a tight correlation (White et al. 2015; Guo et al. 2016). However, the effect of adjusting certain physical prescriptions can be vastly different between models. White et al. (2015) investigated what effect replicating the changes in Henriques et al. 1 INTRODUCTION If models are not able to reproduce ob- servational results it may mean that they are missing key physics which is important in galaxy formation and evolu- tion. Model galaxies can also be separated into ‘star-forming’ and ‘passive’ types, to test for the quenching processes which transform galaxies from star-forming to passive. One way to study this problem is to directly observe galaxies forming and evolving in the distant Universe. At high redshift (z > 1), deep near-infrared observations are vital to select galaxies by rest-frame optical light. Selecting high-redshift galaxies using optical imaging will introduce strong biases against dusty galaxies or those with evolved (i.e. passive) stellar populations (e.g. Cowie et al. 1996). It is only recently that deep near-infrared surveys have been conducted with the required depth and area to produce large galaxy samples at high redshift, sufficient to allow accurate determinations of the galaxy stellar mass function while min- imising the influence of cosmic variance. In particular, the UKIDSS Ultra Deep Survey (UDS) (Lawrence et al. 2007, Almaini et al. in prep.) and UltraVISTA (McCracken et al. 2012) are now deep enough to detect typical (i.e. M∗) galax- ies to z ∼3, over large volumes of the distant Universe (∼100 × 100 projected comoving Mpc at z = 3). Using these surveys, we can directly test model predictions for the build- up of the galaxy populations, rather than inferring their evo- lution by extrapolating back in time. However, each galaxy is only being seen at one point in its life and we cannot infer the full evolutionary history. While it has been shown that SAMs are able to re- produce the SMF at z = 0, they struggle to simultane- ously match observations at both low and high redshift (e.g. Fontanot et al. 2009; Weinmann et al. 2012; Guo et al. 2011; Knebe et al. 2015). This has only become clearer in re- cent years as observational surveys have been able to probe down to lower masses as well as probing to higher redshifts. Observational evidence appears to point towards a seem- ingly ‘anti-hierarchical’ formation scenario where high-mass In order to get a cohesive picture of what happens to Cosmic CARNage II 3 times and increasing the dependence of stellar feedback on halo mass at high redshift were the most successful at qual- itatively matching the evolution of the number density of low-mass galaxies. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 2 SIMULATION DATA We also note that we compare to different observational data than the combined dataset used to calibrate the models. The data from Davidzon et al. (2017) is more recent than the calibration dataset and also allows us to split our sam- ple into passive and star-forming galaxies. When comparing Davidzon et al. (2017) to the stellar mass function calibra- tion data at z = 0 and z = 2, the two largely agree, although the former has smaller error bars. This is encouraging as it shows good agreement between different observations. The eight SAMs we will be using are DLB07 (De Lucia & Blaizot 2007), Galform (Gonzalez-Perez et al. 2014), GalICS-2.0 (Cattaneo et al. 2017, although the exact version used for this comparison is the one described in the appendix of Knebe et al. (2015)), Lgalaxies (Henriques et al. 2013), Morgana (Monaco et al. 2007), Sag (Cora et al. 2018), Sage (Croton et al. 2016) and ySAM (Lee & Yi 2013). The single HOD model is Mice (Carretero et al. 2015). A brief description of the physical prescriptions used in each model is given in the Appendix of Knebe et al. (2015). Any changes to any of the models since then are included in Appendix A. The eight SAMs we will be using are DLB07 (De Lucia & Blaizot 2007), Galform (Gonzalez-Perez et al. 2014), GalICS-2.0 (Cattaneo et al. 2017, although the exact version used for this comparison is the one described in the appendix of Knebe et al. (2015)), Lgalaxies (Henriques et al. 2013), Morgana (Monaco et al. 2007), Sag (Cora et al. 2018), Sage (Croton et al. 2016) and ySAM (Lee & Yi 2013). The single HOD model is Mice (Carretero et al. 2015). A brief description of the physical prescriptions used in each model is given in the Appendix of Knebe et al. (2015). Any changes to any of the models since then are included in Appendix A. Inspecting the top panels, what is clear is that the ob- servational number counts are evolving, with the high-mass end largely in place by z = 3, while the low-mass end rises at late times. Whilst the models match the observations well at low redshift, the strong evolution at the low-mass end is not seen for most of the galaxy formation models. 1 INTRODUCTION We also note that the stellar masses from the models have been convolved with a 0.08(1+z) dex scatter to account for the observational errors when measuring stellar mass. This value comes from Conroy et al. (2009), who estimate an error of ∼0.2 dex at z = 2 when fixing the stellar population synthesis model. of the required physics or that the underlying observational datasets are incomplete or are physically incompatible with each other. In the Cosmic CARNage mock galaxy comparison project (Knebe et al. 2018, hereafter referred to as Paper I) we sought to address some of these issues by requiring the participants to calibrate their models to the same set of observational data. These data included the galaxy stellar mass function at z = 0 and z = 2, the star formation rate function at z = 0.15, the black-hole bulge-mass relation at z = 0, and the cold gas mass fraction at z = 0. Participants were free to weight these five calibrations as they saw fit, and were asked to generate their “best-fit” model that took all of them into account, i.e. calibration set ‘-c02’ in Paper I. The underlying cosmological dark-matter-only simula- tion was run using the Gadget-3 N-body code (Springel 2005) with parameters given by the Planck cosmology (Planck Collaboration et al. 2014, Ωm = 0.307, ΩΛ = 0.693, Ωb = 0.048, σ8 = 0.829, h = 0.677, ns = 0.96). We use 5123 particles of mass 1.24 × 109h−1M⊙in a box of comoving width 125h−1Mpc. The halo catalogues were extracted from 125 snapshots and identified using Rockstar (Behroozi et al. 2013a). The halo merger trees were then generated using the ConsistentTrees code (Behroozi et al. 2013b). We will build on previous work by investigating the evo- lution of the SMF for the eight SAMs and one HOD model that were used in Paper I. These models are all calibrated to the same observational data and are all run on the same background dark matter only simulation, which means that we can discount the differences due to the underlying cosmo- logical framework when considering the differences between the models. Our aim is then to see if the current physical prescriptions used in any of the galaxy formation models can produce a realistic population of galaxies at both low and high redshift. 1 INTRODUCTION As these mod- els are applied independently at each redshift, the evolution of each galaxy is not tracked, although they can be con- nected to their progenitors and descendants via dark matter merger trees. HOD models by design are able to reproduce the SMF at each redshift and are therefore able to repro- duce the population of galaxies at any given time. This type of model is a very useful tool for learning about the rela- tionship between galaxies and their host dark matter haloes and how this changes as a function of redshift. For example, Berlind et al. (2003) found that low-mass haloes are mainly populated by young galaxies and high-mass haloes by older galaxies. Monte Carlo Markov Chain (MCMC) methods were used by Henriques et al. (2013) in an attempt to fit the stellar mass function at all redshifts, but they could not find a single set of parameters that allowed this. They then changed the reincorporation timescale for ejected gas to be inversely proportional to halo mass and independent of red- shift and found that they were able to fit observed numbers of low-mass galaxies from 0 < z < 3. However, the passive fraction of low-mass galaxies was still too high. Their model was later updated further in Henriques et al. (2015) where they also reduce ram-pressure stripping in low-mass haloes, make radio-mode AGN feedback more efficient at low red- shift, and reduce the gas surface density threshold for star formation. They then find that their model reproduces the observed abundance and passive fraction of low-mass galax- ies, both at high and low redshift. Galaxy formation models such as HODs and SAMs must be calibrated using observational datasets. Varying the calibration dataset, even for the same model may produce significantly different catalogues. Essentially, the calibration datasets introduce tension, and it may not be possible for a single model to fit all the required observational datasets simultaneously. This could be because the model lacks some Another attempt to solve this problem was by White et al. (2015), who tried three different physically mo- tivated methods to decouple the accretion rate in galaxies from their star formation rate. They found that changing the gas accretion to be less efficient in low-mass haloes at early R. Asquith et al. 4 A description of how the models were calibrated to the same observational data is given in Paper I. 3.1 Evolution of the Galaxy Stellar Mass Function We start by examining the evolution of the stellar mass func- tion in Figure 1, shown for the whole sample in the top row. The coloured lines are the stellar mass functions for each of the models, computed for each redshift bin using single snap- shots at z = 0.8,2.0 and 3.0 for each redshift bin respectively. We note that the precise choice of snapshot does not af- fect our conclusions. The observations from Davidzon et al. (2017) are based on the UltraVISTA near-infrared survey of the COSMOS field and are shown as a black line and dark shaded region. When finding the best-fit Schechter param- eters to their stellar mass functions, they take into account the errors in measuring stellar mass, known as Eddington bias. As they have applied this correction, when plotting the stellar mass function we do not apply the 0.08(1+ z) dex scatter to the stellar mass values. In Appendix B we have in- cluded a version of Figure 1 where the model stellar masses do have this scatter applied, to show the differences to the SMF. The rest of this paper is structured as follows: in Section 2 we will briefly explain the underlying dark matter simula- tion and the parameters used. In Section 3 we will present the results for the evolution of the SMF and the passive frac- tion. We will then show how the specific star formation rate of star-forming galaxies in the models evolves. We will also examine the average halo mass as a function of stellar mass and the stellar mass - halo mass relation for all the models. In Section 4 we will present our discussion and in Section 5 we will present our conclusions. 1 INTRODUCTION We will investigate the evolution of the SMF in the redshift range 0.5 < z < 3.0 for all nine galaxy formation models and determine if models still struggle to simultane- ously match observations both at low and high redshift. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 3 RESULTS 3.1 Evolution of the Galaxy Stellar Mass Function 3.1 Evolution of the Galaxy Stellar Mass Function Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 2 SIMULATION DATA The excep- tions to this are Mice, Lgalaxies and Sag, which all show an increasing number density of low-mass galaxies towards low redshift. As Mice is an HOD model it has been designed to match the evolution of the SMF. Lgalaxies and Sag likely do a better job of matching the SMF at high redshift The models have all been run on the same underlying dark matter simulation which may be different to the one used in the above reference papers. This can lead to changes in the predictions of each model, as can varying the initial mass function, yield, stellar population synthesis model and calibration data set used. Cosmic CARNage II 5 5 Cosmic CARNage II 5 −5 −4 −3 −2 log(φ[Mpc−3dex−1]) 0.5 < z < 0.8 All 1.5 < z < 2.0 Davidzon+17 DLB07 Galform GalICS-2.0 LGALAXIES 2.5 < z < 3.0 MICE MORGANA SAG SAGE ySAM −5 −4 −3 −2 log(φ[Mpc−3dex−1]) Passive 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −5 −4 −3 −2 log(φ[Mpc−3dex−1]) Star-forming 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) Figure 1. The evolution of the stellar mass function for all the models over the range 0.5 < z < 3.0. The stellar mass function for the whole, passive and star-forming samples are shown in the top, middle and bottom panels, respectively, as coloured lines. The black line is the observational best-fit mass functions from Davidzon et al. (2017), with the dark grey shaded region showing the 1σ errors. For the models, each redshift bin contains one snapshot, at redshifts z = 0.8, 2.0 and 3.0 respectively. We can see that the models match well at low redshift (by construction), but deviate further from the observations at high redshift. The number density of the lowest mass objects is nearly constant in the models but changes by more than 0.5 dex in the observations. Most of the low-mass galaxies that are not present in the observations at high redshift seem to be star-forming. 2 SIMULATION DATA 2.5 < z < 3.0 MICE MORGANA SAG SAGE ySAM 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) log(M∗[M⊙]) 4 3 2 log(φ[Mpc−3dex−1]) 0.0 10.5 11 log(M∗[M⊙] 0.0 10.5 11. log(M∗[M⊙]) 0.0 10.5 11. log(M∗[M⊙]) Figure 1. The evolution of the stellar mass function for all the models over the range 0.5 < z < 3.0. The stellar mass function for the whole, passive and star-forming samples are shown in the top, middle and bottom panels, respectively, as coloured lines. The black line is the observational best-fit mass functions from Davidzon et al. (2017), with the dark grey shaded region showing the 1σ errors. For the models, each redshift bin contains one snapshot, at redshifts z = 0.8, 2.0 and 3.0 respectively. We can see that the models match well at low redshift (by construction), but deviate further from the observations at high redshift. The number density of the lowest mass objects is nearly constant in the models but changes by more than 0.5 dex in the observations. Most of the low-mass galaxies that are not present in the observations at high redshift seem to be star-forming. ejected mass with redshift to make supernova feedback more efficient at high redshift. due to the physics involved in the treatment of gas. Both follow the prescription suggested in Henriques et al. (2013) of scaling the reincorporation timescale of ejected gas with the inverse of the halo mass. This means the process of gas being reincorpoated back into the halo takes longer for low- mass haloes, shifting the growth of galaxies in these haloes from early to late times. Sag also scales the reheated and At the high-mass end, the models underestimate the number density compared to observations, with Mice and GalICS-2.0 as the exceptions. One alternative reason for this tension at the high-mass end may be due to Davidzon et al. (2017) underestimating their uncertainties R. Asquith et al. 6 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 0.0 0.2 0.4 0.6 0.8 1.0 Passive fraction 0.5 < z < 0.8 Davidzon+17 DLB07 Galform 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 1.5 < z < 2.0 GalICS-2.0 LGALAXIES MICE 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 2.5 < z < 3.0 MORGANA SAG SAGE ySAM 0.0 0.2 0.4 0.6 0.8 1.0 Passive fraction Figure 2. 2 SIMULATION DATA The sSFR of star-forming galaxies in the models matches observations well but there is less of a trend with mass in some models. 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −2.5 −2.0 −1.5 −1.0 −0.5 log(sSFR[Gyr−1]) Elbaz+07 DLB07 Galform GalICS-2.0 LGALAXIES MICE MORGANA SAG SAGE ySAM z = 0.0 Figure 4. As for Figure 3, but for z = 2.0 and observations from Daddi et al. (2007). The model data is taken from one snapshot at z = 2.0. Here all of the models lie almost completely below the observational best-fit range. 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −2.5 −2.0 −1.5 −1.0 −0.5 log(sSFR[Gyr−1]) Elbaz+07 DLB07 Galform GalICS-2.0 LGALAXIES MICE MORGANA SAG SAGE ySAM z = 0.0 Figure 3. The relationship between mass and sSFR at z = 0.0 for star-forming galaxies in the nine models. The model data is taken from one snapshot at z = 0.0. The grey shaded region is taken from Elbaz et al. (2007) and shows the observational best-fit to this relation. The sSFR of star-forming galaxies in the models matches observations well but there is less of a trend with mass in some models. 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −2.5 −2.0 −1.5 −1.0 −0.5 log(sSFR[Gyr−1]) Elbaz+07 DLB07 Galform GalICS-2.0 LGALAXIES MICE MORGANA SAG SAGE ySAM z = 0.0 Figure 4. As for Figure 3, but for z = 2.0 and observations from Daddi et al. (2007). The model data is taken from one snapshot at z = 2.0. Here all of the models lie almost completely below the observational best-fit range. 3.2 Star-forming and Passive Galaxy Stellar Mass Functions We explore the mass growth further in the bottom two rows of Figure 1, splitting the population into passive (middle row) and star-forming (bottom row) galaxies We separate 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −2.5 −2.0 −1.5 −1.0 −0.5 log(sSFR[Gyr−1]) Elbaz+07 DLB07 Galform GalICS-2.0 LGALAXIES MICE MORGANA SAG SAGE ySAM z = 0.0 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −2.5 −2.0 −1.5 −1.0 −0.5 log(sSFR[Gyr−1]) Elbaz+07 DLB07 Galform GalICS-2.0 LGALAXIES MICE MORGANA SAG SAGE ySAM z = 0.0 Figure 3. The relationship between mass and sSFR at z = 0.0 for star-forming galaxies in the nine models. The model data is taken from one snapshot at z = 0.0. The grey shaded region is taken from Elbaz et al. 2 SIMULATION DATA (2007) and shows the observational best-fit to this relation. The sSFR of star-forming galaxies in the models matches observations well but there is less of a trend with mass in some models. Figure 4. As for Figure 3, but for z = 2.0 and observations from Daddi et al. (2007). The model data is taken from one snapshot at z = 2.0. Here all of the models lie almost completely below the observational best-fit range. 2 SIMULATION DATA The evolution of the passive fraction over the range 0.5 < z < 3.0. The coloured lines, black solid lines and grey shaded regions are the same as in Figure 1, as are the snapshots used in each redshift bin for the models. For a few models the passive fraction is too high at low masses, particularly at low redshift. The models match well at high masses at low redshift, but generally underpredict the passive fraction for high-mass galaxies at high redshift. 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 0.0 0.2 0.4 0.6 0.8 1.0 Passive fraction 0.5 < z < 0.8 Davidzon+17 DLB07 Galform 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 1.5 < z < 2.0 GalICS-2.0 LGALAXIES MICE 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 2.5 < z < 3.0 MORGANA SAG SAGE ySAM 0.0 0.2 0.4 0.6 0.8 1.0 Passive fraction Figure 2. The evolution of the passive fraction over the range 0.5 < z < 3.0. The coloured lines, black solid lines and grey shaded regions are the same as in Figure 1, as are the snapshots used in each redshift bin for the models. For a few models the passive fraction is too high at low masses, particularly at low redshift. The models match well at high masses at low redshift, but generally underpredict the Passive fraction 10.0 10.5 11.0 log(M∗[M⊙]) 10.0 10.5 11.0 log(M∗[M⊙]) Figure 2. The evolution of the passive fraction over the range 0.5 < z < 3.0. The coloured lines, black solid lines and grey shaded regions are the same as in Figure 1, as are the snapshots used in each redshift bin for the models. For a few models the passive fraction is too high at low masses, particularly at low redshift. The models match well at high masses at low redshift, but generally underpredict the passive fraction for high-mass galaxies at high redshift. 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −2.5 −2.0 −1.5 −1.0 −0.5 log(sSFR[Gyr−1]) Elbaz+07 DLB07 Galform GalICS-2.0 LGALAXIES MICE MORGANA SAG SAGE ySAM z = 0.0 Figure 3. The relationship between mass and sSFR at z = 0.0 for star-forming galaxies in the nine models. The model data is taken from one snapshot at z = 0.0. The grey shaded region is taken from Elbaz et al. (2007) and shows the observational best-fit to this relation. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 3.2 Star-forming and Passive Galaxy Stellar Mass Functions We explore the mass growth further in the bottom two rows of Figure 1, splitting the population into passive (middle row) and star-forming (bottom row) galaxies. We separate passive and star-forming galaxies using a redshift-dependent specific star formation rate (sSFR) cut of sSFR(z) = 1/(3tH(z)) where tH(z) is the Hubble time at that red- shift. We test the robustness of this cut by examining the change in our results when using slightly different cuts of sSFR(z) = 1/(2tH(z)) and sSFR(z) = 1/(4tH(z)). We find that the shape of the stellar mass function changes very little and when accounting for Eddington bias, as it is very difficult to accurately measure all of the sources of error. Due to the steep slope of the SMF at high masses this would have a greater impact at the high-mass end of the SMF. The im- pact of Eddington bias on the SMF are discussed further in Appendix B. Cosmic CARNage II 7 Cosmic CARNage II 7 Cosmic CARNage II 7 −1.5 −1.0 −0.5 0.0 log(φ/φ0) 9 < log(M∗[M⊙]) < 10 All Davidzon+17 DLB07 Galform GalICS-2.0 −2.5 −2.0 −1.5 −1.0 −0.5 0.0 0.5 log(φ/φ0) Passive 0.5 1.0 1.5 2.0 2.5 3.0 z −1.0 −0.5 0.0 0.5 log(φ/φ0) Star-forming 10 < log(M∗[M⊙]) < 11 LGALAXIES MICE MORGANA 0.5 1.0 1.5 2.0 2.5 3.0 z 11 < log(M∗[M⊙]) < 12 SAG SAGE ySAM 0.5 1.0 1.5 2.0 2.5 3.0 z 2 4 6 8 10 11 tlb[Gyr] 2 4 6 8 10 11 tlb[Gyr] 2 4 6 8 10 11 tlb[Gyr] -1.5 -1.0 -0.5 0.0 log(φ/φ0) -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 log(φ/φ0) -1.0 -0.5 0.0 0.5 log(φ/φ0) Figure 5. The evolution of the number density φ, in bins of stellar mass. This is normalised by the number density at 0.2 < z < 0.5, which we call φ0. We show three mass bins as indicated (left to right panels) for all galaxies (top panels), passive galaxies (middle panels) and star-forming galaxies (bottom panels). The dark grey shaded regions and black lines with circular points show data from Davidzon et al. (2017). The coloured points and lines are for the nine models. In the lowest mass bin, most of the models assemble the galaxies before the observations. The models match the observations well at intermediate masses, but the observational number density increases before many of the models at high mass. 3.2 Star-forming and Passive Galaxy Stellar Mass Functions 11 < log(M∗[M⊙]) < 12 SAG SAGE ySAM 2 4 6 8 10 11 tlb[Gyr] -1.5 -1.0 -0.5 0.0 log(φ/φ0) 0.5 1.0 1.5 2.0 2.5 3.0 z -1.0 -0.5 0.0 0.5 log(φ/φ0) Star-forming Figure 5. The evolution of the number density φ, in bins of stellar mass. This is normalised by the number density at 0.2 < z < 0.5, which we call φ0. We show three mass bins as indicated (left to right panels) for all galaxies (top panels), passive galaxies (middle panels) and star-forming galaxies (bottom panels). The dark grey shaded regions and black lines with circular points show data from Davidzon et al. (2017). The coloured points and lines are for the nine models. In the lowest mass bin, most of the models assemble the galaxies before the observations. The models match the observations well at intermediate masses, but the observational number density increases before many of the models at high mass. SMF, as these galaxies will have very blue and red colours respectively. makes no difference to any of the conclusions that we draw. In the observations the passive and star-forming galaxies are seperated using the (NUV - r) vs (r - J) colour-colour diagram as described in Ilbert et al. (2013), which is best suited to differentiate fully quiescent galaxies from those with residual star formation. In practice, the exact location of the split makes little difference to the low-mass end of the star-forming SMF and the high-mass end of the passive Splitting the galaxy population in this way reveals that the main source of the difference between the observations and the models comes from the star-forming population: low-mass star-forming galaxies appear to be far too com- mon at high redshift in the models and the star-forming SMF evolves little from z = 3 to z = 0.5. The exceptions to this are Mice and Lgalaxies, which appear consistent with R. Asquith et al. 8 8 R. Asquith et al. 3.2 Star-forming and Passive Galaxy Stellar Mass Functions 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) 0.5 < z < 1.0 All 1.0 < z < 2.0 Baryon fraction Hartley+13 DLB07 Galform GalICS-2.0 LGALAXIES 2.0 < z < 3.6 MICE MORGANA SAG SAGE ySAM 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) Passive 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) Star-forming 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) Figure 6. The average halo mass in the models compared to measurements from observations between z = 0.5 and z = 3.6. Observational measurements of the average halo mass from Hartley et al. (2013) are derived from clustering and are shown as stars. The values for each model are shown as coloured lines. For the models, the mass of the main host halo was used rather than the subhalo, to better compare with observational halo mass measurements from clustering. The top panels cover the full galaxy sample, the middle panels are for passive galaxies and the bottom panels are for star-forming galaxies. The black dashed line shows the universal baryon fraction. For the models, we use snapshots at z = 1.0, 2.0 and 3.5 for each redshift bin respectively. For the passive sample, the halo masses from observations are approximately constant, but decrease by up to a factor of 10 in the models with increasing redshift. For the star-forming sample, the observations show halo mass increasing with increasing redshift, whereas in the models there is no real trend with redshift. th b ti t l t 3 F th i t fid h i ll ti t d t d th t f log(M∗[M⊙]) Figure 6. The average halo mass in the models compared to measurements from observations between z = 0.5 and z = 3.6. Observational measurements of the average halo mass from Hartley et al. (2013) are derived from clustering and are shown as stars. The values for each model are shown as coloured lines. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 3.3 Evolution of the Passive Fraction rates of the subset of star-forming galaxies. Figure 3 shows the average sSFR as a function of mass at z = 0.0 for each of the models as a solid coloured line. The grey shaded region is taken from Elbaz et al. (2007), who used SDSS data to find a fit to the correlation between SFR and mass at z = 0. Their sample is made up of 19590 galaxies with redshifts z = 0.04− 0.1 and is complete to MB ≤−20. Brinchmann et al. (2004) used Hα emission to derive the SFR of these galaxies and the stellar masses were derived by Kauffmann et al. (2003), who fit using a library of star formation histories to find the most likely stellar mass. Another way of looking at this result is to examine the pas- sive fraction, which is shown in Figure 2. Again, the shaded regions indicate the observations taken from the same source as used for Figure 1. The passive fraction indicates the ratio of passive to star-forming galaxies. At low masses, some of the models, such as DLB07, Galform and Morgana, tend to overestimate the passive fraction compared to observa- tions. This has been seen previously and appears to be linked to how environmental processes are taken into account in the models (Lagos et al. 2014; Gonzalez-Perez et al. 2018). At low redshift the number of star-forming galaxies matches observations well, so this difference is due to the lack of a turnover or flattening of the passive SMF. At higher red- shifts, the overproduction of low-mass star-forming galaxies would act to decrease the passive fractions. However, this is still too high in some models, again due to the rising number density towards low masses in the passive SMF. Most of the models match the observations well here, with Lgalaxies and Sag lying in the observational region at all masses. Some models appear to evolve less with mass than the observations suggest, with some showing almost no trend, whereas the sSFR implied by the observations de- creases by over 0.5 dex between 109M⊙and 1012M⊙. This means that some of the models, such as Galform, match at low masses but not high masses, and others such as DLB07 and ySAM match at high masses but not low masses. This was also discussed in Guo et al. 3.2 Star-forming and Passive Galaxy Stellar Mass Functions For the models, the mass of the main host halo was used rather than the subhalo, to better compare with observational halo mass measurements from clustering. The top panels cover the full galaxy sample, the middle panels are for passive galaxies and the bottom panels are for star-forming galaxies. The black dashed line shows the universal baryon fraction. For the models, we use snapshots at z = 1.0, 2.0 and 3.5 for each redshift bin respectively. For the passive sample, the halo masses from observations are approximately constant, but decrease by up to a factor of 10 in the models with increasing redshift. For the star-forming sample, the observations show halo mass increasing with increasing redshift, whereas in the models there is no real trend with redshift. to find a physically motivated way to reduce the star forma- tion rates of low-mass galaxies at high redshift. The same galaxies at later times would then have lower stellar masses and star formation rates. This would then act to redistribute the passive SMF in the models to better match the observa- tions. the observations at low masses up to z = 3. For the passive galaxies, the number density at low masses does evolve with redshift in the models, as seen in the observations. However, most of the SAMs show rising number density towards lower masses, in contrast with the observations which appear to show a turnover or flattening of the passive SMF towards lower masses. In order to solve these problems, models need Cosmic CARNage II 9 9 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) z = 0.1 Behroozi+13 Baryon fraction DLB07 Galform 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) z = 1.0 GalICS-2.0 LGALAXIES MICE MORGANA 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) z = 2.0 SAG SAGE ySAM 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) z = 0.1 Behroozi+13 Baryon fraction DLB07 Galform 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) z = 1.0 GalICS-2.0 LGALAXIES MICE MORGANA 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 log(M200[M⊙]) z = 2.0 SAG SAGE ySAM 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) Figure 7. 3.3 Evolution of the Passive Fraction (2016), who used data from two SAMs, Galform and Lgalaxies, and one hydrody- namical simulation, Eagle. They found that the median sSFR remained almost constant with mass, in contrast with observations. At low redshift, the models tend to match the observa- tions well at high masses, but one model, Sage, underpre- dicts the passive fraction. This is mainly due to an under- prediction for the number of high-mass passive galaxies. As shown by Stevens & Brown (2017), detailing the structural evolution of galaxy discs with the Dark Sage variant of the model (Stevens et al. 2016) leads to more sensible pas- sive fractions. In the redshift range 1.5 < z < 2.0 the mod- els tend to underpredict the fraction of high-mass galaxies, mainly due to the lack of high-mass passive galaxies above z ∼1. The model which best matches the observed passive fraction for high-mass galaxies is DLB07, which slightly un- derpredicts the number density of both high-mass passive and star-forming galaxies in this redshift range. The relationship between sSFR and stellar mass at z = 2.0 is then shown in Figure 4. Here the observations are taken from Daddi et al. (2007), who use galaxies in the GOODS-S field to find the correlation between SFR and mass at z = 2. They are complete to K < 22 and use only 24µm selected galaxies in order to exclude passive galaxies. The SFRs were estimated using the UV and the stellar masses were derived by Fontana et al. (2004) using SED fitting. This comparison highlights large differences between the observations and models at this redshift, with the mod- els almost completely outside the observational range. The sSFR of star-forming galaxies in the models is on average around 0.5 dex lower than measured in the observations. 3.2 Star-forming and Passive Galaxy Stellar Mass Functions Comparison of the average stellar mass for each halo mass bin in the models to the abundance matching model of Behroozi et al. (2013c), considering only central galaxies. The results from the models are shown as coloured lines and the stellar mass - halo mass relation from Behroozi et al. (2013c) is shown as a dark grey shaded region and black line. The black dashed line shows the universal baryon fraction. The panels are for each redshift, increasing from left to right, using snapshots at z = 0.0, 1.0 and 2.0 respectively. Looking at the data from Behroozi et al. (2013c), we can see that the average stellar mass stays fairly constant with redshift, but increases in the models towards low redshift, particularly at high masses. log(M200[M⊙]) log(M200[M⊙]) log(M200[M⊙]) Figure 7. Comparison of the average stellar mass for each halo mass bin in the models to the abundance matching model of Behroozi et al. (2013c), considering only central galaxies. The results from the models are shown as coloured lines and the stellar mass - halo mass relation from Behroozi et al. (2013c) is shown as a dark grey shaded region and black line. The black dashed line shows the universal baryon fraction. The panels are for each redshift, increasing from left to right, using snapshots at z = 0.0, 1.0 and 2.0 respectively. Looking at the data from Behroozi et al. (2013c), we can see that the average stellar mass stays fairly constant with redshift, but increases in the models towards low redshift, particularly at high masses. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 3.5 Growth of the Galaxy Stellar Mass Function In Figure 5 we examine the growth of the stellar mass func- tion as a function of mass and redshift. This is found by taking the value of the number density φ at fixed stellar mass for a certain redshift bin and normalising it by the value of φ in the lowest redshift bin 0.2 < z < 0.5, which we call φ0. This allows for easier comparison between the models and observations and will highlight when the num- ber density of different populations increases. The dark grey region and black line with circular points shows data from Davidzon et al. (2017). The coloured lines then show the number density evolution for the nine models. The black dotted line shows where the number density is equal to the number density in the lowest redshift bin. For the models, here we use the mass of the main host halo for each galaxy rather than the mass of its subhalo. Host haloes do not reside within another halo, whereas subhaloes are contained within a host halo. Although us- ing the host halo is not necessarily the usual choice when analysing simulation data, it allows us to compare to obser- vational measurements of halo mass from galaxy clustering, which effectively measure the mass of the main host haloes (Mo & White 2002). For this reason we also include both centrals and satellites, in order to best mimic the observa- tional measurements. Assuming galaxy clustering measure- ments can correctly recover the host halo mass, we can then directly compare the observations and models. Looking at the passive galaxies, we can see that the models struggle to match the observed growth of the mass function at low masses, as the number density of low-mass galaxies increases in the models at higher redshift than the observations. The only exception is Mice, which has very few galaxies with mass below 1010M⊙above z ∼1. At in- termediate masses the models match the observations well, but at high masses the growth of the mass function occurs in observations before many of the models. y For the star-forming galaxies, at low masses there is a similar problem with several of the models; the mass func- tion grows too much at high redshift. Just under half of the models have more low-mass star-forming objects in the highest redshift bin than the lowest redshift one. 3.4 Relationship between Mass and Specific Star Formation Rate In order to better compare star formation in the observations and the models, we also look at the specific star formation 10 R. Asquith et al. of the stellar mass function. Looking at the lower panel of Figure 1, we can see that DLB07 overpredicts the num- ber of low-mass star-forming galaxies at both low and high redshift. Sage agrees well with observations at low redshift but overproduces low-mass star-forming galaxies at high red- shift. However, looking at the lower left panel of Figure 5, we can see that DLB07 matches observations of the growth of the mass function better than Sage. These models there- fore have slightly different problems; DLB07 has too many low-mass galaxies at all redshifts, but the number density in- creases at the correct rate. Conversely, Sage has the correct number at low redshift, but the number density increases too early. The models therefore predict a slower evolution of the sSFR with redshift than observations. This has been previously seen by Mitchell et al. (2014), who find that when they scale the reincorporation time of gas with redshift they are able to better match the evolution of the stellar mass function, but still underestimate the sSFR of high-mass galaxies at z ∼2. Hirschmann et al. (2016) also found that their ejec- tive models predicted lower than observed sSFRs at high redshift, even when they could reproduce the growth of the stellar mass function. The models therefore predict a slower evolution of the sSFR with redshift than observations. This has been previously seen by Mitchell et al. (2014), who find that when they scale the reincorporation time of gas with redshift they are able to better match the evolution of the stellar mass function, but still underestimate the sSFR of high-mass galaxies at z ∼2. Hirschmann et al. (2016) also found that their ejec- tive models predicted lower than observed sSFRs at high redshift, even when they could reproduce the growth of the stellar mass function. Reducing the star formation rates of galaxies above z ∼ 2, as suggested in Section 3.2, may help to solve this problem. If galaxies have a lower star formation rate at higher redshift, their resulting mass at lower redshift will be lower. A galaxy with the same star-formation rate at z = 2 will then have a higher sSFR as it will have a lower stellar mass. 3.6 Average Halo Mass In this section we study the average halo mass the galax- ies reside within, shown in Figure 6. For the models we use single snapshots at z = 1.0,2.0 and 3.5 for each redshift bin respectively. The dashed black line indicates the universal baryon fraction, i.e. where all the baryonic material within the halo has been converted into stars. Each of the coloured lines indicates the average halo mass values for a different model, while the black points with errorbars are average halo mass values taken from Hartley et al. (2013), who use the UDS DR8 data to estimate the halo masses from mea- surements of galaxy clustering (e.g. Mo & White 2002, and references therein). Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 3.5 Growth of the Galaxy Stellar Mass Function However, several of the models are more in line with the growth of the observed mass function, namely Sag and Mice. At interme- diate masses, the number density of star-forming galaxies increases at higher redshift in the models than in the ob- servations. The model that is most discrepant, Morgana, has more intermediate mass star-forming galaxies between 1.0 < z < 1.5 than in the lowest redshift bin. For high-mass galaxies, the number density evolves little since high red- shift in the observations. Mice reproduces this trend well but in other models the number density increases at lower redshift. This may be in part due to the fact that there are low numbers of the highest mass galaxies which will natu- rally introduce more scatter in the proportional change in number density. Splitting the sample into passive and star-forming galaxies in Figure 6 we see that there are marked differ- ences between the observations and the models. For passive galaxies, the average halo mass in observations stays con- stant over redshift in the observations, but rises towards low redshift in the models. For the star-forming population, while the observations indicate a general downsizing trend in halo mass of about an order of magnitude between high and low redshift, all the models show virtually no change. It is clear that the models start significantly below the observa- tions at 2.0 < z < 3.5 and only agree with the observations by 0.5 < z < 1.0. Both passive and star-forming low-mass galaxies are therefore in lower mass haloes on average in the models than in the observations at high redshift. One thing that can affect the average halo mass values in the models is the halo mass definition used, as this can lead to differences of up to 20 percent (Jiang et al. 2014). Al- though this may account for some of the scatter between the models, the differences between the observations and models cannot be explained by this alone. Another factor that could affect the observational measurments of halo mass from clus- We can also see interesting differences between mod- els when comparing the stellar mass function to the growth Cosmic CARNage II 11 Cosmic CARNage II 11 Cosmic CARNage II 11 changes the least with redshift is Mice; as this is an HOD model it naturally matches the SMHM relation better than the SAMs. 3.7 Stellar Mass - Halo Mass Relation In Figure 7 we display measurements of the average stel- lar mass of central galaxies in bins of halo mass, com- paring the models with the abundance matching model of Behroozi et al. (2013c). The dashed black line indicates the universal baryon fraction and the dark grey region and black solid line show the fit to the stellar mass - halo mass (SMHM) relation from Behroozi et al. (2013c). The coloured lines show the average stellar mass values for each different model. In the absence of a new population of low-mass, star- forming galaxies being observed at z ∼2, many of the mod- els would need improvements in order to reproduce obser- vations. They would need to produce far fewer low-mass star-forming galaxies at essentially all but the latest times. Shifting star formation from high-mass haloes at high red- shift to low-mass haloes at low redshift would also produce better agreement with observations of galaxy clustering. Re- ducing the number of low-mass star-forming objects would also have to be achieved without reducing the number of high-mass objects significantly. At low redshift, the results from the models and SMHM relation agree well at low and intermediate halo masses. However, above halo masses of ∼1013.5M⊙the average stel- lar mass of centrals in the models is higher than suggested by the SMHM relation. This means that at low redshift, star formation in high-mass haloes is more efficient in the mod- els. The exceptions to this are Lgalaxies and Mice, which agree with the SMHM relation at nearly all halo masses. For most of the models, the slope of the relation at high halo masses does flatten, but not to the extent seen from the SMHM relation. Some of the models, such as Lgalaxies and Sag, do fit the low-mass end of both the star-forming and passive stellar mass function at high redshift. This is likely due to their implementation of the physics involved in the treat- ment of gas, in particular the reincorporation timescales. Mice also matches observations at high redshift, but as this is a HOD model it matches by construction. However, there are still some observables that even these models struggle to match, such as the relation between stellar mass and specific star formation rate and the average halo mass that galax- ies occupy. 4 DISCUSSION Comparing several galaxy formation models allows us to dis- tinguish areas that are challenging for the current generation of models and therefore provide direction for the future de- velopment of the field as a whole. The main issue highlighted in this paper is the fact that most of the models produce too many low-mass, star-forming galaxies at early times. Obser- vationally these appear either to not exist or to be missed by the surveys. This is a difficult area observationally with the answer to this question only becoming evident when the stellar mass functions are reliably pushed to lower masses. At present they are tantalisingly close to indicating a clear turnover in the space density of passive galaxies at low-mass, which would significantly challenge many of the models fea- tured here. 3.5 Growth of the Galaxy Stellar Mass Function tering is ‘halo assembly bias’, which refers to the fact that halo clustering can depend on other properties besides halo mass. For example, Gao et al. (2005) found that at fixed halo mass, haloes that assembled earlier are more clustered than those that assembled later. Therefore, galaxies in older haloes will be more strongly clustered than they should be for their halo mass, which means that their halo masses will be measured as higher than they actually are. This could alleviate some of the discrepancy between the observations and models. For example, if the passive galaxies observed at low redshift are associated to older haloes, then their halo masses could have been overestimated. changes the least with redshift is Mice; as this is an HOD model it naturally matches the SMHM relation better than the SAMs. 3.7 Stellar Mass - Halo Mass Relation Whilst this could be due to problems with the observational measurements of these quantities, this could point towards areas where the models still need to improve. As we move to higher redshift the SMHM relation changes little. The peak of the relation moves to slightly higher halo masses and the average stellar mass for low-mass haloes decreases by ∼0.4 dex at 1011.5M⊙. In the mod- els the average stellar mass for low-mass haloes decreases slightly with increasing redshift, but is above the SMHM relation by z = 1.0 for most models. This discrepancy can be partially explained by the cut in stellar mass applied at M∗= 109M⊙h−1, which may have skewed the distribu- tion towards higher stellar masses. This might be enough to explain the difference for models such as Lgalaxies or Galform, but the discrepancy is too large for Morgana, DLB07 and ySAM. In these models, the average stellar mass for low-mass haloes at high redshift is too high. This means that star formation in these objects is very efficient, leading to an increase in the number of low-mass galaxies at z ∼2. This is likely due to the way that the physics involved in the gas cycle is implemented in these models. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 ACKNOWLEDGEMENTS We thank Carnegie Observatories for their support and hos- pitality during the workshop ‘Cosmic CARNage’ where all the calibration issues were discussed and the roadmap laid out for the work presented here. • Whilst most of the models are able to match the ob- served stellar mass function at low redshift, they tend to overproduce the number density of low-mass galaxies at high redshift. The authors would further like to express special thanks to the Instituto de Fisica Teorica (IFT-UAM/CSIC in Madrid) for its hospitality and support, via the Centro de Excelencia Severo Ochoa Program under Grant No. SEV- 2012-0249, during the three week workshop ‘nIFTy Cos- mology’ where this work developed. We further acknowl- edge the financial support of the 2014 University of West- ern Australia Research Collaboration Award for ‘Fast Ap- proximate Synthetic Universes for the SKA’, the ARC Cen- tre of Excellence for All Sky Astrophysics (CAASTRO) grant number CE110001020, and the ARC Discovery Project DP140100198. We also recognise support from the Universi- dad Autonoma de Madrid (UAM) for the workshop infras- tructure. • In most of the models the low-mass end of the star- forming stellar mass function is already largely in place at high redshift (z > 1), in contrast to observations. This is be- cause the models appear to produce too many star-forming galaxies below the knee of the stellar-mass function at early times. • In most of the models the low-mass end of the star- forming stellar mass function is already largely in place at high redshift (z > 1), in contrast to observations. This is be- cause the models appear to produce too many star-forming galaxies below the knee of the stellar-mass function at early times. • The passive stellar mass function from the models evolves with redshift as in the observations, but does not have the same turnover or flattening in the number density at the low-mass end. • Whilst most of the models match the passive fraction well at high masses, for some of the models the passive frac- tion is too high at low masses. This is despite the overpro- duction of low-mass star-forming galaxies. We would like to thank Rachel Somerville, Gabriella De Lucia and Pierluigi Monaco for kindly providing useful discussion and comments. 5 CONCLUSIONS In this paper we have contrasted nine different galaxy forma- tion models and compared them to the latest high-redshift observations. In doing so we have highlighted the areas in which the models find particular difficulty in matching the observations. We can see from this project that some of the models still have trouble simultaneously matching the stellar mass function at both low and high redshift. The galaxies look roughly correct at z = 0, but for many models there are too many low-mass galaxies at z ∼2, as has also been seen previously (e.g. Fontana et al. 2006; Fontanot et al. 2009; Weinmann et al. 2012; Henriques et al. 2012; Guo et al. 2016). For intermediate- and high-mass haloes, the average stellar mass generally decreases with increasing redshift in the models and the slope of the relation decreases. This sug- gests that star formation was less efficient in the models at high redshift. At z = 0.1 the models overpredict the stellar mass in high-mass haloes, but slightly underpredict it by z = 2.0. For intermediate-mass haloes, the average stellar mass is too low in the models at z = 2.0 by up to 0.5 dex, as is the case for Galform at 1012.5M⊙. The model that 12 R. Asquith et al. high redshift will help shed light on these issues and identify further areas of improvement for the models. To explore this further, we split galaxies into pas- sive and star-forming populations. We find that there are too many star-forming galaxies with stellar masses below 1011M⊙in many of the models at z ∼2. In summary, while some of the models are remarkably successful at reproducing the evolution of the stellar mass function, there remain significant issues. In particular: Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 ACKNOWLEDGEMENTS • Most of the models are able to reproduce the relation- ship between sSFR and the mass of the star-forming galaxies at low redshift, but underpredict the sSFR at high redshift. RA is funded by the Science and Technology Funding Council (STFC) through a studentship. AK is supported by the Ministerio de Econom´ıa y Competitividad and the Fondo Europeo de Desarrollo Regional (MINECO/FEDER, UE) in Spain through grant AYA2015-63810-P. He fur- ther thanks Denison Witmer for california brown and blue. VGP acknowledges support from a European Re- search Council Starting Grant (DEGAS-259586). This work used the DiRAC Data Centric system at Durham Uni- versity, operated by the Institute for Computational Cos- mology on behalf of the STFC DiRAC HPC Facility (www.dirac.ac.uk). This equipment was funded by BIS Na- tional E-infrastructure capital grant ST/K00042X/1, STFC capital grant ST/H008519/1, and STFC DiRAC Opera- tions grant ST/K003267/1 and Durham University. DiRAC is part of the National E-Infrastructure. FJC acknowl- edges support from the Spanish Ministerio de Econom´ıa y Competitividad project AYA2012-39620. SAC acknowledges funding from Consejo Nacional de Investigaciones Cient´ıfi- cas y T´ecnicas (CONICET, PIP-0387), Agencia Nacional de Promoci´on Cient´ıfica y Tecnol´ogica (ANPCyT, PICT- 2013-0317), and Universidad Nacional de La Plata (G11- 124), Argentina. DJC acknowledges receipt of a QEII Fel- lowship from the Australian Government. FF acknowledges financial contribution from the grants PRIN MIUR 2009 ‘The Intergalactic Medium as a probe of the growth of cosmic structures’ and PRIN INAF 2010 ‘From the dawn of galaxy formation’. The work of BH was supported by Advanced Grant 246797 GALFORMOD from the Euro- pean Research Council. NDP was supported by BASAL PFB-06 CATA, and Fondecyt 1150300. Part of the calcu- lations presented here were run using the Geryon cluster at the Center for Astro-Engineering at U. Catolica, which re- ceived funding from QUIMAL 130008 and Fondequip AIC- 57. CP acknowledges support of the Australian Research • Observational measurements of halo mass, estimated from galaxy clustering, indicate clear downsizing in the av- erage halo mass occupied by star-forming galaxies as a func- tion of redshift. This is not clearly indicated by any of the models; both star-forming and passive galaxies in the mod- els occupy haloes with lower masses than those inferred from observations at z = 2. Cosmic CARNage II 13 Cosmic CARNage II 13 13 Council (ARC) through Future Fellowship FT130100041 and Discovery Project DP140100198. WC and CP acknowl- edge support of ARC DP130100117. PAT acknowledges support from the Science and Technology Facilities Coun- cil (grant number ST/L000652/1). SKY acknowledges sup- port from the Korean National Research Foundation (NRF- 2017R1A2A1A05001116). This study was performed under the umbrella of the joint collaboration between Yonsei Uni- versity Observatory and the Korean Astronomy and Space Science Institute. The supercomputing time for the numer- ical simulations was kindly provided by KISTI (KSC-2014- G2-003). Faber S. M., et al., 2007, ApJ, 665, 265 Fontana A., et al., 2004, A&A, 424, 23 Fontana A., et al., 2006, A&A, 459, 745 Fontanot F., De Lucia G., Monaco P., Somerville R. S., Santini P., 2009, MNRAS, 397, 1776 Gan J., Kang X., van den Bosch F. C., Hou J., 2010, MNRAS, 408, 2201 Gao L., Springel V., White S. D. M., 2005, MNRAS, 363, L66 Gonzalez-Perez V., Lacey C. G., Baugh C. M., Lagos C. D. P., Helly J., Campbell D. J. R., Mitchell P. D., 2014, MNRAS, 439, 264 Gonzalez-Perez V., et al., 2018, MNRAS, 474, 4024 Gunn J. E., Gott J. R. I., 1972, ApJ, 176, 1 ) The authors contributed to this paper in the following ways: RA analysed the data, created the plots and wrote the paper along with FRP and OA. AK & CP formed part of the core team and along with FRP organised the nIFTy workshop where this work was initiated. AB organised the follow-up workshop ‘Cosmic CARNage’ where all the discus- sions about the common calibration took place and out of which this paper emerged. JO supplied the simulation, halo catalogue and merger tree for the work presented here. WH supplied the halo mass measurements from the UDS that were used in this work. The remaining authors performed the SAM or HOD modelling using their codes, in particular FJC, AC, SC, DC, FF, VGP, BH, JL, ARHS, CVM, and SKY actively ran their models. All authors proof-read and commented on the paper. Guo Q., et al., 2011, MNRAS, 413, 101 Guo Q., et al., 2016, MNRAS, 461, 3457 Hartley W. G., et al., 2013, MNRAS, 431, 3045 Henriques B. M. B., White S. D. M., Lemson G., Thomas P. A., Guo Q., Marleau G.-D., Overzier R. A., 2012, MNRAS, 421, 2904 Henriques B. M. B., White S. D. References Kovaˇc K., et al., 2014, MNRAS, 438, 717 Lagos C. D. P., Baugh C. M., Zwaan M. A., Lacey C. G., Baldry I. K., Glazebrook K., Brinkmann J., Ivezi´c ˇZ., Lupton R. H., Nichol R. C., Szalay A. S., 2004, ApJ, 600, 681 Lagos C. D. P., Baugh C. M., Zwaan M. A., Lacey C. G., Gonzalez-Perez V., Power C., Swinbank A. M., van Kampen E., 2014, MNRAS, 440, 920 Gonzalez-Perez V., Power C., Swinbank A. M., van Kampen E., 2014, MNRAS, 440, 920 E., 2014, MNRAS, 440, 920 y Behroozi P. S., Wechsler R. H., Wu H.-Y., 2013a, ApJ, 762, 109 Lawrence A., et al., 2007, MNRAS, 379, 1599 Behroozi P. S., Wechsler R. H., Wu H.-Y., Busha M. T., Klypin A. A., Primack J. R., 2013b, ApJ, 763, 18 Lee J., Yi S. K., 2013, ApJ, 766, 38 Behroozi P. S., Wechsler R. H., Conroy C., 2013c, ApJ, 770, 57 Marchesini D., van Dokkum P. G., F¨orster Schreiber N. M., Franx Marchesini D., van Dokkum P. G., F¨orster Schreib M., Labb´e I., Wuyts S., 2009, ApJ, 701, 1765 Marchesini D., van Dokkum P. G., F¨orster Schreibe Marchesini D., van Dokkum P. G., Forster Schreiber M., Labb´e I., Wuyts S., 2009, ApJ, 701, 1765 M., Labb´e I., Wuyts S., 2009, ApJ, 701, 1765 Benson A. J., 2010, Phys. Rep., 495, 33 Marchesini D., et al., 2010, ApJ, 725, 1277 Benson A. J., Bower R. G., Frenk C. S., Lacey C. G., Baugh C. M., Cole S., 2003, ApJ, 599, 38 McCarthy I. G., Frenk C. S., Font A. S., Lacey C. G., Bower R. G., Mitchell N. L., Balogh M. L., Theuns T., 2008, MNRAS, 383, 593 Berlind A. A., Weinberg D. H., 2002, ApJ, 575, 587 Berlind A. A., et al., 2003, ApJ, 593, 1 McCracken H. J., et al., 2012, A&A, 544, A156 Bower R. G., Benson A. J., Malbon R., Helly J. C., Frenk C. S., Baugh C. M., Cole S., Lacey C. G., 2006, MNRAS, 370, 645 Mitchell P. D., Lacey C. G., Cole S., Baugh C. M., 2014, MNRAS, 444, 2637 Brinchmann J., Charlot S., White S. D. M., Tremonti C., Kauff- mann G., Heckman T., Brinkmann J., 2004, MNRAS, 351, 1151 Mo H. J., White S. D. M., 2002, MNRAS, 336, 112 Monaco P., Fontanot F., Taffoni G., 2007, MNRAS, 3 Mundy C. J., Conselice C. J., Ownsworth J. Cosmic CARNage II 13 M., Thomas P. A., Angulo R. E., Guo Q., Lemson G., Springel V., 2013, MNRAS, 431, 3373 Henriques B. M. B., White S. D. M., Thomas P. A., Angulo R., Guo Q., Lemson G., Springel V., Overzier R., 2015, MNRAS, 451, 2663 Hirschmann M., De Lucia G., Fontanot F., 2016, MNRAS, 461, 1760 Ilbert O., et al., 2010, ApJ, 709, 644 Ilbert O., et al., 2013, A&A, 556, A55 Jiang L., Helly J. C., Cole S., Frenk C. S., 2014, MNRAS, 440, 2115 This research has made use of NASA’s Astrophysics Data System (ADS) and the arXiv preprint server. This research has made use of NASA’s Astrophysics Data System (ADS) and the arXiv preprint server. Kauffmann G., et al., 2003, MNRAS, 341, 33 Kennicutt Jr. R. C., 1998, ARA&A, 36, 189 Kimm T., Yi S. K., Khochfar S., 2011, ApJ, 729, 11 Knebe A., et al., 2015, MNRAS, 451, 4029 Knebe A., et al., 2018, MNRAS, 475, 2936 ACKNOWLEDGEMENTS • Observational measurements of halo mass, estimated from galaxy clustering, indicate clear downsizing in the av- erage halo mass occupied by star-forming galaxies as a func- tion of redshift. This is not clearly indicated by any of the models; both star-forming and passive galaxies in the mod- els occupy haloes with lower masses than those inferred from observations at z = 2. • The average stellar mass is higher in low-mass haloes at high redshift in the models compared to observations, mean- ing that star formation in low-mass haloes is more efficient in the models than in the real Universe. • The average stellar mass is higher in low-mass haloes at high redshift in the models compared to observations, mean- ing that star formation in low-mass haloes is more efficient in the models than in the real Universe. Achieving consistent results at both z = 0 and z = 2 with a population of galaxies that evolves strongly with redshift is clearly difficult. The HOD model, Mice, obtains good results but the galaxies present at z = 2 are not evolved directly into the z = 0 population. Of the SAMs, the Lgalaxies and Sag models best match the growth of the observed mass func- tions, but they share the same trends as the other models for the specific star formation rate and average halo mass within which the objects reside. Both of these models found that they needed to modify the treatment of the gas cycle in order to match the evolution of the low-mass end of the stellar mass function. This is very promising for the galaxy formation modelling community, which has long struggled with this issue. While it is clear that current galaxy formation models can reproduce a variety of observational data, we have iden- tified key areas of tension. Some models still overpredict the number of low-mass galaxies at high redshift, but even the models that can match the evolution of the galaxy stellar mass function underpredict the specific star formation rates of galaxies at early times. Future observational surveys at APPENDIX B: STELLAR MASS FUNCTION INCLUDING EDDINGTON BIAS In Figure 1 we have compared the stellar mass function from the models to observational data from Davidzon et al. (2017). We do not scatter the stellar masses in the models with the 0.08(1 + z) dex scatter used to mimic observational uncertainties, as Davidzon et al. (2017) have accounted for this when finding the best-fit Schechter parameters to their stellar mass function. Here we present an alternative version of Figure 1, shown in Figure B1, where we do apply the scatter to the stellar mass values in the models. We compare to obser- vations from Muzzin et al. (2013), who do not take these uncertainties into account when fitting to the stellar mass function. Like Davidzon et al. (2017), the observations from Muzzin et al. (2013) are based on the UltraVISTA near- infrared survey of the COSMOS field. Supernova feedback and winds The mass reheated by super- nova feedback involves an explicit redshift dependence and an additional modulation with virial velocity, according to a fit to results from FIRE (Feedback in Realistic Environ- ments) hydrodynamical simulations (Muratov et al. 2015). Comparing Figure B1 to Figure 1, we can see that the main difference to the SMF is at the high-mass end and that the low-mass end is largely unaffected. Due to the redshift dependence of the scatter we apply to the stellar masses, the differences are also larger at high redshift. As an example, the value of φ increases by over 0.5 dex at 1011M⊙in the redshift bin 2.5 < z < 3.0 in Lgalaxies when the scatter is applied. Gas ejection and reincorporation The energy input by mas- sive stars eject some of the hot gas out of the halo, according to the energy conservation argument presented by Guo et al. (2011). The energy injected by massive stars is proportional to the mean kinetic energy of supernova ejecta per unit mass of stars formed, and includes the same explicit red- shift dependence and the additional modulation with virial velocity as the reheated mass. The ejected gas mass is re- incorporated back onto the corresponding (sub)halo within a timescale that depends on the inverse of the (sub)halo mass (Henriques et al. 2013). In Figure 1, it appears that most of the models under- predict the number of high-mass galaxies at high redshift, with only Mice and GalICS-2.0 matching observations. 14 R. Asquith et al. 14 Strateva I., et al., 2001, AJ, 122, 1861 Strateva I., et al., 2001, AJ, 122, 1861 Orphans The positions and velocities of orphan galaxies are obtained from the integration of the orbits of subhaloes that will not longer be identified. The orbits are integrated nu- merically, considering the last known position, velocity and virial mass of subhaloes as initial conditions, and taking into account mass loss by TS and dynamical friction effects, fol- lowing some aspects of the works by Gan et al. (2010) and Kimm et al. (2011). A merger event occurs when the halo- centric distance becomes smaller that 10 percent of the virial radius of the host halo. Tecce T. E., Cora S. A., Tissera P. B., Abadi M. G., Lagos C. D. P., 2010, MNRAS, 408, 2008 Weinmann S. M., Pasquali A., Oppenheimer B. D., Finlator K., Mendel J. T., Crain R. A., Macci`o A. V., 2012, MNRAS, 426, 2797 White C. E., Somerville R. S., Ferguson H. C., 2015, ApJ, 799, 201 Zheng Z., et al., 2005, ApJ, 633, 791 A1 Sag The changes implemented in SAG are described in detail in Cora et al. (2018). We summarize them here: The changes implemented in SAG are described in detail in Cora et al. (2018). We summarize them here: Cooling Both central and satellite galaxies experience gas cooling processes. Satellite galaxies keep their hot gas haloes which are gradually removed by the action of ram pressure stripping (RPS), modelled according to McCarthy et al. (2008), and tidal stripping (TS). When the mass of the hot gas halo becomes smaller than 10 percent of the total baryonic mass of the galaxy, it is assumed that it no longer shields the cold gas disc from the action of RPS, which is modelled following the criterion from Gunn & Gott (1972); see Tecce et al. (2010) for more details. Values of ram pres- sure experienced by galaxies in haloes of different mass as a function of halo-centric distance and redshift are obtained from fitting formulae derived from the self-consistent infor- mation provided by the hydrodynamical simulations anal- ysed by Tecce et al. (2010), as described in Vega-Mart´ınez et al. (in prep.). A2 Sage A description of the physical prescriptions of each model is available in the Appendix of Knebe et al. (2015). Here we present a brief description of the changes to any of the models since then: The only change in Sage is to the radio mode AGN feed- back. It is explained in detail in Croton et al. (2016) and summarized here: AGN feedback The radio mode AGN feedback has been modified in Sage since Croton et al. (2006). There is now a heating radius, inside which gas is prevented from cool- ing. This heating radius increases with subsequent heating episodes and can not decrease. AGN feedback The radio mode AGN feedback has been modified in Sage since Croton et al. (2006). There is now a heating radius, inside which gas is prevented from cool- ing. This heating radius increases with subsequent heating episodes and can not decrease. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 References R., 2015, MNRAS, 450, 3696 Carretero J., Castander F. J., Gazta˜naga E., Crocce M., Fosalba P., 2015, MNRAS, 447, 646 Muratov A. L., Kereˇs D., Faucher-Gigu`ere C.-A., Hopkins P. F Quataert E., Murray N., 2015, MNRAS, 454, 2691 Cattaneo A., et al., 2017, MNRAS, 471, 1401 Quataert E., Murray N., 2015, MNRAS, 454, 2691 Cimatti A., Daddi E., Renzini A., 2006, A&A, 453, L29 Cimatti A., Daddi E., Renzini A., 2006, A&A Muzzin A., et al., 2013, ApJ, 777, 18 Cirasuolo M., et al., 2007, MNRAS, 380, 585 Planck Collaboration et al., 2014, A&A, 571, A16 Conroy C., Gunn J. E., White M., 2009, ApJ, 699, 486 Pozzetti L., et al., 2007, A&A, 474, 443 Cora S. A., et al., 2018, preprint (arXiv:1801.03883) Pozzetti L., et al., 2010, A&A, 523, A13 Cowie L. L., Songaila A., Hu E. M., Cohen J. G., 1996, AJ, 112, 839 Rodrigues L. F. S., Vernon I., Bower R. G., 2017, MNRAS, 466, 2418 Croton D. J., et al., 2006, MNRAS, 365, 11 Shankar F., et al., 2015, ApJ, 802, 73 Croton D. J., et al., 2016, ApJS, 222, 22 Somerville R. S., Dav´e R., 2015, ARA&A, 53, 51 Daddi E., et al., 2007, ApJ, 670, 156 Springel V., 2005, MNRAS, 364, 1105 Davidzon I., et al., 2017, A&A, 605, A70 De Lucia G., Blaizot J., 2007, MNRAS, 375, 2 Stevens A. R. H., Brown T., 2017, MNRAS, 471, 447 Di Matteo T., Springel V., Hernquist L., 2005, Nature, 433, 604 Stevens A. R. H., Croton D. J., Mutch S. J., 2016, MNRAS, 461, 859 Elbaz D., et al., 2007, A&A, 468, 33 Elbaz D., et al., 2007, A&A, 468, 33 y y on 17 July 2018 14 R. Asquith et al. by University of on 17 July 2018 Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051 by University of Nottingham user on 17 July 2018 by U e s ty o on 17 July 2018 APPENDIX B: STELLAR MASS FUNCTION INCLUDING EDDINGTON BIAS However, in Figure B1 the models and observations agree better at high redshift for several other models, namely Gal- form and Morgana. The data from Muzzin et al. (2013) form part of the combined dataset used to calibrate the mod- els, so it is natural that the models may match this data better. AGN feedback AGN are produced from the growth of central BHs. When this growth takes place from cold gas accretion during gas cooling, it depends on the mass of the hot gas atmosphere, following Henriques et al. (2015). 15 Cosmic CARNage II 1 Cosmic CARNage II 15 −5 −4 −3 −2 log(φ[Mpc−3dex−1]) 0.5 < z < 1.0 All 1.5 < z < 2.0 Muzzin+13 DLB07 Galform GalICS-2.0 LGALAXIES 2.5 < z < 3.0 MICE MORGANA SAG SAGE ySAM −5 −4 −3 −2 log(φ[Mpc−3dex−1]) Passive 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) −5 −4 −3 −2 log(φ[Mpc−3dex−1]) Star-forming 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) Figure B1. Alternative version of Figure 1, applying the 0.08(1 + z) dex scatter to the stellar mass values in the models. Here we compare to observational data from Muzzin et al. (2013), who do not take into account Eddington bias when finding the best-fit Schechter parameters. Here the models match the observations better at high masses and high redshift. 2.5 < z < 3.0 MICE MORGANA SAG SAGE ySAM 9.0 9.5 10.0 10.5 11.0 11.5 12.0 log(M∗[M⊙]) -5 -4 -3 -2 log(φ[Mpc−3dex−1]) log(M∗[M⊙]) 4 3 2 log(φ[Mpc−3dex−1]) log(φ[Mpc−3dex−1]) 10.0 10.5 11. log(M∗[M⊙]) 10.0 10.5 11 log(M∗[M⊙]) Figure B1. Alternative version of Figure 1, applying the 0.08(1 + z) dex scatter to the stellar mass values in the models. Here we compare to observational data from Muzzin et al. (2013), who do not take into account Eddington bias when finding the best-fit Schechter parameters. Here the models match the observations better at high masses and high redshift.
https://openalex.org/W2194112465
https://europepmc.org/articles/pmc4639602?pdf=render
English
null
Hyponatremia due to Severe Primary Hypothyroidism in an Infant
Frontiers in pediatrics
2,015
cc-by
3,032
Abbreviations: AVP, arginine vasopressin; BUN, blood urea nitrogen; FENa, fractional excretion of sodium in the urine; FEUrate, fractional excretion of urate; TSH, thyroid stimulating hormone. Edited by: Wassim Chemaitilly, St. Jude Children’s Research Hospital, USA Hyponatremia due to Severe Primary Hypothyroidism in an Infant Nickolas T. Agathis1, Ingrid M. Libman2 and Michael L. Moritz2* 1 Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA, 2 Children’s Hospital of Pittsburgh of UPMC, The University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Hyponatremia has been reported in the elderly with hypothyroidism and myxedema, but this has not been a universal finding in clinical studies and there have been only a few reports in children. We report a case of an infant who developed hyponatremia due to severe primary hypothyroidism. A 4-month-old ex-preterm male, who had been euthyroid on the newborn screen, developed unexplained hospital-acquired hyponatremia (serum Na 124 mEq/L) while on full oral feeds. He was euvolemic, appeared well and was without myxedema. An evaluation of hyponatremia was negative with the exception of severe primary hypothyroidism (TSH 315.4 IU/mL, repeat 540 IU/mL). The hyponatremia resolved with thyroxine supplementation. This case demonstrates that severe hypothyroidism can result in hyponatremia in infants. It is critical to consider hypothyroidism in the evaluation of an infant with unexplained hyponatremia as untreated hypothyroidism can lead to profound developmental delays. Specialty section: Specialty section: This article was submitted to Pediatric Endocrinology, a section of the journal Frontiers in Pediatrics This article was submitted to Pediatric Endocrinology, a section of the journal Frontiers in Pediatrics Received: 05 August 2015 Accepted: 23 October 2015 Published: 10 November 2015 CASE REPORT published: 10 November 2015 doi: 10.3389/fped.2015.00096 CASE REPORT CASE REPORT Accepted: 23 October 2015 Published: 10 November 2015 A 4-month-old male was born at 32-week gestation as a result of premature rupture of mem- branes with a birth weight of 1782 g and APGAR scores of 3 and 8 at 1 and 5 min postdelivery, respectively. His newborn screen was reported as normal. His past medical history was significant Keywords: child development, hyponatremia, hypothyroidism, infant, newborn, thyroxine INTRODUCTION Reviewed by: Mohamad Maghnie, University of Genova, Italy Laura Gabriela Gonzalez Briceño, Hôpital Necker – Enfants Malades, France Hyponatremia is the most common electrolyte abnormality encountered in hospitalized patients. It is primarily the result of excess free water intake in conjunction with impaired free water excretion due to arginine vasopressin (AVP) excess. One potential cause of hyponatremia is hypothyroidism. This association has been primarily reported in the elderly with myxedema (1, 2), with only a few reports in children (3–6). The mechanisms linking the two entities are not entirely clear, but there is evidence to support both prerenal and renal mechanisms. There is controversy in the literature whether hypothyroidism produces hyponatremia, as this has not been a universal finding in all studies. *Correspondence: Michael L. Moritz michael.moritz@chp.edu We report a 4-month-old ex-preterm male with a complicated past medical history and who was reported to have a normal newborn screen, who developed unexplained hyponatremia. This led to the diagnosis of severe hypothyroidism with resolution of the hyponatremia following thyroxin supplementation. Citation: At 16 weeks of age, weight 4.57 kg, having been admitted to the hospital since birth, he was switched from total parenteral nutrition to enteral feeds consisting of Neocate and breast milk. At that time, his serum sodium was 136 mEq/L. Serum chemistries were done 4 days later, which revealed a serum sodium level of 124 mEq/L and plasma osmolality of 260 mOsm/kg confirmed on two repeated measurements (Table 1). There was no apparent explanation for the hyponatremia, since there was no apparent volume depletion, vomiting, diarrhea, or gastrostomy tube loss. He was stable from a respiratory standpoint and was not requiring oxygen. His only medications were famotidine, lansoprazole, and methadone for fentanyl withdrawal. He was not receiving diuretics or intravenous fluids. He was neurologically asymptomatic with- out lethargy, irritability, vomiting, or feeding intolerance. His total feeds in the previous 24 h were 110 mL/kg and his weight had increased by 400 g since the previous sodium level 4 days earlier. Family history was remarkable for hypothyroidism in the maternal grandmother and a paternal aunt. To further evaluate the cause of hypothyroidism, a thyroid ultrasound and thyroid scan were done, which were both normal. The scan demonstrated homogeneous uptake at the expected location of the thyroid gland on both sides of the neck with no heterotopic uptake seen. y g p y An evaluation of hyponatremia included serum and urine biochemistries including liver function tests, thyroid function tests, a cortisol level, serum and urine osmolality levels, and a uric acid level (Table 1). Spot urine electrolytes revealed a TABLE 1 | Laboratory investigations. Citation: Front. Pediatr. 3:96. doi: 10.3389/fped.2015.00096 November 2015 | Volume 3 | Article 96 1 Frontiers in Pediatrics | www.frontiersin.org Hyponatremia due to Hypothyroidism Agathis et al. low urine sodium level of 23 mEq/L and a low fractional excre- tion of sodium (FENa) of 0.2%; this combination of findings was suggestive of a prerenal state. He had severe hypouricemia with a uric acid level of 0.9 mg/dL and an elevated fractional excretion of urate (FEUrate) of 45%. This combination of find- ings was suggestive of a Syndrome of Inappropriate Antidi- uretic Hormone secretion (SIADH)-like state. Thyroid function tests revealed severe hypothyroidism (TSH 315.4 IU/mL) (normal range 1.7–9.1 IU/mL), which was confirmed on repeat measure- ment with a free T4 of 0.52 ng/dL (normal range 0.8–1.8 ng/dL) and a TSH of 540 IU/mL, with a normal cortisol. The hypona- tremia was initially corrected with a 24-h infusion of 0.9% sodium chloride at a rate of 4 mL/kg/h and thyroid supplementation, to a sodium level of 138 mEq/L. The hypothyroidism was treated with 37.5 mcg daily of levothyroxine on Day 1 and 2, which was increased to 50 mcg daily on day 3. The intravenous fluids were discontinued on day 1 and the sodium level decreased to 133 mEq/L on day 2. The serum sodium then normalized without further intravenous fluids and without oral sodium supplemen- tation. On day 4, repeat urine and serum chemistries revealed a serum sodium of 136 mEq/L, a spot urine sodium of 63 mEq/L with FENa of 0.7% and resolving hypouricemia with an FEUrate that decreased to 22% and eventually to 18% on day 73 (Table 1). Due to mild hyperkalemia an ACTH stimulation test was done which was normal. for an omphalocele repaired at 2 days of life. Between 14 and 15 weeks of life, he had a pyloric stenosis treated with a pyloromy- otomy, gastroesophageal reflux treated with a Nissen fundoplica- tion and gastrostomy tube, and a bilateral herniorrhaphy. Other complications included influenza-A pneumonia during the first month of life requiring 17 days of mechanical ventilation, chronic lung disease, and a grade I intraventricular hemorrhage noted at 6 weeks of age on head ultrasound, which had resolved at 4 months of age. An echocardiogram revealed a resolved patent ductus arteriosus with a clinically insignificant patent foramen ovale and a normal renal sonogram. November 2015 | Volume 3 | Article 96 DISCUSSION We report a case of an ex-preterm infant found to have hospital- acquired hyponatremia most likely due to hypothyroidism as other causes of hyponatremia were excluded. While hyponatremia from SIADH can be due to medications, methadone and lan- soprazole are extremely infrequent causes of hyponatremia and the hyponatremia resolved promptly following treatment of the hypothyroidism despite continuing these medications. The exact etiology of the hypothyroidism has not yet been identified. Con- genital hypothyroidism can not be excluded as the newborn screen for congenital hypothyroidism may have given a false negative result as the child was an ex-preterm infant of 32-week gestation and was also ill requiring an omphalocele repair at 2 days of age. Also the neonatal screening was not repeated 2 weeks after the first screening as recommended by consensus guidelines (7). The associated features of omphalocele, pyloric stenosis, and cardiac anomalies are suggestive of a genetic cause, which would merit further evaluation. Acquired hypothyroidism is unlikely; however, thyroid autoantibodies were not checked. This case report supports the assertion that severe hypothy- roidism is associated with hyponatremia, yet other investigators have not been able to find an association. In a sample of 445 hypothyroid adults, the frequency of hyponatremia was no dif- ferent than in euthyroid controls (17). Similarly, there was no difference in sodium concentrations or the incidence of hypona- tremia in a homogenous group of 32 congenital hypothyroid infants compared to age matched controls (18). Warner et al. was able to demonstrate a statistically significant, but seemingly clinically insignificant, relationship between hypothyroidism and hyponatremia, with a fall in serum sodium concentration of 0.14 mEq/L for every 10 IU/L rise in TSH (19). Based on these findings, our patient’s TSH of 539 IU/L is associated with an aver- age fall in serum sodium concentration of approximately 7 mEq/L from baseline. Therefore, this model was partially accurate in predicting that our patient could develop clinically significant hyponatremia. The association between hyponatremia and hypothyroidism has been recognized primarily in adults with severe myxedema (1, 2), but is an extremely unusual finding in an infant. There have been four previous reports in children (3–6), three of whom were infants with congenital hypothyroidism, of which one was water intoxicated (5), and another an 8-year-old child with hypothy- roidism as a result of brain injury from status epilepticus (4). Citation: Day -4 Day 0 Day 1 Day 2 Day 4 Day 7 Day 73 SERUM (REFERENCE RANGES) Na (mEq/L) 136 124 138 133 136 134 137 K (mEq/L) 5.2 4.9 5.3 5.4 5.4 6.3 6.3 Cl (mEq/L) 106 88 103 100 107 97 96 CO2 (mEq/L) 23.0 31.0 26.0 28.0 26.0 29.0 34 BUN (mEq/L) 25 15 12 13 19 21 16 Cr (mg/dL) 0.3 0.2 0.2 0.2 0.2 0.3 0.2 Glucose (mg/dL) 89 69 94 100 62 Uric acid (mg/dL) 0.9 1.4 Osmolality (mOsm/kg) 260 285 Cortisol (ug/dL) (3–23) 11.5 2.2 TSH (IU/mL) (1.7–9.1) 315.4 539.86 92.9 0.27 T3 (ng/dL) (1.03–2.29) 0.85 1.50 2.50 T4 (ug/dL) (7–15) 2.3 5.8 12.3 Free T4 (ng/dL) (0.8–1.8) 0.52 2.09 URINE Specific gravity 1.006 pH 8.0 Osm (mOsm/kg) 389 349 172 Na (mEq/L) 23 63 21 K (mEq/L) 57.2 39.2 15.8 Cl (mEq/L) 32 69 <15 Cr (mg/dL) 15 14 6 Uric acid (mg/dL) 30.3 21.4 9.4 FENa (%) 0.2 0.7 0.5 FEUrate (%) 45 22 18 2 Hyponatremia due to Hypothyroidism Agathis et al. at 0.2% with an elevated BUN-to-creatinine ratio of 80, reflective of appropriate renal compensation secondary to decreased effec- tive circulating volume. The decreased urinary sodium excretion might also partially reflect decreased sodium intake as the patient was on a low sodium enteral diet. Patients with SIADH can have a low urine sodium concentration and FENa, if they are sodium restricted (13). Our patient also had significant hypouricemia and an elevated fractional excretion of urate of 45% (normal 13–26%) (14), which is consistent with proximal tubular dysfunction from natriuretic peptides (15). This phenomenon may also be encoun- tered in SIADH and cerebral salt wasting (16). After levothyroxine was started, the hyponatremia, hypouricemia and elevated frac- tional excretion of urate resolved. This suggests that the hypona- tremia was related to hypothyroidism. Thyroid autoantibodies and genetic testing were not performed. His levothyroxine requirements decreased overtime and at 2-year follow-up, he was on 25 mcg daily. His development at 2 years of age is almost normal with the exception of an oral aversion requiring gastrostomy tube feedings and mild speech delay. Thyroid autoantibodies and genetic testing were not performed. His levothyroxine requirements decreased overtime and at 2-year follow-up, he was on 25 mcg daily. His development at 2 years of age is almost normal with the exception of an oral aversion requiring gastrostomy tube feedings and mild speech delay. DISCUSSION This case report supports the association between hypothy- roidism and hyponatremia and supports the physiologic hypoth- esis of extracellular volume depletion with proximal tubular dys- function. While hyponatremia may be an uncommon conse- quence of severe hypothyroidism in infants, it is nevertheless an important cause. Hypothyroidism in infancy can lead to sig- nificant neurological damage and delays in intellectual devel- opment, which can be prevented with appropriate thyroxine treatment (20, 21). Therefore, if not for the development of hyponatremia in this patient, the diagnosis of hypothyroidism would likely have been missed or delayed leading to possible irreversible neurological damage and developmental delay in our patient. We therefore recommend that thyroid function tests be included in the evaluation of unexplained hyponatremia, partic- ularly in the pediatric population. The test is of minimal cost and could rule out an easily treatable condition with significant morbidity. There are two possible mechanisms described in the literature in which hypothyroidism could lead to hyponatremia; our patient had features of both. The prevailing view is that of a prerenal mechanism and compensatory dilutional hyponatremia (8). It has been shown that hypothyroidism initially causes a significant increase in peripheral vascular resistance, decrease in cardiac output, and concomitant decrease in glomerular filtration rate (8). There is also evidence to suggest impaired sodium reabsorp- tion in the proximal and distal tubules as a result of decreased Na–K ATPase activity; this also would lead to volume depletion and aggravate the pre-renal mechanisms (9, 10). Supporting this hypothesis are studies, which have demonstrated a relationship between hypothyroidism and subclinical and clinical pre-renal acute kidney injury (11). Effective circulating volume deple- tion from hypothyroidism would lead to up-regulation of both the rennin–angiotensin–aldosterone system and AVP, leading to hyponatremia. ETHICS STATEMENT Another possible mechanism of hyponatremia due to hypothyroidism is the SIADH. Some studies have demonstrated inappropriately elevated AVP levels, possibly due to impaired osmoregulation or decreased metabolic clearance of AVP in hypothyroid patients (12). A formal consent is not required for case reports at our institution. Verbal consent was obtained prior to working on the case and was again obtained after completion of the report. Frontiers in Pediatrics | www.frontiersin.org REFERENCES of antidiuretic hormone. Am J Med (1957) 23(4):529–42. doi:10.1016/0002- 9343(57)90224-3 of antidiuretic hormone. Am J Med (1957) 23(4):529–42. doi:10.1016/0002- 9343(57)90224-3 14. Passwell JH, Modan M, Brish M, Orda S, Boichis H. Fractional excretion of uric acid in infancy and childhood. Index of tubular maturation. Arch Dis Child (1974) 49(11):878–82. doi:10.1136/adc.49.11.878 1. Macaron C, Famuyiwa O. Hyponatremia of hypothyroidism. Appropriate sup- pression of antidiuretic hormone levels. Arch Intern Med (1978) 138(5):820–2. doi:10.1001/archinte.138.5.820 1. Macaron C, Famuyiwa O. Hyponatremia of hypothyroidism. Appropriate sup- pression of antidiuretic hormone levels. Arch Intern Med (1978) 138(5):820–2. doi:10.1001/archinte.138.5.820 15. Espiner EA. Physiology of natriuretic peptides. J Intern Med (1994) 235(6):527–41. doi:10.1111/j.1365-2796.1994.tb01261.x 2. Nakano M, Higa M, Ishikawa R, Yamazaki T, Yamamuro W. Hyponatremia with increased plasma antidiuretic hormone in a case of hypothyroidism. Intern Med (2000) 39(12):1075–8. doi:10.2169/internalmedicine.39.1075 16. Berendes E, Walter M, Cullen P, Prien T, Van Aken H, Horsthemke J, et al. Secretion of brain natriuretic peptide in patients with aneurysmal subarachnoid haemorrhage. Lancet (1997) 349(9047):245–9. doi:10.1016/S0140-6736(96) 08093-2 3. Chelimsky G, Davis ID, Kliegman RM. Neonatal hyponatremia associated with congenital hypothyroidism. Clin Pediatr (1997) 36(3):177–80. doi:10.1177/ 000992289703600310 17. Croal BL, Blake AM, Johnston J, Glen AC, O’Reilly DS. Absence of relation between hyponatraemia and hypothyroidism. Lancet (1997) 350(9088):1402. doi:10.1016/S0140-6736(05)65181-1 4. Schutt-Aine JC. Hypothyroid myxedema and hyponatremia in an eight-year- old child: a case report. J Natl Med Assoc (1980) 72(7):705–8. 5. Buhler UK, Savary A, Krauer B, Stalder GR. Water intoxication in a cretinoid infant. J Clin Endocrinol Metab (1966) 26(1):111–6. doi:10.1210/jcem-26-1-111 18. Asami T, Uchiyama M. Sodium handling in congenitally hypothyroid neonates. Acta Paediatr (2004) 93(1):22–4. doi:10.1111/j.1651-2227.2004. tb00668.x 6. Robles-Valdes C, Ramirez Mayans JA, Alcantara Lomeli JI. Severe hypona- tremia in congenital hypothyroidism. J Pediatr (1979) 94(4):631–2. doi:10.1016/ S0022-3476(79)80037-2 19. Warner MH, Holding S, Kilpatrick ES. The effect of newly diagnosed hypothy- roidism on serum sodium concentrations: a retrospective study. Clin Endocrinol (2006) 64(5):598–9. doi:10.1111/j.1365-2265.2006.02489.x 7. Léger J, Olivieri A, Donaldson M, Torresani T, Krude H, van Vliet G, et al. Euro- pean Society for Paediatric Endocrinology consensus guidelines on screening, diagnosis, and management of congenital hypothyroidism. J Clin Endocrinol Metab (2014) 99(2):363–84. doi:10.1210/jc.2013-1891 20. Oerbeck B, Sundet K, Kase BF, Heyerdahl S. Congenital hypothyroidism: influ- ence of disease severity and L-thyroxine treatment on intellectual, motor, and school-associated outcomes in young adults. Pediatrics (2003) 112(4):923–30. doi:10.1542/peds.112.4.923 8. Ojamaa K, Balkman C, Klein IL. Acute effects of triiodothyronine on arterial smooth muscle cells. ACKNOWLEDGMENTS The pathogenesis of hyponatremia in this patient appears to be most consistent with the theory of a pre-renal state and concomi- tant proximal tubular dysfunction. Our patient’s FENa was low NA performed this work as a medical student at New York University. November 2015 | Volume 3 | Article 96 3 Hyponatremia due to Hypothyroidism Agathis et al. Frontiers in Pediatrics | www.frontiersin.org November 2015 | Volume 3 | Article 96 REFERENCES Ann Thorac Surg (1993) 56(1 Suppl):S61–6. doi:10.1016/ 0003-4975(93)90556-W discussion S66-67, 21. Van Vliet G. Neonatal hypothyroidism: treatment and outcome. Thyroid (1999) 9(1):79–84. doi:10.1089/thy.1999.9.79 9. Bautista AA, Duya JE, Sandoval MA. Salt-losing nephropathy in hypothy- roidism. BMJ Case Rep (2014) 2014:1–4. doi:10.1136/bcr-2014-203895 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 10. Holmes EW Jr, DiScala VA. Studies on the exaggerated natriuretic response to a saline infusion in the hypothyroid rat. J Clin Invest (1970) 49(6):1224–36. doi:10.1172/JCI106336 11. Hanna FW, Scanlon MF. Hyponatraemia, hypothyroidism, and role of arginine- vasopressin. Lancet (1997) 350(9080):755–6. doi:10.1016/S0140-6736(05) 62563-9 Copyright © 2015 Agathis, Libman and Moritz. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 12. Kimura T. Potential mechanisms of hypothyroidism-induced hyponatremia. Intern Med (2000) 39(12):1002–3. doi:10.2169/internalmedicine.39.1002 13. Schwartz WB, Bennett W, Curelop S, Bartter FC. A syndrome of renal sodium loss and hyponatremia probably resulting from inappropriate secretion November 2015 | Volume 3 | Article 96 Frontiers in Pediatrics | www.frontiersin.org
https://openalex.org/W4307309250
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0271425&type=printable
English
null
A multimodal neuroimaging study of brain abnormalities and clinical correlates in post treatment Lyme disease
PloS one
2,022
cc-by
14,341
A multimodal neuroimaging study of brain abnormalities and clinical correlates in post treatment Lyme disease Cherie L. MarvelID1,2*, Kylie H. Alm2, Deeya Bhattacharya1, Alison W. Rebman3, Arnold Bakker2, Owen P. Morgan1¤, Jason A. Creighton1, Erica A. Kozero3, Arun Venkatesan1, Prianca A. Nadkarni1, John N. Aucott3 Cherie L. MarvelID1,2*, Kylie H. Alm2, Deeya Bhattacharya1, Alison W. Rebman3, Arnold Bakker2, Owen P. Morgan1¤, Jason A. Creighton1, Erica A. Kozero3, Arun Venkatesan1, Prianca A. Nadkarni1, John N. Aucott3 1 Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America, 2 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America, 3 Division of Rheumatology, Department of Medicine, Lyme Disease Research Center, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America ¤ Current address: Department of Psychology, Cornell University, Martha Van Rensselaer Hall, Ithaca, NY, United States of America * cmarvel1@jhmi.edu Abstract Citation: Marvel CL, Alm KH, Bhattacharya D, Rebman AW, Bakker A, Morgan OP, et al. (2022) A multimodal neuroimaging study of brain abnormalities and clinical correlates in post treatment Lyme disease. PLoS ONE 17(10): e0271425. https://doi.org/10.1371/journal. pone.0271425 Lyme disease is the most common vector-borne infectious disease in the United States. Post-treatment Lyme disease (PTLD) is a condition affecting 10–20% of patients in which symptoms persist despite antibiotic treatment. Cognitive complaints are common among those with PTLD, suggesting that brain changes are associated with the course of the ill- ness. However, there has been a paucity of evidence to explain the cognitive difficulties expressed by patients with PTLD. This study administered a working memory task to a care- fully screened group of 12 patients with well-characterized PTLD and 18 healthy controls while undergoing functional MRI (fMRI). A subset of 12 controls and all 12 PTLD participants also received diffusion tensor imaging (DTI) to measure white matter integrity. Clinical vari- ables were also assessed and correlated with these multimodal MRI findings. On the work- ing memory task, the patients with PTLD responded more slowly, but no less accurately, than did controls. FMRI activations were observed in expected regions by the controls, and to a lesser extent, by the PTLD participants. The PTLD group also hypoactivated several regions relevant to the task. Conversely, novel regions were activated by the PTLD group that were not observed in controls, suggesting a compensatory mechanism. Notably, three activations were located in white matter of the frontal lobe. DTI measures applied to these three regions of interest revealed that higher axial diffusivity correlated with fewer cognitive and neurological symptoms. Whole-brain DTI analyses revealed several frontal lobe regions in which higher axial diffusivity in the patients with PTLD correlated with longer duration of ill- ness. Together, these results show that the brain is altered by PTLD, involving changes to white matter within the frontal lobe. Higher axial diffusivity may reflect white matter repair and healing over time, rather than pathology, and cognition appears to be dynamically affected throughout this repair process. PLOS ONE PLOS ONE Introduction Lyme disease is a vector-borne infectious disease initiated by the bite of a tick infected with various genospecies of the bacteria Borrelia burgdorferi sensu lato [1]. In recent decades, both the geographic range and the number of cases have increased significantly, and the Centers for Disease Control and Prevention (CDC) recently estimated an incidence of 476,000 cases annu- ally in the US [2]. Untreated Lyme disease can manifest clinically as the skin rash erythema migrans, or cause cardiac, neurologic, or joint signs of infection resulting from the dissemina- tion of the bacteria [1,3]. Lyme disease is treated with antibiotics, after which such symptoms typically resolve. However, a subset of patients (10–20%) who are appropriately treated for Lyme disease develop a chronic illness consisting of persistent or recurrent symptoms [4,5]. A specific, research-based definition for post-treatment Lyme disease (PTLD) has been operationalized to identify patients with symptoms linked temporally to strong evidence of prior exposure to B. burgdorferi [5–7]. Although fatigue, widespread musculoskeletal pain, and cognitive difficul- ties are the most prominent symptoms of PTLD, patients also often report a constellation of other neurologic, sleep, ocular, mood, and other symptoms [7–9]. Symptom severity and course can be variable, yet PTLD often significantly impacts cognition and health-related qual- ity of life [7,10–13]. There is currently no sensitive or specific test to aid diagnosis of PTLD, nor are there FDA-approved treatment options for patients. Research in individuals suffering from PTLD has been relatively sparse, in part, due to the complexity of the disease and the dif- ficulty in confirming a PTLD diagnosis in the absence of additional underlying or co-morbid diseases that would complicate the interpretation of research results. The underlying mechanisms of brain changes that may impact cognition in people with PTLD are largely unknown. Few neuroimaging studies that have been reported in people with PTLD, and there is a lack of consistent findings explaining neurological deficits [14–17]. Sev- eral brain perfusion and metabolism studies have shown abnormal patterns in patients who underwent antibiotic treatment [14–17]. It has been noted that brain changes associated with Lyme disease may involve abnormal white matter perfusion that impacts cognition [18,19]. Moreover, microglial activation of patients with PTLD has been suggested as a contributing factor of PTLD-related neurological deficits [20]. Introduction However, state-of-the-art magnetic reso- nance imaging (MRI) methods that measure the structural and functional integrity of gray and white matter in PTLD have not been reported to date, limiting our understanding of neuro- logic deficits related to PTLD. The aim of the current study was to use functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI) methods to examine brain function and structure in people with PTLD. We sought to test the hypothesis that people with PTLD show altered task-related activations as revealed by fMRI, and white matter abnormalities as revealed by DTI. Editor: Yangming Ou, Harvard Medical School, UNITED STATES Editor: Yangming Ou, Harvard Medical School, UNITED STATES Received: June 29, 2022 Accepted: September 15, 2022 Published: October 26, 2022 Copyright: © 2022 Marvel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: June 29, 2022 Accepted: September 15, 2022 Published: October 26, 2022 Copyright: © 2022 Marvel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data and analysis code are available on the Open Science Framework (https://osf.io/kshq7). Funding: Funding for this project was generously provided by an anonymous donor to JNA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 1 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Participants Twelve adult participants with PTLD ( 18 years) were originally recruited from a referral- based clinic population. Those providing permission to contact for future studies as part of the consent process were approached for the current MRI study. The median time between partic- ipation in both studies was approximately 2.5 weeks (range <1 week– 9.4 weeks). Study participant selection methods replicated many of the criteria set forth in the Infec- tious Diseases Society of America’s (IDSA) proposed case definition for PTLD [6]. A rigorous chart review process confirmed study eligibility to validate PTLD in the absence of PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 2 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease confounding factors. Confirmation of PTLD was determined by: 1) physician-documented erythema migrans rash, or 2) evidence of new-onset objective signs (e.g., joint swelling, facial palsy) and laboratory evidence of infection following CDC recommendations for test interpre- tation [21], or 3) evidence of new-onset symptoms not attributable to another cause and labo- ratory evidence of infection following CDC recommendation for test interpretation. Along with PTLD confirmation, eligibility required a history of appropriate antibiotic treatment and post treatment symptoms specified in the IDSA case definition; fatigue, musculoskeletal pain, and/or cognitive difficulty. Additionally, at least one symptom had been experienced in the past two weeks that limited daily functioning at least half the time when present. Further eligi- bility inclusion and exclusion criteria for this study have been published previously [7]. Participants were excluded for a range of specific, co-morbid conditions with significant symptom overlap with PTLD such as fibromyalgia, chronic fatigue syndrome, major psychiat- ric disease conditions (except non-suicidal depression that manifested after Lyme infection), malignancy, and autoimmune disease. Exclusion criteria also consisted of: history of Lyme vaccine, sleep apnea, cirrhosis, hepatitis B/C, HIV, dementia, cancer (past 2 years), illicit sub- stance abuse, prescription drug abuse, and alcoholism. A total of 18 adult control participants were recruited through community flyers and included in the fMRI analysis. Control participants were additionally screened for any co-mor- bid conditions with significant symptom overlap with PTLD, exclusion criteria as described above, or a past diagnosis of Lyme disease. Participants In a final screening stage, both participants with PTLD and controls were excluded from study participation if they endorsed the following characteristics that might confound data interpretation: major neurologic disorders (including stroke and seizures); head injury result- ing in loss of consciousness of > 5 minutes, significant learning disability, left-handedness, or being a non-native English speaker (i.e., acquired English post puberty). Participants were also excluded for reasons of safety concerns within the MRI environment, such as: current or possi- ble pregnancy, metal inside or attached to the body, and claustrophobia. Demographic and clinical characteristics are summarized in Table 1. A subset of 12 controls who were matched demographically to the PTLD group also had DTI data available and were included in the DTI analysis. The Institutional Review Board of the Johns Hopkins University School of Medicine approved this study, and written informed consent was obtained from all study participants prior to initiation of study activities. The planning and conduct of this research were in accor- dance with the Helsinki Declaration as revised in 2013. PLOS ONE f symptoms reported at the moderate or severe level over the past two weeks on the Post-Lyme Questionnaire of Symptoms interpreted using CDC criteria for positivity, which incorporates duration of illness at the time of the test. bThe total number of symptoms reported at the moderate or severe level over the past two weeks on the Post-Lyme Questionnaire of Symptoms (range of 0 to 36). cTwo-tier tests were interpreted using CDC criteria for positivity, which incorporates duration of illness at the time of the test. dConfirmed through medical record review. Participants presenting with erythema migrans rash were not required to have a concurrent positive two-tier serology. Those with neurologic disease (n = 2 with Bell’s Palsy, n = 1 with meningitis/encephalitis), late Lyme arthritis, or an initial flulike illness were required to have a concurrent positive two-tier test. Two tier tests were interpreted using CDC criteria for positivity, which incorporates duration of illness at the time of the test. dConfirmed through medical record review. Participants presenting with erythema migrans rash were not required to have a concurrent positive two-tier serology. Those with neurologic disease (n = 2 with Bell’s Palsy, n = 1 with meningitis/encephalitis), late Lyme arthritis, or an initial flulike illness were required to have a concurrent positive two-tier test. eBeck Depression Inventory: Cognitive/Affective Subscale score. https://doi.org/10.1371/journal.pone.0271425.t001 binary responses for the following 3 items only; memory impairment, difficulty finding words, and difficulty focusing or concentrating (range 0–3). binary responses for the following 3 items only; memory impairment, difficulty finding words, and difficulty focusing or concentrating (range 0–3). Clinical data collection Participants were asked to complete a self-administered 36-item post-Lyme questionnaire of symptoms (PLQS) which was developed based on prior clinical and research experience among patients with PTLD. The list of individual symptom items has been previously pub- lished [22]. For each item, participants indicated severity over the past 2 weeks (0 = absent, 1 = mild, 2 = moderate, 3 = severe), and a binary response was created (absent/mild = 0 vs. moderate/severe = 1). A total symptom score was generated by summing each binary item (range 0–36). Additionally, in order to focus on specific symptoms of interest to the current study, a ‘neurologic’ symptom score was generated a priori by summing the binary responses for the following 12 symptoms; fatigue, numbness in hands/feet, numbness in face/scalp, head- ache, photophobia, drooping facial muscle, drooping eyelid, neck pain, poor coordination, memory impairment, difficulty finding words, and difficulty focusing or concentrating (range 0–12). Finally, a more narrow ‘cognitive’ symptom score was generated by summing the PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 3 / 22 Neuroimaging of brain abnormalities in post treatment Lyme disease PLOS ONE PLOS ONE Table 1. Baseline demographic and clinical characteristics. N (%) are presented for categorical variable; mean (standard deviation), 95% confidence interval [lower limit, upper limit] are presented for continuous variables; Shapiro-Wilk tests were used for tests of normality. Significant values of the Shapiro-Wilk tests are denoted in bold, p .05, two-tailed. Control groups did not differ from PTLD for age, education, or gender. Group PTLD n = 12 Controls (for fMRI) n = 18 Matched Controls (for DTI) n = 12 Age (years) 45.16 (13.62) [36.51, 53.82], W = .96, p = .72 47.01 (13.10) [40.49, 53.52], W = .86, p = .01 45.33 (13.76) [36.60, 54.08], W = .91, p = .20 Male Gender 7 (58.33%) 6 (33.33%) 4 (33.33%) Education (years) 16.17 (2.21) [14.76, 17.57], W = .97, p = .92 16.44 (2.01) [15.45, 17.44], W = .90, p = .06 16.00 (1.91) [14.79, 17.21], W = .86, p = .05 Duration of illnessa (days) 944.83 (1043.13) [282.06, 1607.61], W = .75, p = .003 Antibiotic exposurea (days) 190.17 (309.37) [-6.40, 386.73], W = .55, p < .001 Number of symptoms at MRI scanb 8.17 (4.39) [5.38, 10.95], W = .94, p = .48 Neurologic symptoms 4.50 (2.02 [3.21, 5.79], W = .90, p = .16 Cognitive symptoms 2.00 (1.35) [1.14, 2.86], W = .71, p = .001 Two-tier antibody positive at MRI scanc 4 (33.33%) Initial Lyme disease clinical presentationd Erythema migrans rash 4 (33.33%) Neurologic Lyme disease 3 (25.00%) Late Lyme arthritis 1 (8.33%) Flulike Illness 4 (33.33%) Beck Depression Inventorye 18.42 (12.05) [10.76, 26.07], W = .79, p = .008 Table 1. Baseline demographic and clinical characteristics. N (%) are presented for categorical variable; mean (standard deviation), 95% confidence interval [lower limit, upper limit] are presented for continuous variables; Shapiro-Wilk tests were used for tests of normality. Significant values of the Shapiro-Wilk tests are denoted in bold, p .05, two-tailed. Control groups did not differ from PTLD for age, education, or gender. aTotal days from Lyme disease onset (or start of antibiotics) until MRI scan. aTotal days from Lyme disease onset (or start of antibiotics) until MRI scan. aTotal days from Lyme disease onset (or start of antibiotics) until MRI scan. MRI procedures The num- ber of target letters (1 or 2), rehearsal duration (4 or 6 seconds), expected response (yes or no), and duration of ITI (6–9 seconds) were pseudorandomized so that presentation of identical parameters was limited to three consecutive trials. Participants were instructed to respond as quickly and accurately as possible while complet- ing all operations silently ‘‘in your head.” Button press responses were recorded if conducted within six seconds following probe onset (yes = right index finger; no = right middle finger). Trials were jittered with an inter-trial interval (ITI) of six to nine seconds. Response time (RT) and accuracy were recorded for each trial. In order to familiarize subjects with the rules of the task, subjects practiced 10 trials of each condition prior to entering the MRI environment. The control and forward conditions were completed during two separate blocks, with the order counter-balanced across participants. Each block contained 64 trials (~ 16 minutes). Each trial consisted of pseudorandom presentations of targets such that letters were unique within a trial. Probes matched a target (or newly derived target) on 50% of the trials. The num- ber of target letters (1 or 2), rehearsal duration (4 or 6 seconds), expected response (yes or no), and duration of ITI (6–9 seconds) were pseudorandomized so that presentation of identical parameters was limited to three consecutive trials. An additional event-related finger tapping task was administered in order to compute indi- vidualized hemodynamic response functions (HRFs) [23,25]. This task consisted of a button press with the right index finger every 29–31 seconds upon presentation of a 1-second cue to “tap” followed by “rest”, which lasted for 10 minutes total. The individualized HRFs were used in the convolution step of MRI processing, rather than a canonical HRF, in case the HRFs in the PTLD group differed from that of control participants. Stimuli were delivered using E-Prime 2.0 software (Psychology Software Tools, Pittsburgh, PA) on a Dell Optiplex SX9202 workstation running Windows 7. The stimuli were rear-pro- jected onto a screen in the MRI scanner, which was then reflected into a head coil-mounted mirror within the participant’s line of sight. Responses were collected using two fiber optic button boxes (MRA, Inc., Washington, PA) that were held in the participant’s right hand. MRI data acquisition. MRI data were acquired on a Philips 3 Tesla scanner using a 32-channel head coil. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 MRI procedures Behavioral task. Participants were asked to perform a working memory task in the MRI scanner consisting of two conditions, previously described in detail [23,24]. Briefly, in the con- trol condition, participants viewed one or two uppercase consonants (one second), followed by a blank screen (four or six seconds). Participants held these letters in mind through silent rehearsal. Finally, a single lowercase letter was presented (one second). Participants indicated via button press whether the single probe item matched either of the targets presented at the start of the trial. In the “forward” condition, rather than rehearse the original target letters pre- sented at the start of the trial, participants were required to count two alphabetical letters PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 4 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease forward of each target letter(s) and hold the new letters in mind. For example, if the target let- ters were ‘‘f” and ‘‘q”, participants would count forward to the letters ‘‘h” and ‘‘s”. When the probe letter appeared, participants indicated whether the probe matched the newly derived let- ters instead of the original target letters. Therefore, the two conditions differed specifically dur- ing the rehearsal phase of the trial, in which target letters were simply rehearsed as presented (control condition) or rehearsed by counting two alphabetical letters forward (forward condition). Participants were instructed to respond as quickly and accurately as possible while complet- ing all operations silently ‘‘in your head.” Button press responses were recorded if conducted within six seconds following probe onset (yes = right index finger; no = right middle finger). Trials were jittered with an inter-trial interval (ITI) of six to nine seconds. Response time (RT) and accuracy were recorded for each trial. In order to familiarize subjects with the rules of the task, subjects practiced 10 trials of each condition prior to entering the MRI environment. The control and forward conditions were completed during two separate blocks, with the order counter-balanced across participants. Each block contained 64 trials (~ 16 minutes). Each trial consisted of pseudorandom presentations of targets such that letters were unique within a trial. Probes matched a target (or newly derived target) on 50% of the trials. MRI procedures Structural images were collected using a sagittal magnetization prepared gradient-echo (MPRAGE) sequence aligned to the anterior-posterior commissure (AC-PC) axis: repetition time (TR)/echo time (TE) = 6.9/3.3 ms; field of view = 240 x 240; 170 slices; slice thickness 1.0 mm; 0 mm gap; flip angle = 8 degrees; voxel size = 0.75 x 0.75 x 1.0 mm. The total scan duration was 6 minutes. FMRI data were collected using a T2-weighted gradient echo EPI pulse sequence (TR = 1000 msec; TE = 30 ms; flip = 61; in-plane resolution = 3.75 mm; slice thickness = 6 mm with a 1 mm gap; 20 oblique-axial slices; FOV = 240 mm). T2-weighted images were acquired in the oblique-axial plane rotated 25 degrees clockwise with respect to the AC-PC line in order to optimize imaging of the cerebellum and neocortex. The number of acquired volumes within each block ranged from 917 to 922 for the working memory tasks and 600 for the tapping task. The start of the fMRI scan was triggered by E- prime software at the beginning of each block. Functional MRI data analysis. The SPM12 software package (Wellcome Department of Cognitive Neurology) was used for preprocessing and statistical computations. High temporal resolution fMRI in conjunction with neocortical-specific HRFs were used to ensure maximum accuracy in characterizing phase-specific blood oxygen level dependent (BOLD) responses [23,25]. Individual HRF regressors were convolved with reference waveforms for the target 5 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease encoding (1 sec), rehearsal (4 or 6 sec) and probe retrieval (6–9 sec) phases of the task for each subject within the first-level event-related analysis. In this report, we only focus on the rehearsal phase of analyses. Standard image preprocessing steps were performed, including slice timing correction (reference = middle slice), motion correction, anatomical co-registra- tion, normalization to the Montreal Neurological Institute (MNI) stereotaxic space, and spatial smoothing (FWHM = 8 mm). Due to a technical error, one PTLD participant’s functional MRI data was corrupted and could not be processed. It was excluded from the functional imaging stages of analysis. Individual statistical maps were computed for each subject using the general linear model approach as implemented in SPM12, with high pass filtering of 128 s. MRI procedures A random effects analysis was then performed to map the average responses to the rehearsal phase of the task on correct trials only. Incorrect trials were not given a regressor and were considered as residual variance. This analysis was performed by computing a contrast volume per subject and using these volumes to calculate one-sample t-test values at every voxel. Of par- ticular interest were within-group contrasts comparing the BOLD signal difference between the 2-target forward minus 2-target control working memory conditions that were then com- pared between groups. MNI coordinates were transformed into the coordinate system of the Talairach and Tourneaux stereotaxic atlas [26] using the MNI to Talairach transformation described by Lancaster et al. [27] in order to make anatomical determinations of the activa- tions. However, MNI coordinates are reported in the tables and figures. Significance levels were set to p < .005, uncorrected, with a minimum cluster size of 10 voxels. Functionally defined regions of interest (ROIs) were circumscribed on each participant’s scan based on the activation clusters observed in the between-groups contrast using the Mars- BaR toolbox for SPM [28]. The resultant contrast values per participant were then entered into subsequent analyses to test for correlations with behavioral task performance and diffusion weighted imaging (DTI) measures. DWI data acquisition and preprocessing. Abnormal white matter fMRI findings led us to investigate the relationship to white matter structural integrity by adding diffusion tensor imaging (DTI) methods to the protocol already in progress, which was administered to a sub- set of participants (n = 12 per group). Diffusion-weighted images (DWI) were acquired using a spin echo sequence with TR = 7012 ms, TE = 75 ms, FOV = 212 x 212 mm2, 0.83 x 0.83 x 2.2 mm voxels, flip angle = 90˚, b-value = 700, number of gradients = 33, and 70 axial slices. Two sequences were acquired. The DWI data were preprocessed using FSL [29] to correct for eddy current-induced distortions and subject motion using affine registration. The b-vector matrix was adjusted based on rigid body registration and skull stripping was performed using FSL’s automated brain extraction tool (BET) to remove non-brain tissue. A standard least squares diffusion ten- sor fitting model was applied to the data to derive whole brain maps for the following diffusion tensor imaging (DTI) metrics: fractional anisotropy (FA), mean diffusivity (MD), radial diffu- sivity (RD), and axial diffusivity (AD). MRI procedures These estimates were computed on a voxel-by-voxel basis using a three-dimensional Gaussian distribution model that yielded a single mean ellip- soid for each voxel. For each participant, the two runs of DWI data were preprocessed sepa- rately, and the scalar maps resulting from each run were averaged to improve signal-to-noise ratio. Tissue class segmentation analysis. To determine the proportion of white matter within each significant cluster of activation derived from the fMRI analysis, a tissue class segmenta- tion analysis was used. First, a study-specific T1 modal model template was created from all participants in the study using Advanced Normalization Tools (ANTs), and a 3D vector field transformation for each subject was calculated to align the individual’s structural scan to the template modal model based on the entire sample [30,31]. Tissue class segmentation analysis PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 6 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease was then completed on the template modal model using FSL’s FAST automated segmentation tool [32], a well-validated automated tissue segmentation tool [33]. FAST utilizes a hidden Markov random field model and an associated expectation-maximization algorithm to seg- ment brain images into three tissue classes: gray matter, white matter, and cerebrospinal fluid. To compute the overlap between the resulting binary white matter segmentation mask and each significant functional activation cluster, each cluster of activation was first transformed into a binary ROI. The binary ROIs were subsequently resampled into a 1 x 1 x 1 mm space using nearest neighbor interpolation, given that both the tissue class segmentation analysis and the DTI analyses were completed in this space. Following registration, each functional ROI mask was multiplied by the binary white matter segmentation mask to identify voxels in each ROI that fell within the white matter segmentation mask. The number of voxels in the subsequent overlap image was then calculated as a percentage relative to the ROI size, yielding the percentage of voxels within a particular ROI that were classified as white matter. This method was repeated for each of the significant activation clusters from the fMRI analysis. ROIs classified as greater than 50% white matter were selected for further analysis. g y DTI data analysis. FSL’s Tract-based Spatial Statistics (TBSS) pipeline [34] was used to derive a mean white matter skeleton from participants’ DTI data that represents the center of all white matter tracts common to the sample. MRI procedures First, each participant’s FA map was registered to every other participant’s FA map using the nonlinear registration tool, FNIRT [35,36]. The “most representative” image, i.e. the image that required the least warping to align every other image to it, was chosen as the target for registration and affine-transformed into MNI152 stan- dard 1 x 1 x 1 mm space. Then, all other FA maps were transformed into this standard space by combining the individual nonlinear transforms to the target FA map with the affine trans- form from the target to MNI space. Next, the co-registered FA maps were merged into one 4D image, where each volume is a specific participant’s standard space FA map. From this 4D image, the mean FA image was computed and thinned using an FA threshold of 0.2, which retained only the center of all fiber pathways common to the group, generating a mean white matter skeleton. This skeletonization process ensures that subsequent analyses are restricted to tracts that are well-aligned across participants, thereby reducing potential misregistrations as a source of false positives. It also ensures subsequent analyses are less susceptible to partial vol- ume effects. Finally, each participant’s aligned FA map was projected onto the mean skeleton to generate skeletonized FA data for each participant. The previously computed warps and skeleton projections were also applied to MD, RD, and AD maps in order to align them into MNI152 1 x 1 x 1 mm standard space and create participant level skeletonized MD, RD, and AD data. The entire TBSS process was repeated for a patient-only whole brain analysis to examine whether voxel-wise DTI metrics correlated with duration of illness (DOI) in the patients with PTLD. We held an agnostic interest in DOI due to the practical question of whether longer exposure to PTLD led to relevant brain changes. The TBSS processing steps were the same as detailed above, with only the PTLD patient group included in this iteration. The resulting skel- etonized DTI data were used to generate voxel-wise cross-subject statistics using “randomise” [37], FSL’s tool for nonparametric permutation inference testing (using 2000 permutations). GLM contrasts were constructed to test for both positive and negative correlations between the voxel-wise skeletonized DTI data and the DOI variable. Family-wise error (FWE) correc- tion was performed using threshold-free cluster enhancement [38], which avoids the use of an arbitrary threshold for the initial cluster formation. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 MRI procedures A p-value < .05, FWE-corrected for multi- ple comparisons was considered statistically significant. For visualization of results, the “tbss_fill” script was used to enhance ease of viewing. 7 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Mean white matter microstructure was computed for the functional activation ROIs that consisted of greater than 50% white matter, as determined by the tissue class segmentation analysis. Using each binarized functional activation ROI as a mask, mean FA, MD, RD, and AD values were extracted from each individual participant’s skeletonized DTI data. To compute the percentage of overlap between the functional activation ROIs and the white matter skeleton, each binarized functional activation ROI mask was multiplied by the mean white matter skeleton generated from the TBSS pipeline to isolate the voxels from the functional ROIs that fell within the white matter skeleton. The number of overlapping voxels for each ROI was then computed, and results were expressed as a percentage relative to the ROI size. This analysis was only computed for ROIs consisting of greater than 50% white mat- ter, as determined by the tissue class segmentation analysis. To investigate the white matter surrounding the frontal lobe activation ROIs, we sought to quantify overlap between the observed functional ROIs and long-range white matter pathways. For this analysis, we examined the 42 standard white matter tracts generated from the XTRACT atlas [39]. Each tract mask was binarized and then multiplied by each binarized functional activation ROI to isolate ROI voxels that fell within the associated long-range white matter tract. The percent overlap for each ROI with each long-range tract was then computed and expressed as a percentage relative to ROI size. Statistical analyses of clinical and behavioral variables. The clinical, behavioral, MRI, and DTI data collected in this study contained continuous variables, with the exception of gen- der (categorical data). T-tests were used to compare continuous variables (e.g., age and educa- tion), and Pearson chi-square tests were used to compare categorical data (e.g., gender). Shapiro-Wilk tests were used to determine if continuous variables followed a normal distribu- tion. If a variable was not normally distributed, Mann-Whitney U tests were conducted to compare groups. Mixed-design ANOVAs were used to compare repeated measures between the two groups (e.g., fMRI task performance). Results The total healthy control group did not differ from the PTLD group in terms of age, (Mdn = 51.1), U = 101, p = .76, r = 18.4, or education level, t(22) = .35, p = .730, d = .133. The matched control group also did not differ from the PTLD group in terms of age, t(22) = .03, p = .975, d = .013, or education level, (Mdn = 16.0), U = 74.0, p = .91., r = 15.1. Pearson’s Chi Square tests were used to determine that gender counts also did not differ between the healthy control and PTLD groups, χ2(1, 30) = 1.83, p = .176, or between the matched control and PTLD groups, χ2(1, 24) = 1.51, p = .219. MRI procedures [No ANOVAs contained a within-subjects fac- tor with more than two levels; sphericity corrections were, therefore, not needed.] Pearson cor- relations were used when the Shapiro-Wilk’s normality tests indicated a normal data distribution (e.g., correlating fMRI data with the PLQS). Otherwise, Spearman’s rho non- parametric correlations were used. All tests were two-tailed, with an alpha level  .05 to define statistical significance. Statistics were performed using IBM SPSS Statistics, Macintosh, version 27.0 (IBM Corp., Armonk, NY, USA). Behavioral results Mean accuracy and RT (for accurate trials only) were computed for the following trial types: control condition, 1 stimulus; control condition, 2 stimuli; forward condition, 1 stimulus; and forward condition, 2 stimuli for each group. Due to a technical error, we were unable to collect behavioral data from one PTLD participant, which also removed them from the fMRI analyses, but their data were included in the DTI analyses. (S3 File, Table 1) A 2(condition: control vs. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 8 / 22 Neuroimaging of brain abnormalities in post treatment Lyme disease PLOS ONE Fig 1. Behavioral performance on the fMRI working memory task. (A) Accuracy performance is presented, broken down by condition (control vs. forward), stimulus number (1 vs. 2 letters), and study group. Accuracy was particularly low for participants in the forward condition with two letters. However, accuracy performance did not differ overall between groups. (B) Response times for accurate trials are presented, as in (A). Response times were slowest in the forward condition with two letters. Overall, the PTLD group responded more slowly than did controls, as indicated by the overhead bracket.  indicates condition x stimulus interaction, p < .001; bracket indicates overall group difference, p = .037. Error bars denote one standard error. https://doi org/10 1371/journal pone 0271425 g001 Fig 1. Behavioral performance on the fMRI working memory task. (A) Accuracy performance is presented, broken down by condition (control vs. forward), stimulus number (1 vs. 2 letters), and study group. Accuracy was particularly low for participants in the forward condition with two letters. However, accuracy performance did not differ overall between groups. (B) Response times for accurate trials are presented, as in (A). Response times were slowest in the forward condition with two letters. Overall, the PTLD group responded more slowly than did controls, as indicated by the overhead bracket.  indicates condition x stimulus interaction, p < .001; bracket indicates overall group difference, p = .037. Error bars denote one standard error. https://doi.org/10.1371/journal.pone.0271425.g001 https://doi.org/10.1371/journal.pone.0271425.g001 forward) x 2(stimulus number: 1 vs. 2) x 2(group: controls vs. PTLD) mixed-design ANOVA yielded a main effect of condition F(1, 27) = 6.51, p = .017, ηp 2 = .019, and stimulus number F (1, 27) = 20.6, p < .001, ηp 2 = .43, indicating that participants’ accuracy decreased as a function of higher working memory load requirements. Behavioral results This was confirmed by an interaction of stimu- lus number x condition, F(1, 27) = 13.9, p = .001, ηp 2 = .34, indicating that trials were least accurate in the forward, 2 stimuli trial type. There were no main effect or interactions involv- ing group, all p-values > .416 (Fig 1A). A 2(condition) x 2(stimulus number) x 2(group) mixed-design ANOVA was also conducted for the RT measure. As with the accuracy measure, there were main effects of condition, F(1, 27) = 51.3, p < .001, ηp 2 = .66, and stimulus number, F(1, 27) = 60.9, p < .001, ηp 2 = .69. There was also an interaction of condition x stimulus num- ber, F(1, 27) = 35.3, p < .001, ηp 2 = .57. There were no interactions involving group. However, there was a main effect of group, showing that the PTLD group responded more slowly overall than did controls, F(1, 27) = 4.80, p = .037, ηp 2 = .15 (Fig 1B). Thus, participants found the for- ward, 2 stimuli trial type to be disproportionately more difficult than other trial types, as evi- denced by decreased accuracy and slowed RTs. Moreover, the PTLD group showed general motor slowing. Functional MRI results Functional imaging analyses focused on the rehearsal phase of each trial. Within this phase, we focused on activations during the most difficult condition (2 stimuli, forward condition). To do so, we computed the contrast values of the “2-target forward” minus “2-target control” con- ditions for each participant. To validate the results of our task, we first examined the healthy control fMRI data and compared it to a prior study that characterized this task in young, healthy adults [23] (S1 Table). Results generally overlapped with those original findings (the current study included healthy participants who were about 20 years older), with increased BOLD signal in association with verbal working memory rehearsal in the frontal lobe (BA 9 and 32), premotor cortex, caudate, thalamus, inferior parietal lobe, and superior cerebellum. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 9 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease The large degree of overlap with the original study supported the fMRI task’s validity. The PTLD group also revealed regions of overlap with those original findings (S2 Table), with increased BOLD signal in the frontal lobe (BA 9, supplementary motor area, and left inferior frontal gyrus BA 45), premotor cortex, caudate, and precuneus. A number of activated regions, however, were observed in the PTLD group but not observed in the controls’ data or in the original study. This suggested that the PTLD group was unable to fully utilize a typical verbal working memory circuit and compensated to maintain high accuracy. Notably, some frontal lobe activations appeared to be primarily in white matter, surpassing a threshold of p < .001 uncorrected, which warranted further examination when it appeared again in the between- groups comparison, as described below. We applied a double subtraction approach to compare BOLD signal activations between the groups. We used the contrast values obtained from the within-groups comparison (first subtraction) to compare differences between groups (second subtraction). Fig 2 shows positive BOLD signals that represented greater “forward minus control” activation differentials in the PTLD group than in the control group (exceptions are noted in Table 2 where activations were higher in the control condition for the control group, yielding a “false” hyperactivation in the PTLD group). PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease PLOS ONE Table 2. Task-related BOLD activations between PTLD and control participants. Regions are based on Talairach coordinates27 and listed anterior-posterior (y-plane). Cluster size T-value x, y, z (MNI) Brain region GM/WM p-value Controls > PTLD 17 3.14 58, 14, 26 Right inferior frontal gyrus (BA 9) GM < .005 10 3.01 40, 12, 30 Right inferior frontal gyrus (BA 9) GM < .005 124 3.69 48, 0, 44 Right precentral gyrus (BA 6) WM (64%) < .001 95 3.68 8, -24, 8 Right thalamus GM < .001 21 3.08 44, -36, 44 Right inferior parietal lobule (BA 40) GM < .005 PTLD > Controls 120 3.37 0, 50, 24 Left medial frontal gyrus (BA 9) GM < .005 33 3.22 -18, 48, 26 Left superior frontal gyrus (BA 9) WM (89%) < .005 40 3.82 -22, 38, 36 Left middle frontal gyrus (BA 8) WM (41%) < .001 56 3.97 -18, 24, 40 Left middle frontal gyrus (BA 8) WM (80%) < .001 46 3.72 -34, 18, 38 Left precentral gyrus (BA 9) WM (99%) < .001 26 3.31 -56, -4, 6 Left precentral gyrus (BA 6) GM < .005 45 3.61 62, -10, 8 Right superior temporal gyrus (BA 42) GM < .001 18 3.15 2, -14, -4 Right thalamus GM < .005 23 3.33 -62, -16, 6 Left superior temporal gyrus (BA 41) GM < .005 A Brodmann Area; GM gray matter; WM white matter.  = positive BOLD signal indicates greatest activity in the control participants during the control condition, which reversed the BOLD signal interpretation in this double subtraction method. The percentage of WM computed within a cluster is noted in parentheses in the GM/WM column (reporting those with WM > 50%). Cluster information is reported at p < .005, uncorrected. that group differences were due to the PTLD group showing hypo-activation (or not activating at all) in brain regions normally associated with the task, even though their accuracy was normal. Increased task-related activity in PTLD participants versus controls was observed in the frontal lobe (BA 8 and 9). While frontal lobe involvement would be expected in this working memory task, the clusters of task-related activation were located primarily within white matter, as opposed to the regions of relative hypoactivation noted above which were predominantly in gray matter. PLOS ONE One white matter region was activated more robustly in controls than in the PTLD group (in BA 6). Closer inspection of this region at the more conservative p < .001 threshold, however, indicated that it was actually comprised of two smaller gray matter activa- tions within close proximity that, when smoothed, bridged white matter. Additional analyses were conducted to compute the percentage of white matter included in each ROI for these four frontal lobe activations, as described below. Based on these findings, masks were created for each ROI that allowed us to compute MRI signal contrast values within these circum- scribed regions for correlations with DTI and clinical variables (described below). A = Brodmann Area; GM = gray matter; WM = white matter. https://doi.org/10.1371/journal.pone.0271425.t002 g y  = positive BOLD signal indicates greatest activity in the control participants during the control condition, which reversed the BOLD signal interpretation in this double subtraction method. The percentage of WM computed within a cluster is noted in parentheses in the GM/WM column (reporting those with WM > 50%). Cluster information is reported at p < .005, uncorrected. Functional MRI results Increased task-related activity in the controls versus PTLD participants was observed in regions that were consistent with the original study and other similar paradigms, such as the premotor cortex, thalamus, and inferior parietal lobe [23–25,40]. These results also indicated Fig 2. FMRI activation differences between study groups during the working memory task. Activations represent a double subtraction between groups (i.e., the difference between-groups of the difference within-groups [(forward, 2 stimulus) minus (control, 2 stimulus)]). Red indicates activity in PTLD > controls, except for where indicated in Table 2. Blue indicates activity in controls > PTLD. Areas of greater activation in control participants (blue) compared to PTLD participants were consistent with localized activity previously documented as relevant to task performance and reflected hypoactivity in these regions by the PTLD group [23]. Unexpectedly, three frontal lobe activations demonstrated by the PTLD group (red) were located primarily within white matter. Numbers denote y-axis on the MNI template. Color scale represents .005 < p < .0005. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g002 Fig 2. FMRI activation differences between study groups during the working memory task. Activations represent a double subtraction between groups (i.e., the difference between-groups of the difference within-groups [(forward, 2 stimulus) minus (control, 2 stimulus)]). Red indicates activity in PTLD > controls, except for where indicated in Table 2. Blue indicates activity in controls > PTLD. Areas of greater activation in control participants (blue) compared to PTLD participants were consistent with localized activity previously documented as relevant to task performance and reflected hypoactivity in these regions by the PTLD group [23]. Unexpectedly, three frontal lobe activations demonstrated by the PTLD group (red) were located primarily within white matter. Numbers denote y-axis on the MNI template. Color scale represents .005 < p < .0005. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g002 10 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 Tissue class segmentation results Neuroimaging of brain abnormalities in post treatment Lyme diseas PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Fig 3. Localization of frontal task-related fMRI activations in white matter. Significant frontal clusters of elevated activation in PTLD participants compared to controls from the contrast values of the forward minus control conditions were transformed into binary regions of interest (ROIs) labeled by their Brodmann area location. Blue: Brodmann area 8 (BA 8) ROI, red: Anterior Brodmann area 9 (BA 9 anterior) ROI, pink: Posterior Brodmann area 9 (BA 9 posterior) ROI. Labeled ROIs were used to compute the percent overlap with each of the following measures. (A) White matter mask (white) derived from a study-specific T1 template using tissue class segmentation analysis. The BA 8 ROI showed 80.06% overlap, the BA 9 anterior ROI showed 89.39% overlap, and the BA 9 posterior ROI showed 99.73% overlap with white matter. (B) Mean fractional anisotropy (FA) and white matter skeleton (green) maps derived using diffusion tensor imaging (DTI) analysis showing overlap with fMRI ROIs. The BA 8 ROI showed 25.00% Fig 3. Localization of frontal task-related fMRI activations in white matter. Significant frontal clusters of elevated activation in PTLD participants compared to controls from the contrast values of the forward minus control conditions were transformed into binary regions of interest (ROIs) labeled by their Brodmann area location. Blue: Brodmann area 8 (BA 8) ROI, red: Anterior Brodmann area 9 (BA 9 anterior) ROI, pink: Posterior Brodmann area 9 (BA 9 posterior) ROI. Labeled ROIs were used to compute the percent overlap with each of the following measures. (A) White matter mask (white) derived from a study-specific T1 template using tissue class segmentation analysis. The BA 8 ROI showed 80.06% overlap, the BA 9 anterior ROI showed 89.39% overlap, and the BA 9 posterior ROI showed 99.73% overlap with white matter. (B) Mean fractional anisotropy (FA) and white matter skeleton (green) maps derived using diffusion tensor imaging (DTI) analysis showing overlap with fMRI ROIs. The BA 8 ROI showed 25.00% overlap, the BA 9 anterior ROI showed 22.35% overlap, and the BA 9 posterior ROI showed 24.46% overlap with skeletonized white matter. For reference, the white matter skeleton accounts for 34.47% of the overall white matter mask from the tissue class segmentation. (C) Mean FA map overlaid with three long range white matter pathways obtained from DTI. Tissue class segmentation results Calculation of the overlap between the white matter mask derived from the tissue segmenta- tion analysis and the ROIs derived from the fMRI working memory contrast (forward minus control conditions) resulted in the identification of four significant functional activation clus- ters that were primarily localized to white matter (i.e. over 50% of the voxels within the func- tional ROI mask overlapped with the white matter tissue segmentation mask). In three frontal activation areas, the patients with PTLD showed elevated activation compared to controls (Fig 3A) localized to the left BA 8 ROI (80.06% white matter), the left BA 9 anterior ROI (89.39% PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 11 / 22 Fig 3. Localization of frontal task-related fMRI activations in white matter. Significant frontal clusters of elevated activation in PTLD participants compared to controls from the contrast values of the forward minus control conditions were transformed into binary regions of interest (ROIs) labeled by their Brodmann area location. Blue: Brodmann area 8 (BA 8) ROI, red: Anterior Brodmann area 9 (BA 9 anterior) ROI, pink: Posterior Brodmann area 9 (BA 9 posterior) ROI. Labeled ROIs were used to compute the percent overlap with each of the following measures. (A) White matter mask (white) derived from a study-specific T1 template using tissue class segmentation analysis. Th BA 8 ROI showed 80.06% overlap, the BA 9 anterior ROI showed 89.39% overlap, and the BA 9 posterior ROI showed 99.73% overlap with white matter. (B) Mean fractional anisotropy (FA) and white matter skeleton (green) maps derived using diffusion tensor imaging (DTI) analysis showing overlap with fMRI ROIs. The BA 8 ROI showed 25.00% overlap, the BA 9 anterior ROI showed 22.35% overlap, and the BA 9 posterior ROI showed 24.46% overlap with skeletonized white matter. For reference, the white matter skeleton accounts for 34.47% of the overall white matter mask from the tissue class segmentation. (C) Mean FA map overlaid with three long range white matter pathways obtained from DTI. The BA 8 ROI showed 41.57% overlap with the frontal aslant tract (FAT; orange), the BA 9 anterior ROI showed 49.62% overlap with anterior thalamic radiation (ATR; yellow), and the BA 9 posterior ROI showed 57.61% overlap with the superior longitudinal fasciculus 2 (SLF 2; cyan). L = left, R = right hemispheres. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 Tissue class segmentation results The BA 8 ROI showed 41.57% overlap with the frontal aslant tract (FAT; orange), the BA 9 anterior ROI showed 49.62% overlap with anterior thalamic radiation (ATR; yellow), and the BA 9 posterior ROI showed 57.61% overlap with the superior longitudinal fasciculus 2 (SLF 2; cyan). L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g003 https://doi.org/10.1371/journal.pone.0271425.g003 12 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease white matter), and the left BA 9 posterior ROI (99.73% white matter). By contrast, in one sig- nificant cluster, controls showed increased activation relative to patients localized to the right BA 6 ROI (63.56% white matter). The remaining 10 activation clusters obtained from func- tional imaging analysis were categorized as non-majority white matter, with eight of the ROIs ranging from 0% - 11.81% white matter and two with 40.63% and 41.30% white matter respectively. DTI results A whole-brain between-groups analysis did not show significant group differences (FWE-cor- rected p > .05). However, we were specifically interested in examining the integrity of white matter within the regions identified in the fMRI analysis. Overlap with white matter skeleton. Overlap with the white matter skeleton derived from the TBSS analysis was computed for ROIs obtained from the fMRI analysis that exhibited greater than 50% white matter as determined by the tissue segmentation analysis (see above). The area of task-related activation in the left BA 8 showed 25.00% overlap with the white mat- ter skeleton, while left BA 9 anterior ROI showed 22.35% overlap, and the left BA 9 posterior ROI showed 24.46% overlap (Fig 3B). Overlap with the white matter skeleton was substantially lower for the right BA 6 ROI (13.46%). Overlap with long range white matter tracts. For the three white matter frontal activa- tion ROIs that exhibited elevated task activation in patients, a follow-up analysis was com- pleted to identify long-range fiber pathways that showed overlap with the task-related activation ROIs. The left BA 8 ROI exhibited 41.57% overlap with the frontal aslant tract (FAT, Fig 3C) and 17.70% overlap with the first branch of the superior longitudinal fasciculus (SLF 1). The left BA 9 anterior ROI exhibited 49.62% overlap with the anterior thalamic radia- tion (ATR, Fig 3C), 12.12% overlap with the dorsal cingulum, and 13.26% overlap with the for- ceps minor. Finally, the left BA 9 posterior ROI showed 57.61% overlap with the second branch of the superior longitudinal fasciculus (SLF 2, Fig 3C). Relationship between white matter microstructure and duration of illness. Within the PTLD group, an exploratory whole brain analysis was conducted to identify areas that demon- strated a significant relationship between DTI microstructural measures (FA, MD, AD, and RD) and DOI. Significant positive correlations with DOI were observed in right frontal regions (FWE-corrected p < .05) for both MD and AD (Fig 4). Across both DTI measures, the correla- tions were found in regions consistent with the right ATR and SLF 3. In MD and AD, voxels with a significant positive correlation with DOI primarily overlapped with the right ATR (60.55% and 17.73%, respectively) and the right SLF 3 (23.39% and 54.29%, respectively). No significant correlations emerged for FA or RD, and no significant negative correlations were found. Relationship among fMRI, DTI, and clinical variables In the PTLD group, we explored the relation among the three clusters of activation localized to the white matter and their respective DTI axial diffusivity measures, using fMRI-derived ROI masks applied to the fMRI and DTI skeletonized maps (Fig 5). The fMRI and DTI BA 9 ROIs marginally and negatively correlated, r(11) = -.55, p = .077 (Table 3 in S1 File). Thus, greater axial diffusivity was tentatively associated with less white matter activation in this region. DTI skeletonized axial diffusivity within the three ROIs were correlated with the sum of symptoms on the PLQS (total, cognitive, and neurologic) to assess the clinical relevance of these regions. Axial diffusivity in the BA 9 anterior ROI negatively correlated with all three clinical measures (total: r(12) = -.76, p = .004; neurological: r(12) = -.78, p = .003; cognitive: r In the PTLD group, we explored the relation among the three clusters of activation localized to the white matter and their respective DTI axial diffusivity measures, using fMRI-derived ROI masks applied to the fMRI and DTI skeletonized maps (Fig 5). The fMRI and DTI BA 9 ROIs marginally and negatively correlated, r(11) = -.55, p = .077 (Table 3 in S1 File). Thus, greater axial diffusivity was tentatively associated with less white matter activation in this region. DTI skeletonized axial diffusivity within the three ROIs were correlated with the sum of symptoms on the PLQS (total, cognitive, and neurologic) to assess the clinical relevance of these regions. Axial diffusivity in the BA 9 anterior ROI negatively correlated with all three clinical measures (total: r(12) = -.76, p = .004; neurological: r(12) = -.78, p = .003; cognitive: r PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 13 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Fig 4. Relationship between diffusion tensor imaging (DTI) metrics and duration of illness (DOI) in PTLD. (A) Mean diffusivity (MD) results. Top: Area of significant correlations between MD and DOI overlaid on the mean fractional anisotropy (FA) map and white matter skeleton (green). Positive correlations are displayed in red (FWE- corrected p < .05). There were no significant negative correlations. Bottom: MD results overlaid with two long range white matter pathways for visualization showing overlap with anterior thalamic radiation (ATR; yellow) and superior longitudinal fasciculus 3 (SLF 3; blue). (B) Axial diffusivity (AD) results. Relationship among fMRI, DTI, and clinical variables Top: Area of significant correlations between AD and DOI overlaid on the mean FA map and white matter skeleton (green). Positive correlations are displayed in red (FWE-corrected p < .05). There were no significant negative correlations. Bottom: AD results overlaid with ATR (yellow) and SLF 3 (blue) showing overlap for localization. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g004 Fig 4. Relationship between diffusion tensor imaging (DTI) metrics and duration of illness (DOI) in PTLD. (A) Mean diffusivity (MD) results. Top: Area of significant correlations between MD and DOI overlaid on the mean fractional anisotropy (FA) map and white matter skeleton (green). Positive correlations are displayed in red (FWE- corrected p < .05). There were no significant negative correlations. Bottom: MD results overlaid with two long range white matter pathways for visualization showing overlap with anterior thalamic radiation (ATR; yellow) and superior longitudinal fasciculus 3 (SLF 3; blue). (B) Axial diffusivity (AD) results. Top: Area of significant correlations between AD and DOI overlaid on the mean FA map and white matter skeleton (green). Positive correlations are displayed in red (FWE-corrected p < .05). There were no significant negative correlations. Bottom: AD results overlaid with ATR (yellow) and SLF 3 (blue) showing overlap for localization. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g004 (12) = -.81, p = .001), showing that greater diffusivity was associated with fewer symptoms (Fig 5). The two other ROIs did not correlate with sum of symptoms [all p-values > .29] (Table 4 in S1 File). A similar comparison between the fMRI beta weight contrast values within these PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 14 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Fig 5. Correlation matrix of the relationship among fMRI, DTI, and clinical variables. Brain regions correspond to the regions of interest revealed by fMRI between-groups contrasts that were located in white matter. FMRI beta values and DTI axial diffusivity measures were correlated with clinical variables. Notably, DTI measures of the BA 9 anterior region negatively correlated with PLQS scores (i.e., higher axial diffusivity was associated with fewer symptoms). PLQS = post-Lyme questionnaire of symptoms, fMRI ACC = accuracy on the fMRI task, fMRI RT = response times on the fMRI task, BDI = Beck Depression Inventory;  = p  .05;  = p  .01 (two-tailed). Fig 5. Relationship among fMRI, DTI, and clinical variables Correlation matrix of the relationship among fMRI, DTI, and clinical variables. Brain regions correspond to the regions of interest revealed by fMRI between-groups contrasts that were located in white matter. FMRI beta values and DTI axial diffusivity measures were correlated with clinical variables. Notably, DTI measures of the BA 9 anterior region negatively correlated with PLQS scores (i.e., higher axial diffusivity was associated with fewer symptoms). PLQS = post-Lyme questionnaire of symptoms, fMRI ACC = accuracy on the fMRI task, fMRI RT = response times on the fMRI task, BDI = Beck Depression Inventory;  = p  .05;  = p  .01 (two-tailed). https://doi.org/10.1371/journal.pone.0271425.g005 ROIs and the sum of PLQS measures also did not correlate [all p-values > .45] (Table 4 in S1 File). Thus, these associations point to frontal lobe axial diffusivity, specifically, as a potential indicator of healthy outcomes in PTLD (i.e., higher axial diffusivity with fewer symptoms). We also compared the three ROIs from fMRI beta contrast and DTI axial diffusivity values to accuracy and RT performance on the working memory task. FMRI values and DTI values did not correlate with accuracy or response times [all p-values > .10] (Table 4 in S1 File). Axial diffusivity in these three regions also did not correlate with accuracy [all p-values > .22] (Table 4 in S1 File). Higher axial diffusivity in the left BA 9 posterior ROI correlated with lower (faster) response times, Spearman’s r(11) = -.63, p = .039 [the two other p-values > .85]. Given that depressive symptoms can accompany PTLD [7,41], we probed for the influence of depressive symptoms on fMRI, DTI, and clinical variables using the Beck Depression Inven- tory (BDI) total score [42]. The BDI total score correlated only with fMRI BA 6 beta values, Spearman’s r(11) = -.767, p = .006, and did not correlate with any other fMRI ROI values (gray or white matter) or axial diffusivity measures [all p-values > .05] (Table 4 in S1 File). However, a higher BDI total score positively correlated with the total sum of symptoms, Spearman’s r (12) = .59, p = .044 and sum of neurological symptoms, Spearman’s r(12), p = .024 [but not with cognitive symptoms, p = .24]. The BDI total score did not correlate with accuracy or response times on the working memory task [both p-values > .66] (Table 4 in S1 File). Relationship among fMRI, DTI, and clinical variables Taken together, these associations suggest that increased axial diffusivity impacts clinical variables in a positive way. Notably, white matter axial diffusivity may be a marker of healing during PTLD and represent a healthier outcome. Discussion This study applied multimodal neuroimaging methods to examine brain structure and func- tion in a carefully selected sample of people with well-characterized PTLD in the absence of co-morbid diseases and other factors that could otherwise explain the results. The original hypothesis that the PTLD group would show altered task-related activations, as revealed by fMRI, was supported. The PTLD group activated different gray brain regions relative to con- trols, some of which were not previously associated with this task in a prior study of healthy adults [23]. Moreover, the PTLD group hypoactivated other areas that were relevant to the PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 15 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease task, relative to controls, suggesting that the PTLD group relied on compensatory mechanisms to complete the task, given that they performed as well as controls did. Unexpectedly, three of the PTLD group’s activated regions found in the frontal lobe were located in white matter. Event-related white matter findings are distinctly unusual in fMRI studies because of the relatively low energy demands and blood volume in white matter [43– 46]. These factors render the white matter BOLD signal comparably much less detectable than in gray matter. However, evidence has accumulated in recent years suggesting that hemody- namic changes can be detected in white matter using high magnetic field strength fMRI meth- ods [45,47]. Explanations for the BOLD signal detection in white matter, especially in an event-related manner, range from reduced physiological noise relative to signal, to visualiza- tion of action potentials, to the energy associated with neurovascular coupling of astrocytes [48]. It is notable that the oxygen extraction fraction in white matter has been reported to be comparable to that of gray matter, a finding that may be explained by the need to maintain resting membrane potentials in white matter oligodendrocytes [46,49]. Thus, white matter changes may signify dysfunction- or excessive function- of glial cells [2]. Because this study was not originally designed to examine white matter function, additional studies are needed to further examine the white matter activations observed here. We scrutinized further the locations of three white matter frontal lobe fMRI-guided ROIs using tissue segmentation analyses. Results showed that the left BA 9 anterior, BA 8, and BA 9 posterior ROIs contained 80% or more overlap with white matter tissue. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 Discussion 16 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease The relationship of these unexpected white matter findings to the clinical features of PTLD suggest that white matter abnormalities may have an important role in the symptomatology of PTLD. Prior studies have reported white matter abnormalities in PTLD. Notably, Fallon et al. (2003) reported brain perfusion abnormalities in white matter regions, including increased blood flow to the frontal lobe [18]. The current findings supported those of Fallon et al. using DTI methods that measured white matter integrity, guided by fMRI activations. The conver- gence of data across imaging modalities underscore the role and vulnerability of the frontal lobe in PTLD symptoms. This study was limited by the small sample size. First, this sample size potentially intro- duced a risk of false positive findings by limited statistical power. However, several measures were employed to mitigate this risk by independently confirming our results in multiple ways. We identified white matter activations that passed a threshold of p < .001, uncorrected (Table 2 and S2 Table), a commonly used threshold for fMRI data reporting. We confirmed that the white matter activations from the fMRI analyses were, indeed, located in white matter using a follow-up segmentation analysis. We then created localized regions of interest specifi- cally from these fMRI-guided activations and used these regions exclusively in our correlations with clinical variables. Given that these regions passed several thresholds, using independent multimodal imaging approaches, the converging findings suggest plausible results in this rela- tively small sample size. Second, generalization of these findings to the larger PTLD community is also mitigated by the relatively small sample size and stringent inclusion criteria (e.g., exclusion of atypical early presentation of Lyme disease that did not meet CDC criteria). The homogeneity of the demo- graphics within our sample further limited generalizability of the findings, given that the study included people with a relatively advanced education level, high socioeconomic status, residing within the mid-Atlantic region of the United States. Third, due to the small sample size, we could not statistically control for the time between infection and antibiotic treatment, antibi- otic dose, and duration of treatment across participants. Such factors may have influenced healing and recovery processes that could not be fully accounted for in this study. Discussion Overlaying these three ROIs onto a skeletonized DTI map, which is a highly conservative mapping that accounts for 34% of the overall white matter mask, indicated 22% or more intersection. These results confirmed that task-related activity can be localized to the white matter of the frontal lobe in people with PTLD. These three ROIs were subsequently examined in more detail exploring their white matter structural integrity using DTI methods (i.e., diffusivity measures). We found that, as a group, the PTLD participants’ DTI measures did not differ from that of controls. When we compared clinical and neurologic symptoms measured outside of the scanner environment to DTI mea- sures in the PTLD group, we found that greater axial diffusivity was associated with fewer symptoms. Thus, the white matter DTI changes observed in this study may represent a healthy marker of the neurological repair process. We found that axial and mean diffusivity increased with DOI in regions within the right frontal lobe. It should be noted that DOI per se did not correlate with clinical measures from the PLQS, behavioral measures from the fMRI task, or BDI scores (all p-values > .42), indicat- ing that diffusivity measures were the driving factor behind the correlations. The regions iden- tified by the diffusivity/DOI correlation were discrete from the white matter ROIs derived from the fMRI task, which were instead located in the left frontal lobe. Importantly, both mea- sures implicated white matter changes within the frontal lobe. Cognitive difficulties referable to the frontal lobe are commonly reported in patients with PTLD [7,13,50]. The measure of axial diffusivity is thought to be related to axonal properties, such as diame- ter, count, and density [51–55]. However, it is important to note that the literature reflects ambiguity with respect to whether increased or decreased axial diffusivity is related to axonal injury. Some studies have reported axonal damage associated with axial diffusivity increases [56–59], while others have reported axonal damage associated with axial diffusivity decreases [60]. Moreover, studies have shown that axial diffusivity patterns can differ by region of inter- est [61–64]. While it is not possible to fully elucidate the biological basis of the altered diffusion signal in the data reported here, our findings demonstrate an important link between axial dif- fusivity and clinical outcomes, as well as DOI. Acknowledgments We thank Bronte Wen for assistance with fMRI data processing and Cheryl Novak and Susan Joseph for the recruitment of study participants. Discussion Fourth, this was a cross-sectional study with a wide ranging DOI across participants. Ideally, participants would be followed longitudinally from disease onset in order to better monitor the changes associated with PTLD directly over time. Nonetheless, given the careful selection of partici- pants with PTLD and little else to medically explain these results, these data provide an impor- tant preliminary look at structural and functional brain changes associated with PTLD and guide future neurologically-based research in the field. This study represents an in-depth examination of the integrity of brain structure and function in people with PTLD using more sophisticated neuroimaging measures than has been reported to date. The findings provide quantitative, objective measures of brain changes that can be associated with clinical and cog- nitive measures. Importantly, these findings support and validate PTLD patient reports of cog- nitive difficulties [7]. Future studies will need to be conducted to replicate these results, given the small sample size. Additionally, longitudinal tracking of brain changes from initial infec- tion through development of PTLD will be needed to characterize changes in gray and white matter over time. Results reported here may have implications for other diseases in which white matter pathology has been demonstrated (e.g., multiple sclerosis) or in illnesses in which cognitive complaints follow disease onset in the absence of objective methods to confirm neuropathol- ogy (e.g., chronic fatigue syndrome, fibromyalgia, post acute COVID) [65–68]. The use of multimodal neuroimaging methods, like the ones used in the current study, may be a viable approach for obtaining information on brain function and structure to identify biomarkers of disease burden. 17 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Supporting information S1 Table. FMRI task BOLD activations within healthy controls. Brain regions are based on Talairach coordinates. Bold indicates activations that overlapped with a prior study in young, healthy adults using the same fMRI task by Marvel & Desmond, 2012. All regions met thresh- old criteria for p < .001, uncorrected. (DOCX) S2 Table. FMRI task BOLD activations within PTLD. Brain regions are based on Talairach coordinates. Bold indicates activations that overlapped with a prior study in young, healthy adults using the same fMRI task by Marvel & Desmond, 2012. ^ = regions that were primarily located in white matter. All regions met threshold criteria for p < .001, uncorrected. (DOCX) S2 Table. FMRI task BOLD activations within PTLD. Brain regions are based on Talairach coordinates. Bold indicates activations that overlapped with a prior study in young, healthy adults using the same fMRI task by Marvel & Desmond, 2012. ^ = regions that were primarily located in white matter. All regions met threshold criteria for p < .001, uncorrected. (DOCX) S1 File. FMRI, DTI, and clinical variables. Table 1 in S1 File shows descriptive statistics for fMRI, DTI, and clinical variables. Table 2 in S1 File shows descriptive statistics for gray matter fMRI data and scores on the Beck Depression Inventory (BDI). In both tables, data are shown as: mean (standard deviation), 95% confidence interval [lower limit, upper limit]. Shapiro- Wilk tests were used for tests of normality. Significant values of the Shapiro-Wilk tests are denoted in bold, p  .05, two-tailed. Table 3 in S1 File reports correlations between the fMRI ROIs and their respective DTI ROIs. Pearson’s tests were used for bivariate correlations involving BA 9 values because they have a normal distribution. A Spearman’s test was used for bivariate correlations involving BA 8 values because they had a non-normal distribution. Table 4 in S1 File reports correlations between fMRI and DTI ROIs and fMRI Task Accuracy, RT, Symptoms, and BDI. Spearman’s tests were used for bivariate correlations involving Task Accuracy, RT, Cognitive Symptoms, and BDI because they had a non-normal distribution. Pearson’s tests were used for bivariate correlations involving Total Clinical Symptoms and Neurological Symptoms because they had a normal distribution. (DOCX) Author Contributions Conceptualization: Cherie L. Marvel, Alison W. Rebman, Arun Venkatesan, John N. Aucott. Conceptualization: Cherie L. Marvel, Alison W. Rebman, Arun Venkatesan, John N. Aucott. Data curation: Kylie H. Alm, Deeya Bhattacharya, Alison W. Rebman, Erica A. Kozero. Formal analysis: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Owen P. Morgan, Prianca A. Nadkarni. Formal analysis: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Owen P. Morgan, Prianca A. Nadkarni. Formal analysis: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Owen P. Morgan, Prianca A. Nadkarni. References 1. Steere AC, Strle F, Wormser GP, Hu LT, Branda JA, Hovius JWR, et al. Lyme borreliosis. Nat Rev Dis Primers. 2016; 2:16090. https://doi.org/10.1038/nrdp.2016.90 PMID: 27976670 2. Kugeler K, Schwartz A, Delorey M, Mead P, Hinckley A. Estimating the Frequency of Lyme Disease Diagnoses, United States, 2010–2018. Emerging Infectious Disease journal. 2021; 27(2):616. https:// doi.org/10.3201/eid2702.202731 PMID: 33496229 3. Steere AC. Lyme Disease. New England Journal of Medicine. 2001 2001/07/12; 345(2):115–25. https:// doi.org/10.1056/NEJM200107123450207 PMID: 11450660 4. Mac S, Bahia S, Simbulan F, Pullenayegum EM, Evans GA, Patel SN, et al. Long-Term Sequelae and Health-Related Quality of Life Associated With Lyme Disease: A Systematic Review. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2020; 71(2):440–52. https://doi.org/10.1093/cid/ciz1158 PMID: 31773171 5. Rebman AW, Aucott JN. Post-treatment Lyme Disease as a Model for Persistent Symptoms in Lyme Disease. Frontiers in medicine. 2020; 7:57. https://doi.org/10.3389/fmed.2020.00057 PMID: 32161761 6. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, et al. The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America. Clinical Infec- tious Diseases. 2006; 43(9):1089–134. https://doi.org/10.1086/508667 PMID: 17029130 7. Rebman AW, Bechtold KT, Yang T, Mihm EA, Soloski MJ, Novak CB, et al. The Clinical, Symptom, and Quality-of-Life Characterization of a Well-Defined Group of Patients with Post treatment Lyme Disease Syndrome. Front Med (Lausanne). 2017; 4:224. https://doi.org/10.3389/fmed.2017.00224 PMID: 29312942 8. Lobraico J, Butler A, Petrini J, Ahmadi R. New Insights Into Stages of Lyme Disease Symptoms From a Novel Hospital-Based Registry. Journal of Primary Care & Community Health. 2014 2014/10/01; 5 (4):284–7. https://doi.org/10.1177/2150131914540693 PMID: 24970880 9. Zomer TP, Barendregt JNM, van Kooten B, van Bemmel T, Landman GW, van Hees BC, et al. Non- specific symptoms in adult patients referred to a Lyme centre. Clinical Microbiology and Infection. 2019 2019/01/01/; 25(1):67–70. https://doi.org/10.1016/j.cmi.2018.09.016 PMID: 30287411 10. Klempner MS, Hu LT, Evans J, Schmid CH, Johnson GM, Trevino RP, et al. Two Controlled Trials of Antibiotic Treatment in Patients with Persistent Symptoms and a History of Lyme Disease. New England Journal of Medicine. 2001 2001/07/12; 345(2):85–92. https://doi.org/10.1056/ NEJM200107123450202 PMID: 11450676 11. Chandra AM, Keilp JG, Fallon BA. Correlates of perceived health-related quality of life in post-treatment Lyme encephalopathy. Psychosomatics. 2013 Nov-Dec; 54(6):552–9. https://doi.org/10.1016/j.psym. 2013.04.003 PMID: 23845316 12. Rebman AW, Aucott JN, Weinstein ER, Bechtold KT, Smith KC, Leonard L. Funding acquisition: John N. Aucott. Funding acquisition: John N. Aucott. Investigation: Cherie L. Marvel, Deeya Bhattacharya, Owen P. Morgan, Jason A. Creighton, Erica A. Kozero, John N. Aucott. Methodology: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Arnold Bakker, John N. Aucott. Project administration: Cherie L. Marvel, Deeya Bhattacharya, Jason A. Creighton. Resources: Cherie L. Marvel, Arnold Bakker, John N. Aucott. 18 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Software: Kylie H. Alm, Owen P. Morgan. Software: Kylie H. Alm, Owen P. Morgan. Supervision: Cherie L. Marvel, Arnold Bakker, John N. Aucott. Software: Kylie H. Alm, Owen P. Morgan. Supervision: Cherie L. Marvel, Arnold Bakker, John N. Aucott. Writing – original draft: Cherie L. Marvel, Kylie H. Alm, Deeya Bhattacharya, Alison W. Reb- man, Arnold Bakker, John N. Aucott. Writing – review & editing: Cherie L. Marvel, Kylie H. Alm, Deeya Bhattacharya, Alison W. Rebman, Arnold Bakker, Owen P. Morgan, Jason A. Creighton, Erica A. Kozero, Arun Venkatesan, Prianca A. Nadkarni, John N. Aucott. References Living in Limbo: Contested Narratives of Patients With Chronic Symptoms Following Lyme Disease. Qualitative Health Research. 2015 2017/03/01; 27(4):534–46. 13. Touradji P, Aucott JN, Yang T, Rebman AW, Bechtold KT. Cognitive Decline in Post-treatment Lyme Disease Syndrome. Archives of Clinical Neuropsychology. 2019; 34(4):455–65. https://doi.org/10.1093/ arclin/acy051 PMID: 29945190 14. Newberg A, Hassan A, Alavi A. Cerebral metabolic changes associated with Lyme disease. Nucl Med Commun. 2002 Aug; 23(8):773–7. https://doi.org/10.1097/00006231-200208000-00011 PMID: 12124483 15. Fallon BA, Das S, Plutchok JJ, Tager F, Liegner K, Van Heertum R. Functional brain imaging and neuropsychological testing in Lyme disease. Clin Infect Dis. 1997 Jul; 25 Suppl 1:S57–63. https://doi. org/10.1086/516175 PMID: 9233666 19 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease 16. Sumiya H, Kobayashi K, Mizukoshi C, Aoki T, Koshino Y, Taki J, et al. Brain perfusion SPECT in Lyme neuroborreliosis. J Nucl Med. 1997 Jul; 38(7):1120–2. PMID: 9225802 17. Westervelt HJ, McCaffrey RJ. Neuropsychological functioning in chronic Lyme disease. Neuropsychol Rev. 2002 Sep; 12(3):153–77. https://doi.org/10.1023/a:1020381913563 PMID: 12428915 18. Fallon BA, Keilp J, Prohovnik I, Heertum RV, Mann JJ. Regional cerebral blood flow and cognitive defi- cits in chronic lyme disease. J Neuropsychiatry Clin Neurosci. 2003 Summer; 15(3):326–32. https://doi. org/10.1176/jnp.15.3.326 PMID: 12928508 19. Fernandez RE, Rothberg M, Ferencz G, Wujack D. Lyme disease of the CNS: MR imaging findings in 14 cases. AJNR Am J Neuroradiol. 1990 May; 11(3):479–81. PMID: 2112310 20. Coughlin JM, Yang T, Rebman AW, Bechtold KT, Du Y, Mathews WB, et al. Imaging glial activation in patients with post-treatment Lyme disease symptoms: a pilot study using [(11)C]DPA-713 PET. J Neu- roinflammation. 2018; 15(1):346. https://doi.org/10.1186/s12974-018-1381-4 PMID: 30567544 21. Centers for Disease Control and Prevention [Internet]. Lyme Disease (Borrelia burgdorferi) 2022 Case Definition. [Accessed August 23, 2022]. Available from: https://ndc.services.cdc.gov/case-definitions/ lyme-disease-2022. 22. Rebman AW, Yang T, Aucott JN. Symptom heterogeneity and patient subgroup classification among US patients with post-treatment Lyme disease: an observational study. BMJ Open. 2021; 11(1): e040399. https://doi.org/10.1136/bmjopen-2020-040399 PMID: 33441353 23. Marvel CL, Desmond JE. From storage to manipulation: How the neural correlates of verbal working memory reflect varying demands on inner speech. Brain Lang. 2012 Jan; 120(1):42–51. https://doi.org/ 10.1016/j.bandl.2011.08.005 PMID: 21889195 24. Marvel CL, Faulkner ML, Strain EC, Mintzer MZ, Desmond JE. An fMRI investigation of cerebellar func- tion during verbal working memory in methadone maintenance patients. Cerebellum. 2012 Mar; 11 (1):300–10. https://doi.org/10.1007/s12311-011-0311-0 PMID: 21892700 25. References Chen SH, Desmond JE. Temporal dynamics of cerebro-cerebellar network recruitment during a cogni- tive task. Neuropsychologia. 2005; 43(9):1227–37. https://doi.org/10.1016/j.neuropsychologia.2004.12. 015 PMID: 15949507 26. Talairach J, & Tournoux P. Co-planar Stereotaxic atlas of the human brain 3-D proportional system, An approach to cerebral imaging (M. Rayport, Trans.): New York: Thieme Medical Publishers, Inc.; 1988. 27. Lancaster JL, Rainey LH, Summerlin JL, Freitas CS, Fox PT, Evans AC, et al. Automated labeling of the human brain: a preliminary report on the development and evaluation of a forward-transform method. Hum Brain Mapp. 1997 1997; 5(4):238–42. https://doi.org/10.1002/(SICI)1097-0193(1997) 5:4<238::AID-HBM6>3.0.CO;2-4 PMID: 20408222 28. Brett M, Anton J-L, Valabregue R, Poline J-B. Region of interest analysis using an SPM toolbox [abstract]. Presented at the 8th International Conference on Functional Mapping of the Human Brain. NeuroImage. 2002;16. 29. Smith SM, Jenkinson M, Woolrich MW, Beckmann CF, Behrens TEJ, Johansen-Berg H, et al. Advances in functional and structural MR image analysis and implementation as FSL. NeuroImage. 2004 2004/01/01/; 23:S208–S19. https://doi.org/10.1016/j.neuroimage.2004.07.051 PMID: 15501092 30. Klein A, Andersson J, Ardekani BA, Ashburner J, Avants B, Chiang M-C, et al. Evaluation of 14 nonlin- ear deformation algorithms applied to human brain MRI registration. NeuroImage. 2009; 46(3):786– 802. https://doi.org/10.1016/j.neuroimage.2008.12.037 PMID: 19195496 31. Yushkevich PA, Avants BB, Pluta J, Das S, Minkoff D, Mechanic-Hamilton D, et al. A high-resolution computational atlas of the human hippocampus from postmortem magnetic resonance imaging at 9.4 T. NeuroImage. 2009; 44(2):385–98. https://doi.org/10.1016/j.neuroimage.2008.08.042 PMID: 18840532 32. Zhang Y, Brady M, Smith S. Segmentation of brain MR images through a hidden Markov random field model and the expectation-maximization algorithm. IEEE Transactions on Medical Imaging. 2001; 20 (1):45–57. https://doi.org/10.1109/42.906424 PMID: 11293691 33. Eggert LD, Sommer J, Jansen A, Kircher T, Konrad C. Accuracy and Reliability of Automated Gray Mat- ter Segmentation Pathways on Real and Simulated Structural Magnetic Resonance Images of the Human Brain. PLoS One. 2012; 7(9):e45081. https://doi.org/10.1371/journal.pone.0045081 PMID: 23028771 34. Smith SM, Jenkinson M, Johansen-Berg H, Rueckert D, Nichols TE, Mackay CE, et al. Tract-based spatial statistics: Voxelwise analysis of multi-subject diffusion data. NeuroImage. 2006 2006/07/15/; 31 (4):1487–505. https://doi.org/10.1016/j.neuroimage.2006.02.024 PMID: 16624579 35. Andersson J, Jenkinson M, editors. Non-linear optimisation FMRIB Technial Report TR 07 JA 1. 2007. 36. Jenkinson M, editor. Non-linear registration aka Spatial normalisation. 2007. 35. Andersson J, Jenkinson M, editors. Non-linear optimisation FMRIB Technial Report TR 07 JA 1. 2007. 36. Jenkinson M, editor. Non-linear registration aka Spatial normalisation. 2007. References 20 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease 37. Winkler AM, Ridgway GR, Webster MA, Smith SM, Nichols TE. Permutation inference for the general linear model. NeuroImage. 2014; 92(100):381–97. https://doi.org/10.1016/j.neuroimage.2014.01.060 PMID: 24530839 38. Smith SM, Nichols TE. Threshold-free cluster enhancement: Addressing problems of smoothing, threshold dependence and localisation in cluster inference. NeuroImage. 2009 2009/01/01/; 44(1):83– 98. https://doi.org/10.1016/j.neuroimage.2008.03.061 PMID: 18501637 39. Warrington S, Bryant KL, Khrapitchev AA, Sallet J, Charquero-Ballester M, Douaud G, et al. XTRACT —Standardised protocols for automated tractography in the human and macaque brain. NeuroImage. 2020 2020/08/15/; 217:116923. https://doi.org/10.1016/j.neuroimage.2020.116923 PMID: 32407993 40. Marvel CL, Desmond JE. The contributions of cerebro-cerebellar circuitry to executive verbal working memory. Cortex. 2010 Jul-Aug; 46(7):880–95. https://doi.org/10.1016/j.cortex.2009.08.017 PMID: 19811779 41. Bransfield RC. Neuropsychiatric Lyme Borreliosis: An Overview with a Focus on a Specialty Psychia- trist’s Clinical Practice. Healthcare (Basel). 2018; 6(3):104. https://doi.org/10.3390/healthcare6030104 PMID: 30149626 42. Beck A, Steer R, Brown G. Beck Depression Inventory—Second Edition Manual. San Antonio: The Psychological Corporation; 1996. 43. Logothetis NK, Wandell BA. Interpreting the BOLD signal. Annu Rev Physiol. 2004; 66:735–69. https:// doi.org/10.1146/annurev.physiol.66.082602.092845 PMID: 14977420 44. Rostrup E, Law I, Blinkenberg M, Larsson HBW, Born AP, Holm S, et al. Regional Differences in the CBF and BOLD Responses to Hypercapnia: A Combined PET and fMRI Study. NeuroImage. 2000 2000/02/01/; 11(2):87–97. https://doi.org/10.1006/nimg.1999.0526 PMID: 10679182 45. Gore JC, Li M, Gao Y, Wu T-L, Schilling KG, Huang Y, et al. Functional MRI and resting state connectiv- ity in white matter—a mini-review. Magnetic resonance imaging. 2019; 63:1–11. https://doi.org/10. 1016/j.mri.2019.07.017 PMID: 31376477 46. Harris JJ, Attwell D. The Energetics of CNS White Matter. The Journal of Neuroscience. 2012; 32 (1):356. https://doi.org/10.1523/JNEUROSCI.3430-11.2012 PMID: 22219296 47. Wu X, Yang Z, Bailey SK, Zhou J, Cutting LE, Gore JC, et al. Functional connectivity and activity of white matter in somatosensory pathways under tactile stimulations. NeuroImage. 2017 2017/05/15/; 152:371–80. https://doi.org/10.1016/j.neuroimage.2017.02.074 PMID: 28284801 48. Gawryluk JR, Mazerolle EL, D’Arcy RCN. Does functional MRI detect activation in white matter? A review of emerging evidence, issues, and future directions. Front Neurosci. 2014; 8:239. https://doi.org/ 10.3389/fnins.2014.00239 PMID: 25152709 49. Raichle ME, MacLeod AM, Snyder AZ, Powers WJ, Gusnard DA, Shulman GL. A default mode of brain function. Proceedings of the National Academy of Sciences. 2001; 98(2):676. 50. Keilp JG, Corbera K, Slavov I, Taylor MJ, Sackeim HA, Fallon BA. WAIS-III and WMS-III performance in chronic Lyme disease. References J Int Neuropsychol Soc. 2006 Jan; 12(1):119–29. https://doi.org/10.1017/ S1355617706060231 PMID: 16433951 51. Beaulieu C. What Makes Diffusion Anisotropic in the Nervous System? Diffusion MRITheory, Methods, and Applications. Oxford, UK: Oxford University Press; 2012. 52. Song SK, Sun SW, Ju WK, Lin SJ, Cross AH, Neufeld AH. Diffusion tensor imaging detects and differ- entiates axon and myelin degeneration in mouse optic nerve after retinal ischemia. Neuroimage. 2003 Nov; 20(3):1714–22. https://doi.org/10.1016/j.neuroimage.2003.07.005 PMID: 14642481 53. Budde MD, Kim JH, Liang HF, Schmidt RE, Russell JH, Cross AH, et al. Toward accurate diagnosis of white matter pathology using diffusion tensor imaging. Magn Reson Med. 2007 Apr; 57(4):688–95. https://doi.org/10.1002/mrm.21200 PMID: 17390365 54. Budde MD, Xie M, Cross AH, Song S-K. Axial Diffusivity Is the Primary Correlate of Axonal Injury in the Experimental Autoimmune Encephalomyelitis Spinal Cord: A Quantitative Pixelwise Analysis. The Jour- nal of Neuroscience. 2009; 29(9):2805. https://doi.org/10.1523/JNEUROSCI.4605-08.2009 PMID: 19261876 55. DeBoy CA, Zhang J, Dike S, Shats I, Jones M, Reich DS, et al. High resolution diffusion tensor imaging of axonal damage in focal inflammatory and demyelinating lesions in rat spinal cord. Brain. 2007 Aug; 130(Pt 8):2199–210. https://doi.org/10.1093/brain/awm122 PMID: 17557778 56. Bosch B, Arenaza-Urquijo EM, Rami L, Sala-Llonch R, Junque´ C, Sole´-Padulle´s C, et al. Multiple DTI index analysis in normal aging, amnestic MCI and AD. Relationship with neuropsychological perfor- mance. Neurobiol Aging. 2012 Jan; 33(1):61–74. https://doi.org/10.1016/j.neurobiolaging.2010.02.004 PMID: 20371138 57. Alves GS, Oertel Kno¨chel V, Kno¨chel C, Carvalho AF, Pantel J, Engelhardt E, et al. Integrating retro- genesis theory to Alzheimer’s disease pathology: insight from DTI-TBSS investigation of the white PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 21 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease matter microstructural integrity. Biomed Res Int. 2015; 2015:291658. https://doi.org/10.1155/2015/ 291658 PMID: 25685779 matter microstructural integrity. Biomed Res Int. 2015; 2015:291658. h 291658 PMID: 25685779 matter microstructural integrity. Biomed Res Int. 2015; 2015:291658. https://doi.org/10.1155/2015/ 291658 PMID: 25685779 58. 58. Schmierer K, Wheeler-Kingshott CA, Tozer DJ, Boulby PA, Parkes HG, Yousry TA, et al. Quantitative magnetic resonance of postmortem multiple sclerosis brain before and after fixation. Magn Reson Med. 2008 Feb; 59(2):268–77. https://doi.org/10.1002/mrm.21487 PMID: 18228601 59. Newcombe V, Chatfield D, Outtrim J, Vowler S, Manktelow A, Cross J, et al. Mapping traumatic axonal injury using diffusion tensor imaging: correlations with functional outcome. PLoS One. 2011; 6(5): e19214–e. https://doi.org/10.1371/journal.pone.0019214 PMID: 21573228 60. Wang X, Cusick MF, Wang Y, Sun P, Libbey JE, Trinkaus K, et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425 October 26, 2022 68. Glass JM. Fibromyalgia and cognition. J Clin Psychiatry. 2008; 69 Suppl 2:20–4. PMID: 18537459 References Diffusion basis spectrum imaging detects and distinguishes coexisting subclinical inflammation, demyelination and axonal injury in experi- mental autoimmune encephalomyelitis mice. NMR Biomed. 2014 Jul; 27(7):843–52. https://doi.org/10. 1002/nbm.3129 PMID: 24816651 61. Bennett IJ, Madden DJ, Vaidya CJ, Howard DV, Howard JH Jr., Age-related differences in multiple measures of white matter integrity: A diffusion tensor imaging study of healthy aging. Hum Brain Mapp. 2010; 31(3):378–90. 62. Burzynska AZ, Preuschhof C, Ba¨ckman L, Nyberg L, Li SC, Lindenberger U, et al. Age-related differ- ences in white matter microstructure: region-specific patterns of diffusivity. Neuroimage. 2010 Feb 1; 49 (3):2104–12. https://doi.org/10.1016/j.neuroimage.2009.09.041 PMID: 19782758 63. Isaac Tseng WY, Hsu YC, Chen CL, Kang YJ, Kao TW, Chen PY, et al. Microstructural differences in white matter tracts across middle to late adulthood: a diffusion MRI study on 7167 UK Biobank partici- pants. Neurobiol Aging. 2021 Feb; 98:160–72. https://doi.org/10.1016/j.neurobiolaging.2020.10.006 PMID: 33290993 64. Madden DJ, Bennett IJ, Burzynska A, Potter GG, Chen NK, Song AW. Diffusion tensor imaging of cere- bral white matter integrity in cognitive aging. Biochim Biophys Acta. 2012 Mar; 1822(3):386–400. https://doi.org/10.1016/j.bbadis.2011.08.003 PMID: 21871957 65. Freeman CW, Masur J, Hassankhani A, Wolf RL, Levine JM, Mohan S. Coronavirus Disease (COVID- 19)-Related Disseminated Leukoencephalopathy: A Retrospective Study of Findings on Brain MRI. American Journal of Roentgenology. 2021 2021/04/01; 216(4):1046–7. https://doi.org/10.2214/AJR.20. 24364 PMID: 32903058 66. Hampshire A, Trender W, Chamberlain SR, Jolly AE, Grant JE, Patrick F, et al. Cognitive deficits in peo- ple who have recovered from COVID-19. EClinicalMedicine. 2021 Sep; 39:101044. https://doi.org/10. 1016/j.eclinm.2021.101044 PMID: 34316551 67. Morris G, Berk M, Puri BK. A Comparison of Neuroimaging Abnormalities in Multiple Sclerosis, Major Depression and Chronic Fatigue Syndrome (Myalgic Encephalomyelitis): is There a Common Cause? Mol Neurobiol. 2018 Apr; 55(4):3592–609. https://doi.org/10.1007/s12035-017-0598-z PMID: 28516431 68. Glass JM. Fibromyalgia and cognition. J Clin Psychiatry. 2008; 69 Suppl 2:20–4. PMID: 18537459 22 / 22
https://openalex.org/W1987480136
http://old.scielo.br/pdf/tema/v13n1/08.pdf
Portuguese
null
Produtos de Grafos Z_m-bem-cobertos
Trends in Computational and Applied Mathematics
2,012
cc-by
5,089
Produtos de Grafos Zm-bem-cobertos R.M. BARBOSA1, M.R.C. SANTANA2, Instituto de Informática, INF, UFG - Uni- versidade Federal de Goiás, 74001-970 Goiânia, GO, Brasil. R.M. BARBOSA1, M.R.C. SANTANA2, Instituto de Informática, INF, UFG - Uni- versidade Federal de Goiás, 74001-970 Goiânia, GO, Brasil. Resumo. Um grafo é Zm-bem-coberto se |I| ≡|J| (mod m), m ≥2, para todo I, J conjuntos independentes maximais em V (G). Um grafo G é fortemente Zm-bem- coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm-bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto e G\{v} é Zm-bem- coberto, ∀v ∈V (G). Mostramos que os grafos 1-Zm-bem-cobertos, bem como os fortemente Zm-bem-cobertos, com exceção de K1 e K2, têm cintura ≤5. Mos- tramos uma condição necessária e suficiente para que produtos lexicográficos de grafos sejam Zm-bem-cobertos e algumas propriedades para o produto cartesiano de ciclos. Palavras-chave. Teoria dos Grafos, Conjuntos Independentes em Grafos, Produ- tos de Grafos. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83. doi: 10.5540/tema.2012.013.01.0075 c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83. doi: 10.5540/tema.2012.013.01.0075 c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83. doi: 10.5540/tema.2012.013.01.0075 c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. 1rommel@inf.ufg.br 2marcia@inf.ufg.br Recebido em 02 Maio 2011; Aceito em 24 Fevereiro 2012. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83. doi: 10.5540/tema.2012.013.01.0075 c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. 1. Introdução De forma similar, foi definido em [2, 3] que um grafo G é fortemente Zm-bem-coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm- bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto e G\{v} é Zm-bem-coberto, ∀v ∈V (G). Uma folha é um vértice de grau 1, um talo é um vértice adjacente a uma folha e um arbusto é um subgrafo induzido por um talo e suas folhas. Vértices x e y de um grafo G são ditos ser conectados por uma 2-ponte se existem vértices u e v ∈V (G) com grau de u e v igual a 2 e com N(u) = {x, v} e N(v) = {u, y}. para qualquer e ∈E(G). De forma similar, foi definido em [2, 3] que um grafo G é fortemente Zm-bem-coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm- bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto e G\{v} é Zm-bem-coberto, ∀v ∈V (G). Uma folha é um vértice de grau 1, um talo é um vértice adjacente a uma folha e um arbusto é um subgrafo induzido por um talo e suas folhas. Vértices x e y de um grafo G são ditos ser conectados por uma 2-ponte se existem vértices u e v ∈V (G) com grau de u e v igual a 2 e com N(u) = {x, v} e N(v) = {u, y}. O Teorema 1.1, provado em [10], fornece uma caracterização de grafos Zm-bem- cobertos de cintura > 5. O Teorema 1.1, provado em [10], fornece uma caracterização de grafos Zm-bem- cobertos de cintura > 5. Teorema 1.1. [10] Um grafo é Zm-bem-coberto conexo de cintura > 5 se e somente se G é K1, C7 ou um grafo conexo de cintura pelo menos seis, que consiste de uma união finita de arbustos Bi, cada um com talo xi, onde cada talo tem ri congruente a 1 (mod m) folhas e onde, para cada i e j, uma e somente uma das seguintes condições é satisfeita: 1. xi e xj são unidos por uma aresta e qualquer outro caminho, caso exista, unindo xi e xj deve incluir pelo menos um talo diferente de xi e xj; 2. 1. Introdução Os grafos aqui considerados são grafos simples. As definições e notações utilizadas seguem [7]. Denotamos o conjunto de vértices de um grafo G por V (G) e o conjunto de arestas por E(G). N(v) é o conjunto de vértices adjacentes a v em G. Um con- junto I ⊆V (G) é independente se quaisquer dois vértices de I não são adjacentes. Denotamos por α(G) a cardinalidade do maior conjunto independente de vértices de G. A cintura de um grafo com um ciclo é o tamanho de seu menor ciclo. Um grafo sem ciclo tem cintura infinita. Um grafo é bem-coberto se todo conjunto in- dependente maximal de vértices em G tiver mesma cardinalidade. Um grafo G é um grafo Zm-bem-coberto, m ≥2, se |I| ≡|J| (mod m), para todos I, J conjuntos independentes maximais em V (G). Estes grafos foram introduzidos em [9]. Ca- racterizações de grafos Zm-bem-cobertos cúbicos foram dadas em [4], com cintura ≥6 em [10], cordais, simpliciais e arco-circulares em [6] e livres de K1,3 em [5]. O problema de determinação do número de independência de um grafo é um problema NP-Completo [14] para grafos em geral. Para grafos bem-cobertos este problema torna-se mais simples, pois é suficiente encontrar qualquer conjunto independente maximal, visto que todos tem a mesma cardinalidade. Caro [8] provou que o pro- blema de reconhecimento de grafos bem-cobertos é Co-NP-completo mesmo para grafos Zm-bem-cobertos que são livres de K1,3m+1. Pinter [16] definiu um grafo for- temente bem-coberto G como um grafo que é bem-coberto e G\{e} é bem-coberto Recebido em 02 Maio 2011; Aceito em 24 Fevereiro 2012. 76 Barbosa e Santana Barbosa e Santana Barbosa e Santana para qualquer e ∈E(G). De forma similar, foi definido em [2, 3] que um grafo G é fortemente Zm-bem-coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm- bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto e G\{v} é Zm-bem-coberto, ∀v ∈V (G). Uma folha é um vértice de grau 1, um talo é um vértice adjacente a uma folha e um arbusto é um subgrafo induzido por um talo e suas folhas. Vértices x e y de um grafo G são ditos ser conectados por uma 2-ponte se existem vértices u e v ∈V (G) com grau de u e v igual a 2 e com N(u) = {x, v} e N(v) = {u, y}. para qualquer e ∈E(G). 1. Introdução xi e xj são conectados por km 2-pontes, k ∈N, e qualquer outro caminho unindo xi e xj deve incluir outro talo além de xi e xj; 3. Todo caminho unindo xi e xj contém pelo menos um talo diferente de xi e xj. 3. Todo caminho unindo xi e xj contém pelo menos um talo diferente de xi e xj. Na figura 1 temos um exemplo de um grafo Z3-bem-coberto conexo de cintura 6, com conjuntos independentes maximais de cardinalidades 4, 7 e 10. 3 2-pontes Figura 1: Grafo Z3-bem-coberto de cintura 6 Figura 1: Grafo Z3-bem-coberto de cintura 6 A classe de grafos Zm-bem-cobertos inclui todos os grafos bem-cobertos, já que em todo grafo bem-coberto os conjuntos independentes maximais têm a mesma cardinalidade e, portanto, são congruentes módulo qualquer natural m. Alguns resultados sobre grafos bem-cobertos podem ser estendidos para os grafos Zm-bem- cobertos. Como principais contribuições, mostramos que se G é 1-Zm-bem-coberto e não é K1 nem K2, então G tem cintura ≤5 e apresentamos também uma condição necessária e suficiente para que produtos lexicográficos de grafos sejam Zm-bem- cobertos. Ainda, mostramos que o produto cartesiano de dois ciclos Cn e Cm é Zm-bem-coberto se e somente se ele é bem-coberto. 77 Produtos de Grafos Zm-bem-cobertos Outros resultados válidos para grafos bem-cobertos não podem ser estendidos aos grafos Zm-bem-cobertos. Na Proposição 3.2, mostramos que podemos construir infinitos grafos fortemente Zm-bem-cobertos que sejam planares. Há apenas 4 grafos planares fortemente bem-cobertos, o que foi provado por Pinter [16]. Para um subconjunto S de V (H ◦{G1, G2, . . . , G|V (H)|}), denotamos XH(S) = {x ∈V (H) : ∃y ∈V (Gx)(x, y) ∈S} e XGx(S) = {y ∈V (Gx) : (x, y) ∈S} para todo x ∈XH(S). No grafo da figura 2 (b), se S = {(1, a), (3, c)}, XH(S) = {1, 3}, XG1(S) = {a} e XG3(S) = {c}. 2. Produto Lexicográfico , G|V (H)|)), S é um conjunto independente maximal em H ◦(G1, . . . , G|V (H)|), se e somente se, XH(S) é um conjunto independente maximal em H, e para todo v ∈XH(S), o conjunto XGv(S) é um conjunto independente maximal em Gv. Proposição 2.1. [17] Dados S ⊂V (H ◦(G1, . . . , G|V (H)|)), S é um conjunto independente maximal em H ◦(G1, . . . , G|V (H)|), se e somente se, XH(S) é um conjunto independente maximal em H, e para todo v ∈XH(S), o conjunto XGv(S) é um conjunto independente maximal em Gv. De forma similar a apresentada por Topp e Volkmann [17], o Teorema 2.2 nos fornece uma condição necessária e suficiente para o produto de grafos ser Zm-bem- coberto. Teorema 2.2. Seja H um grafo e {G1, G2, . . . , G|V (H)|} uma família de grafos não vazios. O produto lexicográfico H ◦{G1, G2, . . . , G|V (H)|} é um grafo Zm-bem- coberto, para um dado m ≥2, se e somente se H e {G1, G2, ..., G|V (H)|} satisfazem as seguintes condições: 1. Gi é Zm-bem-coberto para i = 1, . . . , |V (H)|, para um m qualquer ≥2. 2. P v∈I α(Gv) ≡P u∈J α(Gu) (mod m), ∀I, J, conjuntos independentes maxi- mais de H. Demonstração. (=⇒)Suponha que H ◦{G1, G2, . . . , G|V (H)|} é Zm-bem-coberto e que existe Gv0 que não é Zm-bem-coberto. Então Gv0 tem dois conjuntos inde- pendentes maximais de vértices Iv0 e Jv0 tais que |Iv0| ̸≡|Jv0| (mod m). Es- tenda {v0} a um conjunto independente maximal L em V (H). Para qualquer v ∈L\{v0}, seja Iv um conjunto independente maximal em Gv. Então, pela Proposição 2.1, A = ∪v∈L\{v0}{(v, x) : x ∈Iv} ∪{(v0, y) : y ∈Iv0} e B = ∪v∈L\{v0}{(v, x) : x ∈Iv}∪{(v0, t) : t ∈Jv0} são conjuntos independentes maximais em H ◦{G1, G2, . . . , G|V (H)|} tal que |A| ̸≡|B| (mod m). Isto é uma contradição. Logo, Gi deve ser Zm-bem-coberto para todo i = 1, 2, . . . , |V (H)|. Demonstração. (=⇒)Suponha que H ◦{G1, G2, . . . , G|V (H)|} é Zm-bem-coberto e que existe Gv0 que não é Zm-bem-coberto. Então Gv0 tem dois conjuntos inde- pendentes maximais de vértices Iv0 e Jv0 tais que |Iv0| ̸≡|Jv0| (mod m). 2. Produto Lexicográfico , G|V (H)|} são congruentes (mod m) e portanto H ◦{G1, G2, . . . , G|V (H)|} é um grafo Zm-bem-coberto. 2. Produto Lexicográfico Es- tenda {v0} a um conjunto independente maximal L em V (H). Para qualquer v ∈L\{v0}, seja Iv um conjunto independente maximal em Gv. Então, pela Proposição 2.1, A = ∪v∈L\{v0}{(v, x) : x ∈Iv} ∪{(v0, y) : y ∈Iv0} e B = ∪v∈L\{v0}{(v, x) : x ∈Iv}∪{(v0, t) : t ∈Jv0} são conjuntos independentes maximais em H ◦{G1, G2, . . . , G|V (H)|} tal que |A| ̸≡|B| (mod m). Isto é uma contradição. Logo, Gi deve ser Zm-bem-coberto para todo i = 1, 2, . . . , |V (H)|. Sejam I e J dois conjuntos independentes maximais de vértices em H. Provare- mos que P v∈I α(Gv) ≡P v∈J α(Gv) (mod m). Seja Rv um conjunto independente maximal em Gv, ∀v ∈I∪J. Então, S1 = S v∈I{(v, x) : x ∈Rv} e S2 = S v∈J{(v, x) : x ∈Rv} são conjuntos independentes maximais em H ◦{G1, G2, . . . , G|V (H)|}. En- tão |S1| ≡|S2| (mod m). Mas, {|(v, x) : x ∈Rv}| ≡α(Gv)(mod m), ∀u, v ∈I ∪J, segue que P v∈I α(Gv) ≡|S1| ≡|S2| ≡P v∈J α(Gv) (mod m). v∈I v∈J (⇐=)Seja I um conjunto independente maximal em H ◦{G1, G2, . . . , G|V (H)|}. Pela Proposição 2.1, XH(I) é um conjunto independente maximal em H e XGv(I) é um conjunto independente maximal em Gv para todo v ∈XH(I). Já que I = S v∈XH(I){(v, x) : x ∈XGv(I)} e |XGv(I)| ≡α(Gv) (mod m), temos |I| = P v∈XH(I) |{[v, x] : x ∈XGv(I)}| = P v∈XH(I) |XGv(I)| = P v∈XH(I) α(Gv). Então, pela condição (2), quaisquer dois conjuntos independentes maximais em H◦{G1, G2, . . . , G|V (H)|} são congruentes (mod m) e portanto H ◦{G1, G2, . . . , G|V (H)|} é um grafo Zm-bem-coberto. ∈ ∈J (⇐=)Seja I um conjunto independente maximal em H ◦{G1, G2, . . . , G|V (H)|}. Pela Proposição 2.1, XH(I) é um conjunto independente maximal em H e XGv(I) é um conjunto independente maximal em Gv para todo v ∈XH(I). Já que I = S v∈XH(I){(v, x) : x ∈XGv(I)} e |XGv(I)| ≡α(Gv) (mod m), temos |I| = P v∈XH(I) |{[v, x] : x ∈XGv(I)}| = P v∈XH(I) |XGv(I)| = P v∈XH(I) α(Gv). Então, pela condição (2), quaisquer dois conjuntos independentes maximais em H◦{G1, G2, . . . 2. Produto Lexicográfico Propriedades relacionadas a produtos lexicográficos de grafos foram provadas para problemas de coloração [1] e cobertura por ciclos [15]. Alguns resultados sobre produtos de grafos e algumas aplicações podem ser encontradas em [12]. Topp e Volkmann [17] mostram quando o produto lexicográfico de grafos é bem- coberto. Como uma generalização deste resultado, apresentamos uma condição ne- cessária e suficiente para construção de grafos Zm-bem-cobertos a partir do produto lexicográfico de grafos. Dados um grafo H e uma família de grafos não vazios {G1, G2, . . . , G|V (H)|} indexados pelos vértices de H, o produto lexicográfico H◦{G1, G2, . . . , G|V (H)|} de H e {G1, G2, . . . , G|V (H)|} é o grafo tendo o conjunto de vértices S v∈V (H){v}×V (Gv), sendo que dois vértices (v1, v2) e (u1, u2) são adjacentes se {v1, u1} ∈E(H) ou (v1 = u1 e {v2, u2} ∈E(Gvi)). Se todos os grafos da família G são isomorfos entre si, escrevemos o produto lexicográfico entre H e a família G como H ◦G. Um exemplo de produto lexicográfico H ◦{G1, G2, G3} pode ser visto na figura 2, onde H = P3, G1 = K2, G2 = C4 e G3 = C3. 1 2 3 G1 G2 G3 (1, a) (1, b) (3, a) (3, b) (3, c) (2, a) (2, b) (2, c) (2, d) K2 C3 C4 (b) (a) Figura 2: (a) Grafo P3 e (b) Produto lexicográfico P3 ◦{K2, C4, C3} 1 2 3 G1 G2 G3 (1, a) (1, b) (3, a) (3, b) (3, c) (2, a) (2, b) (2, c) (2, d) K2 C3 C4 (b) (a) (a) (b) Figura 2: (a) Grafo P3 e (b) Produto lexicográfico P3 ◦{K2, C4, C3} Para um subconjunto S de V (H ◦{G1, G2, . . . , G|V (H)|}), denotamos XH(S) = {x ∈V (H) : ∃y ∈V (Gx)(x, y) ∈S} e XGx(S) = {y ∈V (Gx) : (x, y) ∈S} para todo x ∈XH(S). No grafo da figura 2 (b), se S = {(1, a), (3, c)}, XH(S) = {1, 3}, XG1(S) = {a} e XG3(S) = {c}. Os conjuntos independentes maximais nos produtos lexicográficos de grafos po- dem ser descritos como na Proposição 2.1. 78 Barbosa e Santana Barbosa e Santana Proposição 2.1. [17] Dados S ⊂V (H ◦(G1, . . . 3. Grafos Zm-bem-cobertos Conforme a Cintura Pinter [16] provou que há somente 4 grafos fortemente bem-cobertos que são pla- nares. Diferentemente, podemos construir infinitos grafos fortemente Zm-bem- cobertos que sejam planares e de cintura 4, para l e m inteiros naturais, m ≥2, 79 Produtos de Grafos Zm-bem-cobertos através do produto (K1,ml+1) ◦2K1. Na figura 3. podemos observar a forma geral destes grafos (a) e em (b) temos um grafo K1,3 ◦2K1 que é fortemente Z2-bem- coberto planar. (b) K1 K1 2ml + 2 . . . (a) (b) Figura 3: (a) Grafo K1,ml+1 ◦2K1 (b) Grafo K1,3 ◦2K1 K1 K1 2ml + 2 . . . (a) (b) (a) Figura 3: (a) Grafo K1,ml+1 ◦2K1 (b) Grafo K1, (b) Grafo K1,3 ◦2K1 Proposição 3.2. Há um número infinito de grafos planares fortemente Zm-bem- cobertos com cintura 4. Proposição 3.2. Há um número infinito de grafos planares fortemente Zm-bem- cobertos com cintura 4. Demonstração. Sejam l e m inteiros naturais, m ≥2. Pela forma de construção, os grafos (K1,ml+1) ◦2K1 são fortemente Zm-bem-cobertos, planares e com cintura 4. Os grafos Zm-bem-cobertos de cintura ≥6 caracterizados no Teorema 1.1, com exceção de K1 e K2, não são 1-Zm-bem-cobertos e nem fortemente Zm-bem- cobertos, e, portanto, os outros grafos com estas propriedades devem ter cintura ≤ 5. Teorema 3.3. K1 e K2 são os únicos grafos conexos fortemente Zm-bem-cobertos com cintura ≥6. Demonstração. Se G é um grafo Zm-bem-coberto com cintura ≥6, G atende ao Teorema 1.1. C7 não é um grafo fortemente Zm-bem-coberto e K1 é. Suponha que G seja diferente de C7 e K1. Logo, G é uma união finita de arbustos. Se G consiste de apenas um talo com uma folha, G ∼= K2 que é um grafo fortemente Zm-bem-coberto. Considere que G é diferente de C7, K1 e K2. Ao removermos uma aresta que une uma folha x a um talo vi, teremos um componente H em G, H = G −{x} com o talo vi e ml folhas, l ≥0, e então H não é Zm-bem-coberto e portanto G também não é. o e Teorema 3.4. K1 e K2 são os únicos grafos conexos 1-Zm-bem-cobertos com cin- tura ≥6. Demonstração. Seja G um grafo Zm-bem-coberto com cintura ≥6. Pelo Teorema 1.1, G é K1, C7 ou uma união finita de arbustos sendo que cada talo tem 1 (mod m) folhas. Por inspeção, C7 não é 1-Zm-bem-coberto e K1 é 1-Zm-bem-coberto. Se G consiste de apenas um talo com uma folha, G ∼= K2 que é um grafo 1-Zm-bem- coberto. Suponha, então, que G é uma união finita de arbustos. Ao removermos 80 Barbosa e Santana uma folha x de um arbusto B qualquer com talo vi, este terá apenas ml folhas l ≥0 e não mais satisfaz ao Teorema 1.1. Portanto, G não é 1-Zm-bem-coberto. 4. Produto Cartesiano O produto cartesiano G1 ×G2 de dois grafos G1 e G2 é o grafo contendo conjunto de vértices V (G1 × G2) = V (G1)× V (G2), e dois vértices (v1, v2) e (u1, u2) de G1 × G2 são adjacentes se ou [(v1, u1) ∈E(G1) e v2 = u2] ou [(v2, u2) ∈E(G2) e v1 = u1]. Na figura 4. temos um grafo C3 × C4. Figura 4: Grafo C3 × C4 Figura 4: Grafo C3 × C4 Figura 4: Grafo C3 × C4 Uma questão sobre o produto cartesiano de dois grafos G e H é saber se é possí- vel que G × H seja bem-coberto quando G e H não são bem-cobertos. Fradkin [11] respondeu parcialmente esta questão para uma grande classe de grafos que inclui todos os grafos não-bem-cobertos livres de triângulo. Topp e Volkman [17] apre- sentaram alguns resultados sobre o produto cartesiano de alguns grafos, incluindo os grafos bipartidos e ciclos. Teorema 4.5. [17] O produto cartesiano G1 × G2 de grafos G1 e G2 bipartidos diferentes de K1 é bem-coberto se e somente se G1 = G2 = K2. Proposição 4.3. [17] O produto cartesiano Cn×Ck de ciclos Cn e Ck é bem-coberto se e somente se n = 3 ou k = 3. O produto cartesiano de um ciclo Cn × K2 é um grafo cúbico que é Z2-bem- coberto e todos os seus conjuntos independentes maximais têm cardinalidade par. Os grafos cúbicos Zm-bem-cobertos foram caracterizados por Barbosa e Ellingham [6]. [ ] Na Observação 1, temos uma forma de obter um conjunto independente maximal em um grafo Cn × Ck. Observação 1. [17] Sejam Cn e Ck dois ciclos com vértices x1, x2, . . . , xn e y1, y2, . . . , yk, respectivamente, e arestas x1x2, x2x3 . . . , xnx1 e y1y2, y2y3 . . . , yky1. Seja In,k o conjunto dos vértices (xi, yj) de Cn × Ck tal que i = 1, . . . , 2 ⌊n/2⌋, j = 1, . . . , 2 ⌊k/2⌋, e i + j é um inteiro par. Se n e k são ambos ímpares, acrescente a In,k o vértice (xn, yk). O conjunto In,k é independente maximal em Cn × Ck. Observação 1. [17] Sejam Cn e Ck dois ciclos com vértices x1, x2, . . . , xn e y1, y2, . 4. Produto Cartesiano Novamente, vamos considerar dois casos. Primeiro, considere que k é ímpar. Podemos verificar que C3 × C3 não é 1-Zm-bem-coberto. Para k ≥5, seja J1 = I3,k\{(x1, y1)} que é maximal em G\{(x1, y1)}. Agora, seja o conjunto J2 = I3,k\{(x1, y1), (x2, y2), (x1, y3)} ∪{(x1, y2), (x2, y1), (x3, y3)}. J2 também é maximal em G\{(x1, y1)}. J1 e J2 tem cardinalidades consecutivas. Considere, agora, que k é par. Sejam J1 = I3,k\{(x1, y1), } ∪{(x3, y1)} e J2 = J1\{(x2, y4), (x3, y1)} ∪{(x3, y4)}. Logo, J2 e J1 são independentes maximais em G\{(x1, y1)}, com cardinalidades consecutivas e, portanto, G não é Zm-bem- coberto. 4. Produto Cartesiano . . , yk, respectivamente, e arestas x1x2, x2x3 . . . , xnx1 e y1y2, y2y3 . . . , yky1. Seja In,k o conjunto dos vértices (xi, yj) de Cn × Ck tal que i = 1, . . . , 2 ⌊n/2⌋, j = 1, . . . , 2 ⌊k/2⌋, e i + j é um inteiro par. Se n e k são ambos ímpares, acrescente a In,k o vértice (xn, yk). O conjunto In,k é independente maximal em Cn × Ck. O produto cartesiano de dois ciclos Cn e Ck é bem-coberto se e somente se n = 3 ou k = 3. Estes grafos possuem tanto C3 quanto C4 induzidos e estão numa 81 Produtos de Grafos Zm-bem-cobertos classe de grafos que ainda não foi caracterizada para grafos bem-cobertos e Zm- bem-cobertos. Este resultado pode ser estendido para os grafos Zm-bem-cobertos, como mostramos na Proposição 4.4. classe de grafos que ainda não foi caracterizada para grafos bem-cobertos e Zm- bem-cobertos. Este resultado pode ser estendido para os grafos Zm-bem-cobertos, como mostramos na Proposição 4.4. Proposição 4.4. O produto cartesiano Cn×Ck de ciclos Cn e Ck é Zm-bem-coberto se e somente se ele é bem-coberto. Demonstração. O produto Cn × Ck, quando n = 3 ou k = 3 é bem-coberto pela Proposição 4.3 e portanto, Zm-bem-coberto. Demonstração. O produto Cn × Ck, quando n = 3 ou k = 3 é bem-coberto pela Proposição 4.3 e portanto, Zm-bem-coberto. É necessário considerar apenas n ou k ímpares, já que, pelo Teorema 4.5, quando os grafos são ambos bipartidos, seu produto cartesiano não é bem-coberto e por- tanto, não é Zm-bem-coberto. Restam, então, dois casos a considerar: se n e k são ímpares e se apenas um deles é ímpar. É necessário considerar apenas n ou k ímpares, já que, pelo Teorema 4.5, quando os grafos são ambos bipartidos, seu produto cartesiano não é bem-coberto e por- tanto, não é Zm-bem-coberto. Restam, então, dois casos a considerar: se n e k são ímpares e se apenas um deles é ímpar. Caso n e k sejam ímpares, podemos encontrar um conjunto independente maxi- mal J1 = In,k\{(x1, y1), (x1, y3), (x2, y2)}∪{(x1, y2), (xn, y3)} em que |J1|=|In,k|−1 e portanto, |J1| e |In,k| não são congruentes módulo m, pois são consecutivos. 4. Produto Cartesiano Caso n e k sejam ímpares, podemos encontrar um conjunto independente maxi- mal J1 = In,k\{(x1, y1), (x1, y3), (x2, y2)}∪{(x1, y2), (xn, y3)} em que |J1|=|In,k|−1 e portanto, |J1| e |In,k| não são congruentes módulo m, pois são consecutivos. , Para o segundo caso, exatamente um de n e k é ímpar. Neste caso, como Cn × Ck é isomorfo a Ck × Cn, vamos assumir que k é ímpar. Sejam J2 = In,k\{(x1, y1), (x1, y3), (x2, y2), (xn, y2)} ∪{(x1, y2), (x1, yk)} e J3 = J2\{(xn−1, y3), (xn, y4)} ∪{(xn, y3)}. Logo, J3 e J2 são independentes maximais em Cn × Ck com cardinalidades consecutivas e, portanto, G não é Zm-bem-coberto. Para o segundo caso, exatamente um de n e k é ímpar. Neste caso, como Cn × Ck é isomorfo a Ck × Cn, vamos assumir que k é ímpar. Sejam J2 = In,k\{(x1, y1), (x1, y3), (x2, y2), (xn, y2)} ∪{(x1, y2), (x1, yk)} e J3 = J2\{(xn−1, y3), (xn, y4)} ∪{(xn, y3)}. Logo, J3 e J2 são independentes maximais em Cn × Ck com cardinalidades consecutivas e, portanto, G não é Zm-bem-coberto. Embora os grafos Cn × Ck Zm-bem-cobertos sejam também bem-cobertos, mos- tramos na Proposição 4.5 que eles não são 1-Zm-bem-cobertos. Embora os grafos Cn × Ck Zm-bem-cobertos sejam também bem-cobertos, mos- tramos na Proposição 4.5 que eles não são 1-Zm-bem-cobertos. Proposição 4.5. Se G é o produto cartesiano Cn × Ck de ciclos Cn e Ck, então G não é 1-Zm-bem-coberto. Demonstração. Pela Proposição 4.4, Cn × Ck é Zm-bem-coberto se e somente se n = 3 ou k = 3. Vamos mostrar que ao removermos um vértice deste grafo, o grafo resultante não é Zm-bem-coberto. Considere o grafo G = C3 × Ck, já que ele é isomorfo a Ck × C3. Remova de G o vértice (x1, y1). Novamente, vamos considerar dois casos. Primeiro, considere que k é ímpar. Podemos verificar que C3 × C3 não é 1-Zm-bem-coberto. Para k ≥5, seja J1 = I3,k\{(x1, y1)} que é maximal em G\{(x1, y1)}. Agora, seja o conjunto J2 = I3,k\{(x1, y1), (x2, y2), (x1, y3)} ∪{(x1, y2), (x2, y1), (x3, y3)}. J2 também é maximal em G\{(x1, y1)}. J1 e J2 tem cardinalidades consecutivas. Considere o grafo G = C3 × Ck, já que ele é isomorfo a Ck × C3. Remova de G o vértice (x1, y1). 5. Considerações Finais They are also the only ones with girth ≥6 and strongly Zm-well-covered. We show a necessary and sufficient condition for the lexicographic product of graphs to be a Zm-well-covered one and some properties for the cartesian product of cycles. Keywords. Graph theory, independent sets in graphs, graph products. Keywords. Graph theory, independent sets in graphs, graph products. 5. Considerações Finais Apresentamos uma forma de construção de grafos Zm-bem-cobertos a partir do produto lexicográfico de um grafo H e uma família de grafos Zm-bem-cobertos. Para os grafos bem-cobertos, o problema foi resolvido por Topp e Volkmann [17]. 82 Barbosa e Santana Há um número finito de grafos planares fortemente bem-cobertos, como provado por Pinter [16]. Porém, mostramos que existe um número infinito de grafos planares que são fortemente Zm-bem-cobertos que tem, ainda, cintura 4 (Proposição 3.2). ( ) Mostramos que um grafo 1-Zm-bem-coberto, diferente de K1 e K2 tem cintura ≤ 5, porém ainda não é conhecida uma caracterização destes grafos. Especificamente para os grafos com cintura 5, o problema é saber se existe um grafo 1-Zm-bem- coberto que não seja 1-bem-coberto. Caso este grafo não exista, vale a conjectura 5.1. Outro problema é saber se há um grafo fortemente Zm-bem-coberto com cintura 5 que não seja fortemente bem-coberto (conjectura 5.1). Conjectura 5.1. Se G é um grafo 1-Zm-bem-coberto com cintura 5, então G é 1-bem-coberto. Conjectura 5.1. Se G é um grafo fortemente Zm-bem-coberto com cintura 5, então G é fortemente bem-coberto. Se G é o produto cartesiano de dois ciclos Cn e Ck, mostramos que G é Zm-bem- coberto se e somente se G é bem-coberto. Estes grafos não são 1-Zm-bem-cobertos. Abstract. A graph is Zm-well-covered if |I| ≡|J| (mod m), for all I, J maximal independent sets in V (G). A graph G is strongly Zm-well-covered if G is a Zm- well-covered graph and G\{e} is Zm-well-covered, ∀e ∈E(G). A graph G is 1-Zm- well-covered if G is Zm-well-covered and G\{v} is Zm-well-covered, ∀v ∈V (G). We prove that K1 and K2 are the only 1-Zm-well-covered graphs with girth ≥6. They are also the only ones with girth ≥6 and strongly Zm-well-covered. We show a necessary and sufficient condition for the lexicographic product of graphs to be a Zm-well-covered one and some properties for the cartesian product of cycles. Abstract. A graph is Zm-well-covered if |I| ≡|J| (mod m), for all I, J maximal independent sets in V (G). A graph G is strongly Zm-well-covered if G is a Zm- well-covered graph and G\{e} is Zm-well-covered, ∀e ∈E(G). A graph G is 1-Zm- well-covered if G is Zm-well-covered and G\{v} is Zm-well-covered, ∀v ∈V (G). We prove that K1 and K2 are the only 1-Zm-well-covered graphs with girth ≥6. Referências [1] M. Asté, F. Havet, C.L. Sales, Grundy number and products of graphs, Discrete Mathematics, 310 (2010), 1482–1490. [2] R.M. Barbosa, On 1-Zm-well-covered graphs and strongly Zm-well-covered graphs, Ars Combinatoria, 57 (2000), 225–232. [3] R.M. Barbosa, “Sobre Conjuntos Independentes Maximais em Grafos”, Tese de Doutorado, COPPE-UFRJ, 1999. [4] R.M. Barbosa, M.N. Ellingham, A characterisation of cubic parity graphs, Australasian Journal of Combinatorics, 28 (2003), 273–293. [5] R.M. Barbosa, B. Hartnell, Almost parity graphs and claw-free parity graphs, J. Combin. Math. Combin. Comput., 27 (1998), 117–122. [6] R.M. Barbosa, B. Hartnell, Characterization of Zm-well-covered graphs for some classes of graphs, Discrete Mathematics, 233 (2001), 293–297. Produtos de Grafos Zm-bem-cobertos 83 [7] J.A. Bondy, U.S.R. Murty, “Graph Theory”, Graduate Texts in Mathematics, Springer, 2008. [8] Y. Caro, Subdivisions, parity and well-covered graphs, J. Graph Theory, 25 (1997), 85–94. [9] Y. Caro, M. Ellingham, J. Ramey, Local structure when all maximal inde- pendent sets have equal weight, SIAM J. Discrete Mathematics, 11 (1998), 644–654. [10] Y. Caro, B. Hartnell, A Characterization of Zm-well-covered graphs of girth 6 or more, J. Graph Theory, 33 (2000), 246–255. [11] A.O. Fradkin, On the well-coveredness of Cartesian products of graphs, Dis- crete Mathematics, 309 (2009), 238–246. [12] R. Hammack, W. Imrich, S. Klavzar “Handbook of Product Graphs”, Second Edition, CRC Press, 2011. [13] B. Hartnell, Well-covered graphs, J. Combin. Math. Combin. Comput., 29 (1999), 107–115. [14] R.M. Karp, Reducibility among combinatorial problems, em “Complexity of Computer Computations” (Yorktown Heights), pp. 85-104, Nova York, 1972. [15] R.J. Nowakowski, K. Seyffarth, Small cycle double covers of products I: Lexi- cographic product with paths and cycles, J. Graph Theory, 57 (2008), 99–123. [16] M.R. Pinter, Strongly well-covered graphs, Discrete Mathematics, 132 (1994), 231–246. [17] J. Topp, L. Volkmann, On the well coveredness of Products of Graphs, Ars Combinatoria, 33 (1992), 199–215. 84
https://openalex.org/W3006761556
https://europepmc.org/articles/pmc7214483?pdf=render
English
null
The use of endoluminal techniques in the revision of primary bariatric surgery procedures: a systematic review
Surgical endoscopy/Surgical endoscopy and other interventional techniques
2,020
cc-by
10,114
The use of endoluminal techniques in the revision of primary bariatric surgery procedures: a systematic review Yan Mei Goh1,2 · Nicole Ellen James1 · En Lin Goh1,3 · Achal Khanna2 Received: 16 November 2019 / Accepted: 19 February 2020 / Published online: 28 February 2020 © The Author(s) 2020, corrected publication 2020 * Yan Mei Goh yanmei.goh@doctors.org.uk and Other Intervent and Other Intervent Surgical Endoscopy (2020) 34:2410–2428 https://doi.org/10.1007/s00464-020-07468-w REVIEW ARTICLE Abstract Background  Weight regain following primary bariatric surgery is attributed to anatomical, behavioural and hormonal fac- tors. Dilation of the gastrojejunal anastomosis is a possible cause of weight regain after roux-en-Y gastric bypass (RYGB). However, surgical revision has significant risks with limited benefits. Endoluminal procedures have been suggested to manage weight regain post-surgery. This systematic review aims to assess efficacy of endoluminal procedures. Methods Studies where endoluminal procedures were performed following primary bariatric surgery were identified Main Background  Weight regain following primary bariatric surgery is attributed to anatomical, behavioural and hormonal fac- tors. Dilation of the gastrojejunal anastomosis is a possible cause of weight regain after roux-en-Y gastric bypass (RYGB). However, surgical revision has significant risks with limited benefits. Endoluminal procedures have been suggested to manage weight regain post-surgery. This systematic review aims to assess efficacy of endoluminal procedures. Methods  Studies where endoluminal procedures were performed following primary bariatric surgery were identified. Main outcome measures were mean weight loss pre- and post-procedure, excess weight loss, recurrence rates, success rates and post procedure complications fi Methods  Studies where endoluminal procedures were performed following primary bariatric surgery were identified. Main outcome measures were mean weight loss pre- and post-procedure, excess weight loss, recurrence rates, success rates and post-procedure complications.i Results  Twenty-six studies were included in this review. Procedures identified were (i) endoluminal plication devices (ii) other techniques e.g. sclerotherapy, mucosal ablation, and Argon Plasma Coagulation (APC) and (iii) combination therapy involving sclerotherapy/mucosal ablation/APC and endoscopic OverStitch device. Endoluminal plication devices show great- est initial weight loss within 12 months post-procedure, but not sustained at 18 months. Only one study utilising sclerotherapy showed greater sustained weight loss with peak EWL (19.9%) at 18 months follow-up. Combination therapy showed the greatest sustained EWL (36.4%) at 18 months. Endoluminal plication devices were more successfully performed in 91.8% of patients and had lower recurrence rates (5.02%) compared to sclerotherapy and APC, with 46.8% success and 21.5% recurrence rates. Both procedures demonstrate no major complications and low rates of moderate complications. Only mild complications were noted for combination therapy.fi Conclusions  The paucity of good quality data limits our ability to demonstrate and support the long-term efficacy of endo- luminal techniques in the management of weight regain following primary bariatric surgery. Future work is necessary to not only clarify the role of endoluminal plication devices, but also combination therapy in the management of weight regain following primary bariatric surgery. 2 Department of General Surgery, Milton Keynes University Hospital, Milton Keynes, UK 1 Imperial College London, London, UK 3 Oxford University Hospitals NHS Foundation Trust, Oxford, UK Abstract Non-surgical management of weight regain following bariatric surgery requires the input of the multidisciplinary team. Despite this, a proportion of patients still experience weight regain following bariatric surgery [9]. Dilatation of the gastrojejunal anastomosis or the gastric pouch is a well-recognised post-operative occur- rence. On the basis that gastric pouch size, distension and transit time following RYGB is a surgical mechanism for early satiety and weight loss, this post-operative event may reduce the restrictive and malabsorptive effects of RYGB. Surgical revision of the gastrojejunal anastomosis is contro- versial as most patients are exposed to major post-operative complications, higher readmission rates and morbidity [9] but do not achieve significant weight reduction [10, 11]. Data extraction and outcome measures Two independent reviewers (YMG, NEJ) screened all titles and abstract manually for inclusion. A third reviewer (ELG) was consulted in the case of a disagreement. Relevant data were entered into Review manager 5.4 (Cochrane Collabo- ration, Oxford, United Kingdom). The following data items were extracted: year of publication, country of origin, study design, number of participants, type of primary procedure, type of endoluminal procedure performed, patient demo- graphics, mean time since initial procedure, selection crite- ria in each study, mean pre-revision weight and BMI, mean weight loss post-procedure, complications post-procedure, average length of procedure, average stoma diameter at the end of the procedure, excess weight loss, length of follow-up and number of successful endotherapy. Literature search The following databases were searched: (a) Medline (1946—present) via OvidSP, (b) MEDLINE Epub ahead of print, in process and other non-indexed citations (latest issue via Ovid SP, last search 19th July 2019); (c) Ovid Embase (1947—19th July 2019). Additionally, all references of included articles were manually reviewed to identify addi- tional studies. Three strings were utilised; these terms were “bariatric surg*.mp. OR metabolic surg*.mp. OR weight loss surg*.mp.”, “revision*”.mp., “endoscopic procedure. mp. OR endosco*.mp.” and truncated search terms using wild card character and “related articles” function were used to broaden search. The references of included articles were also hand-searched to identify any additional studies. i Hence, endoluminal revision procedures have been devel- oped to address this gap. These techniques come in various forms: endoluminal plication devices and other techniques like sclerotherapy, mucosal ablation and argon plasma coagulation. Endoluminal plication devices work by taking superficial or full-thickness bites of the intraluminal pouch mucosa or at the gastrojejunal anastomosis. Sutures or clips are then deployed via endoscope. Meanwhile, other tech- niques like sclerotherapy, mucosal ablation and APC induce scarring at the gastrojejunal anastomosis thus reducing its size. As the number of patients undergoing bariatric surgery continues to grow, the need to consider endoluminal revision procedures becomes increasingly important. Thus, this sys- tematic review aims to assess efficacy of endoluminal tech- niques that attempt to revise primary bariatric procedures. Criteria for considering studies for this review Studies were appraised for rigorousness in methodology using the Newcastle–Ottawa Quality Assessment Scale [12] and risk of bias assessed using the National Institute of Health (NIH) Quality Assessment Tool for Case Series Studies [13]. All published studies that utilised endoluminal or endo- scopic techniques following primary bariatric surgery were evaluated. Inclusion criteria are as follows: (a) studies inves- tigating patients who had undergone endoscopic procedures following a primary bariatric surgery procedure (b) weight regain after surgery (c) presence or recurrence of comor- bidities (d) post-operative complications (e) presence of anatomical cause for weight regain. Exclusion criteria are as follows: studies that did not include revision surgery, endoluminal procedures used in the management of com- plications following primary surgery, articles that assess primary bariatric surgery, non-endoluminal interventions, review articles, studies not written in the English language, animal studies, comment, opinions or letters, case reports Abstract Keywords  Endoluminal techniques · Revision surgery · Bariatric surgery The role of bariatric surgery has grown significantly over the past decade, with an additional 10,000 procedures performed per year from 2011 to 2015, and an increase of 20,000 proce- dures from 2015 to 2016 in the USA [1]. In particular, gastric bypass, sleeve gastrectomy, adjustable gastric banding and biliopancreatic diversion with duodenal switch are frequently performed. These procedures are associated with significant long-term weight loss as well as alterations in gut hormone production and metabolism that suppress appetite and pro- mote satiety. However, weight regain following primary bari- atric surgery remains an ongoing problem. It is estimated that clinically significant weight regain occurs in up to one-third of patients [2–6] who have undergone a Roux-en-Y gastric bypass (RYGB) or vertical banded gastroplasty (VBG). Electronic supplementary material  The online version of this article (https​://doi.org/10.1007/s0046​4-020-07468​-w) contains supplementary material, which is available to authorized users. There are several factors that weight regain post-RYGB can be attributed to; notably a combination of lifestyle, 3 Oxford University Hospitals NHS Foundation Trust, Oxford, UK :.(123456789 3 Surgical Endoscopy (2020) 34:2410–2428 2411 and technical articles with no evidence of patient follow-up post-procedure, and conference abstracts. mental health, hormonal/metabolic and surgical factors. Thus, the need to understand and address these issues with patients in the pre- and post-operative stage is crucial in pre- venting the reemergence of obesity related comorbidities and impaired quality of life [7, 8]. Non-surgical management of weight regain following bariatric surgery requires the input of the multidisciplinary team. Despite this, a proportion of patients still experience weight regain following bariatric surgery [9]. Dilatation of the gastrojejunal anastomosis or the gastric pouch is a well-recognised post-operative occur- rence. On the basis that gastric pouch size, distension and transit time following RYGB is a surgical mechanism for early satiety and weight loss, this post-operative event may reduce the restrictive and malabsorptive effects of RYGB. Surgical revision of the gastrojejunal anastomosis is contro- versial as most patients are exposed to major post-operative complications, higher readmission rates and morbidity [9] but do not achieve significant weight reduction [10, 11]. mental health, hormonal/metabolic and surgical factors. Thus, the need to understand and address these issues with patients in the pre- and post-operative stage is crucial in pre- venting the reemergence of obesity related comorbidities and impaired quality of life [7, 8]. Results Twenty-six studies comprising a total of 1835 patients who had undergone endoluminal procedure following initial pri- mary bariatric procedure were included in this study (Fig. 1). Endoluminal plication devices were used in 1087 patients, other techniques in 721 patients, and a combination of the two types of procedures in 27 patients. All studies were 1 3 1 3 3 2412 Surgical Endoscopy (2020) 34:2410–2428 published over a period of twelve years from 2007 to 2019. There were eight prospective case series and one prospec- tive multicentre randomised control trial. Of the 26 studies, 19 were performed in USA, one in Brazil, two in centres located in USA and Brazil, one in Belgium, one in France and one in Canada. Mean age of patients included in the review was 51.5 years old (range 22.0–71.4 years). The mean time since initial bariatric procedure was 86.7 months (range 12.0–222 months) (Table 1).i • BMI 30–60 kg/m2 greater than six months after RYGB [21] published over a period of twelve years from 2007 to 2019. There were eight prospective case series and one prospec- tive multicentre randomised control trial. Of the 26 studies, 19 were performed in USA, one in Brazil, two in centres located in USA and Brazil, one in Belgium, one in France and one in Canada. Mean age of patients included in the review was 51.5 years old (range 22.0–71.4 years). The mean time since initial bariatric procedure was 86.7 months (range 12.0–222 months) (Table 1). • BMI 30–60 kg/m2 greater than six months after RYGB [21] • BMI 30–60 kg/m2 greater than six months after RYGB [21] One study [38] did not detail the inclusion nor exclusion criteria in patient selection. Endoluminal bariatric procedures The endoluminal procedures identified were (i) endolumi- nal plication devices e.g. StomaphyX™, Restorative Obesity Surgery Endoluminal (ROSE) procedure, Incisionless Oper- ating Platform (IOP), Over-The-Scope Clip (OTSC-Clip), e.g. sutured Transoral Outlet Reduction (TORe), Endoscopic Overstitch device and Endoscopic Gastrojejunal Revision (EJGR) and (ii) other techniques e.g. sclerotherapy, mucosal ablation, and Argon Plasma Coagulation (APC). Initial bariatric procedures performed were the roux-en-Y gastric bypass (RYGB), transected vertical gastric bypass (TVGB), vertical banded gastroplasty (VBG) and laparoscopic sleeve gastrectomy (LSG) (Tables 1,2). Twenty-five of the 26 included studies had clear selection criteria for all patients included in their study. These are as follows: • Greater than 18 months following initial bariatric proce- dure [14–20]fi • Weight regain or failure to lose sufficient weight [17, 19–35] • Aged between 18 to 65 years old [16, 18, 32] • Decreased satiety [19, 29, 30] • Dilated gastrojejunal anastomosis and gastric pouch [19–21, 25, 30, 32, 33, 36, 37] • Reappearing comorbidities [27] Fig. 1   PRISMA chart of the study selection process Records idenfied through EMBASE (n = 486) Screening Included Eligibility Idenficaon Records aer duplicates removed (n = 511) Records screened (n = 511) Records excluded: Non-English (n = 7) Conference abstracts (n = 156) Reviews (n = 51) Arcles assessing primary bariatric surgery (n = 64) Non-endoluminal revision surgery (n = 103) Management of complicaons Full-text arcles assessed for eligibility (n = 34) Full-text arcles excluded due to duplicate paent cohort or on further assessment did not meet inclusion criteria (n = 8) Studies included in qualitave synthesis (n = 26) Records idenfied through Ovid Medline (n = 128) Fig. 1   PRISMA chart of the study selection process Idenficaon Records idenfied through Ovid Medline (n = 128) Records idenfied through EMBASE (n = 486) Records aer duplicates removed (n = 511) Records excluded: Non-English (n = 7) Conference abstracts (n = 156) Reviews (n = 51) Arcles assessing primary bariatric surgery (n = 64) Non-endoluminal revision surgery (n = 103) Management of complicaons Full-text arcles assessed for eligibility (n = 34) Studies included in qualitave synthesis (n = 26) 1 3 Surgical Endoscopy (2020) 34:2410–2428 2413 1 Table 1   Patient and study demographics Study Country Study type No Primary Operation Procedure M:F Mean age (years) Mean time since initial procedure (months) Selection criteria Mean pre- revision weight (kg) Mean pre- revision BMI (kg/m2) Mikami et al. Endoluminal bariatric procedures [14] USA Retro case series 39 RYGB StomaphyX™3:36 47.8 (29–64)  > 2 years post- op > 10% of nadir weight 108 (65.9– 172.2) 39.8 (22.7– 63.2) Manouchehri et al. [26] Canada Pros case series 14 Vertical Banded Gastro- plasty StomaphyX™1:13 47.3 ± 7.9 116.4 ± 73.2 Persistent WG 119.5 ± 25.9 43.4 ± 9.7 Ong’Uti et al. [15] USA Retro case series 27 RYGB StomaphyX™2:25 49 (44–54) 72 (60–96)  > 2 years post-op 103 (88.5– 115)** 37 (32–40) Goyal et al. (39) USA Retro case series 55 RYGB StomaphyX™1:53 49.6 (30–68) 68.4 (12–156) 96.6 36.1 Mullady et al. [29] USA Pros case series 20 RYGB ROSE 1:19 48 (36–62) 63 (24–117) WR/ no WL, satiety 36.7 (28,4– 48.8) Horgan et al. [18] USA Pros case series 116 RYGB ROSE via Incisionless Operating Platform (IOP) 15:101 45.6 ± 8.7  > 2yrs post- op, > 50% EWL after RYGB 110.8 ± 20.5 39.9 ± 6.7 Ryou et al. [30] USA Pros case series 5 RYGB ROSE 0:5 48 (41–55) 56 (24–76) WR, satiety, dilated pouch/ GJA 100.4 36.3 Gallo et al. [17] USA Retro case series 27 RYGB ROSE 2:25 49.2 ± 9.6 (26–68) 142.8 ± 51.6  > 50% EWL, sig WG 2 years post-op 106.2 ± 21.2** 40.6 (30–67) Buttelmann et al. [31] USA Retro case series 8 RYGB ROSE 48 Inadequate/ failure to lose weight 43.7 Thompson et al. [18] USA Retro case series 116 RYGB IOP 15:101 46 ± 9  > 2yrs after RYGB 110.5 ± 20.5 39.9 ± 6.7 Heylen et al. [27] Belgium Pros case series 46 TVGB OTSC-clip 19:75  > 10% WG 2yrs post-op, reappearing comorbidi- ties, volume/ frequency of meals 32.8 1 Surgical Endoscopy (2020) 34:2410–2428 2414 414 Surgical Endoscopy (2020) 34:2410 2 1 3 Table 1   (continued) Study Country Study type No Primary Operation Procedure M:F Mean age (years) Mean time since initial procedure (months) Selection criteria Mean pre- revision weight (kg) Mean pre- revision BMI (kg/m2) Patel et al. [32] USA Retro case series 50 RYGB EGJR IST- 2:32 IST 48.6 ± 10.3 IST 115.2 ± 39.6 WR > 2yrs, stoma dila- tion > 15 mm IST 114.5 ± 20.5 IST 41/7 ± 6/4 PST- 2:14 PST 55.8 ± 10.8 PST 114 ± 42 PST 110.2 ± 22.6 PST 40.7 ± 8.7 Tsai et al. Endoluminal bariatric procedures [22] Switzerland Retro case series 81 RYGB EGJR (OverStitch device) 22:59 48.0 (26.8– 71.4) 84 (12–222)  > 15 kg increase from nadir weight, 10 kg increase within 6–12 months post-op 127.1 (96–225) 44.7 (35.3–67) Catalano et al. [33] USA Retro case series 28 RYGB Sclerotherapy (sodium morrhuate) 10:18 41.1 (27–58) Stoma size > 1.2 cm, WR after RYGB 112 Loewen and Barba [34] USA Retro case series 71 RYGB Sclerotherapy (sodium morrhuate) 4:67 45 (30–64) 34.8 (34.8–66  > 5% WG, inadequate WL < 50% EWL 98.1 ± 21.6** 35.5 Jirapinyo et al. [24] USA Pros case series 43 RYGB Sclerotherapy (sodium morrhuate) (34) 3:31 47 ± 9 72 ± 60  > 1 yr post- op > 20% of nadir weight TORe (9) 1:8 47 ± 13 84 ± 48 Thompson et al. [21] USA Pros, multi- centre RCT​ 50 RYGB TORe 3:47 47.6 ± 9.46 58 BMI 30–60 at > 6 months post-op, Inadequate WL > 50% EWL/ WR > 5% EWL, GJA > 2 cm 101.5 ± 16.4 37.6 ± 4.9 de Moura et al. [39] USA Retro case report 1 RYGB TORe 0:1 55 144 73.35** 27.9 Kumar and Thompson [37] USA Retro case series 59 RYGB ST TORe 3:56 48.8 ± 1.1 Stoma diam- eter > 20 mm 40.4 ± 1.0 59 FT TORe 15:44 49.9 ± 1.3 41.1 ± 1.3 Kumar and Thompson [36] USA Pros case series 150 RYGB TORe (OverStitch device) 27:123 51.2 ± 0.8 103.2 ± 3.6 GJA > 15 mm 110.7 ± 2.2 40.1 ± 0.7 Surgical Endoscopy (2020) 34:2410–2428 2415 1 3 Table 1   (continued) Study Country Study type No Primary Operation Procedure M:F Mean age (years) Mean time since initial procedure (months) Selection criteria Mean pre- revision weight (kg) Mean pre- revision BMI (kg/m2) Jirapinyo et al. [25] USA Retro case series 25 RYGB TORe (OverStitch device) 7:18 48 (34–69) 72 (24–120) WR, GJA > 15 mm 43 Vargas et al. [23] USA, Brazil Retro case series 130 RYGB TORe (OverStitch device) 16:114 47.12 ± 8.55 100.8 ± 57.4 WR 36.8 ± 6.84 Baretta et al. [20] Brazil Pros case series 30 RYGB APC 4:26 42.83 (22–59)  > 18 months post-op, regain of > 10% of nadir weight, stoma diam- eter > 15 mm 121.77 ± 22.50 45.63 ± 7.63 Moon et al. Endoluminal bariatric procedures [19] USA, Brazil Retro case series 558 RYGB APC 103:455 40.9 ± 9.5 90 (60, 120)*  > 18 months post-op, regain of > 10% of nadir weight, satiety, size of GJ stoma > 15 mm 94.5 ± 18.6 34.0 ± 5.7 Riva et al. [35] France Retro case series 22 RYGB Mucosal abla- tion + endo- scopic suturing (OverStitch device) (11) 5:17 52.2 ± 11.7 106.8 ± 99.6 Sig WG > 50% 104.3 ± 27.4 42.4 ± 9.05 Mucosal abla- tion + endo- scopic sutur- ing + scle- rotherapy (OverStitch device) (11) 100.3 ± 27.0 42.4 ± 10.4 Eid [28] USA Retro case series 5 LSG APC + endo- scopic suturing (OverStitch device) 4:1 59.2 (48–63) 37.4 (32.2– 48.2) WR 110.25 (85.05– 130.50)** 37 1 3 Surgical Endoscopy (2020) 34:2410–2428 2416 416 Surgical Endoscopy (2020) 34:2410–2428 ab e (co t ued) Study Mean post-surgical weight (kg) Mean post-revision BMI (kg/m2) Complication post-procedure 3 months 6 months 1 year 2 years 3 years 3 months 6 months 1 year 2 years 3 years Mikami et al. [14] 101.3 99.3 98 Minor: sore throat (87.1), epigastric pain (76.9) Manouchehri et al. [26] 109.6 ± 24.4 (4 months) 39.8 ± 9.1 (4 months) Minor headache, back pain Ong’Uti et al. [15] 101.3 94.5 93.9 (81.6 ± 102)** 33 (29–36) (0 months) Goyal et al. (39) 92.9 (1 month) 92.8 94.9 Nil Mullady et al. [29] Minor: abdomi- nal bloating, mild sore throats Horgan et al. [18] 103 Mild: pharyh- gitis 48 (41), Nausea/vomit- ing 14 (12), Abdo pain 13 (11) Moderate: superficial dis- tal oesophagus tear 3(2.9) Ryou et al. [30] 92.6 33.4 Nil Gallo et al. [17] 39.2 ± 7 39.9 ± 10.1 37.7 ± 6.3 Nil Buttelmann et al. [31] 40.6 40.7 39 38.9 Nil Thompson et al. [18] 104.6 Heylen et al. [27] 29.7 27.4 Mild: sore throat Moder- ate: 5(10.9) dysphagia (repeat OGD), 2 persistent dysphagia had endoscopic dilatation) 416 Surgical Endoscopy (2020) 3 ( ) Study Mean post-surgical weight (kg) Mean post-revision BMI (kg/m2) Complication post-procedure 3 months 6 months 1 year 2 years 3 years 3 months 6 months 1 year 2 years 3 years Mikami et al. [14] 101.3 99.3 98 Minor: sore throat (87.1), epigastric pain (76.9) Manouchehri et al. [26] 109.6 ± 24.4 (4 months) 39.8 ± 9.1 (4 months) Minor headache, back pain Ong’Uti et al. Endoluminal bariatric procedures [15] 101.3 94.5 93.9 (81.6 ± 102)** 33 (29–36) (0 months) Goyal et al. (39) 92.9 (1 month) 92.8 94.9 Nil Mullady et al. [29] Minor: abdomi- nal bloating, mild sore throats Horgan et al. [18] 103 Mild: pharyh- gitis 48 (41), Nausea/vomit- ing 14 (12), Abdo pain 13 (11) Moderate: superficial dis- tal oesophagus tear 3(2.9) Ryou et al. [30] 92.6 33.4 Nil Gallo et al. [17] 39.2 ± 7 39.9 ± 10.1 37.7 ± 6.3 Nil Buttelmann et al. [31] 40.6 40.7 39 38.9 Nil Thompson et al. [18] 104.6 Heylen et al. [27] 29.7 27.4 Mild: sore throat Moder- ate: 5(10.9) dysphagia (repeat OGD), 2 persistent d h i h d 104.6 Ryou et al. [30] 92.6 Gallo et al. [17] Buttelmann et al. [31] Thompson et al. [18] Heylen et al. [27] Ryou et al. [30] 92.6 Gallo et al. [17] Buttelmann et al. [31] Thompson et al. [18] Heylen et al. [27] 3 2417 Surgical Endoscopy (2020) 34:2410–2428 urgical Endoscopy (2020) 34:2410–2428 ued) Mean post-surgical weight (kg) Mean post-revision BMI (kg/m2) Complication post-procedure 3 months 6 months 1 year 2 years 3 years 3 months 6 months 1 year 2 years 3 years 122.6 121.1 119.1 Nil 92.1 (18 month) Mild: 21 (75) post-injection pain. Complications Six studies reported no complications following proce- dures involving endoluminal plication devices [17, 22, 30, 31, 38, 39]. Minor complications reported were abdominal pain (22.5%), sore throat (49.4%), device failure (3.1%), nausea and vomiting (11.0%). A greater range of moderate complications was reported. Specifically, 9.02% of patients reported moderate complications of mucosal tear or dam- age, 4% reported haematemesis, 2.7% reported bleeding and 10.9% had dysphagia following endoluminal plication. No major complications were reported by any study utilising endoluminal plication devices. There were two studies in which endoluminal procedures were performed in patients following VBG and TVGB, respectively [26, 27]. One study utilised the StomaphyX™ for revision of VBG [26]. The authors demonstrated a weight reduction of 9.9 kg at four months post-revisional procedure, with a decrease in BMI of 3.6 kg/m2 (8.28% weight loss) over the same time period [26]. The other study reported a mean decrease in BMI of 3.1 kg/m2 (9.45% weight loss) fol- lowing the use of the OTSC-clip at 3 months post-revisional procedure in a group of TVGB patients [27]. This was sus- tained at 7.01% at 12 months post-revisional procedure. On review of both papers, neither study had reported the EWL following endoluminal revision surgery. Studies utilising other techniques of sclerotherapy and APC reported minor complication of post-injection pain, abdominal pain and nausea (60%) and moderate complica- tion of mucosal ulceration (35.7%). No major complications were reported. 0 4 8 12 16 20 24 28 32 36 40 0 5 10 15 20 25 30 35 40 Months % Excess Weight Loss Endoluminal Plication Device Sclerotherapy and Argon Plasma Coagulation Fig. 2   Graph demonstrating percentage EWL over time in endolumi- nal plication devices and others (sclerotherapy and APC) All endoluminal plication devices post-RYGB showed a mean overall decrease in EWL over the first three months of 13.9% [14–18, 21–25, 29–32, 36–39]. This EWL was sustained at 13.7% at the 12-month follow-up (Fig. 2). Fol- lowing this, the percentage EWL after 12 months post-pro- cedure is demonstrated to show a steady decline to 8.5% 36 months post-procedure. Endoluminal plication devices were shown to be successful in 91.8% of patients in studies which provided data. Definitions of success in the various procedures are outlined where data are available (Table 2). Weight loss Meanwhile, the other techniques used post-RYGB i.e. sclerotherapy and APC showed a much lower weight loss compared to endoscopic plication devices with a 3.87% EWL three months post-procedure [19, 20, 24, 33, 34]. A 19.9% EWL at 18 months post-procedure is reported in Catalano et al.’s study utilising sclerotherapy [33], which is greater than the EWL (13.0%) in endoluminal plication devices. Sclerotherapy and APC were shown to be less suc- cessful in 46.8% of patients when compared to utilisation of endoluminal plication devices (91.8%), and had higher recurrence rates (21.5%). Excess weight is defined as the difference between the patient’s actual weight and ideal weight. The percentage excess weight loss (EWL) is defined as the proportion of weight loss after endoluminal procedures divided by the dif- ference of regained weight from nadir weight. Revision surgery using endoluminal plication devices were performed in 18 studies post-RYGB [14–18, 21–25, 29–32, 36–39]. Results of these studies were analysed together. Of these, Stomaphyx™ was performed in three studies [14, 15, 38], ROSE in five studies [16, 17, 29–31], IOP in one study [18], TORe in seven studies [21, 23–25, 36, 39] and EJGR in two studies [22, 32]. These procedures were performed a mean of 91.2 months (12.0–222 months) after RYGB. Mean pre-revision weight was 105.6  kg (65.9–225 kg). Mean weight loss (6.27 kg) was greatest within the first 3 months post-procedure. This weight loss was sustained for up to two years after the revision endolu- minal procedure. Post-procedure BMI within the first three months after the revision procedure had decreased by a mean of 7.61%, but there are insufficient data to comment on mean post-procedure BMI after two years. Mean EWL was sus- tained at 19.3% six months following the initial procedure. However, this was not maintained in patients two years post- procedure (EWL 10.3%). Endoluminal bariatric procedures Moderate: 10 (35.7) shal- low circumfer- ential ulcers at stoma Pain 1, heartburn 1, hypertensive urgency 1, bleeding 1 Pain 2 95.1 ± 15.22 Mod: gastric mucosal tear, pulmonary oedema 55.8** 49.5** 21.2** 18.8** Nil Bleeding 1 Bleeding 1 101.1 100.1 100.2 90.7 91.5 36.6 36.3 36.3 36.8 36.7 Pain 6 (4.0), bleeding 5 (3.3), nausea 3 (2.0) Hematemesis 1, delayed GI bleeding 1, nausea 4, severe emesis with torn stitches 2, ste- nosis of GJA 1 36.7 1 Surgical Endoscopy (2020) 34:2410–2428 2418 g ) Mean post-surgical weight (kg) Mean post-revision BMI (kg/m2) Complication post-procedure 3 months 6 months 1 year 2 years 3 years 3 months 6 months 1 year 2 years 3 years Nausea 18 (14), Pain 23 (18), Oesophageal tear requiring endoscopic clipping 1 (< 1), balloon dilation of narrowed GJA after TORe 5 (4) 83.29 (4 months) 78.87 31.14 ± 5.81 Severe stenosis (stoma diam- eter < 3 mm) 2, ulcers at stoma 10 Stenosis 9, GJ ulcer 3, vomiting 3, GJ leakage 2, melena 1 36 34 Minor: nausea and mild abdominal pain (44) 100.08 (75.15– 121.5)** 98.1 (72.9– 119.25)** 99.09 (74.25– 119.7)** 33.64 (26.7–44.9) value, interquartile range, ** conversion from lb to kg (1 lb = 0.45) ight loss, ROSE restorative obesity surgery, endoluminal (ROSE) procedure, EGJR endoscopic gastrojejunal revision, RYGB Roux-en-Y gastric bypass, LSG laparoscopic sleeve TSC-clip over-the-scope clip, IOP incisionless operating platform, TORe sutured transoral outlet reduction, ST superficial-thickness, FT full-thickness, APC argon plasma coagu- spective, retro: retrospective, GJA gastrojejunal anastomosis, WR weight regain, WL weight loss, WG weight gain, EWL excess weight loss Study Mean post-surgical weight (kg) Mean post-revision BMI (kg/m2) Complication post-procedure 3 months 6 months 1 year 2 years 3 years 3 months 6 months 1 year 2 years 3 years 31.14 ± 5.81 1 3 Surgical Endoscopy (2020) 34:2410–2428 2419 30, 38, 39]. Recurrence rates and need for further procedure following endoluminal plication devices were 5.02%. Complications These include the ability to reduce the diameter of the gas- trojejunal stoma and pouch length [16, 18, 21, 23, 25, 29, 30, 38], as well as weight loss post-procedure [16, 18, 29, Endoluminal Plication Device Fig. 2   Graph demonstrating percentage EWL over time in endolumi- nal plication devices and others (sclerotherapy and APC) 1 3 Surgical Endoscopy (2020) 34:2410–2428 2420 1 3 yp ( ) Study Procedure Combination therapy (Y/N) Av. procedure length (mins) Av. stoma diameter at end of procedure (mm) % Excess weight loss 1 week 1 month 2 months 3 months 6 months 12 months Mikami [14] StomaphyX™ N 35 (16–62) (2 weeks) 7.4 10.6 13.1 13.1 17.0 19.5 Ong-Uti [15] StomaphyX™ N 20 (20–30) (2 weeks) 24 33 47 20 Goyal [38] StomaphyX™ N 24.1 (10–55) 12.8 7.3 ± 7.1 11.6 ± 12.1 11.5 (17.9) Mullady et al. [29] ROSE N 103 (50–154) 5.8 kg* 8.8 kg** Horgan et al. [16] IOP N 87 11.5 21.5 ± 15.3 Ryou et al. [30] ROSE N 80 (60–100) 4.2 kg** 7.8 kg** Gallo et al. [17] ROSE N 77 ± 30 8 ± 4 8.9 9.3 8 Buttelmann [31] ROSE N 3.9*** 4.1*** 5.4*** Thompson [18] IOP N 87 11.5 14.5 ± 3.1 Patel 2017 [32] EGJR N IST 50.4 ± 25.3 IST 6.6 ± 2.2 6 weeks* 15 (9–22) 19 (9–27)* 13 (5–32)* 10 (− 3.2 to 23.1) PST 42.9 ± 18.1 PST 4.8 ± 1.8 Tsai [22] EGJR N 17.2 (12–33) 6 (4–14) 4.1** 5.8** 8.0** Catalano [33] Sclerotherapy N 10 (8–15) 10.4 Loewen and Barba [34] Sclerotherapy N Jirapinyo [24] Sclerotherapy (sodium mor- rhuate) N 21 ± 6 2.7 ± 5.5**** 6.1 ± 6.8 (9 months)**** TORe 23 ± 6 10.4 ± 2.2**** 12.3 ± 12.6 (9 months)**** Thompson [21] TORe N 107 ± 182.9 15.9 de Moura [40] TORe N 12 20** 14** Kumar and Thompson [37] ST TORe N 6.9 ± 0.2 8.1 ± 2.5 9.1 ± 2.3 FT TORe 7.1 ± 0.3 20.4 ± 3.3 18.9 ± 5.4 Kumar and Thompson [36] TORe (Over- Stitch) N 9.0 ± 0.2 25.0 ± 1.9 28.8 ± 2.7 24.9 ± 2.6 Jirapinyo [25] TORe (Over- Stitch) N 27 (7–80) 6 (3–10) 11.5** 11.7** 10.8** 1 Surgical Endoscopy (2020) 34:2410–2428 2421 1 3 ( ) Study Procedure Combination therapy (Y/N) Av. procedure length (mins) Av. Complications stoma diameter at end of procedure (mm) % Excess weight loss 1 week 1 month 2 months 3 months 6 months 12 months Vargas [23] TORe (Over- Stitch) N 9.31 ± 6.7 20.2 ± 10 Baretta [20] APC N 8.40 ± 1.85 Moon [19] APC N (5–10) 14.0 ± 6.3 6.5** 7.7** Riva [35] Over- Stitch + sutur- ing Y 91 ± 72.4 9.05 Over- Stitch + scle- rotherapy Study % Excess weight loss Recurrence rates n (%) Definition of successful endo- therapy Number of suc- cessful endother- apy n (%) 18 months 24 months 36 months 48 months 60 months 72 months Mikami [14] Ong-Uti [15] 3 (4.7%) Goyal [38] 4.3 ± 29.8 2 (3.6)–pro- gressed to fur- ther procedure 1. Ability to reduce pouch and stoma size 2. Weight loss 35 (63.6) Mullady et al. [29] 1. Ability to reduce stoma diameter and pouch length 2. Weight loss 17 (85) Horgan et al. [16] 1. Ability to reduce stoma diameter and pouch length 2. Weight loss 112 (97) Ryou et al. [30] 1. Ability to reduce stoma diameter and pouch length 2. Weight loss 5 (100) Gallo et al. [17] 6.7  − 10.7  − 13.5  − 5.8  − 4.5 Buttelmann [31] 5.5*** − 4.5 − 13.5 3 Surgical Endoscopy (2020) 34:2410–2428 2422 22 Surgical Endoscopy (2020) 34:2410–242 1 3 Study % Excess weight loss Recurrence rates n (%) Definition of successful endo- therapy Number of suc- cessful endother- apy n (%) 18 months 24 months 36 months 48 months 60 months 72 months Thompson [18] 1. Ability to reduce stoma diameter and pouch length 2. Weight loss 112 (97) Patel 2017 [32] IST 3 (8.8) PST 0 Tsai [22] Catalano [33] 19.9 1. Stoma size < 12 mm 2. Complications Loss of > 75% of weight regained after initial weight loss 18 (64) n and a [34] 2nd session 35 (49), 3rd ses- sion 10(14), 4th session 1(1.4) 21 (29.6) yo [24] Ability to reduce the GJ to < 12 mm pson [21] Ability to reduce the GJ to < 10 mm 89.6% ura [40] Weight mainte- nance/ weight loss 24 (29.6) and mpson [37] and mpson [36] 20.0 ± 6.4 19.2 ± 4.6 yo [25] Ability to reduce the GJ to < 12 mm 25 (100) [23] 8 ± 8.8 (18– 24mths) 11 (8)—repeat EGD per- formed Ability to reduce the GJ to < 10 mm Vargas [23] 1 3 Surgical Endoscopy (2020) 34:2410–2428 2423 Study % Excess weight loss Recurrence rates n (%) Definition of successful endo- therapy Number of suc- cessful endother- apy n (%) 18 months 24 months 36 months 48 months 60 months 72 months Baretta [20] Moon [19] 8.3** Riva [35] 36.4 Key: * median value, interquartile range, ** mean weight loss, *** mean BMI loss, **** mean %TBWL TBWL total body weight loss, EWL excess weight loss, ROSE restorative obesity surgery, endoluminal (ROSE) procedure, EGJR endoscopic gastrojejunal revision, RYGB Roux-en-Y gastric bypass, OTSC-clip over-the-scope clip, IOP incisionless operating platform, TORe sutured transoral outlet reduction, GJA gastrojejunal anastomosis, APC Argon plasma coagulation Outcomes of combination therapy Only two studies assessed the use of combination therapy [28, 35]. Riva et al.’s study [35] involved a combination of mucosal ablation and endoscopic suturing using the OverStitch device or mucosal ablation, endoscopic sutur- ing and sclerotherapy, performed following RYGB in 22 patients. Mean pre-revision weight and BMI were 102.3 kg and 42.4 kg/m2, respectively. Mean post-revision BMI was reduced by 15.1% at 6 months, and this was increased to 19.8% at 1 year. However, no mean weight or excess weight loss following the revision procedure was reported. In terms of complications, only abdominal pain and nausea were reported (44%).i The other study by Eid [28] consisted of five patients, in whom a combination of APC and endoscopic suturing (OverStitch device) was performed. Mean pre-revision weight was 110.3 kg and mean pre-revision BMI 37 kg/ m2. In this group of patients, greatest weight loss was noted 6 months post-procedure (11.0%) and this reduced slightly to 10.1% at 1 year. This is consistent with a fall in 9.1% of mean post-revision BMI measured at 1 year. No complica- tions were reported. Quality of included studies All studies were evaluated for risk of bias using the New- castle–Ottawa Scale (NOS), which allows for a maximum of eight points, and NIH Quality Assessment Tool (Table 3). A score of 6 or more on the NOS is rated “good”, while the NIH tool judges each study to be of “good”, “fair” or “poor” quality. Both the NOS and NIH tool assess risk of bias in the selection of cases, outcome assessment and duration of follow-up. Additionally, the NOS assesses the representa- tiveness of the exposed cohort and adequacy of follow-up. Of the 25 observational studies, two studies achieved a score of 7, seven were scored 6, nine rated 5, five scored 4 and two scored 3 on the NOS (Table 3, Supplementary Table 1). Twenty-three studies (92%) lacked a comparator group. In all studies, a satisfactory length of follow-up is defined as 12 months or longer—this criteria was met by 20 (80%) studies. Four (15.4%) studies were not awarded an “out- come” score on the Newcastle–Ottawa Scale due to high attrition rates. Eleven (42.3%) studies failed to report the rate of patient follow-up. Two studies in particular [14, 16] had very low follow-up rate (< 20%) at 12 months. The authors defined 12 months as the ideal duration of follow-up for post-procedural assessment of weight loss and complications following StomaphyX [14] and ROSE [16], respectively, in their selected patients, all of whom were at least 2 years post-RYGB. However, only 15.4% [14] and 11.2% [16] were successfully followed up for post-procedural evaluations at All studies were evaluated for risk of bias using the New- castle–Ottawa Scale (NOS), which allows for a maximum of eight points, and NIH Quality Assessment Tool (Table 3). A score of 6 or more on the NOS is rated “good”, while the NIH tool judges each study to be of “good”, “fair” or “poor” quality. Both the NOS and NIH tool assess risk of bias in the selection of cases, outcome assessment and duration of follow-up. Additionally, the NOS assesses the representa- tiveness of the exposed cohort and adequacy of follow-up. Of the 25 observational studies, two studies achieved a score of 7, seven were scored 6, nine rated 5, five scored 4 and two scored 3 on the NOS (Table 3, Supplementary Table 1). Twenty-three studies (92%) lacked a comparator group. Complications Loss of > 75% of weight regained after initial weight loss 18 (64) Loewen and Barba [34] 2nd session 35 (49), 3rd ses- sion 10(14), 4th session 1(1.4) 21 (29.6) Jirapinyo [24] Ability to reduce the GJ to < 12 mm Thompson [21] Ability to reduce the GJ to < 10 mm 89.6% de Moura [40] Weight mainte- nance/ weight loss 24 (29.6) Kumar and Thompson [37] Kumar and Thompson [36] 20.0 ± 6.4 19.2 ± 4.6 Jirapinyo [25] Ability to reduce the GJ to < 12 mm 25 (100) Vargas [23] 8 ± 8.8 (18– 24mths) 11 (8)—repeat EGD per- formed Ability to reduce the GJ to < 10 mm 422 Surgical Endoscopy (2020) 34:2410–24 ( ) % Excess weight loss Recurrence rates n (%) Definition of successful endo- therapy Number of suc- cessful endother- apy n (%) 18 months 24 months 36 months 48 months 60 months 72 months pson [18] 1. Ability to reduce stoma diameter and pouch length 2. Weight loss 112 (97) 017 [32] IST 3 (8.8) PST 0 2] no [33] 19.9 1. Stoma size < 12 mm 2. Quality of included studies In all studies, a satisfactory length of follow-up is defined as 12 months or longer—this criteria was met by 20 (80%) studies. Four (15.4%) studies were not awarded an “out- come” score on the Newcastle–Ottawa Scale due to high attrition rates. Eleven (42.3%) studies failed to report the rate of patient follow-up. Two studies in particular [14, 16] had very low follow-up rate (< 20%) at 12 months. The authors defined 12 months as the ideal duration of follow-up for post-procedural assessment of weight loss and complications following StomaphyX [14] and ROSE [16], respectively, in their selected patients, all of whom were at least 2 years post-RYGB. However, only 15.4% [14] and 11.2% [16] were successfully followed up for post-procedural evaluations at 1 3 2424 Surgical Endoscopy (2020) 34:2410–2428 Table 3   Summary of quality and risk of bias assessment using the Newcastle–Ottawa scale and National Institute of Health quality assessment tool for case series studies Study Newcastle–Ottawa scale National Institute of Health quality assessment tool Overall Mikami et al. [14] 4 Fair Fair Manouchehri et al. [26] 6 Good Good Ong’Uti et al. [15] 7 Good Good Goyal et al. (39) 7 Good Good Mullady et al. [29] 5 Fair Fair Horgan et al. [16] 6 Good Good Ryou et al. [30] 4 Poor Poor Gallo et al. [17] 4 Fair Fair Buttelmann et al. [31] 6 Good Good Thompson et al. [18] 5 Fair Fair Heylen et al. [27] 5 Fair Fair Patel et al. [32] 5 Fair Fair Tsai et al. [22] 6 Good Good Catalano et al. [33] 5 Good Fair Loewen and Barba [34] 4 Fair Fair Jirapinyo et al. [24] 6 Good Good de Moura et al. [39] 3 Poor Poor Kumar and Thompson [37] 5 Fair Fair Kumar and Thompson [36] 6 Good Good Jirapinyo et al. [25] 5 Fair Fair Vargas et al. [23] 5 Good Fair Baretta et al. [20] 4 Fair Fair Moon et al. [19] 5 Fair Fair Riva et al. [35] 6 Good Good Eid [28] 3 Poor Poor 12 months, and the authors did not elaborate the reason(s) behind loss to follow-up. to other techniques (i.e. sclerotherapy, APC) post-RYGB. More specifically, analysis of the included studies has shown successful EWL following the use of endoluminal plication devices in the first 12 months after revisional procedure. This EWL was, however, not well-sustained past 12 months. Quality of included studies Greater procedural success and lower recurrence rates are seen in endoluminal plication devices compared to sclero- therapy and APC. Additionally, this review suggests that endoluminal plication devices are associated with lower rates of mild and moderate complications post-procedure compared to sclerotherapy and APC. With regards to the NIH Quality Assessment Tool (Table 3, Supplementary Table 2), 12 (46.2%) studies were subjectively considered to be of “good” quality, while 11 (42.3%) were considered “fair” and three (11.5%) were rated “poor” in the risk of bias assessment. The studies rated “poor” lacked a clear definition for cohort selection and out- come measures, and either failed to describe the results or statistical methods used. Given the high heterogeneity of the studies included in this review, the results and conclusions should be interpreted with caution. Due to the complex nature of weight regain, which involves an interplay between genetic, anatomical, physi- ological and behavioural factors [42, 43], there are different theories as to which factor is most predictive of treatment response. Excess weight loss (EWL) following revision surgery using endoluminal plication devices is likely to be due to anatomical reasons. Horgan et al. describes failure of maintenance of EWL to be due to loss of restriction attrib- uted to the enlargement of the gastric pouch, dilatation of gastrojejunostomy and fistula development between gas- tric pouch and remnant of the stomach [16]. In this review, Vargas et al.’s study was focused on stoma size reduction, Table 3   Summary of quality and risk of bias assessment using the Newcastle–Ottawa scale and National Institute of Health quality assessment tool for case series studies 3 Discussion Weight regain is estimated to range between 5–7% [2, 4, 5] with higher failure rates (20–35%) in the superobese patients (BMI > 50) [6, 40, 41]. This systematic review addresses less invasive techniques for treatment of post-operative weight gain and associated short-term outcomes. The use of endo- luminal plication devices in revisional surgery is associated with greater initial EWL and fewer complications compared 1 3 3 Surgical Endoscopy (2020) 34:2410–2428 2425 where the revision procedure (TORe) was considered suc- cessful if the stoma diameter was reduced to < 10 mm [23]. The authors achieved a mean of 70.4% reduction in stoma diameter post-procedure and a resultant mean EWL of 20.2% at 12 months, and 8.0% at 18 months [23]. Similarly, Jirapinyo et al. showed a mean reduction of 77.3% in stoma diameter post-procedure in their cohort of 25 patients post- procedure (TORe), with a peak mean weight loss of 11.7 kg at 6 months, which later decreased to 10.8 kg at 12 months [25]. The greater mean EWL in the initial 12 months fol- lowing the use of endoluminal plication devices in patients post-RYGB which decreased in the ensuing months may be attributed to the lack of durability of endoscopic sutures in the long term [44]. Follow-up endoscopy after ROSE pro- cedures showed that superior weight loss is associated with reduction in stoma size, with good durability of anchors and tissue fold for up to 12 months post-revision [16, 18]. These studies provide evidence that stoma size does influence weight loss post-revision surgery, where EWL is greatest in the first 12 months. However, long-term data past 18 months are not recorded for most studies utilising endoluminal plica- tion devices post-RYGB in this review. Future studies, which include follow-up evaluations with endoscopy to verify the link between maintenance of stoma reduction and EWL, are warranted. 20]. Hence, these studies focused on the anatomical aspect of causes in weight regain, similar to that in endoluminal plication devices, although Moon et al. did demonstrate a sustained mean weight loss up to 24 months, longer than those noted in revisional procedures utilising endoluminal plication devices [19]. Furthermore, Manouchehri et al. Discussion has shown that endo- luminal plication devices in revision surgery, specifically the StomaphyX™, can effectively contribute to weight loss in patients following VBG, with only minor complications experienced by patients [26], although sustained weight loss is not demonstrated due to limited duration of follow- up (3 months). However, the role of endoluminal plication devices is more skewed towards that in RYGB patients because VBG has largely been supplanted by RYGB as a primary bariatric surgery technique in recent years. Nev- ertheless, outcomes following endoluminal revisional tech- niques in VBG may still be of interest in a small cohort of patients experiencing weight regain requiring revisional surgery [49–51]. A previous meta-analysis by Vargas et al. has demon- strated the safe and efficacious use of TORe (OverStitch device) in revision surgery performed in RYGB patients [23]. The present review builds on this finding, and sum- marises qualitatively the evidence supporting greater long- term post-procedure weight loss when endoscopic suturing with OverStitch device is combined with sclerotherapy or APC, as shown by Riva et al. [35] and Eid [28]. Riva et al.’s study was aimed at investigating a possible additive effect of combined sclerotherapy and endoscopic suturing, where the induced fibrosis could enhance the durability of sutures [35]. Compared with sclerotherapy/APC (EWL 19.9%) or endo- luminal plication device (EWL 12.9%) alone, combination therapy is shown to induce the greatest mean EWL of 36.4% at 18 months in a small study of five patients [28]. Although combination therapy appears to have some benefit in one study, this has not translated to a larger study of 22 patients. Another possible predictor of EWL following revision surgery is ghrelin levels post-procedure. The role of ghre- lin in obesity in previous work appears to be significant, however, its exact mechanism requires further investigation [45, 46]. However, Dayyeh et al. demonstrated a decrease in ghrelin levels in a group of 33 RYGB patients post-sclero- therapy, contrary to what was observed following mechani- cal endoscopic suturing with endoluminal plication devices [42]. The authors postulated that ghrelin-producing cells were destructed as a result of sclerosis, hence modulating the neurohormonal signalling to the brain and other organs, altering satiety, food intake behaviours insulin secretion and energy expenditure [42, 47]. This alteration in neurophysi- ology may account for the greater sustained EWL over a longer period of time in sclerotherapy compared to endolu- minal plication devices. Discussion This is because the latter predomi- nantly depends on the reduction in GJ stoma diameter slow- ing down the activation of gastric wall mechanoreceptors, inhibiting the release of orexigenic gastric peptides such as ghrelin [42, 46–48], which may be less effective compared to a direct destruction of ghrelin-producing cells in sclero- therapy in inducing neurophysiological changes contributing to sustained weight loss. This, compounded by the lack of clear description on patient selection and specification of outcomes, undermines the internal validity of the conclusions. This finding may suggest the potential of combination therapy in managing weight regain following primary bariatric surgery, however, there is currently insufficient evidence to support its supe- riority over endoluminal plication devices, and vice versa. Compliance with ethical standards Disclosures  Miss Yan Mei Goh, Nicole Ellen James, En Lin Goh, and Achal Khanna have no conflicts of interest or financial ties to disclose. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. Additionally, these studies also lacked control of con- founding factors including patients’ nutritional status, main- tenance of diet and exercise, as well as important comorbid conditions such as type 2 diabetes mellitus. Future work may wish to explore the impact of additional routine follow-up addressing these behavioural issues and dietary and lifestyle modifications on maintenance of weight loss. Additionally, all of the studies were conducted in developed countries. These skewed study populations are unlikely to represent faithfully the true populations in less developed countries, thus the generalisability of these findings to the wider pop- ulation in other parts of the world should be treated with caution. With the majority of studies being retrospective in design and the paucity of studies assessing long-term EWL of greater than 12 months following endoluminal procedures, the question whether endoluminal techniques can sustain long-term EWL still remains. Cohort studies or randomised controlled trials should be performed to not only clarify the role of endoluminal plication devices, but also combination therapy in the management of weight regain following primary bariatric surgery. References 1. English WJ, DeMaria EJ, Brethauer SA, Mattar SG, Rosenthal RJ, Morton JM (2018) American Society for metabolic and bari- atric surgery estimation of metabolic and bariatric procedures performed in the United States in 2016. Surg Obes Relat Dis 14:259–263 2. Brolin RE, Kenler HA, Gorman JH, Cody RP (1992) Long-limb gastric bypass in the superobese: a prospective randomized study. Ann Surg 215(4):387–395 3. Capella JF, Capella RF (1996) The weight reduction operation of choice: vertical banded gastroplasty or gastric bypass? Am J Surg 171(1):74–79 4. Fobi MAL, Lee H, Igwe D, Felahy B, James E, Stanczyk M et al (2001) Revision of failed gastric bypass to distal Roux-en-Y gas- tric bypass: a review of 65 cases. Obes Surg 11(2):190–195 yp g 5. Capella JF, Capella RF (1996) The weight reduction operation of choice: vertical banded gastroplasty or gastric bypass? Am J Surg 1:74–79 6. Biron S, Hould FS, Lebel S, Marceau S, Lescelleur O, Simard S et al (2004) Twenty years of biliopancreatic diversion: what is the goal of the surgery? Obes Surg 14(2):160–164 Study limitations The included studies exhibit some limitations, which must be considered when interpreting the findings of this analy- sis. Firstly, there is significant variation in primary bariatric procedure, endoluminal revision techniques, methodology of reporting, follow-up times, outcomes and complications. Theoretically, the modulation in neurohormonal signal- ling may presumably be applied to APC, but the APC pro- cedures that were utilised in two studies included in this review were aimed at reducing the diameter of the GJ stoma and, therefore, reinitiate weight loss in RYGB patients [19, 1 3 Surgical Endoscopy (2020) 34:2410–2428 2426 There are limited data on endoscopic revision procedures following sleeve gastrectomy. Given the rapid increase in use of sleeve gastrectomy in recent years, future research on the generalisability and applicability of endoscopic revision surgery in patients with sleeve gastrectomy will be necessary to overcome the inherent limitations of the currently avail- able evidence. Moreover, the heterogeneity of the studies, especially with regards to the selection criteria of patients for revisional surgery, limits the statistical analysis of demo- graphic and procedural variables that appeared to be predic- tive of maximal weight loss benefit. quality data limits our ability to demonstrate and support the long-term efficacy of endoluminal techniques in the management of weight regain following primary bariatric surgery. However, we suggest that these techniques have an intermediate role in management of weight regain following bariatric surgery, delaying surgical revision or conversion to distal RYGB or biliopancreatic/duodenal switch procedures. Future work is necessary to substantiate the long-term role of endoluminal bariatric procedures in the management of this group of patients. i Most series have small number of patients and some follow-up data were not available which imposes limits on our ability to make a meaningful conclusion. These high attrition rates could be attributed to a poor understanding of patients’ expressed needs, which is central to the develop- ment and delivery of effective longer term follow-up care following revision surgery. Studies have shown that patients who did not attend regular follow-up commonly described unmet perceived expectations as well as fear of disappoint- ing the healthcare professional if they were unable to meet nutritional or physical activity targets set [52]. Conclusion 7. Zalesin KC, Franklin BA, Miller WM, Nori Janosz KE, Veri S, Odom J et al (2010) Preventing weight regain after bariatric sur- gery: an overview of lifestyle and psychosocial modulators. Am J Lifestyle Med 4:113–120 Our study demonstrates the need for detailed discussion and tailoring of techniques and resources to the individ- ual patient. Endoluminal techniques at present affords the patient an opportunity to alter their lifestyle and delay surgi- cal revision or conversion to distal RYGB or biliopancreatic/ duodenal switch procedures. However, the paucity of good 8. Odom J, Zalesin KC, Washington TL, Miller WW, Hakmeh B, Zaremba DL et al (2010) Behavioral predictors of weight regain after bariatric surgery. Obes Surg 20(3):349–356 1 3 Surgical Endoscopy (2020) 34:2410–2428 2427 26. Manouchehri N, Birch DW, Menzes C, Shi X, Karmali S (2011) Natural orifice surgery: endoluminal pouch reduction following failed vertical banded gastroplasty. Obes Surg 21(11):1787–1791 9. Stefanidis D, Malireddy K, Kuwada T, Phillips R, Zoog E, Gersin KS (2013) Revisional bariatric surgery: perioperative morbidity is determined by type of procedure. Surg Endosc 26(Suppl):26–32f 10. Schwartz RW, Strodel WE, Simpson WS, Griffen WO (1988) Gastric bypass revision: lessons learned from 920 cases. Surgery 104(4):806–812 27. Heylen AMF, Jacobs A, Lybeer M, Prosst RL (2011) The OTSC®-clip in revisional endoscopy against weight gain after bariatric gastric bypass surgery. Obes Surg 21(10):1629–1633 ( ) 11. Spaulding L (2003) Treatment of dilated gastrojejunostomy with sclerotherapy. Obes Surg 13(2):254–257 28. Eid G (2017) Sleeve gastrectomy revision by endoluminal sleeve plication gastroplasty: a small pilot case series. Surg Endosc 31:4252–4255 12. Wells G, Shea B, O’Connell D, Peterson J, Welch V, Losos M, et al. The Newcastle-Ottawa Scale (NOS) for assessing the qual- ity of nonrandomised studies in meta-analyses [Internet]. 2019; https​://www.ohri.ca/progr​ams/clini​cal_epide​miolo​gy/oxfor​d.asp, Accessed 23 Aug, 2019 29. Mullady DK, Lautz DB, Thompson CC (2009) Treatment of weight regain after gastric bypass surgery when using a new endoscopic platform: initial experience and early outcomes (with video). Gastrointest Endosc 70(3):440–444 13. National Heart L and BI. Study quality assessment tools [Internet]. 2018; https​://www.nhlbi​.nih.gov/healt​h-topic​s/study​-quali​ty-asses​ sment​-tools​. Accessed 23 Aug, 2018 30. Ryou M, Mullady DK, Lautz DB, Thompson CC (2009) Pilot study evaluating technical feasibility and early outcomes of second-generation endosurgical platform for treatment of weight regain after gastric bypass surgery. Surg Obes Relat Dis 5(4):450–454 14. Conclusion Mikami D, Needleman B, Narula V, Durant J, Melvin WS (2010) Natural orifice surgery: initial US experience utilizing the Stoma- phyXTM device to reduce gastric pouches after Roux-en-Y gastric bypass. Surg Endosc 24(1):223–228 31. Buttelmann K, Linn JG, Denham W, Ruiz M, Yetasook A, Ujiki M (2015) Management options for obesity after bariatric surgery. Surg Laparosc Endosc Percutaneous Tech 25(1):15–18 15. OngUti SK, Ortega G, Onwugbufor MT, Ivey GD, Fullum TM, Tran DD (2013) Effective weight loss management with endo- scopic gastric plication using StomaphyX device: Is it achievable? Surg Obes Relat Dis. 9(1):113–117 32. Patel LY, Lapin B, Brown CS, Stringer T, Gitelis ME, Linn JG et al (2017) Outcomes following 50 consecutive endoscopic gas- trojejunal revisions for weight gain following Roux-en-Y gastric bypass: a comparison of endoscopic suturing techniques for stoma reduction. Surg Endosc Other Interv Tech 31(6):2667–2677 16. Horgan S, Jacobsen G, Weiss GD, Oldham JS, Denk PM, Borao F et al (2010) Incisionless revision of post-Roux-en-Y bypass stomal and pouch dilation: multicenter registry results. Surg Obes Relat Dis 6(3):290–295 33. Catalano MF, Rudic G, Anderson AJ, Chua TY (2007) Weight gain after bariatric surgery as a result of a large gastric stoma: endotherapy with sodium morrhuate may prevent the need for surgical revision. Gastrointest Endosc 66(2):240–245 17. Gallo AS, DuCoin CG, Berducci MA, Nino DF, Almadani M, Sandler BJ et al (2016) Endoscopic revision of gastric bypass: Holy Grail or Epic fail? Surg Endosc 30(9):3922–3927 34. Loewen M, Barba C (2008) Endoscopic sclerotherapy for dilated gastrojejunostomy of failed gastric bypass. Surg Obes Relat Dis 4(4):539–542 18. Thompson CC, Jacobsen GR, Schroder GL, Horgan S (2012) Stoma size critical to 12-month outcomes in endoscopic suturing for gastric bypass repair. Surg Obes Relat Dis 8(3):282–287 35. Riva P, Perretta S, Swanstrom L (2017) Weight regain following RYGB can be effectively treated using a combination of endo- scopic suturing and sclerotherapy. Surg Endosc Other Interv Tech 31(4):1891–1895 19. Moon RC, Teixeira AF, Neto MG, Zundel N, Sander BQ, Ramos FM et al (2018) Efficacy of utilizing argon plasma coagulation for weight regain in Roux-en-Y Gastric bypass patients: a multi-center study. Obes Surg 28(9):2737–2744 36. Kumar N, Thompson CC (2016) Transoral outlet reduction for weight regain after gastric bypass: Long-term follow-up. Gastro- intest Endosc 83(4):776–779i 20. Conclusion Baretta GAP, Alhinho HCAW, Matias JEF, Marchesini JB, de Lima JHF, Empinotti C et al (2015) Argon plasma coagulation of gastrojejunal anastomosis for weight regain after gastric bypass. Obes Surg 25(1):72–79 37. Kumar N, Thompson CC (2014) Comparison of a superficial suturing device with a full-thickness suturing device for transoral outlet reduction (with videos). Gastrointest Endosc 79(6):984–989 21. Thompson CC, Chand B, Chen YK, Demarco DC, Miller L, Sch- weitzer M et al (2013) Endoscopic suturing for transoral outlet reduction increases weight loss after Roux-en-Y gastric bypass surgery. Gastroenterology 145(1):129–137f 38. Goyal V, Holover S, Garber S (2013) Gastric pouch reduction using ­StomaphyXTM in post Roux-en-Y gastric bypass patients does not result in sustained weight loss: a retrospective analysis. Surg Endosc 27(9):3417–3420i 22. Tsai C, Steffen R, Kessler U, Merki H, Zehetner J (2019) Endo- scopic gastrojejunal revisions following gastric bypass: lessons learned in more than 100 consecutive patients. J Gastrointest Surg 23(1):58–66 g 39. de Moura DTH, Jirapinyo P, Thompson CC (2019) Modified-ESD plus APC and suturing for treatment of weight regain after gastric bypass. Obes Surg 29(6):2001–2002 23. Vargas EJ, Bazerbachi F, Rizk M, Rustagi T, Acosta A, Wilson EB et al (2018) Transoral outlet reduction with full thickness endo- scopic suturing for weight regain after gastric bypass: a large mul- ticenter international experience and meta-analysis. Surg Endosc 32(1):252–259 40. Hsu LK, Benotti PN, Dwyer J, Roberts SB, Saltzman E, Shikora S et al (1998) Nonsurgical factors that influence the outcome of bariatric surgery: a review. Psychosom Med 60(3):338–346 41. Nelson DW, Blair KS, Martin MJ (2012) Analysis of obesity- related outcomes and bariatric failure rates with the duode- nal switch vs gastric bypass for morbid obesity. Arch Surg 147(9):847–854 24. Jirapinyo P, Dayyeh BKA, Thompson CC (2016) Gastrojejunal anastomotic reduction for weight regain in roux-en-y gastric bypass patients: physiological, behavioral, and anatomical effects of endoscopic suturing and sclerotherapy. Surg Obes Relat Dis 12(10):1810–1816 42. Abu Dayyeh BK, Jirapinyo P, Weitzner Z, Barker C, Flicker MS, Lautz DB et al (2012) Endoscopic sclerotherapy for the treatment of weight regain after Roux-en-Y gastric bypass: outcomes, com- plications, and predictors of response in 575 procedures. Gastro- intest Endosc 76(2):275–282 25. Jirapinyo P, Slattery J, Ryan MB, Dayyeh BKA, Lautz DB, Thompson CC (2013) Evaluation of an endoscopic suturing device for transoral outlet reduction in patients with weight regain following Roux-en-Y gastric bypass. Endoscopy 45(7):532–536 43. Conclusion Vetter ML, Cardillo S, Rickels MR, Iqbal N (2009) Narrative review: effect of bariatric surgery on type 2 diabetes mellitus. Ann Intern Med 150(2):94–103 1 3 2428 Surgical Endoscopy (2020) 34:2410–2428 44. Eid GM, McCloskey CA, Eagleton JK, Lee LB, Courcoulas AP (2014) StomaphyX vs a sham procedure for revisional surgery toreduceregainedweightinroux-en-ygastric bypass patients a ran- domized clinical trial. JAMA Surg 149(4):372–379 50. Cariani S, Agostinelli L, Leuratti L, Giorgini E, Biondi P, Amenta E (2010) Bariatric revisionary surgery for failed or complicated vertical banded gastroplasty (VBG): comparison of VBG reopera- tion (re-VBG) versus Roux-en-Y Gastric bypass-on-VBG (RYGB- on-VBG). J Obes. https​://doi.org/10.1155/2010/20624​9 g 45. Cummings DE, Shannon MH (2003) Roles for ghrelin in the regu- lation of appetite and body weight. Arch Surg 138(4):389–396 51. van Gemert W, van Wersch M, Greve J, Soeters P (1998) Revi- sional surgery after failed vertical banded gastroplasty: restoration of vertical banded gastroplasty or conversion to gastric bypass. Obes Surg 8(1):21–28 46. Cummings DE, Weigle DS, Scott Frayo R, Breen PA, Ma MK, Patchen Dellinger E et al (2002) Plasma ghrelin levels after diet- induced weight loss or gastric bypass surgery. N Engl J Med 346(21):1662–1663 g 52. Parretti HM, Hughes CA, Jones LL (2019) ‘The rollercoaster of follow-up care’ after bariatric surgery: a rapid review and qualita- tive synthesis. Obes Rev 20(1):88–107 47. Hansotia T, Maida A, Flock G, Yamada Y, Tsukiyama K, Seino Y et al (2007) Extrapancreatic incretin receptors modulate glucose homeostasis, body weight, and energy expenditure. J Clin Invest 117(1):143–152 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 48. Ogiso K, Asakawa A, Amitani H, Inui A (2011) Ghrelin: a gut hormonal basis of motility regulation and functional dyspepsia. J Gastroenterol Hepatol 26(Suppl 3):67–72 49. Iannelli A, Dainese R, Piche T, Facchiano E, Gugenheim J (2008) Laparoscopic sleeve gastrectomy for morbid obesity. World J Gas- troenterol 14(6):821–827 1 3 3
https://openalex.org/W3036016649
https://opus.bibliothek.uni-wuerzburg.de/files/23533/Pemp2020_Article_InfluenceOfBreastCancerRiskFac.pdf
English
null
Influence of breast cancer risk factors and intramammary biotransformation on estrogen homeostasis in the human breast
Archives of toxicology
2,020
cc-by
9,256
Abstract Understanding intramammary estrogen homeostasis constitutes the basis of understanding the role of lifestyle factors in breast cancer etiology. Thus, the aim of the present study was to identify variables influencing levels of the estrogens present in normal breast glandular and adipose tissues (GLT and ADT, i.e., 17β-estradiol, estrone, estrone-3-sulfate, and 2-methoxy-estrone) by multiple linear regression models. Explanatory variables (exVARs) considered were (a) levels of metabolic precursors as well as levels of transcripts encoding proteins involved in estrogen (biotrans)formation, (b) data on breast cancer risk factors (i.e., body mass index, BMI, intake of estrogen-active drugs, and smoking) collected by question- naire, and (c) tissue characteristics (i.e., mass percentage of oil, oil%, and lobule type of the GLT). Levels of estrogens in GLT and ADT were influenced by both extramammary production (menopausal status, intake of estrogen-active drugs, and BMI) thus showing that variables known to affect levels of circulating estrogens influence estrogen levels in breast tissues as well for the first time. Moreover, intratissue (biotrans)formation (by aromatase, hydroxysteroid-17beta-dehydrogenase 2, and beta-glucuronidase) influenced intratissue estrogen levels, as well. Distinct differences were observed between the exVARs exhibiting significant influence on (a) levels of specific estrogens and (b) the same dependent variables in GLT and ADT. Since oil% and lobule type of GLT influenced levels of some estrogens, these variables may be included in tis- sue characterization to prevent sample bias. In conclusion, evidence for the intracrine activity of the human breast supports biotransformation-based strategies for breast cancer prevention. The susceptibility of estrogen homeostasis to systemic and tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the environment possible. Keywords  Estrogens · Human breast · Multiple linear regression * Leane Lehmann leane.lehmann@uni‑wuerzburg.de Influence of breast cancer risk factors and intramammary biotransformation on estrogen homeostasis in the human breast Daniela Pemp1 · Leo N. Geppert2 · Claudia Wigmann2 · Carolin Kleider1 · René Hauptstein1 · Katja Ickstadt2 · Harald L. Esch1   · Leane Lehmann1 Received: 12 February 2020 / Accepted: 15 June 2020 / Published online: 22 June 2020 © The Author(s) 2020 Archives of Toxicology (2020) 94:3013–3025 https://doi.org/10.1007/s00204-020-02807-1 Archives of Toxicology (2020) 94:3013–3025 https://doi.org/10.1007/s00204-020-02807-1 TOXICOKINETICS AND METABOLISM 2 Mathematical Statistics with Applications in Biometrics, TU Dortmund University, Vogelpothsweg 87, 44221 Dortmund, Germany Introduction (E1), and other endogenous steroids in pre- and postmeno- pausal women (Endogenous Hormones Breast Cancer Col- laborative Group 2002, 2013) over a prolonged period of time. Based on these associations as well as an abundance of experiments in vitro and in animal models, the current understanding of the molecular etiology of breast cancer hypothesizes biotransformation of E2/E1 within the breast tissue to catechols and subsequent oxidation to mutagenic quinones possibly initiating tumor formation. Tumor promo- tion is then favored by estrogen receptor (ESR)-mediated stimulation of proliferation of the initiated cells (Yager and Davidson 2006). Thus, both tumor initiation and progres- sion would depend predominately on intramammary levels of reactive products of estrogen biotransformation, whereas tumor promotion would depend predominately on levels of E2. Breast cancer is the most common cancer in women world- wide. Its development is associated with increased levels of circulating estrogens, e.g., 17β-estradiol (E2), estrone Electronic supplementary material  The online version of this article (https​://doi.org/10.1007/s0020​4-020-02807​-1) contains supplementary material, which is available to authorized users. * Leane Lehmann leane.lehmann@uni‑wuerzburg.de 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 2 Mathematical Statistics with Applications in Biometrics, TU Dortmund University, Vogelpothsweg 87, 44221 Dortmund, Germany 2 Mathematical Statistics with Applications in Biometrics, TU Dortmund University, Vogelpothsweg 87, 44221 Dortmund, Germany :(0123 1 3456789) 3 Archives of Toxicology (2020) 94:3013–3025 3014 Consequently, commonly accepted risk factors such as early menarche and/or late menopause, late age at first preg- nancy, small number of pregnancies, and short or no periods of breastfeeding (Colditz and Bohlke 2014) are supposed to increase the duration or extent of the local exposure of the mammary gland to E2 (biotransformation products) by increasing their systemic production. Concurrently, cur- rent risk reduction strategies involve the chemical modula- tion of ESR as well as systemic inhibition of aromatase or salpingo-oophorectomy (Advani and Moreno-Aspitia 2014) aimed to reduce levels of circulating estrogens. Recently, also modifiable risk factors associated with lifestyle such as (postmenopausal) obesity, alcohol consumption (Colditz and Bohlke 2014), smoking (Gaudet et al. 2017; Jones et al. 2017; Gram et al. 2019), and intake of estrogen-active drugs (EADs) for oral contraception (Grosse et al. Origin of biospecimens Breast tissue specimens were obtained from 47 adult women without breast cancer undergoing reduction mam- moplasty between 2010 and 2015. All women participat- ing in the study gave their written informed consent prior to their inclusion in the study. Women with a personal and/or family history of breast cancer were not eligible for participation. Information on age, height, weight, par- ity (parous/nulliparous), smoking habits (never smoker, current smoker, current nonsmoker, and the latter two with daily cigarette consumption) was volunteered by 47 women, and information on the intake of EADs by 45 women. Body mass index (BMI) was calculated in kg/m2. Given the wide range of enzymes present in breast glandular (GLT) and adipose tissues (ADT; Pemp et al. 2019a), additional (biotrans)formation of estrogens within the breast tissue can reasonably be assumed (Labrie 2015; Mueller et al. 2015; Hilborn et al. 2017; Pemp et al. 2019a). Consequently, breast cancer risk factors may also influence tissue levels of E2 and its biotransformation products by affecting estrogen homeostasis in the breast. Thus, to better understand how estrogen homeostasis may affect initiation and promotion of breast cancer, insight into the influence of breast cancer risk factors on both levels of estrogen and estrogen biotransformation in women without breast cancer is needed. 1 3 Introduction 2009) or hor- mone replacement therapy (Collaborative Group on Hor- monal Factors in Breast Cancer 2019) have been associated with both increased breast cancer risk and higher levels of circulating E2 and E1 (Endogenous Hormones Breast Can- cer Collaborative Group 2003, 2011, 2013), suggesting that these risk factors also act by affecting intramammary levels of E2/E1 (biotransformation products). (biotrans)formation in breast GLT and ADT of pre- and postmenopausal women without breast cancer (Pemp et al. 2019a; Fig. 1) providing suitable data to determine vari- ables affecting intramammary levels of estrogens. Further- more, it was shown that levels of most estrogens and ratios thereof as well as levels of transcripts encoding enzymes involved in their (biotrans)formation differed significantly between GLT and ADT (Pemp et al. 2019a), demonstrating that breast GLT and ADT should be considered separately. Thus, the aim of the present study was to identify vari- ables (reproductive history, lifestyle, and transcript levels of enzymes involved in intracrine activity) influencing lev- els of estrogens and ratios thereof in breast GLT and ADT. Sample preparation and characterization Tissue levels of E1 and E2 can be decreased by hydroxylation catalyzed by CYPs and conjugation, i.e., sulfonation and glucuronida- tion by sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs), respectively. However, only E1-G (Pemp et al. 2019a) and E1-S have been detected in breast GLT and ADT up to now. Catecho- lic hydroxy-estrogens (HO-E) can be oxidized to potentially cancer- initiating estrogen quinones which can be reduced back to catechols by NADPH quinone dehydrogenase 1 (NQO1). Detoxification of catechols is catalyzed by catechol-O-methyl transferase (COMT) resulting in the formation of methoxy(MeO-)estrogens. Of all possi- ble MeO-estrogens, only 2-MeO-E1 has been detected mass spectro- metrically (Fleming et al. 2010; Pemp et al. 2019a), predominately in ADT (Pemp et al. 2019a). Framed estrogens were quantified recently in breast tissues (Pemp et  al. 2019a) and are used in this study as dependent variables in multiple linear regression analyses. Gray- colored estrogens were below the limit of detection in breast tissues (Pemp et al. 2019a) by Pemp et al (2019a). Data used in statistical analyses are presented in Online Resource 3. Sample preparation and characterization Biospecimens were prepared as described previously (Pemp et al. 2019a). Briefly, aliquots of apparently plain ADT and GLT with less than 15% adhering ADT were flash-frozen in liquid nitrogen and stored at − 80 °C. From mixed tissues, GLT was isolated from cryosections (40 µm) at maximum − 20 °C using a scalpel. Biospeci- mens were characterized by their mass percentages of oil (oil%), percentage of area covered by intra- and interstro- mal adipocytes, and lobule type: oil% in GLT and ADT were determined gravimetrically after extraction with chloroform. Percentage of area covered by intra- and inter- stromal adipocytes was estimated microscopically (Leica LMD6500) in cryosections (10 µm) of GLT stained with hematoxylin and eosin Y by two different persons and coded slides (Pemp et al. 2019a). The lobule type of GLT was determined microscopically according to Russo and Russo (2004) and Figueroa et al. (2014). However, only two studies in women without breast cancer have been published in this regard of which only one has performed statistical analyses (Online Resource 1, Savolainen-Peltonen et al. 2014). Even considering those analyzing non-tumor tissue of women with breast cancer and investigating the association of risk factors with tissue levels of estrogens, most studies did not provide informa- tion on parameters statistically compared and whether or not all positive/negative correlations were reported (Online Resource 1). Surprisingly, none of the available studies included reproductive history of the participating women in their statistical analyses, or collected data on smoking or analyzed biotransformation products of E2 other than E1 by recommended methods of specific analysis (Online Resource 1). Only recently, we described quantitative estrogen profiles and transcript levels of enzymes involved in E2 1 3 3015 Archives of Toxicology (2020) 94:3013–3025 Fig. 1   Current knowledge on (biotrans)formation of E2 in breast GLT and ADT of women without breast cancer based on recently published information on quantitative estrogen profiles and levels of transcripts encoding enzymes involved in (biotrans)formation of E2 in human breast tissues (Pemp et al 2019a). Intramammary tissue lev- els of E2 or E1 can be increased by cytochrome P450 (CYP)19A1- mediated formation form androgenic precursors, interconversion of E1 and E2 by hydroxysteroid 17-beta dehydrogenases (HSD17Bs), as well as sulfotransferase (STS)- and glucuronidase beta (GUSB)- mediated hydrolysis of estrogen sulfates and glucuronides, respec- tively. Statistical methods E2, E1, and 2-methoxy(MeO)-E1 were determined by GC–MS/MS (Varian 450-GC, 300-MS; Bruker Daltonics, Bremen, Germany), whereas E1 sulfate (E1-S) and E1 glu- curonide was determined by LC–MS/MS ­(QTrap® 5500; AB Sciex, Darmstadt, Germany). Tissue levels of E2, E1, 2-MeO-E1, and E1-S were quantified using their respec- tive deuterated derivatives (Pemp et al. 2019a). Data used in statistical analyses are presented in Online Resource 2. All statistical analyses were performed with the statistical programming language R [https​://www.R-proje​ct.org/], ver- sion 3.5.2, and all tests of statistical significance were two- sided. Whenever multiple comparisons were performed, p values were adjusted using Holm’s method. Instrumental analysis of E2, estrone, 2‑methoxy‑estrone, estrone sulfate, and glucuronide by Pemp et al (2019a). Data used in statistical analyses are presented in Online Resource 3. Identification of dependent variables E2, E1, and E1-S were detected in most biospecimens, whereas 2-MeO-E1 was detected predominately in ADT (Online Resource 2). Thus, the influence of exVARs on lev- els of E2, E1, E1-S, and ratios thereof was analyzed in both GLT and ADT whereas the influence of exVARs on levels of 2-MeO-E1 could be analyzed in ADT only. Furthermore, continuous variables significantly influencing estrogen levels were further analyzed as dependent variables as well. The adjusted coefficients of determination, the num- bers of conspicuous observations removed, the numbers of observations contributing to the final models (maxi- mum of 45 because of two specimens without information on the intake of EADs). In models considering intracrine activity, maximum number of observations was further reduced because of two and one specimens in GLT and ADT, respectively, without information on transcript lev- els), and the ratio of observations per exVAR of each final model is given. To achieve accurate estimation of regres- sion coefficients, at least two observations per exVAR (Austin and Steyerberg 2015) were aimed for.fi Identification of possible exVARs related to the study cohort and to the tissues collected First, the study cohort and the tissues collected were char- acterized and possible exVARs were defined. Results and discussion To identify exVARs (e.g., breast cancer risk factors) influ- encing dependent variables (e.g., tissue levels of E2) by multiple linear regression models, suitable dependent vari- ables were identified first and potential exVARs were chosen subsequently. Then, multiple linear regression models using stepwise forward selection were applied to assess up to 32 exVARs possibly influencing tissue levels of estrogens and ratios thereof in GLT and ADT. Determination of transcript levels Contingency analyses were performed using Chi-square test. In case of categories following a natural order, Chi-square test for trend was used. Analysis of transcript levels of genes encoding enzymes involved in E2 (biotrans)formation and regulation thereof was performed using customized ­Taqman® Low Density Arrays and ­Taqman® Gene Expression Assays as described 1 3 3016 Archives of Toxicology (2020) 94:3013–3025 interval, as well as the p values of each exVAR selected are given in Online Resource 4. To test the association of every possible explanatory variable (exVAR) with the dependent variable, the vari- able explaining the dependent variable best is chosen by an automatic procedure. Subsequently, all possible exVARs are added one after another to the first one, ultimately choosing the one improving the model most, applying the Akaike information criterion. This is repeated until the model cannot be further improved by adding exVARs. Thus, each exVAR selected into the model contributes to modeling the dependent variable. The significance of the association is expressed by p values and the magnitude of impact is expressed by coefficients of regression. The choice of exVARs is discussed in the results section and more detailed information is given in Online Resource 4. If levels of estrogens or transcripts were below limit of quan- tification (LOQ) in > 40% of samples (Online Resources 2 and 3), they were not included as exVARs. Analyses of independence of variables Spearman’s rank correlation analysis was performed to iden- tify collinearity between numerical exVARs which might hinder each other selection and/or influence each other p values within the models. In the case of variables with > 1 level below LOD or LOQ, correlation was calculated with randomly distributed ranks for ties 10,000 times and high- est Spearman correlation coefficients and lowest p values were used to rather overestimate collinearity. Relationship between categorical and numerical exVARs was evaluated by comparison of medians using unpaired Wilcoxon tests. Indications for relationships between variables and possible consequences for the selection of exVARs are given for each model in Online Resource 4. For levels of transcripts or estrogens below the respec- tive limit of detection (LOD) and below the respective LOQ, LOD and LOQ were set, respectively. When lev- els of transcripts or estrogens were < LOQ in > 1 sample and ≤ 40% of samples, the levels of the respective tran- script/estrogen were included additionally to the con- tinuous exVAR as the qualitative presence of the exVAR (binary exVARq, compared to levels < LOQ). If, in the computed model, observations with Cook’s Distance > 1 appeared, they were removed and the model was com- puted anew. This process was repeated until no conspicu- ous observations occurred. To achieve normal distribution, dependent variables were logarithmized. Data distribu- tions were evaluated in Quantile–Quantile plots with sim- ulated confidence bands. Constant standard deviations of the errors were evaluated using scale-location plots. To check the model assumption of independent identically distributed errors, the residual vs. fitted values plot was used.fi Obesity and related variables According to the WHO BMI classification, 53% and 13% of women were pre-obese and obese, respectively. The remain- ing women (34%) were of normal weight (Online Resource 6). Thus, compared to the German adult female population (29% pre-obese, 24% obese, and 45% of normal weight; Mensink et al. 2013), a higher percentage was pre-obese, but lower percentages were obese and of normal weight. BMI was included as continuous exVAR into the linear regres- sion models. Regarding reproductive history, 40% of the women par- ticipating in the study were nulliparous. In the age class 35–54 years old, 27% were nulliparous, compared to 21% in the respective general population (Online Resource 5). The study population was further classified regarding par- ity and lobule type of the GLT, the latter reflecting age- and parity-related histological changes within the breast. These changes are most obvious in parous women, where lobules type 2 and 3 (Lob2/3), previously induced during Median oil% in GLT and ADT were 16% and 85%, respectively (Pemp et al. 2019a). In the following lin- ear regression models, oil% were included as continuous exVAR. Directly isolated GLT and GLT isolated from mixed tissue were compared by means of two variables, oil% and relative areas covered by inter- and intrastromal adipocytes in GLT, and p values were adjusted for two comparisons. Despite detaching adhering ADT, GLT isolated from mixed tissues (n = 18) still exhibited significantly higher oil% (Fig. 3) than GLT which could be isolated directly; indi- cating a higher number and/or size of adipocytes within intra- and interlobular stroma. Consistently, microscopic comparison of GLT isolated directly and GLT isolated from mixed tissues revealed a significantly larger relative area covered by inter- and intrastromal adipocytes in GLT derived from mixed tissues (Fig. 3). Thus, in the following sections, these specimens will be referred to as large-adipocyte-area (laa)GLT and small-adipocyte-area GLT. The occurrence of laaGLT was statistically independent of BMI classification of the women donating the tissues (p = 0.40, Chi-square test for trend) and lobule types of GLT specimens (p = 0.28, Chi- square test, Online Resource 7). The occurrence of laaGLT was tested as binary exVAR. Fig. 2   Distribution of age and lobule type as well as allocation of menopausal status (MP) of the women contributing specimens to the present study. GLT of parous women exhibits lobule type 2/3 (Lob2/3) until age-related regression. Obesity and related variables Lob1 predominates in parous women after age-related regression as well as in nulliparous (np) women (see text) Age, reproductive history, and related variables In addition, the regression coefficients (which repre- sent the mean changes in the dependent variables for one unit of change in the respective exVAR while holding other predictors in the models constant), their confidence The age of the 47 women participating in the study ranged from 18–66 years. Most tissues were derived from women in the age group of 40–49 years (Online Resource 5). 3 3 3017 Archives of Toxicology (2020) 94:3013–3025 Menopausal status of the study population was allocated according to the range of age at menopause (46–52 years) observed in German women participating in the EPIC study (n > 27,000; Tsilidis et al. 2011) instead of assessment based on the women’s menstrual cycle characteristics (information not available). Thus, with a high probability, women > 52 and < 46 years old can be assumed to be post- (19%, Fig. 2) and premenopausal (55%), respectively. Women between 46–52 years were grouped as perimenopausal (26%). How- ever, this group is likely to contain pre-, peri-, and post- menopausal women. To reflect continuous influence of age as well as abrupt influence of menopause on dependent vari- ables, both the potential exVARs age and menopausal status were included into the models. pregnancy, regress back to lobule type 1 (Lob1p; Russo and Russo 2004). Nulliparous women (exhibiting lobule type 1, Lob1np) represented the third group of lobule types. 23%, 36%, and 40% of GLT were classified Lob2/3, Lob1p, and Lob1np, respectively (Fig. 2). In the following linear regres- sion models, lobule type was tested as categorial exVAR. Relationship of lobule type with menopausal status and age cannot be completely excluded, but seems to be unlikely (Fig. 2). Age and menopause Cessation of ovarian estrogen production in menopause decreases blood levels of estrogens (Endogenous Hormones Breast Cancer Collaborative Group 2011, 2013) and is thus considered to affect levels of estrogens in breast tissues. Depypere et al. (2015) observed lower median levels of E2 in GLT derived from postmenopausal than from premeno- pausal women, yet no statistical analysis was performed. In the present study, levels of E2 were not directly influenced by postmenopausal status. However, postmenopausal status influenced levels of E1 in GLT and levels of E1-S in ADT negatively (p < 0.05). Furthermore, the ratio of E2 levels in ADT to E2 levels in GLT (ADT/GLT of E2) was also influenced negatively by postmenopausal status (p < 0.05). Interestingly, ADT/GLT of E1 was rather positively influ- enced by postmenopausal status (0.10 > p ≥ 0.05, Fig. 4). Thus, menopause seems not to affect levels of all estrogens in GLT and ADT and ratios ADT/GLT of estrogens in the same way which cannot be explained by a mere decrease in systemic delivery of estrogens via plasma. The continuous Fig. 3   Comparison of oil% and area covered by adipocytes (aa%) in GLT isolated with and without cryosection. For statistical comparison of medians, unpaired Wilcoxon test was used. Boxplots depict 25th percentile, median, and 75th percentile. P values were adjusted for multiple comparisons (n = 2) using Holm’s method status, lobule type, BMI, intake of estrogen-active drugs, and smoking are depicted in Online Resource 8. Smoking and intake of EADs Twelve women (26%) declared being current smokers (2–25 cigarettes/day) and EADs were used by eight women. These information were included as categorial exVARs into the linear regression models. Fig. 2   Distribution of age and lobule type as well as allocation of menopausal status (MP) of the women contributing specimens to the present study. GLT of parous women exhibits lobule type 2/3 (Lob2/3) until age-related regression. Lob1 predominates in parous women after age-related regression as well as in nulliparous (np) women (see text) Mosaic plots characterizing the study population used in the linear regression models regarding the exVARs menopausal 1 Archives of Toxicology (2020) 94:3013–3025 3018 status, lobule type, BMI, intake of estrogen-active drugs, and smoking are depicted in Online Resource 8. Identification of possible exVARs related to estrogen biotransformation in tissues Fig. 3   Comparison of oil% and area covered by adipocytes (aa%) in GLT isolated with and without cryosection. For statistical comparison of medians, unpaired Wilcoxon test was used. Boxplots depict 25th percentile, median, and 75th percentile. P values were adjusted for multiple comparisons (n = 2) using Holm’s method enzymes directly forming or further metabolizing the dependent variable according to Fig. 1. In models with ratios of levels of different estrogens as dependent vari- ables, further exVAR considered were levels of transcripts encoding enzymes directly forming or further metabolizing at least one of the estrogens involved in the ratio. If levels of transcripts or precursor estrogens were < LOQ in > 1 sam- ple and ≤ 40% of samples, the qualitative presence of the respective precursor estrogen or transcript was included as binary exVARq, as well. Variables influencing tissue levels of estrogens identified by multiple linear regression models Previous studies investigating variables associated with lev- els of estrogens in breast tissues either performed no statisti- cal analysis at all or univariate analysis (i.e., comparisons of medians in case of categorical variables and correla- tion analyses in the case of continuous variables, Online Resource 1). In addition, methods nowadays less recom- mended for biospecimen analysis (Labrie et al. 2015) were applied to determine estrogen levels and/or undefined speci- mens were used without specifying the presence of GLT or ADT. Because of these differences, the outcome of the present study is only compared with previous ones if at least either a specific method or specifically GLT or ADT was used. Moreover, results observed in previous studies using tissues derived from women with breast cancer or from both women with and without breast cancer together for statisti- cal analyses were included in Online Resource 1, but are not discussed in the following sections. Identification of possible exVARs related to estrogen biotransformation in tissues Besides age, lifestyle factors, and tissue characteristics, oil% may be influenced by estrogens affecting lipogenesis and adipogenesis (Gao and Dahlman-Wright 2013), and thus, levels of E2 were also considered as exVAR. Furthermore, the classification of GLT regarding adipocyte area laaGLT was considered as exVAR as well. Oil% in GLT were positively influenced by laaGLT (p < 0.05), but no other exVAR was selected (Fig. 4). Interestingly, oil% in ADT were positively influ- enced by levels of E2, age, and specimens derived from nulliparous women (compared to parous women prior to Lobule type The impact of lobule type on estrogen levels has not been investigated yet. Average levels of E1-S in GLT exhibiting the least developed lobule type 1 (derived from nulliparous women, Lob1np, or from parous women after age-related regression, Lob1p) were lower than levels of E1-S in GLT categorized Lob2/3, yet not significantly (0.10 > p ≥ 0.05 and p > 0.1, respectively; Fig. 4). Furthermore, lobule type 1 influenced levels of E2 and E2/E1 in ADT negatively com- pared to Lob2/3 (p < 0.05, Fig. 4). Since oil% influenced levels of some estrogens and ratios thereof in ADT and GLT, variables affecting oil% were investigated as well. Besides age, lifestyle factors, and tissue characteristics, oil% may be influenced by estrogens affecting lipogenesis and adipogenesis (Gao and Dahlman-Wright 2013), and thus, levels of E2 were also considered as exVAR. Furthermore, the classification of GLT regarding adipocyte area laaGLT was considered as exVAR as well. Oil% in GLT were positively influenced by laaGLT (p < 0.05), but no other exVAR was selected (Fig. 4). Interestingly, oil% in ADT were positively influ- enced by levels of E2, age, and specimens derived from nulliparous women (compared to parous women prior to Identification of possible exVARs related to estrogen biotransformation in tissues Further possible continuous exVARs were tissue levels of the direct precursor estrogen(s) and of transcripts encoding 1 3 Archives of Toxicology (2020) 94:3013–3025 3019 Fig. 4   Influence of various exVARs on levels of estrogens as well as on ratios thereof and oil% in GLT and ADT (dependent variables) identified by multiple linear regression models using stepwise for- ward selection as detailed in Online Resource 4. For each model, the adjusted coefficient of determination (R2), and ratio of the number of observations (i.e., biospecimens) to exVAR (O/exVAR) after forward selection of variables is given adjusted coefficient of determination (R2), and ratio of the number of observations (i.e., biospecimens) to exVAR (O/exVAR) after forward selection of variables is given Fig. 4   Influence of various exVARs on levels of estrogens as well as on ratios thereof and oil% in GLT and ADT (dependent variables) identified by multiple linear regression models using stepwise for- ward selection as detailed in Online Resource 4. For each model, the exVAR age did not directly influence levels of any estrogen or ratio thereof significantly (Fig. 4). influenced levels of E1 in GLT (positively, p < 0.05), levels of E1-S in GLT (negatively, p < 0.05), and E1-S/E1 in both GLT and ADT (negatively, p < 0.05; Fig. 4). Interestingly, levels of 2-MeO-E1 and E2 (which are more and compara- bly lipophilic than E1) were not influenced by oil% in ADT (Fig. 4), thereby rendering a mere physicochemical effect less likely. In line with the influence of oil% on the respec- tive estrogens, ADT/GLT of both E2 and E1 were positively influenced by oil% in ADT but negatively in GLT (p < 0.05). %l influenced levels of E1 in GLT (positively, p < 0.05), levels of E1-S in GLT (negatively, p < 0.05), and E1-S/E1 in both GLT and ADT (negatively, p < 0.05; Fig. 4). Interestingly, levels of 2-MeO-E1 and E2 (which are more and compara- bly lipophilic than E1) were not influenced by oil% in ADT (Fig. 4), thereby rendering a mere physicochemical effect less likely. In line with the influence of oil% on the respec- tive estrogens, ADT/GLT of both E2 and E1 were positively influenced by oil% in ADT but negatively in GLT (p < 0.05). Since oil% influenced levels of some estrogens and ratios thereof in ADT and GLT, variables affecting oil% were investigated as well. Transcripts encoding enzymes involved in estrogen (biotrans)formation Savolainen-Peltonen et al. (2014) observed no correlation of levels of CYP19A1, i.e., aromatase, STS, and HSD17B1, with levels of E2 in ADT and the respective transcript levels did also not significantly influence levels of E2 in ADT in the present study. However, in the present study, levels of E2 in ADT were influenced positively by levels of the transcript encoding GUSB (Fig. 4, p < 0.05), the enzyme hydrolyzing estrogen glucuronides (Fig. 1). Estrogen glucuronides are mostly associated with elimination from tissues and body but may also contribute to intratissue levels of estrogens, even though GUSB and substrates only meet in a highly controlled manner (Naz et al. 2013). Furthermore, levels of E2 in ADT were influenced negatively (p < 0.05) by levels of transcripts encoding HSD17B2, the enzyme forming E1 by oxidation of E2 (Fig. 1). Congruently, levels of E1 in ADT were positively influenced by levels of HSD17B2. Moreover, levels of CYP19A1 influenced levels of E1 in ADT posi- tively (Fig. 4, p < 0.05). The presence of transcripts encoding the conjugating enzyme UGT1A9 and levels of transcripts encoding CYP1A1 influenced the levels of the respective substrates (i.e., E1, 2-MeO-E1, and E2, respectively) in ADT negatively (0.10 > p ≥ 0.05). Likewise, levels of SULT1A3/4 influenced E1-S in GLT positively (0.10 > p ≥ 0.05). Forward selection of exVARs into models describing estrogen levels in ADT and GLT identified levels of further transcripts, yet the associations exhibited p values ≥ 0.10 (Fig. 4). l Besides BMI, smoking has also been associated with higher levels of E2 and E1 in blood of postmenopausal women (Endogenous Hormones Breast Cancer Collabora- tive Group 2011) but not of premenopausal women (Endog- enous Hormones Breast Cancer Collaborative Group 2013). No studies investigating the impact of smoking on levels of estrogens in breast tissues have been identified. In the present study, smoking influenced levels of E1 in ADT positively and, congruently, E2/E1 in ADT negatively (both p < 0.05, Fig. 4). In all studies analyzing estrogens in breast tissues of women without breast cancer, the intake of EADs for oral contraception or hormone replacement therapy was either an exclusion criterium or not considered in statistical analy- ses (Online Resource 1). In the present study, the intake of ethinyl estradiol did not influence the levels of E2 in GLT or ADT (Fig. 4). Lifestyle‑associated variables A positive association between BMI and estrogens in serum has been observed in pre- (E2, E1; Endogenous Hormones Breast Cancer Collaborative Group 2013) and postmeno- pausal women (E2, E1, E1-S; Endogenous Hormones Breast Cancer Collaborative Group 2003, 2011). The common interpretation is that an increase in BMI leads to an increase in the mass of adipose tissue within the whole body, accom- panied by a change in intra- and extramammary function of adipose tissue (Yaghjyan and Colditz 2011; Brown 2014). Consequently, a higher amount of estrogens is produced and distributed within the body via blood (Lønning et al. 2011), contributing to estrogen levels in breast tissue. Yet, no stud- ies investigating associations between BMI and estrogen levels in breast tissues derived from women without breast cancer have been identified. In the present study, BMI influ- enced levels of E2 in GLT (0.10 > p ≥ 0.05) as well as E1 in GLT and ADT (p < 0.05) positively. In contrast, tissue levels of E1-S were not influenced by BMI (Fig. 4). Oil% Median oil% in GLT and ADT were 16% and 85%, respec- tively (Pemp et al. 2019a). Estrogen levels might be affected by oil% via: (i) physicochemical distribution of the lipophilic molecules E2 and E1 and the more hydrophilic E1-S and (ii) cell-specific enzyme expression in stromal adipocytes. Oil% 1 3 3020 Archives of Toxicology (2020) 94:3013–3025 Biotransformation precursors age-related regression, all p < 0.05), and not significantly, by BMI (p > 0.1, with no apparent statistical reason such as collinearity interfering with the exVAR BMI, Online Resource 4). No studies investigating correlations among estrogens in breast tissues derived from women without breast cancer have been identified. In the present study, biotransforma- tion precursors of the respective estrogens influenced tissue levels of E2, E1, E1-S, and 2-MeO-E1 positively (p < 0.05, except for E1-S as precursor for E1 in ADT 0.10 > p ≥ 0.05; Fig. 4). Thus, exVARs affecting levels of, e.g., E1, may indirectly contribute to levels of E2, E1-S, and 2-MeO-E1. Interestingly, whereas levels of E1 in GLT were positively influenced by levels of E1-S, levels of E1 in ADT were posi- tively influenced by the qualitative presence of both E1-G (p < 0.05) and E1-S (0.10 > p ≥ 0.05). Notably, both linear regression models exhibited low R2 values (Fig. 4) suggesting either important variables miss- ing in the model (which seems likely in this case) or large variations within the data set. Transcripts encoding enzymes involved in estrogen (biotrans)formation Yet, intake of ethinyl estradiol influenced ADT/GLT of E2, levels of E1 in GLT (p < 0.05), and E1-S/ E1 in ADT negatively (0.10 > p ≥ 0.05). In contrast, intake of E2-releasing drugs (containing E2 or E2 valerate) used for hormone replacement therapy positively influenced levels of E1-S and E1-S/E1 in ADT (p < 0.05), but did not influence levels of E2 or E1 in GLT or ADT. Regulation of transcription of genes encoding enzymes by the respective substrates (up-) and products (down-), is a common biochemical feedback mechanism. Furthermore, ligand-activated transcription factors are involved in the reg- ulation of transcription of genes of biotransforming enzyme families; e.g., CYP (Tralau and Luch 2013), SULT (Runge- Morris et al. 2013), and UGT (Hu et al. 2014). Positive asso- ciations of levels of STS as well as presence of UGT1A9 and UGT1A10 were observed with the respective substrates of 1 3 3 Archives of Toxicology (2020) 94:3013–3025 3021 the encoded enzymes; i.e., E1-S in ADT, E1 in GLT and E1 in ADT, respectively (p < 0.05; Fig. 4). UGT1A9 (Cho et al. 2016) and UGT1A10 (Starlard-Davenport et al. 2008) may be regulated by activated ESR1, whereas STS may be regulated via G-protein-coupled ESR action (Gilligan et al. 2017). the encoded enzymes; i.e., E1-S in ADT, E1 in GLT and E1 in ADT, respectively (p < 0.05; Fig. 4). UGT1A9 (Cho et al. 2016) and UGT1A10 (Starlard-Davenport et al. 2008) may be regulated by activated ESR1, whereas STS may be regulated via G-protein-coupled ESR action (Gilligan et al. 2017). prerequisites (Yaghjyan and Colditz 2011; Sherman et al. 2012; Rosner et al. 2013) for data acquisition concerning sample characterization as well as specificity and reliability of estrogen analysis (Pemp et al. 2019a). However, mammoplasty specimens raise concern regard- ing sample bias, in particular (i) “young age”, (ii) “obesity”, and (iii) “extremely large fatty breasts” (Sherman et al. 2012), thus putatively reflecting a specific subpopulation. Most specimens were derived from women 40–49, which is also the modal age group of the respective general popula- tion (Online Resource 5). Yet, the study population indeed lacked women older than 66 years and the proportion of pre-obese and obese women was higher and lower than in the general population, respectively (Online Resource 6). Moreover, 38% of specimens were classified as laaGLT and exhibited higher oil% than GLT which could be isolated directly (section “Obesity and related variables”). Transcripts encoding enzymes involved in estrogen (biotrans)formation Since no data on the occurrence of laaGLT in the general female population are available, it is currently unknown whether women undergoing mammoplasty are predisposed to laa- GLT. Oil% significantly influenced levels of E1, E1-S in GLT, E1-S/E1 in GLT and ADT, as well as ADT/GLT of E1 and E2 and should, therefore, be considered in sample characterization of human breast biospecimens. Of note, oil% in GLT were not influenced by any exVAR deducible by questionnaire. Since levels of CYP19A1, GUSB, and HSD17B2 influ- enced levels of estrogens in ADT significantly, exVARs influencing these variables were investigated, as well. Besides exVARs associated with physiology and lifestyle, levels of transcripts known to be directly or indirectly involved in regulation of phase I and phase II biotransfor- mation were included, since little is known about specific regulation of the transcription of the genes encoding these enzymes (Naz et al. 2013; Zhao et al. 2016; Hilborn et al. 2017). Levels of CYP19A1 in ADT were exclusively influ- enced by the intake of ethinyl estradiol (negatively, Fig. 4, p < 0.05). It is known that the ovarial synthesis of estrogens is negatively regulated by estrogen-active compounds (Fleis- chman et al. 2010). Furthermore, CYP19A1 was detected less frequently in the endometrium of women taking oral contraceptives containing ethinyl estradiol than in non-users (Maia et al. 2013). Low R2 value of the model (Fig. 4) sug- gests at least one other important variable missing in the model (e.g., transcript levels of glucocorticoid receptor; Zhao et al. 2016). Intake of exogenous estrogens, menopausal status, BMI, and smoking were previously shown to be associated with levels of estrogens in blood (Endogenous Hormones Breast Cancer Collaborative Group 2003,2011; Fleischman et al. 2010, 2013; Gaudet et al. 2017). The general consensus is that blood levels of estrogens contribute to breast tissue levels, yet whether levels of circulating estrogens serve as surrogate for levels of estrogens in breast tissues, or more precisely, in GLT or ADT, is a matter of debate (Lønning et al. 2011; Bulun et al. 2012; Colditz and Bohlke 2014; Labrie 2015; Stanczyk et al. 2015; Vihma et al. 2016). Levels of GUSB in ADT were significantly influenced by levels of AHR, ESR2 (positively), and qualitative presence of NR1I3 (negatively). Binding sites for transcription fac- tors such as Sp1 and AP-2 (Naz et al. Transcripts encoding enzymes involved in estrogen (biotrans)formation 2013) in the promo- tor of the GUSB gene provide a link to estrogen signaling (Pellikainen and Kosma 2007; Safe and Kim 2008). BMI influenced levels of GUSB positively (0.10 > p ≥ 0.05). Most interestingly, levels of HSD17B2 in ADT were significantly influenced by BMI, smoking, and postmenopausal status as well as by levels of ARNT (negatively) and levels of ESR2 and N1I2 (positively). Transcription of the HSD17B2 gene is regulated by retinoic acid via RAR alpha/RXR alpha teth- ered to transcription factors Sp1 and Sp3 on the HSD17B2 promoter (Cheng et al. 2008), which provides a link to ESR- mediated signaling. An obvious link between ARNT or N1I2 and expression of HSD17B2 has not been described yet. In the present study, variables known to affect levels of circulating estrogens indeed influenced estrogen levels in breast tissues, as well (Fig. 5).f However, these exVARs affecting levels of circulating estrogens by targeting ovarial and adiposal production of estrogens did obviously not suffice to explain the variances in estrogen levels within the breast. In addition, estrogen levels in both GLT and ADT were further influenced by levels or the presence of their precursor estrogens and levels of transcripts encoding enzymes involved in estrogen bio- transformation. Interestingly, whereas (as expected, Mueller et al. 2015) E1-S seems to represent a source of E1 in breast GLT, intratissue levels of E1 in ADT seem additional to be regulated by glucuronidation via UGT1A9 (Fig. 5). Further- more, although E2-3-G was not detected in human breast tissues, in the light of the contribution of levels of GUSB to Concluding this section, it should be emphasized that lack of influence of transcript levels does not exclude the contri- bution of the respective enzyme activities to estrogen levels. Relevance Including E2, E1, E1-S, and 2-MeO-E1, the present study encompasses the major estrogens detectable in breast tis- sues derived from women without breast cancer. In contrast to all previous studies, the present study fulfilled published 1 3022 Archives of Toxicology (2020) 94:3013–3025 Fig. 5   ExVARs associated with intramammary biotransformation pathways, lifestyle, and reproductive history influencing levels of estrogens (framed) as well as transcript levels of CYP19A1, GUSB, HSD17B2 in ADT with p < 0.05 (exVARs written in black color) or 0.10 > p ≥ 0.05 (exVARs written in grey color) identified by multi- ple regression models using stepwise forward selection. Correlations between estrogen levels in GLT and ADT determined by Spearman correlation analyses were described in Pemp et al. (2019a). EE ethi- nyl estradiol, ERD E2-releasing drug, PostMP postmenopausal status Fig. 5   ExVARs associated with intramammary biotransformation pathways, lifestyle, and reproductive history influencing levels of estrogens (framed) as well as transcript levels of CYP19A1, GUSB, HSD17B2 in ADT with p < 0.05 (exVARs written in black color) or 0.10 > p ≥ 0.05 (exVARs written in grey color) identified by multi- ple regression models using stepwise forward selection. Correlations between estrogen levels in GLT and ADT determined by Spearman correlation analyses were described in Pemp et al. (2019a). EE ethi- nyl estradiol, ERD E2-releasing drug, PostMP postmenopausal status E1 in ADT, i.e., levels of CYP19A1, HSD17B2, presence of UGT1A9, E1-S, and E1-G, as well as BMI and smoking (Fig. 5). E1 in ADT, i.e., levels of CYP19A1, HSD17B2, presence of UGT1A9, E1-S, and E1-G, as well as BMI and smoking (Fig. 5). levels of E2 in ADT and a ten times higher LOD for E2-3-G compared to E1-G (Pemp et al. 2019a), E2 glucuronides cannot be excluded to represent a source of estrogens for the human breast, as well. Inhibition of CYP19A1 suggested in breast cancer pre- vention (Advani and Moreno-Aspitia 2014; Colditz and Bohlke 2014) could thus lower intratissue E2 levels not only by systemic but also by intracrine mechanisms. However, systemic alteration of E2 biosynthesis harbors the risk of losing its beneficial biological effects, e.g., in bone health (Advani and Moreno-Aspitia 2014). Thus, drugs in develop- ment for the treatment of endocrine disorders by targeting enzymes involved in more organ-specific E2 homeostasis (e.g., inhibitors of HSD17B2 and STS; Konings et al. 2018) may also be successful in breast cancer chemoprevention. 3 Compliance with ethical standards Depypere HT, Bolca S, Bracke M, Delanghe J, Comhaire F, Blondeel P (2015) The serum estradiol concentration is the main determinant of the estradiol concentration in normal breast tissue. Maturitas 81:42–45. https​://doi.org/10.1016/j.matur​itas.2015.01.014 Conflict of interest  The authors declare that they have no conflict of interest. Endogenous Hormones Breast Cancer Collaborative Group (2002) Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J Natl Cancer Inst 94:606–616 Ethics approval  This study was performed in line with the principles of the Declaration of Helsinki and its later amendments. Approval was granted by the Ethics Committee of the University of Würzburg, Ger- many (reference numbers 74/10 and 168/10). Endogenous Hormones Breast Cancer Collaborative Group (2003) Body mass index, serum sex hormones, and breast cancer risk in postmenopausal women. J Natl Cancer Inst 95:1218–1226 Consent to participate  Informed consent was obtained from all indi- vidual participants included in the study. Endogenous Hormones Breast Cancer Collaborative Group (2011) Cir- culating sex hormones and breast cancer risk factors in postmeno- pausal women: reanalysis of 13 studies. Br J Cancer 105:709–722. https​://doi.org/10.1038/bjc.2011.254 Consent for publication  All individual participants consented to pub- lish their anonymized data. Endogenous Hormones Breast Cancer Collaborative Group (2013) Sex hormones and risk of breast cancer in premenopausal women: a collaborative reanalysis of individual participant data from seven prospective studies. Lancet Oncol 14:1009–1019. https​://doi. org/10.1016/S1470​-2045(13)70301​-2f Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. Relevance Further supporting the role of intratissue biotransforma- tion, levels of E2 and of E1 in ADT were also significantly influenced by levels of HSD17B2. Recently, it was shown that levels of Hsd17b enzymes significantly influenced intramammary levels of E2 in the ACI rat model of tumori- genesis (Pemp et al 2019b), as well, supporting the impor- tant role of HSD17Bs in intramammary estrogen homeosta- sis (Hilborn et al. 2017) across species. Furthermore, the present study not only supports the commonly accepted importance of CYP19A1 in estrogen homeostasis, but suggests its role within the breast tissue in addition to a systemic effect. Avoiding indirect associa- tions in linear regression models, the influence of exVARs present in ADT on levels of E2 in GLT was not tested in the present study. However, due to correlations between estrogen levels in GLT and ADT (Pemp et al. 2019a) and the influ- ence of precursor estrogens on the respective estrogen levels shown in the present study, levels of E2 in GLT might be affected indirectly by any variable significantly influencing In conclusion, a thorough characterization of specimens enabled taking into account variables related to obesity and “extremely fatty breasts” during statistical analyses. Tissue characterization of GLT derived from mammoplasty (and possibly also of biopsy) specimens by oil% as well as by lobule type seems to be advisable to prevent sample bias. Novel insights in estrogen homeostasis in the normal human breast GLT and ADT support contribution of varia- bles affecting both extra- and intratissue (biotrans)formation 1 3 3 Archives of Toxicology (2020) 94:3013–3025 3023 of estrogens and suggest a central role of E1 levels in breast ADT homeostasis. The susceptibility of estrogen homeosta- sis to systemic and tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the environment possible. Brown KA (2014) Impact of obesity on mammary gland inflammation and local estrogen production. J Mammary Gland Biol Neoplasia 19:183–189. https​://doi.org/10.1007/s1091​1-014-9321-0 p g Bulun SE, Chen D, Moy I, Brooks DC, Zhao H (2012) Aromatase, breast cancer and obesity: a complex interaction. Trends Endo- crinol Metab 23:83–89. https​://doi.org/10.1016/j.tem.2011.10.003 Cheng YH, Yin P, Xue Q, Yilmaz B, Dawson MI, Bulun SE (2008) Retinoic acid (RA) regulates 17beta-hydroxysteroid dehydroge- nase type 2 expression in endometrium: interaction of RA recep- tors with specificity protein (SP) 1/SP3 for estradiol metabolism. J Clin Endocrinol Metab 93:1915–1923. Relevance https​://doi.org/10.1210/ jc.2007-1536 Acknowledgements  Open Access funding provided by Projekt DEAL. The authors would like to thank Prof. Peter Eckert, Prof. Rafael Jakubi- etz, Dr. Iva Neshkova, and Dr. Ulrike Waldhofen (mammoplasty specimens). Cho SJ, Ning M, Zhang Y, Rubin LH, Jeong H (2016) 17beta-Estra- diol up-regulates Udp-glucuronosyltransferase 1a9 expression via estrogen receptor alpha. Acta Pharm Sin B 6:504–509. https​://doi. org/10.1016/j.apsb.2016.04.005 Funding  This study was funded by the German Research Founda- tion with a grant to L. Lehmann (DFG LE 1329/10–1) and is part of the joint research project, “Isoflavones: Cross-species comparison of metabolism, estrogen sensitivity, epigenetics and carcinogenesis”. Colditz GA, Bohlke K (2014) Priorities for the primary prevention of breast cancer. CA Cancer J Clin 64:186–194. https​://doi. org/10.3322/caac.21225​ Availability of data and material  The datasets generated during and/or analyzed during this study are included in this published article [and its supplementary information files] or are available from the correspond- ing author on reasonable request. Collaborative Group on Hormonal Factors in Breast Cancer (2019) Type and timing of menopausal hormone therapy and breast can- cer risk: individual participant meta-analysis of the worldwide epidemiological evidence. Lancet. https​://doi.org/10.1016/S0140​ -6736(19)31709​-X Compliance with ethical standards Figueroa JD, Pfeiffer RM, Patel DA, Linville L, Brinton LA, Gierach GL, Yang XR, Papathomas D, Visscher D, Mies C, Degnim AC, Anderson WF, Hewitt S, Khodr ZG, Clare SE, Storniolo AM, Sherman ME (2014) Terminal duct lobular unit involution of the normal breast: implications for breast cancer etiology. J Natl Can- cer Inst 106:286. https​://doi.org/10.1093/jnci/dju28​6 Fleischman DS, Navarrete CD, Fessler DM (2010) Oral contraceptives suppress ovarian hormone production. Psychol Sci 21:750–752. https​://doi.org/10.1177/09567​97610​36806​2 Fleming JM, Miller TC, Quinones M et al (2010) The normal breast microenvironment of premenopausal women differentially influ- ences the behavior of breast cancer cells in vitro and in vivo. BMC Med 8:27. https​://doi.org/10.1186/1741-7015-8-27 References Gao H, Dahlman-Wright K (2013) Implications of estrogen receptor alpha and estrogen receptor beta for adipose tissue functions and cardiometabolic complications. Horm Mol Biol Clin Investig 15:81–90. https​://doi.org/10.1515/hmbci​-2013-0021 Advani P, Moreno-Aspitia A (2014) Current strategies for the pre- vention of breast cancer. Breast cancer 6:59–71. https​://doi. org/10.2147/BCTT.S3911​4 Gaudet MM, Carter BD, Brinton LA, Falk R, Gram IT, Luo J, Milne RL, Nyante SJ, Weiderpass E, Beane Freeman LE, Sandler DP, Robien K, Anderson KE, Giles GG, Chen WY, Feskanich D, Braaten T, Isaacs C, Butler LM, Koh WP, Wolk A, Adami HO, White E, Margolis KL, Thun MJ, Gapstur SM (2017) Pooled g Austin PC, Steyerberg EW (2015) The number of subjects per variable required in linear regression analyses. J Clin Epidemiol 68:627– 636. https​://doi.org/10.1016/j.jclin​epi.2014.12.014 1 3 3024 Archives of Toxicology (2020) 94:3013–3025 replacement therapy. Rejuvenation Res 16:352–363. https​://doi. org/10.1089/rej.2013.1407 analysis of active cigarette smoking and invasive breast cancer risk in 14 cohort studies. Int J Epidemiol 46:881–893. https​://doi. org/10.1093/ije/dyw28​8 Pellikainen JM, Kosma VM (2007) Activator protein-2 in carcinogen- esis with a special reference to breast cancer–a mini review. Int J Cancer 120:2061–2067. https​://doi.org/10.1002/ijc.22648​ g j y Gilligan LC, Gondal A, Tang V, Hussain MT, Arvaniti A, Hewitt AM, Foster PA (2017) Estrone sulfate transport and steroid sulfatase activity in colorectal cancer: implications for hormone replace- ment therapy. Front Pharmacol. https​://doi.org/10.3389/fphar​ .2017.00103​ Pemp D, Kleider C, Schmalbach K, Hauptstein R, Geppert LN, Köll- mann C, Ickstadt K, Eckert P, Neshkova I, Jakubietz R, Esch HL, Lehmann L (2019a) Qualitative and quantitative differences in estrogen biotransformation in human breast glandular and adipose tissues: implications for studies using mammary biospecimens. Arch Toxicol 93:2823–2833. https​://doi.org/10.1007/s0020​4-019- 02564​-w Gram IT, Park S-Y, Maskarinec G, Wilkens LR, Haiman CA, Le Marchand L (2019) Smoking and breast cancer risk by race/eth- nicity and oestrogen and progesterone receptor status: the Multi- ethnic Cohort (MEC) study. Int J Epidemiol 48:501–511. https​:// doi.org/10.1093/ije/dyy29​0 Pemp D, Esch HL, Hauptstein R, Möller FJ, Zierau O, Bosland MC, Geppert LN, Kleider C, Schlereth K, Vollmer G, Lehmann L (2019b) Novel insight in estrogen homeostasis and bioactivity in the ACI rat model of estrogen-induced mammary gland car- cinogenesis. Arch Toxicol 93:1979–1992. https​://doi.org/10.1007/ s0020​4-019-02483​-w Grosse Y, Baan R, Straif K, Secretan B, El-Ghissassi F, Bouvard V, Benbrahim-Tallaa L, Guha N, Galichet L, Cogliano V, W.O Inter- national Agency for Research on Cancer Monograph Working Group (2009) A review of human carcinogens—Part A: phar- maceuticals. References Lancet Oncol 10:13. https​://doi.org/10.1016/s1470​ -2045(08)70286​-9 Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME (2013) Challenges to the measurement of estradiol: an endocrine society position statement. J Clin Endocrinol Metab 98:1376–1387. https​ ://doi.org/10.1210/jc.2012-3780 Hilborn E, Stal O, Jansson A (2017) Estrogen and androgen-converting enzymes 17beta-hydroxysteroid dehydrogenase and their involve- ment in cancer: with a special focus on 17beta-hydroxysteroid dehydrogenase type 1, 2, and breast cancer. Oncotarget 8:30552– 30562. https​://doi.org/10.18632​/oncot​arget​.15547​ Runge-Morris M, Kocarek TA, Falany CN (2013) Regulation of the cytosolic sulfotransferases by nuclear receptors. Drug Metab Rev 45:15–33. https​://doi.org/10.3109/03602​532.2012.74879​4 Hu DG, Meech R, McKinnon RA, Mackenzie PI (2014) Transcrip- tional regulation of human Udp-glucuronosyltransferase genes. Drug Metab Rev 46:421–458. https​://doi.org/10.3109/03602​ 532.2014.97303​7 Russo J, Russo IH (2004) Development of the human breast. Maturitas 49:2–15. https​://doi.org/10.1016/j.matur​itas.2004.04.011 Safe S, Kim K (2008) Non-classical genomic estrogen receptor (ER)/ specificity protein and ER/activating protein-1 signaling path- ways. J Mol Endocrinol 41:263–275. https​://doi.org/10.1677/ JME-08-0103 Jones ME, Schoemaker MJ, Wright LB, Ashworth A, Swerdlow AJ (2017) Smoking and risk of breast cancer in the generations study cohort. Breast Cancer Res 19:118. https​://doi.org/10.1186/s1305​ 8-017-0908-4 Savolainen-Peltonen H, Vihma V, Leidenius M, Wang F, Turpeinen U, Hamalainen E, Tikkanen MJ, Mikkola TS (2014) Breast adipose tissue estrogen metabolism in postmenopausal women with or without breast cancer. J Clin Endocrinol Metab 99:E2661–2667. https​://doi.org/10.1210/jc.2014-2550 Konings G, Brentjens L, Delvoux B, Linnanen T, Cornel K, Koskimies P, Bongers M, Kruitwagen R, Xanthoulea S, Romano A (2018) Intracrine regulation of estrogen and other sex steroid levels in endometrium and non-gynecological tissues; pathology, physi- ology, and drug discovery. Front Pharmacol 9:940. https​://doi. org/10.3389/fphar​.2018.00940​ Sherman ME, Figueroa JD, Henry JE, Clare SE, Rufenbarger C, Storni- olo AM (2012) The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center: a unique resource for defining the “Molecular Histology” of the breast. Cancer Prev Res (Phila) 5:528–535. https​://doi.org/10.1158/1940-6207.CAPR-11-0234 g p Labrie F (2015) All sex steroids are made intracellularly in periph- eral tissues by the mechanisms of intracrinology after meno- pause. J Steroid Biochem Mol Biol 145C:133–138. https​://doi. org/10.1016/j.jsbmb​.2014.06.001 Stanczyk FZ, Mathews BW, Sherman ME (2015) Relationships of sex steroid hormone levels in benign and cancerous breast tissue and blood: a critical appraisal of current science. Steroids 99:91–102. https​://doi.org/10.1016/j.stero​ids.2014.12.011 Labrie F, Ke Y, Gonthier R, Belanger A (2015) Why both LC-MS/MS and FDA-compliant validation are essential for accurate estro- gen assays? J Steroid Biochem Mol Biol 149:89–91. https​://doi. Yager JD, Davidson NE (2006) Estrogen carcinogenesis in breast can- cer. N Engl J Med 354:270–282. https​://doi.org/10.1056/NEJMr​ a0507​76 Yager JD, Davidson NE (2006) Estrogen carcinogenesis in breast can- cer. N Engl J Med 354:270–282. https​://doi.org/10.1056/NEJMr​ a0507​76 Yaghjyan L, Colditz GA (2011) Estrogens in the breast tissue: a sys- tematic review. Cancer Causes Control 22:529–540. https​://doi. org/10.1007/s1055​2-011-9729-4 Zhao H, Zhou L, Shangguan AJ, Bulun SE (2016) Aromatase expres- sion and regulation in breast and endometrial cancer. J Mol Endo- crinol 57:R19–33. https​://doi.org/10.1530/JME-15-0310 References org/10.1016/j.jsbmb​.2015.02.003 Starlard-Davenport A, Lyn-Cook B, Radominska-Pandya A (2008) Novel identification of UDP-glucuronosyltransferase 1A10 as an estrogen-regulated target gene. Steroids 73:139–147. https​://doi. org/10.1016/j.stero​ids.2007.09.007 Lønning PE, Haynes BP, Straume AH, Dunbier A, Helle H, Knapp- skog S, Dowsett M (2011) Exploring breast cancer estrogen dis- position: the basis for endocrine manipulation. Clin Cancer Res 17:4948–4958. https​://doi.org/10.1158/1078-0432.CCR-11-0043f Tralau T, Luch A (2013) The evolution of our understanding of endo-xenobiotic crosstalk and cytochrome P450 regulation and the therapeutic implications. Expert Opin Drug Metab Toxicol 9:1541–1554. https​://doi.org/10.1517/17425​255.2013.82869​2 Maia H Jr, Haddad C, Pinheiro N, Casoy J (2013) The effect of oral contraceptives on aromatase and Cox-2 expression in the endo- metrium of patients with idiopathic menorrhagia or adenomyosis. Int J Womens Health 5:293–299. https​://doi.org/10.2147/IJWH. S4509​3 Tsilidis KK, Allen NE, Key TJ, Dossus L, Lukanova A, Bakken K et al (2011) Oral contraceptive use and reproductive factors and risk of ovarian cancer in the European Prospective Investigation into Cancer and Nutrition. Br J Cancer 105:1436–1442. https​://doi. org/10.1038/bjc.2011.371 Mensink GB, Schienkiewitz A, Haftenberger M, Lampert T, Ziese T, Scheidt-Nave C (2013) Overweight and obesity in Germany: results of the German Health Interview and Examination Sur- vey for Adults (DEGS1). Bundesgesundheitsblatt Gesund- heitsforschung Gesundheitsschutz 56:786–794. https​://doi. org/10.1007/s0010​3-012-1656-3 Vihma V, Wang F, Savolainen-Peltonen H, Turpeinen U, Hamalainen E, Leidenius M, Mikkola TS, Tikkanen MJ (2016) Quantitative determination of estrone by liquid chromatography-tandem mass spectrometry in subcutaneous adipose tissue from the breast in postmenopausal women. J Steroid Biochem Mol Biol 155:120– 125. https​://doi.org/10.1016/j.jsbmb​.2015.10.004 Mueller JW, Gilligan LC, Idkowiak J, Arlt W, Foster PA (2015) The regulation of steroid action by sulfation and desulfation. Endocr Rev 36:526–563. https​://doi.org/10.1210/er.2015-1036 Naz H, Islam A, Waheed A, Sly WS, Ahmad F, Hassan I (2013) Human beta-glucuronidase: structure, function, and application in enzyme 1 3 Archives of Toxicology (2020) 94:3013–3025 3025 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Yaghjyan L, Colditz GA (2011) Estrogens in the breast tissue: a sys- tematic review. Cancer Causes Control 22:529–540. https​://doi. org/10.1007/s1055​2-011-9729-4 Zhao H, Zhou L, Shangguan AJ, Bulun SE (2016) Aromatase expres- sion and regulation in breast and endometrial cancer. J Mol Endo- crinol 57:R19–33. https​://doi.org/10.1530/JME-15-0310 1 3
https://openalex.org/W2154527967
https://www.scielo.br/j/rsp/a/ZNfYvhwcJmnM4gsst96C4xb/?lang=pt&format=pdf
Portuguese
null
Estatísticas de mortalidade por causas múltiplas: novas perspectivas com o sistema ACME
Revista de saúde pública/Revista de Saúde Pública
1,986
cc-by
2,963
INTRODUÇÃO Informações sobre a mortalidade desempenham importante papel no planejamento local, regional e nacional. Dados sobre mortalidade contribuem na identificação da situação demográfica do país e per- mitem fazer inferências sobre o futuro. A par da perspectiva demográfica, os dados sobre mortalidade são medidas importantes das condições sócio-econô- micas e de saúde. Por seu intermédio mostra-se o progresso numa das áreas de maior preocupação do homem, qual seja, a do prolongamento da vida e prevenção da morte prematura. As chamadas esta- tísticas de mortalidade também se constituem em indicadores muito sensíveis de diferenças existentes nas populações, possibilitando a identificação de grupos de maior risco a fim de implementar progra- mas especiais de saúde e de desenvolvimento. São úteis, por outro lado, para a avaliação de resultados dos programas já realizados. Além disso, os padrões de mortalidade relacionam-se com outros aspectos sociais, tais como a capacidade de trabalho e a ferti- lidade11. morte, ou (b) as circunstâncias do acidente ou vio- lência que produziram a lesão fatal"6. Para um determinado óbito, a causa básica de morte deriva das informações dadas pelo médico ao certificar o óbito, utilizando, para sua codificação, a estrutura, as regras de seleção e de modificação e as disposições correlatas da Classificação Internacional de Doenças. As regras de seleção permitem a identi- ficação da causa básica de morte tendo em vista a posição das afecções mencionadas pelo médico no Modelo Internacional de Atestado Médico da Causa de Morte e a relação causal entre as mesmas. As re- gras de modificação têm a finalidade de aprimorar a informação sobre a causa básica de morte dando preferência a determinadas afecções em detrimento de outras ou associando duas ou mais afecções se- gundo uma rubrica da Classificação Internacional de Doenças. Tais regras para a codificação da causa básica de morte vêm sendo apresentadas na Classifi- cação Internacional de Doenças há décadas, com a finalidade de padronizar sua identificação, fato que contribui para a comparabilidade internacional de estatísticas de mortalidade10. Os estudos estatísticos de mortalidade levam em consideração numerosas variáveis, tais como sexo, idade, estado civil, local de residência e ocor- rência, ocupação, entre outras, utilizando-as para a elaboração de análises pormenorizadas. Assim, apenas como exemplo, quanto à variável idade, a mortalidade infantil é um dos mais sensíveis indica- dores de níveis de saúde de populações. Além destas variáveis, a causa de morte constitui-se em outra das mais importantes formas de sua análise. * Trabalho realizado no Centro Brasileiro de Classificação de Doenças, Departamento de Epidemiologia da Faculdade de Saúde Pública da Universidade de São Paulo - Av. Dr. Arnaldo, 715 - 01255 - São Paulo, SP - Brasil. ** Do Departamento de Epidemiologia da Faculdade de Saúde Pública da Universidade de São Paulo - Av. Dr. Arnaldo, 715 - 01255 - São Paulo, SP - Brasil. ESTATÍSTICAS DE MORTALIDADE POR CAUSAS MÚLTIPLAS NOVAS PERSPECTIVAS COM O SISTEMA ACME * Augusto Hasiak Santo ** Ruy Laurenti** INTRODUÇÃO O conheci- mento preciso de padrões de mortalidade segundo a causa de morte permite a pesquisa de eventuais fato- res etiológicos e a tomada de decisões visando a prevenção desses fatores. O trabalho da identificação e atribuição de um código da Classificação Internacional de Doenças à causa básica é realizado pelo codificador de causas de morte que, para tanto, recebe treinamento espe- cializado em cursos apropriados. É um trabalho que está sujeito a falhas que prejudicam a qualidade da codificação; tais falhas devem-se a vários fatores, como os ligados ao tipo de treinamento recebido, diferenças de interpretação de relações causais entre afecções, esquecimento de considerar afecções ou de aplicar regras ou disposições de codificação, erros de registro de códigos e outros9. Tradicionalmente as estatísticas de mortalidade vem sendo apresentadas e analisadas segundo apenas uma causa de morte. A Organização Mundial de Saúde adotou em 1948, para a mesma, a denomina- ção de causa básica de morte, conceituando-a como "(a) a doença ou lesão que iniciou a sucessão de eventos mórbidos que conduziram diretamente a mento de Epidemiologia da Faculdade de Saúde Pública da Universidade de São Paulo - Av. Dr. Arnaldo, - São Paulo, SP - Brasil. CAUSAS MÚLTIPLAS DE MORTE Tendo-se em vista as limitações existentes nas estatísticas de mortalidade que levam em conta somente uma causa e, principalmente, o fato de que nem sempre uma morte depende de uma só causa, há décadas vem sendo sentida a necessidade de estu- dar-se a mortalidade segundo causas múltiplas. Tais estatísticas são obtidas pela classificação de todas as causas - básicas e associadas - informadas nos atestados de óbito, e forneceriam um volume de dados e informações sensivelmente maior que aquele oferecido apenas pela causa básica2,5. Dado que apenas uma causa é selecionada, são desprezadas, em favor da causa básica, as demais afecções informadas no atestado médico. Por vezes esta seleção, se bem que com normas uniformes e padronizadas, é arbitrária e exclui das tabulações es- tatísticas doenças que desempenham importante pa- pel no processo mórbido que conduz à morte. Desta seleção são excluídas as chamadas causas associadas, que incluem as causas conseqüenciais, importantes para a compreensão da cadeia mórbida que leva di- retamente ao óbito, bem como as causas contribuin- tes, isto é, aquelas afecções presentes no momento da morte porém não relacionadas com a causa básica. Deste modo não se capta o quadro global do processo mórbido. Também, nas mortes violentas, apenas são apresentados dados sobre as circunstân- cias de sua ocorrência, as chamadas causas externas, omitindo-se a natureza da lesão. Para a apresentação de causas múltiplas, dois tipos de tabulação têm sido propostos3,5,8. O pri- meiro deles consistiria numa modificação das tabula- ções tradicionais de mortalidade que descrevem o número de óbitos por determinada causa classifica- dos segundo idade e sexo. Nestas tabulações seria incluída a apresentação da freqüência segundo a qual as diferentes categorias diagnósticas viessem a ser informadas como causas associadas no atestado de óbito. A soma de ambas as freqüências, devidas à causa básica e às causas associadas, corresponderia ao total de informações encontradas no atestado de óbito. A descrição da mortalidade segundo uma só causa adequava-se aos padrões de mortalidade do início do século, quando as mortes se deviam a doenças agudas, infecciosas ou a violências. À medi- da que tais afecções passaram a ser controladas, a sobrevida das pessoas numa população aumentou conseqüentemente. Com isso, a proporção de adul- tos e idosos aumenta, e nesta parcela da população aparecem as denominadas doenças crônicas, que se tornam então as causas de morte mais freqüentes. A CAUSA BÁSICA E SUAS LIMITAÇÕES As tabulações de mortalidade segundo a causa básica fazem corresponder a cada óbito uma só causa, constituindo-se em dados estatísticos simples, unidimensionais e de fácil compreensão, sendo portanto medidas bem aceitas de mortalidade. Apre- sentam as causas iniciais do processo mórbido e assim podem ser usadas pelos responsáveis pela saú- de pública para a prevenção ou controle desse pro- cesso. Entretanto, apesar das numerosas vantagens como dado estatístico, algumas limitações vêm sen- do apontadas em relação ao uso da causa básica de morte para a descrição da mortalidade. substituí-la pela metodologia das causas múltiplas, a qual, por outro lado, daria nova dimensão ao estu- do da mortalidade. portanto medidas bem aceitas de mortalidade. Apre- sentam as causas iniciais do processo mórbido e assim podem ser usadas pelos responsáveis pela saú- de pública para a prevenção ou controle desse pro- cesso. Entretanto, apesar das numerosas vantagens como dado estatístico, algumas limitações vêm sen- do apontadas em relação ao uso da causa básica de morte para a descrição da mortalidade. CAUSAS MÚLTIPLAS DE MORTE Não se pretende tipo de associação, num estudo sobre mortalidade de adultos falecidos em hospitais5, pôde-se consta- tar que, em atestados de óbito nos quais o diabetes era mencionado, as causas que compareciam associa- das eram arteriosclerose, doenças cerebrovasculares, hipertensão arterial e doenças isquêmicas do cora- ção. nicamente por meio de tabelas de decisão às quais foram incorporadas toda a estrutura de códigos da Classificação Internacional de Doenças, as relações etiológicas entre as afecções representadas por tais códigos e interpretadas segundo as disposições das 13 regras de classificação da causa básica. Todos esses elementos foram programados logicamente e de tal forma que se relacionassem entre si perfei- tamente. Instruções especiais foram elaboradas para atribuir códigos a todas as afecções, doenças, lesões, circunstâncias de mortes violentas (acidentes, homi- cídios, suicídios), procedimentos médicos e demais informações que podem ser mencionadas nos atesta- dos de óbito. A programação do ACME recebe a mensagem codificada que transcreve as informações dos atestados de óbito e, após processamento, atri- bui um código à causa básica1,4,9. Várias dificuldades têm sido apontadas para o em- prego generalizado de causas múltiplas. O grande volume de dados a ser trabalhado é citado como principal óbice pois, como mencionado anterior- mente, o trabalho seria, no mínimo, duplicado, considerando a média de dois diagnósticos por ates- tado. De modo especial são encontrados problemas de codificação com o uso da Classificação Interna- cional de Doenças que está muito mais estruturada para identificar a causa básica, sendo particular- mente problemático o uso de rubricas corresponden- tes a duas ou mais afecções classificadas de modo conjunto. Além disso, deve ser lembrado que ine- xiste experiência internacional sobre tabulação e análise de causas múltiplas. O Centro Brasileiro de Classificação de Doenças — CBCD (Centro da OMS para a Classificação de Doenças em Português) da Faculdade de Saúde Pú- blica da Universidade de São Paulo, em 1976, tomou conhecimento do sistema ACME e, de imediato, interessou-se pelo mesmo, tendo em vista seu imenso potencial de uso para as estatísticas de mor- talidade. Estabeleceu-se, a seguir, um intercâmbio entre o CBCD e o NCHS, de Washington, para a aquisição de informações pormenorizadas sobre o sistema ACME. Após cuidadosa análise das ques- tões envolvidas, concluiu-se pela conveniência de sua introdução no Brasil. CAUSAS MÚLTIPLAS DE MORTE O CBCD consultou a Fun- dação SEADE (Sistema Estadual de Análise de Da- dos) que também se interessou pelo sistema e ace- deu em testar seu uso no Estado de São Paulo. O NCHS enviou ao CBCD cópia do programa, bem como toda a documentação correlata, que as cedeu à Fundação SEADE. Teve início um trabalho entre o CBCD e aquela Fundação para introduzir o siste- ma ACME na apuração das causas de morte do Estado de São Paulo, trabalho este que compreen- deu, dentre outras atividades, treinamento de codificadores, adaptação do programa e análise de problemas operacionais. A par disso, técnicos do CBCD e da Fundação SEADE estiveram no NCHS a fim de observar o funcionamento do sistema ACME. CAUSAS MÚLTIPLAS DE MORTE Para a descrição destas mortes o conceito de causa básica já não é tão satisfatório, desde que, mesmo o médico, por vezes, não é capaz de identificar a afecção que iniciou uma sucessão de eventos pa- tológicos, observando, antes, uma associação de doenças. As mortes devidas a doenças crônicas são determinadas por diversas afecções presentes no falecido, as quais nem sempre têm entre si relação etiológica com vistas à seleção de uma causa básica claramente determinada. O segundo tipo de tabulação constitui-se em uma nova forma de apresentação de dados sobre mortali- dade por meio de associações de causas. A morte ocorre freqüentemente devido à ação sinérgica de duas ou mais afecções presentes no falecido e tal fato não se reflete nas estatísticas segundo causa básica. Isso pode ser exemplificado para o caso de sarampo, antes do advento da vacinação, quando as taxas de mortalidade em países desenvolvidos eram bem menores que as verificadas nos subdesenvolvi- dos, nos quais as taxas elevadas eram devidas à pre- sença de desnutrição atuando como causa associada. Com maior propriedade, a morte devida a doenças crônicas é melhor descrita considerando-se associa- ções de causas; por exemplo, freqüentemente as doenças cerebrovasculares incidem em pessoas com hipertensão e certas complicações renais em pacien- tes diabéticos. De modo sintético, considerando-se que estudos internacionais e em nosso meio verificam média de cerca de dois diagnósticos informados por atestado de óbito, pode-se dizer que o uso apenas da causa básica determina a perda de praticamente a metade das informações sobre o padrão nosológico da mor- talidade das populações3,5,8. Vários tipos de associação de causas são possíveis, tais como a da causa básica com as demais causas associadas ou de qualquer causa com as demais in- formadas. Como exemplo do primeiro tipo, num estudo sobre mortalidade em doentes mentais8, verificou-se que, nos óbitos em que a cirrose hepática era selecionada como causa básica, as causas associa- das mais freqüentes foram desnutrição, alcoolismo e complicações hepáticas. Relativamente ao segundo Em que pesem as considerações sobre sua limita- ção, o emprego da causa básica de morte continua a ser essencial para análise de tendências históricas e para comparabilidade entre países. O SISTEMA ACME PARA CLASSIFICAÇÃO DE CAUSA BÁSICA E DE CAUSAS MÚLTIPLAS As citadas dificuldades inerentes ao volume de dados foram resolvidas com o advento dos compu- tadores que facultam o seu processamento eletrô- nico. Tal fato permitiu o desenvolvimento por parte do "National Center for Health Statistics" (NCHS), dos Estados Unidos, de um sistema automático para classificar, além da causa básica de morte, todas as demais afecções indicadas no atestado médico da declaração de óbito1,4. Denominado ACME, sigla para "Automated Classification of Medical Entities", este sistema apresenta-se, não só como uma alter- nativa adequada ao trabalho manual para a classifi- cação da causa básica de morte, como também permite o registro de todos os códigos das afecções presentes no atestado médico, tornando disponíveis dados sobre causas múltiplas. Portanto, o sistema ACME realiza basicamente dois trabalhos: identifica a causa básica de morte e arquiva dados sobre as demais causas associadas. Tradicionalmente, a classificação da causa básica de morte tem sido feita manualmente pelos chama- dos codificadores. Esse trabalho é realizado utilizan- do a estrutura da Classificação Internacional de Doenças, suas quatro regras de seleção e nove de modificação. Para tanto, o codificador recebe inten- sivo treinamento, o qual é internacionalmente nor- matizado pela Organização Mundial da Saúde. Entretanto, como foi visto, é um trabalho sujeito a falhas. O sistema ACME dá margem a maior padro- nização e controle sobre o processo de codificação de causa básica de morte, desde que automatiza tais procedimentos. A causa básica é identificada eletro- Em 1976, foi criado no Brasil, pelo Ministério da Saúde, o Subsistema Nacional de Informações sobre Mortalidade integrando todos os serviços estaduais de apuração de dados das declarações de óbito, objetivando o aprimoramento das estatís- ticas de mortalidade. Considerando os trabalhos em relação ao sistema ACME, estabeleceu-se um convê- nio entre o Ministério da Saúde e a Fundação SEADE para instalação preliminar do mesmo no Estado de São Paulo, convênio esse que previa, oportunamente, a cessão de todas as rotinas técni- cas e administrativas ao Ministério, para eventual aplicação em todo o Brasil7. A partir de 1983, o processamento das declara- ções de óbito do Estado de São Paulo, em torno de 180.000, realiza-se ja pelo sistema ACME. O CBCD, atendendo aos objetivos que desde a sua criação vem sendo perseguidos, qual seja o de traba- lhar para a melhoria das informações contidas nas estatísticas, de mortalidade, vem, continuamente, prestando colaboração direta à Fundação SEADE. O SISTEMA ACME PARA CLASSIFICAÇÃO DE CAUSA BÁSICA E DE CAUSAS MÚLTIPLAS Esta colaboração compreende não só treinamento e supervisão de codificadores como também a elaboração de tabelas de decisão e de validade de causas de morte que se adequem para o nosso meio, a promoção de estudos sobre familiarização de pro- cedimentos e a sugestão das formas de tabulação final de dados. A implantação definitiva desse sistema amplia, desse modo, as possibilidades de uso das estatísticas de mortalidade não somente para os administradores de saúde como também para epidemiologistas e demógrafos. REFERÊNCIAS BIBLIOGRÁFICAS 1. CHAMBLEE, R.F. & EVANS, M.C. New dimensions in cause of death statistics. Amer.J.publ.Hlth, 72: 1265-70, 1982. na Reunião de Diretores de Centros Colaboradores da OMS para a Classificação de Doenças, São Paulo, 1978 - mimeografado ]. 2. DORN, H.F. Underlying and contributory causes of death. In: Haenszel, W., ed. Epidemiological appro- aches to the study of cancer and other chronic diseases. Bethesda, Md, National Cancer Institute, 1966. p. 421-30. 8. SANTO, A.H. Estudo crítico das estatísticas de causa de morte em doentes portadores de transtornos mentais. São Paulo, 1980. [ Dissertação de Mestrado - Faculdade de Saúde Pública da USP ] . 9. SANTO, A.H. Causas múltiplas de morte: formas de apresentação e métodos de análise. São Paulo, 1983. [Projeto de tese de doutoramento apresentado à Comissão de Pós-Graduação da Faculdade de Saúde Pública da USP - mimeografado ]. 3. DORN, H.F. & MORIYAMA, I.M. Uses and signifi- cance of multiple cause tabulations for mortality statistics. Amer.J.publ. Hlth, 54: 400-6, 1964. 4. ISRAEL, R.A.; ROSENBERG, H.M. &CURTIN, L.R. Analytical potential for multiple cause-of-death data. [Draft 4/11/84 - National Center for Health Statistics]. 10. SANTO, A.H. & LAURENTI, R. General review on mortality coding rules. Geneva. World Health Orga- nization, 1983. (DES/ICD-10/83.15). [Apresentado ao Preparatory Meeting on ICD-10, Geneva, 1983 ] 5. LAURENTI, R. Causas múltiplas de morte. São Paulo, 1973. [ Tese de Livre-Docência - Faculdade de Saúde Pública da USP ]. 11. UNITED NATIONS. Department of International Economic and Social Affairs. Levels and trends of mortality since 1950; a joint study by the United Nations and the World Health Organization. New York, 1982. Recebido para publicação em 15/08/1986 Aprovado para publicação em 22/08/1986 11. UNITED NATIONS. Department of International Economic and Social Affairs. Levels and trends of mortality since 1950; a joint study by the United Nations and the World Health Organization. New York, 1982. 6. MANUAL of the international statistical classification of diseases, injuries and causes of death; 6th revision. Geneva, World Health Organization, 1948. Recebido para publicação em 15/08/1986 7. MARQUES, R.M.; TRONKOS, J.A. & TARDELLI A.O. Technical report on feasibility testing of "ACME" system. São Paulo, 1978. [Apresentado
https://openalex.org/W2124222588
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0089791&type=printable
English
null
Pleiotropy of Cancer Susceptibility Variants on the Risk of Non-Hodgkin Lymphoma: The PAGE Consortium
PloS one
2,014
cc-by
8,851
Abstract doi:10.1371/journal.pone.0089791 Editor: Ludmila Prokunina-Olsson, National Cancer Institute, National Institutes of Health, United States of America Editor: Ludmila Prokunina-Olsson, National Cancer Institute, National Institutes of Health, United States of America Received July 20, 2013; Accepted January 27, 2014; Published March 5, 2014 Received July 20, 2013; Accepted January 27, 2014; Published March 5, 2014 Copyright:  2014 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2014 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attrib use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2014 Lim et al. This is an open-access article distributed under the term use, distribution, and reproduction in any medium, provided the original author and Funding: The Population Architecture Using Genomics and Epidemiology (PAGE) program is funded by the National Human Genome Research Institute (NHGRI), supported by U01HG004803 (CALiCo), U01HG004798 (EAGLE), U01HG004802 (MEC), U01HG004790 (WHI), and U01HG004801 (Coordinating Center), and their respective NHGRI ARRA supplements. The complete list of PAGE members can be found at http://www.pagestudy.org. The data and materials included in this report result from collaboration between the following studies: The ‘‘Epidemiologic Architecture for Genes Linked to Environment (EAGLE)’’ is funded through the NHGRI PAGE program (U01HG004798-01 and its NHGRI ARRA supplement). Genotyping services for select NHANES III SNPs presented here were also provided by the Johns Hopkins University under federal contract number (N01-HV-48195) from NHLBI. The study participants derive from the National Health and Nutrition Examination Surveys (NHANES), and these studies are supported by the Centers for Disease Control and Prevention. The dataset used for the analyses described were obtained from Vanderbilt University Medical Center’s BioVU which is supported by institutional funding and by the Vanderbilt CTSA grant UL1 TR000445 from NCATS/NIH. The Multiethnic Cohort study (MEC) characterization of epidemiological architecture is funded through the NHGRI PAGE program (U01HG004802 and its NHGRI ARRA supplement). The MEC study is funded through the National Cancer Institute (R37CA54281, R01 CA63, P01CA33619, U01CA136792, and U01CA98758). Funding support for the ‘‘Epidemiology of putative genetic variants: The Women’s Health Initiative’’ study is provided through the NHGRI PAGE program (U01HG004790 and its NHGRI ARRA supplement). Pleiotropy of Cancer Susceptibility Variants on the Risk of Non-Hodgkin Lymphoma: The PAGE Consortium Pleiotropy of Cancer Susceptibility Variants on the Risk of Non-Hodgkin Lymphoma: The PAGE Consortium Unhee Lim1*, Jonathan M. Kocarnik2, William S. Bush3, Tara C. Matise4, Christian Caberto1, Sungshim Lani Park5, Christopher S. Carlson2, Ewa Deelman4, David Duggan6, Megan Fesinmeyer7, Christopher A. Haiman5, Brian E. Henderson5, Lucia A. Hindorff8, Laurence N. Kolonel1, Ulrike Peters2, Daniel O. Stram5, Maarit Tiirikainen1, Lynne R. Wilkens1, Chunyuan Wu2, Charles Kooperberg2, Loı¨c Le Marchand1 , , , , , Sungshim Lani Park5, Christopher S. Carlson2, Ewa Deelman4, David Duggan6, Megan Fesinmeyer7, Christopher A. Haiman5, Brian E. Henderson5, Lucia A. Hindorff8, Laurence N. Kolonel1, Ulrike Peters2, Daniel O. Stram5, Maarit Tiirikainen1, Lynne R. Wilkens1, Chunyuan Wu2, Charles Kooperberg2, 1 1 Epidemiology Program, University of Hawaii Cancer Center, Honolulu, Hawaii, United States of America, 2 Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Center for Human Genetics Research, Vanderbilt University, Nashville, Tennessee, United States of America, 4 Department of Genetics, Rutgers University, Piscataway, New Jersey, United States of America, 5 Department of Preventive Medicine, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America, 6 Translational Genomics Research Institute, Phoenix, Arizona, United States of America, 7 Center for Child Health, Behavior and Development, Seattle Children’s Research Institute, Seattle, Washington, United States of America, 8 Division of Genomic Medicine, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America Abstract Background: Risk of non-Hodgkin lymphoma (NHL) is higher among individuals with a family history or a prior diagnosis of other cancers. Genome-wide association studies (GWAS) have suggested that some genetic susceptibility variants are associated with multiple complex traits (pleiotropy). Objective: We investigated whether common risk variants identified in cancer GWAS may also increase the risk of developing NHL as the first primary cancer. Methods: As part of the Population Architecture using Genomics and Epidemiology (PAGE) consortium, 113 cancer risk variants were analyzed in 1,441 NHL cases and 24,183 controls from three studies (BioVU, Multiethnic Cohort Study, Women’s Health Initiative) for their association with the risk of overall NHL and common subtypes [diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia or small lymphocytic lymphoma (CLL/SLL)] using an additive genetic model adjusted for age, sex and ethnicity. Study-specific results for each variant were meta- analyzed across studies. Results: The analysis of NHL subtype-specific GWAS SNPs and overall NHL suggested a shared genetic susceptibility between FL and DLBCL, particularly involving variants in the major histocompatibility complex region (rs6457327 in 6p21.33: FL OR = 1.29, p = 0.013; DLBCL OR = 1.23, p = 0.013; NHL OR = 1.22, p = 5.96E-05). In the pleiotropy analysis, six risk variants for other cancers were associated with NHL risk, including variants for lung (rs401681 in TERT: OR per C allele = 0.89, p = 3.76E-03; rs4975616 in TERT: OR per A allele = 0.90, p = 0.01; rs3131379 in MSH5: OR per T allele = 1.16, p = 0.03), prostate (rs7679673 in TET2: OR per C allele = 0.89, p = 5.76E-03; rs10993994 in MSMB: OR per T allele = 1.09, p = 0.04), and breast (rs3817198 in LSP1: OR per C allele = 1.12, p = 0.01) cancers, but none of these associations remained significant after multiple test correction. Conclusion: This study does not support strong pleiotropic effects of non-NHL cancer risk variants in NHL etiology; however, larger studies are warranted. Citation: Lim U, Kocarnik JM, Bush WS, Matise TC, Caberto C, et al. (2014) Pleiotropy of Cancer Susceptibility Variants on the Risk of Non-Hodgkin Lymphoma: The PAGE Consortium. PLoS ONE 9(3): e89791. Abstract The WHI program is funded by the National Heart, Lung, and Blood Institute; NIH; and U.S. Department of Health and Human Services through contracts N01WH22110, 24152, 32100-2, 32105-6, 32108-9, 32111-13, 32115, 32118-32119, 32122, 42107- 26, 42129-32, and 44221. A full listing of WHI investigators can be found at: http://www.whiscience.org/publications/WHI_investigators_shortlist.pdf. Additional support for Dr. Kocarnik was provided by R25CA94880 from NCI. Assistance with phenotype harmonization, SNP selection and annotation, data cleaning, data management, integration and dissemination, and general study coordination was provided by the PAGE Coordinating Center (U01HG004801-01 and its NHGRI March 2014 | Volume 9 | Issue 3 | e89791 1 PLOS ONE | www.plosone.org 1 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma ARRA supplement). The National Institutes of Mental Health also contributes to the support for the Coordinating Center. The funders had no role in study design, data collection and analysis, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ulim@cc.hawaii.edu * E-mail: ulim@cc.hawaii.edu patients without any prior or prevalent cancer diagnoses (except non-melanoma skin cancer) and with a similar reported race/ ethnicity and age at clinic visit (within 5 years) as cancer cases. The MEC is a population-based prospective cohort of over 215,000 men and women in Hawaii and Los Angeles, aged 45–75 years at recruitment and primarily of five ancestries (white, African American, Latino, Japanese American or Native Hawaiian) [19,20]. Incident cancer cases in the MEC were identified by linkage with Hawaii and California SEER tumor registries, from 1993 through October 2010. The WHI is a prospective cohort study investigating postmenopausal women’s health in the U.S. [21]. A total of 161,838 women of ages 50–79 and of various race/ ethnic groups (white, African American, Latino, Asian/Pacific Islander or American Indian) were recruited from 40 clinical centers throughout the U.S. in 1993–1998 for three clinical trials and an observational study. Medical history, including cancer incidence, is updated annually by mail and/or telephone questionnaires and confirmed by medical records and pathologic reports [22]. The current WHI analysis includes NHL cases identified through August 2009. Selection of Cases and Controls We limited our analysis to NHL cases and controls with no previous cancer (except non-melanoma skin cancer) in order to assess genetic pleiotropy without the possibility of confounding by previous cancers or treatments on the risk of NHL. MEC defined NHL cases based on the current World Health Organization classification that considered chronic lymphocytic leukemia (CLL) as a different presentation of the same disease as small lymphocytic lymphoma (SLL) [23,24]. BioVU and WHI defined NHL based on the SEER classification and did not include CLL. Histology information based on the International Classification of Disease- Oncology (ICD-O3) was available in BioVU [25] and MEC [19] through linkage with tumor registries and in WHI [26] through systematic morphology coding of medical record information for the classification of the three most common NHL subtypes: diffuse large B-cell lymphoma (DLBCL; 9678–9680, 9684, 9689, 9699), follicular lymphoma (FL; 9690–9691, 9695, 9698) and CLL (9823)/SLL (9670) [24]. DLBCL was not ascertained in BioVU due to a prioritization for more common cancers in their PAGE analyses. For the current NHL analysis, all three studies included controls that were matched to cases of common cancers being investigated in the PAGE consortium (breast, colorectal, ovarian and prostate cancers and melanoma in all studies, and endometrial and lung cancers, and NHL in MEC and WHI). The matching was performed using frequency matching based on age at Abstract All studies were approved by Institutional Review Boards at their respective study sites: the Vanderbilt Institutional Review Board for BioVU, the Human Studies Program at the University of Hawaii and Office for the Protection of Research Subjects at the University of Southern California for MEC, and the Fred Hutchinson Cancer Research Center Institutional Review Board for WHI. All participants of MEC and WHI provided written informed consent. All BioVU participants signed a ‘‘consent-to-treatment’’ form, informing them that anonymized genetic information from their discarded blood, along with de-identified EMR information, will be used for research and were given the choice to check an ‘‘opt-out’’ box if declining to participate [17]. Introduction Non-Hodgkin lymphoma (NHL) is the sixth most common incident cancer in the U.S. [1]. Although immune suppression, autoimmune disorders and certain infectious agents have been identified as strong risk factors for NHL, common host charac- teristics are also likely to be involved in the etiology of NHL [2]. Risk of NHL has been reported to be greater among individuals with a first-degree family history of hematopoietic cancers [3]. NHL is also a common second primary cancer among survivors of adult leukemia, laryngeal/pharyngeal cancer, renal cell carcinoma and melanoma, suggesting common genetic and/or environmen- tal etiology, although it is difficult to rule out a treatment effect from the first cancer [4–6]. In searching for the shared genetic basis of disease, genome-wide association studies (GWAS) have discovered a number of risk variants that demonstrate associations with two or more complex traits (pleiotropy) [7]. A systematic review of the U.S. National Human Genome Research Institute (NHGRI) Catalog of Published GWAS reported that 16.9% of genes and 4.6% of single nucleotide polymorphisms (SNPs) in the catalog have shown such pleiotropic associations [8,9]. The proportion of pleiotropic variants was higher than expected by chance and was particularly high among cancer risk variants, as well as among the variants associated with altered immunity and metabolic syndrome. Thus, genetic variations involved in cancer- related pathways may increase the risk of cancer of multiple types [10], including NHL. A good example is the multiple cancer site associations reported for variants at 8q24 [10,11], a region where some lymphoid malignancies also exhibit translocations and a common susceptibility SNP [12–15]. In this study, we examined whether established risk variants identified in published GWAS of 17 common cancers present pleiotropic associations with the risk of NHL and its histologic subtypes in three well-characterized studies, as part of the Population Architecture using Genomics and Epidemiology (PAGE) consortium [16]. We also explored whether variants identified for specific NHL subtypes are also associated with overall NHL risk, and whether any such associations differ across ethnic groups. Biospecimen Collection, SNP Selection, and Genotyping Biospecimen Collection, SNP Selection, and Genotyping Biospecimen Collection, SNP Selection, and Genotyping BioVU extracted DNA from discarded whole blood samples for patients drawn as part of routine clinical testing [17]. In the MEC, DNA of NHL cases was extracted from pre-diagnostic samples included in its prospective blood repository of over 67,000 cohort participants assembled in 2001–2006. DNA samples for controls were from either the prospective blood repository or from case- control studies of breast, colorectal and prostate cancers [20,27]. The distribution of established cancer risk factors in the biospeci- men sub-cohort was similar to that in the entire MEC cohort. DNA samples in WHI were extracted from pre-diagnostic blood collected at time of enrollment. A total of 113 SNPs were selected and genotyped by one or more of the three PAGE studies based on genome-wide significant associations (p,5.06E-08) [28] in the cancer GWAS literature at the time of the study design (March 2010). These non-NHL cancer SNPs included risk variants for bladder, brain (glioma), breast, colorectal, esophageal, lung, nasopharyngeal, neuroblastoma, ovarian, pancreatic, acute lymphoblastic leukemia, prostate, skin (basal cell carcinoma, melanoma), testicular germ cell, and thyroid cancers. The NHL SNPs, which included one risk variant for follicular lymphoma (FL) [29,30] and 8 variants for chronic lymphocytic leukemia (CLL) [15,30,31], were only considered in associations with NHL and were excluded from the pleiotropy analysis. All samples were additionally genotyped for the ancestry informative markers (AIMs) described by Kosoy et al. [32] BioVU used Sequenom’s iPLEX Gold coupled with MassARRAY MALDI-TOF MS detection and Illumina’s BeadXpress with a custom GoldenGate genotyping assay. MEC used Applied Biosystems Taqman SNP genotyping assays on the OpenArray and the 7900HT Real-Time PCR platforms. WHI used Illumina BeadXpress with the Veracode GoldenGate genotyping assay. All sites used blind duplicate controls. Samples with low overall call rates (,90% of SNPs) were excluded. SNPs were excluded based on deviation from ethnicity-specific Hardy-Weinberg equilibrium (p,0.01), low call rates (,95%) or low concordance rates – range of minimum varied between 96.5 and 99% in the studies. In addition to site-specific quality control as above, all PAGE study sites genotyped the same 360 DNA samples from the International HapMap Project with excellent concordance rates with the published genotype data [16]. Results Characteristics of the NHL cases and controls in the BioVU, MEC and WHI studies are shown in Table 1. Median age of NHL cases and controls was the highest in MEC, followed by WHI and BioVU, and both BioVU and MEC had a slightly higher representation of men over women. Cases in BioVU and WHI were mostly whites, whereas MEC had more even distribution of four ethnic groups. We first investigated nine previously published GWAS risk variants for specific NHL subtypes (one for FL and 8 for CLL) for an association with overall NHL risk to test for a shared genetic susceptibility. The association reported for FL with rs6457327, in 6p21.33, the major histocompatibility complex region (MHC), replicated in our data for FL [summary OR per allele C vs. A = 1.29 (1.05–1.57), p = 0.013; Figure 1(a), Table S1 in File S1] and was also observed for DLBCL [OR = 1.23 (1.04–1.44), p = 0.013] and overall NHL [OR = 1.22 (1.11–1.34), p = 5.92E-05; Figure 1(b)], but not for CLL/SLL [OR = 1.05 (0.81–1.36), p = 0.73]. When the meta-analysis on overall NHL was limited to MEC and WHI, considering that BioVU did not include DLBCL, the association with the published FL risk variant remained the same [OR = 1.22 (1.11–1.33), p = 5.92E-05]. In the MEC, where all main subtypes were examined, including CLL, the OR for the association of allele C of rs6457327 with FL did not differ from the corresponding OR for DLBCL (p-het. from polytomous regression = 0.61), CLL/SLL (p-het. = 0.23) or other subtypes (p-het. = 0.73). We did observe heterogeneity in the association for the FL risk variant (rs6457327) and overall NHL across study sites (Cochran Q = 6.58; p-het. = 0.01), which was eliminated when the analysis was limited to whites only [Figure 1(c); OR for allele C of rs6457327 = 1.30 (1.16–1.47), p = 8.83E-06; Cochran Biospecimen Collection, SNP Selection, and Genotyping After these stringent quality control procedures, 1,441 NHL cases (BioVU, n = 293; MEC, n = 372; WHI, n = 776) and 24,183 controls (BioVU, n = 9,002; MEC, n = 9,091; WHI, n = 6,090) were included in the current analysis. Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma for number of risk alleles). The unconditional logistic regression model was adjusted for age, sex and race/ethnicity. Residual confounding by race/ethnicity was examined by additionally adjusting for principal components of genetic ancestry (top three in BioVU and WHI and top four in MEC). Effect modification by sex was assessed by a Wald test of the cross-product terms of sex and the continuous SNP variable in BioVU and MEC (WHI includes only women). Heterogeneity across race/ethnic groups was tested similarly using a Wald test in the MEC, where cases of non-white ethnic groups were available in substantial numbers. Also, heterogeneity in the SNP-cancer associations across common NHL subtypes (DLBCL, FL, CLL/SLL) was examined in the MEC, where all subtypes were ascertained, by performing polytomous logistic regression using common controls. A risk score was computed to examine the combined effect of 53 cancer variants that were genotyped in all three studies, by summing up the number of risk alleles (0, 1 or 2 for each SNP) in individuals across SNPs. For subjects with missing genotypes for any of the 53 variants, missing genotypes were estimated using the allele frequencies among controls of the same ethnicity in each study. The risk score was examined as both a continuous and a categorical variable (using quartile cut points based on the distribution among controls). To summarize results from the three studies, we carried out a meta-analysis for each variant and for the risk score variable in fixed-effects models using METAL [33]. Heterogeneity across studies was evaluated using Cochran’s Q statistic. Analyses were conducted initially with significance considered at p,0.05 (two-sided). To control for the potentially inflated Type 1 error due to multiple comparisons, we used Bonferroni correction (p = 0.05/113 = 4.42E-04) to determine the statistical significance threshold for results. diagnosis or clinic visit (+/25 yrs), sex and race/ethnicity in BioVU; and individual matching for each case based on age at cohort entry (+/25 yrs), sex and race/ethnicity in MEC; and age at enrollment (+/23 yrs), enrollment date (+/2365 days), race/ ethnicity, and randomization arms (observational study or clinical trial assignment to hormone replacement therapy, dietary modification, or calcium/vitamin D supplement) in WHI. WHI also included additional controls selected from other genetic studies based on the availability of biomarkers. Study Populations The PAGE consortium was established in 2008 by the U.S. National Human Genome Research Institute to investigate well-replicated genetic variants for complex diseases in several large, ethnically diverse studies (https://www.pagestudy.org) [16]. Three PAGE studies participated in this analysis: biorepository of the Vanderbilt University (BioVU), the Multiethnic Cohort Study (MEC) and the Women’s Health Initiative (WHI). BioVU is a study at Vanderbilt University Medical Center that links de-identified electronic medical records (EMR) to a DNA biobank [17,18]. Out of ,130,000 BioVU participants, over 6,098 cancer cases were identified from 2009–2011 through linkage with the hospital tumor registry or search of diagnostic codes in the EMR. Race/ethnicity was recorded by hospital staff in the EMR (white, African American, Latino or Asian American) and confirmed using principal components analysis of ancestry- informative markers (AIMs). Controls included 9,152 BioVU March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 2 Statistical Analysis Unconditional logistic regression analysis was used in each study to estimate the association of cancer risk variants and NHL risk as odds ratios (ORs) and 95% confidence intervals (CIs). For each cancer risk variant, the allele that increased the risk of cancer in the original report was modeled against the low risk allele. Thus, ORs for NHL would be expected to be .1 if the association was in the same direction as the one found in the cancer GWAS study. Each biallelic SNP was coded as a continuous variable (0, 1 or 2 March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 3 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Table 1. Characteristics of non-Hodgkin lymphoma (NHL) cases and controls in the PAGE studies. Statistical Analysis Among the eight GWAS risk variants for CLL, one variant replicated in our data for CLL/SLL [OR per allele G of rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027] and remained significant after Bonferroni correction (p,0.0063; Table S1 in File S1). None of the CLL variants were associated with overall NHL risk (p.0.05; data not shown). For example, in the MEC, the association of rs17483466 with CLL/SLL significantly differed from that with FL (p-het. from polytomous regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). Of the 113 GWAS risk variants for cancers other than NHL that were examined in PAGE, 53 SNPs were genotyped in all three studies, and the other 60 variants were typed in one or two studies (Table S2 in File S1). Six of the 53 SNPs showed nominal associations with the risk of overall NHL, including three risk variants originally identified for lung cancer, two risk variants identified for prostate cancer and one risk variant for breast cancer (Table 2). None of these associations remained significant after multiple test correction (i.e., p.4.4E-04 for 113 SNPs). Two lung cancer risk variants in the TERT region (rs401681, OR per allele C = 0.89 (0.82–0.96), p = 0.0037; rs4975616, OR per allele A = 0.90 (0.83–0.97), p = 0.010), as well as the prostate cancer risk variant (rs7679673 in TET2; OR per allele C = 0.89 (0.82–0.97), p = 0.0057), were associated with a decreased risk of overall NHL. The two TERT variants were in linkage disequilibrium (LD) among whites (R2 = 0.84) and Native Hawaiians (R2 = 0.79) but less so in other ethnic groups (R2 = 0.53 for African Americans; 0.47 for Latinos, 0.30 for Japanese Americans). The breast cancer susceptibility variant rs3817198 in LSP1 [OR per allele C = 1.12 (1.03–1.22), p = 0.011], the lung cancer SNP rs3131379 in the MHC region in chromosome 6 (6p21.33) [OR per allele T = 1.16 (1.01–1.33), p = 0.030] and the prostate cancer variant rs10993994 in MSMB [OR per allele T = 1.09 (1.01–1.18), p = 0.036] were each associated with an increased risk of overall NHL. The associations for the six variants above did not differ significantly across study sites, except for rs401681 (TERT), which showed a stronger inverse association in BioVU and MEC than in WHI (Cochran Q = 6.26, p-het. = 0.04; Table 2). Statistical Analysis These 6 variants showed the same or similar summary ORs when the analysis was limited to MEC and WHI, where overall NHL included DLBCL, with 4 variants showing nominal significance (unadjusted p,0.05; data not shown). Of the other 60 variants genotyped in only two studies or a single study, 7 SNPs showed moderate associations (unadjusted p,0.05; data not shown). In particular, an esophageal cancer variant (rs1229984 in ADH1B) available in MEC and WHI showed an inverse association with NHL risk [OR per allele C = 0.77 (0.66–0.90), p = 4.4E-04]. Q = 0.05, p-het. = 0.82], although the interaction between the variant and race/ethnicity was not significant in the MEC (p- int. = 0.10). Among the eight GWAS risk variants for CLL, one variant replicated in our data for CLL/SLL [OR per allele G of rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027] and remained significant after Bonferroni correction (p,0.0063; Table S1 in File S1). None of the CLL variants were associated with overall NHL risk (p.0.05; data not shown). For example, in the MEC, the association of rs17483466 with CLL/SLL significantly differed from that with FL (p-het. from polytomous regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). Q = 0.05, p-het. = 0.82], although the interaction between the variant and race/ethnicity was not significant in the MEC (p- int. = 0.10). Among the eight GWAS risk variants for CLL, one variant replicated in our data for CLL/SLL [OR per allele G of rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027] and remained significant after Bonferroni correction (p,0.0063; Table S1 in File S1). None of the CLL variants were associated with overall NHL risk (p.0.05; data not shown). For example, in the MEC, the association of rs17483466 with CLL/SLL significantly differed from that with FL (p-het. from polytomous regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). Of the 113 GWAS risk variants for cancers other than NHL that were examined in PAGE, 53 SNPs were genotyped in all three studies, and the other 60 variants were typed in one or two studies (Table S2 in File S1). Six of the 53 SNPs showed nominal associations with the risk of overall NHL, including three risk variants originally identified for lung cancer, two risk variants identified for prostate cancer and one risk variant for breast cancer (Table 2). Statistical Analysis BioVU MEC WHI Type of Study Cross-sectional [17,18] Nested Case-Control in Cohort [19,20] Nested Case-Control in Cohort [21,22] Focus of Study Cancer Cancer Women’s health Years of Data Collection Enrollment and Blood Draw 2007–2011; Diagnoses between 2009–2011 Enrollment 1993–1996; Blood draw 1995–2006; Diagnoses between 1993 and October 2010 Enrollment and Blood Draw 1993– 1998; Diagnoses between 1993 and August 2009 NHL Cases Controls NHL Cases Controls NHL Cases Controls Selection First primary incident NHL diagnoses from hospital tumor registry and electronic medical records (EMR) Combined controls for multiple cancer sites; matched on age, sex, ethnicity First primary incident NHL diagnoses from linkage of cohort with SEER* registries Combined controls for multiple cancer sites; matched on age, sex, ethnicity First primary incident NHL diagnoses from active follow-up (semi/annual) and EMR verification Combined controls for multiple cancer sites; matched on age, enrollment date, ethnicity, randomization Total, n* 293 9,002 372 9,091 776 6,090 Age, median (range) 57 (18–102) 63 (19–110) 71 (45–92) 71 (45–88) 65 (50–79) 65 (50–79) Sex, n (%) women 139 (47%) 3,711 (41%) 166 (45%) 4,321 (48%) 776 (100%) 6,090 (100%) Race/Ethnicity, n (%) White 275 (94%) 8,061 (90%) 102 (27%) 1844 (20%) 718 (93%) 4,763 (78%) African American 16 (5%) 804 (9%) 68 (18%) 2228 (25%) 27 (3%) 714 (12%) Latino 0 56 (0.6%) 80 (22%) 1864 (21%) 16 (2%) 332 (5%) Asian American/ Pacific Islander 2/0 (0.7%) 81/0 (0.9%) 104/18 (33%) 2513/642 (35%) 15/0 (2%) 281/0 (5%) NHL Subtypes, n (%) 293 N/A 372 N/A 776 N/A DLBCL - 102 (27%) 258 (33%) FL 72 (25%) 68 (18%) 178 (23%) CLL/SLL 42 (SLL only; 14%) 71 (19%) 66 (SLL only; 9%) Others 179 (61%) 131 (35%) 274 (35%) * Any prior cancer cases were excluded from the NHL cases and controls for the current analysis, based on self-report (BioVU, MEC, WHI), the SEER registry linkage (BioVU, MEC), and medical record reviews (BioVU, WHI). Abbreviations: BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative); CLL/SLL (chronic lymphocytic leukemia/small lymphocytic lymphoma), DLBCL (diffuse large B-cell lymphoma), FL (follicular lymphoma), SEER (Surveillance, Epidemiology and End Results). doi:10 1371/journal pone 0089791 t001 Table 1. Characteristics of non-Hodgkin lymphoma (NHL) cases and controls in the PAGE studies. Q = 0.05, p-het. = 0.82], although the interaction between the variant and race/ethnicity was not significant in the MEC (p- int. = 0.10). Statistical Analysis (a) follicular lymphoma, (b) overall NHL, and (c) overall NHL among whites only. doi:10.1371/journal.pone.0089791.g001 Figure 1. Forest plots for the association between a published follicular lymphoma risk variant (rs6457327) and the risk of follicular lymphoma or overall non-Hodgkin lymphoma (NHL) in the Multiethnic Cohort (MEC) and the Women’s Health Initiative (WHI) in the PAGE consortium. (a) follicular lymphoma, (b) overall NHL, and (c) overall NHL among whites only. doi:10.1371/journal.pone.0089791.g001 The pleiotropy analysis for specific NHL subtypes was conducted on all non-NHL GWAS cancer SNPs (n = 113) (Table S3 in File S1). None of the subtype-specific associations were significant after Bonferroni correction (i.e., p.0.05/113 = 4.4E-04 for 113 tests on each subtype). The most significant association for follicular lymphoma was with a breast cancer risk variant [rs11249433 in EMBP1: summary OR per allele C = 1.29 (1.08– 1.54), p = 0.0095]. For CLL/SLL, the most significant association was with a prostate cancer risk variant [rs2735839 in KLK3-KLK2: OR per allele G = 1.51 (1.10–2.07), p = 0.0099]. The associations of overall NHL described above (Table 2) with the risk variants for lung cancer (rs3131379 in MSH5) and prostate cancer (rs7679673 in TET2) appeared to be due to their associations with the risk of DLBCL subtype [OR per allele T in rs3131379 = 1.41 (1.10– 1.80), p = 0.0061; OR per allele C in rs7679673 = 0.83 (0.71–0.98), p = 0.030]. subtype specifically, as indicated in previous studies for shared etiology [34–36]. The CLL risk variants did not extend to other subtypes or NHL overall, indicating the subtype-specificity of the CLL variants. For non-NHL cancer variants, we found no convincing evidence of pleiotropy, with only weak suggestions that specific risk variants for lung, prostate and breast cancers may also be associated with the risk of developing first primary incident NHL among those without a history of other cancers or prior cancer treatments. The effects of three of the six non-NHL GWAS variants nominally associated with NHL (rs401681, rs7679673, rs4975616) were in the opposite direction compared to the original reports. These variants showed an association with increased risks for lung and prostate cancers in the original reports but a lower risk of NHL in our study. Statistical Analysis None of these associations remained significant after multiple test correction (i.e., p.4.4E-04 for 113 SNPs). Two lung cancer risk variants in the TERT region (rs401681, OR per allele C = 0.89 (0.82–0.96), p = 0.0037; rs4975616, OR per allele A = 0.90 (0.83–0.97), p = 0.010), as well as the prostate cancer risk variant (rs7679673 in TET2; OR per allele C = 0.89 (0.82–0.97), Q = 0.05, p-het. = 0.82], although the interaction between the variant and race/ethnicity was not significant in the MEC (p- int. = 0.10). Among the eight GWAS risk variants for CLL, one variant replicated in our data for CLL/SLL [OR per allele G of rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027] and remained significant after Bonferroni correction (p,0.0063; Table S1 in File S1). None of the CLL variants were associated with overall NHL risk (p.0.05; data not shown). For example, in the MEC, the association of rs17483466 with CLL/SLL significantly differed from that with FL (p-het. from polytomous regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). regression 0.03), DLBCL (p het. 0.02) and others (p het. 0.01). Of the 113 GWAS risk variants for cancers other than NHL that were examined in PAGE, 53 SNPs were genotyped in all three studies, and the other 60 variants were typed in one or two studies (Table S2 in File S1). Six of the 53 SNPs showed nominal associations with the risk of overall NHL, including three risk variants originally identified for lung cancer, two risk variants identified for prostate cancer and one risk variant for breast cancer (Table 2). None of these associations remained significant after multiple test correction (i.e., p.4.4E-04 for 113 SNPs). Two lung cancer risk variants in the TERT region (rs401681, OR per allele C = 0.89 (0.82–0.96), p = 0.0037; rs4975616, OR per allele A = 0.90 (0.83–0.97), p = 0.010), as well as the prostate cancer risk variant (rs7679673 in TET2; OR per allele C = 0.89 (0.82–0.97), March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 4 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Figure 1. Forest plots for the association between a published follicular lymphoma risk variant (rs6457327) and the risk of follicular lymphoma or overall non-Hodgkin lymphoma (NHL) in the Multiethnic Cohort (MEC) and the Women’s Health Initiative (WHI) in the PAGE consortium. Statistical Analysis This may be a chance finding given that none of the associations remained significant after correcting for multiple tests, though such effects in opposite directions for different cancer types have been previously demonstrated in the TERT region and SNP rs401681 in particular [37]. A prostate cancer variant (rs7679673 in TET2) was specifically associated with a lower risk of DLBCL. The risk score based on the 53 non-NHL cancer SNPs was not significantly associated with the risk of overall NHL or subtypes, either as a continuous variable (Table 3) or categorized in quartiles (p.0.05; data not shown). There was no significant heterogeneity in any of the associations for individual SNPs or the risk score by sex, or by ethnic group (p.0.05; data not shown). One of the other three nominally positive associations for overall NHL was found with a breast cancer SNP in the coding region for a lymphocyte-specific protein (rs3817198 in LSP1). This gene encodes an intracellular F-actin binding protein that is expressed in endothelium and various hematopoietic cells (lym- phocytes, neutrophils, macrophages) [38]. As such, this protein may be involved in lymphomagenesis through the regulation of lymphocyte motility and migration, as evidenced by an association of another variant in LSP1 (rs2089910) with NHL in a study of an immune and inflammation SNP panel [39]. Discussion BioVU MEC WHI Summary Mean RS, case/ control OR (95% CI)* Mean RS, case/ control OR (95% CI)* Mean RS, case/ control OR (95% CI)* N, case/control OR (95% CI)* p-value Cochran Q (p-het) Overall NHL 47.4/46.9 0.98 (0.96, 1.01) 44.1/44.2 0.98 (0.94, 1.02) 42.4/42.4 1.00 (0.98, 1.01) 1,414/23,469 1.00 (0.98, 1.01) 0.80 3.27 (0.20) DLBCL - - 43.5/44.2 0.98 (0.94, 1.02) 42.8/42.4 1.01 (0.99, 1.04) 360/23,469 1.00 (0.98, 1.03) 0.79 2.00 (0.16) FL 47.0/46.9 1.00 (0.96, 1.05) 44.7/44.2 1.03 (0.98, 1.09) 42.9/42.4 1.02 (0.98, 1.05) 318/23,469 1.02 (0.99, 1.04) 0.17 0.85 (0.65) CLL/SLL 48.1/46.9 1.05 (0.98, 1.12) 46.2/44.2 1.04 (0.99, 1.09) 42.2/42.4 0.98 (0.93, 1.04) 179/23,469 1.02 (0.99, 1.05) 0.19 2.75 (0.25) * ORs and 95% CIs in individual studies were estimated per risk allele in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated in a meta-analysis of fixed effects models. Abbreviations: p-het. (p-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). doi:10.1371/journal.pone.0089791.t003 Table 2. Pleiotropic association of selected cancer susceptibility variants with the risk of overall non-Hodgkin lymphoma (NHL). Discussion BioVU MEC WHI Summary SNP Gene GWAS Risk (Ref.) Allele Cases/ Controls OR (95% CI)* Cases/ Controls OR (95% CI)* Cases/ Controls OR (95% CI)* OR (95% CI)* p-value (0.00044){ Cochran Q (p-het) rs401681 TERT Lung C (T) 292/8984 0.88 (0.74, 1.04) 372/9053 0.76 (0.66, 0.89) 733/5939 0.97 (0.87, 1.09) 0.89 (0.82, 0.96) 0.0037 6.26 (0.04) rs7679673 TET2 Prostate C (A) 290/8899 1.02 (0.86, 1.22) 372/8946 0.88 (0.75, 1.04) 733/5936 0.84 (0.75, 0.94) 0.89 (0.82, 0.97) 0.0057 3.42 (0.18) rs4975616 TERT Lung A (G) 293/9000 0.85 (0.72, 1.00) 368/9058 0.80 (0.68, 0.95) 732/5938 0.97 (0.87, 1.09) 0.90 (0.83, 0.97) 0.0103 4.06 (0.13) rs3817198 LSP1 Breast C (T) 293/8995 1.06 (0.89, 1.27) 371/9042 1.11 (0.93, 1.32) 733/5942 1.15 (1.02, 1.30) 1.12 (1.03, 1.22) 0.0112 0.56 (0.76) rs3131379 MSH5 Lung T (C) 290/8916 1.08 (0.83, 1.41) 334/9053 1.11 (0.78, 1.59) 733/5946 1.21 (1.01, 1.43) 1.16 (1.01, 1.33) 0.0302 0.51 (0.78) rs10993994 MSMB Prostate T (C) 292/9001 1.20 (1.01, 1.42) 369/9054 1.08 (0.93, 1.26) 732/5944 1.05 (0.94, 1.17) 1.09 (1.01, 1.18) 0.0356 1.68 (0.43) * ORs and 95% CIs in individual studies were estimated in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity (ancestry informative markers). Summary ORs and 95% CIs were estimated in a meta-analysis of fixed-effects models. {The Bonferroni corrected p-value for 53 SNPs/tests is 4.4E-04. Abbreviations: p-het. (P-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). doi:10.1371/journal.pone.0089791.t002 Table 3. Associations between a risk score (RS) for 53 GWAS-identified cancer risk variants and the overall and subtype-specific risks of NHL. Discussion Increasing evidence supports the pleiotropic involvement of common genetic risk variants in multiple diseases or complex traits. Thus, we examined a substantial number of risk variants identified in GWAS of common cancers in relation to overall and subtype-specific risk of NHL. Our analysis extended the associ- ation of the FL risk variant to overall NHL and the DLBCL March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 5 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma able 2. Pleiotropic association of selected cancer susceptibility variants with the risk of overall non-Hodgkin lymphoma (NHL). BioVU MEC WHI Summary P Gene GWAS Risk (Ref.) Allele Cases/ Controls OR (95% CI)* Cases/ Controls OR (95% CI)* Cases/ Controls OR (95% CI)* OR (95% CI)* p-value (0.00044){ Cochran Q (p-het) 01681 TERT Lung C (T) 292/8984 0.88 (0.74, 1.04) 372/9053 0.76 (0.66, 0.89) 733/5939 0.97 (0.87, 1.09) 0.89 (0.82, 0.96) 0.0037 6.26 (0.04) 679673 TET2 Prostate C (A) 290/8899 1.02 (0.86, 1.22) 372/8946 0.88 (0.75, 1.04) 733/5936 0.84 (0.75, 0.94) 0.89 (0.82, 0.97) 0.0057 3.42 (0.18) 975616 TERT Lung A (G) 293/9000 0.85 (0.72, 1.00) 368/9058 0.80 (0.68, 0.95) 732/5938 0.97 (0.87, 1.09) 0.90 (0.83, 0.97) 0.0103 4.06 (0.13) 817198 LSP1 Breast C (T) 293/8995 1.06 (0.89, 1.27) 371/9042 1.11 (0.93, 1.32) 733/5942 1.15 (1.02, 1.30) 1.12 (1.03, 1.22) 0.0112 0.56 (0.76) 131379 MSH5 Lung T (C) 290/8916 1.08 (0.83, 1.41) 334/9053 1.11 (0.78, 1.59) 733/5946 1.21 (1.01, 1.43) 1.16 (1.01, 1.33) 0.0302 0.51 (0.78) 0993994 MSMB Prostate T (C) 292/9001 1.20 (1.01, 1.42) 369/9054 1.08 (0.93, 1.26) 732/5944 1.05 (0.94, 1.17) 1.09 (1.01, 1.18) 0.0356 1.68 (0.43) Rs and 95% CIs in individual studies were estimated in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity (ancestry informative markers). Summary ORs and 95% CIs were imated in a meta-analysis of fixed-effects models. he Bonferroni corrected p-value for 53 SNPs/tests is 4.4E-04. breviations: p-het. (P-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). :10.1371/journal.pone.0089791.t002 able 3. Associations between a risk score (RS) for 53 GWAS-identified cancer risk variants and the overall and subtype-specific risks of NHL. Table 3. Associations between a risk score (RS) for 53 GWAS-identified cancer risk variants and the overall and subtype-specific risks of NHL. Discussion BioVU MEC WHI Summary Mean RS, case/ control OR (95% CI)* Mean RS, case/ control OR (95% CI)* Mean RS, case/ control OR (95% CI)* N, case/control OR (95% CI)* p-value Cochran Q (p-het) Overall NHL 47.4/46.9 0.98 (0.96, 1.01) 44.1/44.2 0.98 (0.94, 1.02) 42.4/42.4 1.00 (0.98, 1.01) 1,414/23,469 1.00 (0.98, 1.01) 0.80 3.27 (0.20) DLBCL - - 43.5/44.2 0.98 (0.94, 1.02) 42.8/42.4 1.01 (0.99, 1.04) 360/23,469 1.00 (0.98, 1.03) 0.79 2.00 (0.16) FL 47.0/46.9 1.00 (0.96, 1.05) 44.7/44.2 1.03 (0.98, 1.09) 42.9/42.4 1.02 (0.98, 1.05) 318/23,469 1.02 (0.99, 1.04) 0.17 0.85 (0.65) CLL/SLL 48.1/46.9 1.05 (0.98, 1.12) 46.2/44.2 1.04 (0.99, 1.09) 42.2/42.4 0.98 (0.93, 1.04) 179/23,469 1.02 (0.99, 1.05) 0.19 2.75 (0.25) * ORs and 95% CIs in individual studies were estimated per risk allele in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated in a meta-analysis of fixed effects models. Abbreviations: p-het. (p-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). Table 2. Pleiotropic association of selected cancer susceptibility variants with the risk of overall non-Hodgkin lymphoma (NHL). Discussion BioVU MEC WHI Summary SNP Gene GWAS Risk (Ref.) Allele Cases/ Controls OR (95% CI)* Cases/ Controls OR (95% CI)* Cases/ Controls OR (95% CI)* OR (95% CI)* p-value (0.00044){ Cochran Q (p-het) rs401681 TERT Lung C (T) 292/8984 0.88 (0.74, 1.04) 372/9053 0.76 (0.66, 0.89) 733/5939 0.97 (0.87, 1.09) 0.89 (0.82, 0.96) 0.0037 6.26 (0.04) rs7679673 TET2 Prostate C (A) 290/8899 1.02 (0.86, 1.22) 372/8946 0.88 (0.75, 1.04) 733/5936 0.84 (0.75, 0.94) 0.89 (0.82, 0.97) 0.0057 3.42 (0.18) rs4975616 TERT Lung A (G) 293/9000 0.85 (0.72, 1.00) 368/9058 0.80 (0.68, 0.95) 732/5938 0.97 (0.87, 1.09) 0.90 (0.83, 0.97) 0.0103 4.06 (0.13) rs3817198 LSP1 Breast C (T) 293/8995 1.06 (0.89, 1.27) 371/9042 1.11 (0.93, 1.32) 733/5942 1.15 (1.02, 1.30) 1.12 (1.03, 1.22) 0.0112 0.56 (0.76) rs3131379 MSH5 Lung T (C) 290/8916 1.08 (0.83, 1.41) 334/9053 1.11 (0.78, 1.59) 733/5946 1.21 (1.01, 1.43) 1.16 (1.01, 1.33) 0.0302 0.51 (0.78) rs10993994 MSMB Prostate T (C) 292/9001 1.20 (1.01, 1.42) 369/9054 1.08 (0.93, 1.26) 732/5944 1.05 (0.94, 1.17) 1.09 (1.01, 1.18) 0.0356 1.68 (0.43) * ORs and 95% CIs in individual studies were estimated in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity (ancestry informative markers). Summary ORs and 95% CIs we estimated in a meta-analysis of fixed-effects models. {The Bonferroni corrected p-value for 53 SNPs/tests is 4.4E-04. Abbreviations: p-het. (P-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiativ doi:10.1371/journal.pone.0089791.t002 PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e89791 6 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma subtypes of non-Hodgkin lymphoma (NHL). Table S2. List of 113 GWAS-based cancer risk variants examined for pleiotropy on NHL in PAGE; the 53 SNPs listed as genotyped in all three studies were included in the risk score analysis. Table S3. Pleiotropic association of selected cancer susceptibility variants with the risk of common subtypes of non-Hodgkin lymphoma (NHL). (DOC) Another non-significant positive association for NHL, especially with DLBCL, was with a lung cancer susceptibility variant, rs3131379, in MSH5 or mutS homolog 5, a gene involved in the DNA mismatch repair pathway [40]. References susceptibility loci at 2p16.1 (REL), 8q24.21 and 10p14 (GATA3). Nat Genet 42: 1126–1130. susceptibility loci at 2p16.1 (REL), 8q24.21 and 10p14 (GATA3). Nat Genet 42: 1126–1130. 1. Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA Cancer J Clin 63: 11–30. 15. Crowther-Swanepoel D, Broderick P, Di Bernardo MC, Dobbins SE, Torres M, et al. (2010) Common variants at 2q37.3, 8q24.21, 15q21.3 and 16q24.1 influence chronic lymphocytic leukemia risk. Nat Genet 42: 132–136. 2. Smedby KE, Hjalgrim H (2011) Epidemiology and etiology of mantle cell lymphoma and other non-Hodgkin lymphoma subtypes. Semin Cancer Biol 21: 293–298. 3. Wang SS, Slager SL, Brennan P, Holly EA, De Sanjose S, et al. (2007) Family history of hematopoietic malignancies and risk of non-Hodgkin lymphoma (NHL): a pooled analysis of 10 211 cases and 11 905 controls from the International Lymphoma Epidemiology Consortium (InterLymph). Blood 109: 3479–3488. 16. Matise TC, Ambite JL, Buyske S, Carlson CS, Cole SA, et al. (2011) The Next PAGE in understanding complex traits: design for the analysis of Population Architecture Using Genetics and Epidemiology (PAGE) Study. Am J Epidemiol 174: 849–859. 17. Roden DM, Pulley JM, Basford MA, Bernard GR, Clayton EW, et al. (2008) Development of a large-scale de-identified DNA biobank to enable personalized medicine. Clin Pharmacol Ther 84: 362–369. 4. Nielsen SF, Bojesen SE, Birgens HS, Nordestgaard BG (2011) Risk of thyroid cancer, brain cancer, and non-Hodgkin lymphoma after adult leukemia: a nationwide study. Blood 118: 4062–4069. 18. Ritchie MD, Denny JC, Crawford DC, Ramirez AH, Weiner JB, et al. (2010) Robust replication of genotype-phenotype associations across multiple diseases in an electronic medical record. Am J Hum Genet 86: 560–572. 5. Chakraborty S, Tarantolo SR, Batra SK, Hauke RJ (2013) Incidence and prognostic significance of second primary cancers in renal cell carcinoma. Am J Clin Oncol 36: 132–142. 19. Kolonel LN, Henderson BE, Hankin JH, Nomura AM, Wilkens LR, et al. (2000) A multiethnic cohort in Hawaii and Los Angeles: baseline characteristics. AmJEpidemiol 151: 346–357. 6. Nielsen SF, Nordestgaard BG, Bojesen SE (2012) Associations between first and second primary cancers: a population-based study. CMAJ 184: E57–69. p 20. Kolonel LN, Altshuler D, Henderson BE (2004) The Multiethnic Cohort study: exploring genes, lifestyle and cancer risk. NatRevCancer 4: 519–527. 7. Hindorff LA, MacArthur J, Wise A, Junkins HA, Klemm AK, et al. A catalog of published genome-wide association studies. U.S. National Human Genome Research Institute. References Available: www.genome.gov/gwastudies. Accessed 2013 Nov 15. 21. The Women’s Health Initiative Study Group (1998) Design of the Women’s Health Initiative clinical trial and observational study. The Women’s Health Initiative Study Group. Control Clin Trials 19: 61–109. 8. Sivakumaran S, Agakov F, Theodoratou E, Prendergast JG, Zgaga L, et al. (2011) Abundant pleiotropy in human complex diseases and traits. Am J Hum Genet 89: 607–618. 22. Curb JD, McTiernan A, Heckbert SR, Kooperberg C, Stanford J, et al. (2003) Outcomes ascertainment and adjudication methods in the Women’s Health Initiative. Ann Epidemiol 13: S122–128. 9. Hindorff LA, Sethupathy P, Junkins HA, Ramos EM, Mehta JP, et al. (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A 106: 9362–9367. 23. Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, et al. (2000) The World Health Organization classification of neoplasms of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting–Airlie House, Virginia, November, 1997. HematolJ 1: 53–66. 10. Hindorff LA, Gillanders EM, Manolio TA (2011) Genetic architecture of cancer and other complex diseases: lessons learned and future directions. Carcinogen- esis 32: 945–954. 24. Morton LM, Turner JJ, Cerhan JR, Linet MS, Treseler PA, et al. (2007) Proposed classification of lymphoid neoplasms for epidemiologic research from the Pathology Working Group of the International Lymphoma Epidemiology Consortium (InterLymph). Blood 110: 695–708. 11. Haiman CA, Patterson N, Freedman ML, Myers SR, Pike MC, et al. (2007) Multiple regions within 8q24 independently affect risk for prostate cancer. Nat Genet 39: 638–644. 12. Au WY, Horsman DE, Viswanatha DS, Connors JM, Klasa RJ, et al. (2000) 8q24 translocations in blastic transformation of mantle cell lymphoma. Haematologica 85: 1225–1227. 25. Bush WS, Boston J, Pendergrass SA, Dumitrescu L, Goodloe R, et al. (2013) Enabling high-throughput genotype-phenotype associations in the Epidemio- logic Architecture for Genes Linked to Environment (EAGLE) project as part of the Population Architecture using Genomics and Epidemiology (PAGE) study. Pac Symp Biocomput: 373–384. 13. Okhowat R, Dorner S, Hinterberger W, Fonatsch C (2003) Unusual karyotype aberrations involving 2p12, 3q27, 18q21, 8q24, and 14q32 in a patient with non-Hodgkin lymphoma/acute lymphoblastic leukemia. Cancer Genet Cyto- genet 142: 60–64. 26. De Roos AJ, Martinez-Maza O, Jerome KR, Mirick DK, Kopecky KJ, et al. (2013) Investigation of epstein-barr virus as a potential cause of B-cell non- hodgkin lymphoma in a prospective cohort. Author Contributions Conceived and designed the experiments: UL UP LLM LAH CAH CK. Performed the experiments: UL JMK WSB TCM CC SLP CSC ED DD MF CAH BEH LAH LNK UP DOS MT LRW CW CK LLM. Analyzed the data: UL JMK WSB CC. Contributed reagents/materials/analysis tools: WSB TCM CC CAH BEH LAH LNK UP DOS MT LRW CW CK LLM. Wrote the paper: UL JMK WSB LLM. Discussion This variant is also located near the major histocompatibility complex (MHC or human leukocyte antigen, HLA) region in chromosome 6 (6p21.33), as is the GWAS variant for FL (rs6457327), and has been associated with the risk of systemic lupus erythematosus in a GWAS [41]. Our findings on MSH5 and MHC variants indicate possible involvement of variants in or near this highly-conserved immune- regulatory region in the etiology of NHL (including FL and DLBCL), in addition to that of lung cancer. Acknowledgments The authors thank the WHI investigators and staff for their dedication, and the study participants for making the program possible. The PAGE consortium thanks the staff and participants of all PAGE studies for their important contributions. The authors also gratefully acknowledge the contribution of Julia Higashio and Rasheeda Williams at the PAGE Coordinating Center and of Dr. Kylee Spencer at Heidelberg University. Disclaimers: The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. This study was nested in three large studies with well- characterized phenotypes and pathology-confirmed histologic information for subtype classification. However, despite the sizeable number of NHL cases included, we had limited power, in part likely due to the heterogeneous nature of NHL. Also, only a subset of total cancer variants was genotyped in all three PAGE studies for the NHL analysis. Our analyses do not provide clear evidence that these common cancer genetic susceptibility loci may play a role in the etiologies of NHL. A more systematic approach in larger pooled analyses of specific subtypes, with larger SNP panels, is warranted in future research. Supporting Information Files S1 Supporting tables. Table S1. Association between established GWAS risk variants for follicular lymphoma (FL) and for chronic lymphocytic leukemia (CLL) with the risk of these Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma 27. Lim U, Wilkens LR, Monroe KR, Caberto C, Tiirikainen M, et al. (2012) Susceptibility variants for obesity and colorectal cancer risk: the multiethnic cohort and PAGE studies. Int J Cancer 131: E1038–1043. 35. Cerhan JR, Fredericksen ZS, Novak AJ, Ansell SM, Kay NE, et al. (2012) A two- stage evaluation of genetic variation in immune and inflammation genes with risk of non-Hodgkin lymphoma identifies new susceptibility locus in 6p21.3 region. Cancer Epidemiol Biomarkers Prev 21: 1799–1806. J 28. Hoggart CJ, Clark TG, De Iorio M, Whittaker JC, Balding DJ (2008) Genome- wide significance for dense SNP and resequencing data. Genet Epidemiol 32: 179–185. 36. Slager SL, Camp NJ, Conde L, Shanafelt TD, Achenbach SJ, et al. (2012) Common variants within 6p21.31 locus are associated with chronic lymphocytic leukaemia and, potentially, other non-Hodgkin lymphoma subtypes. Br J Haematol 159: 572–576. 29. Skibola CF, Bracci PM, Halperin E, Conde L, Craig DW, et al. (2009) Genetic variants at 6p21.33 are associated with susceptibility to follicular lymphoma. Nat Genet 41: 873–875. 37. Mocellin S, Verdi D, Pooley KA, Landi MT, Egan KM, et al. (2012) Telomerase reverse transcriptase locus polymorphisms and cancer risk: a field synopsis and meta-analysis. J Natl Cancer Inst 104: 840–854. 30. Conde L, Halperin E, Akers NK, Brown KM, Smedby KE, et al. (2010) Genome-wide association study of follicular lymphoma identifies a risk locus at 6p21.32. Nat Genet 42: 661–664. 38. Easton DF, Pooley KA, Dunning AM, Pharoah PD, Thompson D, et al. (2007) Genome-wide association study identifies novel breast cancer susceptibility loci. Nature 447: 1087–1093. 31. Di Bernardo MC, Crowther-Swanepoel D, Broderick P, Webb E, Sellick G, et al. (2008) A genome-wide association study identifies six susceptibility loci for chronic lymphocytic leukemia. Nat Genet 40: 1204–1210. 39. Cerhan JR, Ansell SM, Fredericksen ZS, Kay NE, Liebow M, et al. (2007) Genetic variation in 1253 immune and inflammation genes and risk of non- Hodgkin lymphoma. Blood 110: 4455–4463. 32. Kosoy R, Nassir R, Tian C, White PA, Butler LM, et al. (2009) Ancestry informative marker sets for determining continental origin and admixture proportions in common populations in America. Hum Mutat 30: 69–78. 40. Kazma R, Babron MC, Gaborieau V, Genin E, Brennan P, et al. (2012) Lung cancer and DNA repair genes: multilevel association analysis from the International Lung Cancer Consortium. Carcinogenesis 33: 1059–1064. p p p p 33. References Cancer Epidemiol Biomarkers Prev 22: 1747–1755. 14. Enciso-Mora V, Broderick P, Ma Y, Jarrett RF, Hjalgrim H, et al. (2010) A genome-wide association study of Hodgkin’s lymphoma identifies new March 2014 | Volume 9 | Issue 3 | e89791 March 2014 | Volume 9 | Issue 3 | e89791 7 PLOS ONE | www.plosone.org Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Willer CJ, Li Y, Abecasis GR (2010) METAL: fast and efficient meta-analysis of genomewide association scans. Bioinformatics 26: 2190–2191. genomewide association scans. Bioinformatics 26: 2190–2191 g g 41. Harley JB, Alarcon-Riquelme ME, Criswell LA, Jacob CO, Kimberly RP, et al. (2008) Genome-wide association scan in women with systemic lupus erythema- tosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci. Nat Genet 40: 204–210. 34. Smedby KE, Foo JN, Skibola CF, Darabi H, Conde L, et al. (2011) GWAS of follicular lymphoma reveals allelic heterogeneity at 6p21.32 and suggests shared genetic susceptibility with diffuse large B-cell lymphoma. PLoS Genet 7: e1001378. March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8
https://openalex.org/W3087992083
https://molecular-cancer.biomedcentral.com/track/pdf/10.1186/s12943-020-01258-7
English
null
Targeting STAT3 in Cancer Immunotherapy
Molecular cancer
2,020
cc-by
15,442
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Targeting STAT3 in Cancer Immunotherapy Sailan Zou1†, Qiyu Tong1†, Bowen Liu2, Wei Huang3, Yan Tian1* and Xianghui Fu1* Abstract As a point of convergence for numerous oncogenic signaling pathways, signal transducer and activator of transcription 3 (STAT3) is central in regulating the anti-tumor immune response. STAT3 is broadly hyperactivated both in cancer and non-cancerous cells within the tumor ecosystem and plays important roles in inhibiting the expression of crucial immune activation regulators and promoting the production of immunosuppressive factors. Therefore, targeting the STAT3 signaling pathway has emerged as a promising therapeutic strategy for numerous cancers. In this review, we outline the importance of STAT3 signaling pathway in tumorigenesis and its immune regulation, and highlight the current status for the development of STAT3-targeting therapeutic approaches. We also summarize and discuss recent advances in STAT3-based combination immunotherapy in detail. These endeavors provide new insights into the translational application of STAT3 in cancer and may contribute to the promotion of more effective treatments toward malignancies. Keywords: STAT3, Cancer, Immunosuppression, Immunotherapy, Immune checkpoint blockade, CAR-T identified and developed for immunotherapy appropriate for the clinical use. REVIEW Open Access Zou et al. Molecular Cancer (2020) 19:145 https://doi.org/10.1186/s12943-020-01258-7 Zou et al. Molecular Cancer (2020) 19:145 https://doi.org/10.1186/s12943-020-01258-7 Introduction Dysregulation of immune checkpoints is a protective mechanism used by a number of malignancies to es- cape from the immune surveillance allowing for can- cer development [1]. This has inspired the idea of boosting the host immune response as an anti-cancer therapy. Indeed, the blockage of immune checkpoints, including programmed cell death protein 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1) and cyto- toxic T-lymphocyte-associated protein 4 (CTLA-4), improves clinical outcomes in subsets of patients with cancers previously considered to be essentially un- treatable [2–4]. In order to expand the array of treat- able cancers as well as increase the number of patients that respond to the therapy, novel therapeutic targets and new molecules/strategies should be urgently The signal transducer and activator of transcription (STAT) proteins are a family of cytoplasmic transcription factors which share an overall general structure, organized into functional modular domains. The mammalian STAT family comprises STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6 that mediate multiple intra- cellular signaling pathways [5]. Among them, STAT3 is in- volved in numerous biological processes including cell proliferation, survival, differentiation, and angiogenesis [6, 7]. In normal cells, transient activation of STAT3 (predom- inantly by phosphorylation) transmits transcriptional sig- nals from cytokines and growth factor receptors at the plasma membrane to the nucleus [5]. In contrast, STAT3 becomes hyperactivated in the majority of human cancers and is generally associated with poor clinical prognosis [8]. Therefore, it is not surprising that STAT3 signaling path- way has long been recognized as a potential therapeutic tar- get for cancer therapy owing to their roles in tumor formation, metastasis and drug resistance [9–12]. More- over, accumulating evidence reveals that STAT3 hyperacti- vation can mediate tumor-induced immunosuppression at * Correspondence: tyfxh@163.com; xfu@scu.edu.cn †Sailan Zou and Qiyu Tong are co-first author. 1Division of Endocrinology and Metabolism, National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610041, Sichuan, China Full list of author information is available at the end of the article The STAT3 signaling pathway g g p y STAT3 is a protein consisting of 770 amino acids and characterized by the presence of 6 functionally con- served domains, including the amino-terminal domain (NH2), the coiled-coil domain (CCD), the DNA-binding domain (DBD), the linker domain, the SRC homology 2 (SH2) domain, and the carboxyl-terminal transactivation domain (TAD) (Fig. 1a). Among them, SH2 is the most highly conserved STAT domain and plays a crucial role in signaling via binding to specific phosphotyrosine mo- tifs [15]. In an unstimulated cell, STAT3 is tightly regu- lated by negative modulators to maintain an inactive state in the cytoplasm. These modulators include mem- bers of the protein inhibitor of activated STAT (PIAS), suppressor of cytokine signaling (SOCS) families, protein tyrosine phosphatases (SHP1, SHP2, PTPN1, PTPN2 PTPRD, PTPRT and DUSP22), and ubiquitin enzymes [8]. In response to stimuli, STAT3 becomes activated mainly by direct phosphorylation at tyrosine (705) and serine (727) residues induced by its upstream ligands in- cluding Janus kinases (JAKs), tyrosine kinases, cytokines and several non-receptor tyrosine kinases such as SRC and ABL; the phosphorylation induces dimerization of STAT3 proteins followed by nuclear translocation, DNA binding, and eventually execution of their nuclear func- tions [15]. y Additionally, increasing evidence suggests that non- coding RNAs (ncRNAs) can directly or indirectly modu- late STAT3 activity (Fig. 1c). As the most extensively studied ncRNAs, numerous microRNAs (miRNAs) have been shown to target STAT3 directly and certain com- ponents of STAT3 signaling pathway (IL-6, JAK2, SOCS1, PIAS3, etc.), thereby modulating STAT3 expres- sion and activation [8, 25–29]. For instance, miR-125b- 5p can directly target STAT3 and inhibit its expression [27], while miR-218 indirectly suppresses STAT3 activa- tion by targeting IL-6 receptor and JAK3 [28]. More re- cently, it has shown that exosome-mediated transfer of certain miRNAs, such as miR-193a-3p, miR-210-3p and miR-5100, can promote metastasis of lung cancer by en- hancing STAT3 activity [29], although the molecular mechanisms await further investigation. Likewise, long non-coding RNAs (lncRNAs) can modulate the expres- sion and activation of STAT3 directly or indirectly through multiple mechanisms [30–33]. For instance, lincRNA-p21 can inhibit the STAT3 transcriptional ac- tivity via directly binding to STAT3 [30]. Lnc-BM can bind to the JH2 domain of JAK2, which increases JAK2 activation, and thus indirectly enhances activity of STAT3 [32]. Zou et al. Molecular Cancer (2020) 19:145 Page 2 of 19 important regulatory mechanisms for STAT3 activity. SMYD2-dependent methylation of STAT3 contributes to the hyperphosphorylation of STAT3, whereas EZH2- and SET9-dependent dimethylation of STAT3 inhibits the activity of DNA-bound STAT3 dimers [20–22]. Sumoylation at the lysine 451 of STAT3 by SUMO2/3 can promote its interaction to the nuclear phosphatase TC45, thereby restraining phosphorylated STAT3 in the nucleus, while de-sumoylation by SENP3 leads to the hyperphosphorylation of STAT3 [23]. However, andro- gen receptor degradation enhancer ASC-J9 can inhibit the STAT3 phosphorylation via inducing the sumoyla- tion of STAT3 at lysine 679 [24]. many levels [13, 14]. Given the similarities between tumori- genesis and STAT3-dependent immunity, new therapeutic strategies that target STAT3 signaling pathway may open up new avenues for long-lasting and multilayered tumor control. This review outlines the role of the STAT3 pathway in tumor immunity, summarizes the recent progress in STAT3-centered anti-cancer approaches, and highlights future directions for the clinical immunotherapy. The STAT3 signaling pathway STAT3 is characterized by the presence of six different functional domains, including an amino-terminal domain (NTD) for cooperative binding of STAT proteins to multiple consensus DNA sites, a coiled-coil domain (CCD) for recruitment of STAT3 to the receptor as well as subsequent phosphorylation, dimerization and nuclear translocation, a DNA-binding domain (DBD) for recognizing and binding to a specific consensus DNA sequence, a linker domain for connecting the DBD with the SRC homology 2 (SH2) domain, a SH2 domain for recruitment and activation as well as dimerization of the STAT3 molecule by interacting with phosphorylated tyrosine residues in the opposing subunit, and a carboxyl-terminal transactivation domain (TAD). b STAT3 signaling pathway. STAT3 is activated by upstream growth factor kinases and cytokine receptors. Non-receptor tyrosine kinases such as SRC and ABL can also lead to constitutive activation of STAT3. Phosphorylated STAT3 dimerizes and translocates to nucleus, which causes the transcription of target genes including immunosuppression, angiogenesis, metastasis, proliferation and survival. The signaling pathway can be inhibited by SOCS proteins, PIAS proteins, and protein tyrosine phosphatases (PTPases), etc. c Interplay between noncoding RNAs and STAT3 signaling pathway. On the one hand, miRNAs and lncRNAs can regulate STAT3 activation through not only directly targeting STAT3, but also targeting the components of the STAT3 signaling pathway, such as IL-6, JAK2, SOCS1 and PIAS3; CircRNAs usually regulate STAT3 by acting as sponges for miRNAs. On the other hand, STAT3 is able to regulate miRNAs and lncRNAs expression in many ways. [53]. For instance, STAT3 can suppress the secretion of type 1 IFNs (IFN-Is) and IFN-I-responsive genes via multiple actions, such as attenuating the activation of IFN-I signaling, reducing the expression of ISGF3 components, and impairing the potential of ISGF3 transactivation [54, 55]. other hand, STAT3 can upregulate miR-21 through in- creasing IL-6 expression [40]. The regulatory effect of STAT3 in lncRNAs is also emerging. STAT3 can dir- ectly bind to the promoter region of certain lncRNAs, such as SNHG17, DUXAP8 and HAGLROS, and thus contributes to their overexpression in cancers [41–43]. In tumor cells, STAT3 often interacts with other signaling pathways, such as NF-κB, to confer robust- ness for tumor progression [44, 56, 57]. NF-κB signal- ing is of importance for both inflammation-induced carcinogenesis and anti-tumor immunity [57]. NF-κB (especially RELA) can upregulate a spectrum of tar- gets involved in chronic inflammation and cancer ini- tiation such as cyclooxygenase 2, IL-6, IL-23, and IL- 1β [44]. The STAT3 signaling pathway FLANC, a novel primate-specific lncRNA, has shown to upregulate and prolong the half-life of phosphorylated STAT3, but not total STAT3, albeit the underlying mechanism remains unknown [34]. Intri- guingly, a recent study revealed that LINC00908- encoded polypeptide ASRPS can directly bind to the CCD domain of STAT3, and thus reduce STAT3 phos- phorylation [35]. In general, circular RNAs (circRNAs) can modulate gene expression by acting as sponges of endogenous miRNAs. It has shown that circ-HIPK3, circ_0076305 and circ-STAT3 positively modulate STAT3 signaling by sponging miR-124-3p, miR-296-5p, and miR-29a/b/c-3p, respectively [36–38]. In parallel, STAT3 has a capacity of regulating ncRNAs directly or indirectly. The regulation of miR-21 by STAT3 has been extensively studied. On the one hand, STAT3 can dir- ectly regulate miR-21 transcription in myeloma cells by binding to its upstream enhancer region [39]. On the Beyond phosphorylation, other posttranslational modi- fications (i.e. acetylation, methylation, and sumoylation) can also regulate STAT3 transcriptional activity through altering STAT3 phosphorylation, and thus add another layer of complexity for STAT3 hyperphosphorylation in cancers (Fig. 1b). For instance, acetylation at several ly- sine residues within both the NH2 and SH2 domains, primarily mediated by the CBP/p300 acetyltransferase, can enhance STAT3 transactivating potential, which is associated with increased dimer stabilization, tyrosine 705 phosphorylation, nuclear translocation, and localized histone hyperacetylation of target promoters [16]. In contrast, deacetylation by several deacetylases, such as HADC1-3, SIRT1 and Loxl3, inhibits transcription of STAT3 targets [17–19]. The dynamic balance of acetyl- ation and deacetylation plays a role in STAT3 activation and is involved in various cellular events. Similarly, methylation and sumoylation are also emerging as Page 3 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer Fig. 1 (See legend on next page.) Fi 1 (S l d t ) Fig. 1 (See legend on next page.) Page 4 of 19 Zou et al. Molecular Cancer (2020) 19:145 (See figure on previous page.) Fig. 1 The domain structure and signaling pathway of STAT3. a Schematic domain structure of STAT3. STAT3-driven tumor immunosuppression STAT3-driven tumor immunosuppression The tumor microenvironment (TME) is a highly com- plex and heterogenous ecosystem consisting tumor- infiltrating immune cells, cancer-associated fibroblasts (CAFs), smooth muscle cells, endothelial cells, and the tumor cells [45, 46]. It is becoming increasingly evident that TME can promote the progression of cancer and mediate therapeutic resistance, particularly against can- cer immunotherapy [47, 48]. Gathered evidence suggests that STAT3 becomes hyperactivated not only in cancer cells themselves but also in immune cells and CAFs within the TME [13, 49–52]. The hyperactivation of STAT3 in TME compartments might have a significant impact on anti-tumor immunity through various mecha- nisms (described below in more detail). The STAT3 signaling pathway Several layers of STAT3-NF-κB crosstalk have been identified thus far: (1) both NF-κB and STAT3 are frequently activated in the same tumor cells and TME-associated infiltrating immune cells, and share a wide range of common targets that par- ticipate in cell proliferation, metastasis, anti-apoptosis, and angiogenesis [56]; (2) STAT3 can prolong nuclear retention of RELA through p300-mediated acetylation, leading to the persistent activation of NF-κB [58]; (3) many cytokines (i.e. IL-6) can in turn simultaneously activate STAT3 and NF-κB [57]; (4) it has recently been demonstrated that NF-κB activity in pancreatic CAFs shielded cancer cells from immune attack by increasing CXCL12 expression [59]. Given the well- known feedforward loop between CXCL12 and STAT3 [60, 61], it is possible that STAT3 contributes to NF-κB-mediated immune evasion by this vicious cycle. As described above, STAT3 activity can be influenced by many factors such as numerous post-translational modifications and multiple ncRNAs regulation. These complexities, together with the fact that the STAT3 sig- naling pathway is responsive to a great variety of cellular stresses and stimuli [44], pose difficulties in our under- standing of abnormal hyperactivated STAT3 in cancers. Future investigations delineating the regulatory network of STAT3 will likely facilitate the clinical translation of STAT3-based therapies for human malignancy. STAT3-mediated crosstalk between cancer cells and diverse cell subsets in the TME Hyperactivation of STAT3 in tumor-infiltrating im- mune cells causes immunosuppression by inhibiting both innate and adaptive immune responses. In brief, excessive STAT3 activity in innate immune cell sub- sets may impair the production of pro-inflammatory mediators such as IFNγ, dampen antigen presenta- tion, and inhibit the tumor-killing activities of ef- fector cells. In adaptive immune subsets, elevated STAT3 activity has the ability to inhibit the accumu- lation of effector T cells, thereby restraining their anti-tumor effects [62–64]. Interestingly, some recent studies suggest previously unknown functions of STAT3 in tumor immunity. For instance, placental growth factor (PlGF) [65] and Cxxc finger protein 1 (Cxxc1) [66] can act as key upstream regulators of STAT3 signaling, which subsequently contributes to the differentiation and function of Th17 cells. STAT3 mediates the major impact of β2 adrenergic receptor on the immunosuppressive potential of myeloid-derived suppressor cells (MDSCs) in the TME [67]. In glioblastoma-infiltrating tumor- associated macrophages, STAT3 acts as a positive regulator of aryl hydrocarbon receptor (AHR) and thus increases the recruitment of tumor-associated macrophages and tumor growth [68]. It has been shown that STAT3 modulates the abundance and function of regulatory T (Treg) cells in response to radiation therapy in head and neck cancer, suggest- ing that STAT3 inhibition may be beneficial for pa- tients receiving radiation [69]. Aberrantly activated STAT3 can lead to tumor-induced immunosuppression via propagating the crosstalk between cancer cells and their immunological microenvironment. In tumor cells, hyperactivated STAT3 promotes the ex- pression of immunosuppressive factors such as VEGF, IL- 6, and IL-10 [53]. Meanwhile, these tumor-derived factors that also happen to be STAT3 activators could be trans- ited to the TME, and thus enhance STAT3 signaling in various immune cell subsets and CAFs (Fig. 2). ( g ) In particular, STAT3 hyperactivation in tumor cells has a vital role in dendritic cells (DCs) maturation. DCs essentially are monocytes at a differentiated stage and the key antigen presenting cells of the immune system. As immune sentinels, DCs play an important role in the initiation of T-cell response against tumors, while imma- ture DCs generally induce immune tolerance [75]. Hyperactivated STAT3 in tumor cells can suppress the expression of IL-12 and TNF-α, leading to a decrease in Bcl-2 expression in DCs [53]. STAT3 also represses the expression of major histocompatibility complex (MHC) class II complexes and co-stimulatory signals (CD80 and CD86), which are essential to the antigen presenting function of DCs [13]. STAT3-mediated crosstalk between cancer cells and diverse cell subsets in the TME Meanwhile, STAT3 inhibits DC maturation and innate immunity through negatively regulating the expression of CXCL10 and CCL5 [53]. Furthermore, the immunosuppressive factors such as IL- 6, IL-10, and VEGF induced by STAT3 can inhibit DC generation through reducing protein kinase C beta II (PKCβII) expression [76]. Given that immature DCs can- not activate antigen-specific CD8+ T cells, activated STAT3 signaling in tumor cells reduces the anti- tumorigenic effector functions of CD8+ T cells. g What’s more, certain factors released by CAFs can modulate STAT3 signaling in other cell types in the tumor milieu. TGFβ, an evolutionarily conserved regula- tor of tumorigenesis, is a crucial driver of the activity of CAFs. Accumulating evidence suggests that TGFβ- stimulated CAFs increase the secretion of IL-6 and IL- 11, which trigger GP130/STAT3 signaling in cancer cells and thus promote cancer metastasis and progression [77–80]. STAT3 is also involved in the crosstalk between CAFs and immune cells. For example, CCL2 secreted from CAFs with STAT3 hyperactivation can promote the recruitment of immunosuppressive MDSCs and hepatocarcinogenesis [72]. Moreover, the differentiation of these recruited MDSCs has been shown to be con- trolled in an IL-6/STAT3-dependent manner [81]. In addition, IL-6 derived from CAFs can activate STAT3 in DCs, which subsequently induce liver cancer immune escape through impairing T-cell proliferation and pro- moting Treg cells expansion [82]. STAT3 signaling in CAFs and other cells orchestrates stromal remodeling of STAT3 in immune cells In tumor cells per se, hyperactivated STAT3 decreases the expression of immune-stimulating factors includ- ing interferons (IFNs), pro-inflammatory cytokines (IL-12, TNF-α) and chemokines (CCL5, CXCL10), while increases the expression of certain cytokines and growth factors (IL-6, IL-10, TGFβ, and VEGF), thereby exerting profound immune effects (Fig. 1b) STAT3 also plays a pivotal role in a plethora of tumor- infiltrating immune cells that predominantly comprise the TME and recent comprehensive reviews have cov- ered this topic [62–64]. Here we would only like to briefly mention the diverse functions of STAT3 in im- mune cell milieu, together with some recent advances Page 5 of 19 Zou et al. Molecular Cancer (2020) 19:145 Page 5 of 19 STAT3-mediated crosstalk between cancer cells and diverse cell subsets in the TME that throw new lights on our understanding of its ex- tremely sophisticated regulation. STAT3 in CAFs CAFs are the key component of the tumor stroma and contribute to cancer progression and treatment failure by modifying the extracellular matrix, secreting soluble factors, supporting angiogenesis and metasta- sis, and inhibiting anti-tumor immune responses [70]. There is a growing body of evidence to support that STAT3 can be activated in CAFs by numerous cyto- kines including leukemia inhibitory factor (LIF) [71]. This STAT3 hyperactivation enables CAFs to produce various immunosuppressive factors such as IL-6, TGFβ, EGF, VEGF, and CCL2, thereby contributing to the pro-oncogenic phenotype of these fibroblasts [72, 73]. Moreover, a recent study revealed that in- creased phosphorylation of STAT3 in CAFs is associ- ated with reduced overall survival in colorectal cancer patients, and STAT3 activation in CAFs enhances in- testinal tumor growth in vivo [74], exemplifying the importance of STAT3 activation in CAFs for cancer initiation and progression. Zou et al. Molecular Cancer (2020) 19:145 Page 6 of 19 Fig. 2 STAT3 induces the immunosuppression in the TME. STAT3 activity in tumor cells supports multiple hallmarks of cancer, including increased secretion of immunosuppressive factors such as IL-6, IL-10 and EGFR, which can activate STAT3 in the innate and adaptive immune cell subsets as well as CAFs in the TME. Likewise, immune cells and CAFs within the TME can release certain factors including IL-6, which subsequently enhance STAT3 signaling in tumor cells. Elevated STAT3 in the TME has dual effects. On the one hand, STAT3 favors the accumulation and enrichment of immunosuppressive Treg cells and B cells, as well as the polarization of M2-like macrophages, which instigate immune evasion. Particularly, STAT3 is a major driver for increased expression of immune checkpoint molecules (such as PD-L1, PD-L2 and CTLA-4) in these cells. On the other hand, STAT3 in CD8+ T cells, NK cells and neutrophils evokes restrained anti-tumor cytolytic activities. STAT3 can also inhibit the anti-tumor ability of DCs through dampening their maturation, activation and antigen presentation. Besides, STAT3 in CAFs can promote their proliferation, survival and migration, and drive the remodeling of tumor stroma for tumor progression. Collectively, STAT3 induces the immunosuppression in the TME, thereby promoting tumor progression with diminishing the anti-tumor immunity. Fig. 2 STAT3 induces the immunosuppression in the TME. STAT3 in CAFs STAT3 activity in tumor cells supports multiple hallmarks of cancer, including increased secretion of immunosuppressive factors such as IL-6, IL-10 and EGFR, which can activate STAT3 in the innate and adaptive immune cell subsets as well as CAFs in the TME. Likewise, immune cells and CAFs within the TME can release certain factors including IL-6, which subsequently enhance STAT3 signaling in tumor cells. Elevated STAT3 in the TME has dual effects. On the one hand, STAT3 favors the accumulation and enrichment of immunosuppressive Treg cells and B cells, as well as the polarization of M2-like macrophages, which instigate immune evasion. Particularly, STAT3 is a major driver for increased expression of immune checkpoint molecules (such as PD-L1, PD-L2 and CTLA-4) in these cells. On the other hand, STAT3 in CD8+ T cells, NK cells and neutrophils evokes restrained anti-tumor cytolytic activities. STAT3 can also inhibit the anti-tumor ability of DCs through dampening their maturation, activation and antigen presentation. Besides, STAT3 in CAFs can promote their proliferation, survival and migration, and drive the remodeling of tumor stroma for tumor progression. Collectively, STAT3 induces the immunosuppression in the TME, thereby promoting tumor progression with diminishing the anti-tumor immunity. Overall, the outcome of STAT3-mediated crosstalk between cancer cells and tumor-infiltrating cells within the TME is to promote tumor growth and de- velopment, along with diminished anti-tumor immun- ity (Fig. 2). the TME characterized by collagen fibrogenesis, collagen disorganization and fibroblast contractility; the remodel- ing of the TME is not only important for cancer cell mi- gration and invasion, but also plays a critical role in resistance to therapeutic intervention [83, 84]. Zou et al. argeting STAT3 for cancer immunotherapy recently emerged to overcome this dilemma, and show great promise to yield therapeutic agents to targeting transcription factors, including STAT3. For instance, the small-molecule proteolysis-targeting chimera (PRO- TAC)-based strategy has attracted a lot of attention because it can inhibit target protein function as well as counteract increased target protein expression [128]. The studies on PROTAC-mediated degradation of oncogenic proteins such as BRD4 [129], BCR-ABL [130], receptor tyrosine kinase (RTK) [131], and BCL- XL [132] have shown encouraging results, suggesting the potential clinical applicability of this ingenious ap- proach. SD-36, a novel inhibitor identified by the PROTAC-based strategy, exhibits high selectivity for STAT3 and high cell permeability [98]. Moreover, SD-36 treatment can cause a profound and long- lasting suppression of tumor in mouse models of leukemia and lymphoma [98], suggesting that PROTAC-based strategy may be a promising and reli- able avenue for searching small molecule inhibitors against STAT3. Further, the outstanding performance of SD-36 in cancer treatment suggests that the strat- egy of targeting STAT3 protein degradation may be superior to suppress STAT3 expression. Although targeting STAT3 has been extensively investi- gated for decades, this field still remains largely unex- plored. The most common approach in targeting STAT3 directly is to prevent the formation of functional STAT3 dimers through disrupting the domains of SH2, DBD, or NTD [85, 86]. In general, direct inhibitors of STAT3 can be classified into three categories: peptides, small mole- cules and oligonucleotides. Studies of these inhibitors on pre-clinical cancer models are summarized in Table 1 [87–112] and relevant ongoing clinical trials are intro- duced in Table 2 [117–119]. Peptides are usually designed based on the structure of amino acid residues in STAT3 protein and can be di- rected towards different domains. Phosphopeptide in- hibitor (PY*LKTK), derived from the binding peptide sequence of the STAT3-SH2 domain, represents the first successful attempt to disrupt STAT3 dimerization [89]. However, the further development of peptide for the clinical use is currently limited due to their poor cellular permeability and lack of stability in vivo, and even the second-generation peptidomimetics are largely suffering from similar limitations [127]. Oligonucleotides represent a new treatment strategy for ‘undruggable’ cancer targets such as STAT3. STAT3-binding decoy oligodeoxynucleotides, can se- quester STAT3 and thus decrease its binding to cog- nate DNA sites within target genes [133]. Antisense oligonucleotides (ASOs) are designed to block STAT3 activity by targeting STAT3 mRNA. STAT3 in CAFs Molecular Cancer (2020) 19:145 Page 8 of 19 Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued) Therapy Type Agent Cell line tested Mouse model Functional outcome Ref VEGFR2 antibody DC101 n/d Xenograft: LLC, CT26 ↓Proliferation; ↑Anti-tumor immunity, Vascular normalization [116] STING agonist cGAMP, RR-CDA AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued) Therapy Type Agent Cell line tested Mouse model Functional outcome Ref VEGFR2 antibody DC101 n/d Xenograft: LLC, CT26 ↓Proliferation; ↑Anti-tumor immunity, Vascular normalization [116] STING agonist cGAMP, RR-CDA AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued) Therapy Type Agent Cell line tested Mouse model Functional outcome Ref VEGFR2 antibody DC101 n/d Xenograft: LLC, CT26 ↓Proliferation; ↑Anti-tumor immunity, Vascular normalization [116 STING agonist cGAMP, RR-CDA AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued) AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide STAT3 in CAFs Molecular Cancer (2020) 19:145 Page 7 of 19 Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models Therapy Type Agent Cell line tested Mouse model Functional outcome Ref Direct inhibitors Peptides DBD-1 Melanoma, Myeloma n/d ↑Apoptosis; ↓Proliferation [87] ISS-610 prodrugs BC n/d ↑Apoptosis [88] PY*LKTK NIH3T3/v-Src or v-Ras n/d ↓Transformation [89] Small molecules 6o BC, PC, PCa, NSCLC n/d ↑Apoptosis; ↓Proliferation [90] FLLL32 BC, PC Xenograft: MDA-MB-231, PANC-1 ↑Apoptosis; ↓Proliferation, Vascularization [91] HJC0152 Glioblastoma Xenograft: U87 ↑Apoptosis; ↓Metastasis, Proliferation [92] LL1 CRC Xenograft: HCT116 ↑Apoptosis; ↓Metastasis, Proliferation [93] LLL-3 BC, Glioblastoma Xenograft: U87 ↑Apoptosis; ↓Metastasis, Proliferation [94] LLL12 HCC Xenograft: SNU398 ↑Apoptosis; ↓Proliferation [95] LYW-6 CRC AOM/DSS induced CRC model; Xenograft: HCT116 ↑Apoptosis; ↓Metastasis, Proliferation [96] Nitidine chloride Oral cancer Xenograft: HSC3 ↑Apoptosis; ↓Proliferation [97] SD-36 BC, CRC, Leukemia, Lymphoma Xenograft: MOLM-16, SUP- M2, SU-DHL-1 ↑Apoptosis; ↓Proliferation [98] Stattic BC n/d ↑Apoptosis [99] STX-0119 n/d Humanized NOG-dKO model ↑Anti-tumor immunity; ↓Proliferation [100] S3I-1757 Melanoma Xenograft: B16-F10 ↓Proliferation [101] S3I-201 BC Xenograft: MDA-MB-231 ↑Apoptosis; ↓Proliferation [102] CPA-7 BC, CRC, Melanoma, PCa, NSCLC Xenograft: CT26 ↑Apoptosis; ↓Proliferation [103] C48 BC, CML, Melanoma, PCa Xenograft: MDA-MB-468, C3L5 ↓Proliferation [104] GPA512 PCa Xenograft: DU145 ↓Proliferation [105] MMPP BC, CRC, PCa, HCC, Lung, Ovary and Skin cancer Xenograft: Patient-derived NSCLC, NCI-H460 ↑Apoptosis; ↓Proliferation [106] Oligonucleotides InS3- 54A18 BC, NSCLC Xenograft: A549 ↓Metastasis, Proliferation [107] STAT3 hpdODN CRC n/d ↓Proliferation [108] Indirect inhibitors JAK2 INCB16562 Leukemia MPLW515L model ↓Proliferation [109] TG101209 Leukemia AML1-ETO9a leukemia model ↑Apoptosis; ↓Proliferation [110] EGFR JND3229 BaF3 Xenograft: BaF3-EGFR ↓Proliferation [111] FGFR, VEGFR ODM-203 Bladder cancer, NSCLC, GC Xenograft: H1581, KMS11, RT4, SNU16 ↑Anti-tumor immunity; ↓Metastasis, Proliferation [112] Combination Direct inhibitor HJC0152 BC, THP1 Xenograft: 4T1 ↑Anti-tumor immunity; ↓Proliferation [113] STING agonist c-diAM (PS)2 JAK1/2 inhibitor Ruxolitinib PC Xenograft: PANC02-H7 ↑Anti-tumor immunity; ↓Proliferation [114] Anti-PD-1 antibody RMP1-14 SRC, ABL inhibitor Dasatinib n/d Tgfbr1/Pten 2cKO model ↑Anti-tumor immunity; ↓Proliferation [115] Anti-CTLA-4 antibody 9D9 Zou et al. argeting STAT3 for cancer immunotherapy For example, AZD9150, a second-generation STAT3 ASO, targets the 3'-untranslated region (3'-UTR) of the STAT3 gene [134]. Preclinical testing and clinical evaluation have revealed the high efficacy and low toxicity of AZD9150 in oncotherapy [135, 136]. Although oligo- deoxynucleotides inhibitors of STAT3 provide exquis- ite specificity and potency, their poor cell membrane penetrance, rapid degradation, and the lack of effect- ive targeted delivery carriers, remain the major obsta- cles that impede their use in solid tumors. Aptamers have also emerged as useful targeted delivery agents for conventional drugs and small RNAs including siR- NAs and miRNAs due to several advantages, such as small physical size, high stability and low immunogenicity [137]. Non-peptide small molecules capable of disrupting phos- phorylation of STAT3 or STAT3-STAT3 dimerization have recently emerged as an attractive alternative approach to the above. These small molecule inhibitors usually select- ively bind to the SH2, the DBD, or the NTD domain of STAT3 to block transcription of target genes [85]. BBI608 (Napabucasin), a small molecule inhibitor that selectively binds to the DBD domain of STAT3, is the only direct STAT3 inhibitor that has advanced into phase III trials thus far. The excellent outcome of a recent phase III monother- apy trial suggested that BBI608 has potential implication in advanced colorectal cancer [117]. Moreover, FDA has ap- proved BBI608 as an orphan drug for treatment of gastric and pancreatic cancer based on the promising results in phase I/II clinical trials. Numerous small molecule inhibitors of STAT3 have been identified by virtual screening. Of note, although these inhibitors exhibit excellent physicochemical prop- erties in vitro, most of them show poor clinical efficacy, which might be due to low aqueous solubility and low cell permeability [86]. Several novel approaches have Page 9 of 19 Zou et al. argeting STAT3 for cancer immunotherapy Molecular Cancer (2020) 19:145 Table 2 STAT3 inhibitors in currently on-going clinical trials Therapy Type Agent Indication Phase NCT number Ref Direct inhibitors Small molecules BBI608 (FDA approved) Advanced malignancies I/II NCT01775423 NA CRC III NCT01830621 [117] Celecoxib* (FDA approved) CRC III NCT00087256 NA C188-9 BC, CRC, HNSCC, HCC, NSCLC, GAC, Melanoma, Advanced cancer I NCT03195699 NA OPB-111077 Acute myeloid leukemia I NCT03197714 NA Advanced HCC I NCT01942083 NA OPB-31121 Advanced cancer, Solid tumors I NCT00955812 NA HCC I/II NCT01406574 NA OPB-51602 Malignant solid tumors I NCT01184807 NA Hematological malignancies I NCT01344876 NA Nasopharyngeal carcinoma I NCT02058017 NA Pyrimethamine* (FDA approved) CLL, Small lymphocytic lymphoma I/II NCT01066663 NA Oligonucleotides AZD9150 Lymphoma I/II NCT01563302 [118] STAT3 decoy Head and neck cancer 0 NCT00696176 [119] Indirect inhibitors JAK1/2 AZD-1480 Solid tumors I NCT01112397 NA CYT 387 Myelofibrosis I/II NCT01423058 [120] PMF, Post-PV, Post-ET MF III NCT02101268 NA Ruxolitinib (FDA approved) Myelofibrosis II NCT03427866 NA JAK2 LY2784544 Myeloproliferative neoplasms II NCT01594723 [121] SB1518 Myelofibrosis III NCT02055781 [122] EGFR Cetuximab (FDA approved) Metastatic CRC I/II NCT02117466 NA Panitumumab (FDA approved) Advanced CRC II NCT03311750 NA Metastatic CRC IV NCT02301962 NA FGFR Ponatinib (FDA approved) CML II NCT04043676 NA CML, ALL II NCT04233346 NA IL-6R Siltuximab (FDA approved) Multiple myeloma II NCT03315026 NA Tocilizumab (FDA approved) HCC I/II NCT02997956 NA VEGF Bevacizumab (FDA approved) Metastatic CRC II NCT02226289 NA VEGFR Apatinib Lung carcinoma II NCT03709953 NA VEGFR, PDGFR Sorafenib (FDA approved) Advanced HCC IV NCT02733809 NA VEGFR, PDGFR, c-KIT Sunitinib (FDA approved) Clear cell renal carcinoma II NCT03066427 NA Pancreatic neuroendocrine tumor metastatic II NCT02713763 NA SRC, ABL Dasatinib (FDA approved) Chronic-phase CML IV NCT01660906 [123] SRC Bosutinib (FDA approved) CML II NCT02810990 NA KX2-391 Bone-metastatic, Castration-resistant PCa II NCT01074138 [124] Combination Direct inhibitors and ICB AZD9150, Durvalumab (anti- PD-L1) NSCLC II NCT03334617 NA PC, CRC, NSCLC II NCT02983578 NA Advanced solid tumors, Metastatic HNSCC I/II NCT02499328 NA Diffuse large B-cell lymphoma I NCT02549651 NA BBI608, Nivolumab (anti-PD- L1) Metastatic CRC II NCT03647839 NA BBI608, Pembrolizumab (anti- PD-1) Metastatic CRC I/II NCT02851004 NA Phase NCT number Ref Page 10 of 19 Zou et al. argeting STAT3 for cancer immunotherapy Molecular Cancer (2020) 19:145 Table 2 STAT3 inhibitors in currently on-going clinical trials (Continued) Therapy Type Agent Indication Phase NCT number Ref Indirect inhibitors and ICB Apatinib, SHR-1210 (anti-PD-1) Melanoma II NCT03955354 NA Bevacizumab, Atezolizumab (anti-PD-L1) Unresectable HCC III NCT03434379 [125] Cetuximab, Pembrolizumab (anti-PD-1) Recurrent or metastatic HNSCC II NCT03082534 NA Dasatinib, Ipilimumab (anti- CTLA-4) GIST, Stage III /IV soft tissue sarcoma I NCT01643278 [126] Dasatinib, Nivolumab (anti- PD-L1) Philadelphia chromosome positive ALL I NCT02819804 NA Ruxolitinib, Pembrolizumab (anti-PD-1) Hematological malignancies II NCT04016116 NA Metastatic stage IV TNBC I NCT03012230 NA Sorafenib, BGB-A317(anti-PD- 1) HCC III NCT03412773 NA Sorafenib, Nivolumab (anti- PD-L1) Advanced or metastatic HCC II NCT03439891 NA Indirect inhibitor and CAR-T Tocilizumab, CAR-T 19 Lymphoblastic leukemia NA NCT02906371 NA ALL Acute lymphoblastic leukemia, BC Breast cancer, Celecoxib* An FDA approved nonsteroidal anti-inflammatory drug, CML Chronic myelogenous leukemia, CLL Chronic lymphocytic leukemia, CRC Colorectal cancer, HNSCC Head and neck squamous cell carcinoma, NA Not available, NSCLC Non-small cell lung cancer, HCC Hepatocellular carcinoma, GAC Gastric adenocarcinoma, Pyrimethamine* An FDA approved anti-parasitic drug, PMF Primary myelofibrosis, Post-PV Post- polycythemia vera, Post-ET MF Post-essential thrombocythemia myelofibrosis, PC Pancreatic cancer, PCa Prostate cancer, GIST Gastrointestinal stromal tumor, TNBC Triple negative breast cancer ALL Acute lymphoblastic leukemia, BC Breast cancer, Celecoxib* An FDA approved nonsteroidal anti-inflammatory drug, CML Chronic myelogenous leukemia, CLL Chronic lymphocytic leukemia, CRC Colorectal cancer, HNSCC Head and neck squamous cell carcinoma, NA Not available, NSCLC Non-small cell lung cancer, HCC Hepatocellular carcinoma, GAC Gastric adenocarcinoma, Pyrimethamine* An FDA approved anti-parasitic drug, PMF Primary myelofibrosis, Post-PV Post- polycythemia vera, Post-ET MF Post-essential thrombocythemia myelofibrosis, PC Pancreatic cancer, PCa Prostate cancer, GIST Gastrointestinal stromal tumor, TNBC Triple negative breast cancer ALL Acute lymphoblastic leukemia, BC Breast cancer, Celecoxib* An FDA approved nonsteroidal anti-inflammatory drug, CML Chronic myelogenous leukemia, CLL Chronic lymphocytic leukemia, CRC Colorectal cancer, HNSCC Head and neck squamous cell carcinoma, NA Not available, NSCLC Non-small cell lung cancer, HCC Hepatocellular carcinoma, GAC Gastric adenocarcinoma, Pyrimethamine* An FDA approved anti-parasitic drug, PMF Primary myelofibrosis, Post-PV Post- polycythemia vera, Post-ET MF Post-essential thrombocythemia myelofibrosis, PC Pancreatic cancer, PCa Prostate cancer, GIST Gastrointestinal stromal tumor, TNBC Triple negative breast cancer Recently, STAT3 silencing by aptamer-siRNA chimera ob- tained excellent inhibition in the therapy of glioblastoma [138, 139], suggesting that the improved oligonucleotides might offer translational potential for the treatment of solid tumors. argeting STAT3 for cancer immunotherapy downstream components of the STAT3 signaling path- way, and hundreds of leading compounds have been identified [144–148]. Out of those, Ruxolitinib, Dasatinib and Siltuximab that target JAK, SRC/ABL, and IL-6 re- spectively, have been approved by FDA for cancer ther- apy. Indirect STAT3 inhibitors in currently on-going clinical trials are summarized in Table 2 [120–124]. Of note, indirect STAT3 inhibitors lack specificity for STAT3 and may cause fairly extensive kinase inhibition because the targeted molecules are often involved in in- tricate signaling pathways. Since STAT3 is a transcription factor, it is traditionally regarded as an undruggable target. Direct targeting of STAT3 has proven to be considerably challenging, owing in part to high sequence similarity with the other STAT members [86, 140]. Moreover, several issues such as high toxicity and poor bioavailability have become sig- nificant impediments to the clinical development of dir- ect STAT3 inhibitors [86]. Interestingly, some FDA- approved compounds, such as Pyrimethamine and Cele- coxib, have been identified as STAT3 inhibitors through drug-repositioning screening [141, 142]. These findings not only provide another source for searching STAT3 inhibitors, but also suggest potential applications of these drugs in cancer therapy. In addition, similar to combined therapy, certain bifunctional compounds are emerging and may represent a new generation of highly efficacious STAT3 inhibitors for cancer therapy in the future. For example, the compound 8u has dual immu- notherapeutic and anticancer efficacy through simultan- eously inhibiting indoleamine-2,3-dioxygenase 1 (IDO1) and STAT3 [143]. Intriguingly, it has recently been shown that phosphory- lated STAT3 is present in exosomes from 5-fluorouracil (5-FU) resistant colorectal cancer cells, which contributes to acquired 5-FU resistance [149]. Given the importance and efficiency of exosomes in intercellular and interorgan communication [150, 151], these findings not only add an- other complexity to STAT3 regulation, but also pave a new way to inhibit the oncogenic function of STAT3, as well as to delivery STAT3 inhibitors via exosomes. Integrating STAT3 in combination cancer immunotherapy Immunotherapy is currently among the most promising approaches for cancer treatment. This therapeutic strat- egy, represented mainly by immune checkpoint blockade (ICB) and chimeric antigen receptor T cells (CAR-T), has obtained unprecedented results in patients with pre- viously incurable cancers [3, 152]. However, there are some key challenges that need to be resolved urgently, Indirect targeting of STAT3 Preclinical and clinical data suggest that combination cancer im- munotherapies have enhanced therapeutic efficacy and reduced drug resistance compared with monotherapy [155, 156]. These encouraging data has triggered many investigations of combination strategies, and the com- bination of STAT3 inhibitors with other immunotherapy agents are also emerging (Fig. 3). p Combined blockade of STAT3 and immune check- point has shown encouraging results, whereby the addition of STAT3 inhibitors can enhance therapeutic efficacy, and reduce resistance to ICB immunotherapy in parallel. Dasatinib, an indirect STAT3 inhibitor against SRC/ABL, significantly facilitated anti-CTLA-4 immuno- therapy in head and neck squamous cell carcinoma [115], while the combined blockade of IL-6 and PD-L1 remarkably inhibited the growth of pancreatic ductal adenocarcinoma and hepatocellular carcinoma (HCC) [167, 168]. The resistance to anti-PD-1 antibodies can be overcome by treatment with JAK inhibitor in mice with pancreatic orthotopic tumors [114]. Niclosamide blocked STAT3-induced PD-L1 transcription, and thus enhanced the efficacy of anti-PD-1/PD-L1 antibodies in non-small cell lung cancer [169]. More recently, a phase III trial reported exciting results of STAT3-based com- bination therapy in treatment of advanced HCC. Com- pared to the first-line drug sorafenib, the combination of bevacizumab (a monoclonal antibody targeting VEGF) and atezolizumab (a PD-L1 inhibitor) can significantly prolong the overall survival and progression-free survival of patients with unresectable HCC, along with compar- able adverse effects [125]. In addition, certain STAT3 in- hibitors, such as BBI608 and AZD9150, combining with immune checkpoint inhibitors, are currently being tested in pre-clinical (Table 1) and clinical trials (Table 2) [126]. The promising early phase clinical trials encour- age further clinical development of this combination strategy. Combined blockade of STAT3 and immune checkpoint Up-regulated expression of the immune checkpoint mol- ecules, including CTLA-4, PD-1, and PD-L1, has been documented to facilitate tumor immune escape. A sub- stantial amount of evidence has shown that STAT3 is able to directly or indirectly regulate these immune checkpoint molecules. As a transcription factor, STAT3 can increase expression of PD-1, PD-L1, and PD-L2 by direct binding to their promoters [157–160]. Meanwhile, STAT3 has been identified to indirectly induce expres- sion of immune checkpoint molecules through modulat- ing diverse signaling pathways. For example, STAT3 increased CTLA-4 expression in tumor-associated B cells in a JAK-dependent manner [161] and enhanced CTLA-4 expression in Treg cells through IL-10 [162]. In addition, STAT3 mediated HDAC6-induced PD-L1 ex- pression in osteosarcoma cells [163]. Indirect targeting of STAT3 In parallel with direct inhibitors, indirect inhibitors of STAT3 have been pursued by targeting the upstream or Zou et al. Molecular Cancer (2020) 19:145 Page 11 of 19 Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Page 12 of 19 Zou et al. Molecular Cancer (2020) 19:145 (See figure on previous page.) Fig. 3 Targeting STAT3 in combination cancer immunotherapy. a Summary of the key steps in the development of STAT3-targeting therapeutics. The first step in the development of STAT3-targeting therapeutics involves the systematic selection of STAT3 inhibitors (including direct or indirect inhibitors) and STAT3 inhibitors-based combined immunotherapy, and then elucidating the biology and effects of these candidates to cancer using tumor cell lines and patient samples. The next major challenge involves the in vivo model-based validation that these therapeutic candidates must undergo rigorous disease-specific in vivo testing using rodents and non-human primate models. Key challenges in translating STAT3 inhibitors into the clinic are low bioavailability and the lack of specific targeting of the tumor site. b Targeting STAT3 in combination cancer immunotherapy. Targeting STAT3 in combination cancer immunotherapy can not only enhance the anti-tumor effects, but also reduce drug resistance. Besides, combined STAT3 inhibitors with CAR-T cells can reduce excessive expansion of CAR-T cells and alleviate cytokine release syndrome (CRS), resulting in lower occurrence of immune-related adverse effects. CD4+ T cells [165]. Given PI3K/AKT as a known repres- sor of STAT3 transcription [166], it is likely that PD-1 indirectly enhances STAT3 expression through inhib- ition of PI3K. The reciprocal regulation of STAT3 and immune checkpoint molecules not only suggests an in- volvement of STAT3 in anti-tumor immunity, but also provides a promising strategy to improve the efficacy of current immune checkpoint inhibitors. including limited clinical response rates and significant autoimmune-related side effects [3, 153]. For instance, ICB has shown remarkable effectiveness in solid tumors including melanoma, non-small cell lung cancers and renal cancer, however, even in these cancers, the major- ity of patients still do not respond to the treatment [3]. Furthermore, certain types of cancer such as pancreatic cancer and prostate cancer show resistance to immune checkpoint inhibition therapy [3, 154]. Thus, combin- ation therapy is considered to be a promising direction for improving outcomes for cancer treatment. Indirect targeting of STAT3 Conversely, recent evidence also suggests a role of immune checkpoint molecules in STAT3 expression. Celada et al. reported that PD-1 upregulation in CD4+ T cells leads to an in- crease in STAT3 mRNA expression by undescribed mechanism, and the latter is required for IL-17 and TGFβ1 production [164]. Interestingly, an early study from the same research group showed PD-1 can attenu- ate TCR-dependent activation of PI3K/AKT pathway in Combined STAT3 inhibitors and CAR-T For example, CAR-T cells targeting B7-H3, a transmembrane protein belonging to the B7 immune fam- ily, inhibited the growth of neuroblastoma, pancreatic and ovarian cancer in vitro and in xenograft mouse models without evident toxicity [171, 172]. The involvement of STAT3 signaling in CAR-T ther- apy is emerging. Transcriptomic profiling showed that anti-CD19 CAR-T cells from responsive patients with chronic lymphocytic leukemia had an increased IL-6/ STAT3 signature, which promoted the expansion of CAR-T cells [173]. In line with this, a novel anti-CD19 CAR-T cells with constitutive activation of STAT3 showed increased proliferation and reduced terminal dif- ferentiation of CAR-T cells, and conferred superior anti- tumor effects [174]. Similarly, CAR-T cells expressing the ectodomain of the IL-4 receptor and the end domain of the IL-21 receptor activated the STAT3 pathway and enhanced Th17-like polarization, representing a poten- tial clinical CAR-T therapy for solid tumors enriching IL-4 [175]. These studies suggest a beneficial effect of STAT3 activation in CAR-T cells. Several previous studies have suggested potential inter- actions between STING signaling and STAT3-driven oncogenic pathways [181–183]. It has been observed that rapid colorectal cancer progression in STING- deficient mice is associated with STAT3 hyperactivation [181]. Further research found that STING plays a vital role in regulation of MDSC differentiation and anti- tumor immunity in nasopharyngeal carcinoma by in- creasing the expression of SOCS1, a classic repressor of STAT3 [182]. Besides, TBK1, which is activated by cyto- solic DNA in a STING-dependent manner, can restrain activation of STAT3 through direct phosphorylation of STAT3 at serine 754 in the TAD [183]. As mentioned above, STAT3 hyperactivation in tumor stroma is immunosuppressive and can increase the ex- pression of certain cytokines and growth factors. Ac- cordingly, constitutive expression of an array of cytokines such as IL-6 and IL-10 potentially could in- crease the risk of serious adverse events of CAR-T ther- apy including cytokine release syndrome [176]. Thus, there are some attempts to combine CAR-T therapy with STAT3 inhibitors for improving the persistence and anti-tumor effects, as well as negating toxicities of CRA-T cells in vivo. For instance, the JAK2/STAT3 axis is a crucial driver of liver-associated MDSCs and inhib- ition of STAT3 increased the efficacy of CAR-T cells in liver cancer metastasis [177]. In addition, a clinical study is currently on-going, which tests the efficacy and ad- ministration of the anti-IL-6 therapy (tocilizumab) on anti-CD19 CAR-T cells associated cytokine release syn- drome (NCT02906371). Combined STAT3 inhibitors and CAR-T Combined STAT3 inhibitors and CAR-T CAR-T cell therapy, a rapidly emerging and effective im- munotherapeutic approach, has revolutionized anti-cancer therapies for hematologic malignancies, especially acute lymphoblastic leukemia and lymphoma [152]. Two Anti- Page 13 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 [178]. Activated STING can propagate interferon recep- tor signaling in tumor-infiltrating DCs and elicit CD8+ T cells against tumor-associated antigens in vivo [179]. Therefore, STING agonists are of continuing research interest as novel adjuvants to boost cancer immunother- apy. A recent study showed that STING-activating nano- particles (STING-NPs) can convert immunosuppressive tumors to immunogenic microenvironments and then induce anti-tumor immune responses and immuno- logical memory in mice with melanoma [180]. In an- other study, Ramanjulu et al. reported that STING agonist can lead to complete and lasting regression of tumors in mice with colon tumors [179]. These encouraging results of preclinical studies point towards the potential for improving clinical outcomes of im- munotherapy, and some STING agonists such as c- diAM (PS)2 and cGAMP are currently being evaluated in clinical trials (NCT03937141, NCT02986867). [178]. Activated STING can propagate interferon recep- tor signaling in tumor-infiltrating DCs and elicit CD8+ T cells against tumor-associated antigens in vivo [179]. Therefore, STING agonists are of continuing research interest as novel adjuvants to boost cancer immunother- apy. A recent study showed that STING-activating nano- particles (STING-NPs) can convert immunosuppressive tumors to immunogenic microenvironments and then induce anti-tumor immune responses and immuno- logical memory in mice with melanoma [180]. In an- other study, Ramanjulu et al. reported that STING agonist can lead to complete and lasting regression of tumors in mice with colon tumors [179]. These encouraging results of preclinical studies point towards the potential for improving clinical outcomes of im- munotherapy, and some STING agonists such as c- diAM (PS)2 and cGAMP are currently being evaluated in clinical trials (NCT03937141, NCT02986867). CD19 CAR-T therapies are currently approved by FDA for the treatment of CD19-positive leukemia or lymph- oma. Although the efficacy of CAR-T therapy in solid tu- mors has lagged far behind, a great number of CAR-T trials are ongoing and positive outcomes are increasingly being reported for multiple solid tumors, including glio- blastoma, gastrointestinal, genitourinary, breast, and lung cancer [170]. Combined STAT3 inhibitors and CAR-T Recent evidence indicates that the combination of STING agonists with STAT3 inhibitors can enhance tumor im- munogenicity and optimize the immunotherapeutic effects [113, 116]. For example, combined STAT3 direct inhibitor HJC0152 with STING agonist c-diAM (PS)2 increased CD8+ T cells, reduced Treg cells and MDSCs in the TME, and thus effectively enhanced anti-tumor immunity in mice with breast cancer [113]. A preclinical study demonstrated the combination of STING agonist (cGAMP or RR-CDA) with the indirect STAT3 inhibitor VEGFR2 was maximally effect- ive for immunotherapy-resistant tumors in breast and lung cancer [116]. STAT3 blockade can also markedly improve other ef- fective immunotherapeutic approaches including cancer vaccines and immunostimulatory Toll-like receptor (TLR) agonists (such as CpG oligodeoxynucleotides). For example, a novel strategy combining STAT3 ASO with TLR9 stimulation (CpG oligonucleotide) has been shown to enhance the anti-tumor immunity and overcome tumor immune tolerance in prostate cancer [184]. The combination of STAT3 inhibitor and DC-based vaccine led to improved therapeutic outcomes in mouse colon cancer [185]. The beneficial outcomes of these Combined STAT3 inhibitors and STING agonists Stimulator of interferon genes (STING) is a major adaptor protein that plays an important role in anti-viral and anti-tumor immunity [178]. When stimulated by cytosolic DNA, STING activates TANK-binding kinase 1 (TBK1), which subsequently phosphorylates interferon regulatory factor 3 (IRF3) to promote IFN expression Page 14 of 19 Page 14 of 19 Page 14 of 19 Zou et al. Molecular Cancer (2020) 19:145 immunotherapy combinations about STAT3 inhibitors warrant further clinical validation. Notably, STAT3 an- tagonists, either direct or indirect STAT3 inhibitors, are generally less likely to completely block STAT3 signaling and might not trigger severe autoimmune disorders. However, it cannot be ignored that the use of STAT3 in- hibitors and other immunotherapy agents in combin- ation may result in more frequent and severe immune- related adverse events (irAEs) compared to monother- apy. Accordingly, risk evaluations for irAEs should be part of the decision criteria for determining immuno- therapy combinations. Likewise, early recognition and adequate management for irAEs are indispensable to minimize treatment-related serious complications. predictive biomarkers that can provide a basis for im- proved precision medicine, though the related studies are currently not explored in depth. Abbreviations AHR A l h d AHR: Aryl hydrocarbon receptor; ALL: Acute lymphoblastic leukemia; AOM: Azoxymethane; ASOs: Antisense oligonucleotides; BC: Breast cancer; Bcl-2: B-cell lymphoma-2; BRD4: Bromodomain-containing protein 4; CAFs: Cancer-associated fibroblasts; CAR-T: Chimeric antigen receptor T cells; CCD: Coiled-coil domain; CCL: C-C motif ligand; CD80/86: Cluster of differentiation 80/86; circRNAs: Circular RNAs; CLL: Chronic lymphocytic leukemia; CML: Chronic myelogenous leukemia; CRS: Cytokine release syndrome; CTLA-4: Cytotoxic T-lymphocyte-associated protein 4; CXCL: C-X-C motif chemokine ligand; Cxxc1: Cxxc finger protein 1; DBD: DNA-binding domain; DCs: Dendritic cells; DSS: Dextran sodium sulfate; DUSP22: Dual specificity phosphatase 22; EGF: Epidermal growth factor; EGFR: Epidermal growth factor receptor; EZH2: Enhancer of zeste homolog 2; FGFR: Fibroblast growth factor receptor; GAC: Gastric adenocarcinoma; GC: Gastric cancer; GIST: Gastrointestinal stromal tumor; GP130: Glycoprotein 130; HADC: Histone deacetylase; HCC: Hepatocellular carcinoma; HIF1α: Hypoxia-inducible factor 1α; HNSCC: Head and neck squamous cell carcinoma; hpdODN: Hairpin decoy oligodeoxynucleotide; ICB: Immune checkpoint blockade; IDO1: Indoleamine-2,3-dioxygenase 1; IFNs: Interferons; IFN-Is: Type I interferons; IL: Interleukin; Loxl3: Lysyl oxidase-like 3; irAEs: Immune-related adverse events; IRF3: Interferon regulatory factor 3; ISGF3: Interferon- stimulated gene factor 3; JAKs: Janus kinases; LLC: Lewis lung carcinoma; lncRNAs: Long non-coding RNAs; LIF: Leukemia inhibitory factor; MDSCs: Myeloid-derived suppressor cells; MHC class II: Major histocompatibility complex class II; MMPs: Matrix metalloproteinases; MPLW515L: Somatic mutations at codon 515 of the thrombopoietin receptor; ncRNAs: Non-coding RNAs; NH2: Amino-terminal domain; NSCL C: Non-small cell lung cancer; NF-κB: Nuclear factor-κB; NTD: N-terminal domain; PC: Pancreatic cancer; PCa: Prostate cancer; PD-1: Programmed cell death protein 1; PDGFR: Platelet-derived growth factor receptor; PD- L1: Programmed cell death 1 ligand 1; PKCβII: Protein kinase C β II; PMF: Primary myelofibrosis; PIAS: Protein inhibitor of activated STAT; PlGF: Placenta growth factor; PI3K: Phosphatidylinositol 3 kinase; Post-ET MF: Post-essential thrombocythemia myelofibrosis; Post-PV: Post- polycythemia vera; PROTAC: Proteolysis-targeting chimera; PTPases: Protein tyrosine phosphatases; PTPN1: Protein tyrosine phosphatase non-receptor type 1; PTPRD: Protein tyrosine phosphatase receptor type D; PTPRT: Protein tyrosine phosphatase receptor type T; RTK: Receptor tyrosine kinase; SH2: Src homology 2 domain; SHP: Src homology 2 domain containing protein tyrosine phosphatase; SIRT1: Sirtuin 1; SMYD2: SET (Suppressor of variegation, Enhancer of Zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF1) domain containing 2; SOCS: Suppressor of cytokine signaling; STAT: Signal transducer and activator of transcription; STING: Stimulator of interferon genes; STING- NPs: STING-activating nanoparticles; SUMO2/3: Small ubiquitin-like modifier 2/3; TAD: Transactivation domain; TBK-1: TANK-binding kinase 1; TCR: T cell receptor; TGFβ: Transforming growth factor β; Th1: T helper 1; TLR: Toll-like receptor; TME: Tumor microenvironment; TNFα: Tumor necrosis factor α; Treg: Regulatory T-cell; VEGF: Vascular endothelial growth factor; VEGF R: Vascular endothelial growth factor receptor; 3ʹ-UTR: 3ʹ-untranslated region; 5-FU: 5-fluorouracil Combined STAT3 inhibitors and CAR-T In summary, therapeutically targeting mediators of the STAT3 signaling, which has already been shown to be beneficial in the restoration of anti-tumor immunity, provides attractive avenues that are currently being ex- plored for the immunotherapy of cancers both as mono- therapy and in combination therapies. Y.T. and X.F. conceived the idea; S.Z., Q.T. and B.L. performed the literature search and draft the manuscript; W.H. revised and edited the manuscript; X.F. and Y.T. supervised and revised the manuscript. The author(s) read and approved the final manuscript. Conclusions and perspectives STAT3 becomes excessively activated in multiple human cancers, and acts as a crucial signaling node for tumor cells and TME comprising cells, especially tumor- infiltrating immune cells. Therefore, targeting STAT3 is expected to offer multiple benefits, including reduced tumor cell intrinsic proliferation, enhanced anti-tumor effects of tumor-infiltrating immune cells, and improved the immunosuppressive crosstalk within the TME. These effects have positioned STAT3 as an arisen potential promising target for cancer therapy. To date, many endeavors have been made to target STAT3 for the development and application of new drugs. These approaches are devised to inhibit STAT3 directly by peptides, small molecules and decoy oligonu- cleotides, or indirectly by blocking upstream signaling pathways such as IL-6 and JAK2 pathways. Currently, the core idea of direct targeting STAT3 is to prevent the formation of functional STAT3 dimers through disrupt- ing phosphorylation of STAT3. Beyond phosphorylation, other posttranslational modifications, such as acetyl- ation, methylation and sumoylation, are emerging to modulate STAT3 activation through diverse mecha- nisms, providing a broadened list of candidate regulatory targets for the STAT3 inhibitors. To improve the response rate and the number of responding cancer types, combined immunotherapies are now being undertaken. Combination therapies of STAT3 inhibitors with currently therapeutic anti-tumor drugs including the immune-checkpoint inhibitors may open up new possibilities for long-lasting and multi- layered tumor control. Although preclinical studies and early clinical trials on combined blockade of STAT3 and immune checkpoint have shown encouraging results, their clinical outcomes await further investigation. Moreover, predictive biomarkers are urgently required to rationally incorporate STAT3 inhibitors into the com- bination immunotherapy. The ncRNAs, particularly miRNAs, might prove to be potentially promising References Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–21. 31. Luo Z, Cao P. Long noncoding RNA PVT1 promotes hepatoblastoma cell proliferation through activating STAT3. Cancer Manag Res. 2019;11:8517–27. 6. Hanlon MM, Rakovich T, Cunningham CC, Ansboro S, Veale DJ, Fearon U, et al. STAT3 Mediates the Differential Effects of Oncostatin M and TNFalpha on RA Synovial Fibroblast and Endothelial Cell Function. Front Immunol. 2019;10:2056. 32. Wang S, Liang K, Hu Q, Li P, Song J, Yang Y, et al. JAK2-binding long noncoding RNA promotes breast cancer brain metastasis. J Clin Invest. 2017;127:4498–515. 7. Rawlings JS, Rosler KM, Harrison DA. The JAK/STAT signaling pathway. J Cell Sci. 2004;117:1281–3. 33. Liang C, Zhao T, Li H, He F, Zhao X, Zhang Y, et al. Long Non-coding RNA ITIH4-AS1 Accelerates the Proliferation and Metastasis of Colorectal Cancer by Activating JAK/STAT3 Signaling. Mol Ther Nucleic Acids. 2019;18:183–93. 8. Johnson DE, O'Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018;15:234–48. 9. Ishibashi K, Koguchi T, Matsuoka K, Onagi A, Tanji R, Takinami-Honda R, et al. Interleukin-6 induces drug resistance in renal cell carcinoma. Fukushima J Med Sci. 2018;64:103–10. 34. Pichler M, Rodriguez-Aguayo C, Nam SY, Dragomir MP, Bayraktar R, Anfossi S, et al. Therapeutic potential of FLANC, a novel primate-specific long non- coding RNA in colorectal cancer. Gut. 2020. Fukushima J Med Sci. 2018;64:103–10. 10. Priego N, Zhu L, Monteiro C, Mulders M, Wasilewski D, Bindeman W, et al. STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis. Nat Med. 2018;24:1024–35. 35. Wang Y, Wu S, Zhu X, Zhang L, Deng J, Li F, et al. LncRNA-encoded polypeptide ASRPS inhibits triple-negative breast cancer angiogenesis. J Exp Med. 2020;217. metastasis. Nat Med. 2018;24:1024–35. 11. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, et al. Stat3 as an oncogene. Cell. 1999;98:295–303. 36. Hu D, Zhang Y. Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p. Gene. 2019;690:81–9. 12. Wang T, Fahrmann JF, Lee H, Li YJ, Tripathi SC, Yue C, et al. JAK/STAT3- Regulated Fatty Acid beta-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance. Cell Metab. 2018;27:136–50 e5. 37. Dong Y, Xu T, Zhong S, Wang B, Zhang H, Wang X, et al. Circ_0076305 regulates cisplatin resistance of non-small cell lung cancer via positively modulating STAT3 by sponging miR-296-5p. Life Sci. 2019;239:116984. Received: 20 May 2020 Accepted: 4 September 2020 Received: 20 May 2020 Accepted: 4 September 2020 25. Zhang L, Li J, Wang Q, Meng G, Lv X, Zhou H, et al. The relationship between microRNAs and the STAT3-related signaling pathway in cancer. Tumour Biol. 2017;39:1010428317719869. Competing interests The authors declare that they have no competing interests. 21. Yang J, Huang J, Dasgupta M, Sears N, Miyagi M, Wang B, et al. Reversible methylation of promoter-bound STAT3 by histone-modifying enzymes. Proc Natl Acad Sci U S A. 2010;107:21499–504. Consent for publication 20. Kim E, Kim M, Woo DH, Shin Y, Shin J, Chang N, et al. Phosphorylation of EZH2 activates STAT3 signaling via STAT3 methylation and promotes tumorigenicity of glioblastoma stem-like cells. Cancer Cell. 2013;23:839–52. Authors’ contributions Y.T. and X.F. conceived the idea; S.Z., Q.T. and B.L. performed the literature search and draft the manuscript; W.H. revised and edited the manuscript; X.F. and Y.T. supervised and revised the manuscript. The author(s) read and approved the final manuscript. Page 15 of 19 Page 15 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 Ethics approval and consent to participate Not applicable. 19. Ma L, Huang C, Wang XJ, Xin DE, Wang LS, Zou QC, et al. Lysyl Oxidase 3 Is a Dual-Specificity Enzyme Involved in STAT3 Deacetylation and Deacetylimination Modulation. Mol Cell. 2017;65:296–309. References 26. Huan L, Liang LH, He XH. Role of microRNAs in inflammation-associated liver cancer. Cancer Biol Med. 2016;13:407–25. 1. Christofi T, Baritaki S, Falzone L, Libra M, Zaravinos A. Current Perspectives in Cancer Immunotherapy. Cancers (Basel). 2019;11. 1. Christofi T, Baritaki S, Falzone L, Libra M, Zaravinos A. Current Perspectives in Cancer Immunotherapy. Cancers (Basel). 2019;11. 27. Guo R, Wu Z, Wang J, Li Q, Shen S, Wang W, et al. Development of a Non- Coding-RNA-based EMT/CSC Inhibitory Nanomedicine for In Vivo Treatment and Monitoring of HCC. Adv Sci (Weinh). 2019;6:1801885. 2. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. 2. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711–23. 2. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711–23. 28. Yang Y, Ding L, Hu Q, Xia J, Sun J, Wang X, et al. MicroRNA-218 functions as a tumor suppressor in lung cancer by targeting IL-6/STAT3 and negatively correlates with poor prognosis. Mol Cancer. 2017;16:141. 3. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455–65. 3. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455–65. 29. Zhang X, Sai B, Wang F, Wang L, Wang Y, Zheng L, et al. Hypoxic BMSC- derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT. Mol Cancer. 2019;18:40. 4. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443–54. 30. Jin S, Yang X, Li J, Yang W, Ma H, Zhang Z. p53-targeted lincRNA-p21 acts as a tumor suppressor by inhibiting JAK2/STAT3 signaling pathways in head and neck squamous cell carcinoma. Mol Cancer. 2019;18:38. 5. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–21. 5. Darnell JE Jr, Kerr IM, Stark GR. Availability of data and materials Not applicable. 18. Nie Y, Erion DM, Yuan Z, Dietrich M, Shulman GI, Horvath TL, et al. STAT3 inhibition of gluconeogenesis is downregulated by SirT1. Nat Cell Biol. 2009; 11:492–500. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Author details 1 1Division of Endocrinology and Metabolism, National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610041, Sichuan, China. 2College of Life Sciences, Sichuan University, Chengdu 610041, Sichuan, China. 3Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, China. 22. Li LX, Fan LX, Zhou JX, Grantham JJ, Calvet JP, Sage J, et al. Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease. J Clin Invest. 2017;127:2751–64. 23. Zhou Z, Wang M, Li J, Xiao M, Chin YE, Cheng J, et al. SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer. Oncogene. 2016; 35:5826–38. Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, China. Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, China. 24. Lin W, Luo J, Sun Y, Lin C, Li G, Niu Y, et al. ASC-J9((R)) suppresses prostate cancer cell invasion via altering the sumoylation-phosphorylation of STAT3. Cancer Lett. 2018;425:21–30. Received: 20 May 2020 Accepted: 4 September 2020 Funding Th k 14. Villarino AV, Kanno Y, O'Shea JJ. Mechanisms and consequences of Jak-STAT signaling in the immune system. Nat Immunol. 2017;18:374–84. Funding This work was supported by the Ministry of Science and Technology of China (2018ZX09201018-005), the National Natural Science Foundation of China (81970561, 81502631, 81570527, 91540113), the 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC18049), and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University (Z20191005, Z20201003). 15. Sgrignani J, Garofalo M, Matkovic M, Merulla J, Catapano CV, Cavalli A. Structural Biology of STAT3 and Its Implications for Anticancer Therapies Development. Int J Mol Sci. 2018;19. 16. Nadiminty N, Lou W, Lee SO, Lin X, Trump DL, Gao AC. Stat3 activation of NF-{kappa} B p100 processing involves CBP/p300-mediated acetylation. Proc Natl Acad Sci U S A. 2006;103:7264–9. 16. Nadiminty N, Lou W, Lee SO, Lin X, Trump DL, Gao AC. Stat3 activation of NF-{kappa} B p100 processing involves CBP/p300-mediated acetylation. Proc Natl Acad Sci U S A. 2006;103:7264–9. 17. Gambi G, Di Simone E, Basso V, Ricci L, Wang R, Verma A, et al. The Transcriptional Regulator Sin3A Contributes to the Oncogenic Potential of STAT3. Cancer Res. 2019;79:3076–87. References 13. Kortylewski M, Kujawski M, Wang T, Wei S, Zhang S, Pilon-Thomas S, et al. Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity. Nat Med. 2005;11:1314–21. 38. Liu Y, Song J, Liu Y, Zhou Z, Wang X. Transcription activation of circ-STAT3 induced by Gli2 promotes the progression of hepatoblastoma via acting as Page 16 of 19 Page 16 of 19 Page 16 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 a sponge for miR-29a/b/c-3p to upregulate STAT3/Gli2. J Exp Clin Cancer Res. 2020;39:101. 61. Shaim H, Estrov Z, Harris D, Hernandez Sanabria M, Liu Z, Ruvolo P, et al. The CXCR4-STAT3-IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide. Front Immunol. 2017;8:1773. a sponge for miR-29a/b/c-3p to upregulate STAT3/Gli2. J Exp Clin Cancer Res. 2020;39:101. 39. Iliopoulos D, Jaeger SA, Hirsch HA, Bulyk ML, Struhl K. STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer. Mol Cell. 2010;39:493–506. 62. Rebe C, Ghiringhelli F. STAT3, a Master Regulator of Anti-Tumor Immune Response. Cancers (Basel). 2019;11. 40. Loffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C, Hackermuller J, Kretzschmar AK, et al. Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Blood. 2007;110:1330–3. 63. Huynh J, Chand A, Gough D, Ernst M. Therapeutically exploiting STAT3 activity in cancer - using tissue repair as a road map. Nat Rev Cancer. 2019; 19:82–96. 64. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nat Rev Immunol. 2007;7: 41–51. 41. Gao H, Liu R, Sun X. STAT3-induced upregulation of lncRNA SNHG17 predicts a poor prognosis of melanoma and promotes cell proliferation and metastasis through regulating PI3K-AKT pathway. Eur Rev Med Pharmacol Sci. 2019;23:8000–10. 65. Yoo SA, Kim M, Kang MC, Kong JS, Kim KM, Lee S, et al. Placental growth factor regulates the generation of TH17 cells to link angiogenesis with autoimmunity. Nat Immunol. 2019;20:1348–59. 42. Du C, Wang HX, Chen P, Chen CH. STAT3-induced upregulation of lncRNA DUXAP8 functions as ceRNA for miR-577 to promote the migration and invasion in colorectal cancer through the regulation of RAB14. Eur Rev Med Pharmacol Sci. 2019;23:6105–18. 66. Lin F, Meng X, Guo Y, Cao W, Liu W, Xia Q, et al. References Epigenetic initiation of the TH17 differentiation program is promoted by Cxxc finger protein 1. Sci Adv. 2019;5:eaax1608. 43. Chen JF, Wu P, Xia R, Yang J, Huo XY, Gu DY, et al. STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells via mTOR signal-mediated inhibition of autophagy. Mol Cancer. 2018;17:6. 67. Mohammadpour H, MacDonald CR, Qiao G, Chen M, Dong B, Hylander BL, et al. beta2 Adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells. J Clin Invest. 2019. 44. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer. 2009;9:798–809. 68. Takenaka MC, Gabriely G, Rothhammer V, Mascanfroni ID, Wheeler MA, Chao CC, et al. Control of tumor-associated macrophages and T cells in glioblastoma via AHR and CD39. Nat Neurosci. 2019;22:729–40. 45. Pearce OMT, Delaine-Smith RM, Maniati E, Nichols S, Wang J, Bohm S, et al. Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers. Cancer Discov. 2018;8:304–19. 69. Oweida AJ, Darragh L, Phan A, Binder D, Bhatia S, Mueller A, et al. STAT3 Modulation of Regulatory T Cells in Response to Radiation Therapy in Head and Neck Cancer. J Natl Cancer Inst. 2019. 46. Azizi E, Carr AJ, Plitas G, Cornish AE, Konopacki C, Prabhakaran S, et al. Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment. Cell. 2018;174:1293–308 e36. 70. Chen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov. 2019;18:99–115. 47. Martin JD, Cabral H, Stylianopoulos T, Jain RK. Improving cancer immunotherapy using nanomedicines: progress, opportunities and challenges. Nat Rev Clin Oncol. 2020. 71. Albrengues J, Bertero T, Grasset E, Bonan S, Maiel M, Bourget I, et al. Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts. Nat Commun. 2015;6:10204. 48. Phuengkham H, Ren L, Shin IW, Lim YT. Nanoengineered Immune Niches for Reprogramming the Immunosuppressive Tumor Microenvironment and Enhancing Cancer Immunotherapy. Adv Mater. 2019;31:e1803322. 72. Yang X, Lin Y, Shi Y, Li B, Liu W, Yin W, et al. FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3-CCL2 Signaling. Cancer Res. 2016;76:4124–35 49. Herrmann A, Kortylewski M, Kujawski M, Zhang C, Reckamp K, Armstrong B, et al. Targeting Stat3 in the myeloid compartment drastically improves the in vivo antitumor functions of adoptively transferred T cells. Cancer Res. 2010;70:7455–64. 73. References Li X, Xu Q, Wu Y, Li J, Tang D, Han L, et al. A CCL2/ROS autoregulation loop is critical for cancer-associated fibroblasts-enhanced tumor growth of oral squamous cell carcinoma. Carcinogenesis. 2014;35:1362–70. 50. Iwata-Kajihara T, Sumimoto H, Kawamura N, Ueda R, Takahashi T, Mizuguchi H, et al. Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors. J Immunol. 2011;187:27–36. 74. Heichler C, Scheibe K, Schmied A, Geppert CI, Schmid B, Wirtz S, et al. STAT3 activation through IL-6/IL-11 in cancer-associated fibroblasts promotes colorectal tumour development and correlates with poor prognosis. Gut. 2019. 51. Siegel AM, Heimall J, Freeman AF, Hsu AP, Brittain E, Brenchley JM, et al. A critical role for STAT3 transcription factor signaling in the development and maintenance of human T cell memory. Immunity. 2011;35:806–18. 75. Giovanelli P, Sandoval TA, Cubillos-Ruiz JR. Dendritic Cell Metabolism and Function in Tumors. Trends Immunol. 2019;40:699–718. 76. Farren MR, Carlson LM, Netherby CS, Lindner I, Li PK, Gabrilovich DI, et al. Tumor-induced STAT3 signaling in myeloid cells impairs dendritic cell generation by decreasing PKCbetaII abundance. Sci Signal. 2014;7:ra16. 52. Gotthardt D, Putz EM, Straka E, Kudweis P, Biaggio M, Poli V, et al. Loss of STAT3 in murine NK cells enhances NK cell-dependent tumor surveillance. Blood. 2014;124:2370–9. 77. Calon A, Espinet E, Palomo-Ponce S, Tauriello DV, Iglesias M, Cespedes MV, et al. Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation. Cancer Cell. 2012;22:571–84. 53. Wang T, Niu G, Kortylewski M, Burdelya L, Shain K, Zhang S, et al. Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells. Nat Med. 2004;10:48–54. 78. Ohlund D, Handly-Santana A, Biffi G, Elyada E, Almeida AS, Ponz-Sarvise M, et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J Exp Med. 2017;214:579–96. 54. Icardi L, Lievens S, Mori R, Piessevaux J, De Cauwer L, De Bosscher K, et al. Opposed regulation of type I IFN-induced STAT3 and ISGF3 transcriptional activities by histone deacetylases (HDACS) 1 and 2. FASEB J. 2012;26:240–9. 79. Huang H, Zhang Y, Gallegos V, Sorrelle N, Zaid MM, Toombs J, et al. Targeting TGFbetaR2-mutant tumors exposes vulnerabilities to stromal TGFbeta blockade in pancreatic cancer. EMBO Mol Med. 2019:e10515. y y 55. Tsai MH, Lee CK. STAT3 Cooperates With Phospholipid Scramblase 2 to Suppress Type I Interferon Response. Front Immunol. 2018;9:1886. 56. References Napabucasin versus placebo in refractory advanced colorectal cancer: a randomised phase 3 trial. Lancet Gastroenterol Hepatol. 2018;3:263–70. 97. Kim LH, Khadka S, Shin JA, Jung JY, Ryu MH, Yu HJ, et al. Nitidine chloride acts as an apoptosis inducer in human oral cancer cells and a nude mouse xenograft model via inhibition of STAT3. Oncotarget. 2017; 8:91306–15. 118. Reilley MJ, McCoon P, Cook C, Lyne P, Kurzrock R, Kim Y, et al. STAT3 antisense oligonucleotide AZD9150 in a subset of patients with heavily pretreated lymphoma: results of a phase 1b trial. J Immunother Cancer. 2018;6:119. 98. Bai L, Zhou H, Xu R, Zhao Y, Chinnaswamy K, McEachern D, et al. A Potent and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor Regression In Vivo. Cancer Cell. 2019;36:498–511 e17. 119. Sen M, Thomas SM, Kim S, Yeh JI, Ferris RL, Johnson JT, et al. First-in-human trial of a STAT3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy. Cancer Discov. 2012;2:694–705. 99. Schust J, Sperl B, Hollis A, Mayer TU, Berg T. Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem Biol. 2006;13:1235–42. 120. Gupta V, Mesa RA, Deininger MW, Rivera CE, Sirhan S, Brachmann CB, et al. A phase 1/2, open-label study evaluating twice-daily administration of momelotinib in myelofibrosis. Haematologica. 2017;102:94–102. 100. Akiyama Y, Nonomura C, Ashizawa T, Iizuka A, Kondou R, Miyata H, et al. The anti-tumor activity of the STAT3 inhibitor STX-0119 occurs via promotion of tumor-infiltrating lymphocyte accumulation in temozolomide- resistant glioblastoma cell line. Immunol Lett. 2017;190:20–5. 121. Berdeja J, Palandri F, Baer MR, Quick D, Kiladjian JJ, Martinelli G, et al. Phase 2 study of gandotinib (LY2784544) in patients with myeloproliferative neoplasms. Leuk Res. 2018;71:82–8. 101. Soleimani AH, Garg SM, Paiva IM, Vakili MR, Alshareef A, Huang YH, et al. Micellar nano-carriers for the delivery of STAT3 dimerization inhibitors to melanoma. Drug Deliv Transl Res. 2017;7:571–81. 122. Mascarenhas J, Hoffman R, Talpaz M, Gerds AT, Stein B, Gupta V, et al. Pacritinib vs Best Available Therapy, Including Ruxolitinib, in Patients With Myelofibrosis: A Randomized Clinical Trial. JAMA Oncol. 2018;4: 652–9. 102. Siddiquee K, Zhang S, Guida WC, Blaskovich MA, Greedy B, Lawrence HR, et al. Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity. Proc Natl Acad Sci U S A. 2007;104:7391–6. 123. Kim DW, Saussele S, Williams LA, Mohamed H, Rong Y, Zyczynski T, et al. References Ji P, Xu X, Ma S, Fan J, Zhou Q, Mao X, et al. Novel 2-Carbonylbenzo [b] thiophene 1,1-Dioxide Derivatives as Potent Inhibitors of STAT3 Signaling Pathway. ACS Med Chem Lett. 2015;6:1010–4. 112. Holmstrom TH, Moilanen AM, Ikonen T, Bjorkman ML, Linnanen T, Wohlfahrt G, et al. ODM-203, a Selective Inhibitor of FGFR and VEGFR, Shows Strong Antitumor Activity, and Induces Antitumor Immunity. Mol Cancer Ther. 2019;18:28–38. 91. Lin L, Hutzen B, Zuo M, Ball S, Deangelis S, Foust E, et al. Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells. Cancer Res. 2010;70:2445–54. 113. Pei J, Zhang Y, Luo Q, Zheng W, Li W, Zeng X, et al. STAT3 inhibition enhances CDN-induced STING signaling and antitumor immunity. Cancer Lett. 2019;450:110–22. 92. Li Z, Zhu T, Xu Y, Wu C, Chen J, Ren Y, et al. A novel STAT3 inhibitor, HJC0152, exerts potent antitumor activity in glioblastoma. Am J Cancer Res. 2019;9:699–713. 114. Lu C, Talukder A, Savage NM, Singh N, Liu K. JAK-STAT-mediated chronic inflammation impairs cytotoxic T lymphocyte activation to decrease anti- PD-1 immunotherapy efficacy in pancreatic cancer. Oncoimmunology. 2017; 6:e1291106. 93. Liu Z, Wang H, Guan L, Lai C, Yu W, Lai M. LL1, a novel and highly selective STAT3 inhibitor, displays anti-colorectal cancer activities in vitro and in vivo. Br J Pharmacol. 2020;177:298–313. 94. Fuh B, Sobo M, Cen L, Josiah D, Hutzen B, Cisek K, et al. LLL-3 inhibits STAT3 activity, suppresses glioblastoma cell growth and prolongs survival in a mouse glioblastoma model. Br J Cancer. 2009;100:106–12. 115. Yu GT, Mao L, Wu L, Deng WW, Bu LL, Liu JF, et al. Inhibition of SRC family kinases facilitates anti-CTLA4 immunotherapy in head and neck squamous cell carcinoma. Cell Mol Life Sci. 2018;75:4223–34. 95. Zuo M, Li C, Lin J, Javle M. LLL12, a novel small inhibitor targeting STAT3 for hepatocellular carcinoma therapy. Oncotarget. 2015;6:10940–9. 116. Yang H, Lee WS, Kong SJ, Kim CG, Kim JH, Chang SK, et al. STING activation reprograms tumor vasculatures and synergizes with VEGFR2 blockade. J Clin Invest. 2019;130:4350–64. 96. Wang H, Liu Z, Guan L, Li J, Chen S, Yu W, et al. LYW-6, a novel cryptotanshinone derived STAT3 targeting inhibitor, suppresses colorectal cancer growth and metastasis. Pharmacol Res. 2020;153:104661. 117. Jonker DJ, Nott L, Yoshino T, Gill S, Shapiro J, Ohtsu A, et al. References Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell. 2013;4:176–85. 57. Taniguchi K, Karin M. NF-kappaB, inflammation, immunity and cancer: coming of age Nat Rev Immunol 2018;18:309–24 55. Tsai MH, Lee CK. STAT3 Cooperates With Phospholipid Scramblase 2 to Suppress Type I Interferon Response. Front Immunol. 2018;9:1886. Suppress Type I Interferon Response. Front Immunol. 2018;9:1886. 56. Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell. 2013;4:176–85. 57. Taniguchi K, Karin M. NF-kappaB, inflammation, immunity and cancer: 80. Ham IH, Oh HJ, Jin H, Bae CA, Jeon SM, Choi KS, et al. Targeting interleukin- 6 as a strategy to overcome stroma-induced resistance to chemotherapy in gastric cancer. Mol Cancer. 2019;18:68. 56. Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell. 2013;4:176–85. y ; 57. Taniguchi K, Karin M. NF-kappaB, inflammation, immunity and cancer: coming of age. Nat Rev Immunol. 2018;18:309–24. 81. Lin Q, Ren L, Jian M, Xu P, Li J, Zheng P, et al. The mechanism of the premetastatic niche facilitating colorectal cancer liver metastasis generated from myeloid-derived suppressor cells induced by the S1PR1-STAT3 signaling pathway. Cell Death Dis. 2019;10:693. 58. Lee H, Herrmann A, Deng JH, Kujawski M, Niu G, Li Z, et al. Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer Cell. 2009;15:283–93. 82. Cheng JT, Deng YN, Yi HM, Wang GY, Fu BS, Chen WJ, et al. Hepatic carcinoma-associated fibroblasts induce IDO-producing regulatory dendritic cells through IL-6-mediated STAT3 activation. Oncogenesis. 2016;5:e198. 59. Garg B, Giri B, Modi S, Sethi V, Castro I, Umland O, et al. NFkappaB in Pancreatic Stellate Cells Reduces Infiltration of Tumors by Cytotoxic T Cells and Killing of Cancer Cells, via Up-regulation of CXCL12. Gastroenterology. 2018;155:880–91 e8. 83. Nagathihalli NS, Castellanos JA, Shi C, Beesetty Y, Reyzer ML, Caprioli R, et al. Signal Transducer and Activator of Transcription 3, Mediated Remodeling of the Tumor Microenvironment Results in Enhanced Tumor Drug Delivery in a Mouse Model of Pancreatic Cancer. Gastroenterology. 2015;149:1932–43 e9. 60. Saha A, Ahn S, Blando J, Su F, Kolonin MG, DiGiovanni J. Proinflammatory CXCL12-CXCR4/CXCR7 Signaling Axis Drives Myc-Induced Prostate Cancer in Obese Mice. Cancer Res. 2017;77:5158–68. Page 17 of 19 Page 17 of 19 Page 17 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. References Molecular Cancer (2020) 19:145 84. Sanz-Moreno V, Gaggioli C, Yeo M, Albrengues J, Wallberg F, Viros A, et al. ROCK and JAK1 signaling cooperate to control actomyosin contractility in tumor cells and stroma. Cancer Cell. 2011;20:229–45. 106. Son DJ, Zheng J, Jung YY, Hwang CJ, Lee HP, Woo JR, et al. MMPP Attenuates Non-Small Cell Lung Cancer Growth by Inhibiting the STAT3 DNA-Binding Activity via Direct Binding to the STAT3 DNA-Binding Domain. Theranostics. 2017;7:4632–42. 85. Gelain A, Mori M, Meneghetti F, Villa S. Signal Transducer and Activator of Transcription Protein 3 (STAT3): An Update on its Direct Inhibitors as Promising Anticancer Agents. Curr Med Chem. 2019;26:5165–206. 107. Huang W, Dong Z, Chen Y, Wang F, Wang CJ, Peng H, et al. Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo. Oncogene. 2016;35:783–92. 86. Beebe JD, Liu JY, Zhang JT. Two decades of research in discovery of anticancer drugs targeting STAT3, how close are we? Pharmacol Ther. 2018; 191:74–91. 108. Souissi I, Ladam P, Cognet JA, Le Coquil S, Varin-Blank N, Baran-Marszak F, et al. A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line. Mol Cancer. 2012;11:12. 87. Nagel-Wolfrum K, Buerger C, Wittig I, Butz K, Hoppe-Seyler F, Groner B. The interaction of specific peptide aptamers with the DNA binding domain and the dimerization domain of the transcription factor Stat3 inhibits transactivation and induces apoptosis in tumor cells. Mol Cancer Res. 2004; 2:170–82. 109. Koppikar P, Abdel-Wahab O, Hedvat C, Marubayashi S, Patel J, Goel A, et al. Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L- induced thrombocytosis and myelofibrosis. Blood. 2010;115:2919–27. 88. Miccoli A, Dhiani BA, Mehellou Y. Phosphotyrosine prodrugs: design, synthesis and anti-STAT3 activity of ISS-610 aryloxy triester phosphoramidate prodrugs. Medchemcomm. 2019;10:200–8. 110. Lo MC, Peterson LF, Yan M, Cong X, Hickman JH, Dekelver RC, et al. JAK inhibitors suppress t(8;21) fusion protein-induced leukemia. Leukemia. 2013; 27:2272–9. 89. Turkson J, Ryan D, Kim JS, Zhang Y, Chen Z, Haura E, et al. Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation. J Biol Chem. 2001;276:45443–55. 111. Lu X, Zhang T, Zhu SJ, Xun Q, Tong L, Hu X, et al. Discovery of JND3229 as a New EGFR(C797S) Mutant Inhibitor with In Vivo Monodrug Efficacy. ACS Med Chem Lett. 2018;9:1123–7. 90. References Xu H, Du X, Xu J, Zhang Y, Tian Y, Liu G, et al. Pancreatic beta cell microRNA-26a alleviates type 2 diabetes by improving peripheral insulin sensitivity and preserving beta cell function. PLoS Biol. 2020;18:e3000603. 129. Winter GE, Buckley DL, Paulk J, Roberts JM, Souza A, Dhe-Paganon S, et al. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science. 2015;348:1376–81. 152. June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359:1361–5. 130. Lai AC, Toure M, Hellerschmied D, Salami J, Jaime-Figueroa S, Ko E, et al. Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL. Angew Chem Int Ed Eng. 2016;55:807–10. 153. Kennedy LB, Salama AKS. A review of cancer immunotherapy toxicity. CA Cancer J Clin. 2020. 154. O'Reilly EM, Oh DY, Dhani N, Renouf DJ, Lee MA, Sun W, et al. Durvalumab With or Without Tremelimumab for Patients With Metastatic Pancreatic Ductal Adenocarcinoma: A Phase 2 Randomized Clinical Trial. JAMA Oncol. 2019. 131. Burslem GM, Smith BE, Lai AC, Jaime-Figueroa S, McQuaid DC, Bondeson DP, et al. The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study. Cell Chem Biol. 2018;25:67–77 e3. 132. Khan S, Zhang X, Lv D, Zhang Q, He Y, Zhang P, et al. A selective BCL-XL PROTAC degrader achieves safe and potent antitumor activity. Nat Med. 2019;25:1938–47. 155. Zhou Y, Fei M, Zhang G, Liang WC, Lin W, Wu Y, et al. Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP. Immunity. 2020;52:357–73 e9. 133. Njatcha C, Farooqui M, Kornberg A, Johnson DE, Grandis JR, Siegfried JM. STAT3 Cyclic Decoy Demonstrates Robust Antitumor Effects in Non-Small Cell Lung Cancer. Mol Cancer Ther. 2018;17:1917–26. 156. Hellmann MD, Paz-Ares L, Bernabe Caro R, Zurawski B, Kim SW, Carcereny Costa E, et al. Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer. N Engl J Med. 2019;381:2020–31. 134. Hong D, Kurzrock R, Kim Y, Woessner R, Younes A, Nemunaitis J, et al. AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer. Sci Transl Med. 2015;7:314ra185. 157. Atsaves V, Tsesmetzis N, Chioureas D, Kis L, Leventaki V, Drakos E, et al. PD- L1 is commonly expressed and transcriptionally regulated by STAT3 and MYC in ALK-negative anaplastic large-cell lymphoma. Leukemia. 2017;31: 1633–7. 135. References PD- 1 up-regulation on CD4(+) T cells promotes pulmonary fibrosis through STAT3-mediated IL-17A and TGF-beta1 production. Sci Transl Med. 2018;10. 142. Li H, Liu A, Zhao Z, Xu Y, Lin J, Jou D, et al. Fragment-based drug design and drug repositioning using multiple ligand simultaneous docking (MLSD): identifying celecoxib and template compounds as novel inhibitors of signal transducer and activator of transcription 3 (STAT3). J Med Chem. 2011;54: 5592–6. 165. Celada LJ, Rotsinger JE, Young A, Shaginurova G, Shelton D, Hawkins C, et al. Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/ Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4(+) T Cell Proliferation. Am J Respir Cell Mol Biol. 2017;56:74–82. 166. Krasilnikov M, Ivanov VN, Dong J, Ronai Z. ERK and PI3K negatively regulate STAT-transcriptional activities in human melanoma cells: implications towards sensitization to apoptosis. Oncogene. 2003;22:4092–101. 143. Huang R, Jing X, Huang X, Pan Y, Fang Y, Liang G, et al. Bifunctional Naphthoquinone Aromatic Amide-Oxime Derivatives Exert Combined Immunotherapeutic and Antitumor Effects through Simultaneous Targeting of Indoleamine-2,3-dioxygenase and Signal Transducer and Activator of Transcription 3. J Med Chem. 2020. 167. Mace TA, Shakya R, Pitarresi JR, Swanson B, McQuinn CW, Loftus S, et al. IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut. 2018;67:320–32. 144. Buchert M, Burns CJ, Ernst M. Targeting JAK kinase in solid tumors: emerging opportunities and challenges. Oncogene. 2016;35:939–51. 168. Liu H, Shen J, Lu K. IL-6 and PD-L1 blockade combination inhibits hepatocellular carcinoma cancer development in mouse model. Biochem Biophys Res Commun. 2017;486:239–44. 145. Kang S, Tanaka T, Narazaki M, Kishimoto T. Targeting Interleukin-6 Signaling in Clinic. Immunity. 2019;50:1007–23. 146. Liu L, Nam S, Tian Y, Yang F, Wu J, Wang Y, et al. 6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells. Cancer Res. 2011;71:3972–9. 169. Luo F, Luo M, Rong QX, Zhang H, Chen Z, Wang F, et al. Niclosamide, an antihelmintic drug, enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer. J Immunother Cancer. 2019;7:245. 170. Bagley SJ, O'Rourke DM. Clinical investigation of CAR T cells for solid tumors: Lessons learned and future directions. Pharmacol Ther. 2019;107419. 147. Tian Y, Nam S, Liu L, Yakushijin F, Yakushijin K, Buettner R, et al. Spirooxindole derivative SOID-8 induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells. PLoS One. 2012;7:e49306. 171. References Odate S, Veschi V, Yan S, Lam N, Woessner R, Thiele CJ. Inhibition of STAT3 with the Generation 2.5 Antisense Oligonucleotide, AZD9150, Decreases Neuroblastoma Tumorigenicity and Increases Chemosensitivity. Clin Cancer Res. 2017;23:1771–84. 158. Song TL, Nairismagi ML, Laurensia Y, Lim JQ, Tan J, Li ZM, et al. Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T- cell lymphoma. Blood. 2018;132:1146–58. 136. Shastri A, Choudhary G, Teixeira M, Gordon-Mitchell S, Ramachandra N, Bernard L, et al. Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells. J Clin Invest. 2018;128:5479–88. 159. Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez GA, et al. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. Cell Rep. 2017;19:1189–201. 137. Zhou J, Rossi J. Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov. 2017;16:181–202. 160. Yoyen-Ermis D, Tunali G, Tavukcuoglu E, Horzum U, Ozkazanc D, Sutlu T, et al. Myeloid maturation potentiates STAT3-mediated atypical IFN-gamma signaling and upregulation of PD-1 ligands in AML and MDS. Sci Rep. 2019; 9:11697. 138. Esposito CL, Nuzzo S, Catuogno S, Romano S, de Nigris F, de Franciscis V. STAT3 Gene Silencing by Aptamer-siRNA Chimera as Selective Therapeutic for Glioblastoma. Mol Ther Nucleic Acids. 2018;10:398–411. 161. Herrmann A, Lahtz C, Nagao T, Song JY, Chan WC, Lee H, et al. CTLA4 Promotes Tyk2-STAT3-Dependent B-cell Oncogenicity. Cancer Res. 2017;77: 5118–28. 139. Yoon S, Wu X, Armstrong B, Habib N, Rossi JJ. An RNA Aptamer Targeting the Receptor Tyrosine Kinase PDGFRalpha Induces Anti-tumor Effects through STAT3 and p53 in Glioblastoma. Mol Ther Nucleic Acids. 2019;14:131–41. 162. Hsu P, Santner-Nanan B, Hu M, Skarratt K, Lee CH, Stormon M, et al. IL-10 Potentiates Differentiation of Human Induced Regulatory T Cells via STAT3 and Foxo1. J Immunol. 2015;195:3665–74. 140. Heppler LN, Frank DA. Targeting Oncogenic Transcription Factors: Therapeutic Implications of Endogenous STAT Inhibitors. Trends Cancer. 2017;3:816–27. 163. Keremu A, Aimaiti A, Liang Z, Zou X. Role of the HDAC6/STAT3 pathway in regulating PD-L1 expression in osteosarcoma cell lines. Cancer Chemother Pharmacol. 2019;83:255–64. 141. Khan MW, Saadalla A, Ewida AH, Al-Katranji K, Al-Saoudi G, Giaccone ZT, et al. The STAT3 inhibitor pyrimethamine displays anti-cancer and immune stimulatory effects in murine models of breast cancer. Cancer Immunol Immunother. 2018;67:13–23. 164. Celada LJ, Kropski JA, Herazo-Maya JD, Luo W, Creecy A, Abad AT, et al. References Outcomes of switching to dasatinib after imatinib-related low-grade adverse events in patients with chronic myeloid leukemia in chronic phase: the DASPERSE study. Ann Hematol. 2018;97:1357–67. 103. Turkson J, Zhang S, Palmer J, Kay H, Stanko J, Mora LB, et al. Inhibition of constitutive signal transducer and activator of transcription 3 activation by novel platinum complexes with potent antitumor activity. Mol Cancer Ther. 2004;3:1533–42. 124. Antonarakis ES, Heath EI, Posadas EM, Yu EY, Harrison MR, Bruce JY, et al. A phase 2 study of KX2-391, an oral inhibitor of Src kinase and tubulin polymerization, in men with bone-metastatic castration-resistant prostate cancer. Cancer Chemother Pharmacol. 2013;71:883–92. 104. Buettner R, Corzano R, Rashid R, Lin J, Senthil M, Hedvat M, et al. Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development. ACS Chem Biol. 2011;6:432–43. 125. Finn RS, Qin S, Ikeda M, Galle PR, Ducreux M, Kim TY, et al. Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma. N Engl J Med. 2020;382:1894–905. 105. Escobar Z, Bjartell A, Canesin G, Evans-Axelsson S, Sterner O, Hellsten R, et al. Preclinical Characterization of 3beta-(N-Acetyl l-cysteine methyl ester)-2abeta,3- dihydrogaliellalactone (GPA512), a Prodrug of a Direct STAT3 Inhibitor for the Treatment of Prostate Cancer. J Med Chem. 2016;59:4551–62. 126. D'Angelo SP, Shoushtari AN, Keohan ML, Dickson MA, Gounder MM, Chi P, et al. Combined KIT and CTLA-4 Blockade in Patients with Refractory GIST Page 18 of 19 Page 18 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 149. Zhang Q, Liu RX, Chan KW, Hu J, Zhang J, Wei L, et al. Exosomal transfer of p-STAT3 promotes acquired 5-FU resistance in colorectal cancer cells. J Exp Clin Cancer Res. 2019;38:320. and Other Advanced Sarcomas: A Phase Ib Study of Dasatinib plus Ipilimumab. Clin Cancer Res. 2017;23:2972–80. and Other Advanced Sarcomas: A Phase Ib Study of Dasatinib plus Ipilimumab. Clin Cancer Res. 2017;23:2972–80. 127. Turkson J, Kim JS, Zhang S, Yuan J, Huang M, Glenn M, et al. Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity. Mol Cancer Ther. 2004;3:261–9. 150. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367. 128. Lai AC, Crews CM. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev Drug Discov. 2017;16:101–14. 151. References Du H, Hirabayashi K, Ahn S, Kren NP, Montgomery SA, Wang X, et al. Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells. Cancer Cell. 2019;35: 221–37 e8. 148. Wen W, Wu J, Liu L, Tian Y, Buettner R, Hsieh MY, et al. Synergistic anti- tumor effect of combined inhibition of EGFR and JAK/STAT3 pathways in human ovarian cancer. Mol Cancer. 2015;14:100. Page 19 of 19 Page 19 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 172. Majzner RG, Theruvath JL, Nellan A, Heitzeneder S, Cui Y, Mount CW, et al. CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. Clin Cancer Res. 2019;25:2560–74. 173. Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018;24: 563–71. 174. Kagoya Y, Tanaka S, Guo T, Anczurowski M, Wang CH, Saso K, et al. A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nat Med. 2018;24:352–9. 175. Wang Y, Jiang H, Luo H, Sun Y, Shi B, Sun R, et al. An IL-4/21 Inverted Cytokine Receptor Improving CAR-T Cell Potency in Immunosuppressive Solid-Tumor Microenvironment. Front Immunol. 2019;10:1691. 176. Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol. 2019;16:372–85. 177. Guha P, Gardell J, Darpolor J, Cunetta M, Lima M, Miller G, et al. STAT3 inhibition induces Bax-dependent apoptosis in liver tumor myeloid-derived suppressor cells. Oncogene. 2019;38:533–48. 178. Corrales L, McWhirter SM, Dubensky TW Jr, Gajewski TF. The host STING pathway at the interface of cancer and immunity. J Clin Invest. 2016;126: 2404–11. 179. Ramanjulu JM, Pesiridis GS, Yang J, Concha N, Singhaus R, Zhang SY, et al. Design of amidobenzimidazole STING receptor agonists with systemic activity. Nature. 2018;564:439–43. 180. Shae D, Becker KW, Christov P, Yun DS, Lytton-Jean AKR, Sevimli S, et al. Endosomolytic polymersomes increase the activity of cyclic dinucleotide STING agonists to enhance cancer immunotherapy. Nat Nanotechnol. 2019; 14:269–78. 181. Zhu Q, Man SM, Gurung P, Liu Z, Vogel P, Lamkanfi M, et al. Cutting edge: STING mediates protection against colorectal tumorigenesis by governing the magnitude of intestinal inflammation. J Immunol. 2014;193:4779–82. 182. References Zhang CX, Ye SB, Ni JJ, Cai TT, Liu YN, Huang DJ, et al. STING signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion. Cell Death Differ. 2019;26:2314–28. 183. Hsia HC, Hutti JE, Baldwin AS. Cytosolic DNA Promotes Signal Transducer and Activator of Transcription 3 (STAT3) Phosphorylation by TANK-binding Kinase 1 (TBK1) to Restrain STAT3 Activity. J Biol Chem. 2017;292:5405–17. 184. Moreira D, Adamus T, Zhao X, Su YL, Zhang Z, White SV, et al. STAT3 Inhibition Combined with CpG Immunostimulation Activates Antitumor Immunity to Eradicate Genetically Distinct Castration-Resistant Prostate Cancers. Clin Cancer Res. 2018;24:5948–62. 185. Nefedova Y, Nagaraj S, Rosenbauer A, Muro-Cacho C, Sebti SM, Gabrilovich DI. Regulation of dendritic cell differentiation and antitumor immune response in cancer by pharmacologic-selective inhibition of the janus- activated kinase 2/signal transducers and activators of transcription 3 pathway. Cancer Res. 2005;65:9525–35. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2466588769
http://www.ajnr.org/content/ajnr/37/11/2123.full.pdf
English
null
Orbital Fat Volumetry and Water Fraction Measurements Using T2-Weighted FSE-IDEAL Imaging in Patients with Thyroid-Associated Orbitopathy
American journal of neuroradiology
2,016
cc-by
5,230
Thyroid-Associated Orbitopathy FSE-IDEAL Imaging in Patients with Measurements Using T2-Weighted Orbital Fat Volumetry and Water Fraction Takahashi, Y. Akiyama and K. Awai Y. Kaichi, K. Tanitame, H. Itakura, H. Ohno, M. Yoneda, Y. http://www.ajnr.org/content/37/11/2123 https://doi.org/10.3174/ajnr.A4859 doi: 2016, 37 (11) 2123-2128 AJNR Am J Neuroradiol of October 23, 2024. This information is current as of October 23, 2024. This information is current as http://www.ajnr.org/content/37/11/2123 https://doi.org/10.3174/ajnr.A4859 doi: 2016, 37 (11) 2123-2128 AJNR Am J Neuroradiol ORIGINAL RESEARCH HEAD & NECK ORIGINAL RESEARCH HEAD & NECK Received January 28, 2016; accepted after revision May 8. From the Department of Diagnostic Radiology (Y.K., K.A.), Graduate School and Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Ja- pan; Department of Radiology (K.T.), Chugoku Rosai Hospital, Kure, Japan; Depart- ment of Ophthalmology and Visual Science (H.I.) and Department of Molecular and Internal Medicine (H.O., M.Y.), Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; and Department of Clinical Radiology (Y.T., Y.A.), Hiroshima University Hospital, Hiroshima, Japan. Please address correspondence to Yoko Kaichi, MD, Diagnostic Radiology, Gradu- ate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan; e-mail: kaichi@hiroshima-u.ac.jp http://dx.doi.org/10.3174/ajnr.A4859 Orbital Fat Volumetry and Water Fraction Measurements Using T2-Weighted FSE-IDEAL Imaging in Patients with Thyroid-Associated Orbitopathy X Y. Kaichi, X K. Tanitame, X H. Itakura, X H. Ohno, X M. Yoneda, X Y. Takahashi, X Y. Akiyama, and AJNR Am J Neuroradiol 37:2123–28 Nov 2016 www.ajnr.org http://dx.doi.org/10.3174/ajnr.A4859 Received January 28, 2016; accepted after revision May 8. ABSTRACT BACKGROUND AND PURPOSE: The quantitative evaluation of orbital fat proliferation and edema and the assessment of extraocular muscles are useful for diagnosing and monitoring thyroid-associated orbitopathy. To evaluate therapy-induced quantitative changes in the orbital fat of patients with thyroid-associated orbitopathy, we performed volumetric and water fraction measurements by using T2-weighted FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation (FSE-IDEAL) imaging. MATERIALS AND METHODS: Orbital FSE-IDEAL images of 30 volunteers were acquired twice within 1 week. Nine patients with thyroid- associated orbitopathy underwent FSE-IDEAL imaging before and after methylprednisolone pulse therapy, and the treatment results were assessed by using their pre- and post-methylprednisolone pulse therapy clinical activity scores. We performed volumetric and water fraction measurements of orbital fat by using FSE-IDEAL imaging and evaluated interscan differences in the volunteers. In patients with thyroid-associated orbitopathy, we compared pre- and posttherapy orbital fat measurements and assessed the correlation between the pretherapy values and clinical activity score improvement. RESULTS: The reproducibility of results obtained by the quantitative evaluation of orbital fat in volunteers was acceptable. After methylprednisolone pulse therapy, the water fraction in the orbital fat of patients with thyroid-associated orbitopathy was significantly decreased (P  .001). There was a significant positive correlation between the pretherapy water fraction and clinical activity score improvement (right, r  0.82; left, r  0.79) and a significant negative correlation between the pretherapy volume and clinical activity score improvement (bilateral, r  0.84). CONCLUSIONS: Volumetric and water fraction measurements of orbital fat by using FSE-IDEAL imaging are feasible and useful for monitoring the effects of therapy and for predicting the response of patients with thyroid-associated orbitopathy to methylprednisolone pulse therapy. CONCLUSIONS: Volumetric and water fraction measurements of orbital fat by using FSE-IDEAL imaging are feasible and useful for monitoring the effects of therapy and for predicting the response of patients with thyroid-associated orbitopathy to methylprednisolone pulse therapy. Please address correspondence to Yoko Kaichi, MD, Diagnostic Radiology, Gradu- ate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan; e-mail: kaichi@hiroshima-u.ac.jp ABSTRACT Two of them underwent local injection of triamcinolone acetonide into the or- bit, and 5 of them followed a conserva- tive wait-and-see approach because their TAO was slight and the risk of MPPT would exceed the benefit. Conse- quently, the 9 patients (2 men, 7 women; median age, 57  12.5 years; 18 eyes) who received MPPT (500 mg of methyl- prednisolone administered intrave- nously once a day for 3 consecutive days) were included in this study and underwent FSE-IDEAL imaging again 1.6  0.7 months thereafter. Re-evalua- tion of the TAO activity was based on the latest CAS obtained around the time of g study (the interval, 1–15 days; median, FIG 1. Axial water (A) and fat (B) images of a healthy volunteer. The ROIs are encircled in yellow. We also recruited 16 patients older than 20 years of age who had a clinical diagnosis of TAO between December 2013 and November 2015. The recorded activity of their TAO was based on their clinical activity score (CAS), including pain, eyelid erythema or edema, con- junctival hyperemia and chemosis, and a swollen caruncle.20 All 16 patients un- derwent orbital FSE-IDEAL imaging. Two of them underwent local injection of triamcinolone acetonide into the or- bit, and 5 of them followed a conserva- tive wait-and-see approach because their TAO was slight and the risk of MPPT would exceed the benefit. Conse- quently, the 9 patients (2 men, 7 women; median age, 57  12.5 years; 18 eyes) who received MPPT (500 mg of methyl- prednisolone administered intrave- nously once a day for 3 consecutive days) were included in this study and underwent FSE-IDEAL imaging again 1.6  0.7 months thereafter. Re-evalua- tion of the TAO activity was based on the latest CAS obtained around the time of he second MR imaging study (the interval, 1–15 days; median, 5 days). e encircled in yellow. at is light green. We manually removed ex- ge SI of 207 and an SD of 88. Therefore the EAL images, we then asured the orbital fat FIG 1. Axial water (A) and fat (B) images of a healthy volunteer. The ROIs are encircled in yellow. FIG 2. Axial fat image (A) of a healthy volunteer. The selected orbital fat is light green. We separated fat tissue by using an adequate signal-intensity threshold and manually removed ex- traorbital fat. ABSTRACT ABBREVIATIONS: CAS  clinical activity score; FSE-IDEAL  FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation; ABBREVIATIONS: CAS  clinical activity score; FSE-IDEAL  FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation; MPPT  methylprednisolone pulse therapy; SI  signal intensity; TAO  thyroid-associated orbitopathy ABBREVIATIONS: CAS  clinical activity score; FSE-IDEAL  FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation; MPPT  methylprednisolone pulse therapy; SI  signal intensity; TAO  thyroid-associated orbitopathy ABBREVIATIONS: CAS  clinical activity score; FSE-IDEAL  FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation; MPPT  methylprednisolone pulse therapy; SI  signal intensity; TAO  thyroid-associated orbitopathy T hyroid-associated orbitopathy (TAO) is the most common extrathyroid manifestation of Graves disease. The enlarge- ment of orbital fat and extraocular muscles in the relatively fixed volume space imposed by the bony orbit may produce proptosis, ocular motility loss, and decreased visual acuity.1 Histologic stud- ies showed lymphocytic infiltration and edema due to the accu- mulation of hydrophilic, interstitial glycosaminoglycans in the orbital fat and extraocular muscles,2,3 attributable to antigenic cross-reactivity between thyroid proteins and orbital fibroblasts.4 T The course of TAO can be divided into the active, regressing, and a burnt-out phase.5 Early active disease is treated with com- bined immunosuppression.6,7 The identification of patients who stand to benefit from this therapy is important but clinically dif- ficult, despite the availability of activity scoring systems. Conse- quently, objective methods are needed. CT has been used to quantify the volume of extraocular mus- cles and orbital fat in TAO.8-11 However, ocular radiation expo- sure limits its clinical use. Because MR imaging without ionizing radiation yields orbital images with excellent soft-tissue contrast in any plane, it has been used for evaluating TAO.12-15 The quantitative evaluation of the orbital fat volume on T1- weighted images was reported useful for assessing the severity of TAO and for monitoring the treatment response.12,16 Recently, 2123 We also recruited 16 patients older than 20 years of age who had a clinical diagnosis of TAO between December 2013 and November 2015. The recorded activity of their TAO was based on their clinical activity score (CAS), including pain, eyelid erythema or edema, con- junctival hyperemia and chemosis, and a swollen caruncle.20 All 16 patients un- derwent orbital FSE-IDEAL imaging. ABSTRACT In this volunteer, the internal rectus muscle showed an average SI of 207 and an SD of 112, whereas the orbital fat showed an average SI of 1636 and an SD of 88. Therefore the threshold value was [(207  112)  (1636  88) / 2  933]. Using FSE-IDEAL images, we then produced 3D reconstruction images of the bilateral orbital fat (B) and measured the orbital fat volume on a workstation. FIG 2. Axial fat image (A) of a healthy volunteer. The selected orbital fat is light green. We separated fat tissue by using an adequate signal-intensity threshold and manually removed ex- traorbital fat. In this volunteer, the internal rectus muscle showed an average SI of 207 and an SD of 112, whereas the orbital fat showed an average SI of 1636 and an SD of 88. Therefore the threshold value was [(207  112)  (1636  88) / 2  933]. Using FSE-IDEAL images, we then produced 3D reconstruction images of the bilateral orbital fat (B) and measured the orbital fat volume on a workstation. Higashiyama et al17 demonstrated that after methylprednisolone pulse therapy (MPPT), the orbital fat volume was unchanged, while the total volume of extraocular muscles was decreased on T2-weighted images. However, in our search of the literature, we found no reports on quantitative changes in orbital fat edema after MPPT or orbital irradiation. Consequently, the correlation between the volume and edema of orbital fat and the treatment response of patients with TAO remains to be elucidated. Orbital Fat Water Fraction and Volume Measurements Orbital Fat Water Fraction and Volume Measurements We placed an ROI in the orbital fat on FSE-IDEAL images of water and fat and measured the average signal intensity (SI) in the ROIs. Next, we defined and calculated the water fraction of the orbital fat as [SI Water / (SI Water  SI Fat)] to assess the orbital fat edema on the basis of the fat fraction (SI Fat / [SI Water  SI Fat]),18 defined to quantify fatty infiltration (Fig 1). The orbital fat volume of both eyes was measured on a workstation (Vir- tual Place Raijin; AZE Ltd, Tokyo, Japan). We first separated fat tissue from other structures by using the threshold value, recorded as the mean value between the average SI plus the SD of the ROI in the internal rectus muscle and the average SI minus the SD of the ROI in the orbital fat, considering some dispersion of signal intensities in the ROIs. Then we manually removed the fatty marrow of the orbital bone and outer fat; the orbital fat volume was automatically measured on the work- station (Fig 2). MR Imaging All i All images were acquired on a 3T scanner (Signa Excite HD 3.0; GE Healthcare, Milwaukee, Wisconsin; gradient strength, 40 mT/m; slew rate, 150 T/m/s) by using an 8-channel phased array brain coil. We optimized the T2-weighted FSE-IDEAL sequence (TR/TE, 6000/100 ms; flip angle, 90°; image matrix, 288  160; FOV, 160  160 mm; section thickness/gap, 2/0 mm; asymmetric echo shifts, /6, /2, 7/6; number of acquisitions, 3; number of sections, 32; scan time, 2 minutes 42 seconds) and obtained orbital water and fat images for all subjects. FSE iterative decomposition of water and fat with echo asym- metry and least-squares estimation (FSE-IDEAL), a novel 3-point Dixon method, is useful for separating the fat signal from the water signal18,19 but has not been used to quantify the orbital structures in TAO. To evaluate the feasibility of FSE-IDEAL im- aging for the precise quantitative evaluation of orbital fat, we sub- jected healthy volunteers to 2 FSE-IDEAL imaging studies per- formed during 1 week. We measured the volume and the water fraction of their orbital fat and ascertained the reproducibility of the measurement results. We also acquired FSE-IDEAL images in patients with TAO to assess MPPT-induced changes in the volume and water fraction of their orbital fat and evaluated the correlation between the quantitative values of orbital fat and the improvement in their symptoms. 2124 Kaichi Nov 2016 www.ajnr.org MATERIALS AND METHODS Subjects Thi i d This prospective study was approved by the Ethics Committee of Hiroshima University. Informed consent was obtained from all participants before entry into the study. We recruited 30 healthy volunteers (15 men, 15 women; me- dian age, 29  7.6 years). They underwent orbital FSE-IDEAL imaging twice within 1 week to evaluate the reproducibility of the water fraction and the volume measurements of the orbital fat. 2124 Kaichi Nov 2016 www.ajnr.org 2124 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.15 0.17 0.19 0.21 0.23 0.25 0.27 CI (95%) 1st scan – 2nd scan Mean of 1st and 2nd scan Bias -4 -3 -2 -1 0 1 2 3 4 9 11 13 15 17 19 21 CI (95%) 1st scan – 2nd scan (ml) Mean of 1st and 2nd scan (ml) Bias A B FIG 3. Bland-Altman analysis confirming the interscan reproducibility of the water fraction (A) and the volume of orbital fat (B) on FSE-IDEAL images of the volunteers. -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.15 0.17 0.19 0.21 0.23 0.25 0.27 CI (95%) 1st scan – 2nd scan Mean of 1st and 2nd scan Bias A -4 -3 -2 -1 0 1 2 3 4 9 11 13 15 17 19 21 CI (95%) 1st scan – 2nd scan (ml) Mean of 1st and 2nd scan (ml) Bias B 1st scan – 2nd scan (ml) B A FIG 3. Bland-Altman analysis confirming the interscan reproducibility of the water fraction (A) and the volume of orbital fat (B) on FSE-IDEAL images of the volunteers FIG 3. Bland-Altman analysis confirming the interscan reproducibility of the water fraction (A) and the volume of orbital fat (B) on FSE-IDEAL images of the volunteers. Patient profiles Characteristics Median age (yr) (range) 57 (43–82) Sex: male/female 2/7 Median duration of the treatment for GD (mo) (range) 11 (1–168) Median duration from the onset of TAO (mo) (range) 4 (1–8) Smoking: yes/no 3/6 Median pretherapy CAS (range) 2 (1–4) Involvement of extraocular muscle: yes/no 7/2 Note:—GD indicates Graves disease. Patient profiles Characteristics Median age (yr) (range) 57 (43–82) Sex: male/female 2/7 Median duration of the treatment for GD (mo) (range) 11 (1–168) Median duration from the onset of TAO (mo) (range) 4 (1–8) Smoking: yes/no 3/6 Median pretherapy CAS (range) 2 (1–4) Involvement of extraocular muscle: yes/no 7/2 Note:—GD indicates Graves disease. Volunteers The difference in the water fraction and the volume of orbital fat on the first and second scans was 1.1%  11.9% and 0.5%  3.6%, respectively. Bland-Altman analysis of the measurement results showed that their reproducibility was adequate (water fraction: r  0.71, bias  0.002; 95% CI bias, 0.012–0.007; 95% CI, 0.052–0.047; volume: r  0.99, bias  0.066; 95% CI bias, 0.235–0.102; 95% CI, 0.951–0.819) (Fig 3). DISCUSSION To the best of our knowledge, this is the first quantitative eval- uation of orbital fat by using FSE-IDEAL imaging. We docu- ment an MPPT-induced reduction in the water fraction of or- bital fat in patients with TAO and report the positive correlation between the pretreatment water fraction and the Statistical Analyses All statistical analyses were performed with commercially avail- able software (XLSTAT, version 2015.6.01.244, Addinsoft; https://www.xlstat.com/en/company/about-us) and Excel 2010 (Microsoft, Redmond, Washington). We used Bland-Altman analysis and the Pearson correlation coefficient to evaluate interscan differences in the volunteers and used the mean value of the water fraction and the volume of the bilateral orbit in our analyses. In patients with TAO, we performed paired-sample t tests to compare the water fraction and the volume of orbital fat before and after MPPT. The right and left orbits were examined separately because patients with unilateral TAO manifested unilateral excess fat exophthalmos.10 To ascertain that the values obtained before and aftertreatmentweresignificantlydifferent,weappliedthe2-samplet test to compare the therapy-induced changes with the values ob- tained in the volunteers. We defined the therapeutic effect of MPPT as the following formula: [CAS Improvement Ratio  (Pretherapy CAS  Posttherapy CAS) / Pretherapy CAS)] and determined the correlation between the measured values before MPPT and the CAS improvement ratio with the Pearson correlation coefficient test. Dif- ferences of P  .05 were considered statistically significant. 12.4%  3.8%, P  .001; left, 13.6%  6.5%, P  .001; Fig 4A). The treatment-induced reduction in the water fraction was sig- nificantly larger than the interscan difference observed in the volunteers (right, P  .002; left, P  .001). There was no significant difference in the orbital fat volume pre- and posttreatment (right, 2.0%  7.8%, P  .37; left, 1.9%  3.8%, P  .17, Fig 4B). The pre- and posttreatment CAS fell by 3 points in 2 patients, by 2 points in 1 patient, and by 1 point in 3 patients. In the other 3, there was no change. The positive correlation between the pre- treatment water fraction and the CAS improvement rate (right, r  0.82, P  .007; left, r  0.79, P  .012) showed that the higher the pretreatment water fraction, the greater was the posttreatment CAS improvement. On the other hand, there was a negative cor- relation between the fat volume and CAS improvement (right, r  0.84, P  .005; left, r  0.84, P  .005), indicating that the larger the pretreatment orbital fat volume, the lower the post- treatment CAS improvement rate. Patients The reproducibility of water fraction and fat volume measurements on all iteratively acquired FSE-IDEAL images was sufficient. In Japan, MPPT is generally applied in patients with TAO with low CAS scores because the Japanese patients with TAO often have orbital inflammation despite low CAS scores.30 We found that the higher the pretreatment water fraction, the greater the posttreatment decrease in the CAS, despite the low pretreatment CAS scores of our study patients. An increase in the water fraction is indicative of an increase in the tissue water content and may reflect acute inflammatory changes. Earlier studies on extraocular muscles found a correlation between the SI increase on pretreat- ment STIR images and a good therapeutic response31-33 and be- tween prolonged pretreatment T2 values and a good response to systemic corticosteroids or orbital radiation therapy.33 These findings indicate that treatment at an early and immunologi- cally active stage of orbitopathy is important.11,34,35 Our quan- titative MR imaging study by using the FSE-IDEAL sequence showed that MPPT was useful in patients with TAO whose orbital fat contained an elevated water fraction. On the other hand, the larger the pretreatment orbital fat volume, the lower the posttreatment CAS improvement rate. Because a longer duration of TAO is associated with a larger orbital fat mass and fibrosis,36 we think that a response to steroids is less likely when the disease is in its late, inactive stage with more fibrosis.33,34 Others12,21 used a 1.5T MR imaging scanner and a receiver surface coil to measure orbital fat volumes. Although surface coils can yield orbital images with high spatial resolution, the signal strength decreases as the distance from the coil increases. Our use of a 3T MR imaging scanner and an 8-channel brain coil resulted in orbital images with a high enough signal-to-noise ratio for the estimation of the orbital fat volume and the water fraction. Orbital images with high spatial and temporal resolution and fewer motion artifacts due to eye movement can be acquired on multidetector row CT scanners.11 However, the low soft-tissue contrast on multidetector row CT images makes it difficult to separate out orbital fat. In addition, ocular radiation exposure is a serious concern. Therefore, MR imaging with high soft-tissue contrast is safer for orbital imaging. We found that MPPT reduced the water fraction in the orbital fat of patients with TAO, possibly because it decreased the severity of edema. Patients The clinical characteristics of the 9 patients with TAO are sum- marized in the Table. After MPPT, the water fraction in the orbital fat on both sides was significantly decreased (right, AJNR Am J Neuroradiol 37:2123–28 Nov 2016 www.ajnr.org 2125 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 right, p < 0.001 le, p < 0.001 pre post pre post right, p = 0.37 10 20 30 40 50 60 10 20 30 40 50 60 Le, p = 0.17 ml ml pre post pre post A B FIG 4. Comparison of the pre- and posttreatment water fraction (A) and the volume of orbital fat (B) in patients with TAO. Methylprednisolone pulse therapy significantly decreased the water fraction. The volume was unchanged. right, p = 0.37 10 20 30 40 50 60 ml pre post B le, p < 0.001 pre post 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 right, p < 0.001 pre post A 10 20 30 40 50 60 Le, p = 0.17 ml pre post B A FIG 4. Comparison of the pre- and posttreatment water fraction (A) and the volume of orbital fat (B) in patients with TAO. Methylprednisolone pulse therapy significantly decreased the water fraction. The volume was unchanged. CAS improvement rate and the negative correlation between the pretreatment orbital fat volume and the CAS improvement rate. found no difference in the degree of exophthalmos, which is related to an increase in orbital fat, in patients with TAO who had undergone MPPT. Expansion of the adipose tissue volume is elicited by glycosaminoglycan-related edema and the emergence of a population of newly differentiated fat cells in these tissues.29 Although MPPT can reduce orbital fat edema, the increase in the number of fat cells may persist after therapy. We used FSE-IDEAL imaging to acquire axial images of the bilateral orbits and measured the water fraction and the volume of orbital fat in healthy volunteers and patients with TAO. Although many images contained motion artifacts due to voluntary and involuntary eye movements, it was possible to differentiate the SI of orbital fat and other intraorbital structures by applying an adequate threshold in the orbital fat volumetry. To measure the water fraction in orbital fat, we placed ROIs on the intraor- bital fat; measurement of the SI was not hampered by motion artifacts. REFERENCES 1. KendlerDL,LippaJ,RootmanJ.Theinitialclinicalcharacteristicsof Graves’ orbitopathy vary with age and sex. Arch Ophthalmol 1993; 111:197–201 CrossRef Medline 1. KendlerDL,LippaJ,RootmanJ.Theinitialclinicalcharacteristicsof Graves’ orbitopathy vary with age and sex. Arch Ophthalmol 1993; 111:197–201 CrossRef Medline 21. Tian S, Nishida Y, Isberg B, et al. MRI measurements of normal extraocular muscles and other orbital structures. Graefes Arch Clin Exp Ophthalmol 2000;238:393–404 CrossRef Medline 2. Winand RJ, Cornet G, Etienne-Decerf J, et al. Original acquisition in the pathogenesis and the treatment of endocrine ophthalmopathy. Metab Pediatr Syst Ophthalmol 1988;11:126–32 Medline 2. Winand RJ, Cornet G, Etienne-Decerf J, et al. Original acquisition in the pathogenesis and the treatment of endocrine ophthalmopathy. Metab Pediatr Syst Ophthalmol 1988;11:126–32 Medline 22. Hoh HB, Laitt RD, Wakely C, et al. The STIR sequence MRI in the assessment of extraocular muscles in thyroid eye disease. Eye 1994; 8:506–10 CrossRef Medline 3. Kahaly G, Hansen C, Beyer J, et al. Plasma glycosaminoglycans in endocrine ophthalmopathy. J Endocrinol Invest 1994;17:45–50 CrossRef Medline 23. Laitt RD, Hoh B, Wakeley C, et al. The value of the short tau inver- sion recovery sequence in magnetic resonance imaging of thyroid eye disease. Br J Radiol 1994;67:244–47 CrossRef Medline 4. Ludgate M, Baker G. Unlocking the immunological mechanisms of orbitalinflammationinthyroideyedisease.ClinExpImmunol2002; 127:193–98 CrossRef Medline 24. Bailey CC, Kabala J, Laitt R, et al. Magnetic resonance imaging in thyroid eye disease. Eye 1996;10:617–19 CrossRef Medline 5. Rundle FF, Wilson CW. Development and course of exophthal- mos and ophthalmoplegia in Graves’ disease with special refer- ence to the effect of thyroidectomy. Clin Sci 1945;5:177–94 Medline 25. Higashiyama T, Nishida Y, Morino K, et al. Use of MRI signal inten- sity of extraocular muscles to evaluate methylprednisolone pulse therapy in thyroid-associated ophthalmopathy. Jpn J Ophthalmol 2015;59:124–30 CrossRef Medline 6. Claridge KG, Ghabrial R, Davis G, et al. Combined radiotherapy and medical immunosuppression in the management of thyroid eye disease. Eye 1997;11:717–22 CrossRef Medline 6. Claridge KG, Ghabrial R, Davis G, et al. Combined radiotherapy and medical immunosuppression in the management of thyroid eye disease. Eye 1997;11:717–22 CrossRef Medline 26. Bartalena L, Baldeschi L, Dickinson AJ, et al. Consensus statement of the European Group on Graves’ Orbitopathy (EUGOGO) on man- agement of Graves’ orbitopathy. Thyroid 2008;18:333–46 CrossRef Medline y 7. Mayer EJ, Herdman G, Burnett C, et al. Serial STIR magnetic reso- nance imaging correlates with clinical score of activity in thyroid disease. Eye 2001;15:313–18 CrossRef Medline 27. Patients Earlier studies7,22-25 reported that the SI of extraocular muscles on short  inversion recovery sequences correlated well with the disease activity assessed with clinical methods. The cor- relation was maintained as disease activity changed. Although the reversibility of the relaxation time in muscles with prolonged T2 values was explained as a therapy-induced decrease in the water content,8 treatment-induced changes in orbital fat have not been documented. Our study has several limitations. The number of patients with TAO who underwent MPPT was small, and the lack of age- matched controls limits the comparison between patients with TAO and healthy individuals. In addition, we used the T2- weighted FSE-IDEAL sequence provided by GE Healthcare. It is a novel, 3-point Dixon method that applies iterative algorithms and the region-growing technique to estimate local field inhomoge- neities.37-39 Application of the 3-point Dixon method developed by different vendors may result in significantly different measure- ment results with respect to the water fraction of orbital fat. In addition, the demarcation between orbital and upper or lower Like Higashiyama et al,17 we detected no significant difference in the pre- and posttreatment orbital fat volume. Others26-28 2126 Kaichi Nov 2016 www.ajnr.org Like Higashiyama et al,17 we detected no significant difference in the pre- and posttreatment orbital fat volume. Others26-28 eyelid fat is ambiguous, and this feature may introduce some in- terobserver variability in the orbital fat volume. Last, the correct cutoff value for the water fraction and the volume of the orbital fat between patients with TAO and their controls remains to be determined. ophthalmopathy. Clin Endocrinol (Oxf) 2001;54:205–09 CrossRef Medline 15. Mayer EJ, Fox DL, Herdman G, et al. Signal intensity, clinical activ- ity and cross-sectional areas on MRI scans in thyroid eye disease. Eur J Radiol 2005;56:20–24 CrossRef Medline 16. Comerci M, Elefante A, Strianese D, et al. Semiautomatic regional segmentation to measure orbital fat volumes in thyroid-associated ophthalmopathy: a validation study. Neuroradiol J 2013;26:373–79 CrossRef Medline CONCLUSIONS The estimation of the water fraction and the volume of orbital fat on FSE-IDEAL images is feasible, and the reproducibility of these measurements is adequate. FSE-IDEAL imaging is useful for monitoring the effects of therapy and for predicting the response of patients with TAO to methylprednisolone pulse therapy. 17. Higashiyama T, Nishida Y, Ohji M. Changes of orbital tissue vol- umes and proptosis in patients with thyroid extraocular muscle swelling after methylprednisolone pulse therapy. Jpn J Ophthalmol 2015;59:430–35 CrossRef Medline 18. Hu HH, Kim HW, Nayak KS, et al. Comparison of fat-water MRI and single-voxel MRS in the assessment of hepatic and pancreatic fat fractions in humans. Obesity (Silver Spring) 2010;18:841–47 CrossRef Medline Disclosures: Kazuo Awai—UNRELATED: Consultancy: GE Healthcare; Grants/ Grants Pending: Toshiba Medical Systems,* Hitachi Medical Corporation,* Bayer Yakuhin,* Eizai,* Daiichi Sankyo,* Comments: research grants. *Money paid to the institution. 19. Takasu M, Tani C, Sakoda Y, et al. Iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) imaging of multiple myeloma: initial clinical efficiency results. Eur Radiol 2012;22:1114–21 CrossRef Medline 20. Bartalena L, Baldeschi L, Dickinson A, et al; European Group on Graves’ Orbitopathy (EUGOGO). Consensus statement of the European Group on Graves’ Orbitopathy (EUGOGO) on man- agement of GO. Eur J Endocrinol 2008;158:273–85 CrossRef Medline 2128 Kaichi Nov 2016 www.ajnr.org REFERENCES Kauppinen-Ma¨kelin R, Karma A, Leinonen E, et al. High dose intra- venous methylprednisolone pulse therapy versus oral prednisone for thyroid-associated ophthalmopathy. Acta Ophthalmol Scand 2002;80:316–21 CrossRef Medline 8. Kahaly GJ. Imaging in thyroid-associated orbitopathy. Eur J Endo- crinol 2001;145:107–18 CrossRef Medline 9. Feldon SE, Lee CP, Muramatsu SK, et al. Quantitative computed tomography of Graves’ ophthalmopathy: extraocular muscle and orbital fat in development of optic neuropathy. Arch Ophthalmol 1985;103:213–15 CrossRef Medline 28. van Geest RJ, Sasim IV, Koppeschaar HP, et al. Methylprednisolone pulse therapy for patients with moderately severe Graves’ orbitopathy: a prospective, randomized, placebo-controlled study. Eur J Endocrinol 2008;158:229–37 CrossRef Medline 10. Peyster RG, Ginsberg F, Silber JH, et al. Exophthalmos caused by excessivefat:CTvolumetricanalysisanddifferentialdiagnosis.AJR Am J Roentgenol 1986;146:459–64 CrossRef Medline 29. Bahn RS, Heufelder AE. Pathogenesis of Graves’ ophthalmopathy. N Engl J Med 1993;329:1468–75 CrossRef Medline 11. FangZJ,ZhangJY,HeWM.CTfeaturesofexophthalmosinChinese subjects with thyroid-associated ophthalmopathy. Int J Ophthalmol 2013;6:146–49 CrossRef Medline 30. Watanabe N, Noh JY, Kozaki A, et al. Radioiodine-associated exac- erbation of Graves’ orbitopathy in the Japanese population: ran- domized prospective study. J Clin Endocrinol Metab 2015;100: 2700–08 CrossRef Medline 12. Nishida Y, Tian S, Isberg B, et al. Significance of orbital fatty tissue for exophthalmos in thyroid-associated ophthalmopathy. Graefes Arch Clin Exp Ophthalmol 2002;240:515–20 CrossRef Medline 31. Hiromatsu Y, Kojima K, Ishisaka N, et al. Role of magnetic resonance imaging in thyroid-associated ophthalmopathy: its predictive value for therapeutic outcome of immunosuppressive therapy. Thyroid 1992;2:299–305 CrossRef Medline 13. Kvetny J, Puhakka KB, Rohl L. Magnetic resonance imaging deter- mination of extraocular eye muscle volume in patients with thy- roid-associated ophthalmopathy and proptosis. Acta Ophthalmol Scand 2006;84:419–23 CrossRef Medline 32. Just M, Kahaly GJ, Higer HP, et al. Graves ophthalmopathy: role of MR imaging in radiation therapy. Radiology 1991;179:187–90 CrossRef Medline 32. Just M, Kahaly GJ, Higer HP, et al. Graves ophthalmopathy: role of MR imaging in radiation therapy. Radiology 1991;179:187–90 CrossRef Medline 14. Prummel MF, Gerding MN, Zonneveld FW, et al. The usefulness of quantitative orbital magnetic resonance imaging in Graves’ 33. Ohnishi T, Noguchi S, Murakami N, et al. Extraocular muscles in 33. Ohnishi T, Noguchi S, Murakami N, et al. Extraocular muscles in AJNR Am J Neuroradiol 37:2123–28 Nov 2016 www.ajnr.org 2127 AJNR Am J Neuroradiol 37:2123–28 Nov 2016 www.ajnr.org 2127 is a rather late phenomenon. Eur J Endocrinol 2015;173:149–53 CrossRef Medline Gravesophthalmopathy:usefulnessofT2relaxationtimemeasure- ments. Radiology 1994;190:857–62 CrossRef Medline is a rather late phenomenon. REFERENCES Eur J Endocrinol 2015;173:149–53 CrossRef Medline ments. Radiology 1994;190:857–62 CrossRef Medline 37. Costa DN, Pedrosa I, McKenzie C, et al. Body MRI using IDEAL. AJR Am J Roentgenol 2008;190:1076–84 CrossRef Medline 34. Kendall-Taylor P, Crombie AL, Stephenson AM, et al. Intravenous methylprednisolone in the treatment of Graves’ ophthalmopathy. BMJ 1988;297:1574–78 CrossRef Medline g 38. Gerdes CM, Kijowski R, Reeder SB. IDEAL imaging of the musculo- skeletal system: robust water fat separation for uniform fat sup- pression, marrow evaluation, and cartilage imaging. AJR Am J Roentgenol 2007;189:284–91 Medline 35. Bartalena L, Marcocci C, Bogazzi F, et al. Use of corticosteroids to prevent progression of Graves’ ophthalmopathy after radioiodine therapy for hyperthyroidism. N Engl J Med 1989;321:1349–52 CrossRef Medline g 39. Humbert IA, Reeder SB, Porcaro EJ, et al. Simultaneous estimation of tongue volume and fat fraction using IDEAL-FSE. J Magn Reson Imaging 2008;28:504–08 CrossRef Medline 36. Potgieser PW, Wiersinga WM, Regensburg NI, et al. Some studies on the natural history of Graves’ orbitopathy: increase in orbital fat 128 Kaichi Nov 2016 www.ajnr.org 2128
https://openalex.org/W2088735728
https://europepmc.org/articles/pmc2409882?pdf=render
English
null
The utility of a multimedia education program for prostate cancer patients: a formative evaluation
British journal of cancer
2,004
cc-by
6,612
Received 12 February 2003; revised 17 February 2004; accepted 14 June 2004; published online 3 August 2004 *Correspondence: Dr P van Schaik; E-mail: P.Van-Schaik@tees.ac.uk The utility of a multimedia education program for prostate cancer patients: a formative evaluation D Flynn1, P van Schaik*,1, A van Wersch1, T Ahmed2 and D Chadwick2 1School of Social Sciences and Law, University of Teesside, Middlesbrough TS1 3BA, UK; 2Department of Urology, James Cook University Hospital, Middlesbrough TS4 3BW, UK D Flynn , P van Schaik , A van Wersch , T Ahmed and D Chadwick 1School of Social Sciences and Law, University of Teesside, Middlesbrough TS1 3BA, UK; 2Department of Urology, James Cook University Hospital, Middlesbrough TS4 3BW, UK A multimedia program (MMP) was developed to educate patients with prostate cancer about their disease. A within-subjects design was used to investigate the changes in levels of cancer-related knowledge, psychosocial functioning, treatment decision-making role and information needs immediately after browsing the MMP. The participants were 67 men recently diagnosed with prostate cancer. Psychosocial functioning was assessed with 20 items describing common emotional states and coping strategies employed by cancer patients. Treatment decision-making role was assessed with the Control Preference Scale. A principle component analysis of the 20 psychosocial items yielded three components: distress, positive approach and nonacceptance. After browsing the MMP significant increases in knowledge and reductions in distress were reported. Marital status was significantly associated with knowledge gain. Married men and those attending the study session with their spouse displayed a significant shift towards a more active role in treatment decisions. The majority of information needs were fulfilled by the MMP; however, information related to the likelihood of a cure, treatment side effects, coping strategies and aetiology were not completely satisfied by the MMP. Implications of the findings and suggestions for future work on the design and evaluation of the MMP are discussed. gg g British Journal of Cancer (2004) 91, 855–860. doi:10.1038/sj.bjc.6602071 www.bjcancer.com P bli h d li 3 A t 2004 gg g British Journal of Cancer (2004) 91, 855–860. doi:10.1038/sj.bjc.6602071 www.bjcancer.com Published online 3 August 2004 & 2004 C R h UK eywords: prostate cancer; knowledge; psychosocial functioning; decision-making; multimedia; information needs In the UK, one in 14 men are at a lifetime risk of developing prostate cancer, which is the second leading cause of death in men after lung cancer, accounting for 9280 deaths in 2000 (Cancer Research UK, 2002a, b). Treatments for prostate cancer include surgery, hormone therapy, radiotherapy, chemotherapy and active monitoring (or watchful waiting). British Journal of Cancer (2004) 91, 855 – 860 & 2004 Cancer Research UK All rights reserved 0007 – 0920/04 $30.00 www.bjcancer.com British Journal of Cancer (2004) 91, 855 – 860 & 2004 Cancer Research UK All rights reserved 0007 – 0920/04 $30.00 www.bjcancer.com British Journal of Cancer (2004) 91, 855 – 860 & 2004 Cancer Research UK All rights reserved 0007 – 0920/04 $30.00 www.bjcancer.com Patient education Patient education (information provision) has been proven to be an effective strategy for alleviating psychosocial problems in both men and women with cancer (Fallowfield et al, 1997; Davison et al, 2003). Information provides a sense of control, reduces distress, facilitates adaptive coping, and increases participation in shared decision-making (SDM) with physicians (Gregoire et al, 1997; van Wersch et al, 1997a, b; Davison et al, 2003). Effective information provision should enrich doctor–patient interactions by transform- ing consultations into negotiations between expert patients and expert physicians; however, in reality it is clinicians, not patients, who are in possession of the knowledge required to make an informed decision (Crawford et al, 1997). Despite the general The utility of a multimedia education program for prostate cancer patients: a formative evaluation Treatment side effects are numerous and can occur for short periods, whereas others such as incontinence and impotence have long-term effects that impact negatively upon quality of life. However, the probability of experiencing side effects associated with particular treatments is unclear from the literature, with large differences in frequency, duration and severity between studies. Furthermore, the relative survival benefit of different treatments has yet to be elucidated; consequently, there is still no unequivocal evidence to support one treatment over another (Holmberg et al, 2002). problems such as social role changes, financial worries, anger, depression and anxiety regarding treatment and potential death (Gregoire et al, 1997; Gray et al, 1999). Research has also reported that men with cancer rarely seek help for psychosocial problems (Jorm, 1994); have little awareness of coping strategies (Whitrod, 1996); are often denied information on positive coping by clinicians (Fitch et al, 1999); and are prone to relying upon avoidance-coping strategies associated with poor psychological outcomes and decreased survival rates (Shrock et al, 1999). Furthermore, men appear to receive little emotional support other than from their spouse (Helgason et al, 2001) who also experience psychosocial problems in response to their partner’s diagnosis (Gray et al, 1999). Participants The participants were 67 men recently diagnosed (1 week or less) with prostate cancer. The men were selected based on consultant urologists’ assessment of their suitability for inclusion in the study. The age range was 48–89 with a mean age of 65.7 years (SD ¼ 7.95). The percentage of participants with secondary (school, aged p16), further (college, aged X16) and higher education (university, ages X18) was 50, 36 and 14% respectively. The majority were married (90%), retired (76%), resided in their own homes with at least one other person (84%) and attended the study session with their spouse (70%). Multimedia patient education A multimedia program (MMP) is a computer-based application that combines text, sound, graphics, video and interactivity, which serve to reinforce and complement one another to facilitate learning. Multimedia programs presently offer the most compre- hensive method of information provision that address several of the shortcomings associated with other media such as printed information. Multimedia programs can be easily and quickly updated to incorporate new treatment approaches and evidence from clinical trails that may refute previous information. Inter- activity can provide autonomy as it enables patients to dictate the pace, type and the order information is viewed in the MMP, which enables more knowledgeable patients to access salient information more quickly without attending to previously accessed informa- tion. Multimedia programs provide all the benefits of patient education without increasing staff costs or time, and are capable of being accessed at home via the Internet or CD-ROM. Disadvan- tages of MMPs are initial development and start-up costs and technology acceptance by clinicians and patients. Design A within-subjects design was used to evaluate the utility of the MMP. The independent variables were study condition (pretrial – immediately before using the MMP and post-trial – immediately after using the MMP), patient age, education, living circumstances and employment status. The outcome measures were the level of cancer-related knowledge, psychosocial functioning, treatment decision-making role and information needs. The main disadvantages of printed information (and other media such as audio and videotapes) are that reading level is often inappropriate; these media possess limited information for patients who wish to pursue a deeper understanding; they are unable to adapt quickly to new information; salient topics are often missing; uncertainties are ignored; and they fail to provide a balanced account of the effectiveness of available treatments (Smith and Timoney, 1997; Coulter et al, 1999). Psychosocial functioning Verbal consultations may fulfil these information needs but this information is subject to poor recall and understanding by patients (Michie et al, 1997). Consequently, patients are increas- ingly given printed information to reinforce, or in many cases to replace, verbal information provided by clinicians (Frank-Strom- borg and Cohen, 1991). Currently, a combination of information provision (verbal and printed) with support from healthcare professionals is considered ‘good clinical practice’. This enables the healthcare professional to respond according to an individual patient’s information needs. Multimedia program An MMP was developed using previous research on the informa- tion needs of prostate cancer patients (e.g. Davison et al, 1995) and a working committee consisting of two consultant urologists, a health psychologist, a psychologist specialising in human– computer interaction and a multimedia developer. The MMP combined text with sound, narration, images, animation and streaming video. The MMP was comprised of six cancer-related modules: (a) prostate anatomy, (b) disease stages, aetiology and symptoms, (c) diagnostic techniques, (d) treatment options (surgery, hormone therapy and radiotherapy) and side effects, which included a research update, (e) coping strategies and (f) further information (self-help groups, prostate cancer organisa- tions, further reading and a cancer glossary). The MMP was operated on a stand-alone PC and participants navigated through the MMP using a mouse. The interface used a selection of on- screen buttons (forward, back, exit) that controlled interaction and navigation through the MMP. Participants were instructed how to use the MMP by a research assistant who was present throughout the study session. gy p y p Clinical trials of MMPs as health education tools have reported positive results for increasing patients’ knowledge, information- seeking and participation in SDM (Krishna et al, 1997). Multi- media programs for patients with benign prostatic hyperplasia have reported positive results for reducing self-assessed prostate symptoms (van Schaik et al, 1999) and facilitating SDM (Barry et al, 1995; Shepperd et al, 1995; Wagner et al, 1995; Murray et al, 2001). Pilot studies of MMPs for prostate cancer have reported patient satisfaction with navigability, layout and content (Jenkin- son et al, 1998; Brink et al, 2000). Patient outcomes such as participation in SDM were neglected, although Brink et al (2000) reported increased knowledge of cancer staging and brachytherapy (the only treatment that was included in the MMP) including increased patient self-efficacy for discussing brachytherapy with physicians. These studies demonstrated that MMPs can be effective media for increasing knowledge of the entire spectrum of treatment options for prostate cancer. Psychosocial functioning Psychosocial problems experienced by men with cancer have received sparse attention compared to women in the research literature. This difference in attention is hard to justify as research has shown that men with prostate cancer experience psychosocial Multimedia education program for prostate cancer D Flynn et al Multimedia education program for prostate cancer D Flynn et al 856 agreement that men should be involved in treatment decisions, the type and amount of information needed for SDM has failed to reach a consensus (Feldman-Stewart et al, 1998). Davison et al (1995) reported the following hierarchical structure of information needs of men recently diagnosed with prostate cancer: likelihood of cure, stage of disease, available treatments, side effects on usual social activity, self-care, treatment side effects, hereditary risks, effects upon family and friends, and treatment effects upon sexual activity. Verbal consultations may fulfil these information needs but this information is subject to poor recall and understanding by patients (Michie et al, 1997). Consequently, patients are increas- ingly given printed information to reinforce, or in many cases to replace, verbal information provided by clinicians (Frank-Strom- borg and Cohen, 1991). Currently, a combination of information provision (verbal and printed) with support from healthcare professionals is considered ‘good clinical practice’. This enables the healthcare professional to respond according to an individual patient’s information needs. patients recently diagnosed with prostate cancer. A formative evaluation is an evaluation that takes place before actual implementation of a final product, and which influences the development of the product (Preece et al, 1994). The results of the formative evaluation will be used to conduct a future ‘summative evaluation’ (undertaken after implementation of the final product, with the aim of testing the functioning of a product) of the final version of the MMP. Involving patients in the formative evaluation is in line with guidelines for conducting research affecting patients in the UK (DoH, 2001). agreement that men should be involved in treatment decisions, the type and amount of information needed for SDM has failed to reach a consensus (Feldman-Stewart et al, 1998). Davison et al (1995) reported the following hierarchical structure of information needs of men recently diagnosed with prostate cancer: likelihood of cure, stage of disease, available treatments, side effects on usual social activity, self-care, treatment side effects, hereditary risks, effects upon family and friends, and treatment effects upon sexual activity. MATERIALS AND METHODS Clinical British Journal of Cancer (2004) 91(5), 855 – 860 & 2004 Cancer Research UK Study questionnaire Rotation method: direct Oblimin with Kaiser normalisation. The stem question preceding the 20 psychosocial functioning items was ‘please tick one box for each of the following statements to indicate how you feel now about your prostate cancer’ (very much, a little or not at all). 857 Table 2 Percentage and cumulative percentage of variance explained per component, and component loading matrix from the principle components factor analysis of the psychosocial functioning items Clinical which has been used previously to assess treatment decision- making preferences of prostate cancer patients (e.g. Davison et al, 1995). Participants indicated their preferred role in treatment decision-making (active, passive or collaborative) by selecting the category indicative of their status. Overall 12.62 (3.90) 14.38 (3.00) aMean. bs.d. Overall Information needs were assessed with free-response items that asked participants to state their most important information need at pre- and post-trial. Participants were also asked to state the most important knowledge they had acquired at post-trial. aMean. bs.d. Study questionnaire A questionnaire in hard-copy format was used to assess psychosocial functioning, cancer-related knowledge, treatment decision-making roles and information needs. The first part of the questionnaire described the aims and objectives of the research and requested demographical information from the participants. A questionnaire in hard-copy format was used to assess psychosocial functioning, cancer-related knowledge, treatment decision-making roles and information needs. The first part of the questionnaire described the aims and objectives of the research and requested demographical information from the participants. Study questionnaire Cancer-related knowledge was assessed using 20 statements that were representative of the information presented in the six cancer- related modules of the MMP: cancer in general and prostate Therefore, the objective of the current study was to conduct a ‘formative evaluation’ by investigating the effect of the MMP on knowledge acquisition, psychosocial functioning, preference for participating in treatment decisions and information needs of Cancer-related knowledge was assessed using 20 statements that were representative of the information presented in the six cancer- related modules of the MMP: cancer in general and prostate British Journal of Cancer (2004) 91(5), 855 – 860 & 2004 Cancer Research UK Multimedia education program for prostate cancer D Flynn et al Table 1 The 20 statements used to assess cancer-related knowledge Knowledge Pretrial Post-trial Cancer in general and prostate anatomy 2.49a (1.01)b 2.82 (0.75) Male hormones are produced by the brain Cancer is a type of infection of tissue The prostate is part of the penis The prostate surrounds the first part of the tube which carries urine from the bladder to the penis Disease advancement 2.75 (1.29) 3.13 (1.08) Cancer is a lump of cells that may invade and destroy surrounding tissues Prostate cancer can spread to other parts of the body Prostate cancer never spreads outside the prostate Growth of prostate cancer is driven by the male hormones Aims and side effects of surgery 3.07 (0.98) 3.22 (0.94) The aim of prostate surgery is to remove part or all of the tumour in the prostate The aim of prostate surgery is to remove the testicles Possible side effects of prostate surgery include problems in control of the bladder After prostate surgery most prostate patients are incontinent Aims and side effects of radiotherapy 2.39 (1.05) 2.67 (0.68) A possible side effect of radiotherapy is breast enlargement Possible side ffects of radiotherapy include tiredness and nausea The aim of radiotherapy is to destroy cancer cells while doing as little harm as possible to normal cells The aim of radiotherapy is to remove the prostate Aims and side effects of hormone therapy 1.92 (1.27) 2.55 (1.00) The aim of hormone therapy is to increase the amount of male hormones P ibl id ff t f h th i l d Table 2 Percentage and cumulative percentage of variance explained per component, and component loading matrix from the principle components factor analysis of the psychosocial functioning items Component Item Distress Positive approach Nonacceptance Shocked 0.77 I cannot believe this has happened to me 0.53 I expected it Angry Anxious 0.92 Frightened 0.79 Uncertain Blame myself 0.84 Miserable Having a good cry Running away 0.94 Pray to God Talk to other patient Talking to someone I trust 0.52 I feel like going to another doctor to make sure it is true 0.74 Finding out more on prostate cancer Do not think about it Carry on with your life 0.75 Fight this disease 0.74 Enjoy myself as much as I can 0.86 % age of variance explained 24.7 12.6 9.7 Cumulative % 24.7 37.3 47.0 Extraction method: principle components analysis. & 2004 Cancer Research UK British Journal of Cancer (2004) 91(5), 855 – 860 Procedure anatomy; disease advancement; and aims and side effects of surgery, radiotherapy and hormone therapy (see Table 1). Participants responded to each statement as ‘true’, ‘false’ or ‘don’t know’. Ethical approval for the study was granted by the Trust on the basis that it was part of existing practice to provide practical advice, guidance and support to men with newly diagnosed with prostate cancer. After the ‘bad news’ consultation the urologist or prostate cancer nurse informed the participant about the study and provided them with a study information sheet (that detailed the study aims and rationale) and a consent form. Participants were given the choice of participating immediately, or within 1 week after the initial bad news consultation. They were also given the choice of attending the study session with a significant other or alone. A private room situated within the urology department was used to conduct the study. Prior to browsing the MMP, the participants were asked to complete the study questionnaire, Previous research on emotional states and coping strategies employed by cancer patients was used to design a 20-item checklist to assess psychosocial functioning. Six items used adjectives as descriptors for internal emotional states (e.g. angry); the remain- ing 14 items used brief statements related to coping strategies for dealing with cancer (see Table 2). The participants responded to each item on a three-point Likert scale (not at all, a little and very much). The Control Preference Scale developed by Degner and Sloan (1992) was used to assess the treatment decision-making role, British Journal of Cancer (2004) 91(5), 855 – 860 Multimedia education program for prostate cancer D Fl l D Flynn et al D Flynn et al 858 Table 3 Information needs at pre- and post-trial and most important knowledge acquired which was followed by instructions on how to use the MMP. No time limit was imposed on patients for browsing the MMP. After browsing the MMP, participants were requested to complete the study questionnaire for a second time. They were then fully debriefed and thanked for their time, cooperation and patience. Knowledge acquisition Overall numbers of correct responses to the 20 knowledge items increased between the pre- and post-trial conditions (see Table 1). A related t-test revealed that overall levels of correct responses significantly increased between the pre- and post-trial conditions (t [59] ¼ 4.49, Po0.001). A multiple regression analysis showed that being married was a significant predictor of overall knowledge gain between the pre- and post-trial conditions (b ¼ 0.31, R2 ¼ 0.10, Po0.05). Clinical Correct responses for each of the five knowledge domains increased between the pre- and post-trial conditions (see Table 1). A two-factor repeated measures ANOVA revealed a significant main effect of knowledge domain (F [3.5, 206.6] ¼ 15.67, Po0.001, MSknowledge domain ¼ 12.30, Greenhouse–Geisser correction ap- plied) and a significant interaction effect between knowledge domain and study condition (F [3.19, 187.89] ¼ 12.22, Po0.001, MSinteraction ¼ 12.91). A series of simple effect tests revealed that the following knowledge domains increased significantly between the pre- and post-trial conditions: cancer in general and prostate anatomy (t [59] ¼ 2.34, Po0.05), disease advancement (t [59] ¼ 2.92, Po0.01), aims and side effects of radiotherapy (t [59] ¼ 2.25, Po0.05) and hormone therapy (t [59] ¼ 4.51, Po0.001). Knowledge gain for aims and side effects of surgery failed to reach significance. Note: Percentages may not equal 100 due to rounding. exclusive categories (see Table 3). A frequency analysis revealed six categories of primary information needs at pretrial with the following hierarchical structure: likelihood of a cure (28%), treatment side effects (15%), coping strategies (13%), diagnostic tests (12%), treatment duration (7%) and aetiology (4%). In total, 19% stated that they had no information needs at pretrial. exclusive categories (see Table 3). A frequency analysis revealed six categories of primary information needs at pretrial with the following hierarchical structure: likelihood of a cure (28%), treatment side effects (15%), coping strategies (13%), diagnostic tests (12%), treatment duration (7%) and aetiology (4%). In total, 19% stated that they had no information needs at pretrial. At post-trial, five categories of information needs displayed at least a 40% decrease, with only aetiology displaying a negligible increase. Approximately 66% of the participants indicated that they required no further information needs at post-trial. Knowledge acquisition Seven categories were reported as the most important knowledge acquired with the following hierarchical structure: hereditary risks (30%), aetiology (24%), likelihood of a cure (4%), disease advancement (4%), coping strategies (2%), diagnostic tests (2%) and treatment side effects (2%). Approxi- mately 33% could not decide upon the most important knowledge they had acquired. DISCUSSION Consistent with previous research evaluating patient education tools, patients in the current study reported significantly less distress (Gregoire et al, 1997; Davison et al, 2003), more cancer- related knowledge (Glajchen and Moul, 1996), and a desire for a more active role in treatment decisions (if they attended the session with their spouse/partner or were married) immediately after browsing the MMP. Procedure Most important information need Most important information still required Most important knowledge acquired Pretrial Post-trial Post-trial F % F F % F F % F Likelihood of a cure 19 28 10 15 3 4 Treatment side effects 10 15 4 6 1 2 Coping strategies 9 13 4 6 1 2 Diagnostic tests 8 12 0 0 1 2 Treatment duration 5 7 1 2 0 0 Aetiology 3 4 4 6 16 24 Hereditary risks 0 0 0 0 20 30 Disease advancement 0 0 0 0 3 4 Cannot decide 13 19 44 66 22 33 Totals 67 100 67 100 67 100 Note: Percentages may not equal 100 due to rounding. Treatment decision-making In the pre- and post-trial study conditions, 68 and 71% respectively of participants preferred an active or collaborative role in treatment decisions. Wilcoxon tests revealed no significant differences in treatment decision-making roles between the pre- and post-trial study conditions; however, a significant shift in preferences for a more active role in treatment decisions was reported for (a) participants who attended the study session with their spouse or partner (z [42] ¼ 2.49, Po0.05) and (b) participants who were married (z [47] ¼ 1.98, Po0.05). g The current study also demonstrated that men expressed similar patterns of psychosocial problems as women with cancer as they reported anxiety, fear and shock (distress) and utilised both positive (positive approach) and negative (nonacceptance) coping strategies (Fallowfield et al, 1997; van Wersch et al, 1997a, b). The association between distress and nonacceptance was also consis- tent with previous research that found men with cancer are prone to relying upon avoidance coping strategies in response to the stress of a life-threatening disease such as cancer (Vingerhoets and Van Heck, 1990). The reduction in distress is an important finding given that less distressed patients are better able to make sense of their experience with cancer and seek desired information (Leydon et al, 2000). Psychosocial functioning Responses to the 20 psychosocial functioning items at pretrial were subjected to a principle components analysis. A three-component solution was extracted that explained 47% of the variance (see Table 2). Each component possessed adequate factor loadings (0.52–0.94) and internal reliability with Cronbach’s alpha values of X0.68. The three components were subsequently named distress (feelings of shock and fear), positive approach (optimism and a fighting spirit) and nonacceptance (denial) that explained 25, 13 and 10% of the variance respectively. Distress was significantly positively associated with both positive approach (r [61] ¼ 0.32, Po0.05) and nonacceptance (r [60] ¼ 0.31, Po0.05) in both study conditions. A related t-test revealed that distress decreased significantly between the pre- and post-trial conditions (t [58] ¼ 2.35, Po0.05). & 2004 Cancer Research UK Future research The use of the MMP for prostate cancer patients at this stage of evaluation cannot be recommended until prospective randomised control trials to compare the utility of the MMP with good clinical practice have been completed. In a summative evaluation, important factors such as ‘usability’ may have influenced the outcome measures in the current study. Usability refers to the ease of use and acceptability of a product for particular types of user to perform specific tasks in a given context, which is influenced by cost, convenience, availability, prerequisite training and organisa- tional issues (Bevan and McLeod, 1994). Therefore, a combination of performance measures and assessments of user satisfaction is required to determine the usability of the MMP in both clinical and residential environments as a function of style and properties of the interface (e.g. methods used to communicate between the user and computer), dialogue structure, functionality (e.g. browsing content), efficiency (e.g. navigation structure), reliability (e.g. fault tolerance), user characteristics (e.g. age) including the combina- tion of attributes that provide the greatest level of satisfaction for the majority of users. Particular attention should be given to obtaining information from patients who dislike or feel uncom- fortable using computers. Ergonomic factors such as postural demands may also be related to perceived ease of use, especially in elderly men, and deserve consideration in future applications of this MMP. Browsing the MMP developed a need to acquire information other than those anticipated at pretrial and to reprioritise information needs. However, the hierarchical structure of infor- mation needs was inconsistent with previous research (Davison et al, 1995, 2002). The discrepancies with previous research may be due to using free-response questions, only requesting primary information needs, and 19% of men in the pretrial condition indicating they did not have any information needs, which increased to 66% at post-trial. Information needs The information needs reported by the study participants were examined for common themes and coded into mutually British Journal of Cancer (2004) 91(5), 855 – 860 Multimedia education program for prostate cancer D Flynn et al Multimedia education program for prostate cancer 859 disease stage could be followed by a description of how to develop positive coping strategies to deal with treatment side effects. The results of the current study were inconsistent with previous research using the Control Preference Scale (CPS) that reported the majority of men within 0–13 weeks of receiving their diagnosis preferred a passive decision-making role (Davison et al, 1995). However, more recent studies utilising the CPS are congruent with the current study reporting that 68% (Davison and Degner, 1997), 75% (Wong et al, 2000) and as many as 93% (Davison et al, 2002) of men recently diagnosed prefer either an active or collaborative role in treatment decisions. This trend in the current study could be attributed to the relatively low mean age of the study participants (Davison et al, 2002), and/or spousal support that served as a catalyst to learn and take part in shared decision- making (Ptacek et al, 1999). p p g g An assessment of reading level required to comprehend the information presented in the MMP needs to be conducted to ensure understanding by all patients irrespective of educational background. A self-test at the end of each topic that provides feedback on performance (and delivering reassur- ance and support in the case of poor performance) could also enhance learning via the use of positive reinforcement (operant conditioning). The inclusion of a search function would also facilitate learning as patients could pinpoint salient information needs more quickly and avoid the frustration of being unable to locate desired information. Following a summative evaluation, algorithms built into the MMP could suggest an appropriate treatment modality based on a patient’s unique status (clinical profile and preferences regarding both positive and negative treatment outcomes). The MMP adequately fulfilled information needs for treatment side effects, coping strategies, diagnostic tests and treatment duration. The failure of the MMP to completely satisfy information needs related to the likelihood of a cure and aetiology is probably attributable to the state of medical knowledge and the lack of prospective clinical trials of sufficient quality comparing one treatment with another rather than shortcomings of the MMP. Limitations Despite these encouraging results, there are several methodological issues that may have had a confounding influence upon the outcome measures. The sampling method (selection by urologist) employed to select participants may have produced an unrepre- sentative sample as reasons for noninclusion were not recorded. Other methodological issues that potentially reduce generalisa- bility of the results include the failure to record the participants’ disease stage and functional status. Furthermore, given the generally late onset of prostate cancer, the mean age (66 years) of the study participants was relatively young. The psychosocial functioning scale utilised in the current study needs to be further validated in future research, as only internal reliability and internal validity was assessed. A research assistant also supported the participants throughout the study session, which may have impacted upon the participants’ level of distress. Furthermore, the question of whether the level of support provided is necessary in future applications of the MMP needs to be investigated. High usability of the MMP is essential if the MMP is to be used in patients’ homes or accessed via the WWW, to ensure that the MMP can successfully compete with the perennial increase in the number of cancer-related Web sites on the Internet. In particular, it must be established if the MMP confers benefits over time in terms of outcomes assessed in the current study, including satisfaction with care, quality of life and ultimately survival. Furthermore, future work should be conducted to determine if the MMP can fulfil post-treatment information needs and those of partner-caregivers who are reported to have information needs equivalent to those of patients (Davison et al, 2002). Suggested improvements to the MMP The inclusion of a decision-aid to communicate quantitative outcome information to patients could address the shortcomings of the MMP in terms of fulfilling information needs. However, the decision-aid used to present outcome data must be processed with a high degree of accuracy by patients, otherwise it can influence the perception of probability (Chatterton, 1999). According to Feldman-Stewart et al (2000) a 10  10 matrix of shaded ovals is the most efficacious format for presenting information on probabilities to patients, although this must be explained with support from clinicians. & 2004 Cancer Research UK British Journal of Cancer (2004) 91(5), 855 – 860 REFERENCES Br Med J 318: 318–322 f d h h Crawford ED, Bennett CL, Stone NN, Knight SJ, DeAntoni E, Sharp L, Garnick MB, Poterfield HA (1997) Comparison of perspectives on prostate cancer: analyses of survey data. Urology 50: 366–372 Michie S, McDonald V, Marteau TM (1997) Genetic counselling: informa- tion given, recall and satisfaction. Patient Educ Couns 32: 101–106 y y gy Davison BJ, Degner LF (1997) Empowerment of men newly diagnosed with prostate cancer. Cancer Nurs 20: 187–197 Murray E, Davis H, Tai SS, Coulter A, Gray A, Haines A (2001) Randomised control trial of an interactive multimedia decision aid on benign prostatic hypertrophy in primary care. Br Med J 323: 1–6 Davison BJ, Degner LF, Morgan TR (1995) Information and decision- making preferences of men with prostate cancer. Oncol Nurs Forum 22: 1401–1408 Preece J, Sharp H, Benyan D, Holland S, Carey T (1994) Human–Computer Interaction. Reading, MA: Addison-Wesley Davison BJ, Gleave ME, Goldenberg SL, Degner LF, Hoffart D, Berkowitz J (2002) Assessing information and decision preferences of men with prostate cancer and their partners. Cancer Nurs 25: 42–49 Ptacek JT, Pierce GR, Ptack JJ, Nogel C (1999) Stress and coping processes in men with prostate cancer: the divergent views of husbands and wives. J Soc Clin Psychol 18: 299–324 y Shepperd S, Coulter A, Farmer A (1995) Using interactive videos in general practice to inform patients about treatment choices: a pilot study. Fam Pract 12: 443–447 Davison BJ, Goldenberg L, Gleave ME, Degner LF (2003) Provision of individualized information to men and their partners to facilitate treatment decision making in prostate cancer. Oncol Nurs Forum 30: 107–114 Shrock D, Palmer RF, Taylor B (1999) Effects of a psychosocial intervention on survival among patients with stage I breast and prostate cancer: a matched case–control study. Altern Ther Health Med 5: 49–55 Degner LF, Sloan JA (1992) Decision-making during serious illness: what role do patients really want to play? J Clin Epidemiol 45: 941–950 y Smith D, Timoney A (1997) Patient information systems. Br J Urol 80: 27–30 DoH (2001) The Expert Patient: A New Approach to Chronic Disease Management for the 21st Century. London: HMSO Fallowfield LJ, Hall A, McGuire GP, Baum S (1997) Psychological outcomes of different treatment policies in women with early breast cancer outside the clinical trial. REFERENCES 50-80-year-old prostate cancer patients. Scand J Urol Nephrol 35: 97–101 50-80-year-old prostate cancer patients. Scand J Urol Nephrol 35: 97–101 Barry MJ, Fowler JF, Mulley AG, Henderson JV, Wennberg JE (1995) Patient reactions to a program designed to facilitate patient participation in treatment decisions for benign prostatic hyperplasia. Med Care 33: 771–782 Clinical Holmberg L, Bill-Axelson A, Helgesen F, Salo JO, Folmerz P, Haggman M, Andersson SO, Spangberg A, Busch C, Nordling S, Palmgren J, Adami HO, Johansson JE, Norlen BJ (2002) A randomized trial comparing radical prostatectomy with watchful waiting in early prostate cancer. N Engl J Med 347: 781–789 Bevan N, McLeod M (1994) Usability measurement in context. Behav Inform Technol 13: 132–145 f Brink SG, Birney AJ, McFarren AE (2000) Charting your course: formative evaluation of a prostate cancer treatment decision-aid. Int Electron J Health Educ 3: 44–54 Jenkinson J, Wilson-Pauwels L, Jewett MAS, Woolridge N (1998) Development of a hypermedia program designed to assist patients with localised prostate cancer in making treatment decisions. J Biocommun 25: 2–11 Cancer Research UK (2002a) CancerStats: Incidence – UK, Retrieved December 22, 2002 from http://www.cancerresearchuk.org/aboutcancer/ statistics/29375/29902 Jorm AF (1994) Characteristics of Australians who reported consulting a psychologist for a health problem: an analysis of data from the 1989–90 National Health Survey. Aust Psychol 29: 212–215 Cancer Research UK (2002b) CancerStats: Mortality – UK, Retrieved December 22, 2002 from http://www.cancerresearchuk.org/aboutcancer/ statistics/29375/29902 y y Krishna S, Balas EA, Spencer DC, Griffin JZ, Boren SA (1997) Clinical trials of interactive computerised patient education: implications for family practice. J Fam Pract 45: 25–33 Chatterton HT (1999) Efficacy, risk and the determination of value: shared decision-making in the age of information. J Fam Pract 48: 505–507 Coulter A, Entwistle V, Gilbert D (1999) Sharing decisions with patients: is the information good enough? Br Med J 318: 318–322 Chatterton HT (1999) Efficacy, risk and the determination of value: shared decision-making in the age of information. J Fam Pract 48: 505–507 p Leydon GM, Boulton M, Moynihan C, Jones A, Mossman J, Boudioni M, McPherson K (2000) Cancer patients’ information needs and information seeking behaviour: in depth interview study. Br Med J 320: 909–913 g g Coulter A, Entwistle V, Gilbert D (1999) Sharing decisions with patients: is the information good enough? REFERENCES Br Med J 301: 575–580 Van Schaik P, Ahmed T, Suvakovic N, Hindmarsh J (1999) The design of an educational multimedia interactive prostate program for BPH. Cleveland Med J 1: 16–22 Feldman-Stewart D, Brundage MD, Hayter C, Groome P, Nickel JC, Downes H, Mackillop WJ (1998) What prostate cancer patients should know: variation in professionals’ opinions. Radiother Oncol 49: 111–123 Van Wersch A, Bonnema J, Prinsen B, Pruyn J, Wiggers T, van Geel AN (1997a) Continuity of information for breast cancer patients: the development, use and evaluation of a multidisciplinary care-protocol. Patient Educ Couns 30: 175–186 Feldman-Stewart D, Brundage MD, McConnnell BA, MacKillop WJ (2000) Practical issues in shared decision-making. Health Expect 3: 46–54 Van Wersch A, de Boer MF, van der Does E, de Jong P, Knegt P, Meeuwis CA, Stringer P, Pruyn JF (1997b) Continuity of information in cancer care: evaluation of a logbook. Patient Educ Couns 31: 223–236 Fitch MI, Johnson B, Gray R, Franssen E (1999) Survivors’ perspectives on the impact of prostate cancer: implications for oncology nurses. Can Oncol Nurs J 9: 23–28 g Vingerhoets AJ, van Heck GL (1990) Gender, coping and psychosomatic symptoms. Psychol Med 20: 125–135 Frank-Stromborg M, Cohen R (1991) Evaluating written patient education materials. Semin Oncol Nurs 7: 128–134 Wagner EH, Barrett P, Barry MJ, Barlow W, Fowler FJ (1995) The effect of a shared decision-making program on rates of surgery for benign prostate hyperplasia. Med Care 33: 765–770 Glajchen M, Moul JW (1996) Teleconferencing as a method of educating men about managing advanced prostate cancer and pain. J Psychosoc Oncol 14: 73–87 y Weed LL (1997) New connections between medical knowledge and patient care. Br Med J 315: 231–235 Gray RE, Fitch MI, Phillips C, Labrecque M, Klotz L (1999) Presurgery experiences of prostate cancer patients and their spouses. Cancer Pract 7: 130–135 Whitrod R (1996) Improved quality of life for men with advanced prostate cancer: the need for an increased contribution by psychologists. Aust Psychol 31: 127–132 Gregoire I, Kalogeropoulos D, Corcos J (1997) The effectiveness of a professionally led support group for men with prostate cancer. Urol Nurs 17: 58–66 Wong F, Stewart DE, Dancey J, Meana M, McAndrews MP, Bunston T, Cheung AM (2000) Men with prostate cancer: influence of psychological factors on informational needs and decision-making. ACKNOWLEDGEMENTS staff time to deliver the information. Multimedia programs are unlikely to replace the ‘human touch’ associated with traditional doctor–patient interactions, although, in the present climate of health care reform, which is geared toward the cost-effective delivery of quality services, MMPs will become increasingly commonplace tools for patient education if they are demonstrated to be more effective than good clinical practice. We thank all the clinical staff at the department of urology at James Cook University Hospital for their support in conducting this study. We would also like to express our deepest gratitude to all the participants who agreed to take part for their patience, time and co-operation. CONCLUSION The incidence of prostate cancer is expected to increase in developed countries due to ageing populations, increased use of PSA screening and declines in other major causes of mortality. This will result in concomitant cost increases to health care providers and it is unlikely that the slow and expensive process of training biomedical practitioners occupying the central role in health care will meet the increased demand for their ‘expert knowledge’ (Weed, 1997). However, empowered with sufficient knowledge patients can make informed decisions about their treatment in collaboration with clinicians without an investment of Coping knowledge may be enhanced by amalgamating informa- tion on positive coping strategies with other salient topics such as likelihood of cure. To enable men to vicariously learn positive coping strategies, streaming video of a real-patient (or trained actor) describing the probability of a cure associated with each British Journal of Cancer (2004) 91(5), 855 – 860 Multimedia education program for prostate cancer D Flynn et al D Flynn et al 860 REFERENCES J Psychosom Res 49: 13–19 Helgason AR, Dickman PW, Adolfsson J, Steineck G (2001) Emotional isolation: prevalence and the effect on well-being among British Journal of Cancer (2004) 91(5), 855 – 860 & 2004 Cancer Research UK & 2004 Cancer Research UK
https://openalex.org/W4381095299
https://jmedicalcasereports.biomedcentral.com/counter/pdf/10.1186/s13256-023-03982-2
English
null
Spontaneous calcific cerebral embolization revealing a calcified rheumatic mitral stenosis: a case report
Journal of medical case reports
2,023
cc-by
2,969
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background  Cerebral cardiac embolism accounts for an increasing proportion of ischemic strokes and transient ischemic attacks. Calcified cerebral emboli are rare and mostly iatrogenic secondary to heart or aorta catheteriza- tion. However, spontaneous cerebral calcified embolism in the case of calcified aortic valve is very rare and there are less than 10 case reports in the literature. And a more interesting fact is that such an event, in the context of calcified mitral valve disease, has never been reported, at least to our knowledge. We are reporting a case of spontaneous calcified cerebral embolism revealing a calcified rheumatic mitral valve stenosis. Case presentation  We report a case of a 59 year-old Moroccan patient, with a history of rheumatic fever at the age of 14 and no history of recent cardiac intervention or aortic/carotid manipulation, who was admitted to the emer- gency department after a transient ischemic attack. Physical examination at admission found normal blood pressure of 124/79 mmHg and heart rate of 90 bpm. A 12-lead electrocardiogram showed an atrial fibrillation, no other anoma- lies. Unenhanced cerebral computed tomography imaging was performed, revealing calcified material inside both middle cerebral arteries. Transthoracic echocardiography was performed, showing severe mitral leaflets calcification with a severe mitral stenosis, probably due to rheumatic heart disease. Cervical arteries Duplex was normal. A vitamin K antagonist (acenocoumarol) was prescribed, targeting an international normalized ratio of 2–3 and mitral valve replacement surgery was performed using mechanical prosthesis. Short- and long-term health, with a 1-year follow- up, were good and the patient did not experience any stroke. Conclusion  Spontaneous calcified cerebral emboli secondary to mitral valve leaflet calcifications is an extremely rare condition. Replacement of the valve is the only option to prevent recurrent emboli and outcomes are still to be determined. Keywords  Cerebral embolism, Calcified mitral stenosis, Transient ischemic attack Spontaneous calcific cerebral embolization revealing a calcified rheumatic mitral stenosis: a case report M. Haboub1*, S. Abouradi1, H. Mechal1, G. Minko1, A. Moukhliss1, S. Arous1, M. E. G. Benouna1, A. Drighil1, L. Azzouzi1 and R. Habbal1 Open Access Open Access Haboub et al. Journal of Medical Case Reports (2023) 17:254 https://doi.org/10.1186/s13256-023-03982-2 Haboub et al. Journal of Medical Case Reports (2023) 17:254 https://doi.org/10.1186/s13256-023-03982-2 (2023) 17:254 Journal of Medical Case Reports Background Cerebral cardiac embolism accounts for an increasing proportion of ischemic strokes and transient ischemic attacks [1]. Calcified cerebral emboli are rarely reported, but potentially cause of strokes and transient ischemic attacks and may be the first manifestation of vascular or cardiac disease. Identification of the source of embo- lization is crucial to prevent future emboli, neurological damage, and death. Non-contrast computed tomography *Correspondence: M. Haboub haboubmeryem@gmail.com 1 Cardiology Department, Hospital University Ibn Rochd, Casablanca, Morocco *Correspondence: M. Haboub haboubmeryem@gmail.com 1 Cardiology Department, Hospital University Ibn Rochd, Casablanca, Morocco © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Haboub et al. Journal of Medical Case Reports (2023) 17:254 Page 2 of 4 Fig. 1  Chest computed tomography confirming mitral leaflet calcification (CT) scan of the head is the most common imaging pro- cedure used as the initial assessment of suspected stroke or transient ischemic attack [2].i Cerebral calcified embolus can occur after percuta- neous and surgical intervention in the context of calci- fied aortic or mitral valve disease [3]. These emboli are presumed to occur because of valve trauma. However, spontaneous cerebral calcified embolism in the case of calcified aortic valve is very rare and there are less than ten case reports in the literature [4]. And a more interest- ing fact is that such an event in the context of calcified mitral valve disease has never been reported, at least to our knowledge.i We are reporting a case of spontaneous calcified cer- ebral embolism revealing a calcified rheumatic mitral valve stenosis. Fig. 1  Chest computed tomography confirming mitral leaflet calcification Case presentation Cervical arteries Duplex showed normal carotid and vertebral arteries. Cervical arteries Duplex showed normal carotid and vertebral arteries. We are reporting a case of a 59-year-old Moroccan man presenting to the emergency department after a transient ischemic attack (right hemiparesia and left central facial paralysis resolving briefly and spontaneously). There was a history of rheumatic fever at age 14 and stage II New York Heart Association (NYHA) dyspnea on moderate exertion for 2  years, with no history of recent cardiac intervention or aortic/carotid manipulation and no other symptoms. Physical examination on admission found an irregular heart rhythm of 90  bpm, blood pressure (BP) of 124/79 mmHg, mid-diastolic rumble at apex, no signs of heart congestion, and no signs of neurological impair- ment. A 12-lead-electrocardiogram showed an atrial fibrillation without other anomalies. Two-dimensional (2D) transthoracic echocardiograms revealed important mitral valve leaflets calcifications, probably related to rheumatic heart disease; planimetry of the valve was not possible. Continuous wave Doppler interrogation of the mitral valve found a severe mitral stenosis with a mean gradient of 15 mmHg and a continuity equation surface of 1 ­cm2. The aortic valve was thickened, but not calci- fied, a moderate aortic regurgitation was noticed. Left ventricular (LV) function was normal and the LV ejection fraction (LVEF) was at 55%. There was a right ventricu- lar (RV) longitudinal systolic dysfunction: TAPSE 11 mm and S’VD 6  cm/second. Tricuspid valve was thin with a mild tricuspid regurgitation. Continuous wave Dop- pler interrogation of the tricuspid valve allowed to esti- mate systolic pulmonary artery pressure at 69  mmHg. Mitral valve calcification was best shown on a cardiac CT (Fig. 1). A vitamin K antagonist (acenocoumarol) was pre- scribed, targeting an international normalized ratio (INR) of 2–3 and replacement of mitral valve using a mechani- cal prosthesis was performed with a good short-term and long-term outcome. The patient did not experience ischemic stroke during a 1-year follow-up. Discussion In our patient, transient ischemic attack (TIA) was cer- tainly due to atrial fibrillation (AF), and cerebral imaging lead to the discovery of calcified emboli. i Approximately 6%–31% of TIA are caused by a car- diogenic cerebral embolism (cardioembolic TIA) [5, 6]. Determining TIA etiology is important before adminis- tering therapy. Permanent or paroxysmal, valvular and non-valvular, AF is associated with a three- to five-fold increased risk of TIA and stroke [7, 8]. Cardiogenic cer- ebral embolization is common among patients with any cause of AF, but particularly in AF resulting from rheumatic and arteriosclerotic heart disease [9]. It is recommended to prescribe these patients oral antico- agulant therapy in case of valvular AF and, according to CHA2DS2VASc score, in case of non-valvular AF.i Calcified cerebral emboli are an infrequent, but increasingly recognized cause of TIA and ischemic stroke, although recognition among general radiolo- gists and clinicians can be limited. Unenhanced CT and computed tomography angiography (CTA) are the imaging techniques of choice for the diagnosis [10, 11]. First described on CT by Yock in 1981, calcified cere- bral emboli were previously thought to be unusual, and to most commonly arise following instrumentation of calcified cardiac valves or direct aortic/carotid artery Unenhanced cerebral CT was performed, revealing cal- cified emboli in both middle cerebral arteries (M3 and M4 segments) (Fig. 2). Susceptibility weighted magnetic resonance cerebral sequences result is reported in Fig. 3. Page 3 of 4 Haboub et al. Journal of Medical Case Reports (2023) 17:254 Fig. 2  Unenhanced cerebral computed tomography with axial reconstructions showing calcified emboli in both middle cerebral arteries. These arteries were permeable Fig. 2  Unenhanced cerebral computed tomography with axial reconstructions showing calcified emboli in both middle cerebral arteries. These arteries were permeable Fig. 3  Susceptibility weighted cerebral magnetic resonance sequences showing absence of signal inside both middle cerebral arteries Fig. 3  Susceptibility weighted cerebral magnetic resonance sequences showing absence of signal inside both middle cer manipulation [12, 13]. However, there is growing evi- dence that spontaneous calcified cerebral embolism is more common, with a recent study and review of pub- lished cases reporting a 2.7% prevalence among a group of patients presenting with suspected stroke over a 1-year period. In this report, the middle cerebral artery was the site of 83% calcified emboli. Funding Funding The authors have no funding to declare. The authors have no funding to declare. 19. Raghib MF, Mutzenback JS, Rosler C, et al. Acute treatment of stroke due to spontaneous calcified cerebral emboli causing large vessel occlusion. J Clin Neurosci. 2018;47:56–61. Availability of data and materials The published information is available from the corresponding author on reasonable request. 20. Katsamakis G, Lukovits TG, Gorelick PB. Calcific cerebral embolism in systemic calciphylaxis. Neurology. 1998;51:295–7. 20. Katsamakis G, Lukovits TG, Gorelick PB. Calcific cerebral embolism in systemic calciphylaxis. Neurology. 1998;51:295–7. 20. Katsamakis G, Lukovits TG, Gorelick PB. Calcific cerebral embolism in systemic calciphylaxis. Neurology. 1998;51:295–7. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. There are some reported cases of cerebral calcific emboli following open heart mitral valvotomy and per- cutaneous mitral valvuloplasty. Mitral calcification accounts for fewer than 1% of cerebral cardiac embolism, and in all described cases, it was secondary to mitral valve intervention [16]. However, there is not any case report published describing spontaneous calcified cer- ebral emboli in the context of calcified rheumatic mitral stenosis.i Author contributions 14. Salka S, Almassi H, Leitshuh M. Spontaneous coronary artery embolus associated with calcific artery stenosis. Chest. 1994;105:1289–90. 14. Salka S, Almassi H, Leitshuh M. Spontaneous coronary artery embolus associated with calcific artery stenosis. Chest. 1994;105:1289–90. MH conceived the study, participated in its design, acquired the data, per- formed a literature review, and drafted the manuscript. SA participated in the design of the study and helped with the literature review. HM helped with the literature review. GM helped with the literature review. AM helped with the literature review. SA participated in the design of the study. MEGB helped with the literature review, and helped draft and edit the manuscript. AD helped draft the manuscript. LA helped draft and edit the manuscript. RH helped draft and edit the manuscript. All authors read and approved the final manuscript. i 15. Wilson JH, Cranley JJ. Recurrent calcium emboli in a patient with aortic stenosis. Chest. 1989;96:1433–4. 15. Wilson JH, Cranley JJ. Recurrent calcium emboli in a patient with aortic stenosis. Chest. 1989;96:1433–4. 16. Hickey TBM. Iatrogenic embolization following cardiac intervention: postmortem analysis of 110 cases. Cardiovasc Pathol. 2019;40:12–8. 16. Hickey TBM. Iatrogenic embolization following cardiac intervention: postmortem analysis of 110 cases. Cardiovasc Pathol. 2019;40:12–8. 17. O’Cearbhaill RM, Moriarty HK, Crosbie I, et al. Calcified cerebral emboli: a case series and review of the literature. J Syst Int Neurosci. 2016;2:180–3. 17. O’Cearbhaill RM, Moriarty HK, Crosbie I, et al. Calcified cerebral emboli: a case series and review of the literature. J Syst Int Neurosci. 2016;2:180–3. 18. Halloran JI, Bekavac I. Unsuccessful tissue plasminogen activator treat- ment of acute stroke caused by a calcific embolus. J Neuroimaging. 2004;14:385–7. 18. Halloran JI, Bekavac I. Unsuccessful tissue plasminogen activator treat- ment of acute stroke caused by a calcific embolus. J Neuroimaging. 2004;14:385–7. References In case of stroke secondary to calcified emboli, the role of thrombolysis remains uncertain, as there are conflict- ing reports regarding its efficacy in this setting [17–19]. There is debate and very limited experience regarding the place of mechanical thrombectomy [18, 20]. Subsequent imaging evaluation of this subgroup of patients who have suffered from ischemic strokes requires caution because the calcified nature of the embolus may be obscured on CT angiography or magnetic resonance imaging (MRI). Clinical evaluation should include consideration of potential proximal source of calcified material and recent cardiac intervention or aortic/carotid manipula- tion. Although there is no data showing benefit of valve replacement, most authors advocate valve replacement to remove the source of emboli. 1. Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial TOAST Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993;24:35–41. 1. Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial TOAST Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993;24:35–41. 2. Walker BS. Calcified cerebral emboli, a “do not miss” imaging diagno- sis: 22 new cases and review of the literature. AJNR Am J Neuroradiol. 2014;35:1515–9. https://​doi.​org/​10.​3174/​ajnr.​A3892. 3. Kapila A, Hart R. Calcific cerebral emboli and aortic stenosis: detection by computed tomography. Stroke. 1986;17:619–21. 3. Kapila A, Hart R. Calcific cerebral emboli and aortic stenosis: detection by computed tomography. Stroke. 1986;17:619–21. 4. Khetarpal V. Calcific aortic valve and spontaneous embolic stroke: a review of literature. J Neurol Sci. 2009;287:32–5. 5. Bogousslavsky J, Hachinski VC, Boughner DR. Cardiac and arterial lesions in carotid transient ischemic attacks. Arch Neurol. 1986;43:223–8. 3. Kapila A, Hart R. Calcific cerebral emboli and aortic stenosis: detection by computed tomography. Stroke. 1986;17:619–21. 4. Khetarpal V. Calcific aortic valve and spontaneous embolic stroke: a review of literature. J Neurol Sci. 2009;287:32–5. computed tomography. Stroke. 1986;17:619–21. 4. Khetarpal V. Calcific aortic valve and spontaneous embolic stroke: a review of literature. J Neurol Sci. 2009;287:32–5. 5. Bogousslavsky J, Hachinski VC, Boughner DR. Cardiac and arterial lesions in carotid transient ischemic attacks. Arch Neurol. 1986;43:223–8. 6. Sempere AP, Duarte J, Cabezas C, et al. Etiopathogenesis of transient ischemic attacks and minor ischemic strokes: a community-based study in Segovia, Spain. Stroke. References 1998;29:40–5. 7. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation as an independent risk factor for stroke: the Framingham Study. Stroke. 1991;22:983–8. g y 8. Wolf PA, Dawber TR, Thomas HE Jr, Kannel WB. Epidemiologic assessment of chronic atrial fibrillation and risk of stroke: the Framingham study. Neurology. 1978;28:973–7. Acknowledgements Not applicable. 13. Khaw N, Gailloud P. CT of calcific cerebral emboli after carotidmanipula- tion. Am J Roentgenol. 2000;174:1467. 13. Khaw N, Gailloud P. CT of calcific cerebral emboli after carotidmanipula- tion. Am J Roentgenol. 2000;174:1467. Discussion Cardiac valvu- lar disease was more common than carotid atheroma- tous disease, with calcified aortic stenosis three times more common than mitral valve disease as the embolic source [3].i Iatrogenic calcified embolus following cardiac sur- gery or catheterization is common [4, 14, 15]. These emboli are presumed to occur because of valve trauma. According to a most recent postmortem analysis of iatro- genic embolization cases, the source of calcified cerebral emboli was attributed to dislodgement and displace- ment of calcified material from calcified aortic valves and Haboub et al. Journal of Medical Case Reports (2023) 17:254 Page 4 of 4 ulcerated aortic atherosclerotic plaques during therapeu- tic and investigative procedures [16].hi Competing interests The authors declare that they have no competing interests. Received: 11 March 2020 Accepted: 10 May 2023 Received: 11 March 2020 Accepted: 10 May 2023 Conclusion y 9. Stirling J. Cerebral embolism as a cause of stroke and transient ischemic attack. Echocardiography. 1996;13(5):513–8. Spontaneous calcified cerebral emboli, secondary to mitral valve leaflet calcification is an extremely rare con- dition. Replacement of the valve is the only option to prevent recurrent emboli and outcomes are still to be determined. 10. Rancurel G, Marelle L, Vincent D, Catala M, Arzimanaglou A, Vacheron A. Spontaneous calcific cerebral embolus from a calcific aortic stenosis in a middle cerebral artery infarct. Stroke. 1989;20:691–3. 11. Oliveira-Filho J, Massaro AR, Yamamoto F, Bustamante L, Scaff M. Stroke as the first manifestation of calcific aortic stenosis. Cerebrovasc Dis. 2000;10:413–6. 12. Yock DH. CT demonstration of cerebral emboli. J Comput Assist Tomogr. 1981;5:190–6. 12. Yock DH. CT demonstration of cerebral emboli. J Comput Assist Tomogr. 1981;5:190–6.i Publisher’s Note S Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Ethics approval and consent to participate Ethics approval and consent to participate The need for ethics approval was waived. The need for ethics approval was waived.
https://openalex.org/W3197428897
https://www.mdpi.com/1422-0067/22/17/9549/pdf?version=1630751937
English
null
Relationships of Gut Microbiota Composition, Short-Chain Fatty Acids and Polyamines with the Pathological Response to Neoadjuvant Radiochemotherapy in Colorectal Cancer Patients
International journal of molecular sciences
2,021
cc-by
15,705
  p g * Correspondence: jaime.gomez@ibima.eu (J.G.-M.); maribel.queipo@ibima.eu (M.I.Q.-O.) Citation: Sánchez-Alcoholado, L.; Laborda-Illanes, A.; Otero, A.; Ordóñez, R.; González-González, A.; Plaza-Andrades, I.; Ramos-Molina, B.; Gómez-Millán, J.; Queipo-Ortuño, M.I. Relationships of Gut Microbiota Composition, Short-Chain Fatty Acids and Polyamines with the Pathological Response to Neoadjuvant Radiochemotherapy in Colorectal Cancer Patients. Int. J. Mol. Sci. 2021, 22, 9549. https://doi.org/ 10.3390/ijms22179549 Abstract: Emerging evidence has suggested that dysbiosis of the gut microbiota may influence the drug efficacy of colorectal cancer (CRC) patients during cancer treatment by modulating drug metabolism and the host immune response. Moreover, gut microbiota can produce metabolites that may influence tumor proliferation and therapy responsiveness. In this study we have investigated the potential contribution of the gut microbiota and microbial-derived metabolites such as short chain fatty acids and polyamines to neoadjuvant radiochemotherapy (RCT) outcome in CRC patients. First, we established a profile for healthy gut microbiota by comparing the microbial diversity and composition between CRC patients and healthy controls. Second, our metagenomic analysis revealed that the gut microbiota composition of CRC patients was relatively stable over treatment time with neoadjuvant RCT. Nevertheless, treated patients who achieved clinical benefits from RTC (responders, R) had significantly higher microbial diversity and richness compared to non-responder patients (NR). Importantly, the fecal microbiota of the R was enriched in butyrate-producing bacteria and had significantly higher levels of acetic, butyric, isobutyric, and hexanoic acids than NR. In addition, NR patients exhibited higher serum levels of spermine and acetyl polyamines (oncometabolites related to CRC) as well as zonulin (gut permeability marker), and their gut microbiota was abundant in pro-inflammatory species. Finally, we identified a baseline consortium of five bacterial species that could potentially predict CRC treatment outcome. Overall, our results suggest that the gut microbiota may have an important role in the response to cancer therapies in CRC patients. Academic Editors: Rustam I. Aminov and Catalina Carrasco-Pozo Received: 18 August 2021 Accepted: 30 August 2021 Published: 2 September 2021 Received: 18 August 2021 Accepted: 30 August 2021 Published: 2 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Keywords: colorectal cancer; gut microbiota; SCFAs; gut permeability; radiochemotherapy; treat- ment outcome Article Relationships of Gut Microbiota Composition, Short-Chain Fatty Acids and Polyamines with the Pathological Response to Neoadjuvant Radiochemotherapy in Colorectal Cancer Patients Lidia Sánchez-Alcoholado 1, Aurora Laborda-Illanes 1, Ana Otero 2, Rafael Ordóñez 2, Alicia González-González 1 , Isaac Plaza-Andrades 1, Bruno Ramos-Molina 3 , Jaime Gómez-Millán 2,* and María Isabel Queipo Ortuño 1,* Lidia Sánchez-Alcoholado 1, Aurora Laborda-Illanes 1, Ana Otero 2, Rafael Ordóñez 2, Alicia González-González 1 , Isaac Plaza-Andrades 1, Bruno Ramos-Molina 3 , Jaime Gómez-Millán 2,* and María Isabel Queipo-Ortuño 1,* 1 Unidad de Gestión Clínica Intercentros de Oncología Médica, Hospitales Universitarios Regional y Virgen de la Victoria, Instituto de Investigación Biomédica de Málaga (IBIMA)-CIMES-UMA, 29010 Málaga, Spain; l.s.alcoholado@gmail.com (L.S.-A.); auroralabordaillanes@gmail.com (A.L.-I.); agonzalez.bq@gmail.com (A.G.-G.); isaacplazaandrade@gmail.com (I.P.-A.) 1 Unidad de Gestión Clínica Intercentros de Oncología Médica, Hospitales Universitarios Regional y Virgen de la Victoria, Instituto de Investigación Biomédica de Málaga (IBIMA)-CIMES-UMA, 29010 Málaga, Spain; l.s.alcoholado@gmail.com (L.S.-A.); auroralabordaillanes@gmail.com (A.L.-I.); agon ale bq@gmail com (A G G ) isaacpla aandrade@gmail com (I P A ) g p g g 3 Grupo de Obesidad y Metabolismo, Instituto Murciano de Investigación Biosanitaria (IM 30120 Murcia, Spain; brunoramosmolina@gmail.com International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Colorectal cancer (CRC) is the second most common malignant cancer in Western countries. The global burden of CRC is expected to substantially increase in the next two decades as a consequence of adopting Western lifestyles [1]. In recent years, several works have demonstrated that the gut microbiome could be a critical environmental factor that contributes to the tumorigenesis and progression of CRC, potentially by inducing pro-inflammatory responses, by producing microbial oncometabolites, and by interfering with the energy balance in cancer cells. Moreover, CRC is frequently associated with a dysbiosis in the microbial composition of the tumor and adjacent mucosa [2–4]. Several Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9549. https://doi.org/10.3390/ijms22179549 Int. J. Mol. Sci. 2021, 22, 9549 2 of 25 studies have suggested that the composition of the gut microbiota could affect the body’s response to a variety of cancer therapies, including chemotherapy, radiotherapy, and immunotherapy [5–7]. py Preoperative radiochemotherapy (RCT) followed by surgery has become the standard treatment for patients with CRC [8,9]. Recent studies have suggested that the gut microbiota may influence drug response (efficacy and toxicity) in CRC patients through several mechanisms such as immunomodulation, reduced diversity, translocation, metabolism, and ecological variation [10]. Specific gut bacteria have been shown to affect cancer treatment by modulating drug metabolism and the host immune response [11,12]. Thus, several phyla are known to mediate drug metabolism via different reactions such as isoxazole scission, denitration, proteolytic degradation, acetylation/deacetylation, deconjugation, physical adherence to the drugs as well as by amine formation and/or hydrolysis [13]. Scott et al. described that the gut microbiota was able to influence the efficacy of one of the first-line treatments for CRC, such asfluoropyrimidines, through drug interconversion involving bacterial vitamin B6 and B9 and ribonucleotide metabolism [14]. In addition, the effect 5-fluorouracil treatment in CRC cells could be mediated by gut microbial metabolites [15]. Remarkably, Fusobacterium nucleatum is able to promote CRC resistance to chemotherapy by targeting both TLR4 and MYD88 innate immune signaling [16]. Furthermore, radiation may also lead to alterations in gut microbiota composition in animal models [17]. 1. Introduction However, the clinical impact of radiotherapy on gut microbiota in cancer patients remains mostly unexplored although it has been proposed that the gut microbiota might play a role in the immunogenic effect of radiotherapy [18]. On the other hand, the gut microbiome produces bacteria-derived metabolites that could affect cancer proliferation and chemotherapy responsiveness. Thus, previous studies describe that SCFAs (such as butyric acid, isobutyric acid and acetic acid) inhibit the growth of cultured human colorectal cancer cells and that butyric acid is the strongest inhibitor [19]. Ross et al. reported an association between the levels of the short-chain fatty acids (SCFAs) propionate and butyrate in patients with early stage breast cancer with a pathological complete response (pCR) to neoadjuvant chemotherapy [20]. Coutzac et al. suggested that SCFA limits anti-CTLA-4 activity in patients with metastatic melanoma [21]. y p In addition, lower SCFA (especially butyrate) concentrations might induce a dysfunc- tion in the gut epithelial barrier, thereby activating proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which damage epithelial cells and their junctions [22]. Other bacteria-derived metabolites, such as the polyamines (PAs) (spermine, spermi- dine and putrescine), have been involved in almost all the steps of colorectal tumorigenesis. PAs are molecules that are indispensable in normal cell growth and gene expression and are needed in cell proliferation, but their concentrations increase during the transition from a healthy cell to a tumor cell [23]. Recently, it was shown that the level of acetylated PAs is more specific for cancer. For example, N1, N12-diacetylspermine (DiAcSPM) was increased in CRC and in dysplastic colorectal lesions [24]. Therefore, taking all of the evidence together, we hypothesized a bidirectional interac- tion between the neoadjuvant RCT and the gut microbiome in CRC patients: RCT might induce alterations in the gut microbiome, and these alterations might, in turn, influence the effectiveness of RCT by directly interacting with the treatment and/or by stimulating the host’s immune response. In this study, we aimed to identify the possible relationship between the gut micro- biome, the fecal SCFAs levels, the serum levels of the polyamines and acetyl derivatives of polyamines, and the intestinal permeability to neoadjuvant RCT outcome in CRC patients. 2.1. Clinical Characteristics of the Patients and Healthy Controls CRC patients and healthy controls had comparable eating habits to exclude the influ- ence of dietary differences. CRC patients and healthy controls followed a Mediterranean Int. J. Mol. Sci. 2021, 22, 9549 3 of 25 3 of 25 diet consisting in a high consumption of olive oil, fruits, legumes, vegetables, nuts, whole grains, and fish and a low intake of red meat and dairy products. Adherence to the Mediter- ranean diet was assessed by using a validated 14-item food frequency questionnaire in all study patients. All CRC patients completed the neoadjuvant RCT and underwent surgical resection. There was no significant difference between CRC patients and healthy controls in terms of age, sex, BMI, and biochemical data (Table 1). A total of 28 of the 40 CRC patients (70%) had a good response to the neoadjuvant RCT (responders, R) (TGR 1–2), and 12 (30%) had a poor or non-response (non-responders, NR) (TGR 3–5) to therapy. Both R and NR patients were similar in terms of sex, age, BMI, and stage of the cancer, as shown in Table 1. Table 1. Clinical characteristics of study groups. Table 1. Clinical characteristics of study groups. Table 1. Clinical characteristics of study groups. 2.1. Clinical Characteristics of the Patients and Healthy Controls Healthy Controls (N = 20) CRC-Patients (N = 40) * p R Patients (N = 28) NR Patients (N = 12) * p Age (years) 61.42 ± 7.40 63.35 ± 6.97 0.326 62.93 ± 8.27 63.12 ± 6.34 0.928 Gender, n (M/F) 10/10 23/17 0.783 16 /12 7/5 0.780 BMI (kg/m2) 25.45 ± 3.23 26.42 ± 4.71 0.412 26.22 ± 4.22 25.92 ± 3.92 0.835 Constipation, n (%) 6 (20%) 10 (25%) 0.914 7 (25%) 3 (25%) 0.690 Alcohol consumption, n (%) 4 (13.3%) 6 (15%) 0.831 4 (14.28%) 2 (16.16%) 0.740 Current smoking, n (%) 9 (30%) 15 (37.5%) 0.774 11 (39.28%) 4 (33.33%) 0.990 Biochemical data Glucose (mg/dl) 94.85 ± 19.86 104.79 ± 27.94 0.161 102.83 ± 26.38 104.15 ± 23.56 0.882 Total cholesterol (mg/dl) 175.2 ± 33.6 183.95 ± 25.71 0.268 184.17 ± 21.64 181.67 ± 26.12 0.755 Triglycerides (mg/dl) 112.67 ± 34.51 114.85 ± 33.62 0.815 109.25 ± 32.12 118.32 ± 27.12 0.398 HDL-cholesterol (mg/dl) 60.7 ± 15.1 54.83 ± 18.23 0.219 55.32 ± 16.21 53.89 ± 18.34 0.807 LDL-cholesterol (mg/dl) 107.78 ± 27.12 112.07 ± 33.45 0.621 109.68 ± 30.29 112.36 ± 33.21 0.805 Histological variables Disease stage II 22 (55%) - 15 (53.57%) 7 (58.33%) 0.945 III 18 (45%) - 13 (46.42%) 5 (41.66%) 0.950 Tumor depth penetration (T) T2–T3 26 (65%) - 18(64.28%) 8 (66.66%) 0.828 T4 14 (35%) - 10 (35.71%) 4 (33.33%) 0.832 Grade of differentiation G1 18 (45%) - 12 (42.85%) 6 (50%) 0.944 G2 10 (25%) - 7 (25%) 3 (25%) 0.690 G3 7 (17.5%) - 5 (18.85%) 2 (16.16%) 0.806 No differentiation 5 (12.5%) - 3 (10.71%) 2 (16.66%) 0.777 CRC: colorectal cancer; R: responders; NR: non-responders; BMI: body mass index; HDL: high density lipoprotein; LDL: low density lipoprotein. Values are expressed as mean ± SD. * p < 0.05 was considered statistically significant. CRC: colorectal cancer; R: responders; NR: non-responders; BMI: body mass index; HDL: high density lipoprotein; LDL: low density lipoprotein. Values are expressed as mean ± SD. * p < 0.05 was considered statistically significant. 2.2. Differences in Taxonomic Composition and Diversity of Gut Microbiota between CRC Patients and Healthy Controls The analysis of stool samples revealed 17,496,823 reads of the 16S rRNA gene (hyper- variable V2–V9 regions), with an average of 105,632 (±10,825) reads for each sample in a range between 359 and 39,873. After trimming and filtering, 52,844 high-quality reads were selected. 2.1. Clinical Characteristics of the Patients and Healthy Controls A total of 15,326 OTUs were obtained in the OTUs clustering process, and after the alignment of the OTU representative sequences, 2582 OTUs were identified to have a relative abundance >1% in at least four samples (97% similarity cut-off). For the taxonomic assignment of these OTUs, QIIME2 pipeline and Greengenes v13.8 were used, and the OTUs were binned into 7 phyla, 39 families, 45 genera, and 53 species. p y g p We first compared the landscape of the gut microbiome in the stool samples of all CRC patients at baseline and in healthy controls in order to define a normal gut microbiota profile. As expected, we found significantly higher diversity and richness (defined by the Shannon and Chao1 indexes, respectively) in the fecal samples of healthy controls with Int. J. Mol. Sci. 2021, 22, 9549 4 of 25 respect to those of CRC patients (Shannon p = 0.026 and Chao1 p = 0.001) (Figure S1A,B). The beta diversity (Bray–Curtis dissimilarity) comparison of the baseline CRC patients and the healthy controls indicated that the two cohorts had significantly different genus compositions of intestinal bacteria (p = 0.0001, ANOSIM) (Figure S1C). Furthermore, the analysis of the gut microbiota profiles between the CRC patients and the healthy controls at baseline revealed significant differences in the abundance at different taxonomic levels. At phylum level, the relative abundance of Fusobacteria (q < 0.001), Firmicutes (q < 0.001), Lentisphaerae (q = 0.007), and Proteobacteria (q = 0.003) were significantly increased in patients with CRC, while the relative abundance of Bacteroidetes (q < 0.001) and Actinobacteria (q = 0.034) were significantly decreased in CRC patients when compared to the controls (Figure 1A). At the genus level, the results indicated significant differences in the microbial com- position of the dominant genera between the CRC patients at baseline and the healthy controls. Compared to the healthy controls, patients with CRC displayed an enrichment in the genera Prevotella (q < 0.001), Oscillospira (q < 0.001), Fusobacterium (q = 0.001), Enter- obacter (q = 0.020), Victivallis (q = 0.012), Escherichia (q = 0.046), and Desulfovibrio (q < 0.001). Conversely, the abundance of Bacteroides (q = 0.003), Roseburia (q < 0.001), Ruminococcus (q = 0.006), Faecalibacterium (q = 0.01), Bifidobacterium (q = 0.023), and Blautia (q = 0.014) were enriched in the healthy controls compared to in the CRC patients (Figure 1B). 2.1. Clinical Characteristics of the Patients and Healthy Controls y p p g At species level, while healthy subjects showed a significantly higher abundance of Bifidobacterium bifidum (q = 0.034) and Faecalibacterium prausnitzii (q = 0.040) with respect to the CRC patients, Fusobacterium nucleatum (q = 0.020), Bacteroides fragilis (q = 0.024), and Escherichia coli (q = 0.016) were significantly increased in the fecal samples of CRC patients in comparison to the controls. 2.3. Changes in Gut Microbiota Diversity and Composition in Response to Neoadjuvant RCT Treatment in CRC Patients 2.3. Changes in Gut Microbiota Diversity and Composition in Response to Neoadjuvant RCT Treatment in CRC Patients We compared the gut microbiota communities at baseline (T0) versus at post-treatment time points (T1, T2, and T3) to study the effect of neoadjuvant RCT on the gut microbial diversity and composition in CRC patients. The alpha diversity comparison showed no significant differences in the levels of richness (Chao 1) and diversity (Shannon) between the baseline and the different time points (Shannon p = 0.75 and Chao1 p = 0.61) (Figure 2A,B). Moreover, the PCoA plot based on the beta diversity (Bray–Curtis dissimilarity) revealed that the differences in the gut microbial community at T1, T2, and T3 compared to at baseline (T0) were not significant (p = 0.716, ANOSIM) (Figure 2C). ( ) g (p ) ( g ) The main bacterial phyla (Firmicutes and Bacteroidetes) remained stable over time, while other, less abundant phyla, such as Fusobacteria and Proteobacteria, were signif- icantly decreased at T3 compared to at T0 (q = 0.042 and q = 0.039, respectively) in the CRC patients. Although the bacterial family and genera proportions differed between the different time points, they were not significantly altered by the RCT treatment (Wilcoxon test p > 0.05), apart from the genera Fusobacterium (q = 0.015), Escherichia (q = 0.04) and Klebsiella (q = 0.035), which were significantly decreased after treatment, and the genus Bifi- dobacterium (q = 0.049), which was significantly increased at T3 compared to T0 (Figure 3). 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to Neoadjuvant RCT in CRC Patients To evaluate the relationship between the microbial community and the treatment outcome, we classified the patients based on their response to RCT into categories such as responders (R) and non-responders (NR). As shown in Table 1, no significant differences in terms of stage of cancer, sex, age, and BMI were observed between the study groups (R vs. NR). 5 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 1. Relative abundance at phylum (A) and genera (B) levels of differentially abundant bacteria in the stool samples CRC patients at baseline (CRC-T0) and healthy controls (HC). * p < 0.05, ** p < 0.001. Figure 1. Relative abundance at phylum (A) and genera (B) levels of differentially abundant bacteria in the stool samples of CRC patients at baseline (CRC-T0) and healthy controls (HC). * p < 0.05, ** p < 0.001. 6 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 2. Gut microbiota diversity and richness at baseline and during RTC treatment and post- treatment points in CRC patients. (A) Shannon index (p = 0.75); (B) Chao1 index (p = 0.61); (C) princi- pal component analysis representation based on Bray–Curtis distance matrix of patient distribution based on bacterial genera composition at baseline and during RTC treatment and at post-treatment points (p = 0.716). The first two coordinates are plotted with the percentage of variability, which is explained and indicated on the axis. Figure 2. Gut microbiota diversity and richness at baseline and during RTC treatment and post- treatment points in CRC patients. (A) Shannon index (p = 0.75); (B) Chao1 index (p = 0.61); (C) princi- pal component analysis representation based on Bray–Curtis distance matrix of patient distribution based on bacterial genera composition at baseline and during RTC treatment and at post-treatment points (p = 0.716). The first two coordinates are plotted with the percentage of variability, which is explained and indicated on the axis. 7 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 3. Heatmap diagram of the gut microbiota composition at different taxa levels for baseline (CRC-T0), treatment points with neoadjuvant RCT (CRC-T1, CRC-T2 and CRC-T3), and the healthy control subjects (HC). The 29 phylum and genera that were shared by all of the tested samples (core microbiome) are displayed. Figure 3. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to Neoadjuvant RCT in CRC Patients Heatmap diagram of the gut microbiota composition at different taxa levels for baseline (CRC-T0), treatment points with neoadjuvant RCT (CRC-T1, CRC-T2 and CRC-T3), and the healthy control subjects (HC). The 29 phylum and genera that were shared by all of the tested samples (core microbiome) are displayed. Figure 3. Heatmap diagram of the gut microbiota composition at different taxa levels for baseline (CRC-T0), treatment points with neoadjuvant RCT (CRC-T1, CRC-T2 and CRC-T3), and the healthy control subjects (HC). The 29 phylum and genera that were shared by all of the tested samples (core microbiome) are displayed. An analysis of the alpha diversity at T3 revealed that the R group had higher diversity (Shannon index, q < 0.001; Simpson index, q = 0.039) and richness that the NR group (Chao1 index, q = 0.015) at genus level (Figure 4A,B). Furthermore, the ordination plot based on Bray–Curtis dissimilarities and the Jaccard index showed different intestinal microbial compositions at the genus level between both the R and the NR groups at T3 (Bray–Curtis index, q = 0.038; Jaccard index, q = 0.035; non-parametric ANOSIM test) (Figure 4C). Next, we searched for differentially abundant taxa in the gut microbiome of R versus NR at T3. The analysis revealed that at the phylum level, there was a significant enrichment in the Actinobacteria (q = 0.0025) and Firmicutes (q = 0.0017) populations and a significant decrease in the Fusobacterias (q = 0.025) and Proteobacterias (q = 0.037) populations in the R group in comparison to the NR group (Figure 5A,B). At the family level, a significantly higher abundance of Ruminococcaceae (q = 0.004) and Bifidobacteriaceae (q = 0.03) accom- panied with a significantly lower abundance of Prevotellaceae (q = 0.045), Enterobactericeae (q = 0.027), and Fusobacteriaceae (q = 0.014) were shown in the R group compared to in the NR group (Figure 5A,C). 8 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 4. Comparison of alpha and beta diversity in CRC patients according to their response to therapy. (A) Shannon index; (B) Chao1index; (C) principal component plot based on the Bray–Curtis distance matrix and the Jaccard indices from the responder (R) and non-responder (NR) patients at genus-level. The first two coordinates are plotted with the percentage of variability, which is explained and indicated on the axis. Figure 4. Comparison of alpha and beta diversity in CRC patients according to their response to therapy. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to Neoadjuvant RCT in CRC Patients (A) Shannon index; (B) Chao1index; (C) principal component plot based on the Bray–Curtis distance matrix and the Jaccard indices from the responder (R) and non responder (NR) patients at genus level The first two coordinates are plotted with the percentage Figure 4. Comparison of alpha and beta diversity in CRC patients according to their response to therapy. (A) Shannon index; (B) Chao1index; (C) principal component plot based on the Bray–Curtis distance matrix and the Jaccard indices from the responder (R) and non-responder (NR) patients at genus-level. The first two coordinates are plotted with the percentage of variability, which is explained and indicated on the axis. In addition, at the genera level, we identified a significant increase in Ruminococcus (q = 0.035), Bilophila (q = 0.008), Collinsiella (q = 0.015), Bifidobacterium (q = 0.024), Roseburia (q = 0.032), and Faecalibacterium (q = 0.041) in R patients with respect to the NR, while a significant increase in Prevotella (q = 0.05), Fusobacterium (q = 0.045), Escherichia (q = 0.037), Bacteroides (q = 0.027), and Klebsiella (q = 0.035) were observed in the NR patients compared to the R group (Figure 6A,B). Finally, at the species level, we found a significant overabun- dance of Prevotella copri (q < 0.001), Escherichia coli (q = 0.029), Fusobacterium nucleatum (q = 0.015), and Bacteroides fragilis (q = 0.029) in the NR group, while the R group displayed a significantly higher abundance of Bifidobacterium bifidum (q = 0.043), Ruminococcus albus (q = 0.019), Collinsella aerofaciens (q = 0.018), and Faecalibacterium prausnitzii (q = 0.027). 2.5. Baseline Microbiota Composition Could Predict Response to RCT Treatment in CRC Patients After describing the significant differences in the intestinal microbial composition between the R and NR after RCT treatment, we next assessed the predictive power of the gut microbiome related to neoadjuvant RCT response. We used random forest (RF) to build a predictive model based on the overall gut microbiota profile using the species-level abundance data as input. After RF analysis with 500 bootstrap samples, we found that Int. J. Mol. Sci. 2021, 22, 9549 9 of 25 9 of 25 the overall gut microbiota composition data had a significant accuracy of 80% and an area under the curve (AUC) of 0.71. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to Neoadjuvant RCT in CRC Patients The main species accounting for this stratification were Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nucleatum, and Bacteroides fragilis, and when the proportions of these bacterial species were only used for testing the accuracy of the RF classifier, this increased to 96% (AUC = 0.92). Thus, the response to RTC or the lack of it were identified with an accuracy of 94% (AUC = 0.95) and of 91% (AUC = 0.92), respectively (Figure 7A). The validation cohort consisted of 84 CRC patients under neoadjuvant RCT (45 R patients and 39 NR patients) (data collected from the Genome Sequence Archive in National Genomics Data Center, accession number CRA002850). After RF analysis in this validation cohort, an accuracy of 92.0% (AUC = 0.93) and 90.0% (AUC = 0.91) were obtained for the response to RTC or the lack of it, respectively (Figure 7B). Among the five species variables, Fusobacterium nucleatum, and Bacteroides fragilis were biomarkers of R patients, and Ruminococcus albus, Bifidobacterium bifidum, and Faecalibacterium prausnitzii were biomarkers of NR patients. Figure 5. Cont. Figure 5. Cont. Figure 5. Cont. 10 of 25 Int. J. Mol. Sci. 2021, 22, 9549 e 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non- nder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the amples of N patients compared to the NR patients. * p < 0.05. Figure 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non- responder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the stool samples of N patients compared to the NR patients. * p < 0.05. Figure 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non- responder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the stool samples of N patients compared to the NR patients. * p < 0.05. Figure 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non- responder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the stool samples of N patients compared to the NR patients. * p < 0.05. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to Neoadjuvant RCT in CRC Patients 11 of 25 Int. J. Mol. Sci. 2021, 22, 9549 gure 6. Heatmap of the fecal microbiota composition at genera level in the responder (R) and non-responder (NR) patient A). Relative abundance at genera level of differentially abundant OTUs in the stool samples of the N patients compared t e NR patients. * p < 0.05 (B). Figure 6. Heatmap of the fecal microbiota composition at genera level in the responder (R) and non-responder (NR) patients (A). Relative abundance at genera level of differentially abundant OTUs in the stool samples of the N patients compared to the NR patients. * p < 0.05 (B). 12 of 25 12 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed using microbial variables (Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nucleatum, and Bacteroides fragilis). (A) Training cohort. The area under the ROC curve (AUC) was 0.95, and the 95% confidence interval (CI) was 0.901–1 for the R patients (green), and the AUG was 0.92 and 95% the IC was 0.827–1 for the NR patients (red). (B) Validation cohort. The AUG was 0.93 and the 95% IC was 0.877–0.987 for the R patients (green), and the AUG was 0.91 and 95% the IC was 0.835–0.984 for the NR patients (red). er operating characteristic (ROC) curve based on the random forest classifier constructed using microbial Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed using microbial variables (Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nucleatum, and Bacteroides fragilis). (A) Training cohort. The area under the ROC curve (AUC) was 0.95, and the 95% confidence interval (CI) was 0.901–1 for the R patients (green), and the AUG was 0.92 and 95% the IC was 0.827–1 for the NR patients (red). (B) Validation cohort. The AUG was 0.93 and the 95% IC was 0.877–0.987 for the R patients (green), and the AUG was 0.91 and 95% the IC was 0.835–0.984 for the NR patients (red). Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed using microbial variables (Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nucleatum, and Bacteroides fragilis). (A) Training cohort. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to Neoadjuvant RCT in CRC Patients The area under the ROC curve (AUC) was 0.95, and the 95% confidence interval (CI) was 0.901–1 for the R patients (green), and the AUG was 0.92 and 95% the IC was 0.827–1 for the NR patients (red). (B) Validation cohort. The AUG was 0.93 and the 95% IC was 0.877–0.987 for the R patients (green), and the AUG was 0.91 and 95% the IC was 0.835–0.984 for the NR patients (red). 2.6. Differences in the Gut Microbiota Functions between Responder and Non-Responder KEGG pathway enrichment analysis of the metagenomic data showed that genes for energy metabolism such as methane metabolism (q < 0.004), carbohydrate metabolism, such as the pentose phosphate pathway (q = 0.0022), pyruvate metabolism (q-value < 0.001), starch and sucrose metabolism (q = 0.008), galactose metabolism (q = 0.007), butanoate metabolism (q = 0.005), and glycolysis-gluconeogenesis (q = 0.0028); for xenobiotic biodegra- dation and metabolism pathways, including benzoate degradation (q = 0.038) and nitro- toluene degradation (q = 0.005); and membrane transport, such as ABC transporters (q = 0.012) and transporters (q = 0.012), were significantly depleted in NR compared to R patients. Nevertheless, compared to the R patients, in the NR patients, there was a signif- icant over-representation of genes for lipid metabolism, such as for araquidonic acid metabolism (q = 0.006); amino acid metabolism pathways, such as for arginine and pro- line metabolism (q = 0.029); for glycine, serine, and threonine metabolism (q = 0.001); in genes for the metabolism of other amino acids such as glutathione metabolism (q = 0.003); for the metabolism of cofactors and vitamins such as riboflavin metabolism (q = 0.003), ubiquinone, and other terpenoid metabolism (q < 0.001); folate biosynthesis (q = 0.014), glycan biosynthesis, and metabolism, such as lipopolysaccharide biosynthesis (q = 0.007); lipopolysaccharide biosynthesis proteins (q = 0.001); cellular processes and signaling that contain cell motility and secretion (q = 0.0018); oxidative phosphorylation (q < 0.001); and for pathways in cancer (q < 0.001) (Figure 8). 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients Significant differences in the serum levels of several polyamines and acetyl derivatives of polyamines were found in the R and NR patients at post-treatment point (T3). Then, in the NR patients, we found a significant increase in the levels of spermine, N1-acetyl spermine (N1-AcSP), N1, N12-diacetylspermine (N1, N12-DiAcSP), N1-acetylspermidine Int. J. Mol. Sci. 2021, 22, 9549 13 of 25 13 of 25 (N1-AcSPD), N1, N8- diacetylspermidine (N1, N8-DiAcSPD), and N1-acetylputrescine (N1-AcPUT) compared to those in the R patients. On the other hand, within-group, there were also significant changes in the levels of N1-AcSPD and spermine in both the R and NR patients and in the serum levels of N8-AcSPD only in the NR group (Table 2). Figure 8. Heatmap of bacterial gene functional predictions using the PICRUSt algorithm from the fecal samples from the responder (R) patients and the non-responder (NR) patients. Table 2. Serum polyamines levels at baseline (T0) and post-treatment (T3). R Patients (N = 28) NR Patients (N = 12) Between-Group Difference 1 p 2 Agmatine (ng/mL) Baseline Post-treatment Change 0.11 ± 0.13 0.25 ± 0.24 0.14 (−0.27, −0.13) 0.13 ± 0.15 0.17 ± 0.15 0.035 (−0.13, 0.061) 0.025 (−0.11, 0.63) 0.571 Arginine (µg/mL) Baseline Post-treatment Change 23.18 ± 4.20 22.82 ± 4.16 −0.36 (−1.5, 2.27) 24.54 ± 4.76 23.10 ± 4.48 −1.43 (−1.13, 4.0) −1.35 (−4.05, 1.35) 0.319 Table 2. Serum polyamines levels at baseline (T0) and post-treatment (T3). Int. J. Mol. Sci. 2021, 22, 9549 14 of 25 Table 2. Cont. Table 2. Cont. Table 2. Cont. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients R Patients (N = 28) NR Patients (N = 12) Between-Group Difference 1 p 2 Ornithine (µg/mL) Baseline Post-treatment Change 19.46 ± 5.74 20.21 ± 4.16 0.74 (−3.69, 2.19) 23.31 ± 8.06 22.80 ± 7.55 −0.51 (−3.72, 4.74) −3.85 (−8.07, 0,37) 0.073 N1,N12-diacetylspermine (ng/mL) Baseline Post-treatment Change 1.08 ± 0.43 0.90 ± 0.52 −0.18 (0.017, 0.34) 1.68 ± 1.34 1.22 ± 0.57 0.46 (−0.152, 1.07) −0.59 (−1.20, 0.06) 0.015 N1,N8-diacetylspermidine (ng/mL) Baseline Post-treatment Change 0.71 ± 0.26 0.74 ± 0.34 0.03 (−0.13, 0.059) 0.99 ± 1.03 0.88 ± 0.38 −0.11 (−0.34, 0.57) −0.28 (−0.74, 0.17) 0.007 N1-acetylspermidine (ng/mL) Baseline Post-treatment Change 22.47 ± 7.10 23.42 ± 8.26 0.94 (−3.88, 1.99) * 27.68 ± 13.47 28.89 ± 10.38 1.20 (−6.10, 3.68) * −5.21 (−11.73, 1.3) 0.021 N8-acetylspermidine (ng/mL) Baseline Post-treatment Change 14.52 ± 3.48 14.69 ± 3.39 0.16 (−0.90, 0.57) 14.88 ± 3.27 16.10 ± 2.33 1.22 (−2.42, −0.20) * −0.35 (−2.38, 1.67) 0.727 N1-acetylputrescine (ng/mL) Baseline Post-treatment Change 5.04 ± 1.60 4.77 ± 1.70 −0.27 (−1.78, 1.09) 5.92 ± 5.38 5.39 ± 3.79 −0.53 (−1.01, 3.32) −0.88 (−3.29, 1.53) 0.030 Putrescine (ng/mL) Baseline Post-treatment Change 8.84 ± 4.40 8.06 ± 3.89 −0.78 (−0.39, 1.96) 7.95 ± 3.52 7.47 ± 3.09 −0.47 (−1.07, 2.02) 0.89 (−1.49, 3.28) 0.457 Spermidine (ng/mL) Baseline Post-treatment Change 17.14 ± 7.19 20.42 ± 12.40 3.28 (−7.42, 0.85) 22.26 ± 12.69 20.90 ± 10.81 −1.35 (−2.01, 4.73) −4.11 (−11.36, 1.12) 0.106 N1-acetylspermine (ng/mL) Baseline Post-treatment Change 0.89 ± 0.33 1.19 ± 0.63 0.29 (−0.55, −0.046) 1.48 ± 0.70 1.33 ± 0.62 −0.14 (−0.11, 0.40) −0.58 (−0.92, −0.25) 0.014 Spermine (ng/mL) Baseline Post-treatment Change 3.77 ± 1.30 4.80 ± 2.88 1.03 (−2.17, 0.107) * 12.10 ± 7.85 7.35 ± 3.66 −4.74 (1.71, 7.77) * −7.32 (−11.74, −4,89) 0.001 Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC- MS/MS). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model (ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC- MS/MS). 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients NR Patients (N = 12) Between-Group Difference 1 p 2 0.71 ± 0.15 0.77 ± 0.17 0.06 (−0.30, 0.18) 0.26 (−0.03, 0.56) 0.012 2.02 ± 1.35 1.70 ± 1.52 −0.32 (−0.9, 0.36) −0.68 (−0.86, 1.76) 0.102 0.93 ± 0.68 1.02 ± 1.07 0.09 (−0.65, 1.34) 1.33 (−0.04, 2.71) 0.016 0.31 ± 0.33 0.44 ± 0.15 −0.13 (−0.23, 0.76) 0.15 (0.03, 0.26) 0.010 0.61 ± 0.32 0.29 ± 0.19 -0.47 (−0.58, 0.76) −0.25 (−0.38, 0.29) 0.002 0.90 ± 0.44 0.39 ± 0.24 −0.51 (0.66, 1.02) −0.18 (−0.45, 0.29) 0.009 0.37 ± 0.64 0.04 ± 0.01 −0.33 (−0.47, 0.86) 0.20 (−0.35, 0.10) 0.216 0.11 ± 0.08 0.05 ± 0.09 −0.05 (−0.07, 0.13) 0.05 (−0.19, 0.13) 0.007 0.07 ± 0.06 0.05 ± 0.01 −0.02 (−0.04, 0.08) 0.02 (−0.07, 0.04) 0.171 272.6 ± 35.1 298.4 ± 47.5 25.2 (11.3, 37.1) −22.2 (−37.4, 10.2) 0.004 gas chromatography coupled with a flame-ionization detector r; NR: non-responder. 1 Difference between R and NR patients treatment changes was conducted with a covariance model calculate differences in the SCFAs and zonulin between the tatistically significant. Table 3. Fecal SCFAs concentrations and serum zonulin levels at baseline (T0) and post-treatment (T3). Table 3. Fecal SCFAs concentrations and serum zonulin levels at baseline (T0) and post-treatment (T3). 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients R Patients (N = 28) NR Patients (N = 12) Between-Group Difference 1 p 2 Acetic acid (mg/g) Baseline Post-treatment Change 0.83 ± 0.39 1.04 ± 0.40 0.20 (−0.39, 0.31) * 0.71 ± 0.15 0.77 ± 0.17 0.06 (−0.30, 0.18) 0.26 (−0.03, 0.56) 0.012 Propionic acid (mg/g) Baseline Post-treatment Change 1.40 ± 1.27 1.01 ± 1.10 −0.39 (−0.51, 0.59) 2.02 ± 1.35 1.70 ± 1.52 −0.32 (−0.9, 0.36) −0.68 (−0.86, 1.76) 0.102 Butyric acid (mg/g) Baseline Post-treatment Change 1.37 ± 0.45 2.36 ± 1.82 0.99 (−1.2, 2.15) * 0.93 ± 0.68 1.02 ± 1.07 0.09 (−0.65, 1.34) 1.33 (−0.04, 2.71) 0.016 Isobutyric acid (mg/g) Baseline Post-treatment Change 0.58 ± 0.33 0.69 ± 0.05 0.11 (0.07, 0.21) 0.31 ± 0.33 0.44 ± 0.15 −0.13 (−0.23, 0.76) 0.15 (0.03, 0.26) 0.010 Valeric acid (mg/g) Baseline Post-treatment Change 0.30 ± 0.16 0.13 ± 0.07 −0.17 (−0.27, 0.39) 0.61 ± 0.32 0.29 ± 0.19 -0.47 (−0.58, 0.76) −0.25 (−0.38, 0.29) 0.002 Isovaleric acid (mg/g) Baseline Post-treatment Change 0.50 ± 0.49 0.20 ± 0.13 −0.30 (−0.43, 0.31) 0.90 ± 0.44 0.39 ± 0.24 −0.51 (0.66, 1.02) −0.18 (−0.45, 0.29) 0.009 4-methylvaleric acid (mg/g) Baseline Post-treatment Change 0.13 ± 0.23 0.07 ± 0.10 −0.06 (−0.09, 0.15) 0.37 ± 0.64 0.04 ± 0.01 −0.33 (−0.47, 0.86) 0.20 (−0.35, 0.10) 0.216 Hexanoic acid (mg/g) Baseline Post-treatment Change 0.15 ± 0.20 0.10 ± 0.10 −0.04 (−0.09, 0.10) 0.11 ± 0.08 0.05 ± 0.09 −0.05 (−0.07, 0.13) 0.05 (−0.19, 0.13) 0.007 Heptanoic acid (mg/g) Baseline Post-treatment Change 0.09 ± 0.15 0.06 ± 0.06 −0.03 (−0.06, 0.07) 0.07 ± 0.06 0.05 ± 0.01 −0.02 (−0.04, 0.08) 0.02 (−0.07, 0.04) 0.171 Zonulin (ng/mL) Baseline Post-treatment Change 257.6 ± 65.4 218.1 ± 76.4 −39.3 (−52.2, 23.9) 272.6 ± 35.1 298.4 ± 47.5 25.2 (11.3, 37.1) −22.2 (−37.4, 10.2) 0.004 Short-chain fatty acids (SCFAs) in fecal samples were analyzed by means of gas chromatography coupled with a flame-ionization detector (GC-FID). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model (ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in the SCFAs and zonulin between the baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model (ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC- MS/MS). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model (ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC- MS/MS). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model (ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. SCFAs are bacterial-derived metabolites with important physiological functions in the host and that have anti-cancer properties. Analysis of the fecal levels of SCFAs revealed significant differences in the concentrations of acetic, butyric, isobutyric, valeric, isovaleric, and hexanoic acid between the R and NR study groups at post-treatment time point T3. Moreover, we found several significant differences in the within-group comparison of the fecal concentrations of acetic and butyric acid, which significantly increased after RCT treatment in the R group. On the other hand, serum zonulin levels (a circulating marker of gut permeability) were significantly increased in the NR group (but not in R group) after RCT treatment (Table 3). Int. J. Mol. Sci. 2021, 22, 9549 15 of 25 vels at baseline (T0) and post-treatment (T3). 3. Discussion In this study, we have demonstrated the existence of a significant association between the gut microbiota and the anti-cancer response of CRC patients treated with neoadjuvant RCT. Moreover, we have found that some microbial-derived metabolites such as SCFAs could be at least partially responsible for the response to RCT in these CRC patients. Finally, we have identified a baseline consortium of CRC-enriched bacterial species that may potentially serve as diagnostic bacterial markers of a good or bad response to neoadjuvant RCT. Where Ruminococcus albus, Bifidobacterium bifidum, and Faecalibacterium prausnitzii, were overrepresented in R patients and chosen as discriminatory variables in our response- prediction RF model, Fusobacterium nucleatum and Bacteroides fragilis were overrepresented in the NR patients. The loss of microbial diversity has been associated with chronic health conditions [25–27] and cancer [27,28] as well as with poor outcomes to certain forms of cancer therapy [29–31]. Accordingly, recent works have also reported that patients with CRC display a lower bacterial diversity and richness in fecal samples and the intestinal mucosa compared to healthy individuals [32,33]. In this study, we found that compared to healthy controls, the CRC microbiota exhibited a state of dysbiosis with a reduced overall bacterial richness and diversity. In addition, the analysis of the Bray–Curtis PCoA plot for beta diversity revealed that the CRC patients were clustered separately to the healthy controls, suggesting important CRC-mediated microbial changes. Related to gut microbiota composition, several microbes have been found to be differ- entially represented in fecal samples between both study groups. Thus, the gut microbiota in the CRC patients was enriched with pro-inflammatory opportunistic pathogens and was depleted in butyrate-producing bacteria, which have been shown to be essential for the preservation of intestinal homeostasis [34]. In particular, we have shown that some bacteria such as Fusobacterium nucleatum, Escherichia coli, and Bacteroides fragilis had high prevalence in CRC patients in comparison to the healthy controls, whereas genera such as Roseburia, Faecalibacterium, and Bifidobacterium were depleted, demonstrating that microbial dysbiosis was already present in CRC at the time of diagnosis. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients (r = 0.547 p = 0.014; r = 0.752 p < 0.001) in the NR patients. Finally, Prevotella copri was positively associated with the serum zonulin levels in NR patients. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT Treatment in CRC Patients Short-chain fatty acids (SCFAs) in fecal samples were analyzed by means of gas chromatography coupled with a flame-ionization detector (GC-FID). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model (ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in the SCFAs and zonulin between the baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Short-chain fatty acids (SCFAs) in fecal samples were analyzed by means of gas chromatography coupled with a flame-ionization detector (GC-FID). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model (ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in the SCFAs and zonulin between the baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. In addition, pairwise comparisons using Spearman rank correlation analysis were then performed between bacterial species enriched in the gut microbiome of both the R and NR patients and the fecal SCFAs and serum polyamines and zonulin levels. Interestingly, we found a statistically significant positive correlation between the fecal levels of butyrate and the abundance of the Faecalibacterium prausnitzii (r = 0.816 p < 0.001) and Ruminoccocus albus (r = 0.924 p = 0.008) in the R group and between the concentration of propionic acid and Bacteroides fragilis in the NR group. In addition, negative associations of Faecalibacterium prausnitzii with the serum levels of spermine (r = −0.619 p = 0.018) and N1,N12-DiAcSP (r = −0.793 p = 0.01) in the R patients were described, while there was a positive association between the abundance of Bacteroides fragilis and Fusobacterium nucleatum with the levels of N1,N12-DiAcSP (r=0.436 p = 0.043; r = 0.637 p = 0.001, respectively) and N8-AcSPD Int. J. Mol. Sci. 2021, 22, 9549 16 of 25 (r = 0.547 p = 0.014; r = 0.752 p < 0.001) in the NR patients. Finally, Prevotella copri was positively associated with the serum zonulin levels in NR patients. 3. Discussion On the other hand, we observed that gut microbiota composition was relatively stable over treatment time following RCT treatment, with the exception of a significant decrease in the abundance of Fusobacterium, Escherichia, and Klebsiella and a significant increase in Bifidobacterium (probiotic bacteria) at post-treatment time compared to at baseline, show- ing the beneficial effect of RCT on the gut microbiome of CRC patients. Klebsiella and Fusobacterium are pathogens normally found in the human intestine that cause diarrhea and bloodstream infections and that considerably increase the rates of treatment failure and death [35]. After treatment, the CRC patients were classified as responders (N) versus non- responders (NR), based on their good or poor response to the RCT. Interestingly, we found significant differences in the alpha diversity at the genus level, with an increase in the diversity (Shannon) and richness (Chao 1) in the R patients compared to in the NR patients. Similarly, there was a statistically significant difference in B-diversity (Bray–Curtis dissimilarities and Jaccard index), finding a notable clustering effect by response status in the gut microbiome of these patients, indicating a difference in the bacterial community composition between the R and NR patients. At the taxa levels, we found a significant enrichment in probiotic and butyrate producer-bacteria such as Bifidobacterium bifidum, Ruminoccous albus, Roseburia, and Faecal- ibacterium prausnitzii in the R patients, while the NR patients showed an enrichment in unfavorable microbial taxa such as Fusobacterium nucleatum, Bacteroides fragilis, Escherichia coli, Prevotella copri, and Klebsiella. Several studies have shown that butyrate-producing bacteria are negatively related to irritable bowel disease and colorectal cancer [36,37]. Int. J. Mol. Sci. 2021, 22, 9549 17 of 25 17 of 25 Additionally, both Fusobacterium and Prevotella have been related to recurrent CRC after chemotherapy. Given that Fusobacterium nucleatum has been previously correlated with chemoresistance [17], our results may suggest that the higher load of Fusobacterium nucleatum present in NR patients could be a potential promoter of CRC chemoresistance and therefore of a poor response to CRC treatment. Similarly, the enterotoxigenic Bacteroides fragilis, which was also enriched in the NR patients, is a significant source of chronic inflam- mation, and it has previously been associated with the development and aggressiveness of colorectal cancer and poor patient outcome [6,38]. 3. Discussion These data also suggest that the gut microbiota composition of the R patients shifted towards a microbial profile that has great similarity to the gut microbiota of the healthy controls. Next, we sought to gain insight into the mechanism through which the gut micro- biome may influence response to RCT. Regarding the metabolic function of gut microbiota, in the current study, Picrust analysis showed significant differences between the R and NR patients. In the NR patients, we have found an increase in the abundance of genes for lipopolysaccharide biosynthesis as well as for araquidonic acid metabolism, for glu- tathione metabolism, and for the amino acid metabolism pathways (such as arginine and proline metabolism) compared to in the R patients. The significant increase of genes for lipopolysaccharide biosynthesis could be related to the significant increase in the abun- dance of Gram-negative bacteria such as Escherichia coli in the NR patients; these bacteria contain specific enzymes that produce LPS, which can induce Toll-like receptor 4 signal- ing and can promote cell survival and proliferation in CRC patients [39]. Similarly, the arachidonic acid pathway is important in the development and progression of numerous malignant diseases, including CRC, due to the fact that araquidonic acid stimulates key downstream signaling cascades that regulate cell proliferation, apoptosis, angiogenesis, in- flammation, and immune surveillance [40,41]. With respect to the increase in the genes for glutathione metabolism in NR patients, some studies have described that the elevated lev- els of glutathione in tumor cells are able to protect such cells in bone marrow, breast, colon, larynx, and lung cancers by conferring resistance to several chemotherapeutic drugs [42,43]. Other bacterial functions involving the metabolism of cofactors and vitamins and the en- ergy production pathways such as oxidative phosphorylation were also increased in NR patients. These pathways may serve as alternative bioenergetic sources for metabolically stressed cancer cells [44]. Remarkably, a recent metagenomic analysis reported that the CRC-associated micro- biome showed an association with the conversion of amino acids into polyamines (e.g., the biosynthesis of putrescine from the amino acids L-arginine and L-ornithine), indicating that these metabolites could be particularly important in CRC development and progres- sion [45]. In our study, significant differences in the serum levels of several polyamines and acetyl derivatives of polyamines were found between R and NR patients at post-treatment point. 3. Discussion Moreover, we observed that the abundance of N1,N12-DiAcSP and N8-AcSPD were positively associated with the increased abundance of Bacteroides fragilis and Fusobacterium nucleatum in NR patients. p In fact, Bacteroides spp. and Fusobacterium spp. can synthesize putrescine and spermidine in vitro and in vivo [46]. Goodwin et al. demonstrated that the purified Bacteroides fragilis toxin (BFT) up-regulates spermine oxidase in HT29/c1 and T84 colonic epithelial cells, producing the spermine oxidase-dependent generation of ROS and the induction of a marker of DNA damage such as γ-H2A.x. [47]. In another study, Johnson et al. found that antibiotic treatment led to a lower tissue concentration of N1, N12- diacylspermine and that a disturbed bacterial biofilm was observed in resected CRC tissues compare to CRC tissues with negative bacterial biofilm, suggesting the implication of gut microbes in the increase of host generated N1, N12-diacetylspermine [48]. Moreover, the activation of the amino acid metabolic pathways by the intestinal microbiota of the NR patients could contribute to the increase in polyamines, which are actively assimilated by the cells of the intestinal epithelium and induce rapid cell proliferation, favoring the tumorigenesis [49,50]. Int. J. Mol. Sci. 2021, 22, 9549 18 of 25 18 of 25 On the other hand, several works performed in both cellular and animal models have demonstrated that CRC is linked to alterations in the metabolism of SCFAs, which have been shown to exhibit potential anti-carcinogenic effects [51,52]. Here, we have found that R patients displayed a significant over-representation of genes involved in butanoate metabolism and a significant increase in the fecal abundance of several SCFAs such as acetic and butyric acid after RCT treatment. Moreover, there was a positive correlation between the fecal levels of butyrate and the abundance of Faecalibacterium prausnitzii and Ruminoccocus albus in these patients. Faecalibacterium praustnitzi is considered important in health promotion, as it is able to produce butyrate from dietary fibre and possesses anti-inflammatory properties [53]. A decrease in Faecalibacterium prausnitzii and butyrate levels defines microbiota dysbiosis in patients suffering inflammatory bowel disease [54]. In addition, Faecalibacterium is able to use the acetate produced by Bifidobacterium (also increased in N patients) with the subsequent modulation of the intestinal mucus barrier by the modification of goblet cells and mucin glycosylation [55]. Butyrate is required for colonic epithelium repair and the production of Treg cells, which regulate the local immune response and suppress colonic inflammation and carcinogenesis [56]. 3. Discussion Moreover, butyrate has been described to be able to induce the production of IL-18 by the intestinal epithelial cells through the activation of the GPR109a receptor, which stimulates mucosal tissue repair via the regulation of the production and availability of IL-22 [57]. The absence of IL-18 has been associated with gut microbiota dysbiosis, a dysregulation of the homeostatic and mucosal repair and alteration of the inflammatory response, producing an increased susceptibility to carcinogenesis [58]. In addition, after RCT treatment, we found a significant decrease in the fecal levels of acetic, butyric, isobutyric, and hexanoic acid in the NR study group compared to in R patients, indicating the exhaustion of butyric acid-producing microbiota in their colon. In a previous study, hexanoic acid was shown to reduce the colonization and dysbiotic expansion of potentially pathogenic bacteria in the gut [59]. y p p y p g g Finally, we found that plasma zonulin levels were significantly increased in the NR patients compared to in the R patients. A higher zonulin level was correlated with the relaive abundance of Prevotella copri in the R patients. Zonulin is a protein synthesized in intestinal and liver cells that reversibly modulates the intestinal permeability of the intestinal epithelial barrier by modulating intercellular tight junctions [60]. Wright et al. found that Prevotella contains key enzymes implicated in mucin degradation, which are able to disrupt the colonic mucus barrier. A disrupted mucosal barrier may result in increased intestinal permeability, which allows the diffusion of antigens, toxins, and pathogens from the luminal environment into the mucosal tissues and circulatory system [55]. As a consequence, an inflammatory response can be triggered that induces cancer initiation, progression, and response to anticancer treatment [61]. Then, the significant increase in Prevotella abundance found in our study could be associated in party with the poor or non-response to RCT in NR patients. This study has some limitations, such as the relatively small sample size, which could reduce the power of the study. However, despite the relatively small size of our study, sta- tistically significant differences were observed, suggesting that the results presented herein provide solid evidence on the potential contribution of the gut microbiome to RCT out- comes in CRC patients. 3. Discussion Moreover, our study also has several strengths, such as the careful design, the well-matched cohorts of CRC patients and controls, a complete definition of the inclusion and exclusion criteria, and the consideration of lifestyle-associated confounding factors that may affect the gut microbiota composition, such as dietary pattern. 4.3. DNA Extraction and Gut Microbiota Sequencing The frozen fecal samples were thawed at 4 ◦C to avoid dramatic temperature changes that might affect bacterial DNA integrity. Afterwards, the fecal samples were manually homogenized for 30 s with a sterile plastic scoop, and aliquots of 200 mg were used for DNA extraction using the QIAamp DNA Stool Mini kit following the manufacturer’s instructions (Qiagen, Hilden, Germany). DNA concentration (A260) and purity (A260/A280 ratio) were estimated with a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). DNA was amplified using the Ion 16S Metagenomics kit (Thermo Fisher Scientific, Madrid, Spain), which contains a primer pool to amplify multiple variable regions (V2, 3, 4, 6–7, 8 and 9) of the 16S rRNA gene. The Ion PlusTM Fragment Library Kit (Thermo Fisher Scientific, Madrid, Spain) was used to ligate the barcoded adapters to the generated amplicons and to create the barcoded libraries, which were pooled and templated on the automated Ion Chef system (Thermo Fisher Scientific, Madrid, Spain). The sequencing was done on an Ion S5 platform (Thermo Fisher Scientific, Madrid, Spain). 4.2. Laboratory Measurements Fasting venous blood samples were collected, and serum was separated in aliquots and was immediately frozen at −80 ◦C. Serum levels of glucose, total cholesterol, triglycerides, HDL-cholesterol, and LDL-cholesterol were measured in duplicate using a Dimension autoanalyzer (Dade Behring Inc., Deerfield, IL, USA) using enzymatic methods (Randox Laboratories Ltd. Ardmore, UK). 4. Materials and Methods 4.1. Study Patients A total of forty patients aged 35–75-years-old who were newly diagnosed with CRC in stages II–III (T2–T4 and/or N1–N2) from the Radiotherapy Oncology Service at the Virgen de la Victoria Hospital and with no metastatic lesions detected on imaging were enrolled in the study and were followed-up with for at least 1 year. All of the CRC patients Int. J. Mol. Sci. 2021, 22, 9549 19 of 25 19 of 25 received only neoadjuvant treatment for 5 weeks with pelvic radiation therapy (50 Gy in fractions of 2 Gy/session) and oral capecitabine (825 mg/m2/12 h) during radiotherapy treatment. Patients with a history of colorectal cancer or bowel resection, type 2 dia- betes, chronic inflammatory bowel disease, severe active infection, or hereditary colorectal cancer syndromes were excluded from the study. Patients who received pelvic cancer radiation therapy or anti-tumor treatment in the previous 2 years, who used antibiotics or immunosuppressants in the previous 2 months, or who regularly used non-steroidal anti-inflammatory drugs, statins, or probiotics before the study were also excluded. A pathologist examined surgical specimens and tumor response after neoadjuvant RCT was determined in surgical specimens according to the tumor regression grades (TRG) system described by Mandard et al. [62]. We divided the CRC patients into TRG1–2 (patients with good response or responders (R)) and TRG 3–5 (patients with poor or non-response (NR)). Blood and fecal samples were collected at baseline (T0), 2 and 4 weeks after starting RCT (T1 and T2, respectively), and 7 weeks after finishing treatment (T3). In the study, we also included fecal samples from 20 healthy patients that were matched with the CRC patients according to sex, age, and BMI. The healthy controls did not have gastrointestinal tract disorders or other complications and were not administered antibiotics or probiotics during the 2 months prior to sample collection. The study protocol was approved by the Medical Ethics Committee at the Virgen de la Victoria University Hospital and was conducted in accordance with the Declaration of Helsinki. Written informed consent was provided by all study participants. 4.4. Bioinformatics Analysis Analysis of 16S rRNA amplicons was performed using QIIME (2-2019.4 version). The q-dada2 plugin with the DADA2 pipeline was used for the quality filtering and the denoised, dereplicated, and chimera filtering of the raw sequence data. The sequence variants obtained through the DADA2 pipeline were merged into a single feature table using the q2-feature-table plugin. Using the q2-vsearch plugin with 97% sequence simi- larity, all amplicon sequence variants from the merged feature table were clustered into OTU’s using the Open Reference Clustering method against Greengenes version 13_8 with 97% similarity from the OTU reference sequences. The OTUs were aligned with Int. J. Mol. Sci. 2021, 22, 9549 20 of 25 MAFFT (via q2-alignment) and were used to construct a phylogeny with fasttree2 (via q2-phylogeny). The q2-feature-classifier classify-sklearn naive Bayes taxonomy classifier was used to assign taxonomy to the OTUs. Alpha diversity metrics (Shannon and Chao1), beta diversity metrics (Bray–Curtis dissimilarity), and principal coordinate analysis (PCoA) were estimated using a q2-diversity plugin after the samples were rarefied to 994 sequences per sample. Alpha diversity significance was estimated with Kruskal–Wallis test, and beta diversity significance was estimated using the non-parametric ANOSIM test. MAFFT (via q2-alignment) and were used to construct a phylogeny with fasttree2 (via q2-phylogeny). The q2-feature-classifier classify-sklearn naive Bayes taxonomy classifier was used to assign taxonomy to the OTUs. Alpha diversity metrics (Shannon and Chao1), beta diversity metrics (Bray–Curtis dissimilarity), and principal coordinate analysis (PCoA) were estimated using a q2-diversity plugin after the samples were rarefied to 994 sequences per sample. Alpha diversity significance was estimated with Kruskal–Wallis test, and beta diversity significance was estimated using the non-parametric ANOSIM test. 4.5. Analysis of Short-Chain Fatty Acids (SCFAs) in Fecal Samples by Gas Chromatography (GC) Coupled with a Flame-Ionization Detector The fecal concentrations of SCFAs were measured by GC coupled with a flame- ionization detector as previously described [63–66] in the Servicios de Apoyo a la Investi- gación de la Universidad de Extremadura (SAIUEx). Briefly, 20 mg of the fecal samples were homogenized manually using a spatula in 200 µL of distilled water. Subsequently, 100 µL of homogenized fecal samples were mixed with 40 mg of sodium chloride, 20 mg of citric acid, 40 µL of 0.1 M hydrochloric acid, and 200 µL of butanol: tetrahydrofuran: acetonitrile (50:30:20). The samples were then vigorously vortexed for 3 min and were cen- trifuged at 14,870× g at room temperature for 10 min. 4.4. Bioinformatics Analysis The supernatant was transferred to a new plastic tube, and 200 µL of a benzyl alcohol–pyridine mixture (3:2) and 100 µL DMSO were added, and the mixture was vortexed for 5 s. Then, 100 µL of benzyl chloroformate was added carefully. To release the gases generated by the reaction, the tube lid was kept open for 1 min. The tube was then closed, and the mixture was vortexed. After derivati- zation, 200 µL hexane was added to the reaction mixture, and the sample was vortexed for 5 min followed by a centrifugation step at 21,000× g for 2 min. Subsequently, 100 µL of derivative extract (upper hexane layer) was transferred to a glass insert, and 5 µL were injected into the gas chromatograph and were further analyzed using an Agilent 6850 gas chromatograph coupled with a split/spitless injector and a flame-ionization detector (FID) (Agilent Technologies, Santa Clara, CA, USA). The temperature of the injector and detector was adjusted to 250 ◦C, and the samples (5 µL) were injected in a split ratio of 25:1 using a fused-silica capillary DB-23 column Agilent (60 m × 0.25 mm (internal diameter) coated with a 0.15 µm thick layer of 80.2% 1-methylnaphatalene. Nitrogen was used as the carrier gas at 1 mL/min (hold 4 min), reduced to 0.8 mL/min (hold 1 min) and then 0.6 mL/min (hold 1 min), and finally increased to 1 mL/min. The temperature of the FID detector was adjusted and maintained at 260 ◦C, and the flow rates of H2, the air, and the make-up gas N2 were adjusted to 30 mL/min, 350 mL/min, and 25 mL/min, respectively. The initial oven temperature was 100 ◦C (hold 2 min), which was increased to 200 ◦C at a rate of 15 ◦C/min, and was finally maintained at 200 ◦C for 5 min. The identity of the SCFAs detected in the fecal samples was confirmed through the comparison of their retention times and their mass spectra with those of the analytical SCFA standards (Sigma–Aldrich, Madrid, Spain). The standard calibration curves for SCFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 4- methylvaleric acid, hexanoic acid, and heptanoic acid) were prepared in triplicate, with a concentration range of 15–1,000 µg/mL. 4.6. Analysis of Serum Polyamine Levels by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC-MS/MS) 4.6. Analysis of Serum Polyamine Levels by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC-MS/MS) For the analysis of the polyamine concentrations, serum samples were processed as previously described [67]. Briefly, 50 µL of serum (aliquoted in 1.5 mL Eppendorf LoBind tube) were mixed with 5 µL of internal standard and 167 µL of methanol. The mixture was vortexed for 1 min, and 334 µL of chloroform was added, vortexed for 1 min, and centrifuged for 10 min at 15,000 rpm and 4 ◦C. After centrifugation, the upper layer was collected and was transferred to a new tube, where 100 µL of carbonate–bicarbonate buffer (pH 9) and 50 µL of dansyl chloride (10 mg/mL in acetone) were added to derivatize the sample. The mixture was vortexed and was placed in the dark for 1 h at room temperature. A total of two extractions of the compounds were conducted with 250 µL of ethyl acetate, Int. J. Mol. Sci. 2021, 22, 9549 21 of 25 21 of 25 between which 2.5 µL of trifluoroacetic acid were added. A SpeedVac at 45 ◦C was used to evaporat the combined organic phases, which were stored at −20 ◦C until analysis. An amount of 50 µL of ammonium acetate and 0.2 M acetonitrile (30:70) was used to reconstitute the samples. Chromatography of the samples was completed with Agilent UHPLC 1290 series binary pump equipment (Agilent Technologies, Santa Clara, CA, USA), and the separation was performed on a Kinetex EVO C18 column (2.6 µm particle size, 2.1 mm internal diameter × 150 mm length) (Phenomenex, Torrance, CA, USA) held at 25 ◦C. A gradient was established between the water acidified with 0.1% formic acid (A), and acetonitrile acidified with 0.1% formic acid (B) at a flow rate of 400 µL/min was used as a mobile phase for elution. The injected amount was 2.5 µL. MS/MS analysis was conducted in an Agilent QqQ 6490 Series mass spectrometer operating in AJS + ESI. 4.6. Analysis of Serum Polyamine Levels by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC-MS/MS) The optimization of the ionization source parameters was performed as follows: nebulizer gas (nitrogen) with a pressure of 15 psi, a gas flow of 15 L/min at 200 ◦C, a sheath gas flow of 11 L/min at 350 ◦C, a capillary voltage of 2.5 kV, and a nozzle voltage of 1000 V in a MassHunter Optimizer (Agilent Technologies, version 6.0) g p g g An Agilent UHPLC 1290 Infinity II Series coupled to an Agilent QqQ/MS 6490 Series (Agilent Technologies, Sta. Clara, CA, USA) was used for LC-MS/MS analysis, while chromatographic separation was performed using a Kinetex EVO C18 analytical column (2.6 µm; 2.1 mm × 150 mm) (Phenomenex, Torrance, CA, USA) Quantification was completed with the commercial standards ornithine, spermine, arginine, spermidine, putrescine, N1-acetylspermidine, N8-acetylspermidine, N1-acetylsp- ermine, N1-acetylputrescine, N1,N8-diacetylspermidine, and N1,N12-diacetylspermine (Toronto Research Chemicals, North York, ON, Canada). The internal standards of the amino acids were arginine (13C6, 15N4) and lysine (13C6, 15N2) (Cambridge Isotope Laboratories), and for the polyamines, the internal stanfdards comprised putrescine-d8, spermidine-d6, spermine-d20, and N8-acetylspermidine-d3 (Toronto Research Chemicals). 4.7. Intestinal Permeability Analysis Plasma levels of zonulin were measured in duplicate using an ELISA commercial kit (Immunodiagnostik AG, Bensheim, Germany). Mean values were used for data analysis. Intra- and inter-assay coefficients of variation were between 3–10%, and the detection limit was 0.22 ng/mL. 4.8. Statistical Analysis The Kruskal–Wallis rank-sum test was performed to compare the bacterial abun- dance between the study groups, and the false discovery rate (FDR) using the Benjamini– Hochberg method was applied to correct the significant p-values (q < 0.05). The Kruskal– Wallis rank-sum test and subsequent post hoc Bonferroni were used to analyze differences in the clinical and biochemical variables between three study groups, whereas differences between the two groups were analyzed using the Mann–Whitney U test. Inter-group comparison among post-treatment changes in fecal SCFAs and plasma zonulin levels were performed using a covariance model (ANCOVA) adjusted for baseline. A Wilcoxon signed-rank test was used to calculate differences in fecal SCFAs and plasma zonulin between baseline and the post-treatment timepoint T3. The Spearman correlation coeffi- cients were calculated to estimate the correlations between the bacterial taxa and microbial derived-metabolites (SCFAs and polyamines) and the permeability. Statistical analyses were conducted with the statistical software package SPSS version 26.0 (SPSS Inc., Chicago, IL, USA). Random forests (RF) were used to predict baseline bacteria (species-level relative abundance data) related to the neoadjuvant RCT response using the default parameters of the R implementation of the algorithm (R package “randomForest”), and bootstrapping (n = 500) was used to assess the classification accuracy. P values below 0.05 were considered statistically significant. Int. J. Mol. Sci. 2021, 22, 9549 22 of 25 5. Conclusions In this study, we have demonstrated that the gut microbiota in CRC patients differs in intestinal microbiota composition in comparison with healthy controls. In CRC patients, the gut microbiota is characterized by a significantly lower bacterial diversity and richness, a significant increase in proinflammatory opportunistic pathogens, and a decrease in the relative abundance of beneficial or commensal butyrate-producing bacteria. In addition, neoadjuvant RCT treatment did not induce significant changes in gut microbiota diversity and composition, with the exception of a significant decrease in Fusobacterium, Escherichia, and Klebsiella and a significant increase in Bifidobacterium at post-treatment time compared to baseline. Nevertheless, after the classification of CRC patients in the R and NR groups to the neoadjuvant RCT, we observed a significant increase in the diversity and richness in R patients compared to in the NR patients. Additionally, a compositional change was shown between both study patient groups, with a significant enrichment of probiotic and butyrate-producing bacteria in the R patients, accompanied by an enrichment in unfavorable pro-inflammatory bacteria in the NR patients. Moreover, the NR patients had significantly higher levels of spermine and some acetyl derivatives of polyamines and serum zonulin and significantly lower levels of fecal of acetic, butyric, isobutyric, and hexanoic acids than the R patients. These microbial-derived metabolites are important factors that connect the intestinal microbiota to CRC and could be respon- sible for RCT efficiency. Moreover, in the NR patients, the PICRUSt analysis found an over-representation of genes involved in lipopolysaccharide biosynthesis as well as in araquidonic acid and glutathione metabolism, genes from pathways associated with bacte- rial pathogenesis, inflammation, cell survival, proliferation, and therapy response. In addition, we also identified a baseline consortium of CRC-enriched bacterial species (Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nu- cleatum, and Bacteroides fragilis) that potentially could predict cancer treatment outcome, suggesting that the intestinal composition in CRC patients is important in predicting the response of the gut microbiome to neoadjuvant RCT. Altogether, our results suggest that a healthy gut microbiome could be indispensable for an optimum therapeutic response and that dysbiotic microbiota could be the underlying reason for variable responses to similar therapeutic strategies in different patients. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22179549/s1. Author Contributions: Conceptualization, M.I.Q.-O. and J.G.-M.; methodology, L.S.-A., A.L.-I., B.R.-M., I.P.-A. and A.G.-G.; Investigation, L.S.-A., B.R.-M., I.P.-A., A.L.-I., A.O., R.O. and A.G.-G.; validation, L.S.-A., A.O., R.O., I.P.-A. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22179549/s1. Conflicts of Interest: The authors declare no conflict of interests. Conflicts of Interest: The authors declare no conflict of interests. Conflicts of Interest: The authors declare no conflict of interests. References 1. Arnold, D.; Lueza, B.; Douillard, J.Y.; Peeters, M.; Lenz, H.J.; Venook, A.; Heinemann, V.; Van Cutsem, E.; Pignon, J.P.; Tabernero, J.; et al. Prognostic and predictive value of primary tumour side in patients with RAS wild-type metastatic colorectal cancer treated with chemotherapy and EGFR directed antibodies in six randomized trials. Ann. Oncol. 2017, 28, 1713–1729. [CrossRef] 1. Arnold, D.; Lueza, B.; Douillard, J.Y.; Peeters, M.; Lenz, H.J.; Venook, A.; Heinemann, V.; Van Cutsem, E.; Pignon, J.P.; Tabernero, J.; et al. Prognostic and predictive value of primary tumour side in patients with RAS wild-type metastatic colorectal cancer treated with chemotherapy and EGFR directed antibodies in six randomized trials. Ann. Oncol. 2017, 28, 1713–1729. [CrossRef] 2. Liang, Q.; Chiu, J.; Chen, Y.; Huang, Y.; Higashimori, A.; Fang, J.; Brim, H.; Ashktorab, H.; Ng, S.C.; Ng, S.S.M.; et al. Fecal bacteria act as novel biomarkers for noninvasive diagnosis of colorectal cancer. Clin. Cancer Res. 2017, 23, 2061–2070. [CrossRef] 3. Yazici, C.; Wolf, P.G.; Kim, H.; Cross, T.L.; Vermillion, K.; Carroll, T.; Augustus, G.J.; Mutlu, E.; Tussing-Humphreys, L.; Brauschweig, C.; et al. Race-dependent association of sulfidogenic bacteria with colorectal cancer. Gut 2017, 66, 1983–1994. [CrossRef] [PubMed] py 2. Liang, Q.; Chiu, J.; Chen, Y.; Huang, Y.; Higashimori, A.; Fang, J.; Brim, H.; Ashktorab, H.; Ng, S.C.; Ng, S.S.M.; et al. Fecal bacteria act as novel biomarkers for noninvasive diagnosis of colorectal cancer. Clin. Cancer Res. 2017, 23, 2061–2070. [CrossRef] 3. Yazici, C.; Wolf, P.G.; Kim, H.; Cross, T.L.; Vermillion, K.; Carroll, T.; Augustus, G.J.; Mutlu, E.; Tussing-Humphreys, L.; Brauschweig, C.; et al. Race-dependent association of sulfidogenic bacteria with colorectal cancer. Gut 2017, 66, 1983–1994. [CrossRef] [PubMed] 4. Yu, J.; Feng, Q.; Wong, H.S.; Zhang, D.; Liang, Q.Y.; Qin, Y.; Tang, L.; Zhao, H.; Stenvang, J.; Li, Y.; et al. Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer. Gut 2017, 66, 70–78. [CrossRef] [PubMed] [ ] 5. Helmink, B.A.; Khan, M.A.W.; Hermann, A.; Gopalakrishnan, V.; Wargo, J.A. The microbiome, cancer, and cancer therapy. Nat. Med. 2019, 25, 377–388. [CrossRef] [PubMed] 6. McQuade, J.L.; Daniel, C.R.; Helmink, B.A.; Wargo, J.A. Modulating the microbiome to improve therapeutic response in cancer. Lancet Oncol. 2019, 20, e77–e91. [CrossRef] 7. Yi, Y.; Shen, L.; Shi, W.; Xia, F.; Zhang, H.; Wang, Y.; Zhang, J.; Wang, Y.; Sun, X.; Zhang, Z.; et al. References Scott, T.A.; Quintaneiro, L.M.; Norvaisas, P.; Lui, P.P.; Wilson, M.P.; Leung, K.Y.; Herrera-Dominguez, L Clayton, P.T.; et al. Host-microbe co-metabolism dictates cancer drug efficacy in C. elegans. Cell 2017, 1 15. Gonzalez-Sarrias, A.; Tome-Carneiro, J.; Bellesia, A.; Tomas-Barberan, F.A.; Espin, J.C. The ellagic acid-derived gut microbiota metabolite, urolithin A, potentiates the anticancer effects of 5-fluorouracil chemotherapy on human colon cancer cells. Food Funct. 2015, 6, 1460–1469. [CrossRef] 16. Liu, J.; Liu, C.; Yue, J. Radiotherapy and the gut microbiome: Facts and fiction. Radiat. Oncol. 2021, 16, 9. [CrossRef] [PubMed] 17. Yu, T.; Guo, F.; Yu, Y.; Sun, T.; Ma, D.; Han, J.; Qian, Y.; Kryczek, I.; Sun, D.; Nagarsheth, N.; et al. Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Cell 2017, 170, 548–563.E16. [CrossRef] [PubMed] 16. Liu, J.; Liu, C.; Yue, J. Radiotherapy and the gut microbiome: Facts and fiction. Radiat. Oncol. 2021, 16, 17. Yu, T.; Guo, F.; Yu, Y.; Sun, T.; Ma, D.; Han, J.; Qian, Y.; Kryczek, I.; Sun, D.; Nagarsheth, N.; et al. Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Cell 2017, 170, 548–563.E16. [CrossRef] [PubMed] 18. Roy, S.; Trinchieri, G. Microbiota: A key orchestrator of cancer therapy. Nat. Rev. Cancer 2017, 17, 271–285. [CrossRef] 19. Ohara, T.; Suzutani, T. Intake of Bifidobacterium longum (BB536) and Fructo-Oligosaccharides (FOS) prevents colorectal carcinogenesis. Euroasian J. Hepatogastroenterol. 2018, 8, 11–17. [CrossRef] 18. Roy, S.; Trinchieri, G. Microbiota: A key orchestrator of cancer therapy. Nat. Rev. Cancer 2017, 17, 271–2 19 Oh T S t i T I t k f Bifid b t i l (BB536) d F t Oli h id (F 18. Roy, S.; Trinchieri, G. Microbiota: A key orchestrator of cancer therapy. Nat. Rev. y y py 19. Ohara, T.; Suzutani, T. Intake of Bifidobacterium longum (BB536) and Fructo-Oligosaccharides (FOS) prevents colorectal carcinogenesis. Euroasian J. Hepatogastroenterol. 2018, 8, 11–17. [CrossRef] g J p g 20. Ross, K. Gut microbial short chain fatty acids are associated with pathological complete response (pCR) after neoadjuvant chemother- apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 October 2020. 21. Coutzac, C.; Jouniaux, J.M.; Paci, A.; Schmidt, J.; Mallardo, D.; Seck, A.; Asvatourian, V.; Cassard, L.; Saulnier, P.; Lacroix, L.; et al. Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer. Nat. Commun. 2020, 11, 2168. g p g 20. Ross, K. 5. Conclusions and A.G.-G.; writing—original draft preparation, M.I.Q.-O., J.G.-M., L.S.-A. and B.R.-M.; writing—review and editing, all authors; funding acquisition, M.I.Q.-O.; supervision, M.I.Q.-O. and J.G.-M. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.I.Q.-O. and J.G.-M.; methodology, L.S.-A., A.L.-I., B.R.-M., I.P.-A. and A.G.-G.; Investigation, L.S.-A., B.R.-M., I.P.-A., A.L.-I., A.O., R.O. and A.G.-G.; validation, L.S.-A., A.O., R.O., I.P.-A. and A.G.-G.; writing—original draft preparation, M.I.Q.-O., J.G.-M., L.S.-A. and B.R.-M.; writing—review and editing, all authors; funding acquisition, M.I.Q.-O.; supervision, M.I.Q.-O. and J.G.-M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by PI15/00256 from the Institute of Health “Carlos III” (ISCIII), co-funded by the Fondo Europeo de Desarrollo Regional-FEDER. Maria Isabel Queipo-Ortuño was supported by the “Miguel Servet Type II” program (CPI18/00003, ISCIII, Spain, co-funded by the Fondo Europeo de Desarrollo Regional-FEDER) and by the “Nicolas Monardes” research program of the Consejería de Salud (C-0030-2018, Junta de Andalucía, Spain. Bruno Ramos Molina was supported by the “Miguel Servet Type I” program (CP19/00098, ISCIII, Spain, co-funded by the Fondo Europeo de Desarrollo Regional-FEDER). Lidia Sanchez-Alcoholado was the recipient of a predoctoral grant (PE-0106-2019) from the Consejería de Salud y Familia (co-funded by the Fondo Europeo de Desarrollo Regional-FEDER, Andalucia, Spain). Aurora Laborda-Illanes was the recipient of a predoctoral grant, PFIS-ISCIII (FI19-00112), co-funded by the Fondo Europeo de Desarrollo Regional-FEDER, Madrid, Spain. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of Virgen de la Victoria University Hospital (30 October 2015). Int. J. Mol. Sci. 2021, 22, 9549 23 of 25 23 of 25 Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available upon request from the corresponding author. The data are not publicly available, as they contain information that could compromise the privacy of research participants. References Gut microbiome components predict response to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer: A prospective, longitudinal study. Clin. Cancer Res. 2021, 27, 1329–1340. [CrossRef] [PubMed] y 8. Alexander, J.L.; Wilson, I.D.; Teare, J.; Marchesi, J.R.; Nicholson, J.K.; Kinross, J.M. Gut microbiota modulation of chemotherapy y 8. Alexander, J.L.; Wilson, I.D.; Teare, J.; Marchesi, J.R.; Nicholson, J.K.; Kinross, J.M. Gut microbiota modulation of chemotherapy efficacy and toxicity Nat Rev Gastroenterol Hepatol 2017 14 356–365 [CrossRef] [PubMed] 8. Alexander, J.L.; Wilson, I.D.; Teare, J.; Marchesi, J.R.; Nicholson, J.K.; Kinross, J.M. Gut microbiota efficacy and toxicity. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 356–365. [CrossRef] [PubMed] J J J J J efficacy and toxicity. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 356–365. [CrossRef] [PubMed] efficacy and toxicity. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 356 365. [CrossRef] [PubMed] Sauer, R.; Becker, H.; Hohenberger, W.; Rödel, C.; Wittekind, C.; Fietkau, R.; Martus, P.; Tschmelitsch, J.; Hag Preoperative versus postoperative chemoradiotherapy for rectal cancer N Engl J Med 2004 351 1731 1 y y p 9. Sauer, R.; Becker, H.; Hohenberger, W.; Rödel, C.; Wittekind, C.; Fietkau, R.; Martus, P.; Tschmelitsch, J.; Hager, E.; Hess, C.F.; et al. Preoperative versus postoperative chemoradiotherapy for rectal cancer. N. Engl. J. Med. 2004, 351, 1731–1740. [CrossRef] Becker, H.; Hohenberger, W.; Rödel, C.; Wittekind, C.; Fietkau, R.; Martus, P.; Tschmelitsch, J.; Hager, E.; Hes p p p py g J , , [ ] 10. Ma, B.; Gao, P.; Wang, H.; Xu, Q.; Song, Y.; Huang, X.; Sun, J.; Zhao, J.; Luo, J.; Sun, Y.; et al. What has preoperative radio(chemo)therapy brought to localized rectal cancer patients in terms of perioperative and long-term outcomes over the past decades? A systematic review and meta-analysis based on 41,121 patients. Int. J. Cancer 2017, 141, 1052–1065. [CrossRef] y y , p J , , [ ] 11. Pouncey, A.L.; Scott, A.J.; Alexander, J.L.; Marchesi, J.; Kinross, J. Gut microbiota, chemotherapy and the host: The influence of the gut microbiota on cancer treatment. Ecancermedicalscience 2018, 12, 868. [CrossRef] 12. Koppel, N.; Maini Rekdal, V.; Balskus, E.P. Chemical transformation of xenobiotics by the human gut microbiota. Science 2017, 356, eaag2770. [CrossRef] 13. Parida, S.; Sharma, D. The power of small changes: Comprehensive analyses of microbial dysbiosis in breast cancer. Biochim. Biophys. Acta Rev. Cancer 2019, 1871, 392–405. [CrossRef] p y 14. 22. Yoshioka, N.; Taniguchi, Y.; Yoshida, A.; Nakata, K.; Nishizawa, T.; Inagawa, H.; Kohchi, C.; Soma, G. Intestinal macrophages involved in the homeostasis of the intestine have the potential for responding to LPS. Anticancer Res. 2009, 29, 4861–4865. 23. Childs, A.; Mehta, D.; Gerner, E. Polyamine-dependent gene expression. Cell. Mol. Life Sci. 2003, 60, 1394–1406. [CrossRef] g p g Ross, K. Gut microbial short chain fatty acids are associated with pathological complete response (pCR) after n apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 References Gut microbial short chain fatty acids are associated with pathological complete response (pCR) after neoadjuvant chemother- apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 October 2020. , y p g p p (p ) j apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 October 2020. 21. Coutzac, C.; Jouniaux, J.M.; Paci, A.; Schmidt, J.; Mallardo, D.; Seck, A.; Asvatourian, V.; Cassard, L.; Saulnier, P.; Lacroix, L.; et al. Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer. Nat. Commun. 2020, 11, 2168. [CrossRef] [PubMed] 21. Coutzac, C.; Jouniaux, J.M.; Paci, A.; Schmidt, J.; Mallardo, D.; Seck, A.; Asvatourian, V.; Cassard, L.; Saulnier, P.; Lacroix, L.; et al. Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer. Nat. Commun. 2020, 11, 2168. [CrossRef] [PubMed] 22. Yoshioka, N.; Taniguchi, Y.; Yoshida, A.; Nakata, K.; Nishizawa, T.; Inagawa, H.; Kohchi, C.; Soma, G. Intestinal macrophages involved in the homeostasis of the intestine have the potential for responding to LPS. Anticancer Res. 2009, 29, 4861–4865. 23. Childs, A.; Mehta, D.; Gerner, E. Polyamine-dependent gene expression. Cell. Mol. Life Sci. 2003, 60, 1394–1406. [CrossRef] 22. Yoshioka, N.; Taniguchi, Y.; Yoshida, A.; Nakata, K.; Nishizawa, T.; Inagawa, H.; Kohchi, C.; Soma, G. Intestinal macrophages involved in the homeostasis of the intestine have the potential for responding to LPS. Anticancer Res. 2009, 29, 4861–4865. 23. Childs, A.; Mehta, D.; Gerner, E. Polyamine-dependent gene expression. Cell. Mol. Life Sci. Int. J. Mol. Sci. 2021, 22, 9549 24 of 25 24 of 25 24. Nakayama, Y.; Torigoe, T.; Minagawa, N.; Yamaguchi, K. The clinical usefulness of urinary N(1), N(12)-diacetylspermine (DiAcSpm) levels as a tumor marker in patients with colorectal cancer. Oncol. Lett. 2012, 3, 970–974. [CrossRef] (DiAcSpm) levels as a tumor marker in patients with colorectal cancer. Oncol. Lett. 2012, 3, 970–974. [CrossRef] 25. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012, 486, 207–214. [CrossRef] p p 25. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012, 486, 207–214. [CrossRef] [ ] 26. Qin, J.; Li, R.; Raes, J.; Arumugam, M.; Burgdorf, K.S.; Manichanh, C.; Nielsen, T.; Pons, N.; Levenez, F.; MetaHIT Consortium; et al. A human gut microbial gene catalogue established by metagenomic sequencing. References Cell Host Microbe 201 36. Gevers, D.; Kugathasan, S.; Denson, L.A.; Vázquez-Baeza, Y.; Van Treuren, W.; Ren, B.; Schwager, E.; Knights, D.; Song, S.J.; Yassour, M.; et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 2014, 15, 382–392. [CrossRef] 37 W N Y X Zh R Li J Xi X H Y Ch Y Y F L N W Z l D bi i i f f l 36. Gevers, D.; Kugathasan, S.; Denson, L.A.; Vázquez-Baeza, Y.; Van Treuren, W.; Ren, B.; Schwager, E.; Knights, D.; Song, S.J.; Yassour, M.; et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 2014, 15, 382–392. [CrossRef] 37. Wu, N.; Yang, X.; Zhang, R.; Li, J.; Xiao, X.; Hu, Y.; Chen, Y.; Yang, F.; Lu, N.; Wang, Z.; et al. Dysbiosis signature of fecal microbiota in colorectal cancer patients. Microb. Ecol. 2013, 66, 462–470. [CrossRef] 37. Wu, N.; Yang, X.; Zhang, R.; Li, J.; Xiao, X.; Hu, Y.; Chen, Y.; Yang, F.; Lu, N.; Wang, Z.; et al. Dys microbiota in colorectal cancer patients. Microb. Ecol. 2013, 66, 462–470. [CrossRef] 38. Bao, Y.; Tang, J.; Qian, Y.; Sun, T.; Chen, H.; Chen, Z.; Sun, D.; Zhong, M.; Chen, H.; Hong, J.; et al. Long noncoding RNA BFAL1 mediates enterotoxigenic Bacteroides fragilis-related carcinogenesis in colorectal cancer via the RHEB/mTOR pathway. Cell Death Dis. 2019, 10, 675. [CrossRef] [PubMed] 39. Hsu, R.Y.; Chan, C.H.; Spicer, J.D.; Rousseau, M.C.; Giannias, B.; Rousseau, S.; Ferri, L.E. LPS-induced TLR4 signaling in human colorectal cancer cells increases beta1 integrin-mediated cell adhesion and liver metastasis. Cancer Res. 2011, 71, 1989–1998. [CrossRef] [ ] 40. Murff, H.J.; Shu, X.O.; Li, H.; Dai, Q.; Kallianpur, A.; Yang, G.; Cai, H.; Wen, W.; Gao, Y.T.; Zheng, W. A prospective study of dietary polyunsaturated fatty acids and colorectal cancer risk in Chinese women. Cancer Epidemiol. Biomark. Prev. 2009, 18, 2283–2291. [CrossRef] [ ] 41. Wang, D.; Mann, J.R.; DuBois, R.N. The role of prostaglandins and other eicosanoids in the gastrointestinal tract. Gastroenterology 2005, 128, 1445–1461. [CrossRef] 42. Bansal, A.; Simon, M.C. Glutathione metabolism in cancer progression and treatment resistance. J. Cell Biol. 2018, 217, 2291–2298. [CrossRef] [PubMed] 43. Lu, S.C. Regulation of glutathione synthesis. Mol. Asp. Med. 2009, 30, 42–59. [CrossRef] [PubMed] 43. Lu, S.C. Regulation of glutathione synthesis. Mol. Asp. Med. 2009, 30, 42–59. [CrossRef] [PubMed] 44. Coffelt, S.B.; Wellenstein, M.D.; de Visser, K.E. References Nature 2010, 464, 59–65. [CrossRef] 27. Garrett, W.S. Cancer and the microbiota. Science 2015, 348, 80–86. [CrossRef] [PubMed] g g g y g q g 27. Garrett, W.S. Cancer and the microbiota. Science 2015, 348, 80–86. [CrossRef] [PubMed] , , , [ ] [ ] 28. Drewes, J.L.; Housseau, F.; Sears, C.L. Sporadic colorectal cancer: Microbial contributors to disease prevention, development and therapy. Br. J. Cancer 2016, 115, 273–280. [CrossRef] [PubMed] 29. Taghinezhad-S, S.; Mohseni, A.H.; Fu, X. Intervention on gut microbiota may change the strategy for management of colorectal cancer. J. Gastroenterol. Hepatol. 2020, 36, 1508–1517. [CrossRef] [PubMed] 30. Gopalakrishnan, V.; Spencer, C.N.; Nezi, L.; Reuben, A.; Andrews, M.C.; Karpinets, T.V.; Prieto, P.A.; Vicente, D.; Hoffman, K.; Wei, S.C.; et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science 2018, 359, 97–103. [CrossRef] 31. Heshiki, Y.; Vazquez-Uribe, R.; Li, J.; Ni, Y.; Quainoo, S.; Imamovic, L.; Li, J.; Sørensen, M.; Chow, B.K.C.; Weiss, G.J.; et al. Predictable modulation of cancer treatment outcomes by the gut microbiota. Microbiome 2020, 8, 28. [CrossRef] 32. Chen, W.; Liu, F.; Ling, Z.; Tong, X.; Xiang, C. Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer. PLoS ONE 2012, 7, e39743. [CrossRef] [PubMed] 33. Saffarian, A.; Mulet, C.; Regnault, B.; Amiot, A.; Tran-Van-Nhieu, J.; Ravel, J.; Sobhani, I.; Sansonetti, P.J.; Pédron, T. Crypt- and mucosa-associated core microbiotas in humans and their alteration in colon cancer patients. mBio 2019, 10, e1315–e1319. [CrossRef] [PubMed] 34. Gao, Z.; Guo, B.; Gao, R.; Zhu, Q.; Qin, H. Microbiota disbiosis is associated with colorectal cancer. Front. Microbiol. 2015, 6, 20. [CrossRef] [PubMed] 35. Yan, X.; Liu, L.; Li, H.; Qin, H.; Sun, Z. Clinical significance of Fusobacterium nucleatum, epithelial-mes cancer stem cell markers in stage III/IV colorectal cancer patients OncoTargets Ther 2017 10 5031–504 35. Yan, X.; Liu, L.; Li, H.; Qin, H.; Sun, Z. Clinical significance of Fusobacterium nucleatum, epithelial-mesenchymal transition, and cancer stem cell markers in stage III/IV colorectal cancer patients. OncoTargets Ther. 2017, 10, 5031–5046. [CrossRef] , ; , ; , ; Q , ; , g , p y , cancer stem cell markers in stage III/IV colorectal cancer patients. OncoTargets Ther. 2017, 10, 5031–5046. [CrossRef] g p g 36. Gevers, D.; Kugathasan, S.; Denson, L.A.; Vázquez-Baeza, Y.; Van Treuren, W.; Ren, B.; Schwager, E Yassour, M.; et al. The treatment-naive microbiome in new-onset Crohn’s disease. References Neutrophils in cancer: Neutral no more. Nat. Rev. Cancer 2016, 16, 431–446. [CrossRef] 44. Coffelt, S.B.; Wellenstein, M.D.; de Visser, K.E. Neutrophils in cancer: Neutral no more. Nat. Rev. Cancer 2016, 16, 431–446. [CrossRef] 44. Coffelt, S.B.; Wellenstein, M.D.; de Visser, K.E. Neutrophils in cancer: Neutral no more. Nat. R [CrossRef] [ ] 45. Thomas, A.M.; Manghi, P.; Asnicar, F.; Pasolli, E.; Armanini, F.; Zolfo, M.; Beghini, F.; Manara, S.; Karcher, N.; Pozzi, C.; et al. Metagenomic analysis of colorectal cancer datasets identifies cross-cohort microbial diagnostic signatures and a enlace with choline degradation. Nat. Med. 2019, 25, 667–678. [CrossRef] [PubMed] g 46. Noack, J.; Dongowski, G.; Hartmann, L.; Blaut, M. The human gut bacteria Bacteroides thetaiotaomicron and Fusobacterium varium produce putrescine and spermidine in cecum of pectin-fed gnotobiotic rats. J. Nutr. 2000, 130, 1225–1231. [CrossRef] 47. Goodwin, A.C.; Destefano Shields, C.E.; Wu, S.; Huso, D.L.; Wu, X.; Murray-Stewart, T.R.; Hacker-Prietz, A.; Rabizadeh, S.; Woster, P.M.; Sears, C.L.; et al. Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis. Proc. Natl. Acad. Sci. USA 2011, 108, 15354–15359. [CrossRef] 48. Johnson, C.H.; Dejea, C.M.; Edler, D.; Hoang, L.T.; Santidrian, A.F.; Felding, B.H.; Ivanisevic, J.; Cho, K.; Wick, E.C.; Hechen- bleikner, E.M.; et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015, 21, 891–897. [CrossRef] 48. Johnson, C.H.; Dejea, C.M.; Edler, D.; Hoang, L.T.; Santidrian, A.F.; Felding, B.H.; Ivanisevic, J.; Cho, K.; Wick, E.C.; Hechen- bleikner, E.M.; et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015, 21, 891–897. [CrossRef] 49. Mendez, R.; Kesh, K.; Arora, N.; Di Martino, L.; McAllister, F.; Merchant, N.; Banerjee, S.; Banerjee, S. Microbial dysbiosis and 48. Johnson, C.H.; Dejea, C.M.; Edler, D.; Hoang, L.T.; Santidrian, A.F.; Felding, B.H.; Ivanisevic, J.; Cho, K.; Wick, E.C.; Hechen bleikner, E.M.; et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015, 21, 891–897. [CrossRef] 49. Mendez, R.; Kesh, K.; Arora, N.; Di Martino, L.; McAllister, F.; Merchant, N.; Banerjee, S.; Banerjee, S. Microbial dysbiosis and polyamine metabolism as predictive markers for early detection of pancreatic cancer. Carcinogenesis 2020, 41, 561–570. [CrossRef] [PubMed] 49. Mendez, R.; Kesh, K.; Arora, N.; Di Martino, L.; McAllister, F.; Merchant, N.; Banerjee, S.; Banerjee, S. Microbial dysbiosis and polyamine metabolism as predictive markers for early detection of pancreatic cancer. Carcinogenesis 2020, 41, 561–570. [CrossRef] [PubMed] Int. J. Mol. Sci. 2021, 22, 9549 25 of 25 50. References Arruabarrena-Aristorena, A.; Zabala-Letona, A.; Carracedo, A. Oil for the cancer engine: The cross-talk between oncogenic signaling and polyamine metabolism. Sci. Adv. 2018, 4, eaar2606. [CrossRef] 51. Bhat, M.I.; Kapila, R. Dietary metabolites derived from gut microbiota: Critical modulators of epigenetic changes in mammals. Nutr. Rev. 2017, 75, 374–389. [CrossRef] 52. Jahani-Sherafat, S.; Alebouyeh, M.; Moghim, S.; Amoli, H.A.; Safaei, H.G. Role of gut microbiota in the pathogenesis of colorectal cancer; a review article. Gastroenterol. Hepatol. Bed Bench. 2018, 11, 101–109. p 53. Sokol, H.; Seksik, P.; Furet, J.P.; Firmesse, O.; Nion-Larmurier, I.; Beaugerie, L.; Cosnes, J.; Corthier, G.; Marteau, P.; Doré., J. Low counts of Faecalibacterium prausnitzii in colitis microbiota. Inflamm. Bowel Dis. 2009, 15, 1183–1189. [CrossRef] 54. Lopez-Siles, M.; Martinez-Medina, M.; Abellà, C.; Busquets, D.; Sabat-Mir, M.; Duncan, S.H.; Aldeguer, X.; Flint, H.J.; Garcia-Gil, L.J. Mucosa-associated Faecalibacterium prausnitzii phylotype richness is reduced in patients with inflammatory bowel disease. Appl. Environ. Microb. 2015, 81, 7584–7592. [CrossRef] 55. Rios-Covian, D.; Gueimonde, M.; Duncan, S.H.; Flint, H.J.; de los Reyes-Gavilan, C.G. Enhanced butyrate formation by cross- feeding between Faecalibacterium prausnitzii and Bifidobacterium adolescentis. FEMS Microbiol. Lett. 2015, 362, fnv176. [CrossRef] 56. Singh, N.; Gurav, A.; Sivaprakasam, S.; Brady, E.; Padia, R.; Shi, H.; Thangaraju, M.; Prasad, P.D.; Manicassamy, S.; Munn, D.H.; et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity 2014, 40, 128–139. [CrossRef] g y 57. Salcedo, R.; Worschech, A.; Cardone, M.; Jones, Y.; Gyulai, Z.; Dai, R.M.; Wang, E.; Ma, W.; Haines, D.; O’hUigin, C.; et al. MyD88-mediated signaling prevents development of adenocarcinomas of the colon: Role of interleukin 18. J. Exp. Med. 2010, 207, 1625–1636. [CrossRef] 58. Vaishnava, S.; Behrendt, C.L.; Ismail, A.S.; Eckmann, L.; Hooper, L.V. Paneth cells directly sense gu homeostasis at the intestinal host-microbial interface. Proc. Natl. Acad. Sci. USA 2008, 105, 20858–2 59. Van Immerseel, F.; De Buck, J.; Boyen, F.; Bohez, L.; Pasmans, F.; Volf, J.; Sevcik, M.; Rychlik, I.; Haesebrouck, F.; Ducatelle, R. Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with Salmonella enterica serovar Enteritidis. Appl. Environ. Microbiol. 2004, 70, 3582–3587. [CrossRef] pp 60. Sturgeon, C.; Fasano, A. Zonulin, a regulator of epithelial and endothelial barri inflammatory diseases. Tissue Barriers 2016, 4, e1251384. [CrossRef] [PubMed] pp 60. Sturgeon, C.; Fasano, A. References Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases. Tissue Barriers 2016, 4, e1251384. [CrossRef] [PubMed] 61. Wright, D.P.; Rosendale, D.; Roberton, A.M. Prevotella enzymes involved in mucin oligosaccharide degr a small operon of genes expressed during growth on mucin. FEMS Microbiol. Lett. 2000, 190, 73–79. [C Rosendale, D.; Roberton, A.M. Prevotella enzymes involved in mucin oligosaccharide degradation and evide f d d i th i FEMS Microbiol Lett 2000 190 73 79 [C R f] [P bM d 62. Mandard, A.M.; Dalibard, F.; Mandard, J.C.; Marnay, J.; Henry-Amar, M.; Petiot, J.F.; Roussel, A.; Jacob, J.H.; Segol, P.; Samama, G. Pathologic assessment of tumor regression after preoperative chemoradiotherapy of esophageal carcinoma. Clinicopathologic correlations. Cancer 1994, 73, 2680–2686. [CrossRef] 63. Ribeiro, W.R.; Vinolo, M.A.R.; Calixto, L.A.; Ferreira, C.M. Use of gas chromatography to quantify serum, colonic luminal content and feces of mice. Bio-Protocol 2018, 8, e3089. [CrossRef] o, M.A.R.; Calixto, L.A.; Ferreira, C.M. Use of gas chromatography to quantify short chain fatty acids in the nal content and feces of mice. Bio-Protocol 2018, 8, e3089. [CrossRef] 64. Mendes, E.; Acetturi, B.G.; Thomas, A.M.; Martins, F.; Crisma, A.R.; Murata, G.; Braga, T.T.; Camâra, N.; Franco, A.; Setubal, J.C.; et al. Prophylactic supplementation of Bifidobacterium longum 51A protects mice from ovariectomy-induced exacerbated 64. Mendes, E.; Acetturi, B.G.; Thomas, A.M.; Martins, F.; Crisma, A.R.; Murata, G.; Braga, T.T.; Camâra, N.; Franco, A.; Setubal, J.C.; et al. Prophylactic supplementation of Bifidobacterium longum 51A protects mice from ovariectomy-induced exacerbated allergic airway inflammation and airway hyperresponsiveness. Front. Microbiol. 2017, 8, 1732. [CrossRef] [PubMed] allergic airway inflammation and airway hyperresponsiveness. Front. Microbiol. 2017, 8, 1732. [CrossRef] [PubMed] 65. Fellows, R.; Denizot, J.; Stellato, C.; Cuomo, A.; Jain, P.; Stoyanova, E.; Balázsi, S.; Hajnády, Z.; Liebert, A.; Kazakevych, J.; et al. Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases. Nat. Commun. 2018, 9, 105. [CrossRef] 66. Liang, J. A sensitive method for the quantification of short-chain fatty acids by benzyl chloroformate derivatization combined with GC-MS. Analyst 2020, 145, 2692–2700. [CrossRef] 67. Samarra, I.; Ramos-Molina, B.; Queipo-Ortuño, M.I.; Tinahones, F.J.; Arola, L.; Delpino-Rius, A.; Herrero, P.; Canela, N. Gender- related differences on polyamine metabolome in liquid biopsies by a simple and sensitive two-step liquid-liquid extraction and LC-MS/MS. Biomolecules 2019, 9, 779. [CrossRef]
W4376867143.txt
https://www.nature.com/articles/s41599-023-01708-9.pdf
en
Temporal dynamics of faculty hiring in mathematics
Humanities & social sciences communications
2,023
cc-by
8,444
ARTICLE https://doi.org/10.1057/s41599-023-01708-9 OPEN Temporal dynamics of faculty hiring in mathematics 1234567890():,; Cody FitzGerald 1,2 ✉, Yitong Huang2,3 ✉, Katelyn Plaisier Leisman1 & Chad M. Topaz4,5,6 University faculty hiring networks are known to be hierarchical and to exacerbate various types of inequity. Still, a detailed, historical understanding of hiring dynamics lacks in many academic fields. We focus on the field of mathematics, analyzing over 120,000 records from 150 institutions over seven decades to elucidate the temporal dynamics of hiring doctoralgranting (DG) faculty at the individual and departmental levels. We demonstrate that the disparity between the number of mathematics Ph.D.s awarded and the number of DG faculty positions filled has grown over time. Even institutions with the best records of DG faculty placement have experienced a temporal decline in the probability of their graduates obtaining a DG faculty position. By quantifying the mathematical prestige of each department with a network statistic, authority centrality, we find an approximately linear relationship between the log of the prestige of one’s Ph.D. institution and the log of the probability of obtaining a faculty position. Moreover, we observe associations suggesting that the probability of DG faculty placement has decreased over time and is smaller for women than for men. On the departmental level, a group of 14 elite departments dominated the authority centrality of the entire network between 1950 and 2019. Strikingly, one department within this elite group increased its centrality scores consistently, which hints at the possibility for a department to improve its prestige. This analysis highlights the challenges of transitioning from Ph.D. holder to faculty member in mathematics. 1 Department of Engineering Sciences and Applied Mathematics, Northwestern University, Evanston, IL, USA. 2 NSF-Simons Center for Quantitative Biology, Evanston, IL, USA. 3 Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA. 4 Institute for the Quantitative Study of Inclusion, Diversity, and Equity, Williamstown, MA, USA. 5 Williams College, Williamstown, MA, USA. 6 University of Colorado–Boulder, Boulder, CO, USA. ✉email: cody.fitzgerald@northwestern.edu; yitong.huang@northwestern.edu HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 1 ARTICLE A HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 Introduction n estimated two thousand mathematics Ph.D.s are awarded annually in the United States (Reys et al., 2022). At the same time, only around 300 tenure-track positions in doctoral degree-granting mathematics departments are under recruitment each cycle. Moreover, upwards of 70% of these positions are filled by faculty who are not new Ph.D.s (Jahan et al., 2019). The hiring process is not just competitive but is opaque and poorly understood by applicants (Fernandes et al., 2020). The result of a search is eventually known publicly, but the list of applicants and the selection criteria used are not revealed. Success in acquiring a faculty position may not only depend on an individual scholar’s research productivity and academic credentials (Fernandes et al., 2020), but also on demographic factors such as race, gender, and childhood socioeconomic status (Clauset et al., 2015; Morgan et al., 2022; Wapman et al., 2022; White-Lewis, 2020). In recent years there has been growing interest in quantifying the faculty hiring process in various fields (Barnett et al., 2010; Clauset et al., 2015; Cowan and Rossello, 2018; Fernandes et al., 2020; Fowler et al., 2007; Hanneman, 2001; Lee et al., 2021; Mai et al., 2015; Wapman et al., 2022; Zuo et al., 2019). For instance, Clauset et al. (2015) used a network science approach to study the trajectories of 19,000 faculty members in business, computer science, and history. They found that faculty hiring networks have a particularly hierarchical structure. They also found that doctoral program prestige is a strong predictor of faculty placement and that there exists a bias towards men as compared to women with the same training, especially in the fields of business and computer science (Clauset et al., 2015). A follow-up study in 2021, which used the same data set, utilized adaptive rewiring network models to test mechanisms that give rise to the hierarchical nature of the faculty hiring observed in the 2015 study (Lee et al., 2021). It concludes that a mixture of two mechanisms, total faculty production and local homophily, likely drives the dynamics of real-world faculty hiring. Another recent study combined a survey of tenure-track faculty members in eight fields, US census data, and the NSF Survey of Earned Doctorates to study the impact of socioeconomic status on faculty position acquisition and found that faculty members tended to grow up in wealthier homes and were 25 times more likely to have a parent who held a Ph.D., as compared to the general US population (Morgan et al., 2022). An extensive study of faculty hiring between 2011 and 2020 encompassed 295,089 US faculty members in 10,612 departments across 107 fields and eight domains (including mathematics and computing) using data from the Academic Analytics Research Center (Wapman et al., 2022). This study confirmed the hierarchical structure observed in previous studies extends across many fields of academic hiring. The study also uncovered populations of US faculty members with high attrition rates: US faculty members trained outside the US, Canada, and the UK; faculty members that are trained and hired by the same university; and faculty members that are trained at universities from which relatively few graduates acquire faculty positions. Finally, this study found that recent changes in the gender parity of faculty members are explained mainly by the retirement of older faculty members, who tend to be men (Wapman et al., 2022). A survey-based statistical analysis of academic job seekers primarily in the life sciences identified significant associations between receiving an academic job offer and the number of job applications filed, winning a career transition award, total citation count, authorship of high-profile papers, and postdoctoral fellowships (Fernandes et al., 2020). The same study also found significant negative associations between receiving an academic job offer and the number of years an applicant is on the job market and joint industry and academic job searches 2 (Fernandes et al., 2020). Despite these efforts spent investigating academic hiring, a comprehensive understanding of the evolution of the mathematics faculty hiring process over many decades is lacking. Our analysis moves towards filling this gap in understanding. We complement the existing work on faculty hiring by studying doctoral-granting (DG) faculty hiring in mathematics over the past 70 years at the individual and departmental scale using the Mathematics Genealogy Project (MGP, http://www. genealogy.ams.org), which is an extensive database of graduate advisor–advisee relationships. We consider academic hiring as a complex system at two different scales: the individual level and the department level. At the individual level, we examine over 120,000 records from MGP to uncover characteristics that lead to successful DG faculty placements in mathematics. Here, we set out to understand which factors, academic or otherwise, allow a mathematics graduate degree holder to transition to a DG faculty member in mathematics and how generally difficult this transition is to make. We will refer to this transition as the graduate-tofaculty transition (GFT) throughout the paper. We begin our investigation into the GFT by studying 150 US math departments and show that the GFT is decreasing over time. This finding even holds for historically “well-placing” departments, which annually graduated at least one student who eventually acquired a DG faculty position in mathematics between 1950 and 2015. We also find statistically significant academic-based and gender-based factors that influence the GFT. In addition to our individual-level examination of the GFT, we use network analysis methods to investigate the department level. Myers et al. (2011) analyzed the MGP database between 1973 and 2011 and showed that department authority scores correlate with departmental rankings from US News & World Reports and the National Research Council. Hub and authority centrality were originally developed in the context of ranking web pages (Kleinberg, 1999). We join Myers et al. (2011) in using these centralities as proxies for departmental prestige. Similar to the definitions given in Myers et al. (2011), we define a department with a high authority value as one where Ph.D. recipients go on to become DG faculty at prestigious schools and a department with a high hub value as one containing many DG faculty who received their Ph.D. at prestigious schools. Expanding on Myers et al. (2011), we conduct a fine-grained analysis that explores gains and losses in the hub and authority centrality of US math departments between 1950 and 2019. In our analysis, we find that a subset of 14 “elite” departments holds approximately 70% of the authority centrality of the entire network. Moreover, the total centrality held by these elite departments remains relatively constant between 1950 and 2019. However, the share of centrality held individually by each of these elite departments varies over that time frame. Strikingly, we find that one of the 14 elite departments dramatically increased its share of both hub and authority centrality. In summary, our investigations into the GFT and the temporal dynamics of centrality at the departmental scale make three contributions: ● ● ● We perform an analysis of the evolution of academic hiring within the field of mathematics between 1950 and 2019. We begin to uncover academic and demographic factors associated with successfully acquiring a DG faculty position at the individual level in the field of mathematics. We find that an elite group of departments comprise a large portion of network centrality between 1950 and 2019. Furthermore, the portion of centrality held by the elite group remains relatively stable throughout that time span. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 However, we find one math department that has dramatically and consistently increased its share of both hub and authority centrality, the latter of which is a proxy for rank. Data collection We begin with a sample of 150 Ph.D.-granting institutions based on the U.S. News Graduate Schools Top Mathematics Programs rankings. The union of three archived U.S. News rankings from 1998, 2010, and 2018 comprise a final sample of 150 institutions that we analyze. We then proceed to gather data from the Mathematics Genealogy Project (MGP, http://www.genealogy.ams.org). We collected all data on October 4, 2022. For each of the 150 schools, we search MGP for all individuals who ever obtained a Ph.D. from that school. From the search results, we retain each person’s name, the year of their Ph.D., and a hyperlink to their full record within MGP. We acquire 121,521 records of graduates from 150 schools. Of these, 466 records are missing a year, accounting for 0.4% of the data. For the remaining records, the year ranges from 1792 to 2022, with a median year of 1997 and a mean year of approximately 1993. Next, we gather information about which individuals have served as DG faculty. From MGP data, the only means we have to infer whether the target individual became a DG faculty member is to check whether they advised Ph.D. students who also appear in MGP. For each person in our database, we see if there are any students listed on their individual MGP record. If there are no students listed, we assume the individual did not serve as DG faculty. If there are students listed, we list all the schools at which the students received their Ph.D.s and take the mode as the primary school at which the advisor was DG faculty. For example, if an individual advised 8 Ph.D. students at the University of Iowa and 2 at the University of Indiana, we would take the University of Iowa as the institution where the individual was DG faculty. If the mode is not unique, we record the first (in chronology) institution. It is a limitation of our approach that we cannot identify faculty in departments that do not grant Ph.D.’s, nor DG faculty who do not have any Ph.D. students appearing in MGP, either through omission of data or through not having served as advisor. Overall, we identify a DG faculty institution for 24,928 individuals. Finally, in our construction of hiring networks, we restrict attention to those who became DG faculty at one of the 150 US News-ranked schools, which results in a data set of 19,372 individuals. Next, if available, we gather information about the school where each individual’s advisor received a Ph.D., as well as how many students the advisor is listed on MGP as having mentored. Finally, for each record, we extract the individual’s first name and run it through the genderize.io algorithm (Demografix ApS, 2022) to infer the individual’s gender. From the outset, it is important to recognize the limitations of this approach. Most critically, inferred gender is not the same as actual gender. The only accurate source for information on an individual’s gender is the individual themself. However, many of the MGP records correspond to deceased individuals, and even for living individuals, a survey methodology is unlikely to produce much data. Moreover, an additional severe limitation of our approach is the algorithm’s unfortunate use of binary gender. Overall, it would be strongly preferable to have self-identified gender information, and information unrestricted to a binary scale. Lacking this data, we proceed with using the gender inference algorithm, keeping in mind its serious shortcomings. For each name, the algorithm outputs an inferred binary gender based on its internal database of names with self-identified ARTICLE gender as scraped from online sources. For example, consider the first name Kelsey. Suppose that the genderize.io database contains 900 instances of this name, 765 coming from selfidentified women, and 135 coming from self-identified men. Then the algorithm would infer the gender of “woman” and would report a probability of 0.85, based on the frequency 765/ 900. An additional limitation of the algorithm is now apparent: there is no way to know the extent to which the underlying data might be biased. For instance, if we had access to self-identified gender information for all individuals in the world with the name Kelsey, would the observed frequency of women be close to the 0.85 reported by the algorithm? Keeping all of the limitations in mind, we proceed by choosing a gender inference probability below which we are unwilling to accept the algorithm’s inference. We initially experimented with a high threshold, p = 0.95, but manual examination of the excluded data suggested that an overwhelming majority of discarded names were of East or South Asian origin. Discarding so many of these names would result in the erasure of those groups from our study. Thus, to achieve more inclusion, we seek a cutoff probability threshold p* such that the frequencies of inferred men and women in the excluded data are equal to the average probabilities of being a man and being a woman in that same excluded data; that is to say, we strive for aggregate internal consistency in the excluded data. We find a single value of p* satisfying this criterion, namely p* ≈ 0.6, and we exclude data where p < p*. We find 93,882 inferred men (77.2%) and 22,410 inferred women (18.4%). For 5229 individuals (4.3%), our cutoff for probability precluded an inference. Results Focusing on individuals: the graduate to faculty transition rate. We begin our analysis by computing the probability that a mathematics Ph.D. holder becomes a faculty member who advises one or more mathematics Ph.D. students to graduation. For this analysis, we use all 121,055 records in our dataset that have a graduation year listed. Figure 1 shows the total number of mathematics Ph.D. graduates each year from 1900 to 2019, and the number of those Ph.D. graduates who are listed on MGP as having eventually advised students. Both the number of Fig. 1 The number of mathematics Ph.D. degrees awarded from 150 US math departments (green solid curve) and the number of people who later became doctoral-granting (DG) faculty members from this pool (blue dotted curve) between the years 1900 and 2019. The steep decline beginning in 2010 is simply an indication of the necessary time lag between receiving one’s Ph.D. and graduating one’s first Ph.D. student. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 3 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 Fig. 2 The combined graduate to doctoral-granting (DG) faculty transition (GFT) rate for 150 DG US math departments from 1900 to 2019. The GFT rate decays nonlinearly over time. mathematics Ph.D. degrees awarded and the number of people who receive a Ph.D. and go on to become a DG faculty member in mathematics increase between 1900 and 1970, but after roughly 1970 the number of new DG faculty members saturates, and even declines slightly, while total annual graduates continue to rise. Most of the steep decline evident in eventual advisors after about 2010 is due to the fact that many of those who graduated more recently may not have advised students yet (but perhaps will eventually). By dividing the number of people who receive a Ph.D. and go on to become a DG faculty member in mathematics (blue dotted curve in Fig. 1) by the total number of mathematics Ph.D. degrees awarded (green solid curve in Fig. 1), we obtain the graduate to DG faculty transition (GFT) rate. The annual GFT rate for all records, shown in Fig. 2, can be interpreted as a global measure of the relative ease of acquiring a DG faculty position in mathematics. The GFT rate decays nonlinearly from 1900 through 2019. In the past 30 or so years, the GFT rate has trended closer to zero than it previously had over the time scale we considered, though we again note that the data is incomplete for recent graduates who may eventually train future students but have not yet done so. To further investigate the GFT rate, we look at what happens when we restrict the data to graduates from a list of “well-placing” schools between the years of 1950 and 2015. We choose these schools to determine if a subset of math departments were immune from the global behavior of the GFT rate over this time span. We choose these years because the data are less complete before 1950, and graduates after 2015 may not have had enough time to acquire a DG faculty position and advise their first trainee. We define a well-placing school as a school that between these years annually graduated at least one mathematics Ph.D. student who eventually mentored a student at one of the 150 departments we included in our analysis. Ten schools fall into this category, including Harvard University, Massachusetts Institute of Technology, Princeton University, Stanford University, University of Chicago, University of North Carolina–Chapel Hill, University of California–Berkeley, University of Michigan, University of Pennsylvania, and University of Wisconsin–Madison. The minimum, median, and maximum GFT rates for schools in this group are shown in Fig. 3. We can see that the transition rate of “well-placing” mathematics departments declines in a similar manner to the global trend 4 Fig. 3 The minimum, median, and maximum graduate-to-faculty transition (GFT) rates for 10 schools that have annually graduated at least one mathematics Ph.D. student who became a doctoral-granting (DG) faculty member in a department in one of the 150 departments we considered between 1950 and 2015. All three measures decrease, but the maximum GFT rate most sharply declines between the years of 1950-2015. seen in Fig. 2. Note the spread that exists in Fig. 3 in the 1950s and 1960s dies off over time as the minimum, median, and maximum GFT rates each trend downward over time. Centrality scores: hubs and authorities. Here, we apply the network science approach described in Myers et al. (2011) to a larger data set and incorporate temporal dynamics into the centrality analysis. Myers et al. constructed a doctoral-granting faculty hiring network based on data from the MGP database between the years 1973 and 2011. In their network, the nodes represent US math departments and the edges are representative of DG faculty hiring. The edges are weighted by the number of people who were trained as graduate students in a department and acquire DG faculty positions in another department. The edges point from the DG faculty department towards the graduate training department, and the graduate training and DG faculty departments can be the same if the person was trained and hired by the same department. As an example, a professor in the Purdue University Department of Mathematics who received a Ph.D. from the University of Wisconsin–Madison Department of Mathematics is represented as an edge pointing from the Purdue University Department of Mathematics node to the University of Wisconsin-Madison Department of Mathematics node. In Myers et al. (2011), network centrality was analyzed for 58 math departments using hub and authority centrality scores. Hub and authority centrality scores were originally developed in the context of ranking web pages on the internet (Kleinberg, 1999) and were used in the search engine for Ask.com (Newman, 2018). Mathematically, centrality authority and centrality hub vectors x and y are the left and right singular vectors corresponding to the largest singular value of the adjacency matrix, A, scaled so their elements each sum to 1. Thus, they satisfy x ¼ αAy; and y ¼ βAT x; where constants α and β satisfy αβ = 1/σ2 and σ is the largest singular value of A (Newman, 2018). HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 ARTICLE Fig. 4 Graduate-to-faculty transition (GFT) rate by average authority score of graduate training department between the years of 1950 and 2019 on a log–log plot. The dashed line represents a least-squares fit of the data, yielding an r2 value of 0.7 on the log-log axes. Fig. 5 The fraction of doctoral-granting (DG) faculty members who acquire a faculty position in a DG department with a higher authority score than the department where they completed their graduate studies versus the year of Ph.D. graduation. The fraction hovers between 15% and 25% and is relatively stable over seven decades. In Myers et al. (2011), departments with high authority scores are defined as “a strong source of prestigious Ph.D. students,” and departments with high hub scores are defined as “a strong destination.” We refine their definitions for the reader’s clarity. We take a department with a high authority score to mean an institution from which Ph.D. recipients go on to become DG faculty at prestigious schools, and we define a department with a high hub score as a department that hires many DG faculty who received a Ph.D. from a prestigious school. Next, we compute the GFT rate as a function of authority score for each of the 150 schools in our set that graduated students between the years of 1950 and 2019 (see Fig. 4). As expected, higher authority scores, suggesting higher prestige, correlate with higher GFT rates. The data appear linear on a log–log plot of the GFT rate versus the authority score of the Ph.D. training departments (r2 = 0.7 via least-squares fitting on the log-log axes). It is worth pointing out that San Diego State University (GFT rate = 0.5, authority score ≈ 1.36 × 10−5) appears to be an outlier in the group because only two graduates in our dataset obtained a Ph.D. from San Diego State University, and one of them eventually became a DG faculty member. Furthermore, we look at the probability of moving up (higher authority score) when transitioning from Ph.D. holder to DG faculty. Of the graduates who eventually go on to advise students at one of the 150 schools of interest, we compute the fraction of those who have advised students at an institution with a greater authority score than that of their Ph.D. granting institution. This fraction is shown in Fig. 5. It remains relatively stable between 1950 and 2019, largely hovering between 15% and 25%. Note that graduates from 2019 may not have enough time to advise students yet, and in fact, our data only contains two individuals who graduated in 2019 and already have advisees listed. Ph.D.; their inferred gender; prestige measures of their Ph.D. granting institution and their advisor’s Ph.D. granting institution; and finally, the number of Ph.D. students overseen by their advisor. We now explain these choices in detail. Since the academic job market is tighter in some years and less so in others, we include the year of Ph.D. receipt as an explanatory variable. To address differential outcomes depending on gender, we include inferred gender in the model. We also include the interaction of the inferred gender with the year of Ph.D. receipt in order to allow for the possibility of a shifting hiring landscape for women over time. To account for the prestige of the target individual’s Ph.D. granting institution, we (initially) include its hub and authority scores as computed over a 10-year time period up to but not including the Ph.D. year. For that same time period, to account for the influence of the target’s Ph.D. advisor as part of the job search process, we include the hub and authority scores of that person’s Ph.D. granting institution. Finally, to account for the target’s Ph.D. advisor’s advising patterns, albeit in a crude manner, we include the total number of students who received Ph.D.s from the target’s advisor over the same 10-year window. It is important to recall that the target Ph.D. and advisor Ph.D. hub and authority scores are all calculated from the same network and thus may be correlated. In fact, the correlation between hub and authority for the target is r ≈ 0.66 and for the advisor is r ≈ 0.51. Among the four network scores, the two least correlated are the target’s authority score and the advisor’s hub score (r ≈ 0.16), so we include these in our model. While including the other two network scores might increase model fit, it could be at the expense of interpretability, especially if estimates for the effects of strongly correlated variables have opposite signs. Concretely, the model described above is Logistic model: probability of faculty placement. To identify factors associated with a target individual obtaining a doctoralgranting (DG) faculty position, we construct a logistic regression model. The dichotomous outcome variable is whether or not the target individual became a DG faculty. As discussed previously, we infer DG faculty status from the individual’s record of student advising as listed in MGP, should it exist. We include several explanatory variables in our model: the target’s year of receipt of Log Odds  β0 þ β1 ðYearÞ þ β2 ðInferred Gender WomanÞ þ β3 ðYear  Inferred Gender WomanÞ þ β4 ðTarget0 s Ph:D: AuthorityÞ þ β5 ðAdvisor0 s Ph:D: HubÞ þ β6 ðNumber of Students Advised by AdvisorÞ; ð1Þ where since we use a logistic regression framework, Log Odds is the log odds of obtaining a DG faculty position. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 5 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 Table 1 Estimates obtained from fitting model (1), which describes the log odds of an individual becoming a DG faculty member, based on N = 81, 691 records (see the section “Logistic model: probability of faculty placement” for details, including important limitations of the modeling approach). Coefficient of Estimate Standard error z Significant (Intercept) Year Inferred gender woman Year ⋅ inferred gender woman Target’s Ph.D. authority Advisor’s Ph.D. hub Students advised by advisor 72.9 −0.0377 −13.9 0.00688 1.37 0.0714 −0.0479 1.28 0.000644 3.81 0.00192 0.0310 0.0521 0.00229 57.1 −58.6 −3.65 3.59 44.3 1.37 −20.9 *** *** *** *** *** *** Significance column is coded as *p < 0.05, **p < 0.01, ***p < 0.001. Fig. 6 The number of departments that comprise 50% and 90% of the hub and authority centrality between the years 1950 and 2019. Centrality scores are computed using a rolling window of 10 years, indicated on the xaxis by the first year of the decade. Black and blue curves correspond to 50% and 90% of centrality, respectively. Solid and dashed curves correspond to hub and authority centrality, respectively. This analysis includes 106 schools. Before fitting the regression, we perform several data filtering steps. First, to be consistent with the time period we have studied previously, we filter out target individuals who received their Ph.D. prior to 1950 or after 2015, resulting in a data set of 104,674 records. Second, to be consistent with whom we include in our network, we further restrict attention to individuals whose advisor received their Ph.D. at one of the 150 schools we study, reducing the data set to 85,323. While we have not formally coded the geography of the advisor Ph.D. institutions for the excluded records, inspection suggests that the vast majority are schools outside of the United States. Third, we are forced to eliminate any records missing a gender inference. Removing these 3,569 records leaves 81,754 remaining. Fourth and last, in 63 cases (constituting 0.1% of the remaining data) we are unable to compute prestige scores because no one from the target individual’s Ph.D. granting institution became DG faculty during the 10-year time window prior to the year of Ph.D. receipt. We must eliminate these records, resulting in a final data set of 81,691 records. Next, to again be consistent with our previous decisions, we take the outcome variable to be not merely whether the target individual became a DG faculty member, but whether they became a DG faculty member at one of the 150 schools we study. There are 4032 individuals in the data set who did become DG faculty but not at one of the 150 schools, and so along with 64,381 6 individuals who did not become DG faculty at any school in our data set, these individuals have an outcome variable coded as false. In summary, our logistic model investigates whether individuals who received a Ph.D. at one of the 150 schools and whose advisor received their Ph.D. at one of those same schools ended up becoming DG faculty at still one of those same schools. We have 81,691 records, and the observed frequency of a positive outcome (becoming DG faculty at one of the 150 schools) is 0.16. We fit the model using the standard glm command in the RStudio statistical computing environment. In terms of evaluating our model, we steer clear of measures of predictive accuracy because our intention is not to build a classifier. Indeed, with such a limited set of explanatory variables, we have no expectation that our model could be used in this way. However, we can still hope to assess if there are important associations between the outcome variable and the explanatory variables that we do have. Loosely, our situation could be compared to a linear regression that produces a low coefficient of determination but statistically significant coefficients. Thus, to diagnose our model, we set a relatively low bar and compare it to a null model. A likelihood ratio test of our model results in p < 0.001, suggesting that the model is preferable to the null one. Table 1 provides estimates from our model. The signs of the significant coefficients have the following interpretations. The negative coefficient on year means that overall, there is an association between time and a decreased probability of obtaining a DG faculty position. Similarly, the negative coefficient on inferred gender woman indicates an association between being a woman and a decreased probability of obtaining a DG faculty position. The positive coefficient on the interaction of year and inferred gender woman suggests that the decreased probability of hiring for inferred women is becoming less severe over time. The positive coefficient on the authority score of the individual’s Ph.D. granting institution shows an association between that institution’s prestige and its students being hired as DG faculty. This result is perhaps not surprising given the way the authority score is constructed. There is one additional significant coefficient, namely, the negative coefficient on the number of students advised by the individual’s Ph.D. advisor. This coefficient indicates an association that is less intuitive to us and could perhaps be a target for further research. We could hypothesize that when an advisor advises more students, they are able to provide less individualized attention to each student. This reduced attention might decrease the probability of a DG faculty placement, either because the student receives insufficient guidance or because the situation discourages the student from wanting to be in academia. Regardless, these are merely conjectures, and ethnographic data might shed further light on plausible explanations. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 Temporal dynamics of US math departments To begin our department-level analysis, we compute the hub and authority centrality scores of a subset of 106 of the US mathematics departments we considered, starting in 1950 and going through 2019, using a rolling window of 10 years for each network construction, thereby extending the analysis presented in Myers et al. (2011). We determine this subset by computing the centrality scores of our entire set of 150 departments for each rolling decade starting with 1950 and ending with 2019. Any school that does not appear as an institution with DG faculty during each rolling 10-year period returns an authority (hub) score of zero. We omit any school which returned a zero centrality score for any of the rolling decades and re-compute the centrality scores for the remaining 106 departments. From this network analysis, we find that a minority of departments hold the majority of the hub and authority centrality. Figure 6 shows that between five and seven departments hold 50% of the authority centrality and between 11 and 19 departments hold 50% of the hub centrality between 1950 and 2019, calculated over rolling decades, and plotted by the first year of each decade. Roughly one-third of departments hold 90% of the authority centrality and two-thirds of departments hold 90% of the hub centrality between the years of 1950 and 2019. Furthermore, we explore the temporal dynamics of hub and authority centrality associated with elite math departments between the years of 1950 and 2019. To select elite departments to study, we compute the departments with the seven highest authority scores across seven network constructions spanning 1950–1959, 1960–1969, 1970–1979, 1980–1989, 1990–1999, 2000–2009, and 2010–2019 and take the union of the department sets. As a result of this computation, the following departments meet our definition of elite: California Institute of Technology, Carnegie Mellon University, Columbia University, Cornell University, Harvard University, Massachusetts Institute of Technology, Princeton University, Stanford University, University of Chicago, University of California–Berkeley, University of Michigan, University of Washington, University of Wisconsin–Madison, and Yale University. Together this group of departments consistently holds approximately 72% of the authority centrality and approximately 43% of the hub centrality between 1950 and 2019 (see Fig. 7). While the share of hub and authority centrality held by this group of elite departments remains relatively constant between 1950 and 2019, the individual share of hub and authority centrality held by each elite department changes more dramatically over time. We compute the Kendall rank correlation coefficient (Kendall, 1948) of authority and hub centrality time series spanning decades beginning in 1950 and ending in 2019 for pairs of elite departments. In this context, a Kendall rank correlation coefficient close to 1 would indicate that the centrality score trajectories of two departments between 1950 and 2019 move in the same direction. Similarly, a Kendall rank correlation coefficient close to −1 would indicate that the centrality trajectories of two departments between 1950 and 2019 move in opposite directions. Large and significant Kendall rank correlation coefficients associated with pairs of elite departments’ hub and authority centrality time series are summarized in Fig. 8. Strikingly, we find consistent gains between 1950 and 2019 in both hub and authority centrality by the Carnegie Mellon University Departments of Mathematics while the Massachusetts Institute of Technology Department of Mathematics and the Yale University Department of Mathematics fall in both measures of centrality over the same time course, as shown in Figs. 8 and 9. To understand what caused the gains in hub and authority centrality between 1950 and 2019, we plot the rolling averages (using a 10-year window) of graduates from Carnegie Mellon ARTICLE Fig. 7 The fraction of hub (green solid curve) and authority (orange dotted curve) centrality held in total by California Institute of Technology, Carnegie Mellon University, Columbia University, Cornell University, Harvard University, Massachusetts Institute of Technology, Princeton University, Stanford University, University of Chicago, University of California–Berkeley, University of Michigan, University of Washington, University of Wisconsin–Madison, and Yale University between the years of 1950 and 2019. Centrality scores are computed using a rolling window of 10 years, indicated on the x-axis by the first year of the decade. To select elite departments to study, we compute the departments with the seven highest authority scores across seven network constructions spanning 1950–1959, 1960–1969, 1970–1979, 1980–1989, 1990–1999, 2000–2009, and 2010–2019 and take the union of the department sets. Both the hub and authority centrality held by this group of elite departments remains relatively stable over this time frame. University, Massachusetts Institute of Technology, and Yale University who eventually became a doctoral-granting (DG) math faculty, as well as the number of DG math faculty hires at each university, starting with the year 1950 and ending with the year 2019 (see Fig. 10). For Carnegie Mellon University, we observe that the average number of graduates that eventually became a DG faculty and the number of DG faculty hires both initially rise, and then remain somewhat constant. In contrast, for Yale University, we observe an initial rise in the average number of graduates that eventually became DG faculty followed by a decline starting slightly after 1980. We also see a decline in the average number of DG faculty hires at Yale University over time. For 10-year windows near the end of the range considered, such as 2010 to 2019, the graduate student and DG faculty averages are slightly lower, likely due to the fact that graduate students may not have secured a DG faculty position yet, and similarly for faculty members who have been hired but have not yet had a student graduate. Discussion In this study, we analyze the MGP database to understand how academic faculty selection operates in the field of mathematics. In particular, we seek to elucidate the factors that influence the transition from mathematics graduate degree holder to doctoralgranting (DG) faculty member and understand temporal trends related to this transition. We first show a growing disparity between the number of mathematics Ph.D.s awarded and the number of DG faculty positions acquired over time. This disparity is most extreme after 1970. The GFT rate has reached its lowest point during the last 30 years, and even historically wellplacing departments exhibit the same trend. Additionally, we find HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 7 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 Fig. 8 Significant (p ≤ 0.05) and large (∣τ∣ > 0.5) Kendall rank correlation coefficients for time series of the hub (left) and authority (right) scores of elite math departments. We focus mainly on the relationships between the Carnegie Mellon University, Massachusetts Institute of Technology, and Yale University Departments of Mathematics. Fig. 9 Time series of hub and authority centrality scores associated with 106 math departments between the years 1950 and 2019. Centrality scores are computed using a rolling window of 10 years, indicated on the x-axis by the first year of the decade. The hub and authority centrality score time series for Carnegie Melon University, Massachusetts Institute of Technology, and Yale University math departments are highlighted in red, yellow, purple, respectively. The geometric mean of all 106 departments’ temporal hub and authority score is plotted as a dashed blue curve. Other elite departments are plotted in dark gray and all other departments are plotted in light gray. The hub and authority centrality time series for the Carnegie Melon University Department of Mathematics increases as the hub and authority centrality time series associated with the Massachusetts Institute of Technology and Yale University Departments of Mathematics decrease in a significant manner. Fig. 10 Averaged DG faculty hires and averaged graduate production at the Carnegie Mellon University, Massachusetts Institute of Technology, and Yale University Departments of Mathematics over time. (Left): Rolling averages using a 10-year window of DG math faculty hires at Carnegie Mellon University, Massachusetts Institute of Technology, and Yale University, by the year of their first graduated student. (Right): Rolling averages using a 10-year window of graduates from Carnegie Mellon University, Massachusetts Institute of Technology, and Yale University who eventually became a doctoral-granting (DG) math faculty, by year of graduation. Both subplots start with the year 1950 and end with the year 2019. 8 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 that the log of the GFT rate correlates with the log of the authority score of the graduate training institution. A logistic regression model reveals that both time and the authority score of an individual’s Ph.D. granting department are significantly associated with the probability of obtaining a DG faculty position in a department. Moreover, individuals inferred to be women appear to be disadvantaged as compared to men, though the disadvantage narrows over time. At the department level, we find that the Carnegie Mellon University Department of Mathematics increased its hub and authority centrality consistently over time, while the Massachusetts Institute of Technology and Yale University Departments of Mathematics fell in both measures over the same time frame. These gains and losses in centrality correspond to the temporal dynamics of the average number of graduate students produced (who eventually become a DG math faculty) and averaged DG math faculty hires at Carnegie Mellon University and Yale University, respectively. Increasing graduate student output (specifically those that become DG faculty members), and DG faculty hiring, would have resulted in increasing the authority and hub scores, respectively. However, the underlying details of what made these universities more or less attractive to graduate students and faculty remain unclear but may involve some combination of department culture, department research interests, and its relevance to funding initiatives over this period, amongst a suite of other unknown factors. Our study has several limitations regarding the data we used. We do not have information about the number of job applications individuals filed, career transition award recipient status, total citation counts, high-impact journal authorship records, postdoctoral fellowship recipient status, the length of time an individual spends on the job market, or if individuals conducted joint academic-industry job searches. It was previously shown that these factors are significantly associated with receiving an academic job offer in a recent survey analysis (Fernandes et al., 2020). We also do not have information pertaining to an individual’s desire and decision-making process regarding pursuing an academic career versus another type of employment. Additionally, we are only studying the transition from mathematics Ph.D. graduate to DG faculty member in mathematics. It is possible that mathematics departments have hired DG faculty members who are not listed in the MGP database due to their graduate training field. Our study also does not include DG faculty members who do not advise graduate students, for example, mathematics Ph.D. holders who teach at liberal arts colleges, community colleges, or even in non-mathematics departments. Our study also has important limitations regarding the centrality metric we used. Our method for computing temporal trajectories of network centrality is rather straightforward, and other more sophisticated frameworks have been designed for this purpose (see, e.g., Taylor et al., 2017, 2019, 2021; Kawakatsu et al., 2021). In our study, we used hub and authority centrality, which are eigenvector-based centrality metrics, to quantify the importance of math departments in our faculty hiring network. Following Myers et al. (2011), we used hubs and authorities because of their interpretable meaning in the context of math departments. However, eigenvector-based centralities can exhibit a phenomenon known as “localization,” in which centrality is concentrated in a handful of nodes relative to the rest of the network (Martin et al., 2014). This property is known to be related to the structure of the network and “is particularly visible in networks with high-degree hubs1 or power-law degree distributions” (Martin et al., 2014). To address this challenge, the recent work of Taylor et al. (2017) has proposed a generalization of eigenvector centrality for temporal networks. They find that ARTICLE “the strength of the coupling between layers is important for determining multiscale properties of centrality, such as localization phenomenon.” They tested their method on the MGP database in the weak and strong coupling limits and noted, “We believe this weak-coupling regime to be inappropriate for the MGP Ph.D. exchange network, as mathematics department prestige should not fluctuate wildly from one year to the next.” They also comment that “For scenarios in which exploring various [couplings] is not computationally feasible, we highlight that restricting one’s attention to the [strong coupling] limit can still yield very informative results.” While we do not use the exact method in Taylor et al. (2017), we do expect the layers of our temporal network to be relatively strongly coupled due to our use of a 10-year rolling window for each network construction. For example, the network that spans 1950–1959 shares much of the same information with the network that spans 1951–1960. In summary, we find that the Graduate Faculty Transition rate, an imperfect indicator of the relative ease of acquiring a DG faculty position in mathematics, is decaying over time, even for historically well-placing departments. We also uncover statistically significant factors (academic and otherwise) that influence the transition, including gender, year, target’s Ph.D. authority, and the number of students advised by Ph.D. advisor. At the department level, we find that the Carnegie Mellon University Department of Mathematics consistently gained both hub and authority centrality between 1950 and 2019, a rare behavior in our network analysis. Taken together, we find that acquiring a DG faculty position is becoming more difficult and overall the rankings of departments remain relatively constant, though it is not impossible for departments to improve their ranking over time. Data availability The code and an anonymized version of the Mathematics Genealogy Project dataset (https://mathgenealogy.org/index.php) we analyzed is posted here https://github.com/cefitzg/MGP_plots. Received: 17 October 2022; Accepted: 13 April 2023; Note 1 Martin and coworkers state the definition of “hubs” as “nodes of unusually high degree,” not to be confused with the technical definition of hubs and authority centralities in the section “Centrality scores: hubs and authorities”. References Barnett GA, Danowski JA, Feeley TH, Stalker J (2010) Measuring quality in communication doctoral education using network analysis of faculty-hiring patterns. J Commun 60(2):388–411 Clauset A, Arbesman S, Larremore DB (2015) Systematic inequality and hierarchy in faculty hiring networks. Sci Adv 1(1):e1400005 Cowan R, Rossello G (2018) Emergent structures in faculty hiring networks, and the effects of mobility on academic performance. Scientometrics 117:527–562 Demografix ApS. https://genderize.io/. Accessed 8 Oct 2022 Fernandes JD, Sarabipour S, Smite C et al. (2020) A survey-based analysis of the academic job market. eLife 9:1–30 Fowler JH, Grofman B, Masuoka N (2007) Social networks in political science: hiring and placement of Ph. Ds, 1960–2002. Political Sci Politics 40:729–739 Hanneman RA (2001) The prestige of Ph.D. granting departments of sociology: a simple network approach. Connections 24(1):68–77 Jahan N, Barr TH, Rose CA, Macias VP (2022) Academic recruitment, hiring, and attrition during 2018–2019. Not Am Math Soc. https://www.ams.org/ profession/data/annual-survey/advance_release_annsurv-recruitmenthiring_report2019.pdf Katakatsu M, Chodrow PS, Eikmeier N, Larremore DB (2021) Emergence of hierarchy in networked endorsement dynamics. Proc Natl Acad Sci USA 118(16):e2015188118 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9 9 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9 Kendall MG (1948) Rank correlation methods. Griffin Kleinberg JM (1999) Authoritative sources in a hyperlinked environment. J ACM 46(5):604–632 Lee E, Clauset A, Larremore DB (2021) The dynamics of faculty hiring networks. EPJ Data Sci 10(1):48 Mai B, Liu J, González-Bailón S (2015) Network effects in the academic market: Mechanisms for hiring and placing PhDs in communication (2007–2014). J Commun 65(3):558–583 Martin T, Zhang X, Newman MEJ (2014) Localization and centrality in networks. Phys Rev E 90(5):2808 Morgan AC, LaBerge N, Larremore DB et al. (2022) Socioeconomic roots of academic faculty. Nat Hum Behav. https://doi.org/10.1038/s41562-022-01425-4 Myers SA, Mucha PJ, Porter MA (2011) Mathematical genealogy and department prestige. Chaos 21(4):041104 Newman M (2018) Networks. Oxford University Press Reys R, Reys B, Shih J (2022) Some patterns of PhDs in mathematics awarded annually by institutions of higher education in the United States over the last two decades. Not Am Math Soc 69(1):96–107 Taylor D, Myers S, Clauset A, Porter M, Mucha P (2017) Eigenvector-based centrality measures for temporal networks. Multiscale Model Simul 15(1):537–574 Taylor D, Porter MA, Mucha PJ (2019) Supracentrality analysis of temporal networks with directed interlayer coupling. In: Holme P, Saramäki J (eds) Temporal network theory. Computational social sciences. Springer, Cham Taylor D, Porter M, Mucha P (2021) Tunable eigenvector-based centralities for multiplex an temporal networks. Multiscale Model Simul 19(1):113–147 Wapman KH, Zhang S, Clauset A, Larremore DB (2022) Quantifying hierarchy and dynamics in US faculty hiring and retention. Nature 610:120–127 White-Lewis DK (2020) The facade of fit in faculty search processes J High Educ 91(6):833–857 Zuo Z, Zhao K, Ni C (2019) Standing on the shoulders of giants?—Faculty hiring in information schools. J Informetr 13:341–353 Acknowledgements CF was supported by the NSF-Simons Center for Quantitative Biology at Northwestern University (NSF: 1764421 and Simons Foundation/SFARI 597491-RWC) and James S. McDonnell Foundation Postdoctoral Fellowship Award in Complex Systems (https://doi. org/10.37717/2020-1591). 10 Competing interests The authors declare no competing interests. Ethical approval This article does not contain any studies with human participants performed by any of the authors. Informed consent This article does not contain any studies with human participants performed by any of the authors. Additional information Correspondence and requests for materials should be addressed to Cody FitzGerald or Yitong Huang. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2023 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9
https://openalex.org/W2974033835
https://aprp.msal.ru/jour/article/download/1590/1252
Russian
null
New types of the partnership agreement in the context of collective use of goods and services (sharing economy)
Aktualʹnye problemy rossijskogo prava
2,019
cc-by
5,456
© Подузова Е. Б., 2019 * Подузова Екатерина Борисовна, кандидат юридических наук, доцент кафедры гражданского права, доцент кафедры нотариата Московского государственного юридического университета имени О.Е. Ку- тафина (МГЮА) ekaterinak7785@yandex.ru 125993, Россия, г. Москва, ул. Садовая-Кудринская, д. 9 ГРАЖДАНСКОЕ И СЕМЕЙНОЕ ПРАВО Е. Б. Подузова* Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy)1 Аннотация. Современный социально-экономический контекст совместного потребления (sharing economy) ставит новые цели и задачи использования конструкций организации сов­ местной деятельности. В статье представлены основные теоретические и практические проблемы видов договора простого товарищества как организационного договора, формы организации и ведения сов­ местной деятельности. Материал статьи подготовлен с учетом реформы договорного права Российской Федерации, новых тенденций в науке гражданского права и в правоприменительной практике. Также была учтена актуальная судебная практика, в которой содержатся новые подходы к толкованию обязательства и договора. В связи с этим особое значение имеют постановления Пленума Верховного Суда РФ (например, от 24 марта 2016 г. № 7 «О применении судами некоторых положений Гражданского кодекса Российской Федерации об ответственности за нарушение обязательств», от 22 ноября 2016 г. № 54 «О некоторых вопросах применения общих положе- ний Гражданского кодекса Российской Федерации об обязательствах и их исполнении»). Особое внимание было уделено правовой природе, конститутивным признакам отдельных Особое внимание было уделено правовой природе, конститутивным признакам отдельных видов договора простого товарищества, их проявлению в современном гражданском законо- дательстве и практике его применения. Ключевые слова: коллективное использование, договор простого товарищества, сделка, разновидности договора, правовая природа, организация, организующий договор, договор об организации совместной деятельности, имущественный элемент, встречное предоставление, инвестиционное товарищество, горное товарищество. Актуальные проблемы российского права. 2019. № 8 (105) август DOI: 10.17803/1994-1471.2019.105.8.086-094 1 Исследование выполнено при финансовой поддержке РФФИ в рамках научного проекта № 18-29-16054 «Концептуальные основы правового регулирования отношений по коллективному использованию то- варов и услуг (sharing economy) в условиях развития цифровых технологий». Актуальные проблемы российского права. 2019. № 8 (105) август 86 Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) Подузова Е. Б. Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) предполагается, что агрегаторы осуществляют подбор заказчику конкретного исполнителя, устанавливают цены на услуги исполнителей. Во втором случае лишь предоставляется удобный сервис для поиска исполнителя8. З а последнее десятилетие популярность коллективного использования товаров и услуг возросла в значительной мере. В первую очередь большой спрос приобрела бизнес-модель Uber, построенная по прин- ципу экономики совместного потребления — sharing economy firm2 (ride-sharing company3, transportation network companies4, online- enabled car transportation service5). В бизнес-сре- де и вне ее активно используется модель work- sharing6, известная со времен Великой депрес- сии в США в 1920-е гг. Данная модель предпо- лагает вывод ряда работ и услуг за пределы персонала компании либо обмен знаниями, поиск лиц, выполняющих работы и оказываю- щих услуги в конкретной бытовой сфере. Такая модель представлена, например, в сервисах YouDo, profi.ru, а также в сервисах взаимного изучения языков посредством общения с их носителями7. З а Каждая из правовых форм реализации сов­ местного потребления представляет собой совместную деятельность. Использование платформ-агрегаторов не предполагает стро- гих требований к правовой форме данных от- ношений. В судебной практике отмечается, что несоблюдение требований к форме договора при достижении сторонами соглашения по всем его существенным условиям не свидетельствует о том, что договор не был заключен9. Возникает вопрос о возможности применения конструкции договора простого товарищества для правового оформления отношений по коллективному ис- пользованию товаров и услуг. В рамках гражданско-правовой реформы произошли существенные изменения граждан- ского законодательства и практики его приме- В литературе выделяются две разновидности таких компаний-агрегаторов. В первом варианте 4 См.: Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition Policy International. 2015. № LS 15-11 ; № 15-29. 5 См.: Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy International, Forthcoming. November 24, 2015. P. 1. 6 What is work-sharing // URL: https://www.wisegeek.com/what-is-work-sharing.htm (дата обращения: 20 фев- раля 2019 г.). 7 См., например: URL: https://www.rosettastone.com/lp/sbsr/livemocha/?prid=livemocha_com (дата обра- щения: 20 февраля 2019 г.). 8 См. об этом подробнее: Савельев А. И. м.: Rogers B. The Social Costs of Uber // University of Chicago Law Review Dialogue. 2015. № 28. P. 85. 2 См.: Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation of the Sharing Economy // George Mason Law & Economics Research Paper. 2015. № 15-01. P. 1. DOI: 10.17803/1994-1471.2019.105.8.086-094 Электронная коммерция в России и за рубежом: правовое регули- рование. М. : Статут, 2016 ; Бычков А. Посредничество при оказании юридических услуг: практика, риски, перспективы // Новая бухгалтерия. 2016. № 9. С. 130—143 ; Иванов А. А. Бизнес-агрегаторы и право // Закон. 2017. № 5. С. 145—157 ; Молотников А. Е., Архипов Е. В. Социальные сети и компании-агрегаторы: правовые аспекты деятельности // Предпринимательское право. 2017. № 4 ; Чесалина О. В. Работа на основе интернет-платформ (crowdwork и work on demand via apps) как вызов трудовому и социальному праву // Трудовое право в России и за рубежом. 2017. № 1 ; De Stefano V. The rise of the «just-in-time workforce»: On-demand work, crowdwork and labour protection in the «gig-economy» // Conditions of work and employment, Series No. 71. Geneva : ILO, 2016. P. 1. URL: http://www.ilo.org/wcmsp5/groups/public/- --ed_protect/---protrav/---travail/documents/publication/wcms_443267.pdf (дата обращения: 31 января 2019 г.). 9 Постановление Пленума Верховного Суда РФ от 25 декабря 2018 г. № 49 «О некоторых вопросах при- менения общих положений Гражданского кодекса Российской Федерации о заключении и толковании договора». П. 3 // СПС «КонсультантПлюс». 9 Постановление Пленума Верховного Суда РФ от 25 декабря 2018 г. № 49 «О некоторых вопросах при- менения общих положений Гражданского кодекса Российской Федерации о заключении и толковании договора». П. 3 // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 87 Гражданское и семейное право жет быть признан недействительным в судеб- ном порядке по требованию любого другого участника данного договора с возложением на управляющего товарища обязанности по воз- мещению всем участникам данного договора причиненных им в связи с этим убытков. нения10, изменились традиционные теорети- ко-практические подходы к понятию и призна- кам договора простого товарищества, к выявле- нию его новых разновидностей. Среди новых разновидностей этого догово- ра можно назвать в первую очередь договор инвестиционного товарищества11. Базируясь на классической модели договора простого товари- щества, договор инвестиционного товарищества имеет специфические черты, характерные для товариществ — юридических лиц. Предусмотрены квалифицированные поря- док и форма заключения договора инвестицион- ного товарищества. В частности, договор считает- ся заключенным, а внесенные в него изменения считаются вступившими в силу со дня нотари- ального удостоверения данного договора или внесенных в него изменений. Договор инвести- ционного товарищества с новым его участником считается заключенным со дня нотариального удостоверения соглашения о присоединении. DOI: 10.17803/1994-1471.2019.105.8.086-094 К таким чертам относятся: особая цель — осуществление совместной инвестиционной деятельности исключительно для извлечения прибыли; управляющие товарищи осуществля- ют от имени всех товарищей ведение общих дел и несут солидарную ответственность за по- следствия таких действий (бездействия); общее число участников договора инвестиционного товарищества не должно быть более 15, в со- став общего имущества товарищей вносится имущество, являющееся собственностью това- рища, либо имущество, находящееся в его дове- рительном управлении. В судебной практике от- носительно распределения прибыли в данном договоре указывается, что прежде чем требо- вать распределения прибыли, товарищ обязан внести вклад в общее дело12. В судебной практике существенные усло- вия договора инвестиционного товарищества включают в себя условия общей конструкции (договора простого товарищества): условия о со- вместной деятельности участников договора, об их общей цели и об объединении ими вкладов, предметом договора простого товарищества является совместная деятельность по достиже- нию общей цели13. К числу существенных усло- вий договора инвестиционного товарищества также относятся размер и порядок выплаты вознаграждения управляющего товарища (ст. 5 Федерального закона «Об инвестиционном то- вариществе»), политика ведения общих дел (инвестиционная декларация) — ст. 8 данного Федерального закона. Законом установлены ограничения для уча- стия управляющего товарища в договорах: он не вправе участвовать одновременно в двух и бо- лее договорах инвестиционного товарищества, если хотя бы один из них содержит запрет на такое участие, в противном случае договор мо- Однозначно признаваемой судебной практи- кой14 разновидностью договора простого това- 10 См.: постановление Пленума Верховного Суда РФ от 24 марта 2016 г. № 7 «О применении судами не- которых положений Гражданского кодекса Российской Федерации об ответственности за нарушение обязательств» ; постановление Пленума Верховного Суда РФ от 22 ноября 2016 г. № 54 «О некоторых вопросах применения общих положений Гражданского кодекса Российской Федерации об обязательствах и их исполнении» // СПС «КонсультантПлюс». 10 См.: постановление Пленума Верховного Суда РФ от 24 марта 2016 г. № 7 «О применении судами не- которых положений Гражданского кодекса Российской Федерации об ответственности за нарушение обязательств» ; постановление Пленума Верховного Суда РФ от 22 ноября 2016 г. № 54 «О некоторых вопросах применения общих положений Гражданского кодекса Российской Федерации об обязательствах и их исполнении» // СПС «КонсультантПлюс». едеральный закон от 28 ноября 2011 г. № 335‑ФЗ «Об инвестиционном товариществе» // СПС «Ко льтантПлюс». 11 Федеральный закон от 28 ноября 2011 г. № 335‑ФЗ «Об инвестиционном товариществе» // СПС «Кон- сультантПлюс». 12 См.: постановление Арбитражного суда Поволжского округа от 28 мая 2015 г. 10 См.: постановление Пленума Верховного Суда РФ от 24 марта 2016 г. № 7 «О применении судами не- которых положений Гражданского кодекса Российской Федерации об ответственности за нарушение обязательств» ; постановление Пленума Верховного Суда РФ от 22 ноября 2016 г. № 54 «О некоторых вопросах применения общих положений Гражданского кодекса Российской Федерации об обязательствах и их исполнении» // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август DOI: 10.17803/1994-1471.2019.105.8.086-094 № Ф06-22892/2015 по делу м.: постановление Арбитражного суда Поволжского округа от 28 мая 2015 г. № Ф06-22892/2015 по дел А12-29061/2013 // СПС «КонсультантПлюс». м.: постановление Четырнадцатого арбитражного апелляционного суда от 27 января 2016 г. по дел А05-6313/2015 // СПС «КонсультантПлюс». 14 См.: постановление ФАС Московского округа от 13 февраля 2014 г. № Ф05-17103/2013 по делу № А40- 24572/13-25-124 // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 88 Подузова Е. Б. Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) ду Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) движимыми и недвижимыми вещами; 3) дело- вая репутация и деловые связи. рищества (помимо договора инвестиционного товарищества) являются страховые (перестрахо- вочные) пулы (ст. 14.1 Закона РФ от 27 ноября 1992 г. № 4015-1 «Об организации страхового дела в Российской Федерации»). Данные дого- воры простого товарищества имеют специаль- ные цели создания и деятельности — обеспече- ние финансовой устойчивости его участников, исполнение ими обязательств по страховым выплатам, размер которых может превысить собственные средства (капитал) одной страхо- вой организации, — и действуют на принципах сострахования или перестрахования. Существенными условиями договора горного товарищества являются: данные, позволяющие определить участок недр, право пользования которым предоставлено или будет предоставле- но пользователю недр в соответствии с законо- дательством Российской Федерации о недрах и по поводу деятельности на котором заключа- ется договор; доли или порядок определения долей сторон в распределяемой между сторо- нами доходной части добытых при исполнении обязательств по договору горного товарищества углеводородного сырья и попутно извлекаемых ресурсов или в распределяемых между сторона- ми доходах от их реализации. Договор горного товарищества должен быть заключен в простой письменной форме. На стадии законопроекта15 предлагается введение еще двух разновидностей договора простого товарищества со специальной сферой применения и субъектным составом. Так, по договору горного товарищества сто- роны обязуются совместно осуществлять де- ятельность по разработке на определенном участке (участках) недр в соответствии с ус- ловиями лицензии на пользование недрами, которая получена или будет получена одной из них в соответствии с законодательством Российской Федерации о недрах, и распре- делять между собой добытые сырье, ресур- сы или доходы от их реализации. Содержание данного договора всегда открыто для третьих лиц. Стороны договора горного товарищества: лицо — пользователь недр и оператор. Субъ- ектный состав этого договора: российские юри- дические лица, являющиеся коммерческими организациями, а также иностранные юриди- ческие лица. 15 Проект федерального закона «Об особенностях совместной деятельности в сфере недропользования и о внесении изменений в отдельные законодательные акты Российской Федерации» (подготовлен Минприроды России) (не внесен в ГД ФС РФ, текст по состоянию на 21 декабря 2016 г.) // СПС «Консуль- тантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август DOI: 10.17803/1994-1471.2019.105.8.086-094 В то же время положения Гражданского кодекса РФ не оставляют сомнений в том, что сделка является юридическим фактом (см., например, п. 1 ст. 8, 153, п. 2 ст. 307 ГК РФ), порождающим граждан- ское правоотношение, включая обязательство. Как представляется, следует различать су- ществование правоотношения, существование прав, обязанностей и реализацию прав, испол- нение обязанностей, то есть стадию реализации содержания правоотношения. Правоотношение возникает из факта совершения сделки либо из факта наступления отлагательного условия сдел- ки (ст. 157 ГК РФ). Срок исполнения обязатель- ства не оказывает влияния на его существова- ние (ст. 314 ГК РФ), срок (периоды) реализации прав также не оказывает влияния на существо- вание вещного правоотношения. В противном случае, если обязательственное правоотно- шение не существует до момента исполнения обязательства, то и вещное правоотношение не существует в те моменты, когда не проис- ходит его реализации посредством активных К мнению о таком существе договора про- стого товарищества затруднительно присоеди- ниться. Исходя из позиции автора сделка соз- дает возможность возникновения у лица, ее совершившего, обязанностей и утраты им прав помимо его воли, то есть сделка порождает не правоотношение, а возможность его возник- новения, отрицается волевой элемент сделки. В таком случае сделка не может быть квали- фицирована в качестве юридического факта, поскольку конститутивным признаком любого юридического факта является создание, изме- нение либо прекращение правоотношения17. Из факта совершения сделки возникает правовая связь, и эта связь обладает волевой составля- ющей, субъектным составом, содержанием, направлена на определенный объект. В то же время положения Гражданского кодекса РФ не оставляют сомнений в том, что сделка является юридическим фактом (см., например, п. 1 ст. 8, 153, п. 2 ст. 307 ГК РФ), порождающим граждан- ское правоотношение, включая обязательство. В том же законопроекте представлена двух- ступенчатая модель организационных отно- шений, реализуемая посредством заключения и исполнения двух договоров об организации совместной деятельности. Следует отметить не- корректность применения термина «договор совместного инвестирования» в свете наличия легализованной и применяемой на практике конструкции договора инвестиционного това- рищества, имеющей свои особенности и сферу применения. Субъектный состав договора совместного ин- вестирования по общему правилу не ограничен. Кроме того, все стороны договора совместного инвестирования могут выступать на стороне оператора по договору горного товарищества, управляющий товарищ по договору совмест- ного инвестирования выполняет функции от- ветственного оператора по договору горного товарищества. 16 Скловский К. И. Сделка и ее действие. Комментарий главы 9 ГК РФ (понятие, виды и форма сделок. Не- действительность сделок). М. : Статут, 2015. 17 См., например: Российское гражданское право / отв. ред. Е. А. Суханов. М. : Статут, 2011. Т. 1. (автор главы — В. С. Ем). DOI: 10.17803/1994-1471.2019.105.8.086-094 Вторым предлагаемым к введению дого- вором является договор совместного инвести- рования в деятельность по разработке недр (договор совместного инвестирования). Его стороны обязуются определять в пределах, установленных договором, порядок осущест- вления одной из сторон договора от своего име- ни и в общих интересах прав и обязанностей оператора по договору горного товарищества (далее — управляющий товарищ) в целях по- следующего распределения между сторонами договора совместного инвестирования получен- ных управляющим товарищем углеводородно- го сырья и попутно извлекаемых ресурсов или доходов от их реализации, а также совместно осуществлять финансирование участия управ- ляющего товарища в договоре горного товари- щества в качестве оператора. Таким образом, договор совместного инвестирования привязан к договору горного товарищества (если договор горного товарищества, для целей участия управ- ляющего товарища в котором заключается дого- вор совместного инвестирования, не заключен, договор совместного инвестирования вступает в силу с момента заключения договора горного Вкладами сторон договора горного товари- щества в совместную деятельность признается все, что они вносят в общее дело, включая: 1) их действия, направленные на достижение общих целей, включая действия по предоставлению друг другу необходимой информации, а также взаимодействие с третьими лицами; 2) имуще- ство, в том числе имущественные права, вклю- чая исключительные права и право пользования 15 Проект федерального закона «Об особенностях совместной деятельности в сфере недропользования и о внесении изменений в отдельные законодательные акты Российской Федерации» (подготовлен Минприроды России) (не внесен в ГД ФС РФ, текст по состоянию на 21 декабря 2016 г.) // СПС «Консуль- тантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 89 Гражданское и семейное право товарищества на тех условиях, которые прини- мались во внимание сторонами договора со- вместного инвестирования при его заключении, кроме случаев, предусмотренных договором) и заключается для его обслуживания. туре указывается, что он не имеет обязательств и не порождает их, а все товарищи не выступают как кредиторы или должники по отношению друг к другу16. друг к другу . К мнению о таком существе договора про- стого товарищества затруднительно присоеди- ниться. Исходя из позиции автора сделка соз- дает возможность возникновения у лица, ее совершившего, обязанностей и утраты им прав помимо его воли, то есть сделка порождает не правоотношение, а возможность его возник- новения, отрицается волевой элемент сделки. В таком случае сделка не может быть квали- фицирована в качестве юридического факта, поскольку конститутивным признаком любого юридического факта является создание, изме- нение либо прекращение правоотношения17. Из факта совершения сделки возникает правовая связь, и эта связь обладает волевой составля- ющей, субъектным составом, содержанием, направлена на определенный объект. DOI: 10.17803/1994-1471.2019.105.8.086-094 Существенными условиями договора со- вместного инвестирования являются: данные, позволяющие определить участок недр, по по- воду деятельности на котором заключен дого- вор; сведения о договоре горного товарище- ства, для целей участия управляющего товарища в котором заключается договор совместного инвестирования, если такой договор горного товарищества заключен; наименование сторо- ны — управляющего товарища; доли сторон в распределяемой между ними доходной ча- сти добытых углеводородного сырья и попутно извлекаемых ресурсов или в распределяемых между сторонами доходах от их реализации. Договор совместного инвестирования должен быть заключен в простой письменной форме. Как представляется, следует различать су- ществование правоотношения, существование прав, обязанностей и реализацию прав, испол- нение обязанностей, то есть стадию реализации содержания правоотношения. Правоотношение возникает из факта совершения сделки либо из факта наступления отлагательного условия сдел- ки (ст. 157 ГК РФ). Срок исполнения обязатель- ства не оказывает влияния на его существова- ние (ст. 314 ГК РФ), срок (периоды) реализации прав также не оказывает влияния на существо- вание вещного правоотношения. В противном случае, если обязательственное правоотно- шение не существует до момента исполнения обязательства, то и вещное правоотношение не существует в те моменты, когда не проис- ходит его реализации посредством активных Применительно к общей категории договора простого товарищества в юридической литера- Актуальные проблемы российского права. 2019. № 8 (105) август 90 Подузова Е. Б. Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) ду Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) правомерности подобного состава и правового режима общего имущества товарищей выступа- ет лишь специальное правовое регулирование соответствующих общественных отношений. действий. Если вещь, например, украдена в та- кой момент, то нет оснований для применения гражданско-правовых способов защиты (ст. 12, 301—303 ГК РФ), поскольку в этот момент не было и самого правоотношения собственности. Договор простого товарищества, будучи од- ним из видов организующих сделок, обладает ее конститутивными признаками — неимуще- ственной направленностью, организационным основанием. Договор простого товарищества носит невзаимный характер, в нем отсутствует встречное предоставление, он порождает ор- ганизационное обязательство. Имущественный элемент в этом договоре выступает как основа совместной деятельности и не влияет на об- щую организующую направленность. Договор простого товарищества порождает внутренние и внешние правоотношения. Внутренние пра- воотношения — организационные, существуют между сторонами этого договора. Внешние от- ношения возникают на базе организационных между стороной (сторонами) этого договора и третьими лицами и носят как имущественный, так и неимущественный характер. Внутренние правоотношения организуют внешние, посред- ством которых образуется прибыль (убытки) то- варищей. 18 Определение Высшего Арбитражного Суда РФ от 20 августа 2013 г. № ВАС-8174/13 по делу № А70- 3394/2012 ; постановление Арбитражного суда Волго-Вятского округа от 9 декабря 2014 г. № Ф01- 4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс». 20 См.: Companies Act, 2006. д у у 19 Companies Act, 2006 // URL: http://www.legislation.gov.uk/ukpga/2006/46/section/29 (дата обращения: 28 января 2019 г.). 3394/2012 ; постановление Арбитражного суда Волго Вятского округа от 9 декабря 2014 г. № Ф01 4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс». 19 Companies Act, 2006 // URL: http://www.legislation.gov.uk/ukpga/2006/46/section/29 (дата обращения: 28 января 2019 г.). 18 Определение Высшего Арбитражного Суда РФ от 20 августа 2013 г. № ВАС-8174/13 по делу № А70- 3394/2012 ; постановление Арбитражного суда Волго-Вятского округа от 9 декабря 2014 г. № Ф01- 4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс». 19 Companies Act, 2006 // URL: http://www.legislation.gov.uk/ukpga/2006/46/section/29 (дата обращения: 28 января 2019 г.). 20 См : Companies Act 2006 DOI: 10.17803/1994-1471.2019.105.8.086-094 В судебной практике подтверждается организующая природа договора простого то- варищества18. Полагаем, что договор простого товарище- ства — это юридический факт, порождающий правоотношение. Согласно ст. 1041 ГК РФ совер- шение действий по достижению общей цели со- вместной деятельности является обязанностью по договору простого товарищества. Данная обязанность может быть исполнена принуди- тельно при применении такого способа защиты гражданских прав, как присуждение к испол- нению обязанности в натуре (ст. 12 ГК РФ). До- говор простого товарищества порождает два вида правоотношений — обязательственное и вещное, поскольку согласно п. 1 ст. 1043 ГК РФ внесенное товарищами имущество, которым они обладали на праве собственности, а также произведенная в результате совместной дея- тельности продукция и полученные от такой деятельности плоды и доходы признаются их общей долевой собственностью, если иное не установлено законом или договором простого товарищества либо не вытекает из существа обя- зательства. Имущество товарищей — сложный объект гражданских прав, включающий в себя деньги, иное имущество, профессиональные и иные знания, навыки и умения, а также дело- вую репутацию и деловые связи (п. 1 ст. 1042 ГК РФ). Общее имущество товарищей состоит из ряда объектов, не относящихся к числу объектов гражданских прав, а также к числу непереда- ваемых объектов гражданских прав (деловая репутация, ст. 150 ГК РФ). В рамках договора простого товарищества возникает вещное право на исключительное право, если оно входит в со- став общего имущества товарищей, что также вступает в противоречие с положениями ч. IV Гражданского кодекса РФ. Аргументом в пользу Следует обратить внимание, что в системе общего права существуют договоры (соглаше- ния) об организации совместной деятельности (ст. 8 английского Акта о компаниях 2006 г.19, в английской бизнес-сфере используется кон- струкция соглашения о партнерстве20). Дан- ные соглашения относятся к числу договоров, которые возможно принудительно исполнить посредством обращения в суд, в понимании английской доктрины и практики эти договоры обладают встречным предоставлением. Разновидности договора простого товарище- ства, как и сам договор простого товарищества, 18 Определение Высшего Арбитражного Суда РФ от 20 августа 2013 г. № ВАС-8174/13 по делу № А70- 3394/2012 ; постановление Арбитражного суда Волго-Вятского округа от 9 декабря 2014 г. № Ф01- 4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 91 Гражданское и семейное право относятся по своей правовой природе к числу организующих сделок с имущественными эле- ментами, выступающими основой совместной деятельности. Изменение конструкции договора простого товарищества посредством введения и предложения новых его разновидностей явля- ется важным достижением реформы граждан- ского законодательства. DOI: 10.17803/1994-1471.2019.105.8.086-094 Однако современные конструкции этого договора и его разновидно- стей, предлагаемые к введению, не учитывают организующую правовую природу отношений товарищества, особую судьбу имущественной основы деятельности товарищей, взаимосвязь внутренних и внешних правоотношений. Зако- нодательство о договорах простого товарище- ства нуждается в дальнейшем изменении. дукт (товар, услуга), подлежащий совместному потреблению. Для заключения договора про- стого товарищества и его видов используется классическая модель организации контрактных связей. Напротив, для заключения соглашений о совместном использовании товаров и услуг во многих случаях применяются агрегаторы как посредники между сторонами. Договор про- стого товарищества порождает два вида пра- воотношений — обязательственное и вещное, поскольку согласно п. 1 ст. 1043 ГК РФ внесенное товарищами имущество, которым они облада- ли на праве собственности, а также произве- денная в результате совместной деятельности продукция и полученные от такой деятельности плоды и доходы признаются их общей долевой собственностью. Соглашения о совместном ис- пользовании товаров и услуг отношений соб- ственности не порождают. Возникает вопрос в целесообразности адап- тации законодательства о договорах простого товарищества к соглашениям о совместном ис- пользовании товаров и услуг. Договор простого товарищества не тождественен данным согла- шениям, поскольку в соглашениях о совмест- ном использовании товаров и услуг отсутствует единая совместная общая цель, к достижению которой стремятся все участники любого вида договора простого товарищества. В свою оче- редь, в договоре простого товарищества изна- чально отсутствует единый материальный про- Общей чертой договоров простого товарище- ства и соглашений о совместном использовании товаров и услуг является совместная деятель- ность их сторон. В связи с этим при разработке норм, регулирующих отношения по совмест- ному использованию товаров и услуг, возмож- но учитывать субсидиарное применение норм о договорах простого товарищества к таким от- ношениям. БИБЛИОГРАФИЯ ду Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) 9. De Stefano V. The rise of the «just-in-time workforce»: On-demand work, crowdwork and labour protection in the «gig-economy» // Conditions of work and employment. Series No. 71. — Geneva : ILO, 2016. — URL: http://www.ilo.org/wcmsp5/groups/public/—-ed_protect/—-protrav/—-travail/documents/publication/ wcms_443267.pdf (дата обращения: 31 января 2019 г.). 10. Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy International, Forthcoming. — November 24, 2015. 10. Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy International, Forthcoming. — November 24, 2015. // 11. Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition Policy International. — 2015. — № LS 15-11; № 15—29. Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation the Sharing Economy // George Mason Law & Economics Research Paper. — 2015. — № 15—01. 13. Rogers B. The Social Costs of Uber // University of Chicago Law Review Dialogue. — 2015. — № 2 13. Rogers B. The Social Costs of Uber // University of Chicago Law Review Dialogue. — 2015. — № 28. Материал поступил в редакцию 20 февраля 2019 г. БИБЛИОГРАФИЯ 1. Бычков А. Посредничество при оказании юридических услуг: практика, риски, перспективы // Новая бухгалтерия. — 2016. — № 9. — С. 130—143. 1. Бычков А. Посредничество при оказании юридических услуг: практика, риски, перспективы // Новая бухгалтерия. — 2016. — № 9. — С. 130—143. 1. Бычков А. Посредничество при оказании юридических услуг: практика, риски, перспективы // Новая бухгалтерия. — 2016. — № 9. — С. 130—143. у р 2. Иванов А. А. Бизнес-агрегаторы и право // Закон. — 2017. — № 5. — С. 145—157. 3. Молотников А. Е., Архипов Е. В. Социальные сети и компании-агрегаторы: правовые аспекты дея- тельности // Предпринимательское право. — 2017. — № 4. 3. Молотников А. Е., Архипов Е. В. Социальные сети и компании-агрегаторы: правовые аспекты дея- тельности // Предпринимательское право. — 2017. — № 4. 4. Российское гражданское право / отв. ред. Е. А. Суханов. — М. : Статут, 2011. — Т. 1. 4. Российское гражданское право / отв. ред. Е. А. Суханов. — М. : Статут, 2011. — Т. 1. 5. Савельев А. И. Электронная коммерция в России и за рубежом: правовое регулирование. — М. : С 2016. 5. Савельев А. И. Электронная коммерция в России и за рубежом: правовое регулирование. — М. : Статут, 2016. Скловский К. И. Сделка и ее действие. Комментарий главы 9 ГК РФ (понятие, виды и форма сдело Недействительность сделок). — М. : Статут, 2015. 7. Челышев М. Ю. Вопросы межотраслевых связей гражданского права в Концепции развития граж- данского законодательства и проекте Гражданского кодекса Российской Федерации // Гражданское право. — 2011. — № 1. — С. 3—7. 7. Челышев М. Ю. Вопросы межотраслевых связей гражданского права в Концепции развития граж- данского законодательства и проекте Гражданского кодекса Российской Федерации // Гражданское право. — 2011. — № 1. — С. 3—7. 8. Чесалина О. В. Работа на основе интернет-платформ (crowdwork и work on demand via apps) как вызов трудовому и социальному праву // Трудовое право в России и за рубежом. — 2017. — № 1. 8. Чесалина О. В. Работа на основе интернет-платформ (crowdwork и work on demand via apps) как вызов трудовому и социальному праву // Трудовое право в России и за рубежом. — 2017. — № 1. Актуальные проблемы российского права. 2019. № 8 (105) август 92 Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy Подузова Е. Б. 21 The research has been carried out with the financial support of RFBR within the framework of scientific project № 18-29-16054 “Conceptual bases of legal regulation of relations of sharing economy in the era of digital technologies.” Актуальные проблемы российского права. 2019. № 8 (105) август Подузова Е. Б. NEW TYPES OF THE PARTNERSHIP AGREEMENT IN THE CONTEXT OF COLLECTIVE USE OF GOODS AND SERVICES (SHARING ECONOMY)21 PODUZOVA Ekaterina Borisovna, PhD in Law, Associate Professor of the Department of Civil Law, Associate Professor of the Department of Notaries of the Kutafin Moscow State Law University (MSAL) ekaterinak7785@yandex.ru 125993, Russia, Moscow, ul. Sadovaya-Kudrinskaya, d. 9 PODUZOVA Ekaterina Borisovna, PhD in Law, Associate Professor of the Department of Civil Law, Associate Professor of the Department of Notaries of the Kutafin Moscow State Law University (MSAL) Abstract. The modern socio-economic context of shared consumption (sharing economy) sets new goals and objectives of using joint activities. The article presents the main theoretical and practical problems of types of the partnership agreement as an organizational agreement, forms of organization and conduct of joint activity. The article is prepared with due regard to the reform of contract law of the Russian Federation, new trends in the science of civil law and in law enforcement practice. Also, the article takes into account the jurisprudence that contains new approaches to the interpretation of an obligation and agreement under consideration. In this regard, the decisions of the Plenum of the Supreme Court of the Russian Federation (for example, the Decision dated March 24, 2016 No. 7 “On Application by Courts of Certain Provisions of the Civil Code of the Russian Federation Concerning the Breach of the Obligations,» 22 November 2016 No. 54 “On Certain Issues of Application of General Provisions of the Civil Code of the Russian Federation on Obligations and Their Performance”) are subjected to thorough examination. Particular attention is paid to the legal nature, constitutional features of certain types of the partnership agreement, their manifestations in modern civil legislation and practice of application. Keywords: collective use, partnership agreement, transaction, types of contract, legal nature, organization, organizing agreement, agreement on organization of joint activities, property element, consideration, investment partnership, mining partnership. 21 The research has been carried out with the financial support of RFBR within the framework of scientific project № 18-29-16054 “Conceptual bases of legal regulation of relations of sharing economy in the era of digital technologies.” 93 Гражданское и семейное право Актуальные проблемы российского права. 2019. № 8 (105) август REFERENCES (TRANSLITERATION) 1. Bychkov A. Posrednichestvo pri okazanii yuridicheskih uslug: praktika, riski, perspektivy // Novaya buhgalteriya. — 2016. — № 9. — S. 130—143. 2. Ivanov A. A. Biznes-agregatory i pravo // Zakon. — 2017. — № 5. — S. 145—157. 3. Molotnikov A. E., Arhipov E. V. Social’nye seti i kompanii-agregatory: pravovye aspekty deyatel’nosti // Predprinimatel’skoe pravo. — 2017. — № 4. 4. Rossijskoe grazhdanskoe pravo / otv. red. E. A. Suhanov. — M. : Statut, 2011. — T. 1. 5. Savel’ev A. I. Elektronnaya kommerciya v Rossii i za rubezhom: pravovoe regulirovanie. — M. : Statut, 5. Savel ev A. I. Elektronnaya kommerciya v Rossii i za rubezhom: pravovoe regulirovanie. M. : Statut, 2016. 6. Sklovskij K. I. Sdelka i ee dejstvie. Kommentarij glavy 9 GK RF (ponyatie, vidy i forma sdelok. Nedejstvitel’nost’ sdelok). — M. : Statut, 2015. 7. Chelyshev M. Yu. Voprosy mezhotraslevyh svyazej grazhdanskogo prava v Koncepcii razvitiya grazhdanskogo zakonodatel’stva i proekte Grazhdanskogo kodeksa Rossijskoj Federacii // Grazhdanskoe pravo. — 2011. — № 1. — S. 3—7. 8. Chesalina O. V. Rabota na osnove internet-platform (crowdwork i work on demand via apps) kak vyzov trudovomu i social’nomu pravu // Trudovoe pravo v Rossii i za rubezhom. — 2017. — № 1. 9. De Stefano V. The rise of the «just-in-time workforce»: On-demand work, crowdwork and labour protection in the «gig-economy» // Conditions of work and employment. Series No. 71. — Geneva : ILO, 2016. — URL: http://www.ilo.org/wcmsp5/groups/public/—-ed_protect/—-protrav/—-travail/documents/publication/ wcms_443267.pdf (data obrashcheniya: 31 yanvarya 2019 g.). 10. Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy International, Forthcoming. — November 24, 2015. 11. Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition Policy International. — 2015. — № LS 15-11; № 15—29. 11. Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition Policy International. — 2015. — № LS 15-11; № 15—29. 12. Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation of the Sharing Economy // George Mason Law & Economics Research Paper. — 2015. — № 15—01. 12. Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation of the Sharing Economy // George Mason Law & Economics Research Paper. — 2015. — № 15—01. 13. Rogers B. REFERENCES (TRANSLITERATION) The Social Costs of Uber // University of Chicago Law Review Dialogue. — 2015. — № 28. Актуальные проблемы российского права. 2019. № 8 (105) август 94
https://openalex.org/W4366597409
https://www.researchsquare.com/article/rs-2838965/latest.pdf
English
null
Triangulating Moderate Impact of Social Media Marketing Communication Between Performance Expectancy, Effort Expectancy and Social Influence on Business Performance in Cashew Industry in Sri Lanka
Research Square (Research Square)
2,023
cc-by
10,067
Triangulating Moderate Impact of Social Media Marketing Communication Between Performance Expectancy, Effort Expectancy and Social Influence on Business Performance in Cashew Industry in Sri Lanka Triangulating Moderate Impact of Social Media Marketing Communication Between Performance Expectancy, Effort Expectancy and Social Influence on Business Performance in Cashew Industry in Sri Lanka Sandunima Kaluarachchi  (  chamodisandunima@gmail.com ) SLIIT https://orcid.org/0009-0008-4673-4591 1. Introduction With the exponential growth of internet- enabled social media subscribers, advancements in digital media platform have created new value positions in the cashew industry. The multiple benefits of social media marketing (e.g. ubiquity, mobility, internet access convenience, personalization, flexibility and information distribution) enable new marketing services that can address previously unmet retail needs (Yang, 2010, M. Kang, 2014, Duffett, 2017). Social media marketing services can provide e-commerce activities in a different way than traditional shopping services. Unlike the traditional market, SMMC enables salespeople to send personalized information and pinpoint user location services to customers in real-time interactions via an online platform. Users can create personalized online business pages, communicate and interact with customers, and exchange content created by themselves (user-generated content) or information from other cashew business related sources on social media platform such as Facebook, Twitter, LinkedIn, YouTube, WhatsApp, Instagram and Google+ (Duffett, 2017, Tarsakoo and Charoensukmongkol, 2020). Traditional media such as television, radio, newspapers, banners, leaflets and magazines have traditionally communicated business conduct and how sellers think, but in the 21st century, social media has begun to replace traditional media’s enduring and significant role in the business sector. Zhang and Du (2020) stated, from the perspective of entrepreneurs this shift in behavior provides both an opportunity and challenge. Marketers are increasingly relying on social media to market and promote their cashew items among customers. Furthermore, incorporating content that is both amusing and relevant would encourage customers to interact and share the information with their friends (Raji et al., 2019). This important aspect, often known as “word of mouth” (WOM), might be seen as the future of SMMC. According to the suggestion of Chen and Qasim (2021), through seamless communication social media marketing channels can provide the benefits of an optimal e-business experience. For example, majority of famous sports arenas, restaurants, bars, theaters, and catering services in the United States (US) as well as all major supermarkets provide cashew nut-based products. In addition, they serve as bulk supplies for producers of confectionery such as chocolate, ice cream, cakes, and others. Moreover, Feni, a locally created alcoholic beverage made from cashew apples and only accessible in Goa, has a 45 percent higher strength. In Malaysia, Forager cashew milk yogurt was also introduced. The product is available for purchase online. Singh et al. 1. Introduction (2021) study, prove in India, dried fruit sales increased by 20% after they adapted to an online marketing platform and their traditional sales decreased by 9%. Therefore, researchers demonstrated that cashew nut social media marketing can reach a large audience and boost business success. Though these services are promising, consumer adoption of social media marketing channels in Sri Lanka is unclear because rural districts are less likely to use a variety of digital marketing platforms than sellers in India and European countries. Statistics data shows cashew contributes 1% of the national GDP (Gross domestic product) (INC, 2021). Further Kapurubandara (2009) reported that, the majority of Sri Lankan sellers in rural areas are unwilling to use social media for business purposes. This could be the reason the social media marketing platform was developed and positioned without a thorough grasp of rural sellers. Moreover, DCSSL (2020) statistics data proves, the total digital literacy rate is 50.1% in Sri Lanka, with urban 66.3%, rural 48.1%, and estate 25.6%, and also given the rural area representation in the statistics, sellers in the Kajugama district study area should not expect much awareness of digital marketing platforms. Therefore, lack of digital awareness is a big issue in the social media marketing context in Sri Lanka. According to Chambers et al. (2020) E- marketing is another highly affordable and practical strategy for Small & medium enterprises (SME), yet the majority of SMEs lack technological expertise. h i h h i li k d h i l i i i d i i l S di h i h h j i f b i l k Though these services are promising, consumer adoption of social media marketing channels in Sri Lanka is unclear because rural districts are less likely to use a variety of digital marketing platforms than sellers in India and European countries. Statistics data shows cashew contributes 1% of the national GDP (Gross domestic product) (INC, 2021). Further Kapurubandara (2009) reported that, the majority of Sri Lankan sellers in rural areas are unwilling to use social media for business purposes. This could be the reason the social media marketing platform was developed and positioned without a thorough grasp of rural sellers. 1. Introduction Moreover, DCSSL (2020) statistics data proves, the total digital literacy rate is 50.1% in Sri Lanka, with urban 66.3%, rural 48.1%, and estate 25.6%, and also given the rural area representation in the statistics, sellers in the Kajugama district study area should not expect much awareness of digital marketing platforms. Therefore, lack of digital awareness is a big issue in the social media marketing context in Sri Lanka. According to Chambers et al. (2020) E- marketing is another highly affordable and practical strategy for Small & medium enterprises (SME), yet the majority of SMEs lack technological expertise. Furthermore, since the cashew nut sector is linked to the micro cultivation industry, it is also. Studies showing that the majority of business owners lack proper knowledge of social media and how to use it for their business reveal an empirical gap in the literature. However, there has been less advancement in cashew nut social media marketing, and there has been lack of study on the subject in Sri Lanka or other countries. Thus, it is timely to examine the underlying drivers of Sri Lankan sellers’ intention to use digital marketing platforms. Based on sellers' perceptions of online business capabilities, this study will provide implications for business and social media marketing in building e-business services. marketing is another highly affordable and practical strategy for Small & medium enterprises (SME), yet the majority of SMEs lack technological expertise. Furthermore, since the cashew nut sector is linked to the micro cultivation industry, it is also. Studies showing that the majority of business owners lack proper knowledge of social media and how to use it for their business reveal an empirical gap in the literature. However, there has been less advancement in cashew nut social media marketing, and there has been lack of study on the subject in Sri Lanka or other countries. Thus, it is timely to examine the underlying drivers of Sri Lankan sellers’ intention to use digital marketing platforms. Based on sellers' perceptions of online business capabilities, this study will provide implications for business and social media marketing in building e-business services. The rest of this paper is structured as follows. Section 2 examines the literature on industry business performance and technology adaptation as well as the primary antecedent variables. A conceptual model and associated hypotheses are advanced from this point of view. section 3 presents the research methodology. Research Article Keywords: Social media marketing communication, Business performance, Performance expectancy, Effort expectancy, Social influence Posted Date: April 20th, 2023 Keywords: Social media marketing communication, Business performance, Performance expectancy, Effort expectancy, Social influence DOI: https://doi.org/10.21203/rs.3.rs-2838965/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Abstract The theoretical triangulation (TT) in social media marketing communication and business performance is rare to observe. The purpose of this paper is to present a critical review of the contributions of multiple theories of performance expectancy theory (PET), effort expectancy theory (EET), and social influence theory (SIT) and explain whether social media marketing communication (SMMC) moderates the relationship between those theories and business performance (BP). A sample of 218 cashew salespeople from Sri Lanka were surveyed, and the data were analysed using structural equation modelling (SEM) through AMOS 26.0. The finding of the SEM analysis indicated that PE, EE and SI have significant positive relationship with potential sellers’ business performance. The moderation effect results showed that SMMC was a poor moderator of the relation between the EE and BP, as well as the SI and BP, nevertheless, the result showed that SMMC is a partial moderator of the relation between the PE and BP. The current study method necessitates caution in generalizing the findings. More variables may be introduced in future studies to explore the moderation effect between the predictor and BP of the unified theory of acceptance and use of technology (UTAUT) framework. The current study helps cashew sellers widen their horizons while evaluating the practical results of academic research. The findings of this study not only demonstrate that the TT appears to be acceptable in explaining predictors and BP in the cashew industry, but also indicate the resilience of the TT’s ability to predictors and BP in a different research context. Through SEM analysis, this study attempts to add SMMC as a moderator in the UTAUT framework and investigate the moderating effect between predictors and BP. In order to that this study aims to bridge the gap between these moderating effects and how salespeople in SMMC impact BP. 1. Introduction Section 4 presents the data analysis and results. Section 5 contains an in-depth discussion of the results. The various causal factors are highlighted and paper close by discussing some implications for future researchers. 2. Literature Review Page 2/14 Page 2/14 This study contends that there is a relationship between predictor variables and business performance and that there is no moderating impact between social media marketing communication comprehension and predictor variables on business performance. However, researchers provide evidence that theoretical triangulation happened in this study. The literature addressed various variables from the UTAUT model in order to suggest technological aspects of cashew sellers to improve business performance and profit. A type of online marketing that uses social media apps as a marketing tool is knows as social media marketing communication (Duffett, 2017, Raji et al., 2019, Samoggia et al., 2019). For the purposes of this study, the moderating impact of social media marketing communication activities refers to the expectations of sellers who do cashew business through the online. Cashew sellers and business performance Business performance (BP), which is closely related to commercial effectiveness, is determined by an entrepreneur's capacity to put best practices into practice in order to deliver a good or service that meets customers' expectations. As a result, the cashew nut industry is working hard to develop the competencies and skills necessary to satisfy the customers by anticipating and meeting their needs. According to Peterson and Altounian (2019) BP is the ability of a particular business to carry out a plan in order to achieve business goals. Therefore, it was considered an essential tool for merchants and entrepreneurs to evaluate the effectiveness of their business. BP can measure a combination of financial and non-financial measurements. Financial performance can be measured based on sales volume, monthly profit, and total assets; non-financial performance can also be measured based on customer retention rate, business reputation, and product and service innovation. Moreover, the role of cashew salespeople in the convenience and quality cashew marketing and distribution system is a widely debated at the global level. Sellers are crucial players in the promotion of marketing and more sustainable business patterns. They have the ability to influence cashew producers, manufacturers and consumers, as well as guide customers to buy or recommend the product to others. Cashew sales are currently carried out at the retail and grocery level (Priyashantha, 2022). Previous studies have investigated this trend to better understand whether sellers’ marketing activities impact business performance (Liu et al., 2022, Peterson and Altounian, 2019). According to recent statistics Priyashantha (2022), the cashew business’s performance has declined steadily over the last five years. 2. Literature Review Every business’s reputation is important, and they frequently strive to create a trustworthy, responsible and carrying image in the minds of their customers (Ambroise and Prim-Allaz, 2017, Nguyen and Adomako, 2021). Therefore, the presence, innovations, communication patterns, physical location and marketing platform of various product categories might influence customer preferences during purchasing and consumption processes. Furthermore, the previous studies demonstrated, the cashew business sector can create job opportunities and more revenue. However, Sri Lanka still only earns a small portion of the cashew nut value chain. Moreover, Araujo et al. (2021) study proved it should be mentioned that although though some professions appear to be reserved for males, women nonetheless make significant contributions to these fields by managing households, processing cashews, and performing other jobs that generate income. Because the majority of small cashew business owners are women, this sector can see more female employees than male. Many small and medium cashew businesses in the country are still struggling to survive. Small businesses to close their doors due to a lack of raw materials, high production costs, and lack of innovation in comparison to other countries. Unified theory of acceptance and use of technology UTAUT is a well-known theory developed by Venkatesh et al. (2003). This model is used to analyze individuals’ intentions to use technology. The model seeks to comprehend information system use and intention to use as a function of contracts drawn from a variety of theories including performance and effort expectancy (expectancy theory), social influence (social cognitive theory) (Reyes-Mercado, 2018). Social media marketing communication moderates the effects of these variables on use and intention to use technology in present study. UTAUT was created in response to the needs of rural enterprises. Voluntary use in this context refers to the optional use of the e-WOM system, but UTAUT has also been applied to salespeople. Furthermore, the information systems literature has extended and updated the UTAUT model to include factors from marketing process (awareness, motivation and knowledge). This paper employs UTAUT for the sake of technology awareness and because rural business owners in developing countries have a low rate of adaptability to new technology. F i th h th Framing the hypotheses 3. Research Methodology As per Sridharan (2021), triangulation, first used in the social sciences and psychology, is now used in a number of management studies to solve issues with theory formulation and interpretation. According to Modell (2015), there are five basic types of triangulation. First, by using “data triangulation”, several data sources can be used in a single study. Second, “methodological triangulation” entails examining the program using a variety of qualitative and/or quantitative techniques. Third, utilizing numerous separate "investigators" in the analysis process is known as "investigator triangulation." Fourth, using several viewpoints or disciplines to interpret a single set of data is known as “theory triangulation”. Fifth, “Environmental Triangulation” uses a variety of settings, locations, and other crucial aspects of the environment where the study was conducted, such the time of day or season. As a result, this study contributes the fourth element, which is applied to studying more than one theory. Based on conceptual framework those are the performance expectancy, effort expectancy and social influence. Furthermore, many scholars who are interested in social media marketing and business performance have discovered that the employment of multiple theories, also referred to as "theoretical triangulation" or "theoretical pluralism," is useful (Hoque et al., 2015). Therefore, theoretical triangulation has this as one of its espoused benefits. The entire quantitative method is utilized in this study to collect data. According to Mustafa et al. (2020), approaches to quantitative data that use numerical measurement are known as quantitative methods. Researchers use a set of questionnaires built to randomly distribute to cashew sellers in Kajugama district. A total of 218 sets of questionnaires distributed to small and large cashew sellers throughout Kajugama district in Sri Lanka. For analysis, 216 sets of fully filled out questionnaires were received. This amount allows for a 99% response rate to be recorded. Based on the suggestion of over 80% from Mustafa et al. (2020), this response rate is acceptable. The structural equation modeling (SEM) method was used to analyze the questionnaire set. The second generation of multivariate analysis used in the study is called SEM (Pim-Wusu et al., 2022). In order to analyze the data collected by questionnaires, academics and researchers both use this strategy extremely frequently. The usage of AMOS software to adapt this SEM method is highly appropriate because the outcome of the analysis will be more precise. Performance expectancy According to the above arguments SI is hypothesized to be: H3: Social influence has a positive relationship with seller’s business performance. Triangulating the three theories with social media marketing communication Researcher focus on the triangulation theory: the performance expectancy, effort expectancy, and the social influence. In order to that researchers try to understand the moderate impact of triangulating the three theories with business performance through the social media marketing communication. According to Rahi and Abd.Ghani (2019) the extent to which adopting a social media marketing communication (SMMC) as a business platform will assist the business growth and contribute to reach large audience is referred to as PE. Results Chua et al. (2018) proved that as it supports sales targets and maximizes revenue, PE plays a crucial role in influencing sellers. Rahi et al. (2019) proved that PE is the most powerful predictor of behavioral intention to use social media marketing tools. A similar variable to EE is perceived ease of use, which refers to the cognitive effort associated with adapting social media platform. Reyes- Mercado (2018) and Abu Afifa et al. (2022) find that one of the more critical antecedents shaping users’ perceptions toward BP and SMMC is perceived ease of use. One of the primary characteristics of social media is that it allows users to market products to one another while conducting physical business, and it allows users to monitor, reviewing and share business details on the go via online services. There is value in expanding the business to measure it. However, Wu and Ho (2022) claimed that, EE has a less significant impact on the behavioral intentions of SMMC users in Taiwan, as it is insufficient to attract business owners solely through EE factors. People are more likely to use the SMMC platform to communicate and exchange information with their reference groups, such as friends, family, and coworkers. SI indicates how strongly people believe significant others, such as family and friends, believe they should use an SMMC (Zhang and Gong, 2021a, Zhang and Gong, 2021b, Rahi and Abd.Ghani, 2019). They have a tendency to affect people's decisions to adopt or use social media. Chua et al. (2018) proposed in the research of the SMMC, SI is significant in determining users' behavioral intentions. As a result, the authors arrive at the following hypothesis: H4: Social media marketing communication has the moderating impact between three theories and business pe Performance expectancy Performance expectancy The degree to which salespeople believe that adapting technology will boost their individual performance is referred to as performance expectancy (PE) (Abu Afifa et al., 2022). According to the original UTAUT formulation, PE is the most powerful predictor of behavioral intention (Guggemos et al., 2020, Garone et al., 2019). UTAUT models are congruent with studies in the domain of information technology applied to BP. Rahi and Abd.Ghani (2019) analyze the adoption of electronic word of mouth (e-WOM) marketing system, in developing countries is examined, and it is discovered that PE has significant positive relationship with BP. González Bravo et al. (2020); Bui and Fowler (2022) and Burau and Andersen (2014) combine UTAUT with theoretical triangulation to analyze the intent to use technology. They discover that, when technology provides users with credible information, PE influencer user intention to use. As a result, it is reasonable to believe that higher PE will result in a more favorable desire to do business online. In light of the facts presented, the following hypothesis is proposed: H1: Performance expectancy has a positive relationship with seller’s business performance. Effort expectancy Effort expectancy Effort expectancy (EE) is related to the user’s expectation of ease. Authors like Rahi et al. (2019) demonstrated that, when users believe that social media marketing is easy to use and does not require much effort, they are more likely to adopt online business. Joa and Magsamen-Conrad (2022) and Mensah and Page 3/14 Onyancha (2021) argued that users are more likely to adopt online marketing if they perceive is simple. Previous research has found a relationship between EE and user intent to engage in online business (Abu Afifa et al., 2022, Aytekin et al., 2022, Reyes-Mercado, 2018). As a result, EE is hypothesized to be: H2: Effort expectancy has a positive relationship with seller’s business performance. The relationship between social influence (SI) and BP is a hotly disputed topic. The level of societal pressure imposed on individuals to adopt new technologies is characterized as SI (Abu Afifa et al., 2022, Rahi and Abd.Ghani, 2019). According to Chen et al. (2021), SI will positively affect user intent to adopt online business. previous research has found that SI has a significant relationship on online business performance (Reyes-Mercado, 2018, Chan et al., 2022, Wu and Ho, 2022). Demographic characteristics of the sample Demographic characteristics of the sample The demographic characteristics of the sample are shown in Table 1. The study sample’s sellers were mostly female (30-40 years old: 75.2%). This overall age group included the most dynamic sellers portion of all respondents (Akbarov, 2022). Furthermore, by studying seller’s online marketing knowledge and perceptions in female groups, one may be able to predict future trends in their society’s female entrepreneur’s behavior (Akbarov, 2022, Potrich et al., 2016). The sample included 70.6% small business owners and 29.4% large business owners. The majority (88.1%) belonged to the group that promoted their business using social media platforms. Table I provides more detailed information on the sample’s demographic characteristics. Table I: Demographic characteristics  Variables   Categories  N   (%) Gender  Male  Female  66  150 30.3  69.7 Age  Less than 20  20-30  30-40  Above 40  - - 162  54 - - 75.2  24.8 Business type  Small  Large  152  64  70.6  29.4  Social media usage  Yes  No  190  26  88.1  11.9 Total    216 100% Table I: Demographic characteristics Table I: Demographic characteristics  Variables   Categories  N   (%) Gender  Male  Female  66  150 30.3  69.7 Age  Less than 20  20-30  30-40  Above 40  - - 162  54 - - 75.2  24.8 Business type  Small  Large  152  64  70.6  29.4  Social media usage  Yes  No  190  26  88.1  11.9 Total    216 100% Variables   Categories  N   (%) Gender  Male  Female  66  150 30.3  69.7 Age  Less than 20  20-30  30-40  Above 40  - - 162  54 - - 75.2  24.8 Business type  Small  Large  152  64  70.6  29.4  Social media usage  Yes  No  190  26  88.1  11.9 Total    216 100% Reliability and validity test A quantitative research investigation must have validity and reliability (Rajeh et al., 2015). The scales of the items used to measure each construct are tested for reliability prior to data analysis using SEM and to confirm the constructions internal consistency (Alzadjal et al., 2022, Rajeh et al., 2015, Arain et al., 2020). Researchers Rajeh et al. (2015), indicated that consistency of study findings provided by data collection techniques is dependent on reliability. To examine the inter-correlation and reliability of the constructs, Cronbach’s value was measured using SPSS 26.0. Cronbach’s alpha assesses the accuracy with which a set of observed variables measures a single unidimensional latent construct (Reyes-Mercado, 2018). According to Rahi et al. (2019), for a set of observed items, a Cronbach’s α coefficient greater than 0.7 is considered acceptable reliability. 3. Research Methodology In the present study, the IBM SPSS 26.0 and AMOS 26.0 programs was utilized to investigate the hypothesized relationship and validate the measurement model using structural equation modeling. CFA tests were conducted for each factor to determine their compatibility. The loading factor value, which must be positive and must be greater than 0.50, but not greater than 1.00 is the main criterion for assessing this compatibility. The three fitness validity criteria that have been proposed for this fit are fitness index, convergent validity and construct validity. The RMSEA value for fitness index should be less than 0.08 (Agrawal et al., 2021, Senthilkumar and Arulraj, 2011, Al-Fadhali, 2022), while the GFI, CFI and TLI values should be greater than 0.90 (Mustafa et al., 2020, Agrawal et al., 2021). The relative/ normed chi- square value should be approximately less than 5.0 Mustafa et al. (2020), convergent validity ( Average variance extracted or AVE ) and construct validity measurements. According to Anser et al. (2022), a minimum of 0.50 is required for AVE to be able to determine compatibility, while Mustafa et al. (2020), Page 4/14 Page 4/14 suggested that the AVE value is more than 0.5 and complies with the convergent validity measurement standards. The criteria for reliability measurement are next. A composite reliability value of greater than 0.7 is necessary for this criterion. suggested that the AVE value is more than 0.5 and complies with the convergent validity measurement standards. The criteria for reliability measurement are next. A composite reliability value of greater than 0.7 is necessary for this criterion. Demographic characteristics of the sample The present study analysis reveals that all Cronbach’s coefficients obtained are greater than 0.7, implying that the set of observed variables are good measures of single unidimensional latent construct (Rajeh et al., 2015, Wu and Ho, 2022). The test produced alpha values ranging from 0.947 to 0.973, indicating a measurement equipment with an acceptable level of reliability. As depicted in results all constructs have Cronbach's alpha values that are significantly higher than 0.7, demonstrating the validity of the scales. For a good model fit, all factors loading of the measurement items should be more than 0.5 (Ts, 2022). As suggested by, Rahi and Abd.Ghani (2019) factor loading levels had to be bigger than 0.6. However, the present study, Kaiser-Meyer-Olkin (KMO) value represented a range of 0.836 to 0.877. Therefore, following construct reliability and validity testing, the measurement and structural model are validated using confirmatory factor analysis. The model identification, the relative value of x2, and the goodness-of-fit indices are all examined. Multicollinearity test In a multiple regression analysis, multicollinearity is defined as the degree to which two or more independent variables are strongly linearly connected to each other. Ts (2022) stated, the model’s precision decreases as multicollinearity increase. According to Rajeh et al. (2015), VIF reflects the degree of multicollinearity; if VIF is greater than 4, it indicates an problem with multicollinearity. Moreover, VIF was calculated for the suggested five components; it was discovered that VIF varies between 1.005 to 1.015, it falls in the acceptable region, also, VIF is less than 2, which indicates that the proposed five dimensions are free of multicollinearity. Descriptive statistics Descriptive statistics The mean values of the study’s variables are more than the crucial value of 2.99, indicating that the correlation is upright (Sakaya, 2023). As a result, data analysis was required to determine whether they fit the characteristics of a normal distribution and to test skewness and kurtosis. The skewness values for five variables were < 3.00 within the range of (-1 to +1) (Nomran and Haron, 2022, Pattnaik and Pattnaik, 2021), and the kurtosis values are within the range of (-1 to +1), which is appropriate for SEM research (Sakaya, 2023). The common method bias (CBM) was investigated by following the procedures indicated by Sakaya (2023), which included using multiple scales to measure the study’s proposed variables. Confirmatory factor analysis Page 5/14 Page 5/14 Confirmatory factor analysis (CFA) was used to evaluate the constructs item loading, validity and reliability. CFA’s first attempt did not engender good model fit. Due to low factor loadings, the items PE5, EE1, EE2, SI1, SMMC1, BP1 and BP5 were dropped. Therefore, second CFA attempt generated a good model fit (Table II). The result of x2/ df = 2.365 indicates a good model fit because the recommended value for model fit is less than 3 (Anwar et al., 2018, Sakaya, 2023). According to previous research, GFI = 0.908, AGFI = 0.890, CFI = 0.967, TLI = 0.960 and NFI = 0.944 imply good model fitness (Rajeh et al., 2015, Bangwal and Tiwari, 2019, Nomran and Haron, 2022). RMR = 0.009 and RMSEA = 0.078 also generated good model fit (Rajeh et al., 2015, Anwar et al., 2018, Ts, 2022, Sakaya, 2023). PCLOSE greater than 0.05 indicates that the model is well fitted. All of the factor loadings were significantly loaded (P < 0.001), and almost all of the item standardized regression weights (Figure I) were greater than 0.70, which was considered acceptable (Nanjundeswaraswamy et al., 2022, Singh and Srivastava, 2019). As a result, all of the model fit criteria were met. However, before testing the hypothesis, it is required to evaluate the measurement model’s validity and reliability. Notes: * There are no uniform criteria for model fitness. This study relied on Anwar et al. (2018) and Sakaya (2023) because they were commonly utilized in previous studies, model fitness criteria. Descriptive statistics Page 6/14 Notes: β: standardized beta coefficients, S.E.: standard error, C.R.: critical ratio, *P < 0.05, **P < 0.01, ***P < 0.001 Notes: β: standardized beta coefficients, S.E.: standard error, C.R.: critical ratio, *P < 0.05, **P < 0.01, ***P < 0.001 Examining the moderation In answering the proposed H4, Tables V, VI and VII summarized the findings of the hypothesis testing of the moderating effect of the seller’s online business performance in rural area. As prescribed by Alzadjal et al. (2021), interaction effect approach was applied in this study. Interaction terms are generated by multiplying the independent variables by the moderator to examine the moderating effects. According to the previous findings performance expectancy, effort expectancy and social influence were all significant predictors of the intention to deal with business performance, consequently the interaction impact should be tested further. The section that follows outline the outcome of the social media marketing communication moderating effect. The moderating effect of social media marketing communication The moderating effect of social media marketing communication The results show that (Table V) social media marketing communication has a positive direct effect on business performance, with a path coefficient value of 0.033 and a critical ratio of 0.752 and the p- value is insignificant, the value is 0.452 (P- value < 0.05). According to Alzadjal et al. (2021) study, if first hypothesis is significant, then the partial moderation can occur. This suggest that there was a partial moderating effect of social media marketing communication between the link between performance expectancy and business performance. Figure III depicts structural model’s moderating awareness relations. The results indicate that (Table VI) social media marketing communication has a direct positive effect on business performance with a path coefficient value of 0.378 and a critical ratio of 11.473 and p-value is significant the value is *** (p-value < 0.001). with a path coefficient of 0.428 and a critical ratio of 12.912, the direct effect of effort expectancy on business performance is significant, and the significant value is *** (p-value < 0.001). With a negative path coefficient value of -0.032 and a critical ratio of 1.120 the direct effect of the interaction term (EE*SMMC) is not significant, and the insignificant value is 0.268 (p-value < 0.05). This suggests that the exist between the potential sellers EE and BP’s connection. Descriptive statistics Table II: Model fits  Model fit criteria  First order estimate  Second order estimate revised  Acceptable range  X2/ df  7.48  2.365 1-3  GFI  0.926 0.908 > 0.90  AGFI  0.757 0.890  > 0.80  CFI  0.926 0.967 > 0.95 TLI  0.907 0.960 > 0.90 NFI  0.916 0.944 > 0.90 RMR 0.060 0.009 < 0.09 RMSEA  0.142 0.078 < 0.08  PCLOSE  0.97 0.94  > 0.05 Notes: * There are no uniform criteria for model fitness. This study relied on Anwar et al. (2018) and Sakaya (2023) because they we commonly utilized in previous studies, model fitness criteria. Correlation coefficients AMOS was used to measure correlation coefficients, and the findings are shown in Table III. It gives early support for the study’s proposed hypothesis. The findings show that there is a significant positive relationship between performance expectancy and business performance (r = 0.722, P < 0.01), a significant positive relationship between effort expectancy and business performance (r = 0.204, P < 0.01), and a significant positive relationship between social influence and business performance (r = 0.561, P < 0.01). Therefore, these findings supported H1, H2, and H3 in this study. Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Multiple liner regression Table IV and Figure II shows the regression outcome. The analysis reveals that the two variables have positive impact and are significant, with the relationship between performance expectancy and business performance (β = 0.771 and P = 0.000), when PE goes up by 1 standard deviation the dependent variable BP goes up by 0.771 standard deviation, also social media marketing communication with business performance (β = 0.332 and P = 0.000), when SMMC goes up by 1 standard deviation the dependent variable BP goes up by 0.332 standard deviation. However, effort expectancy and social influence not having significant impact on business performance (β = -0.027 and P = 0.561) (β = 0.039 and P = 0.401) respectively. 5. Discussion In this study, researchers provided a forum for vies on the application of theoretical triangulation and quantitative research methods by using AMOS to analyze business performance and social media marketing communication actions among sellers in rural areas. To test the relationship between performance expectancy, effort expectancy, and social influence theories with business performance, as well as the moderating impact of social media marketing communication in those theories on business performance, researchers used performance expectancy, effort expectancy, and social influence theories with business performance to demonstrate how triangulation can extend across many competing theories. As a result, scholars propose theoretical triangulation and diversified research methodologies in order to use multiple theories with competing epistemologies in harmony, in accordance with the premise that different theories should be considered as complimentary rather than adversaries. A reoccurring point that binds this paper is that theoretical triangulation and varied research methodologies have the ability to produce a synergy of being mutually informative, allowing for a deeper portrayal of business reality in rural areas, revealing distinctive online business difficulties or dynamics. In line with Hoque et al. (2015), authors argue that researchers must consider how multiple theories and methods might be synthesized or integrated. The incorporation of several views would link theories into a coherent and intelligible online business performance discourse and practice. Here Hoque et al. (2013) argued that, analysing business ambiguities and conflicts is a significant opportunity provided by field research methods, as they allow for the examination of suggestive themes and counterpoints, interpretations and counter-interpretations, and various voices surrounding the social construction of e-business performance in rural areas. As a results, the findings indicate that three theories concerning business performance among salespeople in rural areas significantly enhance relationship. This results are in line with the study’s conceptual framework, which assumes a direct relationship between performance expectancy, effort expectancy, social influence and business performance (Chua et al., 2018). This is also true in the case of cashew sellers, even if salespeople are selling cashew products through the online their e-marketing awareness effort is critical to increasing their business performance. It also appears that cashew demand, motivates salespeople to expand their market, increasing their profit and leading to improved business performance. This result supports the findings of Wu and Ho (2022) and Reyes-Mercado (2018). Therefore, the authors proved that the proposed H1, H2, and H3 (Table III) supported the study. Descriptive statistics This indicates that, even in the presence of a moderator, both constructs have a strong relationship. Figure IV illustrates the structural model’s moderating awareness relations. Furthermore, in this case, the moderating effect is weak. The weak moderating effect has no effect on the modify of the relationship between the dependent variable (BP) and the predictor variable (EE). As a result, “SMMC” can be considered as a poor moderator of the relationship between EE and BP. Page 7/14 Page 7/14 Page 7/14 Page 7/14 The regression model outputs are detailed in Table VII. The results show that social media marketing communication has a positive direct effect on business performance, with a path coefficient value of 0.552 and a critical ratio of 16.482 and p-value of *** (p-value < 0.001). with a path coefficient value of 0.138 and a critical ratio of 4.001, the direct effect of social influence on business performance is significant, and the significant value is *** (p-value < 0.001). The direct effect of the interaction term (SMMC*SI) is not significant, with a negative path coefficient value of -0.043 and a critical ratio of -1.276, and the significant value is 0.201 (p-value < 0.05). this suggests that the moderating effect of SMMC did not exist between the potential sellers SI and BP’s connection. Figure V depicts the moderating relationships of awareness in the structural model. Furthermore, in this case, the moderating effect is weak. The weak moderating effect has no effect on the modify of the relationship between the dependent variable (BP) and the predictor variable (SI). Thus, “SMMC” is a poor moderator because it had no effect on the relationship between SI and BP. The interaction term effect of the rural area sellers SMMC on the relationship of the PE, EE and SI on the BP is summarized in Table VIII. Limitations and future research direction This study does have numerous limitations. First, the current study uncounted the problem of not knowing the population of the chosen unit of analysis, which is the number of cashew sellers in Sri Lanka. Certain data has been erased, because cashew corporation lacks a good recording system and relies solely on a manual system. Based on information from the cashew corporation’s research department, the authors determined the population. As a result, researchers cannot find cashew salespeople across the entire country. Second, the technology and business performance in the cashew industry were evaluated using subjective measurement. According to Aghajari and Amat Senin (2014) the limitations of psychological biases affect subjective measures. Therefore, to test the validity and generalizability of these findings and to throw fresh light on the postulated causal relationships, future research that replicate these measures and use various measurement techniques are advised. Third, the cashew industry was the focus of the current investigation. The findings may differ depending on the industry. Therefore, the conclusions from this study may not be extrapolated to other industries, such as manufacturing or banks. Fourth, the study solely considered the business owner's perspective, marketing techniques, information value, usability, and expectation fulfillment. By including small factors in a context relevant to an application, the research aims to broaden the scope of the existing theoretical evaluation framework. It also aims to develop more realistic time-based versions by revisiting and re-examining the existing framework. Consequently, it significantly affects how well researcher understand the complex topic of how online business performance affects entrepreneurs. Future researchers should focus on qualitative or mixed-method research because it has a big impact on discovering the in-depth analysis regarding the performance of online businesses. The E-UTAUT model is kindly applied in this study to new online business performance, particularly in Sri Lanka. However, just the three elements PE, EE, and SI are applied based on UTAUT. Consequently, there can be some generalizability problems with the performance of emerging online businesses. Future studies should therefore employ all of the UTAUT model's components in order to further the significant discoveries regarding e-business. This increases the legitimacy of the profit growth, making it more beneficial for SMEs and the nation's emerging economic growth. Other exogenous factors can have an impact on the connection that was investigated. Limitations and future research direction Future studies should concentrate on numerous other significant factors that affect sellers' expectations, such as marketing strategies and high-performance network systems. Despite these limitations, the authors believe that this study will not only benefit cashew sellers by providing answers to exploratory research questions, but will also contribute to the body of knowledge in terms of theoretical enrichment to behavioral studies and theoretical triangulation. Findings from this paper are critical because they pertain to the adaptation of social media marketing communication and business performance studies. The literature revealed that there are a limited number of studies that extended the theoretical triangulation to examine cashew sellers in rural areas. Another important point is that the findings of this study not only demonstrate that theoretical triangulation appears to be acceptable in explaining the relationship between predictors and BP in the field of online business, but also support the robustness of theoretical triangulation's ability to predict variables and BP in a different research context. As a result, the current study indirectly contributes to expanding the existing body of knowledge and behavioral literature. References Abu Afifa, M. M., Vo Van, H. & Le Hoang Van, T. 2022. Blockchain adoption in accounting by an extended UTAUT model: empirical evidence from an emerging economy. Journal of Financial Reporting and Accounting, 17 (1), 1-7. Available at:https://doi.org/10.1108/JFRA-12-2021-0434 Aghajari, N. & Amat Senin, A. 2014. Strategic orientation and dual innovative operation strategies. Asia-Pacific Journal of Business Administration, 6 (2), 127- 147. Available at:https://doi.org/10.1108/APJBA-07-2013-0075 Agrawal, S., Singh, V. & Upadhyay, Y. 2021. Structural model of information quality framework to e-agri supply chain. Journal of Advances in Management Research, 18 (4), 609-634. Available at:https://doi.org/10.1108/JAMR-06-2020-0113 Akbarov, S. 2022. Consumer ethnocentrism and purchasing behavior: moderating effect of demographics. Journal of Islamic Marketing, 13 (4), 898-932. Available at:https://doi.org/10.1108/JIMA-02-2020-0047 Akbarov, S. 2022. Consumer ethnocentrism and purchasing behavior: moderating effect of demographics. Journal of Islamic Marketing, 13 (4), 898-932. Available at:https://doi.org/10.1108/JIMA-02-2020-0047 Akbarov, S. 2022. Consumer ethnocentrism and purchasing behavior: moderating effect of demographics. Journal of Islamic Marketing, 13 (4), 898-932. Available at:https://doi.org/10.1108/JIMA-02-2020-0047 Al-Fadhali, N. 2022. An AMOS-SEM approach to evaluating stakeholders' influence on construction project delivery performance. Engineering, Construction and Architectural Management, 11 (1), 1-17. Available at:https://doi.org/10.1108/ECAM-09-2021-0780 Al-Fadhali, N. 2022. An AMOS-SEM approach to evaluating stakeholders' influence on construction project delivery performance. Engineering, Construction and Architectural Management, 11 (1), 1-17. Available at:https://doi.org/10.1108/ECAM-09-2021-0780 Al-Fadhali, N. 2022. An AMOS-SEM approach to evaluating stakeholders' influence on construction project delivery performance. Engineering, Construction and Architectural Management, 11 (1), 1-17. Available at:https://doi.org/10.1108/ECAM-09-2021-0780 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2021. Moderating the role of religiosity on potential customer intention to deal with Islamic banks in Oman. Journal of Islamic Marketing, 15 (1), 1-25. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2021. Moderating the role of religiosity on potential customer intention to deal with Islamic banks in Oman. Journal of Islamic Marketing, 15 (1), 1-25. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2021. Moderating the role of religiosity on potential customer intention to deal with Islamic banks in Oman. Journal of Islamic Marketing, 15 (1), 1-25. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2022. Moderating the role of religiosity on potential customer intention to deal with Islamic banks in Oman. Journal of Islamic Marketing, 13 (11), 2378-2402. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 & Prim-Allaz, I. 2017. 5. Discussion According to the empirical results, the suggested model provides a comprehensive comprehension of the elements that drive business performance. PE, EE and SI explain approximately 61% of the total variance in potential sellers in rural area. The current study extended the theoretical triangulation with a moderator to examine the interaction effect of SMMC on the relationship between predictors and BP at the theoretical contribution level. The most important Page 8/14 Page 8/14 practical finding of this study is that potential sellers have a poor inclination to deal with social media. These findings should be noted as evidence of a partial moderating effect in PE theory (Table V). But researchers considered there to be no moderating impact between these predictors and BP. Because the other two theories (EE and SI) have no moderating effect (Table VI and VII). As a result, H4 is opposed to the study and the author accept the null hypothesis (Table VIII). However, theoretical triangulation still happens with PE, EE, SI and BP, also there is no moderating effect in this study. practical finding of this study is that potential sellers have a poor inclination to deal with social media. These findings should be noted as evidence of a partial moderating effect in PE theory (Table V). But researchers considered there to be no moderating impact between these predictors and BP. Because the other two theories (EE and SI) have no moderating effect (Table VI and VII). As a result, H4 is opposed to the study and the author accept the null hypothesis (Table VIII). However, theoretical triangulation still happens with PE, EE, SI and BP, also there is no moderating effect in this study. References Reputation Risk: Anticipation and Management of Reputation Failure. Risk Management. Ambroise, L. & Prim-Allaz, I. 2017. Reputation Risk: Anticipation and Management of Reputation Failure. Risk Management. Anser, M. K., Yousaf, Z., Yasir, M., Sharif, M., Nasir, M. H., Rasheed, M. I., Waheed, J., Hussain, H. & Majid, A. 2022. How to unleash innovative work behavior of SMEs' workers through knowledge sharing? Accessing functional flexibility as a mediator. European Journal of Innovation Management, 25 (1), 233-248. Available at:https://doi.org/10.1108/EJIM-11-2019-0332 Page 9/14 Anwar, M., Shah, S. Z. A. & Khan, S. Z. 2018. The role of personality in SMEs internationalization: empirical evidence. Review of International Business and Strategy, 28 (2), 258-282. Available at:https://doi.org/10.1108/RIBS-12-2017-0113 Anwar, M., Shah, S. Z. A. & Khan, S. Z. 2018. The role of personality in SMEs internationalization: empirical evidence. Review of International Business and Strategy, 28 (2), 258-282. Available at:https://doi.org/10.1108/RIBS-12-2017-0113 Arain, G. A., Bhatti, Z. A., Hameed, I. & Fang, Y.-H. 2020. Top-down knowledge hiding and innovative work behavior (IWB): a three-way moderated-mediation analysis of self-efficacy and local/foreign status. Journal of Knowledge Management, 24 (2), 127-149. Available at:https://doi.org/10.1108/JKM-11-2018- 0687 Arain, G. A., Bhatti, Z. A., Hameed, I. & Fang, Y.-H. 2020. Top-down knowledge hiding and innovative work behavior (IWB): a three-way moderated-mediation analysis of self-efficacy and local/foreign status. Journal of Knowledge Management, 24 (2), 127-149. Available at:https://doi.org/10.1108/JKM-11-2018- 0687 Araujo, T. D. S., Da Costa, J. M. A. R., De Oliveira Silva Ribeiro, F., De Jesus Oliveira, A. C., Do Nascimento Dias, J., De Araujo, A. R., Barros, A. B., Da Paixão Brito, M., De Oliveira, T. M., De Almeida, M. P., De Carvalho Castro, K. N., Dos Santos Fogaça, F. H., Da Silva, D. A. & De Souza, B. W. S. 2021. Nanoemulsion of cashew gum and clove essential oil (Ocimum gratissimum Linn) potentiating antioxidant and antimicrobial activity. International Journal of Biological Macromolecules, 193 (1), 100-108. Available at:https://www.sciencedirect.com/science/article/pii/S0141813021021243 Aytekin, A., Özköse, H. & Ayaz, A. 2022. Unified theory of acceptance and use of technology (UTAUT) in mobile learning adoption : Systematic literature review and bibliometric analysis. COLLNET Journal of Scientometrics and Information Management, 16 (1), 75-116. Available at:https://doi.org/10.1080/09737766.2021.2007037 Bangwal, D. & Tiwari, P. 2019. Workplace environment, employee satisfaction and intent to stay. International Journal of Contemporary Hospitality Management, 31 (1), 268-284. Available at:https://doi.org/10.1108/IJCHM-04-2017-0230 Bui, B. & Fowler, C. 2022. Carbon controls in a New Zealand electricity utility: An application of theoretical triangulation. Accounting & Finance, 62 (4), 4423- 4451. References Available at:https://doi.org/10.1111/acfi.12942 Burau, V. & Andersen, L. B. 2014. Professions and Professionals: Capturing the Changing Role of Expertise Through Theoretical Triangulation. The American Journal of Economics and Sociology, 73 (1), 264-293. Available at:https://doi.org/10.1111/ajes.12062 Chambers, M., Bliss, K. & Rambur, B. 2020. Recruiting Research Participants via Traditional Snowball vs Facebook Advertisements and a Website. Western Journal of Nursing Research, 42 (10), 846-851. Available at:https://doi.org/10.1177/0193945920904445 Chan, R., Troshani, I., Rao Hill, S. & Hoffmann, A. 2022. Towards an understanding of consumers’ FinTech adoption: the case of Open Banking. International Journal of Bank Marketing, 40 (4), 886-917. Available at:https://doi.org/10.1108/IJBM-08-2021-0397 Chen, L., Rashidin, M. S., Song, F., Wang, Y., Javed, S. & Wang, J. 2021. Determinants of Consumer’s Purchase Intention on Fresh E-Commerce Platform: Perspective of UTAUT Model. SAGE Open, 11 (2), 1-14. Available at:https://doi.org/10.1177/21582440211027875 Chen, X. & Qasim, H. 2021. Does E-Brand experience matter in the consumer market? Explaining the impact of social media marketing activities on consumer- based brand equity and love. Journal of Consumer Behaviour, 20 (5), 1065-1077. Available at:https://doi.org/10.1002/cb.1915 Chua, P. Y., Rezaei, S., Gu, M.-L., Oh, Y. & Jambulingam, M. 2018. Elucidating social networking apps decisions. Nankai Business Review International, 9 (2), 118-142. Available at:https://doi.org/10.1108/NBRI-01-2017-0003 DCSSL. 2020. Computer Literacy Statistics. Sri Lanka LANKA, D. O. C. A. S. S. Available CSSL. 2020. Computer Literacy Statistics. Sri Lanka LANKA, D. O. C. A. S. S. Available DCSSL. 2020. Computer Literacy Statistics. Sri Lanka LANKA, D. O. C. A. S. S. Available at:http://www.statistics.gov.lk/Resource/en/ComputerLiteracy/Bulletins/AnnualBuletinComputerLiteracy-2020. at:http://www.statistics.gov.lk/Resource/en/ComputerLiteracy/Bulletins/AnnualBuletinComputerLiteracy-2020. Duffett, R. G. 2017. Influence of social media marketing communications on young consumers’ attitudes. Young Consumers, 18 (1), 19-39. Available at:https://doi.org/10.1108/YC-07-2016-00622 Garone, A., Pynoo, B., Tondeur, J., Cocquyt, C., Vanslambrouck, S., Bruggeman, B. & Struyven, K. 2019. Clustering university teaching staff through UTAUT: Implications for the acceptance of a new learning management system. British Journal of Educational Technology, 50 (5), 2466-2483. Available at:https://doi.org/10.1111/bjet.12867 González Bravo, L., Fernández Sagredo, M., Torres Martínez, P., Barrios Penna, C., Fonseca Molina, J., Stanciu, I. D. & Nistor, N. 2020. Psychometric analysis of a measure of acceptance of new technologies (UTAUT), applied to the use of haptic virtual simulators in dental students. European Journal of Dental Education, 24 (4), 706-714. Available at:https://doi.org/10.1111/eje.12559 Guggemos, J., Seufert, S. & Sonderegger, S. 2020. Humanoid robots in higher education: Evaluating the acceptance of Pepper in the context of an academic writing course using the UTAUT. British Journal of Educational Technology, 51 (5), 1864-1883. References Available at:https://doi.org/10.1111/bjet.13006 Hoque, Z., A. Covaleski, M. & N. Gooneratne, T. 2013. Theoretical triangulation and pluralism in research methods in organizational and accounting research. Accounting, Auditing & Accountability Journal, 26 (7), 1170-1198. Available at:https://doi.org/10.1108/AAAJ-May-2012-01024 Hoque, Z., Covaleski, M. A. & Gooneratne, T. N. 2015. A response to “theoretical triangulation and pluralism in accounting research: a critical realist critique”. Accounting, Auditing & Accountability Journal, 28 7, 1151-1159. Available at:https://doi.org/10.1108/AAAJ-05-2015-2058 INC. 2021. The nuts & dried fruits statistical yearbook report USA. FRUIT, I. N. A. D. Available at:https://www.nutfruit.org/files/tech/1651579968_Statistical_Yearbook_2021-2022.pdf. Page 10/14 Page 10/14 Joa, C. Y. & Magsamen-Conrad, K. 2022. Social influence and UTAUT in predicting digital immigrants’ technology use. Behaviour & Information Technology, 41 (8), 1620-1638. Available at:https://doi.org/10.1080/0144929X.2021.1892192 Joa, C. Y. & Magsamen-Conrad, K. 2022. Social influence and UTAUT in predicting digital immigrants’ technology use. Behaviour & Information Technology, 41 (8), 1620-1638. Available at:https://doi.org/10.1080/0144929X.2021.1892192 Kapurubandara, M. 2009. A Framework to e-Transform sMEs in developing countries. THE ELECTRONIC JOURNAL OF INFORMATION SYSTEMS IN DEVELOPING COUNTRIES, 39 (1), 1-24. Available at:https://doi.org/10.1002/j.1681-4835.2009.tb00277.x Liu, Y., Dilanchiev, A., Xu, K. & Hajiyeva, A. M. 2022. Financing SMEs and business development as new post Covid-19 economic recovery determinants. Economic Analysis and Policy, 76 (1), 554-567. Available at:https://www.sciencedirect.com/science/article/pii/S0313592622001485 M. Kang, J.-Y. 2014. Augmented reality and motion capture apparel e-shopping values and usage intention. International Journal of Clothing Science and Technology, 26 (6), 486-499. Available at:https://doi.org/10.1108/IJCST-05-2013-0055 Mensah, M. & Onyancha, O. B. 2021. Demographic factors influencing the adoption and use of social media in university libraries in Ghana: A unified theory of acceptance and use of technology (UTAUT) approach. Journal of Electronic Resources Librarianship, 33 (3), 170-194. Available at:https://doi.org/10.1080/1941126X.2021.1949157 Modell, S. 2015. Theoretical triangulation and pluralism in accounting research: a critical realist critique. Accounting, Auditing & Accountability Journal, 28 (7), 1138-1150. Available at:https://doi.org/10.1108/AAAJ-10-2014-1841 Mustafa, M., Nordin, M. N. & Razzaq, A. 2020. Structural Equation Modelling Using AMOS: Confirmatory Factor Analysis for Taskload of Special Education Integration Program Teachers. Universal Journal of Educational Research, 8 (1), 127-133. Available ustafa, M., Nordin, M. N. & Razzaq, A. 2020. Structural Equation Modelling Using AMOS: Confirmatory Factor Ana tegration Program Teachers. Universal Journal of Educational Research, 8 (1), 127-133. Available at:https://www.researchgate.net/publication/339453426_Structural_Equation_Modelling_Using_AMOS_Confirmatory_Factor_Analysis_for_Taskload_of_Specia :https://www.researchgate.net/publication/339453426_Structural_Equation_Modelling_Using_AMOS_Confirmato Nanjundeswaraswamy, T. S., Bharath, S. & Nagesh, P. 2022. Employer branding: design and development of a scale. Journal of Economic and Administrative Sciences, 17 (1), 1-26. Available at:https://doi.org/10.1108/JEAS-01-2022-0012 Nguyen, N. P. & Adomako, S. 2021. References Environmental proactivity, competitive strategy, and market performance: The mediating role of environmental reputation. Business Strategy and the Environment, 30 (4), 2008-2020. Available at:https://doi.org/10.1002/bse.2729 Nomran, N. M. & Haron, R. 2022. Validity of zakat ratios as Islamic performance indicators in Islamic banking: a congeneric model and confirmatory factor analysis. ISRA International Journal of Islamic Finance, 14 (1), 41-62. Available at:https://doi.org/10.1108/IJIF-08-2018-0088 Pattnaik, S. & Pattnaik, S. 2021. Exploring employee performance dimensionality in Indian public sector units. International Journal of Productivity and Performance Management, 70 (3), 657-674. Available at:https://doi.org/10.1108/IJPPM-08-2019-0374 Peterson, R. A. & Altounian, D. 2019. Self-perceived Performance of Female-owned Firms and Male-owned Firms: Insights from Black and Mexican-American Entrepreneurs. In: CRITTENDEN, V. L. (ed.) Go-to-Market Strategies for Women Entrepreneurs. Emerald Publishing Limited. Pim-Wusu, M., Aigbavboa, C. & Thwala, W. D. 2022. Adaptability capacity framework for sustainable practices in the Ghanaian construction industry. Built Environment Project and Asset Management, 12 (1), 1-16. Available at:https://doi.org/10.1108/BEPAM-01-2022-0016 Potrich, A. C. G., Vieira, K. M. & Mendes-Da-Silva, W. 2016. Development of a financial literacy model for university students. Management Research Review, 39 (3), 356-376. Available at:https://doi.org/10.1108/MRR-06-2014-0143 iyashantha, A. K. H. 2022. Gifted but Not Tasted? [Online]. Daily News Available: https://www.dailynews.lk/2022/ Priyashantha, A. K. H. 2022. Gifted but Not Tasted? [Online]. Daily News Available: https://www.dailynews.lk/2022/02/10/features/272229/gifted-not-tasted Rahi, S. & Abd.Ghani, M. 2019. Investigating the role of UTAUT and e-service quality in internet banking adoption setting. The TQM Journal, 31 (3), 491- 506.Available at:https://doi.org/10.1108/TQM-02-2018-0018 Rahi, S., Othman Mansour, M. M., Alghizzawi, M. & Alnaser, F. M. 2019. Integration of UTAUT model in internet banking adoption context. Journal of Research in Interactive Marketing, 13 (3), 411-435. Available at:https://doi.org/10.1108/JRIM-02-2018-0032 Rajeh, M., Tookey, J. E. & Rotimi, J. O. B. 2015. Estimating transaction costs in the New Zealand construction procurement. Engineering, Construction and Architectural Management, 22 (2), 242-267. Available at:https://doi.org/10.1108/ECAM-10-2014-0130 Raji, R. A., Rashid, S. & Ishak, S. 2019. The mediating effect of brand image on the relationships between social media advertising content, sales promotion content and behaviuoral intention. Journal of Research in Interactive Marketing, 13 (3), 302-330. Available at:https://doi.org/10.1108/JRIM-01-2018-0004 Reyes-Mercado, P. 2018. Adoption of fitness wearables. Journal of Systems and Information Technology, 20 (1), 103-127. Available at:https://doi.org/10.1108/JSIT-04-2017-0025 Sakaya, A. J. 2023. Fear of COVID-19 and green bank service purchase intention: the mediating effect of customer empowerment and customers’ perceived value of digital service transactions. Arab Gulf Journal of Scientific Research, 27 (1), 1-22. Available at:https://doi.org/10.1108/AGJSR-08-2022-0137 Sakaya, A. J. 2023. References Fear of COVID-19 and green bank service purchase intention: the mediating effect of customer empowerment and customers’ perceived value of digital service transactions. Arab Gulf Journal of Scientific Research, 27 (1), 1-22. Available at:https://doi.org/10.1108/AGJSR-08-2022-0137 Samoggia, A., Bertazzoli, A. & Ruggeri, A. 2019. Food retailing marketing management: social media communication for healthy food. International Journal of Retail & Distribution Management, 47 (9), 928-956. Available at:https://doi.org/10.1108/IJRDM-08-2018-0178 Samoggia, A., Bertazzoli, A. & Ruggeri, A. 2019. Food retailing marketing management: social media communication for healthy food. International Journal of Retail & Distribution Management, 47 (9), 928-956. Available at:https://doi.org/10.1108/IJRDM-08-2018-0178 Page 11/14 Page 11/14 Senthilkumar, N. & Arulraj, A. 2011. SQM-HEI – determination of service quality measurement of higher education in India. Journal of Modelling in Management, 6 (1), 60-78 Singh, N., Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and pharmacological review of Cinnamomum verum J. Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available ngh, N., Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and ph erum J Presl a versatile spice used in food and nutrition Food Chemistry 338 (1) 1 15 Available Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and pharmaco Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available ngh, N., Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and ph erum J. Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available verum J. Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available at:.https://www.sciencedirect.com/science/article/pii/S0308814620316356 /www.sciencedirect.com/science/article/pii/S0308814620316356 Singh, S. & Srivastava, S. 2019. Engaging consumers in multichannel online retail environment. Journal of Modelling in Management, 14 (1), 49-76. Available at:https://doi.org/10.1108/JM2-09-2017-0098 Sridharan, V. G. 2021. Methodological Insights Theory development in qualitative management control: revisiting the roles of triangulation and generalization. Accounting, Auditing & Accountability Journal, 34 2, 451-479. Available at:https://doi.org/10.1108/AAAJ-09-2019-4177 Tarsakoo, P. & Charoensukmongkol, P. 2020. Dimensions of social media marketing capabilities and their contribution to business performance of firms in Thailand. Journal of Asia Business Studies, 14 (4), 441-461. Available at:https://doi.org/10.1108/JABS-07-2018-0204 Ts, N. 2022. Nurses quality of work life: scale development and validation. Journal of Economic and Administrative Sciences, 38 (2), 371-394. References Available at:https://doi.org/10.1108/JEAS-09-2020-0154 Venkatesh, V., Morris, M. G., Davis, G. B. & Davis, F. D. 2003. User Acceptance of Information Technology: Toward a Unified View. MIS Quarterly, 27 (3), 425-478. Available at:http://www.jstor.org/stable/30036540 Wu, C.-G. & Ho, J. C. 2022. The influences of technological characteristics and user beliefs on customers' perceptions of live chat usage in mobile banking. International Journal of Bank Marketing, 40 (1), 68-86. Available at:https://doi.org/10.1108/IJBM-09-2020-0465 Yang, K. 2010. Determinants of US consumer mobile shopping services adoption: implications for designing mobile shopping services. Journal of Consumer Marketing, 27 (3), 262-270. Available at:https://doi.org/10.1108/07363761011038338 Zhang, H. & Gong, X. 2021a. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European Journal of Marketing, 55 5, 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, H. & Gong, X. 2021a. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European Journal of Marketing, 55 5, 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, H. & Gong, X. 2021b. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European Journal of Marketing, 55 (5), 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, H. & Gong, X. 2021b. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European Journal of Marketing, 55 (5), 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, J. & Du, M. 2020. Utilization and effectiveness of social media message strategy: how B2B brands differ from B2C brands. Journal of Business & Industrial Marketing, 35 (4), 721-740. Available at:https://doi.org/10.1108/JBIM-06-2018-0190 Zhang, J. & Du, M. 2020. Utilization and effectiveness of social media message strategy: how B2B brands differ from B2C brands. Journal of Business & Industrial Marketing, 35 (4), 721-740. Available at:https://doi.org/10.1108/JBIM-06-2018-0190 Declarations Since the cashew nut industry is related to the micro cultivation industry. Most of the business owners do not have proper awareness of social media and how to use social media for their business. Since the cashew nut industry is related to the micro cultivation industry. Most of the business owners do not have proper awareness of social media and ho to use social media for their business. Authors do not have any conflict of interest as far as this study is concerned. Authors do not have any conflict of interest as far as this study is concerned. Figures Page 12/14 Page 12/14 Page 12/14 Page 12/14 Figure 1 Measurement model Figure 2 Structural model Figure 1 Measurement model Figure 2 Figure 2 Structural model Page 13/14 Page 13/14 Figure 3 SMMC Moderating effect between PE and BP Figure 4 SMMC Moderating effect between EE and BP Figure 5 SMMC Moderating effect between SI and BP Figure 3 SMMC Moderating effect between PE and BP Figure 4 Figure 3 g SMMC Moderating effect between PE and BP Figure 4 SMMC Moderating effect between PE and BP SMMC Moderating effect between PE and BP SMMC Moderating effect between PE and BP Figure 4 SMMC Moderating effect between EE and BP Figure 5 SMMC Moderating effect between EE and BP Figure 5 SMMC Moderating effect between SI and BP Page 14/14
https://openalex.org/W4253340719
https://alzres.biomedcentral.com/track/pdf/10.1186/s13195-020-00668-5
English
null
Effects of long-term sleep disruption on cognitive function and brain amyloid-β burden: a case-control study
Research Square (Research Square)
2,020
cc-by
9,436
Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 https://doi.org/10.1186/s13195-020-00668-5 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 https://doi.org/10.1186/s13195-020-00668-5 (2020) 12:101 RESEARCH Open Access Effects of long-term sleep disruption on cognitive function and brain amyloid-β burden: a case-control study Jana Thomas1,2,3* , Sharon J. Ooms2,3, Lara J. Mentink1,2,3, Jan Booij4,5, Marcel G. M. Olde Rikkert1,2,3, Sebastiaan Overeem6,7†, Roy P. C. Kessels2,3,8† and Jurgen A. H. R. Claassen1,2,3* Jana Thomas1,2,3* , Sharon J. Ooms2,3, Lara J. Mentink1,2,3, Jan Booij4,5, Marcel G. M. Olde Rikkert1,2,3, Sebastiaan Overeem6,7†, Roy P. C. Kessels2,3,8† and Jurgen A. H. R. Claassen1,2,3* Abstract 1Department of Geriatric Medicine, Radboud University Medical Center, 6525, GC, Nijmegen, The Netherlands Full list of author information is available at the end of the article * Correspondence: Jana.Thomas@radboudumc.nl; Jurgen.Claassen@radboudumc.nl †Sebastiaan Overeem and Roy P. C. Kessels contributed equally to this work. 1Department of Geriatric Medicine, Radboud University Medical Center, 6525, GC, Nijmegen, The Netherlands Full list of author information is available at the end of the article Abstract Background: Recent evidence indicates that disrupted sleep could contribute to the development of Alzheimer’s disease by influencing the production and/or clearance of the amyloid-β protein. We set up a case-control study to investigate the association between long-term work-induced sleep disruption, cognitive function, and brain amyloid-β burden. Methods: Nineteen male maritime pilots (aged 48–60 years) with chronic work-related sleep disruption and a sex-, age-, and education-matched control sample (n = 16, aged 50–60 years) with normal sleep completed the study. Primary sleep disorders were ruled out with in-lab polysomnography. Additional sleep measurements were obtained at home using actigraphy, sleep-wake logs, and a single-lead EEG device. Cognitive function was assessed with a neuropsychological test battery, sensitive to early symptomatic Alzheimer’s disease. Brain amyloid-β burden was assessed in maritime pilots using 18F-flutemetamol amyloid PET-CT. Results: Maritime pilots reported significantly worse sleep quality (Pittsburgh Sleep Quality Index (PSQI) = 8.8 ± 2.9) during work weeks, compared to controls (PSQI = 3.2 ± 1.4; 95% CI 0.01 to 2.57; p = 0.049). This was confirmed with actigraphy-based sleep efficiency (86% ± 3.8 vs. 89.3% ± 4.3; 95% CI 0.43 to 6.03; p = 0.03). Home-EEG recordings showed less total sleep time (TST) and deep sleep time (DST) during work weeks compared to rest weeks (TST 318.56 (250.21–352.93) vs. TST 406.17 (340–425.98); p = 0.001; DST 36.75 (32.30–58.58) vs. DST 51.34 (48.37–69.30); p = 0.005)). There were no differences in any of the cognitive domains between the groups. For brain amyloid-β levels, mean global cortical standard uptake value ratios of 18F-flutemetamol were all in the normal range (1.009 ± 0.059; 95% CI 0.980 to 1.037), confirmed by visual reads. Conclusions: Capitalizing on the particular work-rest schedule of maritime pilots, this study with a small sample size observed that long-term intermittent sleep disruption had no effects on global brain amyloid-β levels or cognitive function. Keywords: Sleep disruption, Shift work, Alzheimer’s disease, Amyloid-β, Cognitive function Keywords: Sleep disruption, Shift work, Alzheimer’s disease, Amyloid-β, Cognitive function * Correspondence: Jana.Thomas@radboudumc.nl; Jurgen.Claassen@radboudumc.nl †Sebastiaan Overeem and Roy P. C. Kessels contributed equally to this work. 1Department of Geriatric Medicine, Radboud University Medical Center, 6525, GC, Nijmegen, The Netherlands Full list of author information is available at the end of the article * Correspondence: Jana.Thomas@radboudumc.nl; Jurgen.Claassen@radboudumc.nl †Sebastiaan Overeem and Roy P. C. Kessels contributed equally to this work. Background rest week with unrestricted sleep. Their sleep disruptions are caused by external, occupational factors, which re- duce the bias of intrinsically caused sleep problems that could represent an early symptom preceding the clinical manifestation of dementia due to AD (i.e., reverse caus- ality). In this group of maritime pilots, we sought to ex- plore the effects of long-term, externally induced sleep disruption on cognitive function and brain amyloid-β burden, as biomarker of AD. g Sleep loss has been associated with increased risk of de- mentia in later life, specifically dementia caused by Alz- heimer’s disease (AD). In a meta-analysis of 27 studies with nearly 70.000 participants, sleep loss—mostly de- fined as self-reported sleep of < 6 h per night—carried an average relative risk of 1.68 (95% CI 1.45 to 1.86) of de- veloping dementia caused by AD [1]. This finding is relevant, because the etiology of late-onset AD remains unknown and therapeutic options are limited, making sleep a potential target for prevention or treatment of AD [2, 3]. The association between sleep loss and AD could be explained by reverse causality, wherein sleep loss is an early, preclinical manifestation of Alzheimer’s pathology [4–6]. However, the association may also be causal, wherein sleep loss contributes to the develop- ment of the disease. The latter hypothesis is based on a small number of animal and human studies that have identified mechanisms that could explain how sleep loss may increase the risk of AD. In mouse models of genetic and sporadic AD, for example, sleep loss increased brain amyloid-β accumulation [7–10]. In humans, we have previously showed that a single night of full sleep deprivation impaired the overnight reduction in CSF amyloid-β, causing 10% higher levels of CSF amyloid-β the next morning [11]. Also in humans, selective disrup- tion of slow wave sleep (SWS), without affecting other sleep stages, led to a comparable overnight difference in CSF amyloid-β [12]. Additionally, acute increases (5%) in PET-amyloid-β levels in the hippocampus and thalamus were observed after a single night of full sleep deprivation [13]. Two mechanisms have been proposed to explain the relationship between sleep loss and amyloid-β accumulation. First, the clearance of soluble toxic waste (including amyloid-β) from the central ner- vous system, characterized by exchange of interstitial and cerebrospinal fluids through the glymphatic path- way, appears more effective during sleep than in wake- fulness [14–18]. Study design The SCHIP study (Sleep Cognition Hypothesis In mari- time Pilots) is a case-control study in healthy volunteers under the hypothesis that repeated nights of sleep loss may contribute to the risk of dementia due to AD by gradually increasing amyloid-β levels. The study took place between December 2016 and May 2019 and was conducted and reported according to the STROBE guidelines for case-control studies. The timing between measurements was consistent across all participants. Sample size calculations were performed using G*power [21] and published previously [22]. The study protocol was peer-reviewed and published in BMJ Open [22]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 2 of 12 Page 2 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Background Second, the production of amyloid-β may be increased during wakefulness and reduced dur- ing sleep (especially SWS) [5, 11, 17, 19, 20]. These find- ings have led to the hypothesis that long-term sleep loss, through repetitive episodes of amyloid-β accumulation, may contribute to AD. However, evidence from human studies is lacking, and it remains unknown which quan- tity of sleep loss, both in terms of duration and intensity, would be required to raise the risk of developing demen- tia due to AD. The unique work of maritime pilots in the Netherlands offers an opportunity to study the associ- Study population We included 19 middle-aged (mean age = 53; age range: 48 to 60 years) men from the national organization of Dutch maritime pilots (Nederlandse Loodswezen). The profession of maritime pilots in the Netherlands is almost exclusively (99%) male. Their profession is char- acterized by sleep disruptions caused by external, occupational factors, wherein every other week is char- acterized by sleep disruption. We recruited maritime pi- lots with a work history of an average of 20 years (mean = 19.8; range 10 to 30 years). Details of the study population and their occupation have been described in our methods paper [22] and can be found in the add- itional information file (see Additional file 1). Maritime pilots were compared to age-, sex-, and education-matched healthy volunteers (n = 16; mean age = 57; age range 51 to 62 years) with occupations comparable in intellectual demand, but with regular working hours (no shift work). Control participants had normal sleep, confirmed by a Pittsburg Sleep Quality Index (PSQI) of < 5 as well as regular bed times (be- tween 8 p.m. and midnight), and regular wake-up times (between 5 a.m. and 9 a.m.). Participants were excluded from taking part in the study if they were using neuroac- tive medications, consumed > 30 alcoholic beverages per week, had a body mass index of > 30 kg/m2, suffered from intrinsic sleeping disorders (i.e., insomnia, REM sleep behavioral disorder; ruled out by PSG) or, for con- trols only, if they had self-reported cognitive complaints The unique work of maritime pilots in the Netherlands offers an opportunity to study the associ- ation between long-term sleep disruption and AD risk. Maritime pilots have a work schedule characterized by one week of irregular and unpredictable working hours, leading to reduced and fragmented sleep, followed by a Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 3 of 12 Page 3 of 12 Thomas et al. Alzheimer's Research & Therapy (indicated by Cognitive Failure Questionnaire (CFQ) and general health questionnaire). Vascular health was assessed during study visits, using a history of health- related events, i.e., high cholesterol, smoking, diabetes, and hypertension in addition to blood pressure measure- ments in maritime pilots. Baseline characteristics are listed in Table 1; Fig. 1 provides a study flow chart. headband that records EEG signals and can differentiate between light and deep sleep. Amyloid PET-CT imaging with 18F-flutemetamol Amyloid PET-CT imaging with F-flutemetamol Brain PET-CT scans were acquired in 2019 (Fig. 1) in maritime pilots only, since outcomes can be compared to normative data from the literature. We used the vali- dated tracer 18F-flutemetamol [26], a tracer that performs comparable to 11C-PIB [27]. Previous studies suggested that CSF and PET measurements of amyloid-β are in high concordance [28–31], while some suggest that PET is more powerful and more specific to AD pathology [32, 33]. Static brain images were acquired 90–110 min post-injection (four frames of 5 min) after bolus injection of approximately 185 MBq 18F-flutemeta- mol on a Siemens Biograph mCT. To measure tissue up- take ratios, PET scans of the PET-CT session were registered to the CT scan of the PET-CT session by rigid body linear registration with nearest neighbor interpolation using FSL’s FLIRT (FMRIB’s Linear Image Registration Tool) [34–36]. CT scans were then registered to the MNI152 2mm skull template by affine Polysomnography (2016/2017) To ascertain that neither maritime pilots nor controls had intrinsic sleep disorders, we performed full polysom- nography (PSG) in a sleep lab (Kempenhaeghe, Heeze, The Netherlands), registering total sleep time (TST), sleep stages (N1, N2, N3, REM), wake time after sleep onset (WASO), sleep efficiency (SEF), and sleep onset la- tency (SOL). The PSG took place on Sunday nights dur- ing rest weeks for maritime pilots and normal weekend days for controls, between 2016 and 2017 (Fig. 1). PSQI PSQI was filled in twice by maritime pilots (work and rest week) and once by controls. Cognitive assessment g The aim of the cognitive assessment was to explore ef- fects of long-term exposure to sleep disruption in mari- time pilots on AD-related cognitive impairment. Therefore, we applied a cognitive test battery that was designed to detect cognitive dysfunction in preclinical AD [24]. Tests focused on episodic memory (Logical Memory Subtest from the Wechsler Memory Scale – Fourth Edition (WMS-IV LM), Rey Auditory Verbal Learning Test (RAVLT)), semantic memory and lan- guage (letter and semantic fluency, Boston Naming Test), working memory and executive function (Digit Span subtest from the Wechsler Adult Intelligence Scale – Fourth Edition (WAIS-IV), Trail Making Test (TMT) parts A and B, WAIS-IV Coding), and attention (Test of Attentional Performance 2.0, TAP). Overnight memory consolidation was assessed using a novel paradigm based on the Doors Test [25], assessing visual recognition memory after short delay (10 min) and memory consoli- dation after long delay (after sleep). An overview about the cognitive tasks is summarized in the additional infor- mation file (see Additional file 2) and described in detail in our methods paper [22]. Cognitive tests were per- formed in the morning following PSG (in 2016 and 2017). Ethical approval Ethical approval The SCHIP study was approved by the institutional re- view board (CMO Region Arnhem-Nijmegen, NL55712.091.16, file number 2016-2337) and performed in accordance with good clinical practice guidelines and the world medical associations code of ethics (Declar- ation of Helsinki). Written informed consent was ob- tained from all participants after they received detailed study information. Participants received a stipend of 50 euros for participating, travel costs were compensated. Study population Furthermore, it measures total sleep time (TST), sleep onset latency (SOL), num- ber of short awakenings (< 5 min awake), and number of arousals (> 5 min awake). Maritime pilots were instructed to wear the headband for 20 consecutive days (10 workdays and 10 rest days). These measurements took place in 2019, before the amyloid PET scan (Fig. 1). Sleep measurements We combined several complementary methods to assess sleep quality. Actigraphy and sleep log Accelerometer-based sleep measurements (Actiwatch 2; Philips Respironics, Eindhoven, The Netherlands) in maritime pilots and controls were collected for a period of 10 consecutive days. Data was validated with sleep- wake diaries. For maritime pilots, these days contained a mixture of work and rest days. Measurements were taken during the screening phase of the study (Fig. 1), before cognitive testing and amyloid PET scans. Home-EEG (2019) We performed objective measurements of sleep quality during work and rest weeks in 13 of the 19 maritime pi- lots. For this, we used a novel and innovative device with a dry, single-lead EEG electrode (SmartSleep; Philips, Eindhoven, The Netherlands) [23]. This device is a Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 4 of 12 Fig. 1 Flow diagram SCHIP study. Abbreviations: PSQI, Pittsburgh Sleep Quality Index; CFQ, Cognitive Failure Questionnaire; HADS, Hospital Anxiety and Depression Scale; TAP, Test of Attentional Performance; RBD, REM sleep behavior disorder; PSG, polysomnography. Superscript letter “a” indicates that over the course of the study, four maritime pilots went into retirement; therefore, analysis for the work days is based on the remaining, employed maritime pilots (n = 13) Fig. 1 Flow diagram SCHIP study. Abbreviations: PSQI, Pittsburgh Sleep Quality Index; CFQ, Cognitive Failure Questionnaire; HADS, Hospital Anxiety and Depression Scale; TAP, Test of Attentional Performance; RBD, REM sleep behavior disorder; PSG, polysomnography. Superscript letter “a” indicates that over the course of the study, four maritime pilots went into retirement; therefore, analysis for the work days is based on the remaining, employed maritime pilots (n = 13) cerebellum as reference region [37, 38]. Normal global SUVR in a cognitively healthy population (aged 30– 60) was 1.3 (± 0.09) [27], comparable to mean SUVR of 1.29 (± 0.2) reported by Thurfjell et al. [39]. As additional step, all scans were visually rated as posi- tive/negative for the presence of amyloid-β deposition by an experienced and trained [40] nuclear medicine physician (JB) using validated criteria [37]. linear registration with a mutual information cost func- tion and nearest neighbor interpolation using FSL’s FLIR T and by non-linear registration using FSL’s FNIRT (FMRIB’s Non-linear Registration Tool) [34, 35]. These transformations were combined to align the PET scan to the MNI152 space in one single step. Tissue ratio was used as outcome measure, which is equivalent to the standard uptake value ratio (SUVR). The global cortical areas as well as prefrontal and temporal cortex and the cerebellum were selected with the MNI152 2 mm cor- tical atlas. Home-EEG (2019) Subsequently mean uptake values of these regions of interest (ROIs) and the tissue ratio, equiva- lent to the SUVR, were calculated using the linear registration with a mutual information cost func- tion and nearest neighbor interpolation using FSL’s FLIR T and by non-linear registration using FSL’s FNIRT (FMRIB’s Non-linear Registration Tool) [34, 35]. These transformations were combined to align the PET scan to the MNI152 space in one single step. Tissue ratio was used as outcome measure, which is equivalent to the standard uptake value ratio (SUVR). The global cortical areas as well as prefrontal and temporal cortex and the cerebellum were selected with the MNI152 2 mm cor- tical atlas. Subsequently mean uptake values of these regions of interest (ROIs) and the tissue ratio, equiva- lent to the SUVR, were calculated using the In the original protocol, an additional MRI scan was planned for PET-MRI co-registration to allow more de- tailed regional analyses of amyloid-β uptake [22]. Be- cause of limitations in funding, this could not be performed for this study after all. Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 5 of 12 Page 5 of 12 Statistical analysis differences between the groups at baseline (Table 1). All participants were Dutch, of white European descent, and had the same level of education. y Statistical analyses were performed using IBM SPSS Sta- tistics for Windows, version 20.0 (IBM Corp., Armonk, NY, USA). Alpha was set at 0.05 and tested two-sided. All continuous variables were assessed for normal distribution by inspection of histograms and the Shapiro-Wilk test. Normally distributed data are shown as mean ± SD. Not normally distributed data are pre- sented with median and interquartile ranges (IQR). For primary outcomes of cognitive assessment, raw test scores were transformed into z-scores for each neuro- psychological test. Z-scores were computed in SPSS and were based on mean and SD of the whole sample. For the other primary outcome measure, amyloid-β burden, we used the mean global standard value uptake ratio (SUVR) and the dichotomous visual read of the amyloid PET scans (positive/negative) [37]. An independent sam- ples t-test was performed to compare normally distrib- uted outcome measures between the groups (shown as mean ± SD); not normally distributed outcome measures were assessed with Mann-Whitney U tests (shown as median (IQR)). With regard to the home-EEG data, a Wilcoxon signed rank test was performed to compare deep sleep time (DST) during work weeks to DST dur- ing rest periods. Home-EEG (2019) We were able to analyze EEG-based sleep measurements during work and rest days in 13 maritime pilots (of the n = 19 maritime pilots, 4 had retired by 2019 when these measurements were scheduled, and could therefore no longer be measured during workdays; 2 could not be an- alyzed due to technical issues) (Fig. 1). Maritime pilots showed less TST during work weeks compared to rest weeks (Z = −3.18; p = 0.001) as well as less DST during work weeks compared to rest weeks (Z = −2.83; p = 0.005) (Table 3). Based on the home-EEG measure- ments, we created hypnograms of one maritime pilot for a work week and a rest week, illustrated in Fig. 2. Maritime pilots were on average 4 years younger than controls (Table 1; 95% CI −6.139 to −1.716). Results from the independent t-test did not indicate other Table 1 Baseline characteristics Characteristics Controls, n = 16 Maritime pilots, n = 19 Age, years 57 ± 2.9 53 ± 3.4 Educational attainment, years 17.4 ± 7.3 18 ± 0 BMI, kg/m2 25.5 ± 2.7 25.7 ± 2.7 History of diabetes 0 (0) 0 (0) SBP, mmHg NA 148.0 ± 16.4 DBP, mmHg NA 90.16 ± 11.7 Medication (“yes/no”) 2 (10.5) 4 (21.1) Smoking (“yes/no”) 3 (15.8) 3 (15.8) History of hypertension 0 (0) 0 (0) History of high cholesterol 0 (0) 1 (5.3) CFQ 26.4 ± 10.8 29 ± 7.8 HADS Anxiety 4.8 ± 3 4.0 ± 1.7 HADS Depression 3.6 ± 2.5 3.7 ± 2.7 Data is shown as mean ± SD or no. (%) (for normally distributed data) Abbreviations: BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, CFQ Cognitive Failure Questionnaire, HADS Hospital Anxiety and Depression Scale, NA not applicable Table 1 Baseline characteristics Results After exclusion and drop-out of participants (see Fig. 1), 19 maritime pilots and 16 controls completed the study (Fig. 1, Table 1). Sleep characteristics PSQI Maritime pilots reported worse sleep quality on the PSQI compared to controls, during rest weeks but espe- cially during work weeks (Table 2). When comparing PSQI scores between work week and rest week within maritime pilots, results of the t-test revealed that the average PSQI score for work weeks was almost twice the score for rest weeks, with values exceeding the validated cutoff point (≥7) for abnormal sleep behavior (Table 2). PSG (2016/2017) Both maritime pilots and controls had normal sleep pat- terns, including normal amount of DST (Table 2), ruling out intrinsic sleep disorders and indicating undisturbed sleep in maritime pilots during rest days. Actigraphy Subjective reports (PSQI) of poor sleep was confirmed by data from 10 days of actigraphy (mix of workdays and rest days), which indicated more awakenings and less sleep efficiency in the maritime pilot group compared to controls (Table 2). Cognitive assessment PSQI was administered twice for maritime pilots, including one work week and one rest week Abbreviations: PSG polysomnography, TST total sleep time, DST deep sleep time, REM rapid eye movement sleep, WASO wake after sleep onset, SEF sleep efficiency, SOL sleep onset latency, PSQI Pittsburgh Sleep Quality Index *Significant at p <0 .05 **Significant at p < 0.001 participants. There were no correlations between SUVRs and sleep quality (PSQI overall score and DST (for rest and work weeks)). the groups. Maritime pilots performed slightly better on the visual recognition memory after short delay com- pared to controls. Long-term memory consolidation, however, did not differ between the groups. All test scores were within normal age- and education-adjusted ranges based on available normative data (data not shown). All results can be found in Table 4. Discussion We investigated global brain amyloid-β levels and cogni- tive function in a unique population experiencing long- term sleep disruption, wherein every other week was characterized by sleep disruption due to irregular work- ing hours. Cognitive assessment For cognitive assessment, we transformed all raw neuro- psychological test scores into z-scores. Results from the independent t-test did not indicate differences between maritime pilots and controls on tests of episodic mem- ory (WMS-IV LM recognition, RAVLT total median). Small differences were observed on semantic memory and language, in which maritime pilots performed slightly better on the Boston Naming Test compared to controls. Performance on working memory and execu- tive function (WAIS-IV, TMT, WAIS-IV Coding) and attention (TAP 2.0) did not differ significantly between Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 6 of 12 Table 2 Comprehensive sleep characteristics of maritime pilots and controls Measures Controls, n = 16 Maritime pilots, n = 19 p value PSG TST, min 406 ± 44 403 ± 51 0.86 N1, min 46 ± 18 41 ± 14 0.40 N2, min 232 ± 36 215 ± 36 0.20 DST, min 50 ± 25 66 ± 28 0.10 REM, min 68 ± 17 79 ± 17 0.10 WASO, min 61 ± 26 53 ± 39 0.48 SEF, % 85.8 ± 7.1 86.1 ± 9.4 0.91 SOL, min 8 ± 7 11 ± 9 0.32 Actiwatch No. awakenings 33.5 ± 11.1 37.8 ± 10.3 0.24 SEF, % 89.3 ± 4.3 86 ± 3.8 0.03* PSQI (rest week vs. control Overall score 3.2 ± 1.4 4.5 ± 2.2* 0.049* PSQI (work week vs. control) Overall score 3.2 ± 1.4 8.8 ± 2.9** < 0.001** Data is shown as mean ± SD (for normally distributed data) Actiwatch data and PSQI were collected in 2016 and 2017. Actiwatch data was collected for a period of 10 consecutive days; for maritime pilots, these 10 days were a mix of work and rest days. PSQI was administered twice for maritime pilots, including one work week and one rest week Abbreviations: PSG polysomnography, TST total sleep time, DST deep sleep time, REM rapid eye movement sleep, WASO wake after sleep onset, SEF sleep efficiency, SOL sleep onset latency, PSQI Pittsburgh Sleep Quality Index *Significant at p <0 .05 **Significant at p < 0.001 Table 2 Comprehensive sleep characteristics of maritime pilots and controls ( y ) Actiwatch data and PSQI were collected in 2016 and 2017. Actiwatch data was collected for a period of 10 consecutive days; for maritime pilots, these 10 days were a mix of work and rest days. 18F-flutemetamol PET-CT Amyloid PET scans were administered in maritime pi- lots only (n = 19, Fig. 1). SUVRs in healthy populations were reported as 1.29 (± 0.2) [39] and 1.3 (± 0.09) [27]. The global cortical SUVR in maritime pilots was 1.009 (± 0.059; 95% CI 0.980 to 1.037) and therefore below normal values for a cognitively healthy population in this age range [27, 39]. More specifically, we detected a SUVR of 0.860 (± 0.098; 95% CI 0.813 to 0.907) for frontal lobes and a SUVR of 0.996 (± 0.06; 95% CI 0.967 to 1.025) for temporal lobes. In addition, all scans were rated negative for the presence of amyloid-β depos- ition on visual reading. Figure 3 shows examples of amyloid PET images from two representative Our main finding is that, in this relatively small, but deeply phenotyped sample, this intensity and pattern of sleep disruption was not associated with elevated brain amyloid-β levels, nor with cognitive decline. In previous studies, a single night of full sleep deprivation, or selective restriction of deep sleep, and chronic partial sleep fragmentation (rodents only) increased brain amyloid-β levels [8–13]. These observations have fueled the hypothesis that re- peated nights of sleep loss may contribute to the risk of dementia due to AD by gradually increasing amyloid-β levels. Table 3 Results from the home-EEG measurements (maritime pilots only) Measures Rest week, n = 13 Work week, n = 13 p value Home EEG TST†, min 406.17 (340–425.98) 318.56 (250.21–352.93) 0.001* DST†, min 51.34 (48.37–69.30) 36.75 (32.30–58.58) 0.005* Data is shown as median (IQR) (for not normally distributed data) Home-EEG recordings were performed in 2019 in maritime pilots only using a dry single-lead EEG device (Philips, Eindhoven, The Netherlands) Abbreviations: TST total sleep time, DST deep sleep time *Significant at p < 0.05 †Means calculated based on sleep periods within work week or rest week respectively Page 7 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Fig. 2 Example of a maritime pilots’ sleep schedule. a Hypnogram is based on 7 consecutive working days of sleep measurements with a dry electrode single-lead home-EEG device. b Hypnogram is based on 7 rest days of sleep measurements with a dry electrode single-lead home-EEG device Fig. 2 Example of a maritime pilots’ sleep schedule. a Hypnogram is based on 7 consecutive working days of sleep measurements with a dry electrode single-lead home-EEG device. 18F-flutemetamol PET-CT b Hypnogram is based on 7 rest days of sleep measurements with a dry electrode single-lead home-EEG device Fig. 2 Example of a maritime pilots’ sleep schedule. a Hypnogram is based on 7 consecutive working days of sleep measurements with a dry electrode single-lead home-EEG device. b Hypnogram is based on 7 rest days of sleep measurements with a dry electrode single-lead home-EEG device Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 8 of 12 Table 4 Results of cognitive assessment and memory consolidation Measures Controls, n = 16 Maritime pilots, n = 19 p value WMS-IV LM I 0.16 ± 1.07 −0.08 ± 0.99 0.49 LM II 0.29 (−0.93–1.01) 0.11 (−0.61–0.83) 0.72 LM recognition 0.25 ± 1.10 −0.12 ± 0.94 0.29 RAVLT Total −0.08 (−0.63–0.61) 0.77 (−1.14–1.09) 0.41 Del. recall −0.08 ± 0.76 0.06 ± 1.25 0.70 Del. recognition −0.10 ± 1.15 0.21 ± 0.85 0.37 Sensitivity A’ 0.07 (−0.85–0.82) 0.22 (−0.30–0.82) 0.41 WAIS-IV Coding −0.10 ± 0.59 0.21 ± 1.26 0.37 Digit span −0.21 ± 0.62 0.23 ± 1.24 0.21 TMT Part A −0.09 ± 0.75 −0.06 ± 1.15 0.94 Part B −0.33 (−0.57–0.75) −0.38 (−0.96–0.50) 0.24 Fluency D-A-T 0.07 ± 0.89 −0.29 ± 1.13 0.78 Animal 0.38 (−0.88–0.73) 0.20 (−0.70–0.91) 0.84 Profession −0.26 ± 0.85 0.26 ± 1.13 0.14 BNT Short version −0.11 (−0.39–0.31) 0.20 (0.10–0.62) 0.02* TAP evening Cued −0.05 (−0.72–0.44) −0.39 (−0.91–0.50) 0.37 Un-cued −0.05 (−0.87–0.89) −0.17 (−0.76–0.45) 0.84 TAP morning Cued −0.10 (−0.50–0.66) −0.29 (−0.71–0.12) 0.27 Un-cued −0.13 (−0.63–1.09) −0.40 (−0.74–0.33) 0.22 Visual recognition—short delay (10 min) Sensitivity, A’ −0.27 ± 0.90 0.46 ± 0.58 0.007* Hits −0.33 (−1.18–0.15) 0.64 (0.15–0.64) 0.03* False alarms 0.19 ± 1.06 −0.27 ± 0.84 0.16 Memory consolidation—long delay (after sleep) Sensitivity, A′ −0.08 ± 0.95 0.35 ± 0.73 0.14 Hits 0.50 (−0.21–0.70) −0.08 (−0.69–0.54) 0.20 False alarms 0.15 ± 0.90 −0.41 ± 0.76 0.06 Data is shown as mean ± SD (for normally distributed data) or median (IQR) (for not normally distributed data) Test results are expressed in z-scores. TAP: z-scores are based on median reaction-time. Visual recognition—short-term: assessed approximately 10 min after targets were presented. 18F-flutemetamol PET-CT Memory consolidation after long-term took place after one night of sleep (approximately 10 h) Abbreviations: WMS Wechsler Memory Scale, LM logical memory, RAVLT Rey Auditory Verbal Learning Test, WAIS Wechsler Adult Intelligent Scale, TMT Trail Making test, BNT Boston Naming Test, TAP Test of Attentional Performance *Significant at p < 0.05 Table 4 Results of cognitive assessment and memory consolidation Data is shown as mean ± SD (for normally distributed data) or median (IQR) (for not normally distributed data) Test results are expressed in z-scores. TAP: z-scores are based on median reaction-time. Visual recognition—short-term: assessed approximately 10 min after targets were presented. Memory consolidation after long-term took place after one night of sleep (approximately 10 h) Abbreviations: WMS Wechsler Memory Scale, LM logical memory, RAVLT Rey Auditory Verbal Learning Test, WAIS Wechsler Adult Intelligent Scale, TMT Trail Making test, BNT Boston Naming Test, TAP Test of Attentional Performance *Significant at p < 0.05 The sample of maritime pilots offered a unique oppor- tunity to explore if long-term, externally induced sleep disruptions increase dementia risk in terms of AD- related impaired cognitive function and amyloid-β burden. Their sleep behavior is characterized by work weeks with disrupted sleep, alternating with rest weeks of unrestricted sleep. This pattern was confirmed using a combination of methods: self-reported disrupted sleep during work weeks was objectified by sleep diaries, acti- graphy, and home-EEG measurements. Relatively normal sleep during rest weeks of maritime pilots was further- more confirmed with PSG (compared to controls) and home-EEG measurements. Moreover, using PSG we were able to exclude intrinsic sleep disorders in this group, which is important because sleep loss may be an early manifestation of Alzheimer’s pathology and could lead to a reverse causality association [41, 42]. To explore possible AD-related impaired cognitive perform- ance, we applied a cognitive test battery that was chosen for its sensitivity to cognitive changes in early, preclinical AD [24]. On all cognitive domains, maritime pilots showed normal cognitive performance, compared not only to the control group, but also to normative values. This was also the case for overnight episodic memory consolidation, which is dependent on deep sleep [5, 43]. We considered that normal cognitive function would not rule out increased amyloid-β levels, since early stages of amyloid-β accumulation (indicated by PET or CSF) can have a long asymptomatic stage. Therefore, we performed additional global brain amyloid-β imaging in maritime pilots. 18F-flutemetamol PET-CT None of the maritime pilots had evi- dence of elevated amyloid-β levels, with SUVR values remaining below the values established for a healthy population [27, 39]. In a recent meta-analysis, the Page 9 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Fig. 3 Representative transversal slides from 18F-flutemetamol PET scans of two participants. Scans were acquired 90–110 min post-injection and show normal subcortical nonspecific uptake in the brain Fig. 3 Representative transversal slides from 18F-flutemetamol PET scans of two participants. Scans were acquired 90–110 min post-injection and show normal subcortical nonspecific uptake in the brain levels. This argument is, however, not supported by recent work demonstrating that a reduction in total sleep time, but not SWS, determined the increase in amyloid-β production [20]. estimated prevalence of PET amyloid-positivity in cogni- tively healthy men aged 55–60 years was 13% (95% CI 10.3 to 16%) [44]. This indicates that our observation of a prevalence of 0/19 confidently rules out elevated amyloid- β levels, even considering the relatively small sample size. Second, it is possible that sleep disruption alone is in- sufficient to increase AD risk, but requires the presence of other risk factors, such as impaired glucose metabol- ism [45], oxidative stress [46], depression [47], or general poor vascular health [48]. Our study population was healthy and had a low vascular risk (Table 1). What could explain the observed absence of elevated amyloid-β levels or impaired cognitive function, despite evidence of long-term sleep disruptions? First, assuming that the hypothesis that sleep dis- ruption may cause AD is correct, the alternating pat- tern of a week with unrestricted sleep following a week of disrupted sleep may be insufficient to cause elevated brain amyloid-β levels. Either sleep disrup- tion during ≈50% of nights for ≈20 years is insuffi- cient to affect amyloid-β clearance/production or the week of normal sleep following a week of sleep dis- ruption provides compensatory reductions in brain amyloid-β levels. This latter option would then sug- gest that disrupted sleep is a modifiable risk factor and that it may not be necessary to achieve full normalization of sleep to reduce AD risk. Whether this can be extrapolated to the general population is uncertain however. The maritime pilots may, due to their profession, be better able to compensate normal sleep in their rest weeks. 18F-flutemetamol PET-CT While most studies link re- duced total sleep time (< 6 h) to increased AD risk [1, 20], other work suggests that the risk is specifically linked to reduced deep sleep [12, 17]. The maritime pilots had reduced total sleep time during work weeks, but the home-EEG recordings indicate that they still achieved an average of 37 min of deep sleep per sleep period. Therefore, SWS may have been in- sufficiently impaired to result in abnormal amyloid-β Current research on this topic is still in its very early stage, with limited evidence supporting a causal relation- ship between sleep loss and risk of AD dementia. The association between sleep loss and AD may be driven by reverse causality (sleep loss as an early manifestation of AD) or by a shared common pathway that causes sleep loss and increases AD risk. There is also recent evidence that found no association between sleep (subjective sleep quality) and risk of dementia [49]. Previous evidence suggesting a link between sleep and AD has been limited to a small number of studies in ro- dents and humans, with variations in methodology and study population selection. Furthermore, the human studies focused on the relationship between poor sleep for a short period of time (1 or 2 nights) and its effects on amyloid-β (or tau), but have not studied actual devel- opment of AD dementia. Longitudinal studies are available but lack rigorous assessment of sleep and biomarker evidence of AD. Our study adds information on the long-term associ- ation between poor sleep and AD, combining object- ive sleep measures with established biomarkers for AD. Page 10 of 12 Page 10 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Acknowledgements One could argue that the absence of tau measure- ments is a limitation, as recent evidence now also sug- gests that sleep affects tau in a similar manner as amyloid-β [50]. We did not perform tau measurements because the maritime pilots had no evidence of cognitive impairment. Since tau pathology is strongly correlated with cognitive decline [51–53], it is highly unlikely to find evidence of tau accumulation in subjects with nor- mal cognitive function, even more so when they are amyloid-negative. A final limitation is that amyloid-β status was not obtained from the controls. Instead, we compared our outcomes to normative values from the literature, which were acquired with additional MRI measurements for co-registration of the amyloid PET- CT scans. Since we used CT to identify global amyloid-β instead of MRI, this difference in methodology has to be kept in mind when interpreting our results. We would like to thank all participants for taking part in this study and the secretary of the Dutch Maritime Pilot Association for helping with recruitment of participants. Furthermore, a special thanks to the interns who helped with data collection and to J. Doornbosch for helping with the neuropsychological test administration. Dr. T. Tsoneva and S. Pastoor from Philips helped us with data storage and raw data-extraction from the home- EEG devices. J.M.F. Liebregts provided advice on and assistance with design- ing graphics and figures. We would also like to thank Dr. K.V. Haak for his ad- vice on the PET-CT imaging analyses. Abbreviations AD: Alzheimer’s disease; CFQ: Cognitive Failure Questionnaire; DBP: Diastolic blood pressure; DST: Deep sleep time; HADS: Hospital Anxiety and Depression Scale; IQR: Interquartile range; NA: Not applicable; NRA: Number of arousals; NRI: Number of interruptions; PSG: Polysomnography; PSQI: Pittsburgh Sleep Quality Index; RAVLT: Rey Auditory Verbal Learning Test; REM: Rapid eye movement; ROI: Region of interest; SBP: Systolic blood pressure; SCHIP: Sleep Cognition Hypothesis In maritime Pilots; SD: Standard deviation; SEF: Sleep efficiency; SOL: Sleep onset latency; SUVR: Standard uptake value ratio; SWS: Slow wave sleep; TAP: Test of Attentional Performance; TMT: Trail Making Test; TST: Total sleep time; WAIS-IV: Wechsler Adult Intelligence Scale (4th edition); WASO: Wake after sleep onset; WMS-IV LM: Wechsler Memory Scale (4th edition) Logical Memory Another limitation is that controls, although matched for sex, age, education, and general health, might not have been matched entirely with regard to personality, resilience, physical activity, or cognitive skills/general intelligence. Supplementary information S l i f i i pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13195-020-00668-5. Additional file 1. Details of the study population and their occupation. Additional file 2. Supplemental Box 1: Overview Neuropsychological Test Battery. Additional file 1. Details of the study population and their occupation. Additional file 2. Supplemental Box 1: Overview Neuropsychological Test Battery. The uniqueness of the population may also cause bias. Maritime pilots are healthy, have no cardiovascular risk, and are physically active in their work, factors that may reduce their AD risk. They may be resilient to the conse- quences of sleep disruption, because they have success- fully performed this work for > 10 years. One example of such resilience could be their ability to achieve deep sleep even under conditions of fragmented and restricted total sleep during work weeks or their capacity to gener- ate sufficient deep sleep during rest weeks. Conclusions A strength of the study is the comprehensive assess- ments of all outcome measures: cognitive function was assessed with an extensive test battery sensitive to early, preclinical symptoms of AD; sleep was assessed with various measurements including self-reported but also objectively measured sleep, implementing innovative techniques for sleep assessments (home-EEG); sleep dis- orders were ruled out using PSG; and PET-amyloid im- aging was used as a validated AD biomarker. A further strength is the unique cohort of maritime pilots, with prolonged and consistent exposure to sleep loss related to their work, making this a highly valuable population that allowed us to explore poor sleep as isolated variable in relationship with the risk of AD dementia. In this study, we tested the hypothesis that prolonged sleep loss increases the risk of dementia due to AD. We found that a history of work-induced, long-term sleep disruption was not associated with impairment in cogni- tive function, nor with elevated global brain amyloid-β levels, in a group of healthy, middle-aged men. Taking into account the small sample size of our study, our re- sults do not necessarily refute the hypothesis we intended to test, but neither support it. It is possible that amyloid-β accumulation during periods with sleep disruption can be reduced in nights with normal sleep. Alternatively, sleep loss may only increase AD risk in combination with other factors. Finally, the association between sleep loss and AD in epidemiological studies may be driven by reverse causality. These and other hy- potheses have to be tested in future studies. Our study is limited by the small sample size. Home-EEG measurements were available in 13 of the 19 maritime pi- lots. However, outcomes of these sleep measurements con- firmed observations of work-related disrupted sleep based on PSQI, sleep-wake dairy and actigraphy data in the whole sample, and added novel data on total and deep sleep time during work weeks and rest weeks. Funding 9. Rothman SM, Herdener N, Frankola KA, Mughal MR, Mattson MP. Chronic mild sleep restriction accentuates contextual memory impairments, and accumulations of cortical Aβ and pTau in a mouse model of Alzheimer’s disease. Brain Res. 2013;1529:200–8. This work was supported by ISAO (Internationale Stichting Alzheimer Onderzoek (now Alzheimer Nederland), grant number: 15040) awarded to JC. Philips kindly provided the home-EEG devices that were used in this study. The funders had no role in the study design or conduct; collection, analysis, or interpretation of data; nor in the writing of the report or in the decision to submit the manuscript for publication. 10. Zhao HY, Wu HJ, He JL, Zhuang JH, Liu ZY, Huang LQ, et al. Chronic sleep restriction induces cognitive deficits and cortical beta-amyloid deposition in mice via BACE 1-antisense activation. CNS Neurosci Ther. 2017;23(3):233–40. 10. Zhao HY, Wu HJ, He JL, Zhuang JH, Liu ZY, Huang LQ, et al. Chronic sleep restriction induces cognitive deficits and cortical beta-amyloid deposition in mice via BACE 1-antisense activation. CNS Neurosci Ther. 2017;23(3):233–40. 11. Ooms S, Overeem S, Besse K, Rikkert MO, Verbeek M, Claassen JA. Effect of 1 night of total sleep deprivation on cerebrospinal fluid β-amyloid 42 in healthy middle-aged men: a randomized clinical trial. JAMA neurology. 2014;71(8):971–7. Consent for publication Not applicable. 17. Fultz NE, Bonmassar G, Setsompop K, Stickgold RA, Rosen BR, Polimeni JR, et al. Coupled electrophysiological, hemodynamic, and cerebrospinal fluid oscillations in human sleep. Science (80- ). 2019;366(6465):628–31. Competing interests 18. Jessen NA, Munk ASF, Lundgaard I, Nedergaard M. The glymphatic system: a beginner’s guide. Neurochem Res. 2015;40(12):2583–99. 18. Jessen NA, Munk ASF, Lundgaard I, Nedergaard M. The glymphatic system: a beginner’s guide. Neurochem Res. 2015;40(12):2583–99. The authors declare that they have no competing interests. Availability of data and materials d d l d d Data generated and/or analyzed during this study are included in this article and its supplementary information files. Additionally, the datasets used/ analyzed in the current study are available from the corresponding author on reasonable request. 12. Ju Y-ES, Ooms SJ, Sutphen C, Macauley SL, Zangrilli MA, Jerome G, et al. Slow wave sleep disruption increases cerebrospinal fluid amyloid-β levels. Brain. 2017;140(8):2104–11. 12. Ju Y-ES, Ooms SJ, Sutphen C, Macauley SL, Zangrilli MA, Jerome G, et al. Slow wave sleep disruption increases cerebrospinal fluid amyloid-β levels. Brain. 2017;140(8):2104–11. 13. Shokri-Kojori E, Wang G-J, Wiers CE, Demiral SB, Guo M, Kim SW, et al. β- Amyloid accumulation in the human brain after one night of sleep deprivation. Proc Natl Acad Sci. 2018;115(17):4483–8. 13. Shokri-Kojori E, Wang G-J, Wiers CE, Demiral SB, Guo M, Kim SW, et al. β- Amyloid accumulation in the human brain after one night of sleep deprivation. Proc Natl Acad Sci. 2018;115(17):4483–8. Received: 21 April 2020 Accepted: 13 August 2020 Received: 21 April 2020 Accepted: 13 August 2020 Received: 21 April 2020 Accepted: 13 August 2020 23. Garcia-Molina G, Tsoneva T, Jasko J, Steele B, Aquino A, Baher K, et al. Closed-loop system to enhance slow wave activity during sleep. 2018. 24. Weintraub S, Carrillo MC, Farias ST, Goldberg TE, Hendrix JA, Jaeger J, et al. Measuring cognition and function in the preclinical stage of Alzheimer’s disease. Alzheimer’s Dementia. 2018;4:64–75. Authors’ contributions JT, SJO, SO, RPCK, and JAHRC made substantial contributions to conception or design of the work. All authors (JT, SJO, LJM, JB, MGMO, SO, RPCK, JAHRC) were in charge of acquisition, analysis, and interpretation of data for the work. JT, SJO, LJM, JB, RPCK, and JAHRC were responsible for statistical data analyses. Supervision was given by JAHRC. All authors drafted the work or revised it critically for important intellectual content and approved the final version of the manuscript. All authors have agreed both to be personally accountable for their own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. Page 11 of 12 Page 11 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Author details 1 19. Kang DW, Lee CU, Lim HK. Role of sleep disturbance in the trajectory of Alzheimer’s disease. Clin Psychopharmacol Neurosci. 2017;15(2):89. Author details 1Department of Geriatric Medicine, Radboud University Medical Center, 6525, GC, Nijmegen, The Netherlands. 2Donders Institute for Brain, Cognition and Behaviour, 6525, HR, Nijmegen, The Netherlands. 3Radboud Alzheimer Centre, 6525, GA, Nijmegen, The Netherlands. 4Department of Radiology and Nuclear Medicine, Radboud University Medical Center, 6525, GC, Nijmegen, The Netherlands. 5Department of Radiology and Nuclear Medicine, Amsterdam University Medical Centers, Academic Medical Center, 1105, AZ, Amsterdam, The Netherlands. 6Sleep Medicine Centre Kempenhaeghe, 5591, VE, Heeze, The Netherlands. 7Eindhoven University of Technology, 5612, AZ, Eindhoven, The Netherlands. 8Department of Medical Psychology, Radboud University Medical Center, 6525, GA, Nijmegen, The Netherlands. 20. Lucey BP, Hicks TJ, McLeland JS, Toedebusch CD, Boyd J, Elbert DL, et al. Effect of sleep on overnight cerebrospinal fluid amyloid β kinetics. Ann Neurol. 2018;83(1):197–204. 21. Faul F, Erdfelder E, Lang A-G, Buchner A. G* Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39(2):175–91. 22. Thomas J, Ooms S, Verbeek M, Booij J, Rijpkema M, Kessels RPC, et al. Sleep- Cognition Hypothesis In maritime Pilots, what is the effect of long-term work-related poor sleep on cognition and amyloid accumulation in healthy middle-aged maritime pilots: methodology of a case–control study. BMJ Open. 2019;9(6):e026992. References 1. Bubu OM, Brannick M, Mortimer J, Umasabor-Bubu O, Sebastião YV, Wen Y, et al. Sleep, cognitive impairment, and Alzheimer’s disease: a systematic review and meta-analysis. Sleep. 2016;40(1):zsw032. 1. Bubu OM, Brannick M, Mortimer J, Umasabor-Bubu O, Sebastião YV, Wen Y, et al. Sleep, cognitive impairment, and Alzheimer’s disease: a systematic review and meta-analysis. Sleep. 2016;40(1):zsw032. 25. Baddeley A, Emslie H, Nimmo-Smith I. Doors and people: a test of visual and verbal recall and recognition, Thames Valley Test Company, Bury St. Edmunds, UK. 1994. 25. Baddeley A, Emslie H, Nimmo-Smith I. Doors and people: a test of visual and verbal recall and recognition, Thames Valley Test Company, Bury St. Edmunds, UK. 1994. 2. Mander BA, Winer JR, Jagust WJ, Walker MP. Sleep: a novel mechanistic pathway, biomarker, and treatment target in the pathology of Alzheimer’s disease? Trends Neurosci. 2016;39(8):552–66. 26. Curtis C, Gamez JE, Singh U, Sadowsky CH, Villena T, Sabbagh MN, et al. Phase 3 trial of flutemetamol labeled with radioactive fluorine 18 imaging and neuritic plaque density. JAMA neurology. 2015;72(3):287–94. 3. Spira AP, Gottesman RF. Sleep disturbance: an emerging opportunity for Alzheimer’s disease prevention? Int Psychogeriatr. 2017;29(4):529–31. 27. Lowe VJ, Lundt E, Knopman D, Senjem ML, Gunter JL, Schwarz CG, et al. Comparison of [18F] Flutemetamol and [11C] Pittsburgh Compound-B in cognitively normal young, cognitively normal elderly, and Alzheimer’s disease dementia individuals. NeuroImage: Clinical. 2017;16:295–302. 4. Slats D, Claassen JA, Verbeek MM, Overeem S. Reciprocal interactions between sleep, circadian rhythms and Alzheimer’s disease: focus on the role of hypocretin and melatonin. Ageing Res Rev. 2013;12(1):188–200. 28. Hansson O, Seibyl J, Stomrud E, Zetterberg H, Trojanowski JQ, Bittner T, et al. CSF biomarkers of Alzheimer’s disease concord with amyloid-β PET and predict clinical progression: a study of fully automated immunoassays in BioFINDER and ADNI cohorts. Alzheimers Dement. 2018;14(11):1470–81. y g g 5. Ju Y-ES, Lucey BP, Holtzman DM. Sleep and Alzheimer disease pathology [mdash] a bidirectional relationship. Nat Rev Neurol. 2014;10(2):115–9. 6. Roh JH, Huang Y, Bero AW, Kasten T, Stewart FR, Bateman RJ, et al. Disruption of the sleep-wake cycle and diurnal fluctuation of β-amyloid in mice with Alzheimer’s disease pathology. Sci Transl Med. 2012;4(150): 150ra22-ra22. 29. Pannee J, Portelius E, Minthon L, Gobom J, Andreasson U, Zetterberg H, et al. Reference measurement procedure for CSF amyloid beta (Aβ) 1–42 and the CSF Aβ1–42/Aβ1–40 ratio–a cross-validation study against amyloid PET. J Neurochem. 2016;139(4):651–8. 7. Ethics approval and consent to participate The SCHIP study was approved by the institutional review board (CMO, Commissie Mensgebonden Onderzoek, Region Arnhem-Nijmegen, NL55712.091.16, file number 2016-2337) and performed in accordance with good clinical practice guidelines and the world medical associations code of ethics (Declaration of Helsinki). Written informed consent was obtained from all participants after they received detailed study information. Participants re- ceived a stipend of 50 euros for participating; travel costs were compensated. 14. Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, et al. Sleep drives metabolite clearance from the adult brain. Science (80- ). 2013; 342(6156):373–7. 14. Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, et al. Sleep drives metabolite clearance from the adult brain. Science (80- ). 2013; 342(6156):373–7. 15. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012;4(147):147ra11-ra11. 15. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012;4(147):147ra11-ra11. 16. Nedergaard M. Garbage truck of the brain. Science (80- ). 2013; 340(6140):1529–30. 16. Nedergaard M. Garbage truck of the brain. Science (80- ). 2013; 340(6140):1529–30. 7. Kang J-E, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, et al. Amyloid-β dynamics are regulated by orexin and the sleep-wake cycle. Science (80- ). 2009;326(5955):1005–7. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 33. Reimand J, De Wilde A, Teunissen CE, Zwan M, Windhorst AD, Boellaard R, et al. PET and CSF amyloid-β status are differently predicted by patient features: information from discordant cases. Alzheimers Res Ther. 2019;11(1):100. 33. Reimand J, De Wilde A, Teunissen CE, Zwan M, Windhorst AD, Boellaard R, et al. PET and CSF amyloid-β status are differently predicted by patient features: information from discordant cases. Alzheimers Res Ther. 2019;11(1):100. 34. Jenkinson M, Bannister P, Brady M, Smith S. Improved optimization for the robust and accurate linear registration and motion correction of brain images. Neuroimage. 2002;17(2):825–41. 34. Jenkinson M, Bannister P, Brady M, Smith S. Improved optimization for the robust and accurate linear registration and motion correction of brain images. Neuroimage. 2002;17(2):825–41. 35. Jenkinson M, Beckmann CF, Behrens TE, Woolrich MW, Smith SM. Fsl Neuroimage. 2012;62(2):782–90. 36. Andersson JL, Jenkinson M, Smith S. Non-linear registration aka Spatial normalisation FMRIB Technial Report TR07JA2. FMRIB Analysis Group of the University of Oxford 2007. 37. Farrar G, Molinuevo JL, Zanette M. Is there a difference in regional read [18 F] flutemetamol amyloid patterns between end-of-life subjects and those with amnestic mild cognitive impairment? Eur J Nucl Med Mol Imaging. 2019;46(6):1299–308. 38. Vandenberghe R, Van Laere K, Ivanoiu A, Salmon E, Bastin C, Triau E, et al. 18F-flutemetamol amyloid imaging in Alzheimer disease and mild cognitive impairment: a phase 2 trial. Ann Neurol. 2010;68(3):319–29. 39. Thurfjell L, Lötjönen J, Lundqvist R, Koikkalainen J, Soininen H, Waldemar G, et al. Combination of biomarkers: PET [18F] flutemetamol imaging and structural MRI in dementia and mild cognitive impairment. Neurodegener Dis. 2012;10(1–4):246–9. 40. Buckley CJ, Sherwin PF, Smith AP, Wolber J, Weick SM, Brooks DJ. Validation of an electronic image reader training programme for interpretation of [18F] flutemetamol β-amyloid PET brain images. Nucl Med Commun. 2017;38(3):234. 41. Winer JR, Mander BA, Helfrich RF, Maass A, Harrison TM, Baker SL, et al. Sleep as a potential biomarker of tau and β-amyloid burden in the human brain. J Neurosci. 2019:0503–19. 42. Lucey BP, McCullough A, Landsness EC, Toedebusch CD, McLeland JS, Zaza AM, et al. Reduced non–rapid eye movement sleep is associated with tau pathology in early Alzheimer’s disease. Sci Transl Med. 2019;11(474): eaau6550. 43. Mander BA, Marks SM, Vogel JW, Rao V, Lu B, Saletin JM, et al. References Kang J-E, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, et al. Amyloid-β dynamics are regulated by orexin and the sleep-wake cycle. Science (80- ). 2009;326(5955):1005–7. 7. Kang J-E, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, et al. Amyloid-β dynamics are regulated by orexin and the sleep-wake cycle. Science (80- ). 2009;326(5955):1005–7. 30. Palmqvist S, Zetterberg H, Blennow K, Vestberg S, Andreasson U, Brooks DJ, et al. Accuracy of brain amyloid detection in clinical practice using cerebrospinal fluid β-amyloid 42: a cross-validation study against amyloid positron emission tomography. JAMA neurology. 2014;71(10):1282–9. 30. Palmqvist S, Zetterberg H, Blennow K, Vestberg S, Andreasson U, Brooks DJ, et al. Accuracy of brain amyloid detection in clinical practice using cerebrospinal fluid β-amyloid 42: a cross-validation study against amyloid positron emission tomography. JAMA neurology. 2014;71(10):1282–9. 8. Minakawa EN, Miyazaki K, Maruo K, Yagihara H, Fujita H, Wada K, et al. Chronic sleep fragmentation exacerbates amyloid β deposition in Alzheimer’s disease model mice. Neurosci Lett. 2017;653:362–9. 8. Minakawa EN, Miyazaki K, Maruo K, Yagihara H, Fujita H, Wada K, et al. Chronic sleep fragmentation exacerbates amyloid β deposition in Alzheimer’s disease model mice. Neurosci Lett. 2017;653:362–9. Page 12 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 53. Ossenkoppele R, Smith R, Ohlsson T, Strandberg O, Mattsson N, Insel PS, et al. Associations between tau, Aβ, and cortical thickness with cognition in Alzheimer disease. Neurology. 2019;92(6):e601–e12. 31. Palmqvist S, Zetterberg H, Mattsson N, Johansson P, Minthon L, Blennow K, et al. Detailed comparison of amyloid PET and CSF biomarkers for identifying early Alzheimer disease. Neurology. 2015;85(14):1240–9. 32. Bouallègue FB, Mariano-Goulart D, Payoux P, Initiative AsDN. Comparison of CSF markers and semi-quantitative amyloid PET in Alzheimer’s disease diagnosis and in cognitive impairment prognosis using the ADNI-2 database. Alzheimers Res Ther. 2017;9(1):32. Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Publisher’s Note [beta]- amyloid disrupts human NREM slow waves and related hippocampus- dependent memory consolidation. Nat Neurosci. 2015;18(7):1051–7. 44. Jansen WJ, Ossenkoppele R, Knol DL, Tijms BM, Scheltens P, Verhey FR, et al. Prevalence of cerebral amyloid pathology in persons without dementia: a meta-analysis. Jama. 2015;313(19):1924–38. 45. Kuehn BM. In Alzheimer Research. JAMA: Glucose metabolism moves to center stage; 2020. 46. Smith MA, Rottkamp CA, Nunomura A, Raina AK, Perry G. Oxidative stress in Alzheimer’s disease. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 2000;1502(1):139–44. 47. Green RC, Cupples LA, Kurz A, Auerbach S, Go R, Sadovnick D, et al. Depression as a risk factor for Alzheimer disease: the MIRAGE Study. Arch Neurol. 2003;60(5):753–9. 48. Rabin JS, Klein H, Kirn DR, Schultz AP, Yang HS, Hampton O, Pruzin J. Associations of physical activity and β-amyloid with longitudinal cognition and neurodegeneration in clinically normal older adults. JAMA neurology. 2019;76(10):1203-10. 49. Lysen TS, Wolters FJ, Luik AI, Ikram MK, Tiemeier H, Ikram MA. Subjective sleep quality is not associated with incident dementia: the Rotterdam study. J Alzheimers Dis. 2018;64(1):239–47. 50. Holth JK, Fritschi SK, Wang C, Pedersen NP, Cirrito JR, Mahan TE, et al. The sleep-wake cycle regulates brain interstitial fluid tau in mice and CSF tau in humans. Science (80- ). 2019;363(6429):880–4. 51. Bejanin A, Schonhaut DR, La Joie R, Kramer JH, Baker SL, Sosa N, et al. Tau pathology and neurodegeneration contribute to cognitive impairment in Alzheimer’s disease. Brain. 2017;140(12):3286–300. 52. Pontecorvo MJ, Devous MD Sr, Navitsky M, Lu M, Salloway S, Schaerf FW, et al. Relationships between flortaucipir PET tau binding and amyloid burden, clinical diagnosis, age and cognition. Brain. 2017;140(3):748–63.
https://openalex.org/W3018951548
https://hal-cea.archives-ouvertes.fr/cea-03579858/document
English
null
In-Can vitrification of ash
IOP conference series. Materials science and engineering
2,020
cc-by
4,950
To cite this version: Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque. In-Can vitrification of ash. IOP Conference Series: Materials Science and Engineering, 2020, 818 (1), pp.012005. ￿10.1088/1757- 899X/818/1/012005￿. ￿cea-03579858￿ In-Can vitrification of ash Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque To cite this version: Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque. In-Can vitrification of ash. IOP Conference Series: Materials Science and Engineering, 2020, 818 (1), pp.012005. ￿10.1088/1757- 899X/818/1/012005￿. ￿cea-03579858￿ In-Can vitrification of ash Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque To cite this version: Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque. In-Can vitrification of ash. IOP Conference Series: Materials Science and Engineering, 2020, 818 (1), pp.012005. ￿10.1088/1757- 899X/818/1/012005￿. ￿cea-03579858￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: cea-03579858 https://cea.hal.science/cea-03579858v1 Submitted on 18 Feb 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 IOP Publishing doi:10.1088/1757-899X/818/1/012005 THERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste P Conf. Series: Materials Science and Engineering 818 (2020) 012005 doi:10.1088/1757-899X/818/1/01 M Fournier, N Massoni and J F Hollebecque CEA, DEN, DE2D, Univ. Montpellier, Marcoule, F-30207 Bagnols-sur-Cèze M Fournier, N Massoni and J F Hollebecque CEA, DEN, DE2D, Univ. Montpellier, Marcoule, F-30207 Bagnols-sur-Cèze maxime.fournier@cea.fr Abstract. The In-Can Melter is a metallic crucible heated in a refractory furnace using electrical resistors allowing in-container vitrification. The In-Can Melter trial aims to demonstrate the feasibility of the confinement in a glassy matrix of ash coming from existing incineration processes. The ash was pelletised to allow its introduction into the can without dust emissions and then incorporated in a 50 wt.% waste loading confinement matrix. The full-scale trial was preceded by laboratory- and bench-scale tests. The microstructure and chemical durability of the wasteform were characterised. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. nder licence by IOP Publishing Ltd 1 1. Introduction The Thermal treatment for radioactive waste minimisation and hazard reduction (THERAMIN) project is a European Commission programme of work jointly funded by the Horizon 2020 Euratom research and innovation program and various European nuclear waste management organisations (WMOs). The THERAMIN project is being conducted between June 2017 and May 2020. Twelve European WMOs and research and consultancy institutions from seven European countries are participating in THERAMIN. The overall objective of THERAMIN is to provide improved, safe long-term storage and disposal of intermediate-level waste and low-level waste suitable for thermal processing. The work programme provides a vehicle for coordinated European Union-wide research and technology demonstrations designed to provide improved understanding and optimisation of the application of thermal treatment in radioactive waste management programmes across Europe, and moves technologies higher up the Technology Readiness Level (TRL) scale. g g p gy ( ) In the framework of the THERAMIN project, the French Alternative Energies and Atomic Energy Commission (CEA) carried out studies on the treatment and conditioning of ash resulting from the incineration of technological surrogate waste. The trial described in this article is based on the CEA In- Can Melter (ICM) consisting of a metallic crucible melter heated in a refractory furnace. Prior to the full-scale trial, laboratory- and bench-scale tests were conducted to select optimised operating conditions. Wasteforms produced were characterised after the trials. 2. Description of the CEA’s In-Can Melter p The ICM is a metallic crucible heated in a refractory furnace using electrical resistors (Figure 1). The can, with an approximate volume of 50 L, is renewed after each filling. The process can support either liquid or solid waste feeds. With the current off-gas treatment system (not including a post-combustion chamber), it can only tolerate small amounts of organics. It can also accept a small fraction of metal in the waste. The design ensures that the process can operate remotely for high-activity waste. The design can also be adapted for dealing with plutonium-containing material in gloveboxes. The end product can be glass, glass ceramic, or simply a high-density waste product. 1 THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Publishing IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 doi:10.1088/1757-899X/818/ IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 doi:10.1088/1757-899X/818/1/012005 Figure 1. Diagram of the CEA’s In-Can Melter. ICM has been developed for CEA needs since 2005 for military waste vitrification. The technology is at TRL 7 and the ICM has undergone inactive commissioning in France with inactive simulants of Can Solid feeding Dust scrubber Condenser Liquid buffer tank Scrubbing column Recycling Vertical sleeve Furnace Solid feeding Dust scrubber Vertical sleeve Liquid buffer tank Furnace Figure 1. Diagram of the CEA’s In-Can Melter. ICM has been developed for CEA needs since 2005 for military waste vitrification. The technology is at TRL 7 and the ICM has undergone inactive commissioning in France with inactive simulants of alpha effluent. The technology is currently being qualified for solid waste. ICM has been developed for CEA needs since 2005 for military waste vitrification. The technology is at TRL 7 and the ICM has undergone inactive commissioning in France with inactive simulants of alpha effluent. The technology is currently being qualified for solid waste. 3. Ash production The waste selected for this vitrification trial is the ash from multiple incineration tests of surrogate technological waste (polyvinyl chloride, latex, neoprene, polyethylene, cotton, etc.) produced by the CEA’s IRIS process (French acronym for Research facility for solid waste incineration). IRIS is a research facility for the incineration of solids developed to treat organic waste from gloveboxes in the nuclear industry, contaminated with alpha-bearing actinides and containing high quantities of chlorine (Figure 2). Figure 2. Diagram of the CEA’s IRIS process for the treatment of organic waste. Organic waste (4 to 7 kg·h -1) Pyrolyser Post-combustion (1100 °C) Ash Calciner Pitch Air Air Air Cooler High voltage Electrostatic filter HEPA filter Washing column Figure 2. Diagram of the CEA’s IRIS process for the treatment of organic waste. The robustness and efficiency of this process is based on a decoupling of (i) the step of elimination corrosive materials such as chlorine and (ii) the step of combustion of the organic waste. Figure 2 ows that the organic waste first goes into a pyrolysis step at a temperature of 500 °C to remove the st corrosive gaseous compounds and then into a calciner (900 °C) fed with oxygen to complete the mbustion while concentrating the contamination in the mineral ash. The relatively long residence time Organic waste (4 to 7 kg·h -1) Pyrolyser Post-combustion (1100 °C) Ash Calciner Pitch Air Air Air Cooler High voltage Electrostatic filter HEPA filter Washing column Organic waste (4 to 7 kg·h -1) Air Pitch Pyrolyser Post-combustion (1100 °C) Ash Calciner Figure 2. Diagram of the CEA’s IRIS process for the treatment of organic waste. The robustness and efficiency of this process is based on a decoupling of (i) the step of elimination of corrosive materials such as chlorine and (ii) the step of combustion of the organic waste. Figure 2 shows that the organic waste first goes into a pyrolysis step at a temperature of 500 °C to remove the most corrosive gaseous compounds and then into a calciner (900 °C) fed with oxygen to complete the combustion while concentrating the contamination in the mineral ash. The relatively long residence time The robustness and efficiency of this process is based on a decoupling of (i) the step of elimination of corrosive materials such as chlorine and (ii) the step of combustion of the organic waste. 4. Pre-treatment of ash and definition of their vitrification conditions In order to obtain a sufficient amount of ash to carry out the various tests described thereafter, 230 batches, each containing approximately 220 g of ash from various IRIS trials, were homogenised using a Morton Mixers ribbon blender (Figure 3.a-b). This operation resulted in a decrease in the size of the ash agglomerates, with an ash apparent density of 0.6 g·cm-3, compared to 0.2 g·cm-3 before homogenisation, and a true density of 1.9 g·cm-3 measured by helium pycnometry. The composition of the ash thus mixed (Table 1) was analysed by X-ray fluorescence spectroscopy (XRF). Ash is characterised by a high volatility that can lead to clogging of the feeding and off-gas treatment system pipes and large dust carry-over. In order to limit this volatility, a temporary densification by pelletisation was carried out using a Frogerais rotative press. The ash was pressed in the presence of a binder to ensure the mechanical cohesion of the pellets, but without glass frit, being too abrasive for the press. Two types of binder were tested: organic (sorbitol, C6H14O6, or fructose, C6H12O6) and mineral (bentonite, mainly containing montmorillonite (Na,Ca)0.33(Al,Mg)2Si4O10(OH)2 (H2O)n). The addition of 10 wt.% of fructose or bentonite to the ash gave similar and satisfactory results. Bentonite was preferred thereafter to avoid possible COx gas emissions during combustion. Approximately 60,000 pellets (9 kg of ash), reaching a 72% densification, were produced (Figure 3.c). Figure 3. Ash in the mixer (a) before and (b) after homogenisation. (c) Ash pellets. In order to determine the vitrification conditions of the ash, laboratory-scale tests, using small quantities of materials (≈10 g), were carried out with different amounts of ash and glass frit at 1100 °C for 2 h. In the approach proposed in the THERAMIN project, no vitreous matrix formulation study was carried out and FN0C77 glass frit (Table 1), simple and available, was used. At the end of the tests, the crucibles were cut and the materials obtained were observed with a binocular magnifier and evaluated according to two criteria: (i) obtention of a macroscopically homogeneous material and (ii) the absence of foaming during melting. The best results were obtained for a 50:50 mixture between ash and glass frit. Beyond this ratio, the reactivity between ash and glass frit is less efficient and the resulting material becomes less homogeneous. (a) (b) 5 mm (c) (a) 5 mm (c) Figure 3. 4. Pre-treatment of ash and definition of their vitrification conditions Ash in the mixer (a) before and (b) after homogenisation. (c) Ash pellets. (a) (b) 5 mm (c) (b) (a) Figure 3. Ash in the mixer (a) before and (b) after homogenisation. (c) Ash pellets. In order to determine the vitrification conditions of the ash, laboratory-scale tests, using small quantities of materials (≈10 g), were carried out with different amounts of ash and glass frit at 1100 °C for 2 h. In the approach proposed in the THERAMIN project, no vitreous matrix formulation study was carried out and FN0C77 glass frit (Table 1), simple and available, was used. At the end of the tests, the crucibles were cut and the materials obtained were observed with a binocular magnifier and evaluated according to two criteria: (i) obtention of a macroscopically homogeneous material and (ii) the absence of foaming during melting. The best results were obtained for a 50:50 mixture between ash and glass frit. Beyond this ratio, the reactivity between ash and glass frit is less efficient and the resulting material becomes less homogeneous. 3. Ash production Figure 2 shows that the organic waste first goes into a pyrolysis step at a temperature of 500 °C to remove the most corrosive gaseous compounds and then into a calciner (900 °C) fed with oxygen to complete the combustion while concentrating the contamination in the mineral ash. The relatively long residence time 2 HERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 g doi:10.1088/1757-899X/818/1/012005 in furnaces with low gas flow rates makes it possible to produce carbon-free ash, concentrating almost all of the initial activity. The off-gas treatment system, consisting of a post-combustion chamber followed by electrostatic filtration, ensures excellent purification. in furnaces with low gas flow rates makes it possible to produce carbon-free ash, concentrating almost all of the initial activity. The off-gas treatment system, consisting of a post-combustion chamber followed by electrostatic filtration, ensures excellent purification. 5. Bench-scale test and characterisation of the wasteform 5.1. Test conditions Following the laboratory-scale tests, the quantity of treated ash was increased during a bench-scale test. This test was carried out in an Inconel™ 601 crucible having an external diameter of 100 mm and a thickness of 5 mm, filled with 400 g of unpelletised ash and 400 g of FN0C77 glass frit (Figure 4.a). 5.1. Test conditions 3 THERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering (a) IOP Publishing (b) IOP Conf. Series: Materials Science and Engineering (a) g (b) P Conf. Series: Materials Science and Engineering (a) (b) The tapped density of this mixture was estimated at approximately 1 g·cm-3. The mixture was heated at 300 °C·h-1 and maintained at 1100 °C for 8 h. The tapped density of this mixture was estimated at approximately 1 g·cm-3. The mixture was heated at 300 °C·h-1 and maintained at 1100 °C for 8 h. At the end of the test (Figure 4.b), the crucible was cut and the resistance of Inconel™ 601 to corrosion was evaluated. No significant corrosion is seen at the interface between Inconel™ and the wasteform. Above the volume occupied by the wasteform, gaseous species superficially corroded the Inconel™ (Figure 4.c). The crystallised corrosion products are NiFe1.2Cr0.8O4 spinels and nickel oxide. However, no significant reduction in the thickness of the crucible is measured. A mass loss of 2.5 wt.% was measured after the test and a crystallised glass of density 2.63 g·cm-3 was obtained (Figure 4.d). (d) (c) Figure 4. (a) Ash and glass frit mixture introduced into the Inconel™ 601 crucible, (b) crucible cut after the test, (c) inner surface of the crucible after the test and (d) wasteform produced. (a) (b) (d) (c) Concrete cast to facilitate cutting Wasteform (c) (b) Concrete cast to facilitate cutting Wasteform (a) (c) (d) (a) (d) Figure 4. (a) Ash and glass frit mixture introduced into the Inconel™ 601 crucible, (b) crucible cut after the test, (c) inner surface of the crucible after the test and (d) wasteform produced. 5.2. Microstructure and chemical composition of the wasteform 5.2. Microstructure and chemical composition of the wasteform The material is mainly composed of a vitreous matrix consisting of approximately 80 wt.% of Al2O3, B2O3, CaO, Na2O, and SiO2. This matrix includes three types of crystallisation identified by scanning electron microscopy (SEM) and X-ray diffraction (XRD): apatites, zincochromites, and beads composed almost exclusively of bismuth (Figure 5 and Table 1). The stoichiometries of apatites and zincochromites evaluated by energy dispersive X-ray spectroscopy (EDS), respectively Ca4.8P2.9Si0.3Na0.2Al0.1O12.9 and ZnCr2.6Na0.5Mg0.2Al0.1Fe0.1Ni0.1O5.8 (oxygen assumed for stoichiometry), are consistent with the theoretical stoichiometries of these minerals, respectively Ca5(PO4)3(OH) and ZnCr2O4. 5.1. Test conditions The crystals are homogeneously distributed in the matrix, with the exception of a layer approximately one millimetre thick at the interface with the crucible in which they are more numerous. Figure 5. (a) SEM and (b) XRD analysis of the wasteform produced at the bench-scale. (a)     Vitreous matrix 10 µm 10 30 50 70 Counts (a.u.) 2θ (degrees)                 (b)  Apatites  Zincochromites  Bismuth beads Epoxy resin (a)     Vitreous matrix 10 µm Epoxy resin 10 30 50 70 Counts (a.u.) 2θ (degrees)                 (b)  Apatites  Zincochromites  Bismuth beads 70 2θ (degrees) (b) Figure 5. (a) SEM and (b) XRD analysis of the wasteform produced at the bench-scale. THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 IOP Publishing doi:10.1088/1757-899X/818/1/012005 HERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 IOP Publishing doi:10.1088/1757-899X/818/1/012005 P Conf. Series: Materials Science and Engineering 818 (2020) 012005 doi:10.1088/1757-899X/818/1/0 Table 1. Elemental compositions, expressed in wt.%, of ash and glass frit used for the calculation of the theoretical composition of the wasteform. Analysed compositions of the vitreous and crystalline phases composing the wasteform. Element Ash (analysed by XRF) FN0C77 glass frit (specified) Wasteform (calculated) Vitreous matrix  (analysed by EDS) Apatites  (analysed by EDS) Zincochromites  (analysed by EDS) Bismuth beads  (analysed by EDS) Al 11.90 5.95 9.40 0.06 0.15 B 10.05 5.02 n.a.a n.a.a n.a.a n.a.a Ba 0.71 0.36 Bi 4.02 2.01 0.17 9.99 Ca 9.91 4.96 7.10 4.84 0.01 Cl 1.42 0.71 0.08 Cr 0.12 0.06 2.58 Fe 0.50 0.25 0.61 0.05 K 2.25 1.13 1.56 Mg 1.59 0.80 3.00 0.24 Na 0.55 23.74 12.14 13.71 0.16 0.48 Ni 0.69 0.35 0.05 0.17 P 1.62 0.81 0.62 2.88 S 0.31 0.16 0.32 Sb 0.15 0.08 Si 7.45 16.57 12.01 27.71 0.25 0.02 Ti 0.45 0.23 1.04 0.03 Zn 8.13 4.07 5.74 1.00 0.25 a n.a.: not analysed because boron is not detected by EDS. 5.3. Chemical durability On the first day of leaching, the alteration rate of the vitreous matrix is ≥ 2.4 g·m-2·d-1. After 3 days, the rate is ≈1.5 g·m-2·d-1. The alteration therefore decreases over time and quickly becomes non- stoichiometric, showing the typical behaviour of a borosilicate glass of nuclear interest. Si and Al are retained at 30–40% in the alteration layer after one month. Such retention remains relatively low: the amorphous layer formed on the sample surface therefore remains poor in silicon, which explains why the hydrolysis regime remains predominant. The initial dissolution rate of the vitreous matrix leads to a rapid increase in pH90°C to 8.5, the value at which this parameter stabilises. Figure 6. (a) Evolution of NL and pH during wasteform leaching (error bars represent the data dispersion between the duplicates). (b) Compared evolution of NL and pH during the leaching of ISG and In-Can-produced wasteform. The arrows are a visual guide to estimate the drop in alteration rate. 3 4 5 6 7 8 9 0 2 4 6 8 10 12 14 0 10 20 30 pH NLi (g·m-2) time (days) B-ICM B-ISG Si-ICM Si-ISG pH-ICM pH-ISG 6.0 6.5 7.0 7.5 8.0 8.5 0 2 4 6 8 10 12 0 10 20 30 pH NLi (g·m-2) time (days) Al B Na Si pH (a) (b) 6.0 6.5 7.0 7.5 8.0 8.5 0 2 4 6 8 10 12 0 10 20 30 pH NLi (g·m-2) time (days) 3 4 5 6 7 8 9 0 2 4 6 8 10 12 14 0 10 20 30 pH NLi (g·m-2) time (days) (b) (a) Figure 6. (a) Evolution of NL and pH during wasteform leaching (error bars represent the data dispersion between the duplicates). (b) Compared evolution of NL and pH during the leaching of ISG and In-Can-produced wasteform. The arrows are a visual guide to estimate the drop in alteration rate. Comparison of previous results with those acquired for the reference International Simple Glass (ISG) [4, 5] altered under the same conditions (Figure 6.b) shows similar and ‘classical’ trends. Comparison of previous results with those acquired for the reference International Simple Glass (ISG) [4, 5] altered under the same conditions (Figure 6.b) shows similar and ‘classical’ trends.  Because of its composition, the In-Can-produced vitreous matrix has a higher rate of hydrolysis. 5.3. Chemical durability y The chemical durability of the material was studied according to a protocol adapted from the PCT-B standardised test [1]. After crushing the material, 125–250 μm of powder were placed in contact with pure water with a glass-surface-area-to-solution-volume ratio (S/V) of 10 m-1 at 90±2 °C in unstirred perfluoroalkoxy (PFA) reactors. The powder specific surface area of 259 g·cm-2 was estimated by adsorption of krypton on the sample surface (Micromeritics ASAP 2020) according to the Brunauer– Emmett–Teller theory [2]. Solution samples, taken at regular intervals, were filtered with a cut-off of 0.45 μm, acidified with ultrapure grade HNO3 and analysed by inductively coupled plasma-atomic emission spectroscopy (Thermo Scientific iCAP™ 6000 Series). The concentrations were used to calculate normalised mass losses   NL , i i i C x S V   where Ci is the concentration of the element i and xi the mass percentage of i in the glass. The alteration rate, dNL d ,  i r t was calculated by linear regression. Boron is known to be an alteration tracer of the vitreous matrix, which means that it is not retained in alteration products while released from the glass. The evolution of the normalised mass losses of Al, B, Na, and Si is shown in Figure 6.a. Only the elements mainly integrated into the vitreous matrix are considered. The xi considered are those obtained by EDS analysis for Na, Si, Al, and by calculation for B. Therefore, the dissolution rates of the crystalline phases are not considered, because when a wasteform contains crystalline phases deemed to be durable, 5 THERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 IOP Publishing doi:10.1088/1757-899X/818/1/012005 THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 IOP Publishing doi:10.1088/1757-899X/818/1/012005 P Conf. Series: Materials Science and Engineering 818 (2020) 012005 doi:10.1088/1757-899X/818/1/01 like apatites or spinels, the wasteform dissolution is controlled by the properties of the vitreous matrix [3]. This leaching test was duplicated, giving similar results: the average values obtained are discussed hereafter. like apatites or spinels, the wasteform dissolution is controlled by the properties of the vitreous matrix [3]. This leaching test was duplicated, giving similar results: the average values obtained are discussed hereafter. 6. Full-scale trial A full-scale trial was conducted with the CEA’s ICM (Figure 1). The can is made of Inconel™ 601, with an external diameter of 400 mm, a height of 600 mm and a wall thickness of 10 mm. The trial began with pre-loading of the container with 25 kg of FN0C77 glass frit and 17.8 kg of unpelletised ash. This mixture was heated at 300 °C·h-1 up to 1100 °C. Then, 9.2 kg of pelletised ash were fed into the can with a flow of 10 kg·h-1, before the introduction of 1 kg of FN0C77 frit and an additional soaking of 2 h. Thus, the final waste loading was 50 wt.%. The test was continued with 2 h of recycling of the dust recovered in the off-gas treatment system and 2 h of further soaking. The can was then allowed to cool freely. A loss of mass of 2.3 wt.% was measured, consistent with that of the test carried out at bench-scale. The extent of characterisation of the material produced during this trial is more limited than that of the material produced at bench-scale. However, the SEM and XRD characterisations carried out on a sample taken in the central zone of the can show a similar microstructure (Figure 7). The same bismuth beads, apatite, and zincochromite crystals are included in a vitreous matrix. Figure 7. (a) SEM image and (b) XRD analysis of the wasteform produced using the ICM. The XRD results are compared to those of the bench-scale test. 10 30 50 70 Counts (a.u.) 2θ (degrees) Bench-scale test ICM                 (b) (a)    Vitreous matrix   Apatites  Zincochromites  Bismuth beads 10 µm (a)    Vitreous matrix  10 µm 10 30 50 70 Counts (a.u.) 2θ (degrees) Bench-scale test ICM                 (b)  Apatites  Zincochromites  Bismuth beads (a) (b) Figure 7. (a) SEM image and (b) XRD analysis of the wasteform produced using the ICM. The XRD results are compared to those of the bench-scale test. 5.3. Chemical durability Indeed, it contains large fractions of Na2O and B2O3, which are unfavourable to the glass durability. Therefore, in the early stages of dissolution, the evolutions of the concentrations in solution are faster for In-Can-produced wasteform. This conclusion is also valid for the pH evolution.  The growth of the alteration layer is faster for the In-Can-produced wasteform. Thus, its alteration rate drops faster. Between 14 and 28 days, the alteration rate of the In-Can-produced vitreous matrix (0.07 g·m-2·d-1) is almost 1.5 time lower than that of the ISG (0.10 g·m-2·d-1), while the hydrolysis rate of the vitreous phase of the wasteform after 1 day is higher. This result is known for simple glasses [6]: the glasses that are hydrolysed the fastest are also those that have the earliest rate drops. The data trends tend to show that, in the longer term, an alteration layer will form on the In-Can- produced wasteform, causing a decrease of the alteration rate, as for the ISG. 6 THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 IOP Publishing doi:10.1088/1757-899X/818/1/012005 P Conf. Series: Materials Science and Engineering 818 (2020) 012005 doi:10.1088/1757-899X/818/1/01 Acknowledgements This project has received funding from the Euratom research and training programme 2014–2018 under grant agreement No 755480. This paper reflects only the authors’ views, and the European Commission is not responsible for any use that may be made of it. p y y The authors are grateful to Alain Artico, Carine Castaño, and Virginie Lemaitre for carrying out the tests, Thierry Blisson, Florian Emanuel, and Valérie Debono for the wasteform characterisation, and PRIME Verre Company for its technical assistance. 7. Conclusions h l The In-Can Melter trial conducted in the framework of the THERAMIN project demonstrated a successful process for the vitrification of ash generated from the incineration of organic waste from glove boxes in the nuclear industry. This study presents — in a simplified version — the scale up methodology used to demonstrate the feasibility of the thermal treatment of a given waste by a given process. Thus, the laboratory-scale tests assess the waste loading, operating temperature, mixture strategy and melted material quality. Next comes the bench-scale test focusing on crucible corrosion, volatility of some species and wasteform microstructure. Finally, the study concludes with a pilot-scale trial whose main outputs are overall corrosion and kinetic data, process performance, process and package descriptions. p g p A good waste loading of 50 wt.% was achieved in this first approach. The trial also made it possible to begin the technical investigation required for the processing of powdery solids into the can while avoiding dust emission: a temporary densification by pelletising was implemented. The produced wasteform consists of a crystallised glass mainly composed of SiO2, Na2O, B2O3, Al2O3, and CaO. The term ‘crystallised glass’ refers to a vitreous matrix including crystals of apatite, zincochromite, and bismuth alloy. The crystals are distributed homogeneously in the wasteform. The crystalline phases being durable, the durability of the wasteform is controlled by that of the vitreous matrix. The hydrolysis rate of this glass is relatively high because of its high content of B2O3 and Na2O. Indeed, in the approach proposed in the framework of the THERAMIN project, no optimisation study of the glass additives was conducted. However, the ‘classical’ trends observed suggest that, in the long term, an alteration layer will form, leading to a decrease in the alteration rate.   THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005 IOP Publishing doi:10.1088/1757-899X/818/1/012005 THERAMIN 2020 conference: thermal treatment of radioactive waste References [1] ASTM International 2014 Standard C1285-14. Standard Test Method for Setermining Chemical Durability of Nuclear, Hazardeous, and Mixed Waste Glasses and Multiphase Glass Ceramics: The Product Consistency Test (PCT) y ( ) [2] Brunauer S, Emmett P H and Teller E 1938 Adsorption of gases in multimolecular layers J. Am. Chem. Soc. 60 309–19 [3] Nicoleau E, Angeli F, Schuller S, Charpentier T, Jollivet P and Moskura M 2016 Rare-earth silicate crystallization in borosilicate glasses: Effect on structural and chemical durability properties J. Non-Cryst. Solids 438 37–48 [4] Gin S, Abdelouas A, Criscenti L J, Ebert W L, Ferrand K, Geisler T, Harrison M T, Inagaki Y, Mitsui S, Mueller K T, Marra J C, Pantano C G, Pierce E M, Ryan J V, Schofield J M, Steefel C I and Vienna J D 2013 An international initiative on long-term behavior of high-level nuclear waste glass Mater. Today 16 243–8 [5] Kaspar T C, Ryan J V, Pantano C G, Rice J, Trivelpiece C, Hyatt N C, Corkhill C L, Mann C, Hand R J, Kirkham M A, Crawford C L, Jantzen C M, Du J, Lu X, Harrison M T, Cushman C, Linford M R and Smith N J 2019 Physical and optical properties of the International Simple Glass npj Mater. Degrad. 3 15 pj g [6] Gin S, Beaudoux X, Angéli F, Jégou C and Godon N 2012 Effect of composition on the short- term and long-term dissolution rates of ten borosilicate glasses of increasing complexity from 3 to 30 oxides J. Non-Cryst. Solids 358 2559–70 8 8
W3135309452.txt
https://www.mdpi.com/2227-9091/9/3/49/pdf?version=1615275615
en
Alleviating Class Imbalance in Actuarial Applications Using Generative Adversarial Networks
Risks
2,021
cc-by
17,508
risks Article Alleviating Class Imbalance in Actuarial Applications Using Generative Adversarial Networks Kwanda Sydwell Ngwenduna 1,2, * 1 2 3 *   Citation: Ngwenduna, Kwanda and Rendani Mbuvha 3 School of Computer Science and Applied Mathematics, University of the Witwatersrand, West Campus, Mathematical Sciences Building, Private Bag 3, Wits, Braamfontein 2050, South Africa DSI-NICIS National e-Science Postgraduate Teaching and Training Platform (NEPTTP), Wits, Braamfontein 2050, South Africa School of Statistics and Actuarial Science, University of the Witwatersrand, West Campus, Mathematical Sciences Building, Private Bag 3, Wits, Braamfontein 2050, South Africa; rendani.mbuvha@wits.ac.za Correspondence: sngwenduna@gmail.com Abstract: To build adequate predictive models, a substantial amount of data is desirable. However, when expanding to new or unexplored territories, this required level of information is rarely always available. To build such models, actuaries often have to: procure data from local providers, use limited unsuitable industry and public research, or rely on extrapolations from other better-known markets. Another common pathology when applying machine learning techniques in actuarial domains is the prevalence of imbalanced classes where risk events of interest, such as mortality and fraud, are under-represented in data. In this work, we show how an implicit model using the Generative Adversarial Network (GAN) can alleviate these problems through the generation of adequate quality data from very limited or highly imbalanced samples. We provide an introduction to GANs and how they are used to synthesize data that accurately enhance the data resolution of very infrequent events and improve model robustness. Overall, we show a significant superiority of GANs for boosting predictive models when compared to competing approaches on benchmark data sets. This work offers numerous of contributions to actuaries with applications to inter alia new sample creation, data augmentation, boosting predictive models, anomaly detection, and missing data imputation. Sydwell, and Rendani Mbuvha. 2021. Alleviating Class Imbalance in Actuarial Applications Using Keywords: actuarial science; class imbalance; data augmentation; generative models; generative adversarial network; synthetic sampling; SMOTE Generative Adversarial Networks. Risks 9: 49. https://doi.org/10.3390/ risks9030049 Received: 19 October 2020 Accepted: 21 November 2020 Published: 8 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction 1.1. Background Gaining an advantage in competitive markets through offerings of suitable tailored products on customers relies on building and maintaining adequate predictive models. To build these models, a substantial amount of data and a sizeable number of records is desirable. However, when expanding to new or unexplored markets, that level of information is rarely always available. To build such models, actuarial firms often have to procure data from local providers, use limited unsuitable industry and public research or rely from extrapolations from other better known markets. In this work, we show how an implicit model using the Generative Adversarial Network (GAN) Goodfellow et al. (2014) can alleviate this problem through the generation of adequate quality data even from very limited small samples, from difficult domains, or without alignment, thus handling class imbalance. A GAN is an example of a generative model that is used to create new samples from a latent noise space. A generative model describes how a data set is generated in terms of a probabilistic model. This generative model pmodel mimics the training data distribution pdata as close as possible. If this is achieved, then we can sample from pmodel to generate realistic samples that appear to have been drawn from pdata . We are satisfied if Risks 2021, 9, 49. https://doi.org/10.3390/risks9030049 https://www.mdpi.com/journal/risks Risks 2021, 9, 49 2 of 33 our model can also generate diverse samples that are suitable different from the training data. In some cases, the model can be estimated explicitly, and sometimes it can generate samples implicitly. Other models are capable of doing both. GANs were proposed in a seminal paper by Goodfellow et al. (2014). GANs are useful for learning the structure of the data and can generate new samples without explicitly postulating the model Goodfellow et al. (2014). They are known to be better than other generative models due to the quality of samples they generate. GANs have been highly successful in computer vision Brock et al. (2018); Karras et al. (2019); Zhu et al. (2017); Vondrick et al. (2016), music generation Yang et al. (2017), text generation Reed et al. (2016), missing data imputation Li et al. (2019); Shang et al. (2017); Yoon et al. (2018), time series generation Esteban et al. (2017); Yoon et al. (2019); Fu et al. (2019), and data augmentation Antoniou et al. (2017); Fiore et al. (2019); Mariani et al. (2018); Mottini et al. (2018); Park et al. (2018), with remarkable results, but their application to the actuarial discipline remains largely still unexplored. In this work, we discuss how and where GANs can be used by actuaries. Additionally, we provide an experiment showing how GANs can be used to boost imbalanced samples in actuarial data sets and improve actuarial models. 1.2. Aims and Objectives In this paper, we explain what GANs are and how they can be used to synthesize data in order to accurately enhance very infrequent events, alleviate class imbalance and create better prediction models. This work also provides theoretical and practical applications of GANs. We demonstrate a popular GAN architecture to a typical problem resembling an actuarial use on benchmark data sets using Python Python Software Foundation (2017). Overall, we show a significant superiority of GANs for predictive models and stochastic simulations compared to current approaches. Specifically, this paper covers the following aims and objectives: • • • deep overview of generative models and why GANs are of better quality than other generative models; an overview of GANs with practical applications in a number of areas with emphasis for actuarial use; and provide a practical example of a popular GAN use for alleviating class imbalance, data augmentation, and improving predictive models. 1.3. Contribution This work provides thorough theoretical, empirical and practical applications of GANs, with possible leverage in actuarial science for inter alia new sample creation, data augmentation, boosting actuarial models, anomaly detection, missing data imputation, time series simulations and projections in life insurance, short-term insurance, health and care, banking, investment, enterprise risk management, and other non-traditional actuarial areas, such as telecommunications, economics, medicine, engineering, and other wider fields. For example, actuaries build pricing models in order to determine competitive premiums that customers should pay to be provided adequate insurance coverage O’Malley et al. (2005). These pricing models are dependant on risk events, such as mortality, morbidity, and lapse, which need to be estimated using an adequate and accurate model. However, these risk events are often under-represented in data. In this work, we show how a GAN could be used to alleviate this problem through the generation of adequate quality data from limited or highly imbalanced samples. Essentially, we show that synthetic data generated using GANs can augment imbalanced data sets, leading to significantly higher predictive power of possible actuarial models fitted after. Risks 2021, 9, 49 3 of 33 1.4. Structure of the Paper The rest of the paper is organized as follows. Section 2 describes the problem of class imbalance and its common solutions. Section 3 reviews the literature on generative models, with particular emphasis on GANs, while Section 4 covers GAN applications, especially for actuarial adoption. Section 5 describes the methodology followed. Section 6 outlines the example experiments conducted. Section 7 presents the results, and Section 8 discusses the results, while Section 9 gives conclusions, limitations, and possible future work. 2. Class Imbalance 2.1. Definition Whilst machine learning (ML) has gained significant prevalence in the past few decades, class imbalance, limited data sets, and missing data remain pervasive problems Chawla et al. (2002); Fernández et al. (2018); Longadge and Dongre (2013). These issues occur due to the nature of the data space, data collection costs, data limitations, new markets, and absolute rarity. In binary classification problems, class imbalance occurs when one of the classes has overwhelmingly more instances than others. ML classifiers tend to have skewed accuracy towards the majority class when the data is imbalanced Chawla (2009); Fernández et al. (2018). This is problematic as misclassifying a minority class can result in significant misclassification costs than for the majority case Chawla et al. (2002). Class imbalance arises because ML classifiers do not necessarily take into account unequal class distributions. This problem causes a significant and an unexpected performance behavior for most classifiers. 2.2. Techniques to Alleviate Class Imbalance Techniques as shown in Figure 1 exist to alleviate class imbalance, and these techniques include re-sampling, algorithmic-level solutions, cost-sensitive learning, ensembles, and generative models. Figure 1. Taxonomy of solutions to tackle class imbalance. 2.2.1. Re-Sampling Re-sampling techniques modify the training data such that the distribution of the classes is evenly balanced where the majority or minority class is either under-sampled or over-sampled. Over-sampling has been the most frequently used technique than undersampling since under-sampling eliminates important information in the majority class. Risks 2021, 9, 49 4 of 33 Hybrid sampling techniques combine over-sampling with data cleaning techniques, informed under-sampling techniques, or greedy-filtering approaches Batista et al. (2004), thereby eliminating redundant and noisy instances, boosting the predictive accuracy of models trained after. 2.2.2. Synthetic Sampling A pioneering and popular method to alleviate class imbalance has been Synthetic Minority Over-sampling TEchnique (SMOTE) Chawla et al. (2002). However, SMOTE suffers from over-fitting, over-lapping classes, noisy examples, is less reliant on the true probability distribution, and alters the original distribution of the minority classes, and this may not be desirable Batista et al. (2004); Ganganwar (2012); Longadge and Dongre (2013). There have been few empirical reviews which compare and synthesize SMOTE and its density-based variants Gao et al. (2014). There have been few approaches which create synthetic samples by sampling implicitly from the minority class distribution. Current density-based approaches may be subjective as they need to pre-specify the format and structure of the minority class distribution Das et al. (2015); Zhang and Li (2014). Generative models offer a significant alternative, yet these models have not been thoroughly explored in imbalanced learning. In this work, we show how a popular implicit generative model can be used to handle class imbalance and rival SMOTE. 2.2.3. Ensembles Ensemble is where a classifier’s accuracy is increased by the use of training on different over-sampled data sets or different algorithms and combining outputs to a single outcome. These approaches tend to improve the results of re-sampling techniques Wang and Yao (2009); Chawla et al. (2003). However, they can take a long time to compute and still do not solve the true data distribution issue. 2.2.4. Other Methods Algorithmic-level solutions modify the ML classifier to adjust for the presence of class imbalance in the data. Cost-sensitive learning incorporates mis-classification costs in the evaluation metric Ganganwar (2012). This approach is more computationally efficient than data-level solutions He and Garcia (2008). However, mis-classification costs are often unknown and difficult to set, making this method less popular than sampling techniques López et al. (2013). SMOTE and its variants remain the most studied and widely used solutions, with generative models slowly being adopted in alleviating class imbalance Fernández et al. (2018); Fiore et al. (2019); Gao et al. (2014). Generative models are described in detail in Section 3. 3. Generative Models This research is concerned with handling class imbalance through generative modeling. Other approaches exist, such as synthetic sampling; however, these approaches do not take into account the underlying structure of the data distribution and often lead to over-fitting and over-lapping cases Gao et al. (2014). Generative models are flexible models capable of learning the data distribution and sampling from this data distribution, thereby creating new synthetic cases. In this section, we review generative models and explain why GANs are of better quality than other deep generative models. 3.1. Definition Given a data set with observations X, we assume that X has been generated from an unknown true probability distribution pdata . A generative model pmodel mimics pdata as close as possible. If this is achieved, then we can sample from pmodel to generate realistic samples that appear to have been drawn from pdata . We are satisfied if our model can also generate diverse samples that are suitable different from X. In some cases, the model can be estimated explicitly and sometimes it Risks 2021, 9, 49 5 of 33 can generate samples implicitly. Other models are capable of doing both. GANs provide no estimate of the model but are capable of generating new data without knowing it. Goodfellow (2016) provides a taxonomy of common deep generative models shown in Table 1, divided into implicit and explicit models. GANs are designed to remedy most of the disadvantages that come with explicit models and other Markov chain models. Table 1. Taxonomy of generative models. Approximate Explicit Density Tractable Implicit Density Variational Inference Variational Autoencoder Markov chain Deep Belief Network Restricted Boltzmann Machine Full Visible Belief Net NADE MADE PixelRNN/CNN Change of variable models Nonlinear ICA Minimax GAN Generative Adversarial Network Non-saturating GAN GAN variants Direct Markov Generative Moment Matching Network GMMN Generative Stochastic Network GSN 3.2. Explicit Models Explicit models specify or approximate a parameterized log-likelihood representation of the data Goodfellow et al. (2014). Parameters are then estimated and learned from the data and this requires a maximum likelihood estimation which integrates over the entire data space, and this may be intractable Li et al. (2015). These approximation techniques may not always yield the best results as some of them rely on Markov chains, which are time-consuming Goodfellow et al. (2014). Two popular tractable models are fully visible belief networks (FVBNs) Frey et al. (1996) and nonlinear independent component analysis (ICA). Approximate methods improve on the design of tractable models which can be computational intensive and limited Goodfellow et al. (2014); Makhzani et al. (2015); Rezende et al. (2014). Approximate methods use either deterministic, i.e., variational inference, or stochastic approximations such as Markov chain Monte Carlo (MCMC) Geyer (1992). Variational inference involves the use of Variational Autoencoders (VAEs) Kingma and Welling (2013); Rezende et al. (2014) to approximate pmodel ( x ) using lower bounds. 3.2.1. FVBNs FVBN estimates the probability density of the training data pmodel ( x ) into a decomposed product of one-dimensional probability distributions. This model outputs a probability for each possible value if x is discrete and outputs a network of parameters of a simple distribution if x is continuous. Using the generated model, sampling is done one step at a time, conditioned on all previous steps Goodfellow et al. (2014). The problem with these models is their computational complexities as they need to generate one point at a time. Other problems include poor learning representations, over-emphasizing details over global data, and not closely reflecting the true generation process Goodfellow (2016). Moreover, these models have been more useful for image synthesis than structured data sets, such as tabular data Van den Oord et al. (2016). GANs are known to provide new samples in parallel, thus yielding greater speed of generation Goodfellow (2016); Li et al. (2015). Risks 2021, 9, 49 6 of 33 3.2.2. Non-Linear ICA Non-linear ICA involves defining some continuous non-linear transformations of data between high dimensions and lower dimensional spaces. The distribution of the data pmodel is transformed into a distribution of a latent space z defined by pz ( g), where g is some tractable transformed version of pz . The challenge in ICA is finding tractable distributions in the latent space, and these are limited Goodfellow et al. (2016). GANs are known to have fewer restrictions than these models Goodfellow et al. (2014); Bengio et al. (2014); Goodfellow (2016). 3.2.3. Variational Autoencoders VAEs, along with FVBNs and GANs, are three of the most popular approaches for sample generation. VAEs are an extension to AEs Bellinger et al. (2016); Larsen et al. (2015); Rezende et al. (2014). AE learns useful representations of the data by encoding X into a compressed latent space z using q(z| x ) and then decoding z back into X using p( x |z) by minimizing the reconstruction error between the original data and the deconstructed data Bellinger et al. (2016). VAE maximizes the following function:   log p( x ) ≥ Ez∼q(z| x) log p( x |z) + log p(z) − log q(z) . (1) Unlike auto-regressive models, VAEs are normally easy to run in parallel during training and inference Goodfellow et al. (2016); Larsen et al. (2015); Rezende et al. (2014). Conversely, they are normally harder to optimize than auto-regressive models Goodfellow et al. (2016); Makhzani et al. (2015). The encoder converts the input to latent space representations through the mean and variance, and samples can be created from the learned representation. VAEs have been criticized to be generating blurry samples and are intractable Goodfellow et al. (2016); Salimans et al. (2016). 3.2.4. Boltzmann Machines Boltzmann machines rely on the use of Markov chains to model pmodel ( x ) and to sample from it Ackley et al. (1985); Hinton (2002); Salakhutdinov and Hinton (2009). A Markov chain is a process that is used to generate samples by repeatedly drawing a sample from a transition operator Geyer (1992). A Boltzmann machine is an energy-based function defined as:  pmodel ( x ) = exp − E( x ) /Z, (2) where E( x ) is an energy function, and Z is a normalizing factor to ensure that pmodel ( x ) sums to one Ackley et al. (1985); Goodfellow et al. (2016). These methods include restricted boltzmann machine (RBM) Ackley et al. (1985) and deep belief networks (DBNs) Hinton et al. (2006); Hinton and Salakhutdinov (2006). DBNs and RBMs are generative stochastic neural networks that can estimate a probability distribution Ackley et al. (1985). Samples are obtained through MCMC runs to convergence, and this can be very expensive to run Li et al. (2015). These models were pioneers in early 2006 for deep generative models, but they have been rarely used because of poor scale-ability for higher dimension problems and high computational costs Goodfellow et al. (2016). 3.3. Implicit Models Implicit models learn to model the true distribution and define a stochastic procedure to directly generate new data from a latent space. These models can be trained indirectly without needing an explicit density function to be learned or defined. Some of these models, such as the Generative Stochastic Network (GSN) Bengio et al. (2014), involve MCMC methods, which impose greater computational cost and often fail to scale to higher dimensional spaces Goodfellow et al. (2016). GANs Goodfellow et al. (2014) and Generative Moment Matching Networks (GMMNs) Li et al. (2015) are one of the few implicit probabilistic models capable of sampling in parallel and in a single step. Risks 2021, 9, 49 7 of 33 GANs were designed to remedy most of the issues with explicit and some implicit models. GANs are known to parallelize sample generation, have fewer restrictions, subjectively thought to produce better samples, make no use of Markov chain or maximum likelihood estimation, and assume no variational bounds and no distributional assumptions Arjovsky et al. (2017); Goodfellow et al. (2016). As a result, GANs have generated a lot of interest since 2014, with wide applications in many areas. 3.3.1. GANs GANs were originally invented in a landmark paper by Ian Goodfellow in 2014 Goodfellow et al. (2014). The setup of the framework uses an adversarial process to estimate the parameters of two artificial neural network (ANN) Rumelhart et al. (1986) models by iteratively and concomitantly training a discriminator (D) and a generator (G), as shown in Figure 2. Through multiple cycles of generation and discrimination, both networks train each other, while simultaneously trying to outwit each other Goodfellow et al. (2014); Mariani et al. (2018); Odena et al. (2017); Zhu et al. (2017). GANs have two adversarial ANNs: • • G picks z from the prior latent space Z and then generates samples from this distribution using ANN; D receives generated samples from G and the true data examples, and it must distinguish between the two for authenticity. Both D and G are ANNs which play a zero-sum game, where G learns to produce realistic-looking samples and D learns to get better at discriminating between the generated samples and the true data. Once G is trained to optimality, it can create new samples and augment the training data set. GANs can sample in parallel better than other generative models, have fewer restrictions on the generator function, assume no use of Markov Chains, as well as no variational bounds, unlike VAE, and produce subjectively better quality samples than other generative models Arjovsky et al. (2017); Goodfellow et al. (2016); Goodfellow et al. (2014); Radford et al. (2015); Salimans et al. (2016). Figure 2. Generative Adversarial Network (GAN) operation. Whilst GANs are gaining popularity in many applications, they have notable issues. GANs are notoriously difficult to train properly and difficult to evaluate, the likelihood cannot be easily be computed, and they suffer from the vanishing gradient problem, mode collapse, boundary distortion, and over-fitting Arjovsky et al. (2017); Goodfellow et al. (2016); Salimans et al. (2016). Risks 2021, 9, 49 8 of 33 Mode collapse is when many latent noise values z are mapped to the same data point x, leading to a lack of diversity in the samples that are created, i.e., under-fitting. The vanishing gradient problem occurs when D becomes perfect in its training without giving G the chance to improve. As a result, GANs may fail to converge, thereby leading to poor generated samples Arjovsky et al. (2017). Figure 3 provides a nonexhaustive taxonomy of GAN variants and improved training, including common examples Creswell et al. (2018); Hitawala (2018); Hong et al. (2019); Wang et al. (2017). Figure 3. Taxonomy of GAN variants. For GAN reviews, (Creswell et al. (2018); Hitawala (2018); Hong et al. (2019)) provide a comparative overview. Lucic et al. (2018) conduct an in-depth study on GANs and note no significant performance differences on the GANs studied. There are over 300 GAN variants, and it is impossible to review all of them. In this work, we are interested in exploring GAN applications and showing their potential to actuaries, especially for alleviating class imbalance, data augmentation, and improving the predictive ability of actuarial models. 3.3.2. GMMNs GMMNs minimize the maximum mean discrepancy (MMD) between the moments of pdata and pmodel and are known to be simpler than other generative models Li et al. (2015). Moment matching evaluates whether the moments of the true distribution ptrue ( x ) match those of the data pdata ( x ) through MMD. This approach is similar to GANs in terms of training, except using a different loss function, which leads to faster sampling. However, GMMNs have received less attention than GANs and VAEs, limiting their sample generative scheme Arjovsky et al. (2017); Goodfellow et al. (2016); Hitawala (2018). 3.4. Summary There are a number of deep generative models for synthetic sample generation. Some of the models are explicit with an intractable likelihood and inference. Some models are only approximate and generate blurry samples. Other methods do not sample in parallel, are complex, and rely on Markov chains, which are time-consuming. GANs are attractive as they do not make any explicit density estimation, and they remedy most of these issues. GANs have generated extremely good examples in many domains. Section 4 reviews these GAN applications. 4. Applications of GANs The most successful applications of GANs are in computer vision, but there have been applications in other domains, as well. In this section, we focus on the applications where there is some actuarial use. Risks 2021, 9, 49 9 of 33 4.1. Data Augmentation The availability of sufficient data in many domains is a necessity, especially where predictive models are needed to make business decisions. Such models are built on adequate training data for better generalization and meaningful accuracy Goodfellow et al. (2016). In this work, we are interested in adopting GANs for new sample creation and data augmentation in order to boost predictive models by supplementing training data sets with new samples that are learned from the real data distribution in an adversarial manner. Data augmentation is a procedure to create synthetic cases to augment the training data and increase its size, especially for those data points that are lacking. This is where GAN shines—the ability to create new samples and adequate data sets Goodfellow et al. (2014); Fiore et al. (2019). There are two main strategies to check if this augmentation really helped something: we can train our model on fake data and check how well it performs on real samples. We can also train our model on real data to do some classification task and only after check how well it performs on generated data. If it works well in both cases—you can feel free to add samples from the generative model to your real data and retrain it again—you should expect gain of performance. Recently, a number of papers have applied GANs to augment various data sets, with remarkable results on the performance of the predictive models applied after Antoniou et al. (2017); Douzas and Bacao (2018); Fiore et al. (2019); Mariani et al. (2018); Mottini et al. (2018); Park et al. (2018); Xu et al. (2019); Ding et al. (2019). Similarly, GANs can be used to augment actuarial data sets and boost actuarial models, making them more accurate and less biased. In this work, we demonstrate how this can be done for a number of data sets, described in Section 6. 4.2. Anomaly Detection Anomaly detection is the identification of rare items, events, or observations which raise suspicions by differing significantly from the majority of the data. Anomaly detection finds extensive use in a wide variety of applications, such as fraud detection for credit cards, insurance, or health care. The importance of anomaly detection is due to the fact that anomalies in data translate to significant (and often critical) actionable information in a wide variety of application domains. There are a number of these methods, such as clustering-based, classification-task, nearest neighbor, spectral, or statistical, but most of them have rather strong assumptions and long training times. Main generative models, like VAE or GAN, consist of two parts. VAE has an encoder and the decoder, where the encoder basically models the distribution and the decoder reconstructs from it Larsen et al. (2015). A GAN consists of the generator and the discriminator, where the generator models the distribution and the discriminator judges if it is close to the training data Goodfellow et al. (2014). They are pretty similar in some way—there is modeling and judging part (in VAE, we can consider reconstructing as some kind of judgement). The modeling part is supposed to learn the data distribution. What will happen to the judging part if we give it some sample not from the training distribution? In case of a well trained GAN, the discriminator will tell us 0, and reconstruction error of VAE will be higher than average one on the training data Akcay et al. (2018). Our unsupervised anomaly detector is then easily trained and evaluated. We can feed it with some steroids, like statistical distances, if we want. In medicine, Schlegl et al. (2017) propose an AnoGAN for anomaly detection of medical images, and learn the characteristics of lesions by learning the characteristics of health data sets. Akcay et al. (2018) present GANomaly for anomaly detection in visual noise, noting a significant improvement on detecting anomalies on various data sets. These methods can be leveraged for potential applications in fraud detection, lapse prediction and claiming likelihood in insurance. A GAN useful and leveraged for anomaly detection can rival other anomaly detection techniques. Risks 2021, 9, 49 10 of 33 4.3. Time Series Suppose we wanted to simulate the evolution of a stock price for some particular asset using traditional simulations, such as Monte Carlo. We would need to estimate the mean and volatility of the returns using past price evolution and then simulate new prices under the assumption that the returns follow a Gaussian distribution with the estimated parameters. However, this normality assumption may not be entirely true in practice where there is a tendency for higher observed probabilities for the tail events than those predicted by the Gaussian distribution. We could change our assumption, say, into a student-t distribution, but neither would that assumption completely describe the reality. GANs are capable of replicating the price evolution without making any model assumptions. Time series and stochastic processes are widely used by financial firms for risk management, financial projections, stock prediction, extreme event monitoring, and monetary policy making Fu et al. (2019). Traditionally, autoregressive time series models, exponential smoothing and their variants, and, more recently, deep learning, have been introduced and intensively studied and applied for time series data Esteban et al. (2017). However, most of these models rely on strong dependence on model assumptions and model parameter estimation and, thus, are less effective in the estimation of complex distributions with time-varying features Zhou et al. (2018). GANs do not make any explicit assumptions and are capable of learning the distributions and their dependence structures in a non-parametric fashion. There has been a number of time series GANs proposed, such as the recurrent conditional GAN (RCGAN) Esteban et al. (2017) and time series GAN (TimeGAN) Yoon et al. (2019), for the generation of realistic financial time series. Immediate actuarial uses leveraging these GANs are stochastic simulations, capital modeling, mortality projections, reserving, asset and liability management, solvency projection, and other time series generation tasks. GANs can be used to rival Monte Carlo or stochastic simulations without any distributional assumptions. In insurance, mortality forecasting is an important actuarial task. Typically, mortality forecasting models, such as Lee-Carter Lee and Carter (1992), are used, but these make strong mathematical assumptions which need to be validated by the data. Time series GANs could potentially be used to simulate and project mortality rates into the future, potentially competing with existing models. 4.4. Privacy Preservation Data of a lot of companies can be secretive, confidential, or sensitive. Sometimes, we need to share it with third parties like consultants or researchers. If we want to share a general idea about our data that includes the most important patterns, details, and shapes of the objects, we can use GANs directly to sample examples of our data to share with other people without sharing identifiable features. This way we will not share any exact confidential data, just something that looks exactly like it. Privacy-preservation GANs are capable of accomplishing this task Beaulieu-Jones et al. (2019). In actuarial valuation models where model points are used to determine the amount of money to hold for an individual/groups, such GANs may be useful for the creation of synthetic samples to be fed into the valuation model, without needing the details of any policy. In particular, GANs can be used to share synthesized data and make them publicly available, increasing the scope for actuarial research, collaboration, and comparisons. 4.5. Missing Data Imputation Missing data causes an issue in analysis as most standard data analytic methods are not designed for missing data. Techniques, such as single imputation (SI) and multiple imputation (MI) Rubin (2004), exist, but there is no consensus on which of the MI method is superior, even though MI is known to be better than SI Schafer and Olsen (1998). Generative Adversarial Imputation Net (GAIN) Yoon et al. (2018) provides an alternative generative modeling approach to create new cases that can be used to impute missing information. View Imputation via GANs (VIGAN) Shang et al. (2017) deals with data that Risks 2021, 9, 49 11 of 33 are collected from heterogeneous sources, resulting in multi-view or multi-modal data sets where missing data occurs in a number of these sources. These methods were shown to be better than SI/MI methods, thereby improving the effectiveness of ML algorithms trained after. These GANs can be used to impute missing data points in experience investigations and assumption setting in both short-term and life insurance when conducting valuation or pricing, increasing the number of data points available for boosting the predictive power of models built after. 4.6. Semi-Supervised Learning The purpose of a Semi-Supervised GAN (SGAN) is to train the discriminator into a classifier which can achieve superior classification accuracy from as few labeled examples as possible Sricharan et al. (2017), thereby reducing the dependency of classification tasks on enormous labeled data sets. It has been shown that an SGAN generalizes from a small number of training examples much better than a comparable, fully-supervised classifier Chongxuan et al. (2017); Liu et al. (2019); Miyato et al. (2018). This has been lauded as the most useful GAN application with good performance with a small number of labels on data sets Odena (2016); Salimans et al. (2016). For imbalanced data sets, such as mortality, morbidity, fraud, lapses, extreme events, large claims, and sub-standard risks, SGAN may offer a superior alternative predictive model compared to ML models which require significant training data for improved accuracy. Typically, one has to deal with imbalanced classes either through synthetic sample generation using some heuristic method, such as SMOTE, cost sensitive adjustment to the evaluation metric, or adding uncertainty margins, which can be subjective. Through the training of an SGAN, it is possible to have a sample generative scheme whilst having a classifier, as well. This has tremendous advantages over many generative and ML models. 4.7. Domain Adaptation It is quite possible that the training data used to learn a classifier has a different distribution from the data which is used for testing. This results in degradation of the classifier performance and highlights the problem known as domain adaptation Hong et al. (2018). In domain adaptation, the training and test data are from similar but different distributions. This area has become interesting for GANs in the past few years. These methods include CycleGAN Zhu et al. (2017), Discover GAN (DiscoGAN) Kim et al. (2017), DualGAN Yi et al. (2017), and StarGAN Choi et al. (2018), which can be used for multiple domains. With these methods, one can transfer an algorithm learned from a different data set to a new one and achieve similar performance. Such approaches are also able to learn representation adaptation, which is learning feature representations that a discriminator cannot differentiate which domain they belong to Tzeng et al. (2017). By using synthetic data and domain adaptation, the number of real-world examples that are needed to achieve a given level of performance is reduced significantly, utilizing only randomly generated simulated points Hoffman et al. (2017). Domain adaptation can learn transfers between different domains, by synthesizing different data sets. This can be useful in combing public data sets or other market data with internal company data in actuarial firms. 4.8. Summary Given the above taxonomy of GAN applications, Table 2 depicts specific actuarial areas where GANs can be useful. To our knowledge, there has been limited applications of GANs in actuarial areas, such as insurance, health care, banking, investment, and enterprise risk management. This is compounded by the fact that GANs have been highly successful on computer vision, with less emphasis on tabular data sets. However, there have been recent applications of GANs on other tabular data sets, such as airline passengers Mottini et al. (2018) and medical records Armanious et al. (2018). Risks 2021, 9, 49 12 of 33 Table 2. Potential GAN applications in actuarial disciplines. Actuarial Discipline Description Product Design, Propensity and Customer Behavior Create wider and more model points; Boost propensity models with more data per cell, leading to better and accurate models. Actuarial Models Experience monitoring and experience rates derived using a large credible data set. Boost models using data augmentation, semi-supervised learning, missing data imputation and domain adaptation for pricing, assumption setting, anomaly detection, risk estimation, time series and attention prediction in insurance, reinsurance, banking, investment, healthcare, and enterprise risk management. Projections Network modeling by looking at driving dependencies rather than correlation assumptions, i.e., use generative models. Strategic flexible and more decision-based models based on the environment. More GAN-based time series models driven by the environment. Enhanced solvency projection models and stress tests which are based on rich data sets. Reserving Make projections more predictive through a large enough credible data at all model points, i.e., accurate assumptions per risk cell with less margins. Surplus Distribution More granular individual information from alternative data sources through leveraging generative models. Investment Strategy Granular data for asset/liability modeling, i.e., use GANs to simulate scenarios that depend entirely on the adopted investment strategy and boosting the model. Enhanced market risk monitoring. Improvements to portfolio optimization. Data Cleaning Reduce errors; fill in gaps using imputation; increase the sample size; query other data sets and verify patterns using Cycle GANs. Research Make actuarial data sets more publicly available through synthesized data generated by GANs, boosting industry data. This is helpful for creating accurate and more up-to-date standard tables and encouraging actuarial research. External Data Sources Leverage other data sets through combining multiple data sets. For example, DualGAN or CycleGAN can be leveraged to learn a representation that encompasses different data sets. GANs can equally be adopted or leveraged for similar tasks to boost limited actuarial data sets and improve actuarial models, especially in areas where models are needed to make business decisions. Examples of actuarial applications with limited data and the class imbalance problem include claim frequency modeling, claim amount estimation, lapse prediction, fraud detection, mortality/morbidity rate estimation, catastrophe modeling, extreme event models, and risk estimation. Leveraging GANs to increase the data size on these data sets could lead to better actuarial models. In particular, GANs could allow less reliance on using stochastic simulations that are based on subjective distributions and err less on margins used. 5. Methodology This section describes in detail the theoretical operation of GANs, their challenges, and tricks to improve their training. Throughout this paper, it is assumed that both GAN networks are implemented with ANNs. For comparative purposes, we also implement a popular synthetic data generative mechanism using Synthetic Minority Over-sampling Technique (SMOTE) Chawla et al. (2002). 5.1. SMOTE This section describes the theoretical operation of SMOTE for comparative purposes with the GAN applied in this work. SMOTE creates new synthetic cases by linearly Risks 2021, 9, 49 13 of 33 interpolating between two nearest neighbor (NN) instances of the minority class. Chawla et al. (2002) show that SMOTE improves the effectiveness of ML classifiers compared to random over-sampling and under-sampling approaches. Over time, SMOTE has become the default method for synthetic sample generation and has proven to be popular among researchers, becoming a pioneer in imbalanced learning Fernández et al. (2018). Considering a random minority instance x, a new instance s is generated by considering its k-NNs. These k-NNs are found by using the Euclidean distance metric. Initially, an instance y is generated at random from the k-NNs. Then, a new synthetic minority instance s is generated, as follows:  s = x+α y−x , (3) where α is randomly generated from the Uniform distribution [0, 1]. SMOTE parameters are the value of k and the number of minority cases to generate. These parameters can be tuned to ensure an optimal metric is achieved. SMOTE is the benchmark method for addressing class imbalance in binary classification problems. 5.2. Vanilla GAN This section describes the original GAN formulation, called MiniMax GAN (MMGAN). This is the baseline model over which all other GAN variants are based. 5.2.1. The Discriminator The discriminator (D) receives generated samples from a generator G and the true data examples from pdata ( x ), and must distinguish between the two for authenticity through a deep ANN Goodfellow et al. (2014). The resulting output Dθd ( x ) for an input x is the probability of x being sampled from pdata ( x ) instead of p g , where p g is the implicit distribution defined by G. The vector θd represents learned parameters from D. The discriminator’s goal is to yield D ( x ) near 1 for x ∼ pdata and D ( G (z)) closer to 0 for p ∼ pz (z) using the sigmoid function in the output layer. This is achieved by maximizing D’s loss over θd : h i   MM− GAN JD = EX ∼ pdata (x) log Dθd ( x ) + EZ∼ pz (z) log(1 − Dθd ( Gθg (z)) . (4) 5.2.2. The Generator The generator (G) randomly picks a sample z from the prior latent space defined by p(z) and then generates samples from this distribution using an ANN. This deep ANN must learn the parameters Θ g given an input z ∼ pz (z), that will give the output Gθg (z). G is trained to fool D, i.e., to make D’s output for fake/generated sample D ( G (z)) closer to 1. The parameters of G are learned by minimizing G’ loss over Θ g : h i JGMM−GAN = EZ∼ pz (z) log(1 − Dθd ( Gθg (z)) . (5) 5.2.3. GAN Loss Combining the losses for D and G, GANs solve the following minimax game in alternate steps through Gradient Descent (GD) Ruder (2016): h i   min max EX ∼ pdata ( x) log Dθd ( x ) + EZ∼ pz (z) log(1 − Dθd ( Gθg (z)) . (6) θg θd The above losses for D and G are the original formulation proposed by Goodfellow in 2014, called minimax GAN (MM-GAN). Since we are minimizing over θ g and maximizing over θd , training of GANs alternate between GD on G and gradient ascent on D Goodfellow et al. (2016). Typically, for every training of G, D is trained k times although an optimal choice is debatable among researchers. This is shown in Algorithm 1. Risks 2021, 9, 49 14 of 33 Remark 1. Gradient based updates on the networks can be accomplished using one of the GD optimizers. Typically, Stochastic GD (SGD) with Momentum Qian (1999) for D, Root Mean Square propagation (RMSprop) Hinton and Tieleman (2012), or Adaptive Moment estimation (Adam) Kingma and Ba (2014) for G tend to work well in practice Goodfellow et al. (2014); Radford et al. (2015). Algorithm 1: Mini-batch SG ascent of GANs with the original objective for MM-GAN. The number of steps to apply to D, k, is a hyper-parameter. For every training of G, we train D k times. Goodfellow et al. (2014) used k = 1. 1: for number of epochs do 2: update the discriminator 3: 4: for k steps do • Sample mini-batch of m noise samples {z(1) , . . . , z(m) } from the noise prior p g (z). • Sample mini-batch of m true examples { x (1) , . . . , x (m) } from the training data distribution pdata ( x ). • Update the discriminator D by ascending its stochastic gradient on these mini-batches: " #      1 m ∆θd ∑ log D xi + log 1 − D G (zi ) . m i =1 5: end for 6: update the generator 7: • • 8: Sample mini-batch of m noise samples {z(1) , . . . , z(m) } from the noise prior p g (z). Update the generator by descending its stochastic gradient computed on this mini-batch:    1 m ∆θ g ∑ log 1 − D G (zi ) . m i =1 end for 5.2.4. Non-Saturating GAN While the above loss function is useful for theoretical results, unfortunately, it does not work well in practice, and there are challenges getting the GAN to convergence, stabilize its training, and getting diverse samples Arjovsky et al. (2017); Mirza and Osindero (2014); Radford et al. (2015); Salimans et al. (2016). In practice, rather than training the above loss function for G, to provide better gradients in earlier training, Goodfellow et al. (2014) suggest to maximize the following objective function for G instead:   JGNS−GAN = EZ∼ pz (z) log Dθd ( Gθg (z) . (7) This version of GAN is called non-saturating GAN (NS-GAN) and is typically used as the benchmark in most studies and in practice. This leads to the following NS-GAN loss   function:   max max EX ∼ pdata ( x) log Dθd ( x ) + EZ∼ pz (z) log Dθd ( Gθg (z) . (8) θg θd With this new loss function, we alternate between gradient ascent on D and gradient ascent on G. Algorithm 1 is based on the original MM-GAN formulation; however, it can easily be tweaked to represent NS-GAN. 5.2.5. Optimal Solution Theoretically, it can be shown that for p g = pdata , the GAN zero-sum game in Equation (6) has a global optima. Given enough capacity for both networks and D is trained to optimality for a fixed G, convergence of the GAN algorithm is guaranteed Goodfellow Risks 2021, 9, 49 15 of 33 et al. (2014); Manisha and Gujar (2018); Mirza and Osindero (2014); Nowozin et al. (2016); ∗ ( x ) for a fixed G is: Radford et al. (2015). The optimal discriminator DG ∗ DG (x) = pdata ( x ) . pdata ( x ) + p g ( x ) (9) ∗ ( x ) into Equation (6) for Assuming that D is perfectly trained and if we substitute DG G’s loss, this gives rise to the Jensen-Shannon (JS) divergence Lin (1991). The JS divergence can be written as a function of the Kullback-Leibler (KL) divergence Kullback (1997); Kullback and Leibler (1951). Definition 1. The KL divergence between two probability distributions pdata and p g is defined as   KL( pdata , p g ) = DKL pdata || p g = Z pdata ( x ) log ! pdata ( x ) dx. pg (x) Definition 2. The JS divergence between two probability distributions pdata and p g is defined as  JS( pdata , p g ) = D JS pdata || p g      pdata + p g pdata + p g 1 1 + KL p g , . = KL pdata , 2 2 2 2 ∗ ( x ) into Equation (6), the minimum loss for G is reached if and If we substitute DG only if p g = pdata ; thus, one can show that:   JG = − log 4 + 2JS pdata , p g . (10) This equation tells us that, when D has no capacity limitation and is optimal, the GAN loss function measures the similarity between pdata and p g using JS divergence. However, although the above results provide a nice theoretical result, in practice, D is rarely ever fully optimal when optimizing G Goodfellow et al. (2014). Thus, alternative GAN architectures have been proposed to fix this issue and to get closer to optimality. Below, we describe what causes this failure to convergence and how to fix it. 5.3. Challenges with GANs GANs are notoriously difficult to train properly and to evaluate, the likelihood cannot be easily be computed, and they suffer from the vanishing gradient problem, mode collapse, boundary distortion, and over-fitting Arjovsky et al. (2017); Creswell et al. (2018); Goodfellow et al. (2016); Hitawala (2018); Hong et al. (2019); Salimans et al. (2016). This section describes key challenges on GAN training. 5.3.1. Mode Collapse Mode collapse is when many latent noise values z are mapped to the same data point x, leading to a lack of diversity in the samples that are created, i.e., under-fitting. This is regarded as the most significant problem with GANs Manisha and Gujar (2018). Many studies have spent lots of time in varied contexts to fix this. 5.3.2. Vanishing Gradient This occurs when D becomes perfect in its training without giving G the chance to improve. As a result, GANs may fail to converge, thereby leading to poor generated samples Arjovsky et al. (2017). 5.4. Improved GAN Training There are many GAN architectures which avoid the problems that come with the vanilla GAN. We briefly describe some of the most common and popular GAN solutions. Salimans et al. (2016) look at ways to improve GANs (called hacks), while other authors Risks 2021, 9, 49 16 of 33 propose variants to the vanilla GAN by changing the cost function, adding gradient penalties (GPs), adding labels, avoiding over-fitting, and finding better ways of optimizing GANs. Given the vast number of taxonomies, we are not able to cover all of them but only discuss the most popular and those subsequently used in this work. 5.4.1. Conditional GANs The first extension of GAN was the conditional GAN (cGAN) which gave the generator the label Y in the latent space, making them class conditional Mirza and Osindero (2014). Most of the GAN variants can be modified to include cGAN. cGAN allows to create diversified samples and forcing G to create specific samples, thereby fixing mode collapse problem. 5.4.2. Deep Convolutional GAN Until the introduction of deep convolutional GAN (DCGAN) Radford et al. (2015), training GANs was still unstable. DCGANs provide some further tricks using convolutional and deconvolutional layers. Given that DCGANs use convolutional NNs which are typically used for images, we do not review this architecture in detail as our main focus in on tabular data. Despite this, the structure of the DCGAN is very useful in providing stable training for most GANs Lucic et al. (2018). 5.4.3. Loss Variants There are a number of GAN architectures which change the loss function to improve GAN training and stability. The loss function for GAN measures the similarity between pdata and p g using JS. Unfortunately, JS tends not to be smooth enough to ensure a stable training Hong et al. (2019); Manisha and Gujar (2018). There are a number of GAN loss variants which have been proposed over the years. Broadly, there are two loss function groups with better properties, i.e., f-divergence Nowozin et al. (2016) and Integral Probability Metrics (IPMs) Hong et al. (2019); Müller (1997). Among these loss groups, Wasserstein GAN (WGAN) Arjovsky et al. (2017) is arguably the most popular and well-studied Hitawala (2018); Wang et al. (2017). WGAN is considered a general unified framework under the recently proposed Relativistic GAN (RGAN) Jolicoeur-Martineau (2018). Thus, we adopt to describe WGAN as it has become the most widely used GAN architecture since DCGANs. 5.5. WGAN This section describes WGAN and its improved training using WGAN-GP. 5.5.1. Wasserstein Distance IPM generalizes a critic function f belonging to an arbitrary function class, where IPM measures the maximal distance between two distributions under some functional frame f Hitawala (2018). Among the IPMs, the Wasserstein distance is the most common and widely used metric Manisha and Gujar (2018). Informally, the Earth mover (EM) Rubner et al. (2000) distance W ( pdata , p g ) measures the minimal changes needed to transform p g into pdata . More formally, EM between two probability distributions pdata and p g is:   W pdata , p g = inf γ∼Π( pdata ,p g )   E(x,y)∼γ k x − y k , (11) where Π( pdata ,p g ) represents a set of all joint probability distributions in which marginal distributions are, respectively, pdata ( x ) and p g ( x ). Precisely, γ( x, y) is a transport plan, i.e., percentage of mass that should be moved from x to y to transform p g into pdata . Risks 2021, 9, 49 17 of 33 The infimum in Equation (11) is intractable as it is tricky to exhaust all the elements of Π( pdata ,p g ) Arjovsky et al. (2017). This is solved using the following functional format:       W pdata , p g = sup Ex∼ pdata f ( x ) − Ex∼ pg f ( x ) , (12) k f k L ≤1 where the supremum is taken over a 1-Lipschitz function f . A function f is 1-Lipschitz if for all x1 , x2 : | f ( x1 ) − f ( x2 )| ≤ | x1 − x2 |. 5.5.2. The Critic In WGAN, D’s output is not a probability anymore but can instead be any number, and, for this reason, D is typically called the critic. The WGAN critic tries to maximize the difference between its predictions for real samples and generated samples, with real samples scoring higher. Arjovsky et al. (2017) force the critic to be 1-Lipschitz continuous for the loss function to work well:     JWGAN = max EX ∼ pdata ( x) D ( x ) + EZ∼ pz (z) 1 − D ( G (z)) , (13) w ∈W where W is the set of 1-Lipschitz continuous functions. Typically, to enforce the Lipschitz constraint, the critic weights w are clipped to lie within a small range, usually [−0.01, 0.01] after each training batch Arjovsky et al. (2017); Gulrajani et al. (2017). The critic is trained to convergence so that the gradients of G are accurate, thus removing the need to balance the training of G and D by simply training D several times between G’s updates, to ensure it is close to convergence. Typically, 5 critic updates to 1 generator update is used Arjovsky et al. (2017). WGAN used the RMSProp version of gradient GD with a small learning rate and no momentum Arjovsky et al. (2017). However, Adam may also be used as it is a combination of RMSProp with Momentum. 5.6. Improved WGAN Training Even though WGAN has been shown to stabilize GAN training, it is not generalized for deeper training due to weight clipping which tends to localize most parameters at −0.01 and 0.01 Gulrajani et al. (2017); Manisha and Gujar (2018). This effect dramatically reduces the modeling capacity for D. Gulrajani et al. (2017) amend WGAN through an addition of a gradient-penalty (GP) to the loss function, coming with WGAN-GP. In total, three changes are made to WGAN critic to convert it to WGAN-GP: include a GP to the loss function; do not clip critic weights; and do not use batch normalization layers in the critic. WGAN-GP is defined using the following loss function: h     2 i EX ∼ pdata (x) D ( x ) + EZ∼ pz (z) 1 − D ( G (z)) + λEx̃∼ pdata k ∆D ( x̃ ) k2 −1 , (14) where x̃ samples uniformly along the straight line between points sampled from pdata and p g , and λ is the GP term. Gulrajani et al. (2017) show a better distribution of learned parameters compared to WGAN, and this method has been the default method in most GAN loss variants. We adopt the conditional version of WGAN-GP, called WCGAN-GP, as an alternative to current actuarial/statistical approaches for synthetic sample generation. Once WGANGP is trained to convergence, G can be used to create new samples by feeding it the latent space Z. 6. Experiments This section outlines the experiments conducted, showing a popular GAN application for data augmentation and boosting predictive models. We compare WGAN with SMOTE. This exercise can be similarly adopted for any actuarial modeling problem, such as mortality, Risks 2021, 9, 49 18 of 33 morbidity, medical segmentation, credit risk, extreme events, regression, Value-at-Risk, and anomaly detection in insurance, investment, banking, and healthcare. 6.1. Data Sets We considered 5 publicly available imbalanced data sets from the Center for Machine Learning and Intelligent Systems database Dua and Graff (2017). The data sets are described below and shown in Table 3. Table 3. Imbalanced data sets used in the experiments. Imbalanced Data Set Majority Cases Minority Cases Number of Features Numeric Features Ordinal Features Credit Card Fraud Pima Indians Diabetes Glass Identification German Credit Scoring Breast Cancer Wisconsin 284,807 500 144 700 357 492 268 70 300 212 31 8 9 20 28 31 8 9 14 28 0 0 0 6 0 6.1.1. Credit Card Fraud European public credit card fraud transactions made in 2013 are utilized Pozzolo (2015). This data is highly imbalanced, with 492 fraudulent transactions out of a total of 284,807 transactions, representing a mere 0.172% of fraud cases. This data set contains 31 anonymized features (Time, Amount, V0,V1, . . . V28) and the Class indicator showing 1 for frauds and 0 for non-fraudulent cases. All the variables are numeric. 6.1.2. Pima Indians Diabetes This data set contains the prediction of the onset of diabetes within 5 years in Pima Indians given some medical details, representing 34.90% of diabetic cases out of a total of 768 samples Smith et al. (1988). There are 8 independent variables. 6.1.3. German Credit Scoring This data comes from the German credit scoring from the Machine Learning Repository Dua and Graff (2017). There are 1000 observations with 20 independent variables. The dependent variable is the evaluation of customer’s current credit status, which indicates whether a borrower’s credit risk is good or bad. 6.1.4. Breast Cancer Wisconsin This data represents the characteristics of a cell nuclei that is present in the digitized image of a breast mass Street et al. (1993). The data is used to predict the presence of benign or malignant cancer, with 37.25% being malignant samples from a total of 569 cases. 6.1.5. Glass Identification This data set determines whether the glass type is float or not in term of their oxide content Evett and Spiehler (1987). There are 32.71% of float glass types out of a total of 214 cases. 6.2. Scaling the Data Many ML methods expect data to be of the same scale to avoid the dominance of certain variables and this can affect the accuracy of specific models Ioffe and Szegedy (2015); Mitchell (2006). Normalization re-scales the data to the range between 0 and 1. Standardization centers the data distribution to N (0, 1). We adopt normalization as it does not assume any specific distribution. This will potentially speed up convergence Goodfellow et al. (2016); Mitchell (2006). Risks 2021, 9, 49 19 of 33 6.3. Train-Test Split ML models are usually trained and tested on unseen data. Two approaches to split the data are cross-validation (CV) and train-test split Friedman et al. (2001). CV divides the data into K subsets that can lack sufficient credibility and can result in higher variability of predictions, if the data size is too small Friedman et al. (2001). Train-test split, however, can allow a larger subset of the data to be used for estimating model coefficients and results in more reasonable results Mitchell (2006). Existing literature typically uses a 70–90% train-test split, especially if the data is large. This technique is simple, easy to understand and widely used, despite giving noisy estimates sometimes Friedman et al. (2001); Goodfellow et al. (2016); Mitchell (2006). CV is typically used to optimize parameters of a classifier. This work adopts 75% training data and 25% testing data. Other train-test splits are possible; however, we leave this for future work. 6.4. SMOTE Implementation Over-sampling is performed on the 75% training data using the R imbalance library. The R imbalance library contains functions for performing SMOTE and other variants.1 The two parameters to tune are the number of neighbors and the over-sampling rate. We kept the over-sampling rate the same to ensure balanced class distributions within each data set. Using SMOTE, we create additional synthetic cases to augment the above training data sets. We varied the number of k-NNs for each data set to ensure optimal parameters are chosen through a 10-fold CV. This was done through a grid search scheme, with values of k-NN ranging from 1 to 15, optimized using the Area under the Precision-Recall Curve (AUPRC). The best parameter values for each data set are shown in Table 4 below. Table 4. Optimal parameter values for k-NN for each data set. Data Set Value of k-NN Credit Card Fraud Pima Indians Diabetes Glass Identification German Credit Scoring Breast Cancer Wisconsin 6 9 10 12 10 6.5. GAN Implementation Given its popularity and wide use, WGAN is adopted for an alternative synthetic sample generation. Specifically, we adopt the conditional version of WGAN with GP; thus, we use WCGAN-GP Gulrajani et al. (2017); Mirza and Osindero (2014). Below, we describe how parameters are chosen and results generated. 6.5.1. Software Due to its simplicity and faster computations, the high-level Keras François (2015) library with Tensorflow back-end is chosen to implement WCGAN-GP.2 This is trained using all minority cases of each data set. 6.5.2. The Generator This section describes how the parameters for G are chosen. The random noise for z is generated from N (0, 1) with 100 dimensions. This is based on GAN hacks which suggest to sample from a spherical distribution Salimans et al. (2016). 1 The imblearn package in Python can also do SMOTE and its notable variants. Other packages exist in R, such as ROSE, unbalanced, smotefamily, DMwR, ebmc, and IRIC. 2 Pytorch and Tensorflow are also popular packages available in Python for implementing GANs. Risks 2021, 9, 49 20 of 33 Rectified Linear Unit (ReLU) Glorot et al. (2011) is adopted in the hidden layers Salimans et al. (2016). For G’s output later, hyperbolic tangent (tanh) is adopted. No drop out or batch normalization is applied in the hidden layers, following advice by Gulrajani et al. (2017) for WGAN-GP. The layers are chosen such that they are ordered in an ascending manner for G. For simplicity, after a number of iterations, 3 layers were chosen for each data set. In the first layer, there were 128 units; in the second layer, 256 units; and, in the third layer, 512 units. These layers worked well in the experiments conducted. The output layer had the data dimension of the data as the number of units. Weights are initialized using the He initialization method He et al. (2015). Adam is used to optimize the weights of G Radford et al. (2015); Salimans et al. (2016). For Adam, we used default values with β 1 = 0.5 and β 2 = 0.9 for G Kingma and Ba (2014). We used a batch size of 128 when optimizing the gradients for faster training Ioffe and Szegedy (2015). Initial learning rate η for G was fixed at 0.00004. The number of epochs were found to be 5000 where the GAN training was found to be stable. 6.5.3. The Critic ReLu is adopted with a negative slope of 0.2 Glorot et al. (2011); Radford et al. (2015). Similarly to the generator, 3 layers were used in the hidden layers. The layers were arranged in a descending manner, with 512 units in the first layer, 256 units in the second layer, and 128 units for the last layer. The critic gives the output a single value using a linear function Arjovsky et al. (2017). Adam was used with default parameters in Keras François (2015), as per Table 5. Table 5. Adam parameters for the critic François (2015); Kingma and Ba (2014). Parameter Value η β1 β2 ε 0.00001 0.5 0.90 10−8 Critic weights were also initialized using the He method, and a similar batch size as in the generator was used. We pre-trained the critic 100 times at each adversarial training step Arjovsky et al. (2017). This ensures faster convergence at each step before G is updated. We used WGAN with a GP with the default values as per the original paper Gulrajani et al. (2017). The GP value was left unchanged at 10. We call this model WGAN-GP. We found that, after 5000 epochs, the losses plateaued and did not change much. 6.5.4. Labels Typically, to boost faster training and fix mode collapse, additional information can be incorporated in both G and D using cGAN Mirza and Osindero (2014). We used the conditional version of WGAN-GP where class labels were added to the minority cases. To accomplish this, clustering was done on the minority cases in order to induce class labels on the training data. We explored a number of common mechanisms considering k-means clustering Hartigan and Wong (1979), Agglomerative Hierarchical Clustering (AHC) Voorhees (1986), Hierarchical DBSCAN Ester et al. (1996), and t-distributed Stochastic Neighborhood Embedding (t-SNE) Maaten and Hinton (2008). The details of these algorithms are beyond the scope of this work. Due to its wide use and simplicity, we adopted k-means clustering with 2 clusters for each data set. This yielded labels that could be fed into G and D to induce generated samples. We call the final model WCGAN-GP after incorporating these class labels into the training. Risks 2021, 9, 49 21 of 33 6.5.5. Training WGAN-GP Figure 4 presents the experiments of training WCGAN with GP. For comparative purposes, using similar parameters, we show the quality of samples generated for WCGAN3 with GP, WGAN, cGAN and non-saturating GAN on the credit card fraud data. Figure 4. Comparison of GAN experiments ran on fraud data cases. We consider this for two combinations of the features for illustrative purposes up to 5000 epochs. The results show the superiority of samples generated by WCGAN with GP. There is a clear mode collapse problem on the vanilla GAN and cGAN. WGAN and WCGAN with GP show better samples. There are clear damped oscillations and unstable losses for GAN and cGAN where Wasserstein GANs exhibit stable training and losses, especially after 1000 iterations, where it seems to settle and stabilize. Figure 5 shows the critic loss for each epoch, where, after 1000 epochs, the loss starts to plateau. Thus, we decided to stop the training after 5000 epochs. We repeated this experiment for each data set and adopted WCGAN with GP after 5000 epochs as the model to use for synthetic sample generation. 6.5.6. Generating Synthetic Samples Once the WCGAN with GP is trained to 5000 epochs, the learned generator distribution is used to create more synthetic samples by feeding it the number of samples to output. 3 The version of the WCGAN was incorporated with an improved WGAN training using the GP term as per the paper by Gulrajani et al. (2017). Risks 2021, 9, 49 22 of 33 Figure 5. Difference between generated and real data critic loss. 6.6. Logistic Regression For simplicity, and given the wide use with actuaries, a Logistic Regression (LR) McCullagh (1984) model is trained using Python 3.7 Python Software Foundation (2017) on both the imbalanced training data and over-sampled data sets to predict the likelihood of each minority case using this equation: ! d hθ ( x (d) ) = θ + θi Xi , 0 < hθ ( x (d) ) < 1, (15) log 0 ∑ 1 − hθ ( x (d) ) i =1 where hθ ( x (d) ) is the probability of the given minority case, θi ’s are the estimated coefficients using SGD, Xi is the feature vector for sample i, and d is the number of features to include in the LR model. The coefficients are estimated by minimizing a loss function through a SGD. Typically, classification is such that, when hθ ( x (d) ) ≥ 50% for each instance, assign the minority case and, otherwise, the majority case. 6.7. Evaluation The confusion matrix returns a report showing how predicted classes on unseen test data using the LR model compare to actual observed classes, as depicted in Table 6. Table 6. The confusion matrix. Confusion Matrix Predicted: Minority Predicted: Majority Actual: Minority Actual: Majority True Positive (TP) False Positive (FP) False Negative (FN) True Negative (TN) True Negative (TN) is the number of majority cases that were correctly classified as such. False Positive (FP) is the number of majority cases that were incorrectly classified as minority. True Positive (TP) is the number of minority cases that were correctly classified as minority. False Negative (FN) is the number of minority cases that were incorrectly classified as majority. Using these definitions, Table 7 presents the most well known evaluation metrics for binary problems. Risks 2021, 9, 49 23 of 33 Table 7. Evaluation metrics for binary problems. Metric Accuracy Precision Recall F1-Score Formula   TP + TN  TP + TN+ FP + FN TP TP + FP   TP TP   + FN Precision × Recall 2× Precision + Recall Precision is the ability of the LR model not to label a minority case that is actually majority. Recall is the ability of the LR model to find all minority cases. F1-Score is a harmonic mean between Precision and Recall He and Garcia (2008). F1-Score puts equal weight to both Precision and Recall. Accuracy can be misleading and inappropriate when there are imbalanced classes and, thus, may be biased towards majority cases Chawla et al. (2002); Ganganwar (2012); He and Garcia (2008). Thus, we do not use rely on it in this work. Accuracy, Precision, Recall, and F1-Score should be close to 100% for a LR model to do well on the testing data. However, these scores are influenced by what threshold is used to decide between the two binary classes. The Receiver Operating Characteristic (ROC) curve Bradley (1997); Hanley and McNeil (1982) measures a classifier’s performance on a test set over different decision thresholds by varying the Precision and the FP rate. The Area under the Curve (AUC) measures the performance of the LR model trained on both imbalanced and over-sampled data sets and tested on unseen data with values close to 100% considered excellent performance Bekkar et al. (2013); Hanley and McNeil (1982). We also compute the Precision-Recall curve and compute the Area Under the Precision-Recall Curve (AUPRC) to get a weighted score. A method that gives the highest score is better. 6.8. Statistical Hypothesis Testing Friedman test Friedman (1937), followed by a post-hoc Nemenyi test Nemenyi (1962), are performed to verify the statistical significant differences between WCGAN-GP and SMOTE. 6.8.1. Friedman Test The Friedman test is a non-parametric ranking test to determine whether SMOTE and WCGAN-GP methods perform similarly in mean performance rankings based on the measures above, when normality does not hold Friedman (1937). 6.8.2. Post-Hoc Nemenyi Test If the null hypothesis is rejected, a post-hoc test can be applied where WCGANGP is considered as the control method. The post-hoc Nemenyi test evaluates pairwise comparisons between the over-sampling methods if the Friedman test suggests that there is a difference in performance Nemenyi (1962); Pohlert (2014). We adopt WCGAN-GP as the control method. 6.8.3. Implementation Both tests are conducted using the Pairwise Multiple Comparison Ranks Package (PMCMR) Pohlert (2014) available in R. We assume statistical significance of the alternative hypothesis at p-values < 0.05. In other words, we fail to reject the null hypothesis when the resulting p-value is higher than 0.05, suggesting that there is no difference between SMOTE and WCGAN-GP. Risks 2021, 9, 49 24 of 33 7. Results This section presents the results of all the LR models applied on the baseline and over-sampled data sets, with metrics on Precision, Recall, F1-Score, AUC, and AUPRC computed on the same unseen test data. 7.1. Comparisons Table 8 presents the evaluation metrics (based on the testing set) of the LR model applied on the baseline and over-sampled data sets for a default threshold of 50%. Bold shows an algorithm that performs the best for that data set, i.e., a higher score for that metric. Figure 6 shows the average performance across all data sets from each evaluation metric. Table 8. Evaluation metrics based on a default threshold of 50%. Method Precision Recall F1-Score AUPRC AUC 85.71% 5.11% 86.24% 63.41% 93.33% 76.42% 72.90% 9.69% 81.03% 74.60% 72.28% 81.35% 81.70% 98.36% 88.20% 74.47% 53.54% 75.51% 56.45% 80.30% 59.68% 64.22% 64.24% 66.67% 72.49% 68.18% 74.10% 73.61% 75.48% 75.22% 60.31% 47.83% 46.51% 51.34% 70.51% 81.08% 55.47% 56.99% 59.11% 63.02% 58.84% 66.60% 68.57% 69.61% 70.94% 50.00% 73.91% 55.00% 42.86% 70.83% 78.57% 46.15% 72.34% 64.71% 53.83% 87.29% 69.56% 63.93% 72.86% 78.03% 94.34% 92.59% 96.23% 94.34% 100.00% 96.23% 94.34% 96.15% 96.23% 95.39% 98.56% 96.93% 95.50% 96.45% 97.00% Credit Card Fraud Baseline SMOTE WCGAN-GP Pima Indians Diabetes Baseline SMOTE WCGAN-GP German Credit Scoring Baseline SMOTE WCGAN-GP Glass Identification Baseline SMOTE WCGAN-GP Breast Cancer Wisconsin Baseline SMOTE WCGAN-GP Figure 6. Average performance across all data sets. Risks 2021, 9, 49 25 of 33 In general, SMOTE improves Recall at the expense of a lower Precision. This results in a lower F1-Score than Baseline results. As a result of a much lower Precision for SMOTE, AUPRC is penalized and lower than both Baseline and WCGAN-GP. SMOTE compromises the Precision significantly, whereas WCGAN-GP improves Recall, while not significantly penalizing Precision. Overall, WCGAN-GP shows a higher F1-Score. Thus, using a default threshold, WCGANGP performs the best on F1-Score, followed by Baseline and SMOTE being last (on the average). The lower Precision on SMOTE may be due to the strict assumed probability distributions and possible creation of over-lapping and noisy samples Bellinger et al. (2015); Das et al. (2015); Gao et al. (2014); Mathew et al. (2015); Zhang and Li (2014). While the univariate results on Precision, Recall, and F1-Score are useful, they do not give the entire picture over different thresholds Bekkar et al. (2013). Since AUC and AUPRC are based on varied thresholds, these metrics are typically preferred over one dimension measurements, such as Precision, Recall, and F1-Score Bekkar et al. (2013); Ganganwar (2012); López et al. (2013). Since we are also comparing the above results with the Baseline model, these metrics are impacted by class imbalance He and Garcia (2008). Thus, we rely on the AUC and AUPRC. 7.1.1. AUC The ROC curve represents the trade-off between Precision and the FP rate, while the AUC is the area under the ROC curve Bekkar et al. (2013). SMOTE reports higher AUC values than the Baseline. In general, WCGAN-GP is better on 3 of the 5 data sets except on credit card fraud and diabetes data sets. Overall, the average AUC value is not too different between WCGAN-GP and SMOTE. This result conflicts the AUPRC scores where WCGAN-GP shows a clear dominant superiority over SMOTE. Whilst AUC may be useful, it does not consider Recall, which may be the most important metric for minority cases. AUC may be affected by skewed data sets and the data distribution He and Garcia (2008). ROC curves are appropriate when the data is balanced, whereas Precision-Recall curves are appropriate for imbalanced data sets Bekkar et al. (2013); He and Garcia (2008). AUC may tend to provide an overly optimistic view than AUPRC He and Garcia (2008). In general, an algorithm that dominates in AUC may not necessarily dominate the AUPRC space He and Garcia (2008). Saito and Rehmsmeier (2015) suggest that the Precision-Recall curve and AUPRC are more informative than the ROC curve and AUC. Since we are also comparing with the Baseline which is imbalanced, ROC and AUC may be inappropriate; thus, AUPRC provides a sensible measure for all methods. 7.1.2. AUPRC AUPRC has all the characteristics of the AUC; thus, for the purposes of this work, we rely more on AUPRC than AUC He and Garcia (2008); Saito and Rehmsmeier (2015). Overall, WCGAN-GP shows better improvements over SMOTE. WCGAN-GP is highest on AUPRC, suggesting this algorithm performs the best across many thresholds and all the data sets used. On the average, SMOTE does not provide a superior predictive performance than the Baseline on all the metrics. Below, we further provide conclusive evidence on the statistical significance of the above results on the AUPRC. 7.2. Statistical Hypothesis Testing Table 9 shows the results of the Friedman test applied on AUPRC to verify the statistical significance of WCGAN-GP compared to SMOTE. Risks 2021, 9, 49 26 of 33 Table 9. Results for Friedman’s test. Data Set p-Value Significance Credit Card Fraud Pima Indians Diabetes German Credit Scoring Glass Identification Breast Cancer Wisconsin 2.9560 × 10−23 0.188386 1.0683 × 10−11 0.465622 4.0085 × 10−12 Yes No Yes No Yes There is enough evidence at 5% significance level to reject the null hypothesis on 3 of the data sets, except German credit scoring and glass identification, suggesting that over-sampling methods are not performing similarly and are different. Since the null hypothesis was rejected for 3 of the data sets, a post-hoc test was applied to further determine pairwise comparisons using the Nemenyi test where WCGAN-GP is the control method. Table 10 shows the results of the post-hoc test. Table 10. Results for the post-hoc test. Test Credit Card Fraud German Credit Breast Cancer WCGAN-GP vs. SMOTE 0.001 0.003 0.001 The above results confirm the significant superiority of WCGAN-GP over SMOTE as all the p-values are less than 0.05 for the 3 data sets where Friedman’s test suggested a difference. These results confirm the findings shown in Figure 6 and Table 8 where the average performance seen on both the AUC and AUPRC was lower for SMOTE compared to WCGAN-GP. In general, WCGAN-GP provides statistically significant better performance on 3 of the 5 data sets. 8. Discussion 8.1. Results Overall, SMOTE improves the AUC/AUPRC when applied on the imbalanced data set but significantly penalizes Precision, leading to a lower AUPRC on 2 of the data sets used. SMOTE samples synthetic points along line segments joining minority instances using the Euclidean distance. This approach may end up using majority instances, thus creating noisy examples and over-lapping cases Han et al. (2005). SMOTE is not based on the true distribution of the minority class data Gao et al. (2014). The poor performance of SMOTE (especially on Precision on the credit card fraud data set) may be attributed to these effects. Overall, SMOTE alters the data distribution as was observed by the significant compromise on Precision and generally lower F1-Score, AUPRC, and AUC values. Other SMOTE variants, such as density-based approaches, are meant to improve the above SMOTE weaknesses Bellinger et al. (2015); Gao et al. (2014). However, they make strict assumptions about the structure and distribution of the minority class data. SMOTE was the quickest to over-sample. WCGAN-GP requires a significant pre-training of both the critic and the generator. GANs are well-known for their training and computing powers Creswell et al. (2018); Lucic et al. (2018). Thus, they have expensive run-times. However, current GANs, such as WGAN and WGAN-GP, remedy this impact with stable training. The quality of generated samples may be worth it compared to the training times. In this study, the GANs reached stable training even for small samples, such as credit card fraud cases. This means that GANs may still be used even for smaller data sets with enough training capacity. Using WCGAN-GP to over-sample minority cases provided the best performance on the AUPRC and on 3 of the data used on AUC. GANs do not make explicit assumptions about the probability distribution of the minority class data. This idea has been used to Risks 2021, 9, 49 27 of 33 create new samples in a number of data sets. Recent works on this Douzas and Bacao (2018); Fiore et al. (2019); Mariani et al. (2018); Park et al. (2018); Ding et al. (2019) report GAN superior performances over SMOTE for image data sets. There is a significant potential to create new samples using GANs, leveraging them to augment limited actuarial data sets. Given the current surge in interest for GANs, optimizing and training GANs is becoming straightforward as there are many implementations in Keras, Pytorch, and Tensorflow. Thus, running times for GANs might not necessarily be an issue, enabling GANs to provide a superior over-sampling approach to supplement imbalanced data sets. Because GANs have become so popular, their limitations have been improved tremendously. However, there are still open challenges for GANs. GANs rely on the generated examples being completely differentiable with respect to the generative parameters. As a result, GANs cannot product discrete data directly. Another key challenge is the evaluation of GANs after training, even though there are measures to compute the quality of results generated. Research for GANs grows each year. Practitioners may need to add GANs to their toolkit as this will significantly improve their models and aid on decision-making as GANs will be characterized by advancements in deep learning, training process maturity, and open acceptance and their wide use in commercial applications. 8.2. Implications for Actuaries Given the superiority of GANs over other generative models and their wide applications, there is scope for actuarial use. The most obvious use is data augmentation and boosting predictive models used for assumption setting, propensity modeling, pricing, reserving, capital, and solvency projections, as demonstrated in the experiments conducted. Using synthetic data sets created through GANs could allow actuaries to share salient features of their data without sharing the full data set, enabling actuarial data sets to be more widely available for public use and research purposes. Given the surge in marketing and social promotions, info-graphics are the main ingredient of social media marketing. GANs can help marketers and designers in the creative process. Other applications include anomaly detection, joint distribution learning, discriminative modeling, semi-supervised learning, domain adaptation, attention prediction, data manipulation, missing data imputation, time series generation, privacy preservation, and computer vision. These GAN applications have potential actuarial adoption in insurance, banking, health and care, and other non-traditional areas, as GANs have been shown to provide better alternatives to current approaches. Research for GANs grows each year, and actuaries may need to add GANs to their toolkit, as this will significantly improve their models and aid on decision-making. 9. Conclusions, Limitations and Future Research 9.1. Conclusions Gaining an advantage in competitive markets through offerings of suitable tailored products on customers relies on building and maintaining adequate predictive models. To build these models, a substantial amount of data and a sizeable number of records is desirable. However, when expanding to new or unexplored markets, that level of information is rarely always available. As a result, actuarial firms have to procure data from a local provider, through purchasing reinsurance from a re-insurer, through limited unsuitable industry and public research or rely from extrapolations from other betterknown markets. In this work, we show how an implicit model using GANs can alleviate this problem through the generation of adequate quality data even from very limited small samples, from difficult domains, or without alignment. This example is a classic data augmentation application of GANs where we showed their superiority of SMOTE and improving the original results. SMOTE improved the classification performance. However, SMOTE is not based on the true underlying minority Risks 2021, 9, 49 28 of 33 class distribution. SMOTE density estimation approaches remedy this issue; however, they are not based on the true data distribution as they make strong data assumptions. Using WCGAN-GP, it is possible to create synthetic cases implicitly, and this turned out to offer a significantly better improvement over SMOTE. This work comprehensively reviews GAN theory and applications in a number of domains, with possible adoption for actuarial use. These applications have scope for actuarial science, and actuaries can add them to their toolkit to aid predictive models. 9.2. Summary of Applications In our opinion, the future of GANs will be characterized by open acceptance of GANs and their applications by the research community and being used in commercial applications. Given their impressive results and advancement in deep learning techniques, we expect a wider use of GANs. The training instability of GANs will soon be done without any problems as the maturity of the training improves with new techniques being invented at a rapid speed. There are potential applications of GANs for actuarial use in insurance, health and care, banking, investment, and enterprise risk management to inter alia new sample creation, data augmentation, boosting predictive models, anomaly detection, semi-supervised learning, attention prediction, time series generation, and missing data imputation. In conclusion, GANs have the potential to boost actuarial models and make better business decisions. 9.3. Limitations and Future Research We repeated training and testing of each over-sampling method 30 times to minimize stochastic effects—this sample size can be increased for more robustness. Alternatively, a bootstrapping approach can be applied to better understand the distributional attributes of the model errors. Given that we considered train-test split to split the data, changing this could potentially change the outcome of the results. Given that we have provided the significance of the results using 30 multiple samples, this adds further comfort to the outcome. However, the potential use of other data splitting methods, such as bootstrap, CV, and different train-test splits, can enhance this work further. Different data splitting methods for different data sets may provide further research work. Our future work includes comparing current traditional actuarial approaches, such as stochastic simulations and pricing models versus each GAN approach in each domain, extensively. Time series GANs have been gaining interest in the past few years. An interesting research area is using recurrent conditional GANs to simulate and project mortality, compared with the traditional Lee-Carter model and its variants. Below are possible future research to improve this work: • • • • Consideration on other data sets to apply the same techniques, especially complex data sets that include small disjuncts, over-lapping, mixed data types, and multiple classes, particularly actuarial data sets. Alternative consideration for other ML algorithms would show which ML technique is best and for which data set and domain. Empirical comparison of these results with other tabular data sets where GANs were applied. Implementation and leveraging of the GANs in R or Python for actuarial use. Author Contributions: Conceptualization, K.S.N. and R.M.; methodology, K.S.N.; software, K.S.N.; validation, K.S.N.; formal analysis, K.S.N.; investigation, K.S.N. and R.M.; resources, K.S.N.; writing— original draft preparation, K.S.N.; writing—review and editing, R.M.; visualization, K.S.N.; supervision, R.M.; project administration, K.S.N. and R.M.; and funding acquisition, K.S.N. All authors have read and agreed to the published version of the manuscript. Risks 2021, 9, 49 29 of 33 Funding: This research was funded by the DSI-NICIS National e-Science Postgraduate Teaching and Training Platform (NEPTTP) (http://www.escience.ac.za/). Acknowledgments: The authors would like to thank the reviewers for their helpful comments. The support of the DSI-NICIS National e-Science Postgraduate Teaching and Training Platform (NEPTTP) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NEPTTP. Conflicts of Interest: The authors declare that there are no conflict of interest regarding the publication of this paper. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References Goodfellow, Ian, Jean Pouget-Abadie, Mehdi Mirza, Bing Xu, David Warde-Farley, Sherzil Ozair, Aaron Courville, and Yoshua Bengio. 2014. Generative adversarial nets. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 2672–80. Ackley, David H., Geoffrey Everest Hinton, and Terrence Joseph Sejnowski. 1985. A learning algorithm for boltzmann machines. Cognitive Science 9: 147–69. [CrossRef] Akcay, Samet, Amir Atapour-Abarghouei, and Toby P. Breckon. 2018. Ganomaly: Semi-supervised anomaly detection via adversarial training. In Asian Conference on Computer Vision. Cham: Springer, pp. 622–37. Antoniou, Antreas, Amos Storkey, and Harrison Edwards. 2017. Data augmentation generative adversarial networks. arXiv, arXiv:1711.04340. Arjovsky, Martin, Soumith Chintala, and Leon Bottou. 2017. Wasserstein gan. arXiv, arXiv:1701.07875. Armanious, Karim, Chenming Jiang, Marc Fischer, Thomas Küstner, Tobias Hepp, Konstantin Nikolaou, Sergio Gatidis, and Bin Yang. 2018. Medgan: Medical image translation using gans. arXiv, arXiv:1806.06397. Batista, Gustavo Enrique, Ronaldo Cristiano Prati, and Maria-Carolina Monard. 2004. A study of the behavior of several methods for balancing machine learning training data. ACM SIGKDD Explorations Newsletter 6: 20–29. [CrossRef] Beaulieu-Jones, Brett K., Zhiwei Steven Wu, Chris Williams, Ran Lee, Sanjeev P. Bhavnani, James Brian Byrd, and Casey S. Greene. 2019. Privacy-preserving generative deep neural networks support clinical data sharing. Circulation: Cardiovascular Quality and Outcomes 12: e005122. [CrossRef] Bekkar, Mohamed, Hasiba Kheliouane Djemaa, and Akrouf Alitouche Taklit. 2013. Evaluation measures for models assessment over imbalanced data sets. Journal of Information Engineering and Applications 3: 27–38. Bellinger, Collin, Chris Drummond, and Nathalie Japkowicz. 2016. Beyond the boundaries of smote. In Joint European Conference on Machine Learning and Knowledge Discovery in Databases. Berlin: Springer, pp. 248–63. Bellinger, Collin, Nathalie Japkowicz, and Chris Drummond. 2015. Synthetic oversampling for advanced radioactive threat detection. Paper presented at the IEEE 14th International Conference on Machine Learning and Applications (ICMLA), Miami, FL, USA, December 9–11, pp. 948–53. Bengio, Yoshua, Eric Thibodeau-Laufer, Guillaume Alain, and Jason Yosinski. 2014. Deep generative stochastic networks trainable by backprop. Paper presented at the International Conference on Machine Learning, Bejing, China, June 22–24, pp. 226–34. Bradley, Andrew P. 1997. The use of the area under the roc curve in the evaluation of machine learning algorithms. Pattern Recognition 30: 1145–59. [CrossRef] Brock, Andrew, Jeff Donahue, and Karen Simonyan. 2018. Large scale gan training for high fidelity natural image synthesis. arXiv, arXiv:1809.11096. Chawla, Nitesh Vijay. 2009. Data mining for imbalanced datasets: An overview. In Data Mining and Knowledge Discovery Handbook. Berlin: Springer, pp. 875–86. Chawla, Nitesh Vijay, Kevin Bowyer, Lawrence Hall, and W. Phillip Kegelmeyer. 2002. Smote: synthetic minority over-sampling technique. Journal of Artificial Intelligence Research 16: 321–57. [CrossRef] Chawla, Nitesh Vijay, Alecsander Lazarevic, Lawrence Hall, and Kevin Bowyer. 2003. Smoteboost: Improving prediction of the minority class in boosting. In European Conference on Principles of Data Mining and Knowledge Discovery. Berlin: Springer, pp. 107–19. Choi, Yunjei, Minje Choi, Munyoung Kim, Jung-Woo Ha, Sunghun Kim, and Jaegul Choo. 2018. Stargan: Unified generative adversarial networks for multi-domain image-to-image translation. Paper presented at the IEEE Conference on Computer Vision and Pattern Recognition, Salt Lake City, UT, USA, June 18–23, pp. 8789–97. Chongxuan, Lee, Kun Xu, Jun Zhu, and Bo Zhang. 2017. Triple generative adversarial nets. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 4088–98. Creswell, Antonia, Tom White, Vincent Dumoulin, Kai Arulkumaran, Biswa Sengupta, and Anil Bharath. 2018. Generative adversarial networks: An overview. IEEE Signal Processing Magazine 35: 53–65. [CrossRef] Das, Barnan, Narayana Chatapuram Krishnan, and Diane J. Cook. 2015. Racog and wracog: Two probabilistic oversampling techniques. IEEE Transactions on Knowledge and Data Engineering 27: 222–34. [CrossRef] [PubMed] Ding, Zihan, Xiao-Yang Liu, Miao Yin, Wei Liu, and Linghe Kong. 2019. Tgan: Deep tensor generative adversarial nets for large image generation. arXiv, arXiv:1901.09953. Risks 2021, 9, 49 30 of 33 Douzas, George, and Fernando Bação. 2018. Effective data generation for imbalanced learning using conditional generative adversarial networks. Expert Systems With Applications 91: 464–71. [CrossRef] Dua, Dheeru, and Graff Casey. 2017. UCI Machine Learning Repository. University of California, Irvine, School of Information and Computer Sciences. Available online: https://archive.ics.uci.edu/ml (accessed on 18 May 2019). Esteban, Cristóbal, Stephanie L. Hyland, and Gunnar Rätsch. 2017. Real-valued (medical) time series generation with recurrent conditional gans. arXiv, arXiv:1706.02633. Ester, Martin, Hans-Peter Kriegel, Joerg Sander, and Xiaowei Xu. 1996. A density-based algorithm for discovering clusters in large spatial databases with noise. Kdd 96: 226–31. Evett, I. W., and E. J. Spiehler. 1987. Rule induction in forensic science. In KBS in Goverment. Burlington: Jones & Bartlett Publishers, pp. 107–18. Fernández, Alberto, Salvador Garcia, Francisco Herrera, and Nitesh Vijay Chawla. 2018. Smote for learning from imbalanced data: progress and challenges, marking the 15-year anniversary. Journal of Artificial Intelligence Research 61: 863–905. [CrossRef] Fiore, Ugo, Alfredo De Santis, Francesca Perla, Paolo Zanetti, and Francesco Palmieri. 2019. Using generative adversarial networks for improving classification effectiveness in credit card fraud detection. Information Sciences 479: 448–55. [CrossRef] François, Chollet. 2015. keras. Available online: https://github.com/fchollet/keras (accessed on 20 August 2019). Frey, Brendan J., Geoffrey Everest Hinton, and Peter Dayan. 1996. Does the wake-sleep algorithm produce good density estimators? In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 661–667. Friedman, Jerome, Trevor Hastie, and Robert Tibshirani. 2001. The Elements Of Statistical Learning. Springer Series in Statistics New York. New York: Springer, vol. 1. Friedman, Milton. 1937. The use of ranks to avoid the assumption of normality implicit in the analysis of variance. Journal of the American Statistical Association 32: 675–701. [CrossRef] Fu, Rao, Jie Chen, Shutian Zeng, Yiping Zhuang, and Agus Sudjianto. 2019. Time series simulation by conditional generative adversarial net. arXiv, arXiv:1904.11419. Ganganwar, Vaishali. 2012. An overview of classification algorithms for imbalanced datasets. International Journal of Emerging Technology and Advanced Engineering 2: 42–47. Gao, Ming, Xia Hong, Sheng Chen, C. J. Harris, and Emad Khalaf. 2014. Pdfos: Pdf estimation based over-sampling for imbalanced two-class problems. Neurocomputing 138: 248–59. [CrossRef] Geyer, Charles J. 1992. Practical markov chain monte carlo. Statistical Science 7: 473–83. [CrossRef] Glorot, Xavier, Antoine Bordes, and Yoshua Bengio. 2011. Deep sparse rectifier neural networks. Paper presented at the Fourteenth International Conference on Artificial Intelligence and Statistics, Fort Lauderdale, FL, USA, April 11–13, pp. 315–23. Goodfellow, Ian. 2016. Nips 2016 tutorial: Generative adversarial networks. arXiv, arXiv:1701.00160. Goodfellow, Ian, Yoshua Bengio, and Aaron Courville. 2016. Deep Learning. Cambridge: MIT Press, vol. 1. Gulrajani, Ishaan, Faruk Ahmed, Martin Arjovsky, Vincent Dumoulin, and Aaron Courville. 2017. Improved training of wasserstein gans. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 5767–77. Han, Hui, Wenyuan Wang, and Binghuan Mao. 2005. Borderline-smote: A new over-sampling method in imbalanced data sets learning. In International Conference on Intelligent Computing. Berlin: Springer, pp. 878–87. Hanley, James A, and Barbara J. McNeil. 1982. The meaning and use of the area under a receiver operating characteristic (roc) curve. Radiology 143: 29–36. [CrossRef] [PubMed] Hartigan, J. A., and M. A. Wong. 1979. Algorithm as 136: A k-means clustering algorithm. Journal of the Royal Statistical Society. Series c (Applied Statistics) 28: 100–8. [CrossRef] He, Haibo, and Edwardo A. Garcia. 2008. Learning from imbalanced data. IEEE Transactions on Knowledge & Data Engineering 21: 1263–84. He, Kaiming, Xiangyu Zhang, Shaoqing Ren, and Jain Sun. 2015. Delving deep into rectifiers: Surpassing human-level performance on imagenet classification. Paper presented at the IEEE International Conference on Computer Vision, Santiago, Chile, December 7–13, pp. 1026–34. Hinton, Geoffrey Everest. 2002. Training products of experts by minimizing contrastive divergence. Neural Computation 14: 1771–800. [CrossRef] Hinton, Geoffrey Everest, Simon Osindero, and Yee-Why Teh. 2006. A fast learning algorithm for deep belief nets. Neural Computation 18: 1527–54. [CrossRef] Hinton, Geoffrey Everest, and Ruslan Russ Salakhutdinov. 2006. Reducing the dimensionality of data with neural networks. Science 313: 504–7. [CrossRef] Hinton, Geoffrey Everest, and Tiejman. Tieleman. 2012. Lecture 6.5—Rmsprop, Coursera: Neural Networks for Machine Learning. Available online: https://www.cs.toronto.edu/~tijmen/csc321/slides/lecture_slides_lec6.pdf (accessed on 28 August 2019). Hitawala, Saifuddin. 2018. Comparative study on generative adversarial networks. arXiv, arXiv:1801.04271. Hoffman, Judy, Eric Tzeng, Taesung Park, Jun-Yan Zhu, Phillip Isola, Kate Saenko, Alexei Efros, and Trevor Darrell. 2017. Cycada: Cycle-consistent adversarial domain adaptation. arXiv, arXiv:1711.03213. Hong, Weixiang, Zhenzhen Wang, Ming Yang, and Junsong Yuan. 2018. Conditional generative adversarial network for structured domain adaptation. Paper presented at the IEEE Conference on Computer Vision and Pattern Recognition, Salt Lake City, UT, USA, June 18–23, pp. 1335–44. Risks 2021, 9, 49 31 of 33 Hong, Yongjun, Uiwon Hwang, Jaeyoon Yoo, and Sungroh Yoon. 2019. How generative adversarial networks and their variants work: An overview of GAN. ACM Computing Surveys (CSUR) 52: 10. [CrossRef] Ioffe, Sergey, and Christian Szegedy. 2015. Batch normalization: Accelerating deep network training by reducing internal covariate shift. arXiv, arXiv:1502.03167. Jolicoeur-Martineau, Alexia. 2018. The relativistic discriminator: A key element missing from standard gan. arXiv, arXiv:1807.00734. Karras, Terro, Samuli Laine, and Timo Aila. 2019. A style-based generator architecture for generative adversarial networks. Paper presented at the IEEE Conference on Computer Vision and Pattern Recognition, Long Beach, CA, USA, June 15–20, pp. 4401–10. Kim, Taeskoo, Moonsu Cha, Hyunsoo Kim, Jungkwon Lee, and Jiwon Kim. 2017. Learning to discover cross-domain relations with generative adversarial networks. Paper presented at 34th International Conference on Machine Learning, Sydney, Australia, August 6–11, vol. 70, pp. 1857–65. Kingma, Diederik Pieter, and Jimmy Ba. 2014. Adam: A method for stochastic optimization. arXiv, arXiv:1412.6980. Kingma, Diederik Pieter, and Max Welling. 2013. Auto-encoding variational bayes. arXiv, arXiv:1312.6114. Kullback, Solomon. 1997. Information Theory and Statistics. North Chelmsford: Courier Corporation. Kullback, Solomon, and Richard Arthur Leibler. 1951. On information and sufficiency. The Annals of Mathematical Statistics 22: 79–86. [CrossRef] Larsen, Anders Boesen Lindbo, Søren Kaae Sønderby, Hugo Larochelle, and Ole Winther. 2015. Autoencoding beyond pixels using a learned similarity metric. arXiv, arXiv:1512.09300. Lee, Ronald D., and Lawrence R. Carter. 1992. Modeling and forecasting us mortality. Journal of the American Statistical Association 87: 659–71. Li, Stevan Cheng-Xian, Bo Jiang, and Benjamin Marlin. 2019. Misgan: Learning from incomplete data with generative adversarial networks. arXiv, arXiv:1902.09599. Li, Yujia, Kevin Swersky, and Richard Zemel. 2015. Generative moment matching networks. Paper presented at the 32nd International Conference on Machine Learning, Lille, France, July 6–11, pp. 1718–27. Lin, Jianhua. 1991. Divergence measures based on the shannon entropy. IEEE Transactions on Information Theory 37: 145–51. [CrossRef] Liu, Zhiyue, Jiahai Wang, and Zhiwei Liang. 2019. Catgan: Category-aware generative adversarial networks with hierarchical evolutionary learning for category text generation. arXiv, arXiv:1911.06641. Longadge, Rushi, and Snehalata Dongre. 2013. Class imbalance problem in data mining review. arXiv, arXiv:1305.1707. López, Victoria, Alberto Fernández, Salvado García, Vasile Palade, and Francisco Herrera. 2013. An insight into classification with imbalanced data: Empirical results and current trends on using data intrinsic characteristics. Information Sciences 250: 113–41. [CrossRef] Lucic, Mario, Karol Kurach, Marcin Michalski, Sylvain Gelly, and Olivier Bousquet. 2018. Are gans created equal? A large-scale study. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 700–9. Van der Maaten, Laurens, and Geoffrey Everest Hinton. 2008. Visualizing data using t-sne. Journal of Machine Learning Research 9: 2579–605. Makhzani, Alireza, Jonathon Shlens, Navdeep Jaitly, Ian Goodfellow, and Brendan Frey. 2015. Adversarial autoencoders. arXiv, arXiv:1511.05644. Manisha, Padala, and Sujit Gujar. 2018. Generative adversarial networks (gans): What it can generate and what it cannot? arXiv, arXiv:1804.00140. Mariani, Giovanni, Florian Scheidegger, Roxana Istrate, Costas Bekas, and Cristiano Malossi. 2018. Bagan: Data augmentation with balancing gan. arXiv, arXiv:1803.09655. Mathew, Josey, Ming Luo, Chee Khiang Pang, and Hian Leng Chan. 2015. Kernel-based smote for svm classification of imbalanced datasets. Paper presented at the IECON 2015-41st Annual Conference of the IEEE Industrial Electronics Society, Yokohama, Japan, November 9–12, pp. 1127–32. McCullagh, Peter. 1984. Generalized linear models. European Journal of Operational Research 16: 285–92. [CrossRef] Mirza, Mehdi, and Simon Osindero. 2014. Conditional generative adversarial nets. arXiv, arXiv:1411.1784. Mitchell, Tom M. 2006. The Discipline of Machine Learning. Pittsburgh: Machine Learning Department, School of Computer Science, Carnegie Mellon University, vol. 9. Miyato, Takeru, Shin-ichi Maeda, Masanori Koyama, and Shin Ishii. 2018. Virtual adversarial training: a regularization method for supervised and semi-supervised learning. IEEE Transactions on Pattern Analysis and Machine Intelligence 41: 1979–93. [CrossRef] [PubMed] Mottini, Alejandro, Alix Lheritier, and Rodrigo Acuna-Agost. 2018. Airline passenger name record generation using generative adversarial networks. arXiv, arXiv:1807.06657. Müller, Alfred. 1997. Integral probability metrics and their generating classes of functions. Advances in Applied Probability 29: 429–43. Nemenyi, Peter. 1962. Distribution-free multiple comparisons. In Biometrics. Princeton: Princeton University, vol. 18, p. 263. Nowozin, Sebastian, Botond Cseke, and Ryota Tomioka. 2016. f-gan: Training generative neural samplers using variational divergence minimization. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 271–279. Odena, Augustus 2016. Semi-supervised learning with generative adversarial networks. arXiv, arXiv:1606.01583. Odena, Augustus, Christopher Olah, and Jonathon Shlens. 2017. Conditional image synthesis with auxiliary classifier gans. Paper presented at the 34th International Conference on Machine Learning, Sydney, Australia, August 6–11, vol. 70, pp. 2642–51. Risks 2021, 9, 49 32 of 33 O’Malley, B. R., Robert E. Dorrington, Stephen C. Jurisich, J. A. Valentini, T. M. Cohen, and B. J. Ross. 2005. An investigation of the mortality of south african assured lives, 1995–1998. South African Actuarial Journal 5: 27–59. [CrossRef] Park, Noseong, Mahmoud Mohammadi, Kshitij Gorde, Sushil Jajodia, Hongkyu Park, and Youngmin Kim. 2018. Data synthesis based on generative adversarial networks. Proceedings of the Very Large Data Bases Endowment 11: 1071–83. [CrossRef] Pohlert, Thorsten 2014. The pairwise multiple comparison of mean ranks package (pmcmr). R Package 27: 10. Pozzolo, Andrea Dal. 2015. Adaptive Machine Learning for Credit Card Fraud Detection. Edited by Machine Learning Group. Brussels: Université Libre de Bruxelles. Python Software Foundation. 2017. Python Language Reference (Version 3.6. 3.). Amsterdam: Python Software Foundation. Qian, Ning. 1999. On the momentum term in gradient descent learning algorithms. Neural Networks 12: 145–51. [CrossRef] Radford, Alec, Luke Metz, and Soumith Chintala. 2015. Unsupervised representation learning with deep convolutional generative adversarial networks. arXiv, arXiv:1511.06434. Reed, Scott, Zeynep Akata, Xinchen Yan, Lajanugen Logeswaran, Bernt Schiele, and Honglak Lee. 2016. Generative adversarial text to image synthesis. arXiv, arXiv:1605.05396. Rezende, Danilo Jimenez, Shakir Mohamed, and Daan Wierstra. 2014. Stochastic backpropagation and approximate inference in deep generative models. arXiv, arXiv:1401.4082. Rubin, Donald Bruce. 2004. Multiple Imputation for Nonresponse in Surveys. Hoboken: John Wiley & Sons, vol. 81. Rubner, Yossi, Carlo Tomasi, and Leonidas Guibas. 2000. The earth mover’s distance as a metric for image retrieval. International Journal of Computer Vision 40: 99–121. [CrossRef] Ruder, Sebastian. 2016. An overview of gradient descent optimization algorithms. arXiv, arXiv:1609.04747. Rumelhart, David Everett, Geoffrey Everest Hinton, and Ronald J. Williams. 1986. Learning representations by back-propagating errors. Nature 323: 533. [CrossRef] Saito, Takaya, and Mark Rehmsmeier. 2015. The precision-recall plot is more informative than the roc plot when evaluating binary classifiers on imbalanced datasets. PLoS ONE 10: e0118432. [CrossRef] Salakhutdinov, Ruslan Russ, and Geoffrey Everest Hinton. 2009. Deep boltzmann machines. In Artificial Intelligence and Statistics. Berlin: Springer, pp. 448–55. Salimans, Tim, Ian Goodfellow, Wojciech Zaremba, Vicki Cheung, Alec Radford, and Xi Chen. 2016. Improved techniques for training gans. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 2234–42. Schafer, Joseph L., and Maren K. Olsen. 1998. Multiple imputation for multivariate missing-data problems: A data analyst’s perspective. Multivariate Behavioral Research 33: 545–71. [CrossRef] Schlegl, Thomas, Philipp Seeböck, Sebastian M. Waldstein, Ursula Schmidt-Erfurth, and Georg Langs. 2017. Unsupervised anomaly detection with generative adversarial networks to guide marker discovery. In International Conference on Information Processing in Medical Imaging. Cham: Springer, pp. 146–157. Shang, Chao, Aaron Palmer, Jiangwen Sun, Ko-Shin Chen, Jin Lu, and Jinbo Bi. 2017. Vigan: Missing view imputation with generative adversarial networks. Paper presented at 2017 IEEE International Conference on Big Data (Big Data), Boston, MA, USA, December 11–14, pp. 766–75. Smith, Jack W., James E. Everhart, W. C. Dickson, William C. Knowler, and Robert Scott Johannes. 1988. Using the adap learning algorithm to forecast the onset of diabetes mellitus. In Proceedings of the Annual Symposium on Computer Application in Medical Care. Bethesda: American Medical Informatics Association, pp. 261–65. Sricharan, Kumar, Raja Bala, Matthew Shreve, Hui Ding, Kumar Saketh, and Jin Sun. 2017. Semi-supervised conditional gans. arXiv, arXiv:1708.05789. Street, W. Nick, William H. Wolberg, and O. L. Mangasarian. 1993. Nuclear feature extraction for breast tumor diagnosis. In Biomedical Image Processing and Biomedical Visualization. Washington, DC: International Society for Optics and Photonics, vol. 1905, pp. 861–70. Tzeng, Eric, Judy Hoffman, Kate Saenko, and Trevor Darrell. 2017. Adversarial discriminative domain adaptation. Paper presented at the IEEE Conference on Computer Vision and Pattern Recognition, Honolulu, HI, USA, July 21–26, pp. 7167–76. Van den Oord, Aaron, Nal Kalchbrenner, Lasse Espeholt, Oriol Vinyals, and Alex Graves. 2016. Conditional image generation with pixelcnn decoders. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 4790–4798. Vondrick, Carl, Hamed Pirsiavash, and Antonio Torralba. 2016. Generating videos with scene dynamics. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 613–621. Voorhees, ELLEN M. 1986. Implementing agglomerative hierarchic clustering algorithms for use in document retrieval. Information Processing & Management 22: 465–76. Wang, Kunfeng, Chao Gou, Yanjie Duan, Yilun Lin, Xinhu Zheng, and Fei-Yue Wang. 2017. Generative adversarial networks: Introduction and outlook. IEEE/CAA Journal of Automatica Sinica 4: 588–98. [CrossRef] Wang, Shuo, and Xin Yao. 2009. Diversity analysis on imbalanced data sets by using ensemble models. Paper presented at 2009 IEEE Symposium on Computational Intelligence and Data Mining, Nashville, TN, USA, March 30–April 2, pp. 324–31. Xu, Lei, Maria Skoularidou, Alfredo Cuesta-Infante, and Kalyan Veeramachaneni. 2019. Modeling tabular data using conditional gan. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 7335–45. Yang, Li-Chia, Szu-Yu Chou, and Yi-Hsuan Yang. 2017. Midinet: A convolutional generative adversarial network for symbolic-domain music generation. arXiv, arXiv:1703.10847. Risks 2021, 9, 49 33 of 33 Yi, Zili, Hao Zhang, Ping Tan, and Minglun Gong. 2017. Dualgan: Unsupervised dual learning for image-to-image translation. Paper presented at the IEEE International Conference on Computer Vision, Venice, Italy, October 22–29, pp. 2849–57. Yoon, Jinsung, Daniel Jarrett, and Mihaela Van der Schaar. 2019. Time-series generative adversarial networks. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 5508–18. Yoon, Jinsung, James Jordon, and Mihaela Van Der Schaar. 2018. Gain: Missing data imputation using generative adversarial nets. arXiv, arXiv:1806.02920. Zhang, Huaxiang, and Mingfang Li. 2014. Rwo-sampling: A random walk over-sampling approach to imbalanced data classification. Information Fusion 20: 99–116. [CrossRef] Zhou, Xingyu, Zhisong Pan, Guyu Hu, Siqi Tang, and Cheng Zhao. 2018. Stock market prediction on high-frequency data using generative adversarial nets. Mathematical Problems in Engineering 2018: 4907423. [CrossRef] Zhu, Jun-Yan, Taesung Park, Phillip Isola, and Alexei A. Efros. 2017. Unpaired image-to-image translation using cycle-consistent adversarial networks. Paper presented the IEEE International Conference on Computer Vision, Venice, Italy, October 22–29, pp. 2223–32.
https://openalex.org/W1854386788
http://pure-oai.bham.ac.uk/ws/files/22038230/Ambrose_et_al_Use_of_Aleuria_alantia_Proteomes_2015.pdf
English
null
Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer
Proteomes
2,015
cc-by
8,897
Publisher Rights Statement: Publisher Rights Statement: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Publisher Rights Statement: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted reproduction in any medium, provided the original work is properly cited. Checked October 2015 Checked October 2015 General rights U l li g Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. U d l d d/ i t f th bli ti f th U i •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the Univer study or non-commercial research. Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for th study or non-commercial research. •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. played above, please note the terms and conditions of the licence govern your use of this document. here a licence is displayed above, please note the terms and conditions of the licence govern your use of this document When citing, please reference the published version. Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer Ambrose, Sarah; Gordon, Naheema; Goldsmith, James; Wei, Wenbin; Zeegers, Maurice; Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Ambrose, S, Gordon, N, Goldsmith, J, Wei, W, Zeegers, M, James, N, Knowles, M, Bryan, R & Ward, D 2015, 'Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer', Proteomes, vol. 3, no. 3, pp. 266-282. https://doi.org/10.3390/proteomes3030266 Link to publication on Research at Birmingham portal Link to publication on Research at Birmingham portal Publisher Rights Statement: Citation for published version (Harvard): Ambrose, S, Gordon, N, Goldsmith, J, Wei, W, Zeegers, M, James, N, Knowles, M, Bryan, R & Ward, D 2015, 'Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer', Proteomes, vol. 3, no. 3, pp. 266-282. https://doi.org/10.3390/proteomes3030266 Proteomes 2015, 3, 266-282; doi:10.3390/proteomes3030266 Article OPEN ACCESS proteomes ISSN 2227-7382 www.mdpi.com/journal/proteomes Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer Sarah R. Ambrose 1, Naheema S. Gordon 1, James C. Goldsmith 1, Wenbin Wei 1, Maurice P. Zeegers 1,2, Nicholas D. James 3, Margaret A. Knowles 4, Richard T. Bryan 1 and Douglas G. Ward 1,* 1 School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK; E-Mails: SRA295@student.bham.ac.uk (S.R.A.); n.gordon@bham.ac.uk (N.S.G.); jgoldsmith38@googlemail.com (J.C.G.); w.wei@bham.ac.uk (W.W.); m.zeegers@maastrichtuniversity.nl (M.P.Z.); r.t.bryan@bham.ac.uk (R.T.B.) 2 Department of Complex Genetics, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre, Maastricht 6200 MD, The Netherlands 3 Clinical Trials Unit, University of Warwick, Coventry CV4 7AL, UK; E-Mail: N.D.James@warwick.ac.uk 4 Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James’s’ University Hospital, Beckett Street, Leeds LS9 7TF, UK; E-Mail: M.A.Knowles@leeds.ac.uk * Author to whom correspondence should be addressed; E-Mail: d.g.ward@bham.ac.uk; Tel.: +44-121-414-9528. Academic Editor: Michael A. Tainsky Received: 30 June 2015 / Accepted: 27 August 2015 / Published: 3 September 2015 Abstract: Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun i f h l i h d f f bl dd ll li Proteomes 2015, 3, 266-282; doi:10.3390/proteomes3030266 Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 1. Introduction Urothelial bladder cancer is the fourth most common cancer in men and ninth most common cancer in women in western societies [1]. This highly heterogeneous disease presents as a spectrum from low-grade non-invasive tumours with a good prognosis but high recurrence rate through to high-grade muscle invasive tumours with a very poor prognosis. Low grade tumours tend to have a near-normal karyotype with few genomic rearrangements and often have activating mutations in FGFR3 and the MAPK pathway, whereas high-grade tumours typically have inactivating mutations in TP53 and/or other tumour suppressor genes and multiple chromosomal aberrations [2]. Algorithms based on clinicopathological factors can be used to guide treatment, which ranges from transurethral resection and surveillance for recurrence for low-risk disease through to cystectomy and systemic chemotherapy for muscle-invasive and metastatic disease [3]. Bladder tumours are typically detected by flexible cystoscopy, a burdensome and resource-intensive procedure. Patients undergoing surveillance for bladder cancer will require this procedure at regular intervals for many years [4]. There is thus a need for a urine or blood-based test to reduce reliance on cystoscopy. Despite extensive research, most candidate urinary biomarkers for bladder cancer do not show sufficient sensitivity and specificity to replace cystoscopy [5]. Most of the proposed biomarkers are particularly poor at detecting low-grade NMIBC. Indeed, no urinary biomarkers have been validated as having sufficient sensitivity and specificity to be widely adopted in clinical practice [5]. A number of urinary biomarkers have been proposed for the detection of bladder cancer including tests based on miRNA [6], RNA [7], DNA [8], metabolites [9] and proteins [10]. The latter may be measured in exfoliated cells (e.g., ImmunoCyt®) or as soluble proteins in the urine e.g., NMP22 and BTA. Unfortunately, none of these tests are both highly specific and sensitive for early stage and low grade disease. Nucleic acid tests based on DNA methylation and mutations have the advantage over other biomarkers in identifying the presence of a disease-specific variant of the molecule that is being detected, rather than the total level of a molecule which may be released from both cancer and normal cells. Because bladder cancer is highly heterogeneous at the molecular level it is likely that a panel of biomarkers will be required to detect all tumours. Theoretically, highly specific markers can be combined to generate an effective test. Proteomes 2015, 3 267 Proteomes 2015, 3 glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1 as proteins warranting further investigation as urinary biomarkers for low-grade bladder cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring lectin binding to unfractionated proteomes, but it is possible that more specific reagents and/or a focus on individual proteins may produce clinically useful biomarkers. Keywords: bladder cancer; urine; biomarker; lectin; glycoproteome Keywords: bladder cancer; urine; biomarker; lectin; glycoproteome Keywords: bladder cancer; urine; biomarker; lectin; glycoproteome Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer Sarah R. Ambrose 1, Naheema S. Gordon 1, James C. Goldsmith 1, Wenbin Wei 1, Maurice P. Zeegers 1,2, Nicholas D. James 3, Margaret A. Knowles 4, Richard T. Bryan 1 and Douglas G. Ward 1,* Sarah R. Ambrose 1, Naheema S. Gordon 1, James C. Goldsmith 1, Wenbin Wei 1, Maurice P. Zeegers 1,2, Nicholas D. James 3, Margaret A. Knowles 4, Richard T. Bryan 1 and Douglas G. Ward 1,* 1 School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK; E-Mails: SRA295@student.bham.ac.uk (S.R.A.); n.gordon@bham.ac.uk (N.S.G.); jgoldsmith38@googlemail.com (J.C.G.); w.wei@bham.ac.uk (W.W.); m.zeegers@maastrichtuniversity.nl (M.P.Z.); r.t.bryan@bham.ac.uk (R.T.B.) 1 School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK; E-Mails: SRA295@student.bham.ac.uk (S.R.A.); n.gordon@bham.ac.uk (N.S.G.); jgoldsmith38@googlemail.com (J.C.G.); w.wei@bham.ac.uk (W.W.); m.zeegers@maastrichtuniversity.nl (M.P.Z.); r.t.bryan@bham.ac.uk (R.T.B.) 2 Department of Complex Genetics, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre, Maastricht 6200 MD, The Netherlands 3 Clinical Trials Unit, University of Warwick, Coventry CV4 7AL, UK; E-Mail: N.D.James@warwick.ac.uk 4 Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James’s’ University Hospital, Beckett Street, Leeds LS9 7TF, UK; E-Mail: M.A.Knowles@leeds.ac.uk * Author to whom correspondence should be addressed; E-Mail: d.g.ward@bham.ac.uk; Tel.: +44-121-414-9528. Academic Editor: Michael A. Tainsky Academic Editor: Michael A. Tainsky Received: 30 June 2015 / Accepted: 27 August 2015 / Published: 3 September 2015 Received: 30 June 2015 / Accepted: 27 August 2015 / Published: 3 September 2015 Abstract: Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun proteomics. None of the lectins showed a strong preference for bladder cancer cell lines over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over those from non-cancer controls. However, several lectins showed a strong preference for bladder cell line glycans over serum glycans and are potentially useful for enriching Proteomes 2015, 3 Numerous proteomic approaches have been applied to analyse urine in the search for bladder cancer biomarkers (reviewed in [10]). The urinary proteome is challenging to mine in depth, in part due to an abundance of plasma proteins, and many proteomic studies have suggested plasma proteins as biomarkers despite the fact that they are unlikely to be specific for bladder cancer. For example, Chen et al. carried out quantitative shotgun proteomics on pooled bladder cancer urine samples and non-cancer controls using iTRAQ for relative quantitation, followed by MRM quantitation of candidate biomarkers producing a multimarker panel with a ROC AUC of 0.814 [11,12]. The “biomarkers”, however, are moderately abundant plasma proteins rather than cancer-specific proteins. Top-down approaches have the ability to detect proteoforms not readily distinguishable in bottom-up approaches. However, to date, CE-MS and MALDI based profiling have failed to generate a highly sensitive and specific test for bladder cancer [13–15]. A small number of studies have used lectin affinity chromatography in studies of the urinary glycoproteome, however these studies have used broad specificity lectins (expected to capture most glycoproteins) rather than focussing on alterations in glycosylation [16,17]. Kreunin et al. [16] compared the glycoproteome of urine from bladder cancer patients and non-cancer patients using Concanavalin A (ConA) affinity chromatography combined with nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS). Alpha-1β-glycoprotein was identified as the most discriminatory protein, but again, this is a plasma protein. It has been reported that protein glycosylation is significantly altered in many cancers including bladder cancer [18]. Alterations in glycosylation patterns in cancer reflect changes in expression of glycosyltransferases and glycosidases [19]. The changes to glycan structure that can occur in cancer include O-glycan truncation, increased branching of N-glycans, and increased sialylation, sulfation and fucosylation [18]. There have been several studies on the glycosylation state of specific proteins found in the sera of cancer patients. For example, increased fucosylation and sialylation of PSA have been reported in prostate cancer [20]. Wu and colleagues used a fucose specific lectin, Aleuria aurantia lectin (AAL) to characterise the fucosylation of haptoglobin in ovarian cancer and found increased levels of fucosylated haptoglobin in patient sera [21]. Another study observed glycan-specific changes in periostin and thrombospondin in ovarian cancer [22]. All of these studies found that the specific protein glycoforms were able to better differentiate cancer sera from control serum samples than the total concentration of the protein. 1. Introduction However, total levels of protein markers may be influenced by non-malignant conditions and haematuria, limiting specificity and therefore suitability for inclusion in multimarker tests. A test based on bladder cancer-specific variants of proteins would be expected to outperform a test based on the total levels of these proteins. 268 2.2. Patient Samples Patient urine and serum samples were collected as part of the Bladder Cancer Prognosis Programme (BCPP) [25]. Recruitment to BCPP was undertaken between 2005 and 2011 and consists of samples from patients with suspected primary bladder cancer. Midstream urine was collected, centrifuged at 2000 rpm for 10 min and the supernatant stored in aliquots at −80°C. After sample collection, each patient underwent TURBT and definitive diagnosis by histopathological examination of the resected tissue. Urine samples obtained from patients with non-malignant conditions were retained in the BCPP collection and have been used as non-cancer controls. For the lectin ELISA experiments, urine samples were pooled into four control pools (two normal and two from patients with cystitis or inflammation) and six cancer pools (two from each of stages pTa, pT1 and pT2+). For the proteomics experiments, two pools of pTa patient urine were used (n = 75; n = 36) and a non-cancer control urine pool (n = 28). All samples used were negative for haematuria by dipstick test. Further patient information is provided in Supplemental Materials. 2.1. Materials and Cell Lines All lectins were purchased from Vector Laboratories Ltd. (Peterborough, UK). All other chemicals and materials were purchased from Sigma-Aldrich (St. Louis, MI, USA) unless otherwise stated. All lectins were purchased from Vector Laboratories Ltd. (Peterborough, UK). All other chemicals and materials were purchased from Sigma-Aldrich (St. Louis, MI, USA) unless otherwise stated. The bladder cancer cell lines 5637 and HB-CLS-2 were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). NHU-TERT, VM-CUB-1, MGH-U3, RT4, RT112, SW780 and T24 were provided by Professor Margaret Knowles (University of Leeds, Leeds, UK). The UROtsa cell line was a gift from Alexander Dowell (University of Birmingham, Birmingham, UK). Cell lines were cultured as previously described [24]. The bladder cancer cell lines were derived from tumours of different grades as detailed in Supplemental Table S1. Cell lysates were prepared by sonication in PBS containing cOmplete EDTA-free protease inhibitor cocktail followed by a 10 min centrifugation at 13,000 rpm. The protein concentration of the supernatant was determined by Bradford assay. Proteomes 2015, 3 Perhaps the best example of a cancer specific glycoform is AFP-L3. An increase in total AFP concentration in the serum was originally used as an indicator for hepatocellular carcinoma (HCC); however, measuring the total AFP concentration cannot always discriminate between small HCCs and chronic liver disease. Further study of the protein identified a core-fucosylated form of AFP known as AFP-L3 which is specific to HCC and which can be measured in the serum to distinguish between HCC and chronic liver disease, making it a clinically useful biomarker [23]. Identifying a similarly cancer-specific glycoprotein biomarker for bladder cancer could be the answer to finding an accurate non-invasive test for disease detection and long-term surveillance of patients. In the experiments reported here we test the hypothesis that incorporating selective lectin chromatography into urinary proteomics workflows has the potential to uncover urinary biomarkers for bladder cancer. We focus on low grade non-invasive bladder cancer as this is the form of the disease which is most challenging to detect using currently available non-invasive tests. 269 Proteomes 2015, 3 Proteomes 2015, 3 2.6. Filter-Aided Sample Preparation and Tryptic Digestion Up to 200 µg of protein was dissolved in 9 M Urea, 1% CHAPS (Melford Laboratories, Ipswich, UK) in 100 mM triethylammonium bicarbonate (TEAB), and incubated with 20 mM dithiothreitol (DTT) for 30 min at room temp. Following addition of 50 mM iodoacetamide the proteins were captured in 0.5 mL 30 kDa MWCO centrifugal filters, centrifuged at 13,000 rpm for five minutes and washed four times with 100 mM TEAB. Proteins were digested by incubating overnight at 37 °C with 5 μg of sequencing grade trypsin (Promega). Peptides were collected by centrifugation. 2.4. Lectin Dot Blots Samples were diluted (cell lysate or serum diluted to 3 ug protein/mL in PBS and urine diluted ×50 in PBS and 2 μL spotted onto nitrocellulose membrane. Once dry, the membrane was blocked with 1% BSA in PBS for 40 min and then washed three times with PBST. The membrane was incubated in a lectin solution at 10 ug/mL in PBS for 30 min, washed with PBST and incubated with streptavidin-HRP (as above). The membrane was washed with PBST and imaged using ECL and photographic film. 2.5. Lectin Affinity Chromatography Lectin conjugated agarose beads were washed 10 times with PBS to remove sugars in their storage solution and 500 µL of 50% slurry mixed with 5 mL of pooled urine and 500 μL of 12 × PBS. Binding was allowed to occur during a 2 h incubation on a rotating mixer at 4°C. The beads were captured on filters and the flow through was collected and stored. The beads were then washed thoroughly with PBS and bound glycoproteins eluted with 2 × 400 μL of 100 mM L-fucose or 200 mM N-aceytl-D-galactosamine (GalNAc). In experiments using UEA1 and DBA (which require divalent cations) PBS was substituted with 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 100 uM MnCl2, 100 uM ZnSO4 and 20 mM MOPS, pH 7.4. All lectin affinity-chromatography–shotgun-proteomics experiments were performed as 2 independent replicates. Proteomes 2015, 3 Proteomes 2015, 3 270 2.7. Stable Isotope Labelling After tryptic digestion, formaldehyde was added to the control urine peptides to a final concentration of 0.2% w/v and deuterated formaldehyde was added to the pTa patient urine peptides. Sodium cyanoborohydride was added to both samples to a final concentration of 25 mM. After 30 min, 0.5 M ammonium bicarbonate was added to quench the reaction. The control and cancer urine samples were then combined and acidified with 10% trifluoroacetic acid (TFA), the peptides were captured on a C18 cartridge, washed with 0.1% TFA and peptides eluted with 600 μL of 60% acetonitrile (ACN)/0.1% TFA. 2.3. Lectin ELISAs Urine samples were diluted ×50 in PBS and cell lysates and serum samples were diluted to 3 ug protein/mL in PBS and 100 µL added to 96 well Maxisorb Immuno-plates followed by a 1 hour incubation at 37°C to adsorb proteins. The wells were washed three times with 200 μL PBS containing 0.05% w/v Tween® 20 (PBST, Sigma-Aldrich). The plates were blocked with 1% BSA in PBS for one hour. After washing with PBST, 100 μL of biotinylated lectin at 10 μg/mL in PBS was added to the wells and incubated for 30 min. After washing with PBST, 100 μL of a 1 in 200 dilution of streptavidin conjugated to horseradish peroxidase (R&D Systems, Abingdon, UK) in 1% BSA in PBS was added and incubated for 30 min. The plates were then washed five times with PBST and 100 μL of substrate solution (3,3′,5,5′-tetramethlbenzidine) was added. The reaction was stopped with 40 μL of 2M H2SO4 and the absorbance measured at 450 nm. All ELISAs were performed in triplicate and means compared across experimental groups using t-tests. 3.1. Lectin Binding to Urothelial Cell Line Lysates Lectin ELISAs were used to evaluate the ability of 25 different lectins to bind to cell lysates of 2 normal urothelial human cell lines and 8 human bladder cancer cell lines derived from tumours of different grades (potentially allowing us to determine how glycosylation differs between low- and high-grade disease). Although different cell lines show quite different lectin binding profiles, none of the lectins showed consistent, substantially different binding between the normal and cancer cell lines or between cancer cell lines derived from low or high grade tumours (p > 0.05 in all cases). The data are summarised in Figure 1. Figure 2 demonstrates the specificity of AAL for fucose containing substrates: including 100 mM L-Fucose during the incubation of AAL on urothelial cell line lysate coated plates effectively prevents any binding from taking place. Proteomes 2015, 3 used was 0%–50% of buffer B (2 mM ammonium formate, pH 3.0, 80% ACN) for 45 min followed by 50%–100% buffer B over five minutes and then 100%–0% buffer B for the last 10 min. Fractions were dried and reconstituted in 0.1% formic acid (FA) in water. The peptides in each fraction were analysed by LC-MS/MS using a 60 min gradient of 0%–36% ACN in 0.1% FA at a flow rate of 350 nL/min with an Acclaim® Pepmap C18, 3 μm, 100 Å column (25 cm × 75 μm) (Thermo Scientific, Loughborough, UK) attached to an Ultimate 3000 RS HPLC system coupled to an Impact Quadrupole-TOF mass spectrometer (Bruker Daltonics, Coventry, UK) working in data-dependent mode at 5 MS/MS per cycle. 2.9. Peptide Identification and Analysis All MS/MS spectra were searched against a database containing Swissprot human sequences and randomized versions thereof using MASCOT (version 2.3 Matrix Science Ltd, London, UK). Search parameters were as follows: (i) species: Homo sapiens; (ii) enzyme: trypsin; (iii) ≤2 missed cleavages; (iv) 10 ppm precursor ion tolerance; (v) 0.02 Da fragment ion tolerance; (vi) fixed modifications: cysteine carbamidomethylation; (vii) variable modifications: methionine oxidation; (viii) a peptide score of >25. For quantitative experiments light and heavy dimethylation of N-termini and lysine residues were also included as variable modifications. Proteinscape 3 software (Bruker Daltonics) was used to combine multiple search results and filter the data using a protein false discovery rate of 1%. WARP-LC (Bruker Daltonics) was used for relative quantitation based on extracted ion chromatograms and limma used for statistical analysis of differential expression [26]. 2.8. Peptide Fractionation and LC-MS/MS Peptides were dried and dissolved in mixed mode buffer A (110 μL 20 mM ammonium formate, pH 6.5, 3% ACN) and separated into 16 fractions using an Acclaim® Mixed-Mode WAX-1, 3 um, 120 Å (2.1 × 150 mm) column (Dionex, Camberley, UK) at a flow rate of 100 μL/min. The elution gradient 271 3.2. Lectin Binding Properties of Urinary Proteins The binding of the same panel of 25 lectins to the proteins in pooled urine from patients without bladder cancer or with pTa, pT1 or pT2+ bladder cancer was tested by ELISA. None of the lectins demonstrated a statistically significant higher or lower binding (p > 0.05) to the proteins in the urine of cancer patients relative to urine of non-cancer controls (Figure 3). 272 Proteomes 2015, 3 272 teomes 2015, 3 2 Figure 1. Lectin binding to urothelial cell lysates. Cell lines are shown from left to right, non-cancer (NHU-TERT & UROtsa), grade 1 (MGH-U3, RT4, SW780), grade 2 (RT112, VM-CUB-1, 5637) and grade 3 bladder cancer (T24, HB-CLS-2). The relative binding level of each lectin to the cell lines is shown on a sliding scale from high (red) to low (blue). , Figure 1. Lectin binding to urothelial cell lysates. Cell lines are shown from left to right, non-cancer (NHU-TERT & UROtsa), grade 1 (MGH-U3, RT4, SW780), grade 2 (RT112, VM-CUB-1, 5637) and grade 3 bladder cancer (T24, HB-CLS-2). The relative binding level of each lectin to the cell lines is shown on a sliding scale from high (red) to low (blue). Figure 1. Lectin binding to urothelial cell lysates. Cell lines are shown from left to right, non-cancer (NHU-TERT & UROtsa), grade 1 (MGH-U3, RT4, SW780), grade 2 (RT112, VM-CUB-1, 5637) and grade 3 bladder cancer (T24, HB-CLS-2). The relative binding level of each lectin to the cell lines is shown on a sliding scale from high (red) to low (blue). Absorbance 450 nm 0.900 0.800 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 - Fucose + Fucose Absorbance 450 nm 0.900 0.800 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 - Fucose + Fucose Figure 2. Fucose inhibition of AAL binding to cell lysates. Lectin ELISA results are shown for AAL binding (absorbance 450 nm) to cell lysates ±100 mM L-fucose. Figure 2. Fucose inhibition of AAL binding to cell lysates. Lectin ELISA results are shown for AAL binding (absorbance 450 nm) to cell lysates ±100 mM L-fucose. 273 Proteomes 2015, 3 273 Figure 3. Lectin binding to pooled urine samples. Each pooled urine samples consists of urine from >8 patients with: C1&C2 = no abnormality detected, C3&C4 = no malignant disease (cystitis/inflammation), Ta1 & Ta2 = pTa UBC, T1-1 &T1-2 = pT1 UBC, T2-1 & T2-2 = MIBC. 3.3. Lectins with Selectivity for Urothelial Glycans Relative to Plasma Glycans A lectin that could enrich urothelial glycoproteins relative to plasma glycoproteins in urine would be a useful tool in urine proteomics. We therefore compared the binding of the 25 lectins to bladder cell line lysates with their binding to serum from non-cancer control subjects by lectin ELISA and confirmed selected results with lectin dot blots (Figure 4). Whilst RCA1 and PHA-E bound more strongly to serum than to cell lysate, the majority of lectins preferred the lysate with 10 lectins showing very low binding to serum. These lectins, in particular UEA1 and DBA, showed very high cell:serum binding ratios. Lectins that bind mannose, glucose or sialic acid tended to show low binding to urothelial glycans whereas fucose, galactose and N-acetylgalactosamine binding lectins exhibited high binding to urothelial glycans relative to serum. 3.2. Lectin Binding Properties of Urinary Proteins The relative binding level of each lectin to the 8 pooled urines is shown on a sliding scale from high (red) to low (blue). Figure 3. Lectin binding to pooled urine samples. Each pooled urine samples consists of urine from >8 patients with: C1&C2 = no abnormality detected, C3&C4 = no malignant disease (cystitis/inflammation), Ta1 & Ta2 = pTa UBC, T1-1 &T1-2 = pT1 UBC, T2-1 & T2-2 = MIBC. The relative binding level of each lectin to the 8 pooled urines is shown on a sliding scale from high (red) to low (blue). 3.4. Glycoproteome Analysis of Pooled pTa UBC Patient Urine Shotgun proteomics was used to assess the ability of AAL, UEA and DBA affinity chromatography to extract subproteomes from a pooled bladder cancer urine sample (75 patients with G1-G3 pTa disease). UEA1 and DBA were chosen for glycoprotein enrichment due to their striking preference for urothelial proteins over serum proteins and AAL was chosen because it not only displayed a preference for urothelial proteins over serum proteins, but has previously been shown to have an affinity for cancer-related glycoproteins. LC-MS/MS analysis of the proteins bound to UEA1 or DBA and eluted with 100 mM L-fucose or 200 mM N-aceytl-D-galactosamine respectively identified surprisingly few proteins: DBA captured 140 proteins of which 75 were found in two experimental replicates and UEA1 captured 122 proteins of which 69 were common to both replicates. Furthermore, these proteins included keratins and abundant proteins such as uromodulin and albumin indicative of non-specific binding. In contrast, in both experimental replicates, more than 500 proteins were eluted from AAL with 100 mM L-Fucose. There was a high degree of overlap between the proteins identified 274 Proteomes 2015, 3 Proteomes 2015, 3 in the AAL eluates in both replicates with 436 protein identifications common to both (Figure 5). Of these 436 proteins, 285 contain glycosylation sites (65%). Of these 285 glycoproteins, 274 possess N- linked glycans and 24 possess O-linked glycans. Figure 4. Lectin binding to urothelial cell line lysates and serum. The histogram shows data from lectin ELISAs run in triplicate (pooled cell lysate and pooled serum). The inserted panel shows confirmatory dot blots With the exception of PHA E SNA and LCA all Figure 4. Lectin binding to urothelial cell line lysates and serum. The histogram shows data from lectin ELISAs run in triplicate (pooled cell lysate and pooled serum). The inserted panel shows confirmatory dot blots. With the exception of PHA-E, SNA and LCA all lectins showed significantly different binding to lysates and serum (p < 0.05). Figure 4. Lectin binding to urothelial cell line lysates and serum. The histogram shows data from lectin ELISAs run in triplicate (pooled cell lysate and pooled serum). The inserted panel shows confirmatory dot blots. With the exception of PHA-E, SNA and LCA all lectins showed significantly different binding to lysates and serum (p < 0.05). AAL eluate duplicates Flow-through duplicates 114 436 119 130 449 94 161 275 174 Figure 5. The number of proteins identified by LC-MS/MS in the AAL flow-through and eluted fractions. The upper Venn diagrams show the number of proteins identified in the flow-through and eluted fractions of two independent AAL chromatographies of a pooled urine sample from patients with pTa bladder cancer. The lower Venn diagram shows the overlap between the proteins identified in both eluates and both flow-throughs. AAL eluate duplicates Flow-through duplicates 114 436 119 130 449 94 161 275 174 174 161 Figure 5. The number of proteins identified by LC-MS/MS in the AAL flow-through and eluted fractions. The upper Venn diagrams show the number of proteins identified in the flow-through and eluted fractions of two independent AAL chromatographies of a pooled urine sample from patients with pTa bladder cancer. The lower Venn diagram shows the overlap between the proteins identified in both eluates and both flow-throughs. Proteomes 2015, 3 275 The AAL flow throughs (i.e., proteins not captured by AAL) were also analysed by LC-MS/MS and the protein identifications compared with the proteins identified in the AAL eluate to determine which proteins were enriched by the AAL affinity chromatography. We defined proteins as enriched by AAL if they were present in both eluates and were identified by at least twice as many peptides in the eluates as in the flow throughs. Using these criteria, AAL enriched 186 proteins. Of these 186 enriched proteins, 115 (62%) contain glycosylation sites and 84 of these proteins were not identified in the flow-throughs. Of the 186 enriched proteins, 70 are associated with the extracellular space and 71 with the plasma membrane. This suggests that AAL lectin affinity chromatography may be able to identify proteins originating from the urothelial cell surface or proteins released from the urothelium. The levels of abundant plasma proteins were decreased in the AAL eluates relative to the flow-thoughs: albumin decreased from 8307 peptide spectrum matches in the flow-throughs to 585 in the eluates and serotransferrin decreased from 950 to 76 peptide spectrum matches. Information on the protein identifications are provided in Supplemental Materials. 3.5. Quantitative Comparison of AAL Binding Proteins in the Urine of Control Subject and Patients with G1 pTa Bladder Cancer 3.5. Quantitative Comparison of AAL Binding Proteins in the Urine of Control Subject and Patients with G1 pTa Bladder Cancer AAL affinity chromatography was performed on pooled urine samples from patients with G1 pTa bladder cancer (n = 36) and non-cancer controls (n = 28). The eluted proteins were digested and the peptides stable isotope labelled as described in the method section, the samples combined and analysed by shotgun proteomics. Duplicate experiments again proved reproducible with 394 protein identified in both AAL eluates. We also analysed the 2 pooled urine samples without AAL enrichment in duplicate with 501 protein identifications common to replicates. The heavy/light peptide intensity ratios were used to estimate the relative levels of proteins in the two pooled urine samples in the AAL eluate and whole urine datasets. In the AAL eluates the concentrations of 21 proteins were significantly (p << 0.01) and substantially (≥2-fold increase in both experimental replicates) higher in the pooled pTa urine than the control urine. Of these, 12 proteins were also increased in the cancer sample in the whole urine experiments whereas 9 of the proteins were increased in cancer only in the AAL eluates. The 12 proteins elevated in cancer in both the whole urine and AAL eluates are likely to be present at a higher total concentration in the cancer sample whereas the 9 proteins increased in the AAL eluates but not whole urine could be cancer specific glycoforms. Of the 9 proteins with apparent altered glycosylation (rather than simply an increase in total concentration), 6 were previously identified as released by bladder cancer cell lines in vitro [24] (mucin-1 (MUC1), golgi apparatus protein 1 (GLG1), endoplasmin (HSP90B1), prostatic acid phosphatase (ACPP), Ig gamma-2 chain C region (IGHG2), and deoxyribonuclease-2-alpha (DNASE2A)), and 3 were not (voltage-dependent anion-selective channel 1, carbonic anhydrase 1 and bile salt-activated lipase 11) . The cancer:normal intensity ratio in the AAL eluate and the whole urine data for the proteins previously identified as released by bladder cancer cell lines is shown in Figure 6. 276 Proteomes 2015, 3 Cancer: normal ratio 4 3.5 3 2.5 2 1.5 1 0.5 0 Cancer: normal ratios in whole urine and AAL enriched urine MUC1 HSP90B1 GLG1 ACPP IGHG2 DNASE2A Proteins whole urine AAL Figure 6. AAL binding proteins and their cancer: normal peptide intensity ratios for the whole urine experiment and the AAL experiment. 3.5. Quantitative Comparison of AAL Binding Proteins in the Urine of Control Subject and Patients with G1 pTa Bladder Cancer Mucin-1 (MUC1), endoplasmin (HSP90B1), golgi apparatus protein 1 (GLG1), prostastic acid phosphatase (ACPP), Ig gamma-2 chain C region (IGHG2), and deoxyribonuclease-2-alpha (DNASE2A) all have a greater cancer: normal ratio after AAL enrichment. Cancer: normal ratios in whole urine and AAL enriched urine Cancer: normal ratios in whole urine and AAL enriched urine Cancer: normal ratios in whole urine and AAL enriched urine Cancer: normal ratio 4 3.5 3 2.5 2 1.5 1 0.5 0 Cancer: normal ratios in whole urine and AAL enriched urine MUC1 HSP90B1 GLG1 ACPP IGHG2 DNASE2A Proteins whole urine AAL Figure 6. AAL binding proteins and their cancer: normal peptide intensity ratios for the whole urine experiment and the AAL experiment. Mucin-1 (MUC1), endoplasmin (HSP90B1), golgi apparatus protein 1 (GLG1), prostastic acid phosphatase (ACPP), Ig gamma-2 chain C region (IGHG2), and deoxyribonuclease-2-alpha (DNASE2A) all have a greater cancer: normal ratio after AAL enrichment. Proteomes 2015, 3 277 cancer [28]. The literature on lectin binding to bladder cancer tissues is complex with no clear consensus as to which lectins bind preferentially to tumours over normal tissue or relationships of lectin binding to stage, grade and outcome [29,30]. These data indicate that specific glycoprotein markers will be required rather than global changes in glycosylation. Perhaps our most important finding is that several lectins are selective for urothelial proteins over plasma proteins. This is a useful characteristic for bladder cancer urine proteomics because low abundance urothelial proteins can be masked from detection by mass spectrometry by the presence of highly abundant serum proteins in the urine. The broad specificity lectins WGA and ConA that have been used in previous urinary glycoprotoemic studies [16,17] however, are not selective for urothelial proteins over plasma proteins. When we tested the ability of the 2 lectins with the greatest selectivity for urothelial proteins, UEA1 and DBA, to enrich urothelial glycoproteins from urine we identified only a small number of proteins. Perhaps these unexpected results are because the lectins recognise cellular proteins that are not released into the urine or because the eluting sugars were unable to effectively compete with the glycoprotein-lectin interaction. Nonetheless, it would seem that the other lectins with a high lysate/serum ratio (PNA, SJA, GSL I, SBA, PHA-L, STL, VVA) should be considered in urinary glycoproteomic workflows. AAL was selected in this study partly because it showed a small preference for urothelial proteins but more so because it has previously been used to enrich aberrantly glycosylated proteins in HCC [31]. AAL is specific for core and terminal fucose structures including fucose (α-1,6) linked to N-acetylglucosamine and fucose (α-1,3) linked to structures related to N-acetyllactosamine (85). The fucose (α-1,6) residue is a core fucose structure that is present in many mammalian tissues and has been reported to be altered in pathological settings. Of the 580 unique proteins captured by AAL, 6 behave as if they are aberrantly glycosylated in bladder cancer: mucin-1, golgi apparatus protein 1, prostatic acid phosphatase, endoplasmin, deoxyribonuclease-2-alpha and Ig gamma-2 chain C region. The selection was based on the fact that they are all released by bladder cell lines [24] and it appears that it is their glycosylation status rather than the total quantity of these proteins which change in bladder cancer. 4. Discussion Current urinary protein biomarkers for bladder cancer lack the sensitivity and/or specificity required to have utility in the clinic. Aberrant glycosylation has been widely reported in cancer and some cancer biomarkers utilise changes in protein glycosylation to improve biomarker effectiveness. In this study we investigated whether cancer specific glycoforms are a feature of bladder cancer and whether lectin affinity chromatography is a useful tool in urine proteomics. In summary, the binding of 25 different lectins to bladder cancer cell lines or urine from control subjects and patients with bladder cancer did not detect any major global changes in glycosylation. We did, however, find that many lectins have a preference for bladder cancer cell line proteins over plasma proteins and this may be useful for discriminating between proteins that are released into the urine from the urothelium and those that are filtered through the kidney or leak in due to haematuria. A recent paper by Yang et al. reported that the binding of many lectins differed significantly between a normal urothelial cell line (HCV29) and several bladder cancer cell lines, and went on to show increased binding of LCA and SNA and decreased binding of ConA to bladder cancer tissue relative to adjacent normal tissue [27]. The use of different reference cell lines (NHU-TERT and UROtsa) in our study may be the reason that we did not see clear differences between the non-cancer and cancer cell lines, although differences between individual cell lines were observed. The lack of evidence for cancer-specific changes in glycosylation in our experiments does not exclude the possibility that they exist since we looked globally at the whole glycoproteome rather than determining the glycosylation status of individual proteins. The fact that no lectins demonstrated preferential binding to bladder cancer patient urine samples over control urine samples may reflect that the vast majority of the proteins in the urine samples are not tumour derived. Consistent with this finding, a recent study of urinary glycans found only small changes in the N- and O-linked glycomes of patients with bladder 5. Conclusions Our data demonstrate a role for certain lectins in urinary biomarker discovery. The urinary glycoproteome has not been fully explored to date and using the lectins with a strong preference for urothelial proteins over serum proteins (Figure 4) in conjunction with shotgun proteomics may identify much needed biomarkers for bladder cancer. To assess the biomarker potential of the candidate glycoprotein biomarkers suggested by the current work, initial validation could be carried out by lectin affinity chromatography combined with an ELISA using specific antibodies against the target glycoprotein. If the glycoprotein concentration in urine samples from bladder cancer patients is confirmed as significantly greater than the concentration in control samples then a more streamlined assay such as a sandwich ELISA combining lectin and antibody binding would be required for full validation and ultimately clinical use. Proteomes 2015, 3 278 bladder cancer but sensitivity for early disease is poor [38]. An investigation of urinary levels of MUC1 31 patients with TCC and 30 control patients found no significant difference between patient groups [40]. However, total MUC1 was measured whereas our data suggest that it is an alternatively glycosylated form of MUC1 that is increased in the urine of bladder cancer patients. The golgi apparatus protein 1 (GLG1), also known as CFR, ESL-1 and MG-160, is a 135 kDa glycosylated single pass type I transmembrane protein that contains 16 cysteine-rich GLG1 repeats [41]. It is found in the golgi apparatus and on the cell surface membrane. GLG1 is able to bind with many different proteins making it an important regulatory protein and signal transducer. The localisation of GLG1 is a crucial determinant of GLG1 function and is influenced by two mechanisms: the transmembrane domain and cytoplasmic tail retain GLG1 in the golgi apparatus, whereas the cys-rich repeats destabilise the protein and GLG1 is recruited to the cell surface via processes of stability control [41]. It has also been reported that GLG1 can be released from the cell by proteolytic cleavage at the juxtamembrane region [42]. These mechanisms of localisation and proteolytic cleavage may be altered in cancer and may play a role in the increased presence of GLG1 in the urine. GLG1 has been shown by immunohistochemistry to be highly or intermediately expressed in both high and low grade urothelial cancer tissue samples [43]; furthermore, GLG1 was detected on the cell surface of bladder cancer cell lines (Ward, unpublished data). Therefore, it seems plausible that the GLG1 detected in pTa patient urine is tumour derived. Proteomes 2015, 3 For two of these proteins, Deoxyribonuclease-2-alpha (DNASE2) and Ig gamma-2 chain C region (IGHG2) there is a lack of further evidence for a role in bladder. Endoplasmin (HSP90B1) is a ubiquitously expressed molecular chaperone of plasma membrane associated and secreted proteins [32]. Heat shock proteins are widely reported as overexpressed in cancer and one study reported that HSP90B1 is overexpressed in canine bladder cancer [33]. Prostatic acid phosphatase (ACPP) is a 100 kDa tyrosine phosphatase that dephosphorylates a diverse array of substrates under acidic conditions [34]. ACPP exists as intracellular and secreted forms that possess different glycosylation patterns and different hydrophobicities [35]. Although serum ACPP can be used to monitor prostate cancer it is reportedly not expressed in bladder cancer [36] and is not likely be a good biomarker for bladder cancer (because urinary ACPP may be primarily derived from the prostate) unless a genuinely bladder cancer specific glycoform exists. MUC1 and GLG1 appear to be the most interesting of the 6 candidates as discussed below. MUC1 is a transmembrane protein present in normal urothelium on the apical surfaces of umbrella cells and acts to protect the cells from adhesion of bacteria [37]. Overexpression and changes in glycosylation of MUC1 have been reported in lung, breast, ovary, colon and bladder cancer [38]. In an immunohistochemistry study of 539 bladder tumours MUC1 was expressed in 62% of the tumours and increased with tumour grade [39]. Serum levels of MUC1 are elevated in patients with late stage Author Contributions Experimental work: SRA, NSG, JCG and DGW. Data analysis: WW. Provision and characterization of cell lines: MAK. BCPP management team (biospecimen collection): MPZ, NDJ, RTB. Study design and writing: DGW, SJA and RTB. Conflicts of Interest The authors declare no conflict of interest. References 1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 2015, 136, E359–E386. 2. Knowles, M.; Hurst, C. Molecular biology of bladder cancer: New insights into pathogenesis and clinical diversity. Nat. Rev. Cancer 2015, 15, 25–41. 2. Knowles, M.; Hurst, C. Molecular biology of bladder cancer: New insights into pathogenesis and clinical diversity. Nat. Rev. Cancer 2015, 15, 25–41. 3. Sylvester, R.; van der Meijden, A.; Oosterlinck, W.; Witjes, J.; Bouffioux, C.; Denis, L.; Newling, D.W.; Kurth, K. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: A combined analysis of 2596 patients from seven EORTC trials. Eur. Urol. 2006, 49, 466–477. 4. Babjuk, M.; Burger, M.; Zigeuner, R.; Shariat, S.; van Rhijn, B.; Compérat, E.; Sylvester, R.J.; Kaasinen, E.; Böhle, A.; Palou Redorta, J.; et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: Update 2013. Eur. Urol. 2013, 64, 639–653. 4. Babjuk, M.; Burger, M.; Zigeuner, R.; Shariat, S.; van Rhijn, B.; Compérat, E.; Sylvester, R.J.; Kaasinen, E.; Böhle, A.; Palou Redorta, J.; et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: Update 2013. Eur. Urol. 2013, 64, 639–653. 5. Schmitz-Dräger, B.; Droller, M.; Lokeshwar, V.; Lotan, Y.; Hudson, M.; van Rhijn, B.; Marberger, M.J.; Fradet, Y.; Hemstreet, G.P.; Malmstrom, P.U.; et al. Molecular markers for bladder cancer screening, early diagnosis, and surveillance: The WHO/ICUD consensus. Urol. Int. 2015, 94, 1–24. 6. Miah, S.; Dudziec, E.; Drayton, R.; Zlotta, A.; Morgan, S.; Rosario, D.; Hamdy, F.C.; Catto, J.W. An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer. Br. J. Cancer 2012, 107, 123–128. 7. O’Sullivan, P.; Sharples, K.; Dalphin, M.; Davidson, P.; Gilling, P.; Cambridge, L.; Harvey, J.; Toro, T.; Giles, N.; Luxmanan, C.; et al. A multigene urine test for the detection and stratification of bladder cancer in patients presenting with hematuria. J. Urol. 2012, 188, 741–747. 8. Hurst, C.; Platt, F.; Knowles, M. Comprehensive mutation analysis of the TERT promoter in bladder cancer and detection of mutations in voided urine. Eur. Urol. 2014, 65, 367–369. 9. Pasikanti, K.; Esuvaranathan, K.; Ho, P.; Mahendran, R.; Kamaraj, R.; Wu, Q.; Chiong, E.; Chan, E.C. Noninvasive urinary metabonomic diagnosis of human bladder cancer. J. Proteome Res. 2010, 9, 2988–2995. 10. Acknowledgements We thank all the West Midlands Consultant Urologists and their units involved with BCPP, as well as the BCPP research nurses and Margaret Grant, Deborah Bird, Jennifer Barnwell, Duncan Nekeman and Eline van Roekel. BCPP is funded by Cancer Research UK, the University of Birmingham and the Birmingham and The Black Country and West Midlands North and South Comprehensive Local Research Networks, and sponsored by the University of Birmingham. The BCPP biospecimen collection is supported by funding from the Birmingham ECMC. Douglas Ward is funded by a philanthropic donation to the University of Birmingham in support of bladder cancer research. 279 Proteomes 2015, 3 Proteomes 2015, 3 Proteomes 2015, 3 280 11. Chen, Y.; Chen, H.; Domanski, D.; Smith, D.; Liang, K.; Wu, C.; Chen, C.L.; Chung, T.; Chen, M.C.; Chang, Y.S.; et al. Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers. J. Proteomics 2012, 75, 3529–3545. 12. Chen, C.; Lin, T.; Tsai, C.; Wu, C.; Chung, T.; Chien, K.; Wu, M.; Chang, Y.S.; Yu, J.S.; Chen, Y.T. Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics. J. Proteomics 2013, 85, 28–43. 13. Bryan, R.; Wei, W.; Shimwell, N.; Collins, S.; Hussain, S.; Billingham, L.; Murray, P.G.; Deshmukh, N.; James, N.D.; Wallace, D.M.; et al. Assessment of high-throughput high-resolution MALDI-TOF-MS of urinary peptides for the detection of muscle-invasive bladder cancer. Proteomics Clin. Appl. 2011, 5, 493–503. 14. Schiffer, E.; Vlahou, A.; Petrolekas, A.; Stravodimos, K.; Tauber, R.; Geschwend, J.; Neuhaus, J.; Stolzenburg, J.U.; Conaway, M.R.; Mischak, H.; et al. Prediction of muscle-invasive bladder cancer using urinary proteomics. Clin. Cancer Res. 2009, 15, 4935–4943. 15. Vlahou, A.; Schellhammer, P.F.; Mendrinos, S.; Patel, K.; Kondylis, F.; Gong, L.; Nasim, S.; Wright, G.L., Jr. Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine. Am. J. Pathol. 2001, 158, 1491–1502. 16. Kreunin, P.; Zhao, J.; Rosser, C.; Urquidi, V.; Lubman, D.; Goodison, S. Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling. J. Proteome Res. 2007, 6, 2631–2639. 17. Yang, N.; Feng, S.; Shedden, K.; Xie, X.; Liu, Y.; Rosser, C.; Lubman, D.M.; Goodison, S. Urinary glycoprotein biomarker discovery for bladder cancer detection using LC/MS-MS and label-free quantification. Clin. Cancer Res. 2011, 17, 3349–3359. 18. Ohyama, C. Glycosylation in bladder cancer. Int. J. Clin. Oncol. 2008, 13, 308–313. 19. Meany, D.; Chan, D. Aberrant glycosylation associated with enzymes as cancer biomarkers. Clin. Proteomics 2011, 8, 7, doi:10.1186/1559-0275-8-7. 20. Dwek, M.; Jenks, A.; Leathem, A. A sensitive assay to measure biomarker glycosylation demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate cancer compared with benign prostatic hyperplasia. Clin. Chim. Acta 2010, 411, 1935–1939. 21. Wu, J.; Zhu, J.; Yin, H.; Buckanovich, R.; Lubman, D. Analysis of glycan variation on glycoproteins from serum by the reverse lectin-based ELISA assay. J. Proteome Res. 2014, 13, 2197–2204. 22. Abbott, K.; Lim, J.; Wells, L.; Benigno, B.; McDonald, J.; Pierce, M. References Wood, S.; Knowles, M.; Thompson, D.; Selby, P.; Banks, R. Proteomic studies of urinary biomarkers for prostate, bladder and kidney cancers. Nat. Rev. Urol. 2013, 10, 206–218. Proteomes 2015, 3 Proteomes 2015, 3 281 25. Zeegers, M.; Bryan, R.; Langford, C.; Billingham, L.; Murray, P.; Deshmukh, N.; Hussain, S.; James, N.; Wallace, D.M.; Cheng, K.K. The West Midlands Bladder Cancer Prognosis Programme: Rationale and design. BJU Int. 2010, 105, 784–788. 25. Zeegers, M.; Bryan, R.; Langford, C.; Billingham, L.; Murray, P.; Deshmukh, N.; Hussain, S.; James, N.; Wallace, D.M.; Cheng, K.K. The West Midlands Bladder Cancer Prognosis Programme: Rationale and design. BJU Int. 2010, 105, 784–788. 26. Smyth, G. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 2004, 3, 3, doi:10.2202/1544-6115.1027. 27. Yang, G.; Tan, Z.; Lu, W.; Guo, J.; Yu, H.; Yu, J.; Sun, C.; Qi, X.; Li, Z.; Guan, F. Quantitative glycome analysis of N-glycan patterns in bladder cancer vs normal bladder cells using an integrated strategy. J. Proteome Res. 2015, 14, 639–653. 28. Takeuchi, M.; Amano, M.; Kitamura, H.; Tsukamoto, T.; Masumori, N.; Hirose, K.; Ohashi, T.; Nishimura, S.I. N- and O-glycome analysis of serum and urine from bladder cancer patients using a high-throughput glycoblotting method. J. Glycomics Lipidomics 2013, 3, doi:10.4172/2153-0637.1000108. 29. Langkilde, N.; Wolf, H.; Orntoft, T. Binding of wheat and peanut lectins to human transitional cell carcinomas. Correlation with histopathologic grade, invasion, and DNA ploidy. Cancer 1989, 15, 849–853. 30. Neal, D.; Charlton, R.; Bennett, M. Histochemical study of lectin binding in neoplastic and non-neoplastic urothelium. Br. J. Urol. 1987, 60, 399–401. 31. Zhang, S.; Jiang, K.; Zhang, Q.; Guo, K.; Liu, Y. Serum fucosylated paraoxonase 1 as a potential glycobiomarker for clinical diagnosis of early hepatocellular carcinoma using ELISA Index. Glycoconj. J. 2015, 32, 119–125. 32. Melnick, J.; Dul, J.; Argon, Y. Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature 1994, 370, 373–375. 33. LeRoy, B.; Painter, A.; Sheppard, H.; Popiolek, L.; Samuel-Foo, M.; Andacht, T. Protein expression profiling of normal and neoplastic canine prostate and bladder tissue. Vet. Comp. Oncol. 2007, 5, 119–130. 34. Muniyan, S.; Chaturvedi, N.; Dwyer, J.; Lagrange, C.; Chaney, W.; Lin, M. Human prostatic Acid phosphatase: Structure, function and regulation. Int. J. Mol. Sci. 2013, 14, 10438–10464. 35. White, K.; Rodemich, L.; Nyalwidhe, J.; Comunale, M.; Clements, M.; Lance, R.; Schellhammer, P.F.; Mehta, A.S.; Semmes, O.J.; Drake, R.R. Glycomic characterization of prostate-specific antigen and prostatic acid phosphatase in prostate cancer and benign disease seminal plasma fluids. J. Proteome Res. Proteomes 2015, 3 Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis. Proteomics 2010, 10, 470–481. 23. Li, D.; Mallory, T.; Satomura, S. AFP-L3: A new generation of tumor marker for hepatocellular carcinoma. Clin. Chem. Acta 2001, 313, 15–19. 24. Bryan, R.; Regan, H.; Pirrie, S.; Devall, A.; Cheng, K.; Zeegers, M.; James, N.D.; Knowles, M.A.; Ward, D.G. Protein shedding in urothelial bladder cancer: Prognostic implications of soluble urinary EGFR and EpCAM. Br. J. Cancer 2015, 112, 1052–1058. Proteomes 2015, 3 2009, 8, 620–630. 36. Heyderman, E.; Brown, B.; Richardson, T. Epithelial markers in prostatic, bladder, and colorectal cancer: An immunoperoxidase study of epithelial membrane antigen, carcinoembryonic antigen, and prostatic acid phosphatase. J. Clin. Pathol. 1984, 37, 1363–1369. 37. Ahmad, S.; Lam, T.; N’Dow, J. Significance of MUC1 in bladder cancer. BJU Int. 2015, 115, 161–162. 38. Simms, M.; Hughes, O.; Limb, M.; Price, M.; Bishop, M. MUC1 mucin as a tumour marker in bladder cancer. BJU Int. 1999, 84, 350–352. 39. Stojnev, S.; Ristic-Petrovic, A.; Velickovic, L.; Krstic, M.; Bogdanovic, D.; Khanh do, T.; Ristic, A.; Conic, I.; Stefanovic, V. Prognostic significance of mucin expression in urothelial bladder cancer. Int. J. Clin. Exp. Pathol. 2014, 7, 4945–4958. Proteomes 2015, 3 Proteomes 2015, 3 282 40. Xiang, S.; Zhou, S.; Guan, W.; Hu, Z.; Zhuang, Q.; Ye, Z. Value of quantitative examination of urine MUC1 in bladder transitional cell carcinoma. Di Yi Jun Yi Da Xue Xue Bao 2005, 25, 998–1000. 41. Miyaoka, Y.; Kato, H.; Ebato, K.; Saito, S.; Miyata, N.; Imamura, T.; Miyajima, A. Retention in the Golgi apparatus and expression on the cell surface of Cfr/Esl-1/Glg-1/MG-160 are regulated by two distinct mechanisms. Biochem. J. 2011, 440, 33–41. 42. Antoine, M.; Köhl, R.; Tag, C.; Gressner, A.; Hellerbrand, C.; Kiefer, P. Secreted cysteine-rich FGF receptor derives from posttranslational processing by furin-like prohormone convertases. Biochem. Biophys. Res. Commun. 2009, 382, 359–364. 43. Uhlen, M.; Oksvold, P.; Fagerberg, L.; Lundberg, E.; Jonasson, K.; Forsberg, M.; Zwahlen, M.; Kampf, C.; Wester, K.; Hober, S.; et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 2010, 28, 1248–1250. © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3038916867
https://izvestiya.tinro-center.ru/jour/article/download/560/512
Russian
null
Species composition and distribution of fishes and prawns in the Amur River channel
Izvestiâ TINRO/Izvestiâ Tihookeanskogo naučno-issledovatelʹskogo rybohozâjstvennogo centra
2,020
cc-by
10,175
* Кошелев Всеволод Николаевич, кандидат биологических наук, заведующий сектором, e-mail: scn74@mail.ru; Колпаков Николай Викторович, доктор биологических наук, руководитель филиала, e-mail: kolpakov_nv@mail.ru. Koshelev Vsevolod N., Ph.D., head of sector, Khabarovsk branch of VNIRO (KhabarovskNIRO), 13a, Amursky Boulevard, Khabarovsk, 680038, Russia, e-mail: scn74@mail.ru; Kolpakov Nikolai V., D. Biol., director of branch, Khabarovsk branch of VNIRO (KhabarovskNIRO), 13a, Amursky Boulevard, Khabarovsk, 680038, Russia, e-mail: kolpakov_nv@mail.ru. Известия ТИНРО Известия ТИНРО 2020 Том 200, вып. 2 УДК [597.2/.5+595.384.12] (282.257.5) В.Н. Кошелев, Н.В. Колпаков* Хабаровский филиал ВНИРО (ХабаровскНИРО), 680038, г. Хабаровск, Амурский бульвар, 13а ВИДОВОЙ СОСТАВ И РАСПРЕДЕЛЕНИЕ РЫБ И КРЕВЕТОК В РУСЛЕ НИЖНЕГО АМУРА or 292 Видовой состав и распределение рыб и креветок в русле нижнего Амура 24.0 % of total biomass); other 6 fish species with considerable biomass were lizard gudgeon Saurogobio dabryi (12.6 %), ussuri catfish T. ussuriensis (9.7 %), amur white-pinned gudgeon Romanogobio tenuicorpus (7.8 %), amur sturgeon Acipenser schrenckii (5.6 %), amur gudgeon Gobio cynocephalus (2.4 %), and kaluga Huso dauricus (0.6 %). Distribution density varied in the range of 0.01–2.57 g/m2 (on average 0.45 ± 0.27 g/m2) for fish and 0.005–0.044 g/m2 (0.021 ± 0.012 g/m2) for prawns. Abundance of both fish and prawns increased multifold to­ wards the Amur mouth that correlated with increasing of forage benthos biomass in the same direction. So, the main portion of fish biomass (82.4 %) was distributed in the Amur River downstream (40–400 km from the mouth) and the prawn stock was concentrated completely in its lowermost part (50–150 km from the mouth). iii 24.0 % of total biomass); other 6 fish species with considerable biomass were lizard gudgeon Saurogobio dabryi (12.6 %), ussuri catfish T. ussuriensis (9.7 %), amur white-pinned gudgeon Romanogobio tenuicorpus (7.8 %), amur sturgeon Acipenser schrenckii (5.6 %), amur gudgeon Gobio cynocephalus (2.4 %), and kaluga Huso dauricus (0.6 %). Distribution density varied in the range of 0.01–2.57 g/m2 (on average 0.45 ± 0.27 g/m2) for fish and 0.005–0.044 g/m2 (0.021 ± 0.012 g/m2) for prawns. Abundance of both fish and prawns increased multifold to­ wards the Amur mouth that correlated with increasing of forage benthos biomass in the same direction. So, the main portion of fish biomass (82.4 %) was distributed in the Amur River downstream (40–400 km from the mouth) and the prawn stock was concentrated completely in its lowermost part (50–150 km from the mouth). iii p ( ) Key words: Amur River, beam-trawl, fish, prawn, fish distribution, fish abundance. Введение Отлов рыб в русле и крупных протоках Амура на протяжении истории ихти­ ологических исследований проводили с использованием различных орудий лова: мальковых ловушек, плавных, ставных и накидных сетей, закидных и ставных неводов, крючковых снастей [Солдатов, 1915; Пробатов, 1935; Никольский, 1950, 1956; Новомодный и др., 2004]. Однако применение данных орудий в крупных реках возможно только в мелководной прибрежной зоне, основная же часть русла остается не охваченной исследованиями по причине больших глубин и высоких скоростей течения [Zajicek, Wolter, 2018]. Невозможность получить данные о распределении и численности молоди и взрослых рыб, нагул которых приурочен к руслу реки, не позволяет сформировать целостное представление о качественном и количественном составе речного ихтиоцена. р Оценка в крупных реках численности рыб, за исключением покатной молоди, методом площадей [Аксютина, 1968] возможна только для закидного невода и на­ кидной сети. Использование же для оценки плотности рыб ставных и плавных сетей имеет множество допущений, требует наличия достоверной промысловой статистики, проведения трудоемких расчетов [Сечин, 1990; Лобырев, 2008] или экспериментов по мечению [Пасечник, Шмигирилов, 2008]. Вместе с тем в крупных реках и эстуариях для оценки численности рыб и креве­ ток успешно используются тралы, причем предпочтительнее бим-трал, для которого характерна неизменная величина горизонтального раскрытия [Dettmers et al., 2001; Wolter, Freyhof, 2004; Herzog et al., 2005; de Souza et al., 2010; Вдовин, Мизюркин, 2011; Szalóky et al., 2014]. Такие работы проводятся и в России, например, в нижнем течении р. Дон [Живоглядов и др., 2019] и на реках Камчатки [Коваль и др., 2015]. В 2003 г. Хабаровским филиалом ТИНРО-центра (ныне Хабаровский филиал ВНИРО) была выполнена съемка с помощью бим-трала на р. Амур. Цель нашей работы — описание видового состава и анализ пространственного распределения рыб и креветок в русловой части нижнего Амура. ВИДОВОЙ СОСТАВ И РАСПРЕДЕЛЕНИЕ РЫБ И КРЕВЕТОК В РУСЛЕ НИЖНЕГО АМУРА По результатам траловой съемки, выполненной бим-тралом в сентябре-октябре 2003 г. (57 тралений на глубинах 1,5–23,0 м), описано распределение рыб и креветки в русле нижней части Амура (40–960 км). Всего поймано 2584 экз. 22 видов рыб и 1077 экз. одного вида беспозвоночных (креветка Palaemon modestus). Среди рыб по числу видов доминировали представители отрядов Cypriniformes (13 видов) и Siluriformes (4 вида). Общая численность донных и придонных рыб составила 32,161 млн экз., чис­ ленность креветки — 4,887 млн экз. Наиболее многочисленными среди рыб были два промысловых на Амуре вида: косатка Бражникова Tachysurus brashnikowi (16,26 млн экз.) и косатка-скрипун T. sinensis (4,32 млн экз.). Численность рыб и креветки многократно возрастает к устью Амура, что коррелирует с увеличением биомассы кормового бентоса в том же направлении. Общая биомасса рыб и креветки составила соответственно 583,8 и 8,8 т. Биомасса рыб изменялась в пределах 0,012–2,572 г/м2 (в среднем 0,450 ± 0,270 г/м2), биомасса креветки была равна 0,005–0,044 г/м2 (0,021 ± 0,012 г/м2). При этом 82,4 % биомассы рыб было приурочено к нижнему участку (40–400 км от устья), вся биомасса креветки — к участку 50–150 км от устья. По биомассе в уловах преобладали: косатка- скрипун (32,9 %), косатка Бражникова (24,0 %), ящерный пескарь Saurogobio dabryi (12,6 %), косатка-плеть T. ussuriensis (9,7 %), амурский белоперый пескарь Romanogobio tenuicorpus (7,8 %), амурский осетр Acipenser schrenckii (5,6 %), амурский обыкновенный пескарь Gobio cynocephalus (2,4 %) и калуга Huso dauricus (0,6 %). Ключевые слова: река Амур, бим-трал, рыбы, креветка, распределение, численность. DOI: 10.26428/1606-9919-2020-200-292-307. Koshelev V.N., Kolpakov N.V. Species composition and distribution of fishes and prawns in the Amur River channel // Izv. TINRO. — 2020. — Vol. 200, Iss. 2. — P. 292–307.i p , Distribution of fishes and prawns in the Amur River (40–960 km from the mouth) is described on results of the beam-trawl survey conducted in September-October, 2003 (57 trawl stations at the depth of 1.5–23.0 m). In total, 2584 ind. of 22 fish species and 1077 ind. of 1 invertebrate species (prawn Palaemon modestus) were caught. The highest species richness was registered for orders Cypriniformes (13 species) and Siluriformes (4 species). The fish and prawn stocks were evaluated as 32.161 . 106 ind. (583.8 t) for bottom and near-bottom fish and 4.887 . 106 ind. (8.8 t) for prawns. Two commercial fish species were the most abundant: catfishes Tachysurus brashnikowi (16.26 . 106 ind. or 32.9 % of total biomass) and T. sinensis (4.32 . 106 ind. Материалы и методы Съемка в р. Амур выполнена в сентябре-октябре 2003 г. на НИС «Профессор Солдатов» (тип «Ярославец»), в качестве орудия лова использован бим-трал (далее по тексту трал). За основу при его создании взят мальковый бим-трал Расса, кроме того, использовались детали конструкций некоторых других тралов [Ахлынов, 1954; Пахору­ ков, 1980]. Стальная рама трала имеет размеры 2,5 х 0,9 м (площадь сечения — 2,25 м2) (рис. 1). Сетной мешок трала двухслойный: наружный слой выполнен из траловой дели с ячеей 40 х 40 мм, внутренний — из дели с ячеей 10 х 10 мм. Длина сетного мешка 7 м. Буксировка трала осуществлялась по одноваерной схеме на уздечке с двумя поводками. Траления выполнены на 9 участках, расположенных в 40–55 км (I), 65–70 (II), 140–150 (III), 390–397 (IV), 402–407 (V), 519–524 (VI), 590–595 (VII), 865–880 (VIII) и 944–958 км (IX) от устья (рис. 2). 293 Кошелев В.Н., Колпаков Н.В. Рис. 1. Внешний вид бим-трала, использованного в съемке Fig. 1. Configuration of beam-trawl used in survey Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III а Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — бон; VIII — Чепчики; IX — Владимировка Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — l IV Zh bt k h l V K i k I l d VI Ch ki I l d VII Рис. 1. Внешний вид бим-трала, использованного в съемке Fig. 1. Configuration of beam-trawl used in survey Рис. 1. Внешний вид бим-трала, использованного в съемке Fig. 1. Configuration of beam-trawl used in survey Рис. 1. Внешний вид бим-трала, использованного в съемке Fig. 1. Configuration of beam-trawl used in survey Рис. 1. Внешний вид бим-трала, использованного в съемкеi Рис. 1. Внешний вид бим-трала, использованного в съемке Fig. 1. Configuration of beam-trawl used in survey р Fig. 1. Configuration of beam-trawl used in survey Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про­ тока Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока Галбон; VIII — Чепчики; IX — Владимировка Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galbon channel; VIII — Chepchiki; IX — Vladimirovka Рис. 2. Материалы и методы Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про­ а Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока бон; VIII — Чепчики; IX — Владимировка Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про­ тока Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока Галбон; VIII — Чепчики; IX — Владимировка Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galbon channel; VIII — Chepchiki; IX — Vladimirovka Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про­ тока Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока Галбон; VIII — Чепчики; IX — Владимировка Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi ; ; д р Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prok channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galb channel; VIII — Chepchiki; IX — Vladimirovka ; ; р Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galbon channel; VIII — Chepchiki; IX — Vladimirovka Всего на участке Амура длиной 920 км выполнено 57 тралений общей протя­ женностью 115,2 км, суммарная площадь облова составила 287,9 тыс. м2. Скорость тралений варьировала от 5,5 до 9,0 км/ч (1,5–2,5 м/с), глубины — от 1,5 до 23,0 м. 294 Видовой состав и распределение рыб и креветок в русле нижнего Амура Длина ваера составляла от 4 до 6 глубин. Следует отметить наличие в русле Амура большого количества «задевов» (стволов деревьев и т.п.), что создавало определенные сложности при тралениях. Длина ваера составляла от 4 до 6 глубин. Следует отметить наличие в русле Амура большого количества «задевов» (стволов деревьев и т.п.), что создавало определенные сложности при тралениях. Все уловы разбирали до вида, определяли число пойманных особей каждого из них. Биологический анализ рыб проводили по стандартным ихтиологическим мето­ дикам [Правдин, 1966]. Материалы и методы У креветок измеряли общую длину тела от конца рострума до конца тельсона (Lo, n = 30). Массу гидробионтов определяли с точностью ±0,1 г на весах AND HL. Расчет численности вида вели отдельно для каждого из 9 обловленных участков, после чего была определена его численность в реке. Численность на каждом из участков рассчитывали по формуле рассчитывали по формуле N = Sx/(Kq), где S — площадь участка; x — суммарный улов, экз.; K — коэффициент уловистости; q — площадь обловов на участке. Коэффициент уловистости бим-трала для выловлен­ ных видов не определен, поэтому при расчетах он принят равным единице. Биомассу рыб рассчитывали как произведение численности вида на среднюю массу его особей в уловах. Точное определение координат районов тралений и скорости буксировки трала производили с использованием прибора GPS, для измерения глубины воды в ходе тра­ ления использовали эхолот «Wide 3D View». Номенклатура приведена в соответствии с Каталогом рыб Эшмейера [http://researcharchive.calacademy.org/research/]. Результаты и их обсуждение Характеристика условий обитания гидробионтов в Амуре. Исследуемый участок низовьев р. Амур имеет протяженность 960 км и проходит по территории со сложным рельефом и геологическим строением. Большую его часть можно отнести к горной стране со средне- и низкогорным рельефом с большим количеством межгор­ ных впадин и равнин [Мордовин, 1996]. Климат низовьев реки обусловлен движением воздушного потока в зимнее время с континента в сторону океана, а летом — наобо­ рот. Зима характеризуется морозной, сухой и солнечной погодой, лето, как правило, теплое, облачное и дождливое. Наибольшее количество осадков приходится на летний период — 80–95 % годовой суммы [Мордовин, 1996]. В конце лета нижний участок Амура подвергается влиянию тропических циклонов, сопровождающихся затяжными дождями [Крюков и др., 2005]. Среднегодовая температура воздуха составляет в устье Амура –2,4 оС, в г. Комсомольск-на-Амуре –0,6 оС, у Хабаровска +1,4 оС [Многолет­ ние данные…, 1986; Соловьев, 1995; Болдовский, 2006]. Температурные условия в течение года обусловливают длительность ледостава, который продолжается в Амуре у Николаевска-на-Амуре в среднем 183 сут, у Хабаровска — 151 сут [Многолетние данные…, 1986]. Толщина льда в конце зимы, в зависимости от района, варьирует от 0,7 до 1,8 м [Соловьев, 1995]. Река Амур имеет паводочный режим. Основное питание (около 90 %) реки бас­ сейна Амура получают от летне-осенних муссонных дождей. Весенние паводки из-за малоснежности формирует лишь небольшое половодье [Болдовский, 2006]. На теплый период года (май-октябрь) приходится 87 % годового стока вод, на холодный (ноябрь- апрель) — 13 % [Жабин и др., 2010]. Самые низкие уровни воды наблюдаются в конце зимы. Максимальный подъем воды в паводок на Верхнем и Среднем Амуре составляет 10,0–11,0 м, на Нижнем Амуре — 6,0–7,0 м, в устье у Николаевска — 4,3 м [Мордовин, 1996]. Скорости течения на Нижнем Амуре в июне-июле вдоль фарватера составляют в среднем у поверхности 1,12 м/с, у дна 0,67 м/с, в августе — соответственно 1,18 м/с и 0,71 м/с [Соловьев, 1974]. Температура воды в разные месяцы на Нижнем Амуре варьирует в широких пределах (0,1–22,7 оС). Она повышается от устья к границе среднего и нижнего течения 295 Кошелев В.Н., Колпаков Н.В. Кошелев В.Н., Колпаков Н.В. (г. Хабаровск). Средняя сумма тепла (градусо-дни) за год в устье Амура составляет 2389, у Комсомольска-на-Амуре — 2832, у Хабаровска — 3034 [Многолетние данные…, 1986]. у ур у р Ширина реки на исследованном участке варьирует от 0,64 до 3,70 км. Глубины достигают 40 м (р-н пос. Тыр), обычно — 5–10 м. Грунты нижнего течения Амура представлены участками с песками, илами, песчано-илистыми и гравийно-галечнико­ выми фракциями. Результаты и их обсуждение До 35 % (700 км2) площади русла нижнего течения Амура занимают участки с динамически устойчивыми грунтами — с гравийно-галечниковой фракцией [Соловьев, Свирский, 1976]. [ , р , ] Кормовая база придонных и донных рыб Амура состоит главным образом из пред­ ставителей зообентоса (без моллюсков) [Никольский, 1956]. По данным С.Е. Сиротского с соавторами [2009] средняя плотность бентосного населения основного русла нижнего Амура (без моллюсков) в теплое время года составила 2,8 тыс. экз./м2 при средней био­ массе 14,7 г/м2. На песчаных биотопах зафиксированы минимальные значения биомассы бентосных организмов (< 1 г/м2). На песках с примесью иловых отложений биомасса бентоса достигает 5 г/м2, доминирующими группами зообентоса на этих грунтах явля­ ются хирономиды и олигохеты. Максимум биомассы бентосных организмов отмечен на гравийно-галечниковом субстрате у пос. М. Горький (388 км от устья) — 74,3 г/м2 и у пос. Нижняя Гавань (180 км) — 56,6 г/м2. Основу сообществ здесь составляют личинки ручейников. Отмечена общая тенденция увеличения биомассы зообентоса к устью Амура [Сиротский и др., 2009]. Видовой состав и распределение рыб и креветок. Всего за период работ в р. Амур выловлено 2584 экз. 22 видов рыб и 1077 экз. одного вида беспозвоночных (табл. 1). Доминировали в уловах представители отрядов Cypriniformes (13 видов) и Siluriformes (4 вида). Из встреченных в уловах рыб несомненный интерес с точки зрения новых знаний, в частности о распределении и численности на протяжении об­ следованных ≈1000 км, представляют виды, для которых предпочитаемым биотопом является русловая часть реки. По литературным данным [Труды Амурской ихтиоло­ гической экспедиции…, 1952, 1958; Никольский, 1956; Атлас…, 2002; Кошелев и др., 2016] к обитателям русла реки и крупных проток (ширина > 200 м) можно отнести 3 вида из рода Tachysurus — китайскую косатку-скрипуна T. sinensis, косатку Бражни­ кова T. brashnikowi и косатку-плеть T. ussuriensis, а также 4 вида пескарей — ящерного Saurogobio dabryi, амурского обыкновенного Gobio cynocephalus, амурского белоперого Romanogobio tenuicorpus и восьмиусого Gobiobotia pappenheimi. Кроме того, для калуги Huso dauricus и амурского осетра Acipenser schrenckii река является главным местом нагула [Никольский, 1956; Крыхтин, Горбач, 1994; Кошелев, 2010]. Таким образом, из 22 видов рыб 9 донных видов являются типичными обитателями главного русла Амура. Для остальных рыб, отмеченных в уловах, река — второстепенный биотоп, их нагул приурочен большей частью к придаточной системе Амура — озерам и притокам. Из трех видов креветок, обитающих в бассейне р. Амур, в наших сборах отмечен только Palaemon modestus. Этот придонно-пелагический вид встречается как в реках, так и в озерах [Erchardt, Tiffan, 2016; Барабанщиков, Шаповалов, 2019]. Результаты и их обсуждение В Амуре распространен от лимана до низовьев р. Биджан, в бассейне р. Уссури, оз. Ханка, в верховьях Аргуни (озера Далай-нор, Буир-нур и др.) [Бирштейн, Виноградов, 1934; Куренков, 1950; Боруцкий и др., 1952]. Средняя численность рыб в уловах трала на 9 участках работ варьировала от 390 до 63090 (8980) экз./км2. Вверх по течению реки численность рыб снижалась по экспоненте (рис. 3), локальные максимумы обилия (63090 и 52180 экз./км2) отмечены соответственно на участках II (65–70 км от устья) и IV (390–397 км). Креветка в уло­ вах встречалась на расстоянии от 50 до 150 км от устья, ее численность составляла 2970–24080 (16260) экз./км2 и вверх по течению также снижалась. Увеличение численности рыб и креветки к устью Амура, по нашему мнению, об­ условлено прежде всего состоянием кормовой базы данных видов, у которых в составе 296 Видовой состав и распределение рыб и креветок в русле нижнего Амура 297 Видовой состав и распределение рыб и креветок в русле нижнего Амура Таблица 1 Видовой состав и численность рыб и беспозвоночных (N) на 9 участках (I–IX) русла Амура по данным траловой съемки (сентябрь-октябрь 2003 г.) Table 1 Species composition and number of fishes and invertebrates (N) at the 9 parts (I–IX) of the Amur River channel by beam-trawl survey data (September-October, 2003) Вид N, экз./км2 N общая, млн экз. Стация I II III IV V VI VII VIII IX Ср. Результаты и их обсуждение значение Рыбы Acipenser schrenckii Brandt, 1869 880 1420 0 0 0 0 0 10 0 210 0,211 р Huso dauricus (Georgi, 1775) 180 0 0 0 0 0 0 0 0 20 0,018 р Tachysurus sinensis Lacepède, 1803 850 47120 420 710 0 170 0 0 0 2930 4,320 р, о Tachysurus brashnikowi (Berg, 1907) 1450 60 2000 44180 0 560 1430 0 0 2160 16,26 р, о Tachysurus argentivittatus (Regan, 1905) 0 0 0 0 0 40 0 0 0 3,4 0,010 р Tachysurus ussuriensis (Dybowski, 1872) 0 0 0 800 0 560 40 50 80 110 0,435 р Gobio cynocephalus Dybowski, 1869 450 890 210 360 530 300 170 0 10 180 0,530 п, р, о Gobiobotia pappenheimi Kreyenberg, 1911 0 0 0 0 0 3570 840 100 0 390 1,480 р Saurogobio dabryi Bleeker, 1871 3660 6940 2210 3200 3200 2280 1310 30 30 1460 4,430 р, о Romanogobio tenuicorpus (Mori, 1934) 2810 5340 1680 2490 2130 1760 1180 210 260 1250 3,600 р Sarcocheilichthys sinensis Bleeker, 1871 30 0 0 0 0 130 0 0 0 10 0,030 р, о Sarcocheilichthys czerskii (Berg, 1914) 0 120 0 0 0 0 0 0 0 10 0,010 р, о Hemibarbus maculatus Bleeker, 1871 250 1190 210 180 0 40 0 0 0 120 0,250 р, о Cyprinus rubrofuscus Lacepède, 1803 0 0 0 0 0 0 0 0 10 3,4 0,001 р, о Carassius gibelio (Bloch, 1782) 0 0 0 180 530 0 130 0 0 20 0,197 р, о Hemiculter sp. 0 0 0 0 0 40 210 0 0 20 0,177 р, о Rhodeus sp. Результаты и их обсуждение 30 0 0 0 0 0 80 0 0 10 0,070 п, р, о Leuciscus waleckii (Dybowski, 1869) 30 0 0 0 0 40 0 0 0 10 0,012 р, о Xenocypris macrolepis Bleeker, 1871 50 0 0 0 0 0 0 30 0 10 0,011 р, о Lota lota (Linnaeus, 1758) 130 0 0 0 0 40 0 0 0 20 0,023 п, р Pungitius bussei (Warpachowski, 1888) 0 0 110 0 0 0 0 0 0 3,5 0,032 п, р, о Siniperca chuatsi (Basilewsky, 1855) 0 0 0 90 0 0 0 0 0 3,5 0,029 р, о Итого рыбы 10780 63090 6840 52180 6400 9550 5390 430 390 8980 32,161 – Беспозвоночные Palaemon modestus (Heller, 1862) 24080 2970 7050 0 0 0 0 0 0 16260 4,887 р, о Примечание. Стации обитания: п — притоки, р — русло Амура, о — пойменные озера. Для некоторых видов жирным шрифтом выделена пред­ почитаемая стация. 297 Кошелев В.Н., Колпаков Н.В. Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2 Fig. 3. Variation of fish distribution density along the Amur River channel, ind./km2 N = 43127e-0,004L R2 = 0,65 0 10000 20000 30000 40000 50000 60000 70000 0 100 200 300 400 500 600 700 800 900 1000 Плотность (N), экз./км2 Расстояние от устья (L), км Плотность (N), экз./км2 Расстояние от устья (L), км Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2 Fig. 3. Variation of fish distribution density along the Amur River channel, ind./km2 Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2 Fig. 3. Variation of fish distribution density along the Amur River channel, in Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2 Fig. 3. Variation of fish distribution density along the Amur River channel, ind./km2 пищи преобладают донные животные [Константинов, 1950; Егорова, 1952; Пикулева, 1952; Никольский, 1956; Кошелев, 2010]. Известно, что биомасса бентоса в нижнем течении Амура по мере продвижения к устью неуклонно растет [Боруцкий и др., 1952; Сиротский и др., 2009]. пищи преобладают донные животные [Константинов, 1950; Егорова, 1952; Пикулева, 1952; Никольский, 1956; Кошелев, 2010]. Известно, что биомасса бентоса в нижнем течении Амура по мере продвижения к устью неуклонно растет [Боруцкий и др., 1952; Сиротский и др., 2009]. Общая численность донных и придонных рыб в русле Амура по данным трало­ вой съемки составила 32,161 млн экз., численность креветки — 4,887 млн экз. (табл. 1). Результаты и их обсуждение Наиболее массовыми были 5 видов рыб (в сумме 93,6 % по численности): косатка Бражникова (50,6 %), ящерный пескарь (13,8 %), косатка-скрипун (13,4 %), амурский белоперый (11,2 %) и восьмиусый (4,6 %) пескари (рис. 4). Рис. 4. Соотношение (% по численности) массовых видов рыб в уловах трала в русле р. Амур (сентябрь-октябрь 2003 г.) Fig. 4. Percentage of common fish species number in catches of beam-trawl from the Amur River in September-October of 2003       7VLQHQVLV 7EUDVKQLNRZL *SDSSHQKHLPL 6GDEU\L 5WHQXLFRUSXV ɉɪɨɱɢɟ  ɦɥɧɷɤɡ Рис. 4. Соотношение (% по численности) массовых видов рыб в уловах трала в русле р. Амур (сентябрь-октябрь 2003 г.) Fig. 4. Percentage of common fish species number in catches of beam-trawl from the Amur River in September-October of 2003 По нашим расчетам общая биомасса донных и придонных видов рыб в русле Амура на обследованной площади 2234,52 км2 составила 583,8 т (0,261 г/м2), биомасса креветки на площади 486,75 км2 — 8,8 т (0,018 г/м2). По участкам биомасса рыб из­ менялась в пределах 0,012–2,572 г/м2 (в среднем 0,450 ± 0,270 г/м2), биомасса креветок была равна 0,005–0,044 г/м2 (0,021 ± 0,012 г/м2) (рис. 5). При этом 82,4 % биомассы рыб было приурочено к нижнему участку, 40–400 км от устья, вся биомасса креветки — к участку 50–150 км от устья. 298 Видовой состав и распределение рыб и креветок в русле нижнего Амура Рис. 5. Изменчивость биомассы рыб (а) и креветки (б) вдоль русла р. Амур (сентябрь- октябрь 2003 г.) Fig. 5. Результаты и их обсуждение Variation of fish (а) and prawn (б) biomass in the Amur River in September-October, 2003, by I–IX areas along the channel, g/m2 По биомассе на исследуемом участке доминировали: косатка-скрипун (32,9 %), косатка Бражникова (24 0 %) ящерный пескарь (12 6 %) косатка-плеть (9 7 %) бе­ 0,346 2,572 0,629 0,118 0,163 0,067 0,012 0,014 0,109 0,0 0,3 0,6 0,9 1,2 1,5 1,8 2,1 2,4 2,7 I II III IV V VI VII VIII IX Биомасса, г/м2 Участок 0,044 0,005 0,013 0,000 0,010 0,020 0,030 0,040 0,050 I II III IV V VI VII VIII IX Биомасса, г/м2 Участок б а Видовой состав и распределение рыб и креветок в русле нижнего Амура Видовой состав и распределение рыб и креветок в русле нижнего Амура 0,346 2,572 0,629 0,118 0,163 0,067 0,012 0,014 0,109 0,0 0,3 0,6 0,9 1,2 1,5 1,8 2,1 2,4 2,7 I II III IV V VI VII VIII IX Биомасса, г/м2 Участок а а Участок 0,044 0,005 0,013 0,000 0,010 0,020 0,030 0,040 0,050 I II III IV V VI VII VIII IX Биомасса, г/м2 У б б Участок Рис. 5. Изменчивость биомассы рыб (а) и креветки (б) вдоль русла р. Амур (сентябрь- октябрь 2003 г.) Fig. 5. Variation of fish (а) and prawn (б) biomass in the Amur River in September-October, 2003, by I–IX areas along the channel, g/m2 Рис. 5. Изменчивость биомассы рыб (а) и креветки (б) вдоль русла р. Амур (сентябрь- октябрь 2003 г.) Fig. 5. Variation of fish (а) and prawn (б) biomass in the Amur River in September-October, 2003, by I–IX areas along the channel, g/m2 ь 2003 г.) Fig. 5. Variation of fish (а) and prawn (б) biomass in the Amur River in September-October, by I–IX areas along the channel, g/m2 По биомассе на исследуемом участке доминировали: косатка-скрипун (32,9 %), косатка Бражникова (24,0 %), ящерный пескарь (12,6 %), косатка-плеть (9,7 %), бе­ лоперый пескарь (7,8 %), амурский осетр (5,6 %), амурский обыкновенный пескарь (2,4 %) и калуга (0,6 %) (рис. 6). По данным траловой съемки, выполненной сходным по конструкции тралом в оз. Ханка (бассейн р. Амур) в 2018 г., биомасса рыб в придонных слоях в теплый период года составила 0,199 г/м2, биомасса креветок — 0,047 г/м2 [Барабанщиков, Шаповалов, 2019]. Это цифры одного порядка с полученными нами данными. Однако 299 Кошелев В.Н., Колпаков Н.В. Рис. 6. Соотношение (% по массе) рыб в траловых уловах в русле р. Амур (сентябрь- октябрь 2003 г.) Fig. 6. Характеристика массовых видов. В уловах отмечены оба обитающих в реке Амур вида осетровых — калуга (n = 7) и амурский осетр (n = 60). Численность амурского осетра составила 211 тыс. экз., калуги — 18 тыс. экз. В уловах присутствовали только неполовозрелые особи, у амурского осетра в возрасте 0+…2+ лет (длина 22–47 см), у калуги в возрасте 0+…1+ лет (30–40 см) (табл. 3). Результаты и их обсуждение Percentage of fish species biomass in catches of beam-trawl from the Amur River in September-October, 2003 32,9% 24,0% 12,6% 9,7% 7,8% 5,6% 2,4% 0,6% 4,4% T. sinensis T. brashnikowi S. dabryi T. ussuriensis R. tenuicorpus A. schrenckii G. cynocephalus H. dauricus Прочие 583,8 т 32,9% 24,0% 12,6% 9,7% 7,8% 5,6% 2,4% 0,6% 4,4% T. sinensis T. brashnikowi S. dabryi T. ussuriensis R. tenuicorpus A. schrenckii G. cynocephalus H. dauricus Прочие 583,8 т 12,6% Рис. 6. Соотношение (% по массе) рыб в траловых уловах в русле р. Амур (сентябрь- октябрь 2003 г.) Fig. 6. Percentage of fish species biomass in catches of beam-trawl from the Amur River in September-October, 2003 следует отметить довольно существенные различия в составе наиболее массовых видов. В оз. Ханка в уловах рыб по биомассе, так же как и в русле р. Амур, преоб­ ладала косатка Бражникова (60,1 %), вместе с тем здесь существенную долю уловов составляли молодь горбушек Chanodichthys spp. (17,9 %) и уклея Culter alburnus (5,8 %), а также интродуцент китайская лапша-рыба Protosalanx sinensis (8,8 %) и уссурийская востробрюшка Hemiculter lucidus (4,7 %). Биомасса рыб на Нижнем Дону по данным съемки бим-тралом составила от 8,01 до 12,27 г/м2 [Живоглядов и др., 2019], что существенно выше наших оценок. Правда, в последнем случае для бим-трала со сходными характеристиками (1,9 х 0,6 м) ис­ пользован коэффициент уловистости 0,1. С учетом этой особенности методического подхода биомасса рыб в нижнем течении Дона (0,8–1,2 г/м2) будет в 3–6 раз выше, чем в среднем в русле Амура и в оз. Ханка, но вполне сопоставима с биомассой рыб в нижнем течении Амура (табл. 2). Наши данные также весьма близки к оценкам, по­ лученным для нижнего течения рек Пенжина и Таловка (северо-западная Камчатка) [Коваль и др., 2015] (табл. 2). 300 Таблица 2 Биомасса рыб в ряде водоемов России (коэффициент уловистости 1) Table 2 Fish Fish biomass in some water bodies of Russia (catchability coefficient 1.0) Водоем Биомасса рыб, г/м2 Орудие отлова Источник данных Р. Дон 0,8–1,2 Бим-трал Живоглядов и др., 2019 Р. Пенжина (30–70 км от устья) 0,9 Бим-трал, закидной невод Коваль и др., 2015 Р. Таловка (25–45 км от устья) 0,7 Русло р. Амур: 40–960 км 0,3 Бим-трал Наши данные 40–400 км 0,6 40–70 км 1,5 Оз. Ханка 0,2 Бим-трал Барабанщиков, Шаповалов, 2019 Характеристика массовых видов. В уловах отмечены оба обитающих в реке Амур вида осетровых — калуга (n = 7) и амурский осетр (n = 60). Численность амурского осетра составила 211 тыс. Результаты и их обсуждение экз., калуги — 18 тыс. экз. В уловах присутствовали только неполовозрелые особи, у амурского осетра в возрасте 0+…2+ лет (длина 22–47 см), у калуги в возрасте 0+…1+ лет (30–40 см) (табл. 3). Таблица Биомасса рыб в ряде водоемов России (коэффициент уловистости 1) T bl Биомасса рыб в ряде водоемов России (коэффициент уловистости 1) Table 2 Fish Fish biomass in some water bodies of Russia (catchability coefficient 1.0) Водоем Биомасса рыб, г/м2 Орудие отлова Источник данных Р. Дон 0,8–1,2 Бим-трал Живоглядов и др., 2019 Р. Пенжина (30–70 км от устья) 0,9 Бим-трал, закидной невод Коваль и др., 2015 Р. Таловка (25–45 км от устья) 0,7 Русло р. Амур: 40–960 км 0,3 Бим-трал Наши данные 40–400 км 0,6 40–70 км 1,5 Оз. Ханка 0,2 Бим-трал Барабанщиков, Шаповалов, 2019 Характеристика массовых видов. В уловах отмечены оба обитающих в реке Амур вида осетровых — калуга (n = 7) и амурский осетр (n = 60). Численность амурского осетра составила 211 тыс. экз., калуги — 18 тыс. экз. В уловах присутствовали только неполовозрелые особи, у амурского осетра в возрасте 0+…2+ лет (длина 22–47 см), у калуги в возрасте 0+…1+ лет (30–40 см) (табл. 3). 300 Видовой состав и распределение рыб и креветок в русле нижнего Амура Таблица 3 Биологические показатели массовых видов рыб в уловах Table 3 Biological characters of most common fish species in the beam-trawl catches Вид Длина тела, см Масса, г n Калуга 33,30 ± 1,53 30–40 186,4 ± 28,7 130–313 7 Амурский осетр 31,60 ± 0,84 22–47 153,6 ± 13,6 43–474 60 Косатка-скрипун 12,3 ± 1,4 4,5–19,0 44,4 ± 15,8 1,7–158,0 11 Косатка Бражникова 6,8 ± 0,5 3,2–16,5 8,6 ± 1,9 0,6–77,8 50 Амурский обыкновенный пескарь 11,9 ± 0,3 10,6–14,0 25,6 ± 2,9 15,7–39,0 9 Белоперый пескарь 9,7 ± 0,2 6,8–13,6 12,7 ± 0,9 3,7–32,5 55 Ящерный пескарь 10,5 ± 0,5 2,4–18,4 16,6 ± 2,0 0,1–60,5 73 Восьмиусый пескарь 3,8 ± 0,1 2,5–5,8 0,90 ± 0,04 0,2–2,7 108 Примечание. Над чертой — пределы изменчивости параметра, под чертой — среднее значение параметра и ошибка средней. Т Биологические показатели массовых видов рыб в уловах Данные высококормные участки были зафиксированы в диапазоне глубин 8–20 м, что сходно с обследованными глубинами на II и IV участках (7–17 м). Анализ питания косатки-скрипуна и косатки Бражникова показал, что на этом участке реки ручейники составляют 98–100 % их рациона [Никольский, 1956]. Можно полагать, что высокое обилие этих косаток в нижнем течении Амура приурочено именно к локальным участкам с массовыми по­ селениями ручейников. ру Косатка-плеть (0,435 млн экз.) отмечена от района с. Владимировка (20-й км Среднего Амура) до протоки Жеребцовской (398-й км Нижнего Амура). Ее распростра­ нение полностью совпадает с определенным по материалам предыдущих исследований. По-прежнему северная граница распространения данного вида проходит примерно в одном районе. По данным Г.В. Никольского [1956] это с. Сухановка (370-й км Ниж­ него Амура), по нашим данным это район протоки Жеребцовской (398-й км Нижнего Амура). В уловах присутствовали только половозрелые особи (> 20 см) длиной от 22 до 48 см и массой от 107 до 514 г. ру Косатка-плеть (0,435 млн экз.) отмечена от района с. Владимировка (20-й км Среднего Амура) до протоки Жеребцовской (398-й км Нижнего Амура). Ее распростра­ нение полностью совпадает с определенным по материалам предыдущих исследований. нение полностью совпадает с определенным по материалам предыдущих исследований. По-прежнему северная граница распространения данного вида проходит примерно в одном районе. По данным Г.В. Никольского [1956] это с. Сухановка (370-й км Ниж­ него Амура), по нашим данным это район протоки Жеребцовской (398-й км Нижнего Амура). В уловах присутствовали только половозрелые особи (> 20 см) длиной от 22 до 48 см и массой от 107 до 514 г. Такие некрупные рыбы, как пескари, не являются объектами промысла в рос­ сийских водах Амура, в отличие от КНР, где повсеместно встречаются на рыбных рынках. В связи с отсутствием промысла изучение пескарей Амура было эпизоди­ ческим и большей частью прошло в период Амурской экспедиции (1945–1949 гг.) [Никольский, 1956; Труды Амурской ихтиологической экспедиции…, 1958]. По нашим данным 3 из наиболее массовых видов пескарей — ящерный (4,43 млн экз.), белоперый (3,60 млн экз.) и амурский обыкновенный (0,53 млн экз.) — широко распространены в русловой части нижнего течения Амура (см. табл. 1). Размерно- массовые характеристики этих видов приведены в табл. 3. Восьмиусый пескарь (1,48 млн экз.) отмечен в уловах от района с. Владимировка (23–28-й км Среднего Амура) до района о.Ченки (528-й км Нижнего Амура). Основной улов, 87 из 117 экз. (74,3 %), пришелся на участок в районе о. Ченки. Т Биологические показатели массовых видов рыб в уловах Biological characters of most common fish species in the beam-trawl catches Примечание. Над чертой — пределы изменчивости параметра, под чертой — среднее ие параметра и ошибка средней. Из 67 экз. осетровых 60 отловлены на двух самых нижних участках I и II (со­ ответственно 45–50 и 65–70 км от устья). Исходя из полученных данных можно полагать, что оба вида в первые годы жизни скатываются на нагул в предустьевой участок Амура. Впоследствии в возрасте 3–5 лет они осваивают солоноватые воды, выходя для нагула на высококормные участки Амурского лимана [Кошелев, 2006]. За все время изучения амурских осетровых в литературе имеются сведения о поимке 6 мальков (сеголеток) амурского осетра и 27 сеголеток калуги [Солдатов, 1915; Соин, 1951; Юхименко, 1963]. Таким образом, данные о численности молоди начальных возрастных групп осетровых в р. Амур являются первыми в истории изучения этих видов. Более ранние [Крыхтин, 1979] и поздние работы [Кошелев и др., 2016], судя по размерному составу уловов плавных сетей, охватывали наблюдениями рыб крупнее и старше. Наиболее многочисленными в уловах в нижнем течении Амура были косатка Бражникова (16,42 млн экз.) и китайская косатка-скрипун (4,32 млн экз.) (см. табл. 1). Косатка Бражникова отмечена на 6 из 9 обследованных участков на глубинах от 2 до 17 м. По мнению Г.В. Никольского [1956], данный вид косаток широко распространен в среднем и нижнем течении Амура. Плотность ее скоплений варьировала от 0,6 экз./га в районе протоки Тахта до 441,8 экз./га в районе протоки Жеребцовской. В уловах встречались как молодь, так и половозрелые особи (см. табл. 3). Косатка-скрипун присутствовала в уловах почти на 500-километровом участке русла Нижнего Амура от о. Ченки (524-й км) до района Еремейских островов (50-й км). По мнению Г.В. Ни­ кольского [1956], это самая многочисленная косатка нижнего и среднего течения Амура, что не подтверждается нашими данными (см. табл. 1). Плотность скоплений данного вида варьировала от 1,7 экз./га в районе о. Ченки до 471,2 экз./га в районе протоки Тахта. Косатка-скрипун отмечена на глубинах от 3 до 16 м. Формирование плотных скоплений этих двух косаток (табл. 1) на II и IV участ­ ках, по-видимому, обусловлено комплексом благоприятных факторов, к основным из которых можно отнести наличие агрегаций зообентоса и гидрологические условия (скорости течения и глубины). В нижней части Амура (0–400 км) отмечено несколь­ ко мест с биомассой бентоса (основу которого составляют ручейники), во много раз 301 Кошелев В.Н., Колпаков Н.В. превышающей средние показатели [Сиротский и др., 2009]. Т Биологические показатели массовых видов рыб в уловах Все восьмиусые пескари пойманы на глубинах до 10 м, а большинство (74,3 %) — на глубинах от 2 до 7 м. у у Креветка Palaemon modestus встречалась только в уловах на 100-километровом участке реки от протоки Прокми (145-й км) до района Еремейских островов (50-й км). Распределение креветок неоднородно, как уже было отмечено, по мере приближения к устью Амура происходило увеличение уловов. Все креветки пойманы на глубинах от 1,5 до 10,0 м, основная масса — от 3,0 до 6,0 м. В уловах присутствовали как молодь, так и половозрелые особи. р Биологические показатели креветки P. modestus в траловых уловах: длина — 35,0–63,0 (в среднем 51,3 ± 1,5) мм, масса — 0,35–3,45 (1,81 ± 1,50) г, n = 30. Анализ итогов работы на р. Амур свидетельствует, что сконструированный сотруд­ никами Хабаровского ТИНРО бим-трал позволяет проводить работы по отлову рыб и беспозвоночных на большей части русла Амура и в его крупных протоках. Данный трал постоянно идет по дну, не всплывая в толщу воды. Тем самым выполняется основное условие лова донных рыб и креветок — постоянный контакт трала с дном [Кушнарен­ ко, 1975]. Данный трал устойчив к «задевам», перескакивая или выворачивая их. Трал имеет высокую горизонтальную устойчивость и не переворачивается. Более длинная рама по сравнению с мальковым бим-тралом Расса обеспечивает, соответственно, и большую (в 2,5 раза) общую площадь облова при одинаковой длине траления. Особен­ ности конструкции позволяют работать по одноваерной схеме, используя только одну лебедку. Трал достаточно прост и надежен в эксплуатации. Данный трал возможно рекомендовать к использованию на других крупных реках России. Благодарности Авторы выражают благодарность за помощь в сборе и обработке материала со­ трудникам Хабаровского филиала ТИНРО С.А. Иванову и Ж.С. Литовченко. Соблюдение этических стандартов Все применимые международные, национальные и/или институциональные принципы использования животных были соблюдены. Информация о всех пойманных рыбах была включена в статью. Библиографические ссылки на все использованные в работе данные других авторов оформлены в соответствии с правилами данного издания. Информация о вкладе авторов В.Н. Кошелев — сбор биологического материала, систематизация первичных данных, подготовка первой версии статьи, Н.В. Колпаков — систематизация первичных данных, редактирование и подготовка окончательного варианта рукописи. Заключение По данным съемки с использованием бим-трала осенью 2003 г. (сентябрь-октябрь) на нижнем участке русла р. Амур (40–960 км) в уловах встречались 22 вида рыб и 1 вид 302 Видовой состав и распределение рыб и креветок в русле нижнего Амура беспозвоночных. Наибольшим числом видов были представлены отряды Cypriniformes (13 видов) и Siluriformes (4 вида). Суммарная численность донных и придонных рыб на обследованном участке составила 32,161 млн экз., численность креветки Palaemon modestus — 4,887 млн экз. Самыми массовыми среди рыб стали промысловые виды косаток — косатка Бражникова (16,26 млн экз.) и косатка-скрипун (4,32 млн экз.). Вы­ явлен значительный рост численности рыб и креветок по мере приближения к устью Амура, что связано с увеличением биомассы кормового бентоса в том же направлении. В зависимости от участка, биомасса рыб варьировала в пределах от 0,012 до 2,572 г/м2 (в среднем 0,450 ± 0,270 г/м2). Сходная ситуация с креветкой — 0,005–0,044 г/м2 (в среднем 0,021 ± 0,012 г/м2). Общая биомасса рыб и креветки составила соответственно 583,8 и 8,8 т. Подавляющая часть биомассы рыб (82,4 %) была сконцентрирована на нижнем участке реки (40–400 км от устья), вся биомасса креветок — на участке 50–150 км от устья. Среди рыб лидерами по биомассе стали косатка-скрипун (32,9 %), косатка Бражникова (24,0 %), ящерный пескарь (12,6 %) и косатка-плеть (9,7 %). Опробованный в съемке 2003 г. бим-трал позволяет успешно решить проблему количественного учета донных и придонных рыб Амура на большей части его русла и в крупных протоках. Финансирование работы Исследование не имело спонсорской поддержки. Список литературы Аксютина З.М. Элементы математической оценки результатов наблюдений в биологи­ ческих и рыбохозяйственных исследованиях : моногр. — М. : Пищ. пром-сть, 1968. — 288 с. Атлас пресноводных рыб России / под ред. Ю.С. Решетникова. — М. : Наука, 2002. — Т. 1. — 379 с. Ахлынов И.Я. Устройство трала и техника тралового лова : моногр. — М. : Пищепром- издат, 1954. — 84 с. Барабанщиков Е.И., Шаповалов М.Е. Распределение и динамика количественных показателей дальневосточных пресноводных креветок (сем. Palaemonidae) в оз. Ханка в летне- осенний период 2018 года // Чтения памяти Владимира Яковлевича Леванидова. — 2019. — Вып. 8. — C. 23–27. doi.org/10.25221/levanidov.08.03. Бирштейн Я.А., Виноградов Л.Г. Пресноводные Decapoda СССР и их географическое распространение // Зоол. журн. — 1934. — Т. 13, вып. 1. — С. 39–70. Болдовский Н.В. Региональная гидрогеология юга Дальнего Востока России : учеб. по­ ие. — Хабаровск : Тихоокенск. гос. ун-т, 2006. — 101 с. Боруцкий Е.В., Ключарева О.А., Никольский Г.В. Донные беспозвоночные (зообен­ тос) Амура и их роль в питании амурских рыб // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1952. — Т. 3. — С. 5–139. 303 Кошелев В.Н., Колпаков Н.В. Вдовин А.Н., Мизюркин М.А. Сравнение оценок обилия рыб по данным учетных работ лами разных конструкций // Вопр. рыб-ва. — 2011. — Т. 12, № 1(45). — С. 150–161. Егорова М.Н. Материалы по питанию косатки-плети (Liocassis ussuriensis Dyb.) и косатки Герценштейна (Liocassis herzensteini Berg) в бассейне Амура // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1952. — Т. 3. — С. 449–490. Жабин И.А., Абросимова А.А., Дубина В.А., Некрасов Д.А. Влияние стока р. Амур на гидрологические условия Амурского лимана и Сахалинского залива Охотского моря в период весенне-летнего паводка // Метеорол. и гидрол. — 2010. — № 4. — С. 93–100. Живоглядов А.А., Живоглядова Л.А., Жердев Н.А. и др. Результаты комплексных ис­ следований сообществ Нижнего Дона в 2017 г. : препринт / Researchgate. — 2019. — 7 с. DOI: 10.13140/RG.2.2.15295.15521. Коваль М.В., Горин С.Л., Калугин А.А. Экологическая характеристика сообщества мо­ лоди рыб и нектобентоса гиперприливного эстуария рек Пенжина и Таловка (северо-западная Камчатка) в августе 2014 г. // Исслед. водн. биол. ресурсов Камчатки и сев.-зап. части Тихого океана. — 2015. — Вып. 37. — С. 164–191. DOI: 10.15853/2072-8212.2015.37.164-191. Константинов А.С. Хирономиды бассейна р. Амур и их роль в питании амурских рыб // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 147–286. Список литературы h k d ( б Константинов А.С. Хирономиды бассейна р. Амур и их роль в питании амурских рыб // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 147–286. Кошелев В.Н. Амурский осетр Acipenser schrenckii Brandt, 1869 (распределение, биоло­ гия искусственное воспроизводство) : дис канд биол наук Хабаровск 2010 188 с ур д ц , Кошелев В.Н. Амурский осетр Acipenser schrenckii Brandt, 1869 (распределение, биоло­ гия, искусственное воспроизводство) : дис. … канд. биол. наук. — Хабаровск, 2010. — 188 с. у р ) у р Кошелев В.Н. Изучение распределения молоди калуги и амурского осетра в лимане Амура в 2005 г. // Ресурсы и экологические проблемы Дальнего Востока : мат-лы межрегион. науч.-практ. конф. — Хабаровск : ДВГГУ, 2006. — С. 180–184. Кошелев В.Н., Шмигирилов А.П., Рубан Г.И. Распределение, численность и размер­ ная структура популяций калуги Acipenser dauricus и амурского осетра A. schrenckii в нижнем Амуре и Амурском лимане // Вопр. ихтиол. — 2016. — Т. 56, № 2. — С. 156–162. DOI: 10.7868/ S0042875216020090. Крюков В.Г., Воронов Б.А., Гаврилов А.В., Макаров А.В. Река Амур: проблемы и пути их решения (результаты деятельности Координационного комитета по устойчивому развитию бассейна р. Амур) : моногр. — Хабаровск : Приамурское географическое общество, 2005. — 153 с. Крыхтин М.Л. Современное состояние и перспективы развития осетрового хозяйства в бассейне р. Амур // Биологические основы развития осетрового хозяйства в водоемах СССР. — М. : Наука, 1979. — С. 68–74. у Крыхтин М.Л., Горбач Э.И. Осетровые рыбы Дальнего Востока // Экономическая жизнь Дальнего Востока. — 1994. — Т. 1, № 3. — С. 86–91. Куренков И.И. К биологии дальневосточных пресноводных креветок // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 379–390. Кушнаренко А.И. Поведение молоди рыб у сетного полотна и в зоне действия малькового трала // Вопр. ихтиол. — 1975. — Т. 15, вып. 5(94). — С. 871–878. р р ( ) Лобырев Ф.С. Оценивание численности рыб на основе моделирования работы жаберных сетей : автореф. дис. … канд. биол. наук. — М. : МГУ им. М.В. Ломоносова, 2008. — 22 с. р ф д д у , Многолетние данные о режиме и ресурсах поверхностных вод суши. Т. 1, вып. 19 : Бассейны Амура (без бассейнов Шилки, Амгуни, Амазара, Уссури) и Уды / ред. И.С. Якорь. — М. : Гидрометеоиздат, 1986. — 412 с. р ф у Многолетние данные о режиме и ресурсах поверхностных вод суши. Список литературы Т. 1, вып. 1 Бассейны Амура (без бассейнов Шилки, Амгуни, Амазара, Уссури) и Уды / ред. И.С. Якорь М. : Гидрометеоиздат, 1986. 412 с. Мордовин А.М. Годовой и сезонный сток рек бассейна Амура. — Хабаровск, 1996. — 37 с. Никольский Г.В. Амурская ихтиологическая экспедиция 1945–1949 гг. // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 1–18. д ц , Никольский Г.В. Рыбы бассейна Амура : моногр. — М. : АН СССР, 1956. — 553 с. Новомодный ГВ Золотухин С Ф Шаров П О Рыбы Амура: богатство и кризис : д ц , Никольский Г.В. Рыбы бассейна Амура : моногр. — М. : АН СССР, 1956. — 553 с Новомодный Г.В., Золотухин С.Ф., Шаров П.О. Рыбы Амура: богатство и кризи аналит. обзор. — Владивосток : Апельсин, 2004. — 63 с. Пасечник О.И., Шмигирилов А.П. Оценка численности амурской кеты Oncorhynchus keta (Walbaum, 1792) по результатам мечения // Чтения памяти Владимира Яковлевича Лева­ нидова. — Владивосток : Дальнаука, 2008. — Вып. 4. — С. 294–303. Пахоруков А.М. Изучение распределения молоди рыб в водохранилищах и озерах : моногр. — М. : Наука, 1980. — 65 с. Пикулева В.А. Питание косатки-скрипуна (Psevdobagrus fulvidraco Rich.) и малой косатки (Liocassis brashnikovi Berg) в бассейне Амура // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1952. — Т. 3. — С. 435–448. , Правдин И.Ф. Руководство по изучению рыб (преимущественно пресноводных) : моногр. — 4-е изд., перераб. и доп. — М. : Пищепромиздат, 1966. — 375 с. 304 Видовой состав и распределение рыб и креветок в русле нижнего Амура Пробатов А.Н. Материалы по изучению осетровых рыб Амура // Учен. зап. Пермского та. — 1935. — Т. 1, вып. 1. — С. 33–72. Сечин Ю.Т. Методические указания по оценке численности рыб в пресноводных водо­ емах. — М. : ВНИИПРХ, 1990. — 50 с. Сиротский С.Е., Макарченко Е.А., Макарченко М.А. Характеристика бассейна реки Амур по составу зообентоса // Вопр. рыб-ва. — 2009. — Т. 10, № 3(39). — С. 453–467. Соин С.Г. Материлы по развитию осетровых р. Амур // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1951. — Т. 2. — С. 223–232. Солдатов В.К. Обзор исследований, произведенных на Амуре в 1909–1913 гг.; Исследо­ вание осетровых Амура : Материалы к познанию русского рыболовства. — Петроград : тип. В.Ф. Киршбаума (отд-ние), 1915. — Т. 3, вып. 12. — 415 с. р у ( ), , Соловьев И.А. Список литературы 64. — P. 632–642. doi.org/10.1111/j.1095-8649. 2004.00327.x.i Zajicek P., Wolter Ch. The gain of additional sampling methods for the fish-based assessment of large rivers // Fish. Res. — 2018. — Vol. 197. — P. 15–24. Список литературы Амуролиманский русловой процесс и водные пути : моногр. — Влади­ восток : ДВО РАН, 1995. — 271 с. Соловьев И.А. Русловой процесс и водные пути Амурского лимана : моногр. — Влади­ восток, 1974. — 290 с. Соловьев И.А., Свирский В.Г. Гидрологическая обстановка Нижнего Амура и ее роль в воспроизводстве осетра и калуги // Биология рыб Дальнего Востока. — Владивосток : ДВГУ, 1976. — С. 70–74. Труды амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1952. — Т. 3. — 512 с. Труды амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1958. — Т. 4. — 358 с. Юхименко С.С. Питание амурского осетра и калуги в нижнем течении Амура // Вопр. ихтиол. — 1963. — Т. 3, вып. 2(27). — С. 311–318. de Souza M.R., Fagundes L., von Seckendorff R.W. et al. Construction characteristics of a beam trawl for catching young shrimps in estuaries // Rev. Bras. Eng. Pesca. — 2010. — Vol. 5, № 3. — P. 42–50. Dettmers J.M., Gutreuter S., Wahl D.H., Soluk D.A. Patterns and abundance in main channels of the upper Mississippi River system // Can. J. Fish. Aquat. Sci. — 2001. — Vol. 58. — P. 933–942. Erchardt J.M., Tiffan K.F. Ecology of nonnative Siberian prawn (Palaemon modestus) in the l S k Ri W hi t USA // A ti E l 2016 V l 50 №4 P 607 621 Dettmers J.M., Gutreuter S., Wahl D.H., Soluk D.A. Patterns and abundance in main channels of the upper Mississippi River system // Can. J. Fish. Aquat. Sci. — 2001. — Vol. 58. — P. 933–942. y Erchardt J.M., Tiffan K.F. Ecology of nonnative Siberian prawn (Palaemon modestus) in the lower Snake River, Washington, USA // Aquatic Ecology. — 2016. — Vol. 50, № 4. — P. 607–621.i Herzog D.P., Barko V.A., Scheibe J.S. et al. Efficacy of a benthic trawl for sampling small-bod­ ied fishes in large river systems // North American Journal of Fisheries Management. — 2005. — Vol. 25. — P. 594–603. Ái Szalóky Z., György Á.I., Tóth B. et al. Application of an electrified benthic frame trawl for sampling fish in a very large European river (the Danube River) — Is offshore monitoring necessary? // Fish. Res. — 2014. — Vol. 151. — P. 12–19. doi.org/10.1016/j.fishres.2013.12.004.i g ji Wolter C., Freyhof J. Diel distribution patterns of fishes in a temperate large lowland rive J. Fish Biol. — 2004. — Vol. References Aksyutina, Z.M., Elementy matematicheskoi otsenki rezul’tatov nablyudenii v biologicheskikh i rybokhozyaistvennykh issledovaniyakh (Elements of Mathematical Evaluation of the Results of Observations in Biological and Fishery Research), Moscow: Pishchevaya Promyshlennost’, 1968. Atlas presnovodnykh ryb Rossii (Atlas of Freshwater Fishes in Russia), Reshetnikov, Yu.S., Moscow: Nauka, 2002, vol. 1.i , , , Akhlynov, I.Ya., Ustrojstvo trala i tekhnika tralovogo lova (Trawl device and trawl fishing technique), Moscow: Pishchepromizdat, 1954. Barabanshchikov, Ye.I. and Shapovalov, M.Ye., Distribution and dynamics of quantitative indicators of Far Eastern freshwater shrimp (family Palaemonidae) in Lake Hanka in the summer- autumn period of 2018, in Chteniya pamyati Vladimira Yakovlevicha Levanidova (Vladimir Yakov­ levich Levanidov’s Biennial Memorial Meetings), Vladivostok: Dal’nauka, 2019, vol. 8, pp. 23–27. Birshtein, Y.A. and Vinogradov, L.G., Freshwater Decapoda of the USSR and their geographi­ cal distribution, Zool. Zh., 1934, vol. 13, no. 1, pp. 39–70. pp Boldovsky, N.V., Regional’naya gidrogeologiya yuga Dal’nego Vostoka Rossii (Regional hydrogeology of the south of the Russian Far East), Khabarovsk: Tikhookeanskiy. gos. un-t, 2006. 305 Кошелев В.Н., Колпаков Н.В. Borutsky, E.V., Klyuchareva, O.A., and Nikolsky, G.V., Benthic invertebrates (zoobenthos) of Amur and their role in the nutrition of Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 5–139. Borutsky, E.V., Klyuchareva, O.A., and Nikolsky, G.V., Benthic invertebrates (zoobenthos Borutsky, E.V., Klyuchareva, O.A., and Nikolsky, G.V., Benthic invertebrates (zoobenthos Amur and their role in the nutrition of Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–19 y y y ( ) Amur and their role in the nutrition of Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 5–139.i gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 5–139. Vdovin, A.N. and Mizyurkin, M.A. The comparison of estimations of fishes’ abundance, on data of registration works carried out by trawls of different construction, Vopr. Rybolov., 2011, vol. 12, no. 1(45), pp. 150–161. Egorova, M.N., Nutritional information for killer whales (Liocassis ussuriensis Dyb.) And Her­ zenstein killer whales (Liocassis herzensteini Berg) in the Amur basin, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 449–490.l Zhabin, I.A., Abrosimova, A.A., Dubina, V.A., and Nekrasov, D.A., Influence of the Amur River runoff on the hydrological conditions of the Amur Liman and Sakhalin Bay (Sea of Okhotsk) during the spring-summer flood, Russ. Meteorol. Hydrol., 2010, vol. 35, no. 4, pp. 295–300. References g p gl y pp Zhivoglyadov, A.A., Zhivoglyadova, L.А., Zherdev, N.A., Guskova, O.S., Luzhnyak, O.L., and Shlyakhova, N.A., Results of complex investigation of the Lower Don communities in 2017, in Researchgate, 2019. doi 10.13140/RG.2.2.15295.15521 Koval, M.V., Gorin, S.L., and Kalugin, A.A., Ecological characteristic of nectobenthos and juvenile fish community of hypertidal estuary of Penzhina and Talovka Rivers (Northwest Kamchatka) in August 2014, Issled. Vodn. Biol. Resur. Kamchatki i Sev.-Zapadn. Chasti Tikhogo Okeana, 2015, vol. 37, pp. 164–191. doi 10.15853/2072-8212.2015.37.164-191 Konstantinov, A.S., Chironomids of the river basin Cupid and their role in the nutrition of Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1950, vol. 1, pp. 147–286. Koshelev, V.N., Amur sturgeon Acipenser schrenckii Brandt, 1869 (distribution, biology, arti­ ficial reproduction), Cand. Sci. (Biol.) Dissertation, Khabarovsk, 2010. Koshelev, V.N., A study of the distribution of juvenile Kaluga and Amur sturgeon in the Amur estuary in 2005, in Mat-ly Mezhregion. Nauchno-Pract. Conf. “Resursy i ekologicheskiye problemy Dal’nego Vostoka” (Materials of the Inter-Regional Sci. Pract. Conf. “Resources and environmental problems of the Far East”), Khabarovsk: DVGGU, 2006, pp. 180–184. Koshelev, V.N., Shmigirilov, A.P., and Ruban, G.I., Distribution, abundance, and size structure of Amur kaluga Acipenser dauricus and Amur sturgeon A. schrenckii in the Lower Amur and Amur Estuary, Vopr. Ikhtiol., 2016, vol. 56, no. 2, pp. 235–241. y p pp Kryukov, V.G., Voronov, B.A., Gavrilov, A.V., and Makarov, A.V., Reka Amur: problemy i puti ikh resheniya (rezul’taty deyatel’nosti Koordinatsionnogo komiteta po ustoychivomu razvitiyu basseyna r. Amur) (Amur River: problems and solutions (results of the Coordination Committee for Sustainable Development of the Amur River Basin)), Khabarovsk: Priamurskoye geograficheskoye obshchestvo, 2005. Krykhtin, M.L., The current state and development prospects of sturgeon in the river basin. Amur, in Biologicheskiye osnovy razvitiya osetrovogo khozyaystva v vodoyemakh SSSR, Moscow: Nauka, 1979, pp. 68–74.i Krykhtin, M.L. and Gorbach, E.I., Sturgeon fish of the Far East, in Ekonomicheskaya zhizn’ Dal’nego Vostoka, 1994, vol. 1, no. 3, pp. 86–91. Kurenkov, I.I., To the biology of Far Eastern freshwater shrimp, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1950, vol. 1, pp. 379–390.i Kushnarenko, A.I., The behavior of juvenile fish near the net and in the zone of activity of the fry trawl, Vopr. Ikhtiol., 2075, vol. 15, no. 5(94), pp. References (Proceedings of the Amur ich­ ological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. (Proceedings of the Amur ich­ thyological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. (Proceedings of the Amur ich­ g p ) py Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. (Proceedings of the Amur ich­ ological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1958, vol. 4. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945 1949 gg. (Proceedings o thyological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1958, vol. 4. Yukhimenko, S.S., Food of the Amur sturgeon and Kaluga in the lower reaches of the Amur, Vopr. Ikhtiol., 1963, vol. 3, no. 2(27), pp. 311–318. Vopr. Ikhtiol., 1963, vol. 3, no. 2(27), pp. 311–318. de Souza, M.R., Fagundes, L., von Seckendorff, R.W., Tomas, A.W.G., Tutui, S.L., Casarini, L.M., Construction characteristics of a beam trawl for catching young shrimps in estuaries, Rev. Bras. de Souza, M.R., Fagundes, L., von Seckendorff, R.W., Tomas, A.W.G., Tutui, S.L., Casarini, L.M., Construction characteristics of a beam trawl for catching young shrimps in estuaries, Rev. Bras. Eng. Pesca, 2010, vol. 5, no. 3, pp. 42–50. Dettmers, J.M., Gutreuter, S., Wahl, D.H., and Soluk, D.A., Patterns and abundance in main nnels of the upper Mississippi River system, Can. J. Fish. Aquat. Sci., 2001, vol. 58, pp. 933–942. Erchardt, J.M. and Tiffan, K.F., Ecology of nonnative Siberian prawn (Palaemon modestus) in the lower Snake River, Washington, USA, Aquatic Ecology, 2016, vol. 50, no. 4, pp. 607–621.i Herzog, D.P., Barko, V.A., Scheibe, J.S., Hrabik, R.A., and Ostendorf, D.E., Efficacy of a benthic trawl for sampling small-bodied fishes in large river systems, North American Journal of Fisheries Management, 2005, vol. 25, pp. 594–603. Á Szalóky, Z., György, Á.I., Tóth, B., Sevcsik, A., Specziár, A., Csanyi, B., Szekeres, J., and Erős, T., Application of an electrified benthic frame trawl for sampling fish in a very large European river (the Danube River) — Is offshore monitoring necessary?, Fish. Res., 2014, vol. 151, pp. 12–19. doi.org/10.1016/j.fishres.2013.12.004i i Wolter, C. and Freyhof, J., Diel distribution patterns of fishes in a temperate large lowland river, J. Fish Biol., 2004, vol. 64, pp. 632–642. doi.org/10.1111/j.1095-8649.2004.00327.xi , , , , pp g j Zajicek, P. and Wolter, Ch., The gain of additional sampling methods for the fish-based as­ sessment of large rivers, Fish. Res., 2018, vol. 197, pp. 15–24. References 871–878.i y p ( ) pp Lobyrev, F.S., Estimation of fish abundance based on the modeling of gill nets, Extended Ab­ stract of Cand. Sci. (Biol.) Dissertation, Moscow: Mosk. Gos. Univ., 2008. Mnogoletniye dannyye o rezhime i resursakh poverkhnostnykh vod sushi (Long-term data on the regime and resources of land surface water, vol. 1, issue 19: Amur basins (without Shilka, Amguni, Lobyrev, F.S., Estimation of fish abundance based on the modeling of gill nets, Extended Ab­ stract of Cand. Sci. (Biol.) Dissertation, Moscow: Mosk. Gos. Univ., 2008. Mnogoletniye dannyye o rezhime i resursakh poverkhnostnykh vod sushi (Long-term data on the regime and resources of land surface water, vol. 1, issue 19: Amur basins (without Shilka, Amguni, Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986. the regime and resources of land surface water, vol. 1, issue 19: Amur basins (without Shilka, Amguni, Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986.l t e eg e a d esou ces o a d su ace wate , vo . , ssue 9: u bas s (w t out S a, gu , Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986.l Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986. Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986. Mordovin, A.M., Godovoy i sezonnyy stok rek basseyna Amura (The annual and seasonal flow of the rivers of the Amur basin), Khabarovsk, 1996. Mordovin, A.M., Godovoy i sezonnyy stok rek basseyna Amura (The annual and seasonal flow of the rivers of the Amur basin), Khabarovsk, 1996. Nikolsky, G.V., Amur ichthyological expedition 1945–1949, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1950, vol. 1, pp. 1–18.i Nikolsky, G.V., Ryby bassejna Amura (Amur basin fish), Moscow: Аkad. Nauk SSSR, 1956. Novomodny, G.V., Zolotukhin, S.F., and Sharov, P.O., Ryby Amura: bogatstvo i krizis (Cupid Fish: Wealth and Crisis), Vladivostok: Apel’sin, 2004. ) p Pasechnik, O.I. and Shmigirilov, A.P., Estimation of the number of Amur chum salmon On­ corhynchus keta (Walbaum, 1792) based on tagging results, in Chteniya pamyati Vladimira Yakov­ 306 Видовой состав и распределение рыб и креветок в русле нижнего Амура Видовой состав и распределение рыб и креветок в русле нижнего Амура levicha Levanidova (Vladimir Yakovlevich Levanidov’s Biennial Memorial Meetings), Vladiv Dal’nauka, 2008, vol. 4, pp. 294–303. cha Levanidova (Vladimir Yakovlevich Levanidov’s Biennial Memorial Meetings), Vladivostok: ’nauka, 2008, vol. 4, pp. 294–303. References Pakhorukov, A.M., Izucheniye raspredeleniya molodi ryb v vodokhranilishchakh i ozorakh (Study of the distribution of juvenile fish in reservoirs and lakes), Moscow: Nauka, 1980. Pikuleva, V.A., Feeding of a killer whale (Psevdobagrus fulvidraco Rich.) And a small killer whale (Liocassis brashnikovi Berg) in the Amur basin, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 435–448. whale (Liocassis brashnikovi Berg) in the Amur basin, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 435–448. ( g) , y g y p 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 435–448. Pravdin, I.F., Rukovodstvo po izucheniyu ryb (preimushchestvenno presnovodnykh) (Guide to the Study of Fish (Mainly Freshwater)), 4th ed., Moscow: Pishchevaya Promyshlennost’, 1966. P b A N M i l h d f A fi h U h P k Probatov, A.N., Materials on the study of Amur sturgeon fishes, Uchen. zap. Permskogo un-ta, 5, vol. 1, no. 1, pp. 33–72. Sechin, Yu.T., Metodicheskiye ukazaniya po otsenke chislennosti ryb v presnovodnykh vodo- yemakh (Guidelines for assessing the number of fish in freshwater bodies), Moscow: Vses. Nauchno- Issled. Inst. Presnovodn. Rybn. Khoz., 1990. ssled. Inst. Presnovodn. Rybn. Khoz., 1990. Sirotsky, S.E., Makarchenko, E.A., and Makarchenko, M.A., Water quality of the Am River basin based on zoobentos composition, Vopr. Rybolov., 2009, vol. 10, no. 3(39), pp. 453–46 Soin, S.G., Materials for the development of sturgeon p. Amur, Tr. Amurskoy ikhtiologiches ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1951, vol. 2, pp. 223–232. Soldatov, V.K., A review of studies conducted on the Amur River in 1909-1913; The study of sturgeon Amur, in Materialy k poznaniyu russkogo rybolovstva (Materials for the knowledge of Rus­ sian fisheries), Petrograd: tip. V.F. Kirshbauma (otd-niye), 1915, vol. 3, issue 12. Soloviev, I.A., Amurolimanskiy ruslovoy protsess i vodnyye puti (Amurolimanian channel process and waterways), Vladivostok: Dal’nevost. Otd. Ross. Akad. Nauk, 1995. Soloviev, I.A., Ruslovoy protsess i vodnyye puti Amurskogo limana (Channel process and wa­ ways of the Amur estuary), Vladivostok, 1974. Soloviev, I.A. and Svirsky, V.G., The hydrological situation of the Lower Amur and its role in the reproduction of sturgeon and Kaluga, in Biologiya ryb Dal’nego Vostoka (Biology of fish of the Far East), Vladivostok: Dal’nevost. Gos. Univ., 1976, pp. 70–74. East), Vladivostok: Dal nevost. Gos. Univ., 1976, pp. 70 74. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. References Поступила в редакцию 9.12.2019 г. После доработки 7.04.2020 г. Принята к публикации 20.05.2020 г. 307
https://openalex.org/W2950597272
https://repository.ubn.ru.nl/bitstream/handle/2066/202113/1/202113.pdf
English
null
Polyomavirus T Antigen Induces<i>APOBEC3B</i>Expression Using an LXCXE-Dependent and TP53-Independent Mechanism
MBio
2,019
cc-by
13,031
Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/202113 Download date: 2024-10-24 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/202113 Download date: 2024-10-24 Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism Starrett, G.J.; Serebrenik, Artur A.; Roelofs, P.A.; McCann, Jennifer L.; Verhalen, Brandy; Jarvis, Matthew C.; Span, P.N.; Harris, R.S. 2019, Article / Letter to editor (Mbio, 10, 1, (2019), article e02690-18) Doi link to publisher: https://doi.org/10.1128/mBio.02690-18 Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism Starrett, G.J.; Serebrenik, Artur A.; Roelofs, P.A.; McCann, Jennifer L.; Verhalen, Brandy; Jarvis, Matthew C.; Span, P.N.; Harris, R.S. 2019, Article / Letter to editor (Mbio, 10, 1, (2019), article e02690-18) Doi link to publisher: https://doi.org/10.1128/mBio.02690-18 Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism Gabriel J. Starrett,a Artur A. Serebrenik,a Pieter A. Roelofs,b Jennifer L. McCann,a Brandy Verhalen,c Matthew C. Jarvis,a Teneale A. Stewart,a Emily K. Law,a Annabel Krupp,d Mengxi Jiang,c John W. M. Martens,e Ellen Cahir-McFarland,d Paul N. Span,b Reuben S. Harrisa,f on April 15, 2019 b http://mbio.asm.org/ Downloaded from on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from aDepartment of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA bDepartment of Radiation Oncology, Radiotherapy and OncoImmunology Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands cDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA dDepartment of Neuroimmunology, Biogen, Cambridge, Massachusetts, USA eDepartment of Medical Oncology Cancer Genomics Netherlands Erasmus MC Cancer Institute Erasmus University Medical Center Rotterdam The Netherlands bDepartment of Radiation Oncology, Radiotherapy and OncoImmunology Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands cDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA dDepartment of Neuroimmunology Biogen Cambridge Massachusetts USA on April 15, 2019 by guest http://mbio.asm.org/ from eDepartment of Medical Oncology, Cancer Genomics Netherlands, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands fHoward Hughes Medical Institute, University of Minnesota, Minneapolis, Minnesota, USA ABSTRACT APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of mul- tiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated in- duction of APOBEC3B. Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B. Last, global gene expression anal- yses in a wide range of human cancers show significant associations between ex- pression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. January/February 2019 Volume 10 Issue 1 e02690-18 Citation Starrett GJ, Serebrenik AA, Roelofs PA, McCann JL, Verhalen B, Jarvis MC, Stewart TA, Law EK, Krupp A, Jiang M, Martens JWM, Cahir- McFarland E, Span PN, Harris RS. 2019. Polyomavirus T antigen induces APOBEC3B expression using an LXCXE-dependent and TP53-independent mechanism. mBio 10:e02690-18. https://doi.org/10.1128/mBio .02690-18. Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). RESEARCH ARTICLE Host-Microbe Biology crossm RESEARCH ARTICLE Host-Microbe Biology Starrett et al. G enetic diversity is key to virus replication, pathogenesis, and transmission, and particularly for escape from adaptive immune responses in vertebrate species (1–3). Each virus has evolved to maintain an optimized level of genetic diversity for its own unique life cycle through various mechanisms, with some viruses having high mutation rates and others much lower mutation rates, notably the dsDNA viruses (4, 5). Recently, it has been concluded that the genome compositions of multiple DNA tumor viruses, including high-risk human papillomavirus (HPV) types and BK polyomavirus (BKPyV), have been shaped by long-term interactions with the innate, antiviral APOBEC deaminases (6–9). It has also been suggested that acutely occurring mutations by these enzymes in the major capsid gene of polyomaviruses promote antibody escape during polyomavirus-associated nephropathy and progressive multifocal leukoencephalopa- thy (10). The overlap between these disease variants and oncogenic enzymes is striking especially in light of growing evidence linking BKPyV infection and a subset of urothe- lial carcinomas with high levels of APOBEC-signature mutations (11). G g g Several APOBEC enzymes, including APOBEC3B (A3B), bind 5=-TC dinucleotide mo- tifs in single-stranded DNA and catalyze the hydrolytic conversion of cytosine to uracil (12, 13). Left unrepaired, uracil lesions can serve as the templates for new DNA synthesis and directly result in C-to-T mutations. Alternatively, if the uracil base is excised by cellular uracil DNA glycosylase 2 (UNG2), then the resulting abasic site becomes noninstructional and may trigger cellular DNA polymerases to insert an adenine opposite the lesion, except for REV1, which tends to incorporate either adenine or cytosine. Thus, APOBEC-catalyzed DNA deamination of 5=-TC motifs results in both C-to-T and C-to-G mutations (a signature frequently expanded to include the 3’- nucleobases A and T and referred to in the context of trinucleotide motifs 5=-TCA and 5=-TCT). An additional hallmark of virus mutagenesis by APOBEC enzymes is a bias toward occurring on the template of lagging-strand DNA replication (14–16). A likely mechanistic relationship with single-stranded DNA replication intermediates is sup- ported by similar correlations in model yeast and Escherichia coli experiments (17, 18). Human cells have the potential to express up to nine active DNA cytosine deami- nases (AID, APOBEC1, and A3A/B/C/D/F/G/H) (19–22). Seven of these enzymes prefer 5=-TC motifs in single-stranded DNA, whereas AID uniquely prefers 5=-RC and APOBEC3G (A3G) prefers 5=-CC. Starrett et al. A3B is the most likely APOBEC family member to contribute to the mutagenesis and evolution of small DNA tumor viruses because it is specifically upregulated by viral oncoproteins. For high-risk HPV types, the oncopro- teins E6 and E7 have been implicated through various pathways (23–26). For polyo- maviruses, including JC, BK, and Merkel cell (JCPyV, BKPyV, and MCPyV, respectively), the large T antigen (TAg) is sufficient for A3B upregulation through a yet-to-be determined mechanism (6). However, the considerable functional overlap of these proteins, RB inactivation by E7 and TAg and p53 inactivation by E6 and TAg, may indicate limited pathways for A3B modulation by viruses (27, 28). Here we investigate the molecular mechanism by which polyomaviruses promote the transcriptional up- regulation of A3B with results converging on the cellular RB/E2F pathway, which is often deregulated in cancer. January/February 2019 Volume 10 Issue 1 e02690-18 Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells. IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mu- tations in 5=-TC motifs, oncogene activation, acquired drug resistance, and poor clin- ical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this muta- tional mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution. KEYWORDS APOBEC3B, RB/E2F pathway, polyomavirus, virus evolution January/February 2019 Volume 10 Issue 1 e02690-18 ® mbio.asm.org 1 mbio.asm.org 1 ® Starrett et al. Starrett et al. RESULTS Visualization of endogenous APOBEC3B protein in polyomavirus-infected cells. A3B induction by polyomaviruses has been shown at the mRNA level by RT-qPCR and at the protein level by immunoblotting in primary renal proximal epithelial cells (RPTECs) (6). To extend these results to other relevant cell types, RT-qPCR and immu- nofluorescent microscopy were used to ask whether polyomavirus infection causes a general pan-nuclear upregulation of A3B enzyme and/or localization to discrete sub- nuclear regions such as virus replication centers. Immortalized human kidney [HuK(i)G10] cells were infected with BKPyV (Dunlop strain) and JCPyV (MAD1 strain) and subjected to analyses at various days postinfection (dpi). Infected cells have enlarged nuclei and robust expression of TAg and VP1 at 3 to 5 dpi (Fig. 1A). A3B expression was more variable but still clearly and significantly increased after infection with either virus mbio.asm.org 2 January/February 2019 Volume 10 Issue 1 e02690-18 ® Polyomavirus T Antigen Induces APOBEC3B Expression F E C D A B G 0 1000 2000 3000 4000 0 200 400 600 TAg A3B 0 1000 2000 3000 0 100 200 300 400 TAg EdU p < 0.0001 p < 0.0001 Spearman’s rho = 0.42 Spearman’s rho = 0.73 DAPI TAg EdU A3B Merge 20μm A3B expression relative to TBP 1 dpi 5 dpi Mock BKPyV Mock BKPyV Mock BKPyV Mock BKPyV 1dpi 5dpi TAg Percent positive cells VP1 A3B Mock JCPyV (Mad1) TAg VP1 DAPI 100μm A3B Merge 0 0.5 1.0 1.5 p=0.041 p=0.036 p=0.050 p=0.022 p=0.014 BKPyV JCPyV Mock 6dpi (HuK(i)G10) BKPyV BKPyV JCPyV 0 20 40 60 80 100 10 2 4 6 8 0 Percent A3B positive cells 3 5 7 9 Days post infection FIG 1 Visualization and quantification of A3B expression in PyV-infected cells. (A and B) Immunofluorescent images and quantification of TAg, VP1, and A3B in BKPyV-infected HuK(i)G10 cells at 1 and 5 dpi (significance determined using Welch’s two-tailed t test; n  2 biological replicates). (C) Time course of A3B mRNA levels in JCPyV (Mad1 strain) versus mock-infected HuK(i)G10 cells. (D) RT-qPCR quantification of A3B transcripts in mock-, BKPyV-, and JCPyV (Mad1)-infected HuK(i)G10 cells at 6 dpi (significance determined by Welch’s two-tailed t test; n  3 technical replicates). (E) High-resolution immunofluorescent microscopy images of DAPI, A3B, EdU, and TAg in HuK(i)G10 cells infected with JCPyV (Mad1 strain). RESULTS (F and G) Correlation coefficients and P values for EdU and A3B levels versus T antigen intensity in 100 cell images from a single experiment similar to that in panel E. on April 15, 2019 by g http://mbio.asm.org/ Downloaded from C D A3B expression relative to TBP Mock JCPyV (Mad1) 0 0.5 1.0 1.5 p=0.041 p=0.036 BKPyV JCPyV Mock 6dpi (HuK(i)G10) 10 2 4 6 8 0 Percent A3B positive cells 3 5 7 9 Days post infection D A3B expression relative to TBP 0 0.5 1.0 1.5 p=0.041 p=0.036 BKPyV JCPyV Mock 6dpi (HuK(i)G10) A Mock BKPyV Mock BKPyV 1dpi 5dpi TAg VP1 DAPI 100μm A3B Merge BKPyV D A C A Mock BKPyV 1dpi TAg VP1 DAPI 100μm A3B Merge BKPyV E DAPI TAg EdU A3B Merge 20μm JCPyV E on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from Merge G 0 1000 2000 3000 4000 0 200 400 600 TAg A3B 0 p < 0.0001 Spearman’s rho = 0.42 F G 0 1000 2000 3000 0 100 200 300 400 TAg EdU p < 0.0001 Spearman’s rho = 0.73 B 1 dpi 5 dpi Mock BKPyV Mock BKPyV TAg Percent positive cells VP1 A3B p=0.050 p=0.022 p=0.014 BKPyV 0 20 40 60 80 100 G B F FIG 1 Visualization and quantification of A3B expression in PyV-infected cells. (A and B) Immunofluorescent images and quantification of TAg, VP1, and A3B in BKPyV-infected HuK(i)G10 cells at 1 and 5 dpi (significance determined using Welch’s two-tailed t test; n  2 biological replicates). (C) Time course of A3B mRNA levels in JCPyV (Mad1 strain) versus mock-infected HuK(i)G10 cells. (D) RT-qPCR quantification of A3B transcripts in mock-, BKPyV-, and JCPyV (Mad1)-infected HuK(i)G10 cells at 6 dpi (significance determined by Welch’s two-tailed t test; n  3 technical replicates). (E) High-resolution immunofluorescent microscopy images of DAPI, A3B, EdU, and TAg in HuK(i)G10 cells infected with JCPyV (Mad1 strain). (F and G) Correlation coefficients and P values for EdU and A3B levels versus T antigen intensity in 100 cell images from a single experiment similar to that in panel E. compared to mock-infected controls (Fig. 1A to D). Generally, JCPyV is regarded to have slower replication dynamics than BKPyV (Dunlop), so initial JCPyV infections were run out in a time course showing peak A3B expression at 7 dpi (Fig. 1C). January/February 2019 Volume 10 Issue 1 e02690-18 RESULTS A J LXCXE J LXCXE TPPK NLS DBD Helicase HR-AH TPPK NLS p53 Rb BK LTAg BK truncTAg 105 LFCHEDMF 105 ****KK** Rb-binding mutant C A3B Merge truncT truncT pRb- TAg C 50μm 50μm A C Merge Rb-binding mutant B 37 kDa 37 kDa 37 kDa 75 kDa 100 kDa 75 kDa 100 kDa 15 kDa 20 kDa 15 kDa 20 kDa Mock Empty TAg TAg-pRb- truncT truncT-pRb- A3G A3B TAg GAPDH UNG2 TAg truncT truncT Longer exposures Shorter exposures B on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from Mock Empty truncT truncT pRb- D 37 kDa 37 kDa 100 kDa 20 kDa TAg A3G A3B truncT GAPDH Mock Empty truncT truncT pRb- E 0 0.02 0.04 0.06 0.08 0.10 A3B mRNA relative to TBP p=2.05x10-5 p=0.0049 p=0.016 D E FIG 2 RB-binding domain is necessary for A3B induction by polyomavirus T antigen. (A) Diagram of BKPyV T antigen isoforms and the LXCXE mutant used here. (B) Immunoblots for the indicated proteins in RPTECs transduced with a lentiviral vector expressing LTAg, truncT, the indicated mutants, or nothing (empty). Mock-transduced cells were analyzed in parallel as an additional control. (C) Immunofluorescent microscopy images for truncT, truncT LXCXE mutant, and A3B in transduced RPTECs. (D and E) Immunoblots and RT-qPCR results for MCF10A cells transduced with the indicated constructs as in panel B (mean and SEM shown in panel E; n  3 biological replicates; P value determined by Welch’s two-tailed t test). APOBEC3B upregulation by polyomavirus large T antigen requires the canon- ical RB-interacting motif LXCXE. Based on the results above and our previous studies (6), multiple polyomaviruses have the conserved capacity to upregulate A3B expression in primary and immortalized kidney epithelial cells through the functions of large (L) TAg. To investigate the LTAg domains responsible for A3B induction, and thus also implicate associated cellular factors, we tested a naturally occurring splice variant of BKPyV LTAg, known as truncT, which lacks the DNA-binding and helicase domains essential for p53 neutralization (30–32) (schematic in Fig. 2A). In parallel, we also assessed derivatives of LTAg and truncT with a disrupted LXCXE motif, which is required for inhibiting the tumor suppressor protein RB1 as originally shown for SV40 TAg (33). RESULTS Across these experiments, JCPyV-infected HuK(i)G10 cells showed a greater differential expression of A3B mRNA and protein compared to mock-treated cells (Fig. 1B to D). JCPyV-infected cells were also analyzed 7 dpi by high-resolution immunofluorescent microscopy for expression of A3B and viral proteins and for formation of virus replica- tion foci. Cells were stained for DAPI, TAg, A3B, and EdU with virus replication centers appearing as brightly stained puncta positive for both TAg and EdU (representative images in Fig. 1A and E) (29). In infected cells, A3B is strongly induced with a pan-nuclear staining pattern that is sometimes coincident with EdU-positive virus replication foci. Incorporation of EdU into active replication foci is highlighted by strong positive correlations with TAg stain intensity, as expected, whereas A3B showed weaker but still significantly positive correlations (Fig. 1F and G). These data indicate that A3B upregulation may be a general property of polyomavirus infection and that A3B may access at least a subset of virus replication centers. mbio.asm.org 3 ® Starrett et al. Mock Empty truncT truncT pRb- Mock Empty truncT truncT pRb- A B D E J LXCXE J LXCXE TPPK NLS DBD Helicase HR-AH TPPK NLS p53 Rb BK LTAg BK truncTAg 105 LFCHEDMF 105 ****KK** Rb-binding mutant A3B Merge truncT truncT pRb- TAg C 50μm 50μm 0 0.02 0.04 0.06 0.08 0.10 37 kDa 37 kDa 100 kDa 20 kDa TAg A3G A3B truncT GAPDH A3B mRNA relative to TBP 37 kDa 37 kDa 37 kDa 75 kDa 100 kDa 75 kDa 100 kDa 15 kDa 20 kDa 15 kDa 20 kDa Mock Empty TAg TAg-pRb- truncT truncT-pRb- A3G A3B TAg GAPDH UNG2 TAg truncT truncT Longer exposures Shorter exposures p=2.05x10-5 p=0.0049 p=0.016 FIG 2 RB-binding domain is necessary for A3B induction by polyomavirus T antigen. (A) Diagram of BKPyV T antigen isoforms and the LXCXE mutant used here. (B) Immunoblots for the indicated proteins in RPTECs transduced with a lentiviral vector expressing LTAg, truncT, the indicated mutants, or nothing (empty). Mock-transduced cells were analyzed in parallel as an additional control. (C) Immunofluorescent microscopy images for truncT, truncT LXCXE mutant, and A3B in transduced RPTECs. (D and E) Immunoblots and RT-qPCR results for MCF10A cells transduced with the indicated constructs as in panel B (mean and SEM shown in panel E; n  3 biological replicates; P value determined by Welch’s two-tailed t test). January/February 2019 Volume 10 Issue 1 e02690-18 RESULTS MCF10A β-Actin p53 WT 55 kDa 43 kDa 17 kDa p21 ΔTP53 C MCF-7L DMSO Nutlin, 5 µ M PMA Nutlin + PMA B A3B TP53 P21 MDM2 SLC7a11 0 0.2 0.4 0.6 0.8 1.0 10 20 30 40 * * * * * * * * * * * mRNA relative to TBP MCF10A A A3B TP53 P21 MDM2 SLC7a11 0 1 2 3 10 20 30 40 * * * * * * * * * * DMSO PMA DMSO PMA 0 0.02 0.04 0.06 0.08 0.10 0.5 1.0 1.5 2.0 2.5 A3B mRNA relative to TBP WT ΔTP53 MCF10A D D B A on April 15, 2019 b http://mbio.asm.org/ Downloaded from on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from FIG 3 Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells treated with DMSO, 5 M nutlin, PMA, or nutlin  PMA. Statistically significant changes by Student’s t test (P  0.05) are noted by an asterisk (mean and SEM; n  3 technical replicates). (C) Immunoblots of WT and TP53 KO MCF10A cell lines. (D) RT-qPCR results showing the effects of PMA treatment on A3B mRNA levels in WT and TP53 KO MCF10A cell lines. on April 15, 2019 by guest http://mbio.asm.org/ aded from Immunofluorescent microscopy images also show truncT-mediated induction of nu- clear A3B but not by the RB-binding mutant derivative (Fig. 2C). To date, many aspects of A3B regulation and function have been determined using normal-like and cancerous mammary epithelial cell lines due to higher capacities for genetic manipulation over primary cells and greater relevance to cancer (35, 36). To ask whether TAg induction of A3B also occurs in one of these more tractable systems, the normal-like mammary epithelial cell line MCF10A was transduced with constructs expressing BKPyV truncT or the RB-binding mutant and analyzed as described above. Both immunoblotting and RT-qPCR yielded similar results with truncT but not the RB-binding mutant causing A3B induction (Fig. 2D and E). Thus, polyomavirus T antigen appears to possess a conserved, LXCXE-dependent capacity to induce A3B in different epithelial cell types. TP53 inactivation is dispensable for APOBEC3B induction. The aforementioned data comparing LTAg and truncT simultaneously implicate RB1 and demonstrate that p53 inhibition is not required for A3B induction because truncT completely lacks the p53 binding domain (Fig. 2). RESULTS RPTECs were transduced with lentiviruses expressing an empty multiple cloning site as a negative control, BKPyV LTAg as a positive control, BKPyV truncT, and RB-binding site mutant derivatives; incubated 3 days; and assessed by immunoblotting and fluores- cence microscopy. Mock-transduced cells express low levels of A3G, and transduction with empty lentivirus causes a modest increase in this protein and also raises A3B levels to faintly detectable levels (Fig. 2B). In contrast, both LTAg and truncT induce expres- sion of A3B and UNG2, a known target of the RB-E2F axis (34), and all induction for both of these proteins is eliminated by two amino acid substitutions shown to abrogate RB binding in SV40 TAg (LFCHED to LFCHKK) (33) (Fig. 2B). The LTAg and truncT mutants invariably migrate faster than the corresponding wild-type proteins during SDS-PAGE, which is likely due to a charge differential caused by the two amino acid substitutions. mbio.asm.org 4 Polyomavirus T Antigen Induces APOBEC3B Expression ® DMSO PMA DMSO PMA 0 0.02 0.04 0.06 0.08 0.10 0.5 1.0 1.5 2.0 2.5 A3B mRNA relative to TBP WT ΔTP53 MCF10A MCF10A mRNA relative to TBP MCF10A MCF-7L DMSO Nutlin, 5 µ M PMA Nutlin + PMA β-Actin p53 WT 55 kDa 43 kDa 17 kDa p21 ΔTP53 A B D C A3B TP53 P21 MDM2 SLC7a11 A3B TP53 P21 MDM2 SLC7a11 0 1 2 3 10 20 30 40 0 0.2 0.4 0.6 0.8 1.0 10 20 30 40 * * * * * * * * * * * * * * * * * * * * * FIG 3 Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells treated with DMSO, 5 M nutlin, PMA, or nutlin  PMA. Statistically significant changes by Student’s t test (P  0.05) are noted by an asterisk (mean and SEM; n  3 technical replicates). (C) Immunoblots of WT and TP53 KO MCF10A cell lines. (D) RT-qPCR results showing the effects of PMA treatment on A3B mRNA levels in WT and TP53 KO MCF10A cell lines. January/February 2019 Volume 10 Issue 1 e02690-18 RESULTS 0 1 2 3 4 Fold change expression RB1 RBL1 RBL2 0 5 10 15 A3B UNG2 CCNE2 A RPTE RB1 RBL1 RBL2 + - - + + - + + + - - - + - + - + - - + + - - + siRNA * * ** ** ** ** ** ** ** ** ** ** ** ** + + B siRNA 130 95 130 95 RB1 RBL1 - - + - - + 55 43 RB1 RBL1 A3B TUB 40 35 kDa B A Fold change expression on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from on April 15, 2019 by guest http://mbio.asm.org/ aded from RB1 RBL1 RBL2 A3B UNG2 CCNE2 0 1 2 3 4 Fold change expression 0 5 10 15 C MCF10A RB1 RBL1 RBL2 + - - + + - + + + - - - + - + - + - - + + - - + siRNA ** * * ** + + + * * * * * * * * ** D siRNA 55 43 130 95 - - + - RB1 RBL1 40 35 kDa RB1 TUB A3B D C Fold change expression siRNA 55 43 130 95 - - - + RB1 RBL1 40 35 kDa TUB A3B RBL1 FIG 4 Modulation of RB family genes and A3B regulation. (A and C) Bar plots of RT-qPCR quantification of RB-family mRNAs (top) and predicted responsive genes, A3B, UNG2, and CCNE2 (bottom), in RPTEC and MCF10A cells with siRNA-mediated KD of RB-family genes. P values for each siRNA combination compared to control were calculated using Welch’s two-tailed t test and were indicated using the following symbols: , P  0.1; *, P  0.05; **, P  0.01; n  3 biological replicates. (B and D) Immunoblots for A3B, RB1, and RBL1 in RPTEC and MCF10A cells following treatment with the indicated siRNA. tional activities. To investigate the roles of RB1, RBL1, and RBL2 in A3B transcriptional regulation, a series of knockdown experiments was done with siRNAs targeting each of these factors in RPTECs and MCF10A cells (Fig. 4A to D). RT-qPCR showed that 75% knockdown was achieved for each targeted gene (upper panels in Fig. 4A and C). As controls, CCNE2 was upregulated upon RB1 knockdown and UNG2 was moderately upregulated by RBL2 knockdown (lower panels in Fig. 4A and C). RESULTS 0 1 2 3 4 Fold change expression RB1 RBL1 RBL2 0 5 10 15 A3B UNG2 CCNE2 RB1 RBL1 RBL2 A3B UNG2 CCNE2 0 1 2 3 4 Fold change expression 0 5 10 15 A C B D MCF10A RPTE RB1 RBL1 RBL2 + - - + + - + + + - - - + - + - + - - + + - - + siRNA RB1 RBL1 RBL2 + - - + + - + + + - - - + - + - + - - + + - - + siRNA siRNA siRNA siRNA * * ** ** ** ** ** ** ** ** ** ** ** ** + + ** * * ** + + + * * * * * * * * ** 130 95 130 95 RB1 RBL1 - - + - - + 55 43 55 43 55 43 RB1 RBL1 A3B TUB 40 35 130 95 130 95 - - + - RB1 RBL1 - - - + RB1 RBL1 40 35 40 35 kDa kDa kDa RB1 TUB A3B TUB A3B RBL1 FIG 4 Modulation of RB family genes and A3B regulation. (A and C) Bar plots of RT-qPCR quantification of RB-family mRNAs (top) and predicted responsive genes, A3B, UNG2, and CCNE2 (bottom), in RPTEC and MCF10A cells with siRNA-mediated KD of RB-family genes. P values for each siRNA combination compared to control were calculated using Welch’s two-tailed t test and were indicated using the following symbols: , P  0.1; *, P  0.05; **, P  0.01; n  3 biological replicates. (B and D) Immunoblots for A3B, RB1, and RBL1 in RPTEC and MCF10A cells following treatment with the indicated siRNA. RESULTS To further ask whether p53 inactivation might influence A3B gene expression, we quantified A3B mRNA levels in two cell lines that have been used to study A3B regulation, MCF10A and the human estrogen receptor-positive breast cancer cell line MCF-7L (above and references 35 to 38). Each cell line was treated with either DMSO or 5 M nutlin, which is a drug that protects p53 from MDM2-mediated degradation (39). As controls, mRNA levels were quantified for two genes activated by p53 (P21, MDM2) (40–43) and one gene repressed by p53 (SLC7A11) (44, 45). Respectively, the expression of these genes was derepressed or repressed by nutlin treatment (Fig. 3A and B). In comparison, neither steady-state nor PMA-induced A3B mRNA levels were changed by nutlin (Fig. 3A and B). Moreover, Cas9-mediated knockout of TP53 in MCF10A cells also caused no significant effect on basal or PMA-induced A3B expression levels (Fig. 3C and D). These data combine to indicate that p53 by itself has no significant role in the either the PMA-induced pathway or basal-state transcriptional regulation of A3B, discouraging our original hypothesis (23) and conflicting with published data (38) (see Discussion). RB-family knockdown is insufficient to induce APOBEC3B expression. RB1 is argu- ably the most widely studied target of the LXCXE motif of viral proteins such as HPV E7, adenovirus E1A, and polyomavirus LTAg (27, 31, 32, 46). However, the related pocket proteins RBL1 (p107) and RBL2 (p130) also have an LXCXE-binding motif, are similarly targeted and inactivated by LTAg and truncT, and may be involved in A3B regulation (47–50). The aforementioned viral proteins bind to the hypophosphorylated forms of RB1, RBL1, and RBL2, which inhibits phosphorylation by cyclin-dependent kinases (CDKs) and leads to an accelerated cell cycle in part by deregulation of E2F transcrip- mbio.asm.org 5 January/February 2019 Volume 10 Issue 1 e02690-18 ® Starrett et al. January/February 2019 Volume 10 Issue 1 e02690-18 RESULTS (C) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A ll d h lb l b f d h d h / l / l P f d h p p g 0 0.5 1.0 1.5 CCNE2 MCF10A 0 1 4 MCF7 0 1 4 T47D 0 1 4 HCC1143 0 1 4 HCC1954 0 1 4 SUM225CWN 0 1 4 HCC1937 0 1 4 HCC1395 0 1 4 HCC1599 0 1 4 HCC202 0 1 4 Hs578T 0 1 4 Fold chan Hours post palbociclib treatment [Palbo] on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from A3B CCNE2 Fold change expression 0 0.5 1.0 1.5 2.0 0 5 10 15 20 No PMA PMA-treated 0 0.5 1.0 1.5 C 0 1 3 0 1 3 ociclib treatment Hours post PMA treatment [Palbo] A3B CCNE2 Fold change expression , 0 0.5 1.0 1.5 2.0 2.5 0 10 20 30 No PMA 3h PMA-pretreated 0 0.5 1.0 1.5 2.0 B 0 1 3 0 1 3 Hours post palbociclib treatment p p [Palbo] C B 1 3 0 1 Hours post palbociclib treatment Hours post PMA treatment Hours post PMA treatment FIG 5 Palbociclib treatment of cancer cell lines and A3B expression. (A) RT-qPCR quantification of A3B and CCNE2 mRNA expression in the indicated cell lines treated with 0, 0.5, or 2.5 M palbociclib for 0, 1, or 4 h. (B) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0 ng/ml or 25 ng/ml PMA for 3 h prior to treatment with 0, 0.5, or 2.5 M palbociclib for 0, 1, and 3 h. (C) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0, 0.5, or 2.5 M palbociclib for 30 min and then treated with 0 ng/ml or 25 ng/ml PMA for 0, 1, and 3 h. scriptional. These results also suggest that truncT may have at least one additional activity mediated by its LXCXE motif that contributes to A3B upregulation. Pharmacological inhibition of CKD4/6 does not alter APOBEC3B expression. Pal- bociclib is a selective inhibitor of CDK4 and CDK6, which are kinases that function normally to phosphorylate pRB, prevent binding to E2F transcription factors, and stimulate the expression of many genes involved in cell cycle progression (51–53). To corroborate the knockdown experiments above, we treated a panel of transformed cell lines with palbociclib and quantified mRNA expression levels over time. RESULTS Polyomavirus T Antigen Induces APOBEC3B Expression ® on April 15, 2019 http://mbio.asm.org/ Downloaded from ® Polyomavirus T Antigen Induces APOBEC3B Expression 0 0.5 1.0 1.5 A3B 0 0.5 1.0 1.5 CCNE2 MCF10A 0 1 4 MCF7 0 1 4 T47D 0 1 4 HCC1143 0 1 4 HCC1954 0 1 4 SUM225CWN 0 1 4 HCC1937 0 1 4 HCC1395 0 1 4 HCC1599 0 1 4 HCC202 0 1 4 Hs578T 0 1 4 Fold change expression A Hours post palbociclib treatment [Palbo] 0 0.5 1.0 1.5 A3B 1 5 CCNE2 nge expression A A 0 0.5 1.0 1.5 CCNE2 A3B CCNE2 A3B CCNE2 MCF10A 0 1 4 MCF7 0 1 4 T47D 0 1 4 HCC1143 0 1 4 HCC1954 0 1 4 SUM225CWN 0 1 4 HCC1937 0 1 4 HCC1395 0 1 4 HCC1599 0 1 4 HCC202 0 1 4 Hs578T 0 1 4 Fold change Fold change expression Fold change expression , 0 0.5 1.0 1.5 2.0 2.5 0 10 20 30 No PMA 3h PMA-pretreated 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 5 10 15 20 No PMA PMA-treated 0 0.5 1.0 1.5 B C 0 1 3 0 1 3 0 1 3 0 1 3 Hours post palbociclib treatment Hours post palbociclib treatment Hours post PMA treatment [Palbo] [Palbo] [Palbo] FIG 5 Palbociclib treatment of cancer cell lines and A3B expression. (A) RT-qPCR quantification of A3B and CCNE2 mRNA expression in the indicated cell lines treated with 0, 0.5, or 2.5 M palbociclib for 0, 1, or 4 h. (B) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0 ng/ml or 25 ng/ml PMA for 3 h prior to treatment with 0, 0.5, or 2.5 M palbociclib for 0, 1, and 3 h. RESULTS However, no combi- nation of siRNAs resulted in significant upregulation of A3B mRNA levels (lower panels in Fig. 4A and C). Knockdown of RB1 and RBL1 was validated at the protein level, but RBL2 could not be clearly discerned with available commercial antibodies (Fig. 4B and D). In contrast to the RT-qPCR results, protein-level A3B expression did appear to be elevated upon RBL1 knockdown. These results suggest that depletion of each RB family member alone or in combination is insufficient to significantly upregulate A3B mRNA levels, at least in these two different normal-like cell types where A3B is induced by TAg and truncT. However, the observed upregulation at the protein level in RBL1-depleted cells raises the possibility of an additional layer of regulation that may be posttran- mbio.asm.org 6 January/February 2019 Volume 10 Issue 1 e02690-18 0 0.5 1.0 1.5 A3B 0 0.5 1.0 1.5 CCNE2 A3B CCNE2 A3B CCNE2 MCF10A 0 1 4 MCF7 0 1 4 T47D 0 1 4 HCC1143 0 1 4 HCC1954 0 1 4 SUM225CWN 0 1 4 HCC1937 0 1 4 HCC1395 0 1 4 HCC1599 0 1 4 HCC202 0 1 4 Hs578T 0 1 4 Fold change expression Fold change expression Fold change expression , 0 0.5 1.0 1.5 2.0 2.5 0 10 20 30 No PMA 3h PMA-pretreated 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 5 10 15 20 No PMA PMA-treated 0 0.5 1.0 1.5 A B C 0 1 3 0 1 3 0 1 3 0 1 3 Hours post palbociclib treatment Hours post palbociclib treatment Hours post PMA treatment [Palbo] [Palbo] [Palbo] FIG 5 Palbociclib treatment of cancer cell lines and A3B expression. (A) RT-qPCR quantification of A3B and CCNE2 mRNA expression in the indicated cell lines treated with 0, 0.5, or 2.5 M palbociclib for 0, 1, or 4 h. (B) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0 ng/ml or 25 ng/ml PMA for 3 h prior to treatment with 0, 0.5, or 2.5 M palbociclib for 0, 1, and 3 h. (C) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0, 0.5, or 2.5 M palbociclib for 30 min and then treated with 0 ng/ml or 25 ng/ml PMA for 0, 1, and 3 h. January/February 2019 Volume 10 Issue 1 e02690-18 RESULTS This panel of cell lines was constructed based on A3B expression, ranging from low to high (54, 55), TP53 status, and ability to phosphorylate RB. As a positive control for palbociclib efficacy, we analyzed expression of CCNE2, which encodes cyclin E2, promotes entry into S phase, and is a known CDK4/6-RB-regulated gene (56, 57). The majority of cell lines showed a dose- and time-responsive decrease in CCNE2 mRNA expression (Fig. 5A). This effect was minimal in HCC1937 and HCC1599 cells, which are known to display decreased RB phosphorylation (58, 59). In contrast, none of the palbociclib- treated cell lines showed a reproducible or significant change in A3B mRNA expression. In addition, MCF10A cells were treated with PMA to induce A3B mRNA expression by the PKC/ncNF-B pathway, and again, palbociclib had little effect (palbociclib added mbio.asm.org 7 January/February 2019 Volume 10 Issue 1 e02690-18 ® Starrett et al. post- or pre-PMA addition in Fig. 5B and C). These results combine to indicate that the kinase activity of CDK4 and CDK6 is dispensable for A3B expression in multiple different cell lines. Tumor transcriptome analyses support involvement of the RB pathway in APOBEC3B regulation. We next used bioinformatics approaches to mine TCGA data and assess global correlates with A3B mRNA expression in human tumors. First, we conducted pathway analysis using all genes with significant positive correlations between A3B expression in the TCGA breast tumor cohort. This analysis revealed that 11 of the top 20 significantly enriched upstream transcription factors contributing to this expression pattern are part of the CDK4/6-cyclin D-RB-E2F axis (green-labeled genes in Fig. 6A). These regulatory factors were either significantly activated or inhib- ited, generally corresponding with known functions, with the net outcome being accelerated cell cycling (respectively, red and blue bars in Fig. 6A). Upon closer pairwise examination of effectors in this signal transduction pathway, A3B mRNA expression has the strongest positive correlations with expression of RBL1, E2F1, E2F2, E2F7, and E2F8 (Fig. 6B). Last, we expanded this expression correlation analysis to include 22 different tumor types in TCGA and all 11 APOBEC family members. This global approach further highlighted strong correlations between A3B and expression of E2F1, E2F2, E2F7, and E2F8 and indicated that the association between A3B, this signal transduction pathway, and the cell cycle is evident in many cancer types (Fig. 6C). RESULTS Heat map intensities also indicated that A3B is the only APOBEC family member that positively and globally correlates with activation of the RB-E2F axis. To further reconcile our experimental and bioinformatics data sets, we used the meta-analysis regulatory data from TargetGen- eReg (http://www.targetgenereg.org/) to compare the regulation of A3B with known cell cycle-related genes (60). These results, summarized in Table 1, further indicate that the A3B mRNA expression profile is consistent with that of a cell cycle-regulated gene that becomes upregulated during the G2/M phase, which is similar to FOXM1 and distinct from UNG2, CCNE2, and A3C (the last being an APOBEC3 family member likely to be regulated by p53). January/February 2019 Volume 10 Issue 1 e02690-18 DISCUSSION RB1 Spearman: -0.173 q=1.74x10-4 5 6 7 8 9 10 11 12 13 3 4 5 6 7 8 9 10 11 E2F1 Spearman: 0.552 q=3.98x10-74 E2F4 Spearman: 0.228 q=2.32x10-9 E2F7 Spearman: 0.429 q=9.74x10-41 12 13 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 3 4 5 6 7 8 9 10 11 12 0 1 2 A3 mRNA expression (RSEM Log2) -4 -2 0 2 4 6 8 10 12 14 B 0 20 40 60 E2F4 TP53 E2F1 RB1 TBX2 CCND1 CDKN2A E2F3 FOXM1 TCF3 MITF FOXO3 E2F2 RBL1 SMARCB1 YY1 NUPR1 KDM5B E2F6 E2F7 Activated Neutral Inhibited Enrichment (-log p-value) A RB1 Spearman: -0.173 q=1.74x10-4 -4 -2 0 2 4 6 8 10 12 14 5 6 7 8 9 10 11 12 13 3 4 5 6 7 8 9 10 11 E2F1 Spearman: 0.552 q=3.98x10-74 E2F2 Spearman: 0.573 q=2.57x10-81 E2F3 Spearman: 0.301 q=3.74x10-18 E2F4 Spearman: 0.228 q=2.32x10-9 E2F5 Spearman: 0.223 q=8.20x10-9 E2F7 Spearman: 0.429 q=9.74x10-41 E2F6 Spearman: 0.115 q=0.953 E2F8 Spearman: 0.534 q=1.98x10-68 12 13 3 4 5 6 7 8 9 10 11 12 13 2 4 6 8 10 12 14 0 2 4 6 8 10 12 6 7 8 9 10 11 12 6 7 8 9 10 11 12 13 14 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 3 4 5 6 7 8 9 10 11 12 0 1 2 A3B mRNA expression (RSEM Log2) mRNA expression (RSEM Log2) Spearman: 0.353 q=2.89x10-26 0 2 4 6 8 Spearman: -0.134 q=0.0248 7 8 9 RBL1 RBL2 10 12 10 11 12 13 -4 -2 0 2 4 6 8 10 12 14 -4 -2 0 2 4 6 8 10 12 14 B B A A mRNA expression (RSEM Log2) on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from on April 15, 2019 by guest http://mbio.asm.org/ aded from A3B mRNA expression (RSEM Log2) AICDA A1 A2 A3A A3B A3C A3D A3F A3G A3H A4 OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA GBM GBM GBM LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 −0.3 0.0 0.3 0.6 Spearman’s correlation C FIG 6 Evidence for A3B regulation by the RB/E2F pathway in tumors. DISCUSSION (A) Top 20 hits from IPA enrichment analysis of upstream transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. Negative logs of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. A2 A3A A3D A3H GBM OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 C C A1 A3C A3G OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM GBM −0.3 0.0 0.3 0.6 Spearman’s correlation AICDA A3B A3F A4 OV RCA NSC LCA ESC IRP UAD USC EAD AML SCA BM GG IHC AAD RAD ARC TAD GCT HCA CEC CM A3G A4 FIG 6 Evidence for A3B regulation by the RB/E2F pathway in tumors. (A) Top 20 hits from IPA enrichment analysis of upstream transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. Negative logs of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. January/February 2019 Volume 10 Issue 1 e02690-18 DISCUSSION Negative logs of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. DISCUSSION In this study, we investigate the mechanism of A3B upregulation by polyomavirus T antigen through analyses of separation-of-function mutants, genetic knockdowns, pharmacologic treatments, and transcriptomic data. We use high-resolution fluores- cence microscopy to show that polyomavirus infection causes A3B upregulation and protein accumulation in the nuclear compartment with the potential to access viral replication foci. Second, we show that the LXCXE motif of LTAg and truncT, which is well known to inhibit the tumor suppressor RB, is essential for A3B upregulation, whereas the p53-binding domain is dispensable. Further investigation into this path- way using genetic and pharmacologic treatments indicate that solely perturbing RB family members (RB, RBL1, and RBL2) or kinases responsible for their phosphorylation (CDK4 and CDK6) is insufficient to cause A3B mRNA upregulation. However, bioinfor- matics analyses of tumor expression data show strong global correlations between A3B mRNA expression and expression of other genes regulated by the RB-E2F signaling pathway, including several members of the E2F family of transcription factors. Additional analysis of cell cycle-regulatory networks using the TargetGeneReg da- tabase suggest that A3B might be a late cell cycle gene repressed by the RB/E2F family members associated with the DREAM complex in quiescence and activated by other transcription factors. Suppression by the DREAM complex is supported by the mild upregulation of A3B protein observed upon RBL1 knockdown in this study. One putative activating transcription factor is FOXM1, which was significantly enriched as an upstream regulator of A3B and is known to activate genes in late G2/M (61). Interest- ingly, in lymphoblastoid B cells, FOXM1 has been reported to frequently cooccupy NF-B binding sites and form protein complexes with NF-B transcription factors, which have been implicated in A3B regulation (36, 62). DISCUSSION Taken together, these results indicate mbio.asm.org 8 ® Polyomavirus T Antigen Induces APOBEC3B Expression RB1 Spearman: -0.173 q=1.74x10-4 -4 -2 0 2 4 6 8 10 12 14 5 6 7 8 9 10 11 12 13 3 4 5 6 7 8 9 10 11 E2F1 Spearman: 0.552 q=3.98x10-74 E2F2 Spearman: 0.573 q=2.57x10-81 E2F3 Spearman: 0.301 q=3.74x10-18 E2F4 Spearman: 0.228 q=2.32x10-9 E2F5 Spearman: 0.223 q=8.20x10-9 E2F7 Spearman: 0.429 q=9.74x10-41 E2F6 Spearman: 0.115 q=0.953 E2F8 Spearman: 0.534 q=1.98x10-68 12 13 3 4 5 6 7 8 9 10 11 12 13 2 4 6 8 10 12 14 0 2 4 6 8 10 12 6 7 8 9 10 11 12 6 7 8 9 10 11 12 13 14 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 3 4 5 6 7 8 9 10 11 12 0 1 2 A3B mRNA expression (RSEM Log2) mRNA expression (RSEM Log2) Spearman: 0.353 q=2.89x10-26 0 2 4 6 8 Spearman: -0.134 q=0.0248 7 8 9 RBL1 RBL2 10 12 10 11 12 13 -4 -2 0 2 4 6 8 10 12 14 -4 -2 0 2 4 6 8 10 12 14 AICDA A1 A2 A3A A3B A3C A3D A3F A3G A3H A4 OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA GBM GBM GBM LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM OV BRCA HNSC BLCA CESC KIRP LUAD LUSC READ LAML ESCA LGG LIHC PAAD PRAD SARC STAD TGCT THCA UCEC SKCM E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 E2F1 E2F2 E2F3 E2F4 E2F5 E2F6 E2F7 E2F8 RB1 RBL1 RBL2 −0.3 0.0 0.3 0.6 0 20 40 60 E2F4 TP53 E2F1 RB1 TBX2 CCND1 CDKN2A E2F3 FOXM1 TCF3 MITF FOXO3 E2F2 RBL1 SMARCB1 YY1 NUPR1 KDM5B E2F6 E2F7 Activated Neutral Inhibited Enrichment (-log p-value) Spearman’s correlation A C B FIG 6 Evidence for A3B regulation by the RB/E2F pathway in tumors. (A) Top 20 hits from IPA enrichment analysis of upstream transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. DISCUSSION FIG 6 Evidence for A3B regulation by the RB/E2F pathway in tumors. (A) Top 20 hits from IPA enrichment analysis of upstream transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. Negative logs of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. mbio.asm.org 9 January/February 2019 Volume 10 Issue 1 e02690-18 ® Starrett et al. TABLE 1 Cell cycle analysis of A3B and other cell cycle-regulated genes Type of value Value for gene: APOBEC3B APOBEC3C CCNE2 FOXM1 UNG2 Chromosome 22 22 8 12 12 p53 expression score 4 14 14 17 14 No. of cell cycle data sets 2 1 5 2 5 G1/S or G2/M G2/M 0 G1/S G2/M G1/S p53 target No Yes No No No Cell cycle gene Yes No Yes Yes Yes DREAM target Yes No No Yes Yes MMB-FoxM1 target No No No No No RB-E2F target No No Yes No Yes TABLE 1 Cell cycle analysis of A3B and other cell cycle-regulated genes on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from that the RB/E2F pathway, which is commonly modified in cancer, likely contributes to A3B overexpression observed in virus infections and in different tumor types, but additional unknown signals are also likely to be required for full induction (model in Fig. 7). on April 15, 2019 by guest http://mbio.asm.org/ aded from Our original studies with HPV and A3B led us to propose a model in which p53 represses A3B transcription and that p53 inactivation by the viral oncoprotein E6 or by genetic mutation results in derepression of A3B transcription (23). This transcription repression model is consistent with strong correlations in tumors and cell lines between A3B overexpression and TP53 inactivation (54). A recent study confirmed these corre- lations and used pharmacologic and genetic approaches to provide further support for such a model (38). mbio.asm.org 10 DISCUSSION However, three different results in the cell-based systems presented here do not support a dominant role for p53 in A3B repression. Specifically, the p53 binding domain of BKPyV is dispensable for A3B upregulation, nutlin treatment has no effect on basal or induced A3B expression, and p53 knockout alone fails to induce A3B expression (Fig. 2 and 3). Moreover, another recent study showed that p53 inactivation renders cells more permissive for A3B mutagenesis (55). Therefore, we disfavor a transcriptional role and now favor a “tolerance model” in which p53 inactivation (genetic, epigenetic, or viral) is required for cells to be able to tolerate the increased levels of DNA damage caused by A3B overexpression. This model also explains why somatic TP53 mutations were identified as a significant global correlate with A3B overexpression in cancer (11). The RB-E2F signaling axis is one of the most frequently mutated pathways in cancer, which contributes to several hallmarks of cancer by deregulating the cell cycle (63). RB p21 CyclinD1 Rb CDK4/6 Deleted/Suppressed Amplified/Overexpressed PyV LTAg PyV truncT p107 p130 P P P p53 PyV LTAg Palbociclib ncNFκB A3B TEAD Other regulators FIG 7 Model for A3B transcriptional regulation. Schematic of the cell cycle-related proteins affected by T antigen and drug treatments used in this study with implications for A3B transcriptional regulation. Solid lines represent established processes, and dashed lines represent regulatory interactions/pathways implicated by our studies. Deleted/Suppressed Amplified/Overexpressed FIG 7 Model for A3B transcriptional regulation. Schematic of the cell cycle-related proteins affected by T antigen and drug treatments used in this study with implications for A3B transcriptional regulation. Solid lines represent established processes, and dashed lines represent regulatory interactions/pathways implicated by our studies. FIG 7 Model for A3B transcriptional regulation. Schematic of the cell cycle-related proteins affected by T antigen and drug treatments used in this study with implications for A3B transcriptional regulation. Solid lines represent established processes, and dashed lines represent regulatory interactions/pathways implicated by our studies. mbio.asm.org 10 January/February 2019 Volume 10 Issue 1 e02690-18 ® Polyomavirus T Antigen Induces APOBEC3B Expression inactivation is also a common target for viral genes in order to promote the survival of infected cells. The integration and continued expression of viral oncogenes in the host genome are common characteristics of virus-associated tumors. MATERIALS AND METHODS Cell lines, culture conditions, and lentivirus production. Primary renal proximal tubule epithelial cells (RPTECs; Lonza) were grown in REGM (Lonza). MCF10A cells were grown in MEGM (Lonza) containing penicillin (100 U/ml) and streptomycin (100 g/ml). HuK(i)G10 cells were grown in RenaLife epithelial medium (Lifeline Cell Technologies) with 5% FBS. MCF7 and derivative cell lines were grown in Richter’s modification medium containing 5% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 g/ml), and 11.25 nM recombinant human insulin. All cell lines were grown at 37°C in a 5% CO2 incubator. Lentiviruses expressing TAg and mutant derivatives were produced in 293T cells and trans- duced into RPTECs as described previously (23). Antibodies. Large T and truncT forms of BKPyV T antigen were detected using pAb416 (30). JCPyV large T antigen was detected using PAB2000 (68). A Harris lab custom rabbit anti-human A3B monoclonal antibody, 5210.87.13, was used in immunoblotting assays and in high-resolution immunofluorescent microscopy experiments with JCPyV-infected HuK(i)G10 cells (69). Santa Cruz sc-130688 was used for A3B quantification and lower-resolution microscopy of BKPyV- and JCPyV-infected HuK(i)G10 cells (69). UNG2 was detected using the Abcam antibody ab23926. RB1 was detected using Santa Cruz sc-102, and RBL1 was detected using Cell Signaling 89798, whereas RBL2 could not be detected in immunoblots with Abcam antibody ab71143. TP53 (p53) was detected using clone DU-1 (Santa Cruz SC-126), p21 using CST clone 1201 (CST no. 2947), and beta-actin using CST clone 13E5 (CST no. 4970). RNA isolation, RT-qPCR, and immunoblots. Total RNA was harvested by removal of medium and resuspension in TRIzol (Thermo Fisher), and purification was done per the manufacturer’s protocol. RT-qPCR was used to quantify A3B and UNG2 transcripts in siRNA experiments as described previously (11, 23) and these methods were adapted for CCNE2. Protein lysates from virus and siRNA experiments were harvested at 3 or 7 dpi or postransduction, quantified, and immunoblotted as described previously (70). Data were plotted and t tests were calculated using GraphPad Prism 7. Immunofluorescent microscopy experiments. HuK(i)G10 kidney cells were seeded at 6,000 cells/ well in a 96-well plate. Twenty-four hours later, infection with JCPyV was performed as described, and then cells were collected 7 dpi. DISCUSSION For example, HPV- positive tumors frequently express the viral E7 oncoprotein, which also has an LXCXE motif, and this is thought to be critical for tumor development (64, 65). These tumors also tend to have a high burden of APOBEC-associated mutations on the DNA strand that serves as the template for lagging-strand replication, which is synthesized during the S phase of the cell cycle (11, 14, 17). Although some of these effects have been explained by perturbations to the p53 pathway, other pathways affected by E6 have also been shown to alter A3B gene expression, such as those regulated by the TEAD and ZNF384 transcription factors (23, 25, 36, 38). It is therefore not surprising in hindsight that expression of the antiviral enzyme A3B is also induced upon disruption of the RB-E2F pathway. This idea is supported by a study showing that high-risk HPV E7 is capable of upregulating A3B (26). An additional study also found that elevated A3B expression is significantly correlated with proliferative features in breast cancer (66). Last, the LXCXE motif may be acting through another cellular signaling pathway. For instance, the LXCXE of various viral proteins triggers activation of the antiviral cGAS- STING pathway, which could also contribute to A3B transcriptional regulation (67). on April 15, 2019 by guest http://mbio.asm.org/ aded from All of these results combine to indicate that A3B transcriptional regulation is complex and governed by multiple pathways and different transcription factors. Per- haps linkage to the cell cycle evolved to prevent potentially oncogenic mutations of the host genome during normal cellular DNA replication or, alternatively, to maximize antiviral responses during particularly susceptible cell cycle stages. Further experiments using mutant viral oncogenes as molecular probes, such as T antigen, E6, and E7, are likely to continue to provide valuable insights into the regulation of A3B and lead to a greater understanding of its roles in tumorigenesis, virus evolution, and antiviral immunity. January/February 2019 Volume 10 Issue 1 e02690-18 MATERIALS AND METHODS Infected cells were incubated with EdU (Click-iT Plus EdU Alexa Fluor 647 imaging kit; Thermo Fisher Scientific) for 15 min and incubated with CSK buffer (10 mM HEPES-KOH, pH 7.4; 300 mM sucrose; 100 mM NaCl; 3 mM KCl; 0.5% Triton X-100) (71) for 2 min on ice. Cells were then fixed in 4% PFA for 10 min followed by permeabilization with 0.5% Triton X-100 for 20 min. For EdU detection, the Click-iT reagent was added for 30 min in the dark according to the manufacturer’s protocol and washed three times with PBS. Samples were incubated with BlockAid blocking solution (Thermo Fisher) for 1 h at room temperature. T antigen, VP1, and A3B staining was performed using the aforementioned antibodies at 1:1,000, 1:1,000, and 1:100 (1:50 for sc-130688) dilutions in BlockAid, respectively, overnight at 4°C followed by staining with the secondary antibodies for 1 h at room mbio.asm.org 11 ® Starrett et al. temperature. Images were acquired on the Opera Phenix (PerkinElmer) with the confocal 63 water objective. Immunofluorescence in RPTE cells was performed as described using the above-mentioned antibodies and imaged on the Invitrogen EVOS FL Imaging System (72). siRNA and expression construct transfection. siRNAs targeting RB1 (J-003296-23; Dharmacon), RBL1 (SI02629921; Qiagen), and RBL2 (sc-29425; Santa Cruz) and fluorescein-conjugated nontargeting control siRNA (sc-36869; Santa Cruz) were purchased and diluted to a working concentration of 20 M. A final concentration of 20 nM was used for all targets in RPTECs, and 40 nM was used in MCF10A cells. siRNAs were delivered to cells using Lipofectamine RNAiMax (Thermo Fisher) as described previously (73). Drug treatment. Cells were treated with 5 M nutlin (Sigma) for 24 h, and after 18 h of treatment, 25 ng/ml phorbol myristate acetate (PMA; Sigma) was added for the final 6 h prior to RNA extraction. MCF10A cells were treated only with PMA or DMSO, and RNA was isolated 6 h after treatment. For the palbociclib experiments, MCF10A and MCF7 (p53 WT and low A3B expression); HCC1937 and HCC1395 (low A3B expression); T47D, HCC1954, and Hs578T (intermediate A3B expression); and HCC1599, HCC1143, SUM-225-CWN, and HCC202 (high A3B expression) cells were cultured in 6-well plates (Costar 3516; Corning Incorporated) until 70% confluence. Palbociclib (S1116; Selleckchem) was stored as a 5 mM solution in H2O and added to cells at concentrations of 0 M (H2O control), 0.5 M, and 2.5 M. MATERIALS AND METHODS No palbociclib was added to cells of the 0-h time point, which instead was transferred to ice prior to RNA isolation. RNA was also isolated at 0, 1, and 4 h post-palbociclib administration (total RNA purification kit 37500; Norgen), and cDNA was synthesized with 500 ng RNA (iScript 170-8891; Bio-Rad). RT-qPCR assays for A3B and CCNE2 were performed using the C1000 Thermal Cycler (Bio-Rad). For pretreatment with PMA (tlrl-pma; InvivoGen; 1-mg/ml stock in DMSO), cells were treated with 0 ng/ml (DMSO) or 25 ng/ml PMA for 3 h, followed by treatment with 0 M, 0.5 M, and 2.5 M palbociclib. RNA was isolated 0, 1, and 3 h after addition of palbociclib and processed as described above. For pretreatment with palbociclib, cells were treated with 0 M, 0.5 M, and 2.5 M palbociclib for 30 min, followed by treatment with 0 ng/ml (DMSO) or 25 ng/ml PMA. RNA was isolated 0, 1, and 3 h after addition of PMA and processed as described above. on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from on April 15, 2019 by guest http://mbio.asm.org/ d from Bioinformatics. TCGA expression data were downloaded from the Broad GDAC Firehose as of January 2016. Expression correlations against A3B by all other genes in the breast cancer cohort were calculated, and significant correlates were used to determine significant activation or inhibition of upstream regulators using Ingenuity Pathway Analysis (Qiagen). All other Spearman correlations and P values were calculated, and heat maps were plotted using the R statistical environment. P values were adjusted for multiple comparisons using the Bonferroni correction, and resulting q values were reported. Cell cycle data were acquired from http://www.targetgenereg.org/ in May 2018. ACKNOWLEDGMENTS We thank James A. DeCaprio, Diako Ebrahimi, and N. Alpay Temiz for valuable discussions and comments. We thank James A. DeCaprio, Diako Ebrahimi, and N. Alpay Temiz for valuable discussions and comments. This work was supported in part by Biogen and by grants from the National Institutes of Health (NIAID R01 AI123162 to M.J., NCI R21 CA206309 to R.S.H., and NIAID R37 to R.S.H.). J.W.M.M., P.N.S., and R.S.H. received funding from the Dutch Cancer Society (KWF grant no. EMCR-2016-10270). Partial salary support for T.A.S. was provided by the Susan G. Komen Foundation, support for G.J.S. and J.L.M. was provided by a Graduate Research Fellowship from the National Science Foundation, and support for M.C.J. was provided by a training grant from the NCI (T32 CA009138). R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished McKnight University Professor, and an Investigator of the Howard Hughes Medical Institute. R.S.H. is a cofounder, shareholder, and consultant for ApoGen Biotechnologies Inc. Biogen sponsored a portion of the work in the Harris laboratory. 1. Escalera-Zamudio M, Rojas-Anaya E, Kolokotronis SO, Taboada B, Loza- Rubio E, Méndez-Ojeda ML, Arias CF, Osterrieder N, Greenwood AD. 2016. Bats, primates, and the evolutionary origins and diversification of mammalian gammaherpesviruses. mBio 7:e01425-16. https://doi.org/10 .1128/mBio.01425-16. January/February 2019 Volume 10 Issue 1 e02690-18 2. Cuevas JM, Geller R, Garijo R, López-Aldeguer J, Sanjuán R. 2015. Ex- tremely high mutation rate of HIV-1 in vivo. PLoS Biol 13:e1002251. https://doi.org/10.1371/journal.pbio.1002251. Polyomavirus T Antigen Induces APOBEC3B Expression Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity. Nat Commun 8:15024. https://doi.org/10.1038/ncomms15024. 32. Ludlow JW, DeCaprio JA, Huang CM, Lee WH, Paucha E, Livingston DM. 1989. SV40 large T antigen binds preferentially to an underphosphory- lated member of the retinoblastoma susceptibility gene product family. Cell 56:57–65. https://doi.org/10.1016/0092-8674(89)90983-5. Cell 56:57–65. https://doi.org/10.1016/0092-8674(89)90983-5. 33. Christensen JB, Imperiale MJ. 1995. Inactivation of the retinoblastoma susceptibility protein is not sufficient for the transforming function of the conserved region 2-like domain of simian virus 40 large T antigen. J Virol 69:3945–3948. on April 15, 2019 by guest http://mbio.asm.org/ from 14. Seplyarskiy VB, Soldatov RA, Popadin KY, Antonarakis SE, Bazykin GA, Nikolaev SI. 2016. APOBEC-induced mutations in human cancers are strongly enriched on the lagging DNA strand during replication. Ge- nome Res 26:174–182. https://doi.org/10.1101/gr.197046.115. 34. Haug T, Skorpen F, Aas PA, Malm V, Skjelbred C, Krokan HE. 1998. Regulation of expression of nuclear and mitochondrial forms of human uracil-DNA glycosylase. Nucleic Acids Res 26:1449–1457. 15. Haradhvala NJ, Polak P, Stojanov P, Covington KR, Shinbrot E, Hess JM, Rheinbay E, Kim J, Maruvka YE, Braunstein LZ, Kamburov A, Hanawalt PC, Wheeler DA, Koren A, Lawrence MS, Getz G. 2016. Mutational strand asymmetries in cancer genomes reveal mechanisms of DNA damage and repair. Cell 164:538–549. https://doi.org/10.1016/j.cell.2015.12.050. 35. Law EK, Sieuwerts AM, LaPara K, Leonard B, Starrett GJ, Molan AM, Temiz NA, Vogel RI, Meijer-van Gelder ME, Sweep FCGJ, Span PN, Foekens JA, Martens JWM, Yee D, Harris RS. 2016. The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer. Sci Adv 2:e1601737. https://doi.org/10.1126/sciadv.1601737. 16. Morganella S, Alexandrov LB, Glodzik D, Zou X, Davies H, Staaf J, Sieuwerts AM, Brinkman AB, Martin S, Ramakrishna M, Butler A, Kim H-Y, Borg Å, Sotiriou C, Futreal PA, Campbell PJ, Span PN, Van Laere S, Lakhani SR, Eyfjord JE, Thompson AM, Stunnenberg HG, van de Vijver MJ, Martens JWM, Børresen-Dale A-L, Richardson AL, Kong G, Thomas G, Sale J, Rada C, Stratton MR, Birney E, Nik-Zainal S. 2016. The topography of mutational processes in breast cancer genomes. Nat Commun 7:11383. https://doi.org/10.1038/ncomms11383. 36. Leonard B, McCann JL, Starrett GJ, Kosyakovsky L, Luengas EM, Molan AM, Burns MB, McDougle RM, Parker PJ, Brown WL, Harris RS. 2015. The PKC/NF-kB signaling pathway induces APOBEC3B expression in multiple human cancers. Cancer Res 75:4538–4547. https://doi.org/10.1158/0008 -5472.CAN-15-2171-T. 37. Polyomavirus T Antigen Induces APOBEC3B Expression Santiago ML, Pyeon D. 2015. APOBEC3A functions as a restriction factor of human papillomavirus. J Virol 89:688–702. https://doi.org/10.1128/JVI .02383-14. 9. Vartanian J, Guétard D, Henry M, Wain-Hobson S. 2008. Evidence for editing of human papillomavirus DNA by APOBEC3 in benign and precancerous lesions. Science 320:230–233. https://doi.org/10.1126/ science.1153201. 27. 27. Barbosa MS, Edmonds C, Fisher C, Schiller JT, Lowy DR, Vousden KH. 1990. The region of the HPV E7 oncoprotein homologous to adenovirus E1a and Sv40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation. EMBO J 9:153–160. 10. Peretti A, Geoghegan EM, Pastrana DV, Smola S, Feld P, Sauter M, Lohse S, Ramesh M, Lim ES, Wang D, Borgogna C, FitzGerald PC, Bliskovsky V, Starrett GJ, Law EK, Harris RS, Killian JK, Zhu J, Pineda M, Meltzer PS, Boldorini R, Gariglio M, Buck CB. 2018. Characterization of BK polyoma- viruses from kidney transplant recipients suggests a role for APOBEC3 in driving in-host virus evolution. Cell Host Microbe 23:628–635.e7. https://doi.org/10.1016/j.chom.2018.04.005. and casein kinase II phosphorylation. EMBO J 9:153–160. 28. Mietz JA, Unger T, Huibregtse JM, Howley PM. 1992. The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein. EMBO J 11:5013–5020. 29. Low J, Humes HD, Szczypka M, Imperiale M. 2004. BKV and SV40 infection of human kidney tubular epithelial cells in vitro. Virology 323:182–188. https://doi.org/10.1016/j.virol.2004.03.027. g j 11. Burns MB, Temiz NA, Harris RS. 2013. Evidence for APOBEC3B mutagen- esis in multiple human cancers. Nat Genet 45:977–983. https://doi.org/ 10.1038/ng.2701. 30. Abend JR, Joseph AE, Das D, Campbell-Cecen DB, Imperiale MJ. 2009. A truncated T antigen expressed from an alternatively spliced BK virus early mRNA. J Gen Virol 90:1238–1245. https://doi.org/10.1099/vir.0 .009159-0. 12. Shi K, Carpenter MA, Banerjee S, Shaban NM, Kurahashi K, Salamango DJ, McCann JL, Starrett GJ, Duffy JV, Demir Ö, Amaro RE, Harki DA, Harris RS, Aihara H. 2017. Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B. Nat Struct Mol Biol 24:131–139. https://doi.org/10.1038/nsmb.3344. on April 15, 2019 b http://mbio.asm.org/ Downloaded from on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from 31. DeCaprio JA, Garcea RL. 2013. A cornucopia of human polyomaviruses. Nat Rev Microbiol 11:264–276. https://doi.org/10.1038/nrmicro2992. 13. Kouno T, Silvas TV, Hilbert BJ, Shandilya SMD, Bohn MF, Kelch BA, Royer WE, Somasundaran M, Kurt Yilmaz N, Matsuo H, Schiffer CA. 2017. Polyomavirus T Antigen Induces APOBEC3B Expression Sieuwerts AM, Willis S, Burns MB, Look MP, Meijer-Van Gelder ME, Schlicker A, Heideman MR, Jacobs H, Wessels L, Leyland-Jones B, Gray KP, Foekens JA, Harris RS, Martens JWM. 2014. Elevated APOBEC3B correlates with poor outcomes for estrogen-receptor-positive breast cancers. Horm Cancer 5:405–413. https://doi.org/10.1007/s12672-014-0196-8. 17. Hoopes JI, Cortez LM, Mertz TM, Malc EP, Mieczkowski PA, Roberts SA. 2016. APOBEC3A and APOBEC3B preferentially deaminate the lagging strand template during DNA replication. Cell Rep 14:1273–1282. https:// doi.org/10.1016/j.celrep.2016.01.021. pril 15, 2019 by guest 18. Bhagwat AS, Hao W, Townes JP, Lee H, Tang H, Foster PL. 2016. Strand- biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli. Proc Natl Acad Sci U S A 113: 2176–2181. https://doi.org/10.1073/pnas.1522325113. 38. Periyasamy M, Singh AK, Gemma C, Kranjec C, Farzan R, Leach DA, Navaratnam N, Pálinkás HL, Vertessy BG, Fenton TR, Doorbar J, Fuller- Pace F, Meek DW, Coombes RC, Buluwela L, Ali S. 2017. P53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells. Nucleic Acids Res 45:11056–11069. https://doi.org/10.1093/nar/gkx721. g 19. Harris RS, Dudley JP. 2015. APOBECs and virus restriction. Virology 479-480:131–145. https://doi.org/10.1016/j.virol.2015.03.012. 39. Vassilev LT. 2004. Small-molecule antagonists of p53-MDM2 binding: research tools and potential therapeutics. Cell Cycle 3:417–419. https:// doi.org/10.4161/cc.3.4.801. 20. Conticello SG. 2008. The AID/APOBEC family of nucleic acid mutators. Genome Biol 9:229. https://doi.org/10.1186/gb-2008-9-6-229. 21. Vieira VC, Soares MA. 2013. The role of cytidine deaminases on innate immune responses against human viral infections. Biomed Res Int 2013:1. https://doi.org/10.1155/2013/683095. 40. Lahav G, Rosenfeld N, Sigal A, Geva-Zatorsky N, Levine AJ, Elowitz MB, Alon U. 2004. Dynamics of the p53-Mdm2 feedback loop in individual cells. Nat Genet 36:147–150. https://doi.org/10.1038/ng1293. 22. Venkatesan S, Rosenthal R, Kanu N, McGranahan N, Bartek J, Quezada SA, Hare J, Harris RS, Swanton C. 2018. Perspective: APOBEC mutagenesis in drug resistance and immune escape in HIV and cancer evolution. Ann Oncol 29:563–572. https://doi.org/10.1093/annonc/mdy003. 41. Barak Y, Juven T, Haffner R, Oren M. 1993. Mdm2 expression is induced by wild type p53 activity. EMBO J 12:461–468. 42. El-Deiry WS, Harper JW, O’Connor PM, Velculescu VE, Canman CE, Jack- man J, Pietenpol JA, Burrell M, Hill DE, Wang Y, Wiman KG, Mercer WE, Kastan MB, Kohn KW, Elledge SJ, Kinzler KW, Vogelstein B. 1994. WAF1/ CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 54:1169–1174. 23. REFERENCES in viruses: patterns and determinants. Nat Rev Genet 9:267–276. https:// doi.org/10.1038/nrg2323. 5. Sanjuán R, Domingo-Calap P. 2016. Mechanisms of viral mutation. Cell Mol Life Sci 73:4433–4448. https://doi.org/10.1007/s00018-016-2299-6. 5. Sanjuán R, Domingo-Calap P. 2016. Mechanisms of viral mutation. Cell Mol Life Sci 73:4433–4448. https://doi.org/10.1007/s00018-016-2299-6. 6. Verhalen B, Starrett GJ, Harris RS, Jiang M. 2016. Functional upregulation of the DNA cytosine deaminase APOBEC3B by polyomaviruses. J Virol 90:6379–6386. https://doi.org/10.1128/JVI.00771-16. 2. Cuevas JM, Geller R, Garijo R, López-Aldeguer J, Sanjuán R. 2015. Ex- tremely high mutation rate of HIV-1 in vivo. PLoS Biol 13:e1002251. https://doi.org/10.1371/journal.pbio.1002251. 7. Warren CJ, Van Doorslaer K, Pandey A, Espinosa JM, Pyeon D. 2015. Role of the host restriction factor APOBEC3 on papillomavirus evolution. Virus Evol 1:vev015. https://doi.org/10.1093/ve/vev015. 3. Buck CB, Van Doorslaer K, Peretti A, Geoghegan EM, Tisza MJ, An P, Katz JP, Pipas JM, McBride AA, Camus AC, McDermott AJ, Dill JA, Delwart E, Ng TFF, Farkas K, Austin C, Kraberger S, Davison W, Pastrana DV, Varsani A. 2016. The ancient evolutionary history of polyomaviruses. PLoS Pat- hog 12:e1005574. https://doi.org/10.1371/journal.ppat.1005574. 8. Wang Z, Wakae K, Kitamura K, Aoyama S, Liu G, Koura M, Monjurul AM, Kukimoto I, Muramatsu M. 2014. APOBEC3 deaminases induce hyper- mutation in human papillomavirus 16 DNA upon beta interferon stim- ulation. J Virol 88:1308–1317. https://doi.org/10.1128/JVI.03091-13. hog 12:e1005574. https://doi.org/10.1371/journal.ppat.100 y S, Shackelton LA, Holmes EC. 2008. Rates of evolutionary c 4. Duffy S, Shackelton LA, Holmes EC. 2008. Rates of evolutionary change mbio.asm.org 12 mbio.asm.org 12 mbio.asm.org 12 ® Polyomavirus T Antigen Induces APOBEC3B Expression Polyomavirus T Antigen Induces APOBEC3B Expression Vieira VC, Leonard B, White EA, Starrett GJ, Temiz NA, Lorenz LD, Lee D, Soares MA, Lambert PF, Howley PM, Harris RS, Harris S. 2014. Human papillomavirus E6 triggers upregulation of the antiviral and cancer genomic DNA deaminase APOBEC3B. mBio 5:e02234-14. https://doi.org/ 10.1128/mBio.02234-14. 43. Seoane J, Le H-V, Massagué J. 2002. Cell culture Myc suppression of the p21 Cip1 Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 419:729–734. https://doi.org/10.1038/nature01119. 24. Mori S, Takeuchi T, Ishii Y, Kukimoto I. 2015. Identification of APOBEC3B promoter elements responsible for activation by human papillomavirus type 16 E6. Biochem Biophys Res Commun 460:555–560. https://doi.org/ 10.1016/j.bbrc.2015.03.068. 44. Chen D, Tavana O, Chu B, Erber L, Chen Y, Baer R, Gu W. 2017. NRF2 is a major target of ARF in p53-independent tumor suppression. Mol Cell 68:224–232.e4. https://doi.org/10.1016/j.molcel.2017.09.009. 10.1016/j.bbrc.2015.03.068. 25. Mori S, Takeuchi T, Ishii Y, Yugawa T, Kiyono T, Nishina H, Kukimoto I. 2017. Human papillomavirus 16 E6 upregulates APOBEC3B via the TEAD transcription factor. J Virol 91:e02413-16. https://doi.org/10.1128/JVI .02413-16. 45. Jiang L, Kon N, Li T, Wang SJ, Su T, Hibshoosh H, Baer R, Gu W. 2015. Ferroptosis as a p53-mediated activity during tumour suppression. Na- ture 520:57–62. https://doi.org/10.1038/nature14344. 26. Warren CJ, Xu T, Guo K, Griffin LM, Westrich JA, Lee D, Lambert PF, 46. Whyte P, Buchkovich KJ, Horowitz JM, Friend SH, Raybuck M, Weinberg January/February 2019 Volume 10 Issue 1 e02690-18 ® Starrett et al. cycle gene regulatory networks. Nucleic Acids Res 44:6070–6086. https://doi.org/10.1093/nar/gkw523. RA, Harlow E. 1988. Association between an oncogene and an anti- oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature 336:124–129. https://doi.org/10.1038/334124a0. product. Nature 336:124–129. https://doi.org/10.1038/3341 61. Laoukili J, Kooistra MRH, Brás A, Kauw J, Kerkhoven RM, Morrison A, Clevers H, Medema RH. 2005. FoxM1 is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol 7:126–136. https://doi.org/10.1038/ncb1217. 47. Caracciolo V, Reiss K, Khalili K, De Falco G, Giordano A. 2006. Role of the interaction between large T antigen and Rb family members in the oncogenicity of JC virus. Oncogene 25:5294–5301. https://doi.org/10 .1038/sj.onc.1209681. 62. Zhao B, Barrera LA, Ersing I, Willox B, Schmidt SCS, Greenfeld H, Zhou H, Mollo SB, Shi TT, Takasaki K, Jiang S, Cahir-McFarland E, Kellis M, Bulyk ML, Kieff E, Gewurz BE. 2014. The NF-B genomic landscape in lympho- blastoid B cells. Cell Rep 8:1595–1606. https://doi.org/10.1016/j.celrep .2014.07.037. 48. Henley SA, Dick FA. 2012. The retinoblastoma family of proteins and their regulatory functions in the mammalian cell division cycle. Cell Div 7:10. https://doi.org/10.1186/1747-1028-7-10. p g 49. Classon M, Dyson N. 2001. P107 and P130: versatile proteins with interesting pockets. Exp Cell Res 264:135–147. https://doi.org/10.1006/ excr.2000.5135. 63. Sherr CJ, Beach D, Shapiro GI. 2016. Targeting CDK4 and CDK6: from discovery to therapy. Cancer Discov 6:353–367. https://doi.org/10.1158/ 2159-8290.CD-15-0894. 50. Harris KF, Christensen JB, Imperiale MJ. 1996. BK virus large T antigen: interactions with the retinoblastoma family of tumor suppressor pro- teins and effects on cellular growth control. J Virol 70:2378–2386. 64. 10.1016/j.bbrc.2015.03.068. Mirabello L, Yeager M, Yu K, Clifford GM, Xiao Y, Zhu B, Cullen M, Boland JF, Wentzensen N, Nelson CW, Raine-Bennett T, Chen Z, Bass S, Song L, Yang Q, Steinberg M, Burdett L, Dean M, Roberson D, Mitchell J, Lorey T, Franceschi S, Castle PE, Walker J, Zuna R, Kreimer AR, Beachler DC, Hildesheim A, Gonzalez P, Porras C, Burk RD, Schiffman M. 2017. HPV16 E7 genetic conservation is critical to carcinogenesis. Cell 170: 1164–1174.e6. https://doi.org/10.1016/j.cell.2017.08.001. on April 15, 2019 by guest http://mbio.asm.org/ Downloaded from on April 15, 2019 b http://mbio.asm.org/ Downloaded from 51. Whittaker SR, Mallinger A, Workman P, Clarke PA. 2017. Inhibitors of cyclin-dependent kinases as cancer therapeutics. Pharmacol Ther 173: 83–105. https://doi.org/10.1016/j.pharmthera.2017.02.008. 52. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. 2015. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov 14:130–146. https://doi.org/10.1038/nrd4504. 1164–1174.e6. https://doi.org/10.1016/j.cell.2017.08.001. 65. Zhen S, Hua L, Takahashi Y, Narita S, Liu YH, Li Y. 2014. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. Biochem Biophys Res Commun 450: 1422–1426. https://doi.org/10.1016/j.bbrc.2014.07.014. 53. O’Leary B, Finn RS, Turner NC. 2016. Treating cancer with selective CDK4/6 inhibitors. Nat Rev Clin Oncol 13:417–430. https://doi.org/10 .1038/nrclinonc.2016.26. on April 15, 2019 by guest http://mbio.asm.org/ from 54. Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B, Refsland EW, Kotandeniya D, Tretyakova N, Nikas JB, Yee D, Temiz NA, Donohue DE, McDougle RM, Brown WL, Law EK, Harris RS. 2013. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494:366–370. https://doi.org/10.1038/nature11881. 66. Cescon DW, Haibe-Kains B, Mak TW. 2015. APOBEC3B expression in breast cancer reflects cellular proliferation, while a deletion polymor- phism is associated with immune activation. Proc Natl Acad Sci U S A 112:2841–2846. https://doi.org/10.1073/pnas.1424869112. 67. Lau L, Gray EE, Brunette RL, Stetson DB. 2015. DNA tumor virus onco- genes antagonize the cGAS-STING DNA-sensing pathway. Science 350: 568–571. https://doi.org/10.1126/science.aab3291. 55. Nikkilä J, Kumar R, Campbell J, Brandsma I, Pemberton HN, Wallberg F, Nagy K, Scheer I, Vertessy BG, Serebrenik AA, Monni V, Harris RS, Pettitt SJ, Ashworth A, Lord CJ. 2017. Elevated APOBEC3B expression drives a kataegic-like mutation signature and replication stress-related therapeu- tic vulnerabilities in p53-defective cells. Br J Cancer 117:113–123. https:// doi.org/10.1038/bjc.2017.133. 68. Knowles WA, Sharp IR, Efstratiou L, Hand JF, Gardner SD. 1991. 10.1016/j.bbrc.2015.03.068. Prepa- ration of monoclonal antibodies to JC virus and their use in the diag- nosis of progressive multifocal leucoencephalopathy. J Med Virol 34: 127–131. https://doi.org/10.1002/jmv.1890340211. 56. Otto T, Sicinski P. 2017. Cell cycle proteins as promising targets in cancer therapy. Nat Rev Cancer 17:93–115. https://doi.org/10.1038/nrc.2016 .138. 69. Leonard B, Starrett GJ, Maurer MJ, Oberg A, Van Bockstal M, Van Dorpe J, De Wever O, Helleman J, Sieuwerts AM, Berns EMJJ, Martens JWM, Anderson B, Brown WL, Kalli KR, Kaufmann SH, Harris RS. 2016. APOBEC3G expression correlates with T cell infiltration and improved clinical outcomes in high-grade serous ovarian carcinoma. Clin Cancer Res 22:4746–4755. https://doi.org/10.1158/1078-0432.CCR-15-2910. 57. Fry DW, Harvey PJ, Keller PR, Elliott WL, Meade M, Trachet E, Albassam M, Zheng X, Leopold WR, Pryer NK, Toogood PL. 2004. Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol Cancer Ther 3:1427–1438. pril 15, 2019 by guest 70. Jiang M, Abend JR, Tsai B, Imperiale MJ. 2009. Early events during BK virus entry and disassembly. J Virol 83:1350–1358. https://doi.org/10 .1128/JVI.02169-08. 58. Raspé E, Coulonval K, Pita JM, Paternot S, Rothé F, Twyffels L, Brohée S, Craciun L, Larsimont D, Kruys V, Sandras F, Salmon I, Van Laere S, Piccart M, Ignatiadis M, Sotiriou C, Roger PP. 2017. CDK4 phosphorylation status and a linked gene expression profile predict sensitivity to palbociclib. EMBO Mol Med 9:1052–1066. https://doi.org/10.15252/emmm.201607084. 71. Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M. 2009. Mechanism of genomic instability in cells infected with the high-risk human papillo- maviruses. PLoS Pathog 5:e1000397. https://doi.org/10.1371/journal .ppat.1000397. 59. Stoeck A, Lejnine S, Truong A, Pan L, Wang H, Zang C, Yuan J, Ware C, MacLean J, Garrett-Engele PW, Kluk M, Laskey J, Haines BB, Moskaluk C, Zawel L, Fawell S, Gilliland G, Zhang T, Kremer BE, Knoechel B, Bernstein BE, Pear WS, Liu XS, Aster JC, Sathyanarayanan S. 2014. Discovery of biomarkers predictive of GSI response in triple-negative breast cancer and adenoid cystic carcinoma. Cancer Discov 4:1154–1167. https://doi .org/10.1158/2159-8290.CD-13-0830. 72. Jiang M, Entezami P, Gamez M, Stamminger T, Imperiale MJ. 2011. Functional reorganization of promyelocytic leukemia nuclear bodies during BK virus infection. mBio 2:e00281-10. 73. Jiang M, Zhao L, Gamez M, Imperiale MJ. 2012. Roles of ATM and ATR-mediated DNA damage responses during lytic BK polyomavirus infection. PLoS Pathog 8:e1002898. https://doi.org/10.1371/journal.ppat .1002898. 60. Fischer M, Grossmann P, Padi M, DeCaprio JA. 2016. January/February 2019 Volume 10 Issue 1 e02690-18 10.1016/j.bbrc.2015.03.068. Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell mbio.asm.org 14 January/February 2019 Volume 10 Issue 1 e02690-18
https://openalex.org/W4293062697
https://zenodo.org/record/7002276/files/Influence_of_Position_of_Rice_Anthers_At_Plating_on_Callusing_and_Plant_Regeneration.pdf
English
null
Influence of Position of Rice Anthers At Plating on Callusing and Plant Regeneration
Zenodo (CERN European Organization for Nuclear Research)
1,987
cc-by
637
Influence of position of rice anthers at plating on callusing and plant regeneration Callusing response of anthers. 1 =Anther positioned on edge callusing from upper lohc. 2 =One anthe-r on edge at left and the nthcrllat at right. both callusing. 3 =Anther llat.caHusing from hoth lobe~. 4 =Anther flat. callusing from only one lobe. 5 =Anther (JtJ edge, callusing fr{lnl upper and lower lobes. (l =Anther on edge. callusing from lower lohc only. Note calli from lower lobes growing into the medium. S. T Men:r and F./. Zapata, Tissue Culture Lahora/ory. Plalll Breeding Department. I I? Rl S. T Men:r and F./. Zapata, Tissue Culture Lahora/ory. Plalll Breeding Department. I I? Rl We studied the effect of anther orientation at plating on callusing response and plant regeneration in japonica rice variety Taipei 309. Callus induction from anthers was according to standard I R R I procedure. Anthers were plated on semisolid EIO (modified B5 medium. each liter containing I mg 2.4-D. 0.5 mg BAP. 0.5 mg lA A. 20 g sucrose, and 5 g glucose) . Sixty anthers were inoculated at random (specific positioning of anthers is extremely difficult because l)f their small size). Microscopic examination showed that anthers were naturally positioned approximately half on edge (one lobe touching the medium) and half nat (both lobes in contact with the medium). Callusing response of anthers. 1 =Anther positioned on edge callusing from upper lohc. 2 =One anthe-r on edge at left and the nthcrllat at right. both callusing. 3 =Anther llat.caHusing from hoth lobe~. 4 =Anther flat. callusing from only one lobe. 5 =Anther (JtJ edge, callusing fr{lnl upper and lower lobes. (l =Anther on edge. callusing from lower lohc only. Note calli from lower lobes growing into the medium. Plates were incubated at 26-29 °C in dim light and scored for callusing 40 d alter plating. Some calli of both nat and edge-oriented anthers were transferred for regeneration in MS medium (I mg each NAA and kinetin/liter and 30 g sucrose/liter) under continuous light. Percentage of green plant regeneration was recorded. About 15% of the calli transferred ror regeneration produced green or grcr!1 and albino plantlets. or the 2,160 anthers plated, 518 (24%) produced calli. Of the anthers with calli, 60% were plated on edge (see figure) and 40% nat. Most of the anthers plated nat callused on both lobes, but callusing on one lobe was also observed. Most anthers plated on edge produced callus on the upper lobe. Published by the International Rice Research Institute, P. 0. Box 933, Manila, Philippines Genetic Evaluation and utilization TISSUE CULTURE Published by the International Rice Research Institute, P. 0. Box 933, Manila, Philippines Genetic Evaluation and utilization TISSUE CULTURE Published by the International Rice Research Institute, P. 0. Box 933, Manila, Philippin Influence of position of rice anthers at plating on callusing and plant regeneration Only rarely did anthers plated on edge produce callus from both lobes or from the lower lobe only. Observations m<.~dc on rice varieties IRS, Basmati 370, and Pankaj showed the same anther callusing response. No special anther inoculation; technique is needed 10 get satisf:ictory callusing response in rice cxcept'\o sec I I . . • t 1at ant 1ers are not buned 111 the medium. D Observations m<.~dc on rice varieties IRS, Basmati 370, and Pankaj showed the same anther callusing response. Most of the anthers dehisccd within 5 d of plating. liberating a large part of the pollen grains on the medium. None of the liberated pollen grains developed into calli. Calli from pollen grains held within the anther started appearing about 25 d after inoculation. No special anther inoculation; technique is needed 10 get satisf:ictory callusing response in rice cxcept'\o sec I I . . • t 1at ant 1ers are not buned 111 the medium. D IRRN 12:4 (August 1987)
https://openalex.org/W4318700676
https://biblio.ugent.be/publication/01GTM0MMAJ5M120CW3XCHYPTNT/file/01GZXFBKJBZ8PV00WWCEJ566PD.pdf
English
null
Unexpected complexity of the ammonia monooxygenase in archaea
˜The œISME journal
2,023
cc-by
12,891
INTRODUCTION diverse enzymes of the CuMMO (copper-dependent membrane monooxygenase) protein family, with a few notable exceptions. Crystal structures [22–26] and cryo-EM structures [27, 28] of particulate methane monooxygenase (pMMO) from five methano- trophs have consistently confirmed a three-polypeptide protomer (subunits-A, -B and -C) arranged in a trimer of α3β3γ3 configuration with at least two conserved metal sites in each protomer. Even so, the elucidation of the active site has remained ambiguous. It was first proposed to reside in the PmoB subunit of pMMO [29]. More recently, a cryo-EM analysis supports the active site primarily being coordinated by PmoA [27], while differing amino acid conservation in Verrucomicrobia [30], a recent spectroscopic analysis [31], and mutagenesis of a hydrocarbon monooxygenase [32], suggest its localization in the PmoC subunit. Nitrification, the conversion of ammonium to nitrate, is a crucial step in the global nitrogen cycle solely performed by microorganisms. The process has attracted particular attention due to its agricultural and environmental relevance. The first and rate limiting [1] step of nitrification is the oxidation of ammonia via the integral membrane protein complex ammo- nia monooxygenase (AMO) [2, 3]. While ammonia-oxidizing bacteria (AOB) were first discovered over 125 years ago [4] and have been extensively studied, this biological process was also detected in the archaeal domain in the last 20 years [5–7]. Ammonia-oxidizing archaea (AOA) have gained broad attention as they are widespread in nature and are more abundant than their bacterial counterparts in most terrestrial and marine environments, indicating important roles in nitrogen cycling [8–14]. Their central nitrogen and carbon metabolism, however, is distinct from that of AOB [15–18]. In particular, subunits of the AMO complex show only about 40% identity to those of bacteria [19] and archaeal proteins catalyzing the second step in ammonia oxidation, i.e. the conversion of hydroxylamine to nitrite, are still unknown [19–21]. These differences imply important functional differ- entiation in their environmental roles that have yet to be unraveled. Although no AMO structure has been determined experimen- tally, homology modeling for the AMO of the bacterium Nitrosomonas europaea using pMMO as a template supported a homotrimeric structure as well as conservation of the CuB and CuC copper sites [33]. The archaeal AMO complex is the most distantly related of all CuMMO proteins [34, 35] and very little is known so far about its structure or function. Received: 8 July 2022 Revised: 9 January 2023 Accepted: 12 January 2023 Published online: 31 January 2023 1Archaea Biology and Ecogenomics Unit, Department of Functional and Evolutionary Ecology, University of Vienna, Vienna, Austria. 2Mass Spectrometry Facility, Max Perutz Labs, Vienna BioCenter (VBC), Vienna, Austria. 3VIB Center for Inflammation Center and Department of Biochemistry & Microbiology, Ghent University, Ghent, Belgium. 4Molecular Systems Biology Unit, Department of Functional and Evolutionary Ecology, University of Vienna, Vienna, Austria. 5Department of Biochemistry and Cell Biology, Max Perutz Labs, University of Vienna, Vienna, Austria. ✉email: christa.schleper@univie.ac.at ARTICLE OPEN Unexpected complexity of the ammonia monooxygenase in archaea Logan H. Hodgskiss 1, Michael Melcher1, Melina Kerou 1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides 3, Stefanie Wienkoop4, Markus Hartl2,5 and Christa Schleper 1✉ Logan H. Hodgskiss 1, Michael Melcher1, Melina Kerou 1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides 3, Stefanie Wienkoop4, Markus Hartl2,5 and Christa Schleper 1✉ 1, Michael Melcher1, Melina Kerou 1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides 3 Markus Hartl2,5 and Christa Schleper 1✉ Logan H. Hodgskiss 1, Michael Melcher1, Melina Kerou 1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides 3, Stefanie Wienkoop4, Markus Hartl2,5 and Christa Schleper 1✉ © The Author(s) 2023, corrected publication 2023 © The Author(s) 2023, corrected publication 2023 © The Author(s) 2023, corrected publication 2023 Ammonia oxidation, as the first step of nitrification, constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase, is lacking, in particular for the environmentally abundant ammonia-oxidizing archaea (AOA). Here the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Besides the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modeling and in-gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase but also reveals clear differences in extracellular domains of the enzyme. These data open avenues for further structure-function studies of this ecologically important nitrification complex. The ISME Journal (2023) 17:588–599; https://doi.org/10.1038/s41396-023-01367-3 The ISME Journal (2023) 17:588–599; https://doi.org/10.1038/s41396-023-01367-3 www.nature.com/ismej Received: 8 July 2022 Revised: 9 January 2023 Accepted: 12 January 2023 Published online: 31 January 2023 MATERIALS AND METHODS Reactor growth Ladders for each gel are represented at the bottom of the respe ournal (2023) 17:588 – 599 1 2 3 4 5 6 7 8 9 11 13 15 17 18 19 20 21 22 1236 1048 720 480 242 146 66 kDa A Relative intensity 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Gel band number AMO Complex of N. viennensis AmoA AmoB AmoC4/C6 AmoX NVIE_004540 NVIE_004550 A Relative intensity 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Gel band number AMO Complex of N. viennensis AmoA AmoB AmoC4/C6 AmoX NVIE_004540 NVIE_004550 A 1 2 2 3 4 5 6 7 8 9 11 13 15 17 18 19 20 21 22 1236 1048 720 480 242 146 66 kDa 1 3 5 7 9 11 13 17 18 19 20 22 23 24 25 26 27 1236 1048 720 480 242 146 66 kDa B Relative in 0.0 0.1 0.2 0.3 0.4 0.5 0.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Gel band number Relative intensity 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Gel band number AMO Complex of N. cavascurensis AmoA AmoB AmoC AmoX (NCAV_0491) NCAV_0488 NCAV_0486 Fig. 1 Relative intensity patterns of AMO subunits in BN-PAGE gels. Relative abundance of iBAQ normalized intensities of known and putative AMO subunits. iBAQ intensities for each protein are normalized to the highest detected intensity of that protein to create a relative abundance profile for each protein. A Patterns of AMO intensity in N. viennensis. B Patterns of AMO intensity in N. cavascurensis. Bands selected to be cut and analyzed via mass spectroscopy are indicated by numbered brackets from left (top of gel) to right (bottom of gel) and correspond to numbers on the x-axis of respective plots. Ladders for each gel are represented at the bottom of the respective panels. MATERIALS AND METHODS Reactor growth and chemical cross-linking. Beside the three known AmoABC proteins, three additional potential subunits were identified and one of the six predicted AmoC proteins in N. viennensis was recognized as the primary homolog in the protein complex. In addition, the overall subunit composition of the AMO complex was confirmed in the distantly related thermophilic AOA Nitrosocaldus cavascurensis. g Nitrososphaera viennensis was grown as a continuous culture in 2 L bioreactors (Eppendorf) filled with 1.5 L of freshwater medium (FWM) [37, 38] with modified trace element solution [5], 7.5 µM FeNaEDTA, 2 mM NH4Cl, and 1 mM pyruvate at 42 °C and pH 7.5. Carbonate was supplied by gassing the reactors with a 98% air 2% CO2 mixture. The applied dilution rates ranged from 0.035 to 0.07 h−1. 1 2 2 3 4 5 6 7 8 9 11 13 15 17 18 19 20 21 22 1236 1048 720 480 242 146 66 kDa 1 3 5 7 9 11 13 17 18 19 20 22 23 24 25 26 27 1236 1048 720 480 242 146 66 kDa A B Relative intensity 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Gel band number AMO Complex of N. viennensis AmoA AmoB AmoC4/C6 AmoX NVIE_004540 NVIE_004550 Relative intensity 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Gel band number AMO Complex of N. cavascurensis AmoA AmoB AmoC AmoX (NCAV_0491) NCAV_0488 NCAV_0486 ative intensity patterns of AMO subunits in BN-PAGE gels. Relative abundance of iBAQ normalized intensit MO subunits. iBAQ intensities for each protein are normalized to the highest detected intensity of that protein t e profile for each protein. A Patterns of AMO intensity in N. viennensis. B Patterns of AMO intensity in N. cavascurens and analyzed via mass spectroscopy are indicated by numbered brackets from left (top of gel) to right (bo d to numbers on the x-axis of respective plots. INTRODUCTION Based on comparative metagenomics alone, it has been suggested that an additional subunit might be present in the complex, termed AmoX [15, 36]. Due to the difficulty of growing nitrifying organisms and the inherent problems with isolating membrane proteins, no struc- tural studies have been successfully carried out for any AMO complex, bacterial or archaeal. This holds true for most of the To gain insights into the overall architecture of the archaeal AMO complex, membrane protein fractions from the well characterized soil AOA, Nitrososphaera viennensis, were analyzed biochemically using native gel electrophoresis, mass spectrometry, L.H. Hodgskiss et al. 589 MATERIALS AND METHODS Reactor growth 1 2 3 4 5 6 7 8 9 11 13 15 17 18 19 20 21 22 1236 1048 720 480 242 146 66 kDa B Relative intensity 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Gel band number AMO Complex of N. cavascurensis AmoA AmoB AmoC AmoX (NCAV_0491) NCAV_0488 NCAV_0486 B 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Gel band number 1 12 13 14 15 16 Gel band number 2 1 3 5 7 9 11 13 17 18 19 20 22 23 24 25 26 27 1236 1048 720 480 242 146 66 kDa 242 Fig. 1 Relative intensity patterns of AMO subunits in BN-PAGE gels. Relative abundance of iBAQ normalized intensities of known and putative AMO subunits. iBAQ intensities for each protein are normalized to the highest detected intensity of that protein to create a relative abundance profile for each protein. A Patterns of AMO intensity in N. viennensis. B Patterns of AMO intensity in N. cavascurensis. Bands selected to be cut and analyzed via mass spectroscopy are indicated by numbered brackets from left (top of gel) to right (bottom of gel) and correspond to numbers on the x-axis of respective plots. Ladders for each gel are represented at the bottom of the respective panels. The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. MATERIALS AND METHODS Reactor growth 590 C B X A 170 kDa 130 100 70 55 40 35 25 15 10 Most Abundant Proteins (% iBAQ Intensity of Band) (F1) SlaA (90%) (F3) NVIE_028570 (28%), SlaA (26%) (F5) NVIE_024150 (56%) (F6) AmoA (63%) Ladder (F7) AmoC (81%) (F8) NVIE_021780 (31.9%), AmoB (12%) (F9) NVIE_004540 (47%), NVIE_004550 (35%), AmoX (8%) SimplyBlue SafeStain 96% 4% <0.05% AmoC6/C4 AmoC1/C2 AmoC3 Composition of AmoC Peptides (unique peptides identified) C B X A 170 kDa 130 100 70 55 40 35 25 15 10 Most Abundant Proteins (% iBAQ Intensity of Band) (F1) SlaA (90%) (F3) NVIE_028570 (28%), SlaA (26%) (F5) NVIE_024150 (56%) (F6) AmoA (63%) Ladder Ladder Ladder (F7) AmoC (81%) (F8) NVIE_021780 (31.9%), AmoB (12%) (F9) NVIE_004540 (47%), NVIE_004550 (35%), AmoX (8%) 170 kDa 130 100 70 55 40 35 25 15 10 kDa 245 190 135 100 80 58 46 32 25 22 17 Silver Staining (Classic) Silver Staining (Farmer’s Reducer) SimplyBlue SafeStain C B X A C B X A 96% 4% <0.05% AmoC6/C4 AmoC1/C2 AmoC3 Composition of AmoC Peptides (unique peptides identified) Fig. 2 Tricine-SDS-PAGE gels of AMO cut-outs. Tricine-SDS-PAGE of AMO bands from BN-PAGE gels. Comparison of three different staining methods for Tricine-SDS-PAGE gels with size markers on left side. Bands cut for analysis from a gel stained with SimplyBlue SafeStain and digested using trypsin are indicated by brackets. Percentages represent the percentage of iBAQ normalized protein intensities for each individual band. Band identifiers are indicated in parentheses. Green arrows marked A, B, C, and X represent expected heights of bands fo AMO subunits AmoA, AmoB, AmoC, and AmoX, respectively. Orange arrows marked A, B, C, and X represent equivalent bands of AmoA AmoB, AmoC, and AmoX, respectively, from silver-stained gels. A pie chart from the band with the highest amount of AmoC shows the percentage of AmoC bands coming from distinguishable AmoC homologs. 0 Most Abundant Proteins (% iBAQ Intensity of Band) (F5) NVIE_024150 (56%) Fig. 2 Tricine-SDS-PAGE gels of AMO cut-outs. Tricine-SDS-PAGE of AMO bands from BN-PAGE gels. Comparison of three different staining methods for Tricine-SDS-PAGE gels with size markers on left side. Bands cut for analysis from a gel stained with SimplyBlue SafeStain and digested using trypsin are indicated by brackets. Percentages represent the percentage of iBAQ normalized protein intensities for each individual band. Band identifiers are indicated in parentheses. RESULTS C l Complexome analysis of native membrane complexes displays the AMO composition of Nitrososphaera viennensis Nitrososphaera viennensis was grown in continuous culture for several weeks under optimal growth conditions in order to obtain enough biomass for biochemical analyses (Melcher et al. [45]). Between 800–2000 µg of membrane proteins were obtained from 450–550 mg of biomass per preparation, of which approximately 40–50 µg were loaded per lane on blue-native PAGE gels [39]. After optimization of conditions, 22 bands were cut out and subjected to mass spectrometry (see Supplementary Materials and Methods; Fig. S1A). AMO subunits (AmoA, AmoB, and AmoC) were among the most abundant proteins (22% of iBAQ normalized intensity) detected overall in these membrane fractions. The relative intensity profiles of AmoA, AmoB, and AmoC showed three distinct peaks corresponding to bands 4, 7, and 12, with the most prominent peak occurring at band 7 (Fig. 1A). The subunits AmoA, AmoB, and AmoC made up 10%, 5%, and 14%, respectively, of the total protein found in band 7 based on iBAQ normalized intensities. AmoX was also present in band 7 representing 10%. The most intense signals for the AmoC subunit were represented by two of the six AmoC homologs, AmoC6 and AmoC4. These two homologs could not be distinguished based on the peptides identified in the BN-PAGE gel. In denaturing Tricine- SDS-PAGE of cutouts from band 7, all known components of the AMO complex were visualized and confirmed by proteomics (Fig. 2). In addition, this allowed for the identification of unique peptides of the AmoC6 subunit (see Supplementary Discussion). T id if ddi i l i h i h b f h Harvested biomass was concentrated in three centrifugation steps. First with a continuous centrifuge (CEPA model LE) operating at maximum speed. Biomass from the continuous centrifuge was then suspended in 400 mL volumes and concentrated using a Sorvall Lynx 4000 with an F12–6 × 500 rotor for 30 min at 4 °C and 16,000 × g. Finally, biomass was resuspended in small volumes and aliquoted to 1.5 mL Eppendorf tubes and concentrated to a final pellet for 30 min at 4 °C and 16,000 × g using a bench-top centrifuge. Pellets were frozen at −70 °C until further analysis. MATERIALS AND METHODS Reactor growth Green arrows marked A, B, C, and X represent expected heights of bands for AMO subunits AmoA, AmoB, AmoC, and AmoX, respectively. Orange arrows marked A, B, C, and X represent equivalent bands of AmoA, AmoB, AmoC, and AmoX, respectively, from silver-stained gels. A pie chart from the band with the highest amount of AmoC shows the percentage of AmoC bands coming from distinguishable AmoC homologs. PAGE of N. viennensis, BN-PAGE of N. cavascurensis, and cross-linked samples, respectively. Relevant scripts for analysis can be found in the GitHub repository https://github.com/hodgskiss/Archaeal_AMO. Nitrosocaldus cavascurensis was grown as a batch culture in the same reactors, volume, and medium as described for N. viennensis, but at 68 °C with 1 mM NH4Cl, 1 mM pyruvate, and pH 7.0. Carbonate was also supplied by gassing, but with a mixture of air/ N2/ CO2 to achieve a 10% O2 and 2% CO2 mixture. To increase the biomass, NH4Cl was added stepwise with syringes via a septum to increase the final NO2 −concentration to approximately 2.5 mM before harvesting the cultures. Sample and data processing Detailed information for bioinformatic analysis, membrane protein extraction, BN-PAGE methods, Tricine-SDS-PAGE methods, mass spectro- metry preparation, cross-linking, data analysis, and AlphaFold multimer predictions can be found in Supplementary Materials and Methods. Briefly, cells were lysed and membrane fractions were isolated using ultracentrifugation (Beckman Coulter Ultracentrifuge; SW 41 Ti Swinging-Bucket Rotor, kmax = 124; 200,000 × g) for 90 min at 4 °C using 13.2 mL thinwall polypropylene tubes with a level of decelera- tion set to 7. Membrane proteins were extracted using n-dodecyl-β-D- maltoside (DDM; Invitrogen BN2005) and loaded on a 3–12% pre-cast BN-PAGE gel (Invitrogen BN1001). Selected bands were cut out and analyzed via mass spectrometry for protein identification. Procedures for protein extraction and running a BN-PAGE gel were based on previous studies [39, 40] and the NativePAGE Novex Bis-Tris Gel System manual from Life Technologies (MAN0000557). Study design and analysis for membrane extraction and BN-PAGE was guided by previous studies [41, 42] Cross-linking methods were based on protocols from Hevler et al. (2021) [43]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [44] partner repository with the dataset identifiers PXD035349, PXD034632, and PXD034475 for BN- To identify additional proteins that might be part of the archaeal AMO complex, a correlation analysis was conducted to The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 591 Table 1. Correlations of proteins with occurrence of AmoA,B, and C in (A) N. viennensis and (B) N. cavascurensis BN-PAGE gels. Gene Proteina Conserved in Extant AOAb Exclusive to AOAb Correlationsc AmoA AmoB AmoC6 A AMO Correlation Results for N. viennensis NVIE_016740 surface associated S-layer protein X X X X nuoJ Complex I X X X X nuoM Complex I X X X X coxB Complex IV X X X coxA1 Complex IV X NVIE_013530 protein of unknown function X X X X NVIE_027260 conserved protein of unknown function X X X X NVIE_017130 protein of unknown function DUF373 X X X amoX potential AMO subunit X X X X X NVIE_004540 hypothetical protein X X X X X B AMO Correlation Results for N. Sample and data processing cavascurensis coxA Cytochrome c oxidase polypeptide 1 X X NCAV_1739 Uncharacterized protein X X X coxB Putative heme-copper oxidase subunit II X X X NCAV_0011 ABC-1 domain-containing protein X X amt Ammonium transporter X X X petB Putative cytochrome b/b6 X NCAV_1587 Putative heme/copper-type cytochrome/ quinol oxidase, subunit X X X NCAV_0486 Uncharacterized protein (NVIE_004550 homolog) X X X X X NCAV_0488 Uncharacterized protein (NVIE_004540 homolog) X X X X X NCAV_0491 Uncharacterized protein (AmoX) X X X X X aProteins with at least one AmoA, AmoB, or AmoC protein correlation. bConservation and exclusiveness to AOA based on results from Abby et al. (2020) [46]. cCorrelation ≥0.7 and adjusted p value ≤0.001. able 1. Correlations of proteins with occurrence of AmoA,B, and C in (A) N. viennensis and (B) N. cavascurensis BN-PAGE gels. Nitrosocaldaceae and Nitrosopumilaceae were analyzed. Of these genes, 19 were conserved in AOA with five being found exclusively in AOA (Supplementary Dataset 2). The five genes of interest included two canonical amo genes (amoA and amoB) and the genes amoX, NVIE_004540, and NVIE_004550. The absence of amoC in the genes of interest is attributed to a truncated version existing within the genome of “Candidatus Nitrosopumilus koreensis AR1” (likely due to assembly issues) that precluded it from being identified as conserved in all AOA. The amoX gene was previously identified in metagenomic studies [15, 36] and NVIE_004540 was already a candidate identified from the BN-PAGE correlation analysis. The additional conserved protein, NVIE_004550, was newly identified and found to be located directly upstream of NVIE_004540, indicating potential co-transcription. The two new candidates encode for polypeptides of 9.6 kDa and 12.8 kDa respectively, and – like the candidate subunit AmoX - their predicted secondary structure is predominantly helical and their subcellular localization transmembrane. The two new candidate amo genes NVIE_004540 and NVIE_004550 have therefore been termed amoY and amoZ, respectively. find candidates with a similar migration pattern as all three primary AMO subunits AmoA, AmoB, and AmoC4/C6. Patterns of the 50% most abundant proteins were compared to each other using a Kendall correlation to determine the likelihood of dependence between various proteins with a focus on proteins correlated with known AMO subunits. Additional criteria were (i) their presence in fully sequenced AOA, and (ii) their absence in species that do not oxidize ammonia [46]. Sample and data processing The two proteins that initially met these criteria were the putative AMO subunit AmoX and a hypothetical protein, NVIE_004540 (Table 1). The migration patterns for these proteins can be seen in Fig. 1A. While this unbiased selection process produced additional AMO candidates, further analysis was needed to verify the presence of these newly identified and other potential subunits. Linkage analysis in AOA genomes supports proposed and additional AMO subunits Earlier analyses of known subunits within the soil strains, or the family Nitrososphaeraceae (as defined by the Genome Taxonomy Database [47]; used throughout), has shown a general lack of spatial clustering of all earlier known subunit genes. However, within the families Nitrosopumilaceae and Nitrosocaldaceae, the genes for the canonical AMO subunits, AmoABC, and the proposed subunit AmoX are syntenic [36, 48, 49]. To investigate co-localization of potential additional subunit genes, the syntenic status and conservation across AOA of the five genes upstream and downstream of the amo gene cluster in A closer analysis in Nitrosocaldaceae, the earliest diverging lineage in evolutionary reconstructions of AOA [46, 50], revealed that the genes for the three candidate subunits for AMO (AmoX, AmoY-homolog of NVIE_004540, and AmoZ-homolog of NVIE_004550) clustered spatially with the canonical subunits (AmoABC) and were syntenic in Nitrosocaldus cavascurensis and Ca. Nitrosocaldus islandicus. Spatial clustering of all six subunit The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 592 Cenarchaeum symbiosum A Ca. Nitrosopelagicus brevis NP-Theta NP-Gamma NT-Alpha NP-Alpha NP-Delta NP-Eta NP-Iota NC-Alpha NS-Gamma NS-Zeta 164-166 15 1 2 11-12 12-123 9-10 627-733 151-175 3 203-236 4 1 3 2 263 Nitrosocaldus Nitrosopelagicus Nitrosoarchaeum Nitrosomarinus Nitrosopumilus Nitrosospongia Nitrosotenuis Nitrosothermus Nitrosocosmicus Nitrososphaera Nitrosotalea Non-AOA Thermoproteota NS-Alpha 0.1 amoA amoX amoC amoB amoY amoZ Fig. 3 Genomic comparison of AMO subunit synteny in AOA. Left: Phylogenetic tree of AOA based on 32 conserved ribosomal proteins, ultrafast bootstrap values of 100% are indicated as blue circles. Taxonomic labels are colored according to GTDB family identity [47], Nitrosocaldaceae-red, Nitrosopumilaceae-blue, Nitrososphaeraceae-orange. Clades/organisms in bold were included in syntenic analysis. Clades are named according to Alves et al. (2018) [34]. Right: Representation of general syntenic patterns in different clades of AOA. Gaps between genes on the same contig are marked by a zig-zag line A double forward slash indicates separate contigs. Numbers under the zig-zag lines represent number of genes between amo subunit genes. A finer analysis and a full list of species can be found in Fig. S11 and Supplementary Dataset 2, respectively. Non-AOA Thermoproteota amoB amoX amoA 203-236 Fig. 3 Genomic comparison of AMO subunit synteny in AOA. Left: Phylogenetic tree of AOA based on 32 conserved ribosomal proteins, ultrafast bootstrap values of 100% are indicated as blue circles. Taxonomic labels are colored according to GTDB family identity [47], Nitrosocaldaceae-red, Nitrosopumilaceae-blue, Nitrososphaeraceae-orange. Clades/organisms in bold were included in syntenic analysis. BN-PAGE protein gel indicates same AMO composition in the thermophilic archaeon Nitrosocaldus cavascurensis BN-PAGE protein gel indicates same AMO composition in the thermophilic archaeon Nitrosocaldus cavascurensis Expression patterns of AMO subunits in Nitrososphaera viennensis and Nitrosopumilus maritimus Linkage analysis in AOA genomes supports proposed and additional AMO subunits Clades are named according to Alves et al. (2018) [34]. Right: Representation of general syntenic patterns in different clades of AOA. Gaps between genes on the same contig are marked by a zig-zag line A double forward slash indicates separate contigs. Numbers under the zig-zag lines represent number of genes between amo subunit genes. A finer analysis and a full list of species can be found in Fig. S11 and Supplementary Dataset 2, respectively. genes is also found in recently obtained MAGs [51] within the genus Nitrosocaldus. In the case of the newly proposed genus Ca. Nitrosothermus [51], amo genes were split on multiple contigs and synteny could not be definitively determined (Fig. 3). Additionally, all six amo genes are inferred to have been newly acquired by the last common ancestor of AOA [46]. was obtained (Fig. 1B) a correlation of the additional subunits was also observed with AmoA, AmoB, and AmoC in this thermophilic organism (Kendall correlation of proteins, as performed for N. viennensis). The three proteins AmoX, NCAV_0488 (AmoY), and NCAV_0486 (AmoZ) all had migration patterns within the gel that strongly correlated with AmoABC (Table 1). This analysis confirmed that the proposed subunits were translated in N. cavascurensis, and potentially had a physical connection within the AMO complex. The emergence of Nitrosopumilaceae was accompanied by a separation of this genomic region into a primary cluster contain- ing amoABCX and a secondary cluster containing amoYZ (Fig. 3). Within Nitrosotalea sp., these clusters are 11–12 genes apart, while the rest of Nitrosopumilaceae species have these clusters only 1–2 genes apart (with the exception of the sponge symbiont Ca. Cenarchaeum symbiosum). The emergence of the family Nitroso- sphaeraceae led to a scattering of all subunit genes across the genome with the exception of amoA and amoX, which are typically linked. Chemical cross-linking supports physical interaction of additional subunits To estimate the physical proximity of the proposed subunits to known subunits and other proteins within the BN-PAGE gel, in-gel cross-linking [43] was performed using the cross-linker disuccini- midyl sulfoxide (DSSO) on an additional BN-PAGE cut-out from band 7 (Fig. S1B). Mass spectrometry and cross-linking analysis showed multiple cross-links among AmoA, AmoB, AmoC, and AmoX as well as with the two newly proposed subunits AmoY and AmoZ (Fig. 4C). Many cross-links were also connected to NVIE_016740, a putative S-layer protein that likely represents a highly abundant surface layer protein as known from other archaea (SlaA) [52, 53]. As this protein presumably helps establish the pseudo-periplasm in AOA, it is not surprising to find it heavily cross-linked to membrane proteins. Although amoZ was identified in the genomic analysis, the protein AmoZ (NVIE_004550) was not correlated with AmoABC in the BN-PAGE gel of N. viennensis. When examining the relative abundance profile for AmoZ, the general pattern of AMO peptide peaks was followed. However, this remained undetected in the correlation analysis due to a high relative abundance peak occurring at the bottom of the gel peaking at the last band taken at approximately 66 kDa based on the BN-PAGE ladder (Fig. 1A). This is above the predicted mass of 12.8 kDa, but suggests that AmoZ could also be part of the AMO but a weaker association possibly lead to its dissociation from the complex and migration to the bottom of the gel. p AmoX also had individual cross-links to several other proteins (Supplementary Dataset 1). As only single connections were found, and these proteins did not appear in any other syntenic or correlative analyses, they were not taken to represent a structural role in the AMO complex. These cross-links can rather be attributed to the high abundance of those proteins in the cell membrane. BN-PAGE protein gel indicates same AMO composition in the thermophilic archaeon Nitrosocaldus cavascurensis To test the composition of the AMO complex outside of the context of N. viennensis, the BN-PAGE approach was applied to membrane protein fractions of N. cavascurensis, a distantly related thermophilic AOA species of the Nitrosocaldaceae family [48] that was recently obtained in pure culture (Melcher et al. in preparation). Although a slightly different pattern of complexes Expression patterns of AMO subunits in Nitrososphaera viennensis and Nitrosopumilus maritimus cavascurensis AmoZ (NVIE_004550) 1 123 AmoB 1 185 AmoA 1 216 AmoC 1 187 AmoX 1 102 SlaA (NVIE_016740) 1 200 400 600 800 1000 1202 AmoY (NVIE_004540) 1 82 SASD allowed SASD violating Euclidean allowed Euclidean violating 0 2 4 6 8 10 12 14 16 18 5-10 10-15 15-20 20-25 25-30 30-35 35-40 40-45 45-50 50-55 55-80 >80 Cross-links Cα pair distance (Å) Proportion of cross-links (%) Cross-linked chain combinations ABC X(ABC) Y(ABC) Z(ABC) XY XZ YZ intra 0 5 10 15 20 25 SASD allowed SASD violating extracellular cytoplasmic The ISME Journal (2023) 17:588 – 599 C D AmoZ (NVIE_004550) 1 123 AmoB 1 185 AmoA 1 216 AmoC 1 187 AmoX 1 102 SlaA (NVIE_016740) 1 200 400 600 800 1000 1202 AmoY (NVIE_004540) 1 82 D C AmoZ (NVIE_004550) AmoC 7 AmoY (NVIE_004540) E SASD allowed SASD violating Euclidean allowed Euclidean violating 0 2 4 6 8 10 12 14 16 18 5-10 10-15 15-20 20-25 25-30 30-35 35-40 40-45 45-50 50-55 55-80 >80 Cross-links Cα pair distance (Å) F Proportion of cross-links (%) Cross-linked chain combinations ABC X(ABC) Y(ABC) Z(ABC) XY XZ YZ intra 0 5 10 15 20 25 SASD allowed SASD violating F E Cross-linked chain combinations identified for most amo genes, an obvious conserved promoter motif for all six genes was not identified. identified for most amo genes, an obvious conserved promoter motif for all six genes was not identified. identified for most amo genes, an obvious conserved promoter motif for all six genes was not identified. A re-evaluation of these transcriptomic data (see Methods) also revealed amoC6 as the primarily transcribed amoC homolog (Fig. 5), thus confirming the identification of a unique AmoC6 peptide from a Tricine-SDS-PAGE band digested with chymotrypsin (Supplementary Discussion; Supplementary Dataset 1). Together this indicates that copper limitation in N. viennensis [54] confirmed that the genes amoA, amoB, and amoC have some of the highest transcription levels in the cell, as also shown in previous studies [55–57]. A clustering analysis of the same dataset revealed that amoA, amoB, amoC, amoX, amoY, and amoZ all appear to be co-regulated, and fell into the clusters containing the most highly expressed genes. (Fig. S2; Supplementary Dataset 2). While putative transcriptional promoter sequences can be g A re-evaluation of these transcriptomic data (see Methods) also revealed amoC6 as the primarily transcribed amoC homolog (Fig. Expression patterns of AMO subunits in Nitrososphaera viennensis and Nitrosopumilus maritimus Available transcriptomic studies of AOA were inspected to explore whether the expression patterns of the newly predicted subunits would corroborate their involvement in the AMO. A recent study on The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 593 A B N. viennensis N. cavascurensis extracellular cytoplasmic N. cavascurensis N. cavascurensis B A N. viennensis copper limitation in N. viennensis [54] confirmed that the genes amoA, amoB, and amoC have some of the highest transcription levels in the cell, as also shown in previous studies [55–57]. A clustering analysis of he same dataset revealed that amoA, amoB, amoC, amoX, amoY, and amoZ all appear to be co-regulated, and fell into the clusters containing the most highly expressed genes. (Fig. S2; Supplementary Dataset 2). While putative transcriptional promoter sequences can be identified for most amo genes, an obvious conserved promoter motif for all six genes was not identified. A re-evaluation of these transcriptomic data (see Methods) also revealed amoC6 as the primarily transcribed amoC homolog (Fig. 5), thus confirming the identification of a unique AmoC6 peptide from a Tricine-SDS-PAGE band digested with chymotrypsin (Supplementary Discussion; Supplementary Dataset 1). Together this indicates that A C D E F B N. viennensis N. Expression patterns of AMO subunits in Nitrososphaera viennensis and Nitrosopumilus maritimus viennensis showing the location of amo genes and average log2 transformed transcript per million (TPM) values from copper replete conditions in Reyes et al. (2020) [54]. Orange bars on the genome indicate the locations of AMO subunit genes that are strongly expressed. Blue bars on the genome indicate amo genes that have low transcriptional activity. Boxes show amo genes (in bold) and immediate neighbors colored based on average gene expression from copper replete cultures. Red indicates a strong expression while blue represents a low or absent expression. All strongly expressed amo genes were found in clusters of highly expressed genes across both limited and replete conditions (see Fig. S2). 0.0 Mb 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Mb 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 Mb 2.1 2.2 2.3 2.4 2.5 NVIE_2916 amoB NVIE_028420 radA …7 genes… amoC6 NVIE_028550 NVIE_028520 amoC1 nirK NVIE_000550 NVIE_000520 amoC2 NVIE_002340 NVIE_002320 NVIE_004530 amoY (NVIE_004540) amoZ (NVIE_004550) NVIE_0459 amoC3 gds2 NVIE_011550 amoA amoX NVIE_027290 NVIE_027260 NVIE_2798 amoC5 ypfJ NVIE_024070 amoC4 NVIE_019090 NVIE_019070 Nitrososphaera viennensis 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 7.5 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 Fig. 5 Transcription of AMO subunit genes in N. viennensis. Genomic representation of N. viennensis showing the location of amo genes and average log2 transformed transcript per million (TPM) values from copper replete conditions in Reyes et al. (2020) [54]. Orange bars on the genome indicate the locations of AMO subunit genes that are strongly expressed. Blue bars on the genome indicate amo genes that have low transcriptional activity. Boxes show amo genes (in bold) and immediate neighbors colored based on average gene expression from copper replete cultures. Red indicates a strong expression while blue represents a low or absent expression. All strongly expressed amo genes were found in clusters of highly expressed genes across both limited and replete conditions (see Fig. S2). discovered in the Thermoplasmata phylum [61]. A HMMER search using the extended regions of the bacterial homologs against the genomes of collected AOA did not reveal any significant similarities. Therefore, a general structural search using Phobius [62] was carried out with the N. Structural search for missing components in the archaeal AMO complex p As previously observed [60], comparisons of the amino acid sequences of the three subunits AmoA, AmoB, and AmoC from archaea with those of bacteria indicate that the primary differences between the archaeal AMO subunits and the bacterial AMO subunits are missing transmembrane helices, at least one in AmoB and two in AmoC, and a missing C-terminal soluble portion found in bacterial AmoB/PmoB (Figs. S3–S5). These observations also hold true for the new clade of archaeal AMO recently Expression patterns of AMO subunits in Nitrososphaera viennensis and Nitrosopumilus maritimus Blue bars on the genome indicate amo genes that have low transcriptional activity. Boxes show amo genes (in bold) and immediate neighbors colored based on average gene expression from copper replete cultures. Red indicates a strong expression while blue represents a low or absent expression. All strongly expressed amo genes were found in clusters of highly expressed genes across both limited and replete conditions (see Fig. S2). 4 Fig. 4 Structural support of proposed AMO subunits based on BN-PAGE cross-linking and AlphaFold modeling. A, B Cartoon representations of the AlphaFold structure models of the N. viennensis (A) and N. cavascurensis (B) hexamers, indicating their putative membrane orientation based on sequence hydropathy analysis. Subunits are colored as follows: AmoA, light grey; AmoB, black; AmoC, salmon; AmoX, lavender; AmoY, cyan; AmoZ, blue. Residues in the CuB and CuC copper sites are represented in red sticks. Disulfide bonds are indicated in yellow. C Representation of identified cross-links among existing and proposed AMO subunits of an AMO band cut from a BN- PAGE gel of N. viennensis. Green: suspected subunits based on comparative genomics. Blue: newly proposed subunits based on BN-PAGE correlation and syntenic analysis. D Cross-links within the solvent accessible surface distance (SASD) threshold for DSSO, depicted in green, mapped on the N. viennensis AlphaFold model. The single observed cross-link between the AmoZ and AmoB subunits is depicted in magenta, as it violates SASD distance criteria (50.0 Å) but is within range of Euclidean distance (31.8 Å). E Distribution of SASD and Euclidean Cα–Cα distances of unique DSSO cross-links identified with Annika and MeroX. Twenty-seven out of 67 unique cross-links satisfied distance criteria (SASD < 35.0 Å). F Percentage of cross-linked subunit combinations. 0.0 Mb 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Mb 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 Mb 2.1 2.2 2.3 2.4 2.5 NVIE_2916 amoB NVIE_028420 radA …7 genes… amoC6 NVIE_028550 NVIE_028520 amoC1 nirK NVIE_000550 NVIE_000520 amoC2 NVIE_002340 NVIE_002320 NVIE_004530 amoY (NVIE_004540) amoZ (NVIE_004550) NVIE_0459 amoC3 gds2 NVIE_011550 amoA amoX NVIE_027290 NVIE_027260 NVIE_2798 amoC5 ypfJ NVIE_024070 amoC4 NVIE_019090 NVIE_019070 Nitrososphaera viennensis log2 TPM 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 7.5 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 Fig. 5 Transcription of AMO subunit genes in N. viennensis. Genomic representation of N. Expression patterns of AMO subunits in Nitrososphaera viennensis and Nitrosopumilus maritimus viennensis genome to search for genes that could encode a protein with the following criteria: (i) 1–3 transmembrane helices, (ii) conservation across all AOA [46], and (iii) present in the top 100 transcribed genes [54] (similar levels as the primary AMO subunits). This revealed six possible candidates (Table 2). The only candidates to meet the structural requirements while maintaining syntenic and similar patterns of migration in BN-PAGE were amoX, amoY, and amoZ. AmoC6 is the primary structural AmoC homolog in the AMO complex of N. viennensis, at least under the applied growth conditions. Transcriptomics of the marine strain, Nitrosopumilus maritimus, also showed high expression of amoA, amoB, amoC, amoX, and amoY (Nmar_1506). The gene amoZ (Nmar_1507), albeit syntenic with amoY, exhibited lower expression levels [55]. The three newly proposed AMO subunits were also inspected in proteomic datasets that were generated with methods allowing for the improved recovery of membrane proteins. All six of the known and proposed subunits were found in membrane fractions from N. viennensis from a previous study [15] as well as in the proteome of N. maritimus [55]. In other proteomic studies of AOA [58, 59], the three new subunits were not always present, likely due to their small size and limited number of trypsin cleavage sites. The addition of the three proposed subunits in archaea increases the number of transmembrane helices from 10–11 to approximately 14 per protomer making it comparable to the number found in bacterial crystal structures of pMMO where each protomer of the trimer (i.e., one unit of PmoABC), contains 14–15 transmembrane helices [23, 63]. Expression patterns of AMO subunits in Nitrososphaera viennensis and Nitrosopumilus maritimus 5), thus confirming the identification of a unique AmoC6 peptide from a Tricine-SDS-PAGE band digested with chymotrypsin (Supplementary Discussion; Supplementary Dataset 1). Together this indicates that The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 594 Fig. 4 Structural support of proposed AMO subunits based on BN-PAGE cross-linking and AlphaFold modeling. A, B Cartoon representations of the AlphaFold structure models of the N. viennensis (A) and N. cavascurensis (B) hexamers, indicating their putative membrane orientation based on sequence hydropathy analysis. Subunits are colored as follows: AmoA, light grey; AmoB, black; AmoC, salmon; AmoX, lavender; AmoY, cyan; AmoZ, blue. Residues in the CuB and CuC copper sites are represented in red sticks. Disulfide bonds are indicated in yellow. C Representation of identified cross-links among existing and proposed AMO subunits of an AMO band cut from a BN- PAGE gel of N. viennensis. Green: suspected subunits based on comparative genomics. Blue: newly proposed subunits based on BN-PAGE correlation and syntenic analysis. D Cross-links within the solvent accessible surface distance (SASD) threshold for DSSO, depicted in green, mapped on the N. viennensis AlphaFold model. The single observed cross-link between the AmoZ and AmoB subunits is depicted in magenta, as it violates SASD distance criteria (50.0 Å) but is within range of Euclidean distance (31.8 Å). E Distribution of SASD and Euclidean Cα–Cα distances of unique DSSO cross-links identified with Annika and MeroX. Twenty-seven out of 67 unique cross-links satisfied distance criteria (SASD < 35.0 Å). F Percentage of cross-linked subunit combinations. 0.0 Mb 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Mb 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 Mb 2.1 2.2 2.3 2.4 2.5 NVIE_2916 amoB NVIE_028420 radA …7 genes… amoC6 NVIE_028550 NVIE_028520 amoC1 nirK NVIE_000550 NVIE_000520 amoC2 NVIE_002340 NVIE_002320 NVIE_004530 amoY (NVIE_004540) amoZ (NVIE_004550) NVIE_0459 amoC3 gds2 NVIE_011550 amoA amoX NVIE_027290 NVIE_027260 NVIE_2798 amoC5 ypfJ NVIE_024070 amoC4 NVIE_019090 NVIE_019070 Nitrososphaera viennensis log2 TPM 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 7.5 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 Fig. 5 Transcription of AMO subunit genes in N. viennensis. Genomic representation of N. viennensis showing the location of amo genes and average log2 transformed transcript per million (TPM) values from copper replete conditions in Reyes et al. (2020) [54]. Orange bars on the genome indicate the locations of AMO subunit genes that are strongly expressed. DISCUSSION Th h l The archaeal AMO complex is a key enzyme of AOA energy metabolism that is highly expressed in all ammonia oxidizing organisms investigated and has large implications for the environment due to its overwhelming presence in many ecosystems [8–14, 67]. The work here profits from the recent improvements in the cultivation of AOA in continuous cultures (Melcher et al. in preparation) and presents novel biochemical and comparative genomic evidence on the composition of the AMO complex in Nitrososphaera viennensis and other AOA that contrasts with the proposed composition of this complex within AOB. p p p p g To compare the degree of conservation of the predicted hexameric organization of the AMO complex, a structural model of the AMO complex of N. cavascurensis was also obtained (Fig. 4B). The resultant models were similar in their overall arrangement to each other and to the N. viennensis model, with high overall confidence scores (top model, pLDDT = 77.7 and ptm score = 0.591). Differences between the N. viennensis and N. cavascurensis models include the localization of the transmem- brane (TM) helix of AmoZ. In N. viennensis the TM helix is predicted to interact mostly with the TM helix of AmoY, while in N. cavascurensis it is predicted to interact with the TMs of AmoB and AmoA (Figs. 4A, B, S7A, C). This would affect the relative positioning of the N-terminal domain of AmoZ with respect to the AmoB soluble domain, allowing for a more “open” conformation. However, the extended loop connecting the N-terminal pair of helices in AmoZ with the TM domain theoretically allows for some flexibility (additional information in Supplementary Discussion, Fig. S8). The present analysis has verified that AmoX, NVIE_004540, and NVIE_004550 are all likely present within the archaeal AMO complex and proposes the naming of NVIE_004540 and NVIE_004550 as AmoY and AmoZ, respectively. This finding is based on a host of independent analyses including proteomic, genomic, transcriptomic, structural, and modeling approaches. The presence of six subunits rather than three is unique to the archaeal domain and could represent a more complex regula- tory strategy for the AMO complex in archaea. Differences in the ammonia oxidation pathway are already well established between the archaeal and bacterial domains (i.e., unresolved second step in archaea [19, 21]; iron-based c cytochromes [68, 69] and ubiquinone in bacteria [70, 71] vs. copper- based plastocyanins [15, 16] and menaquinone in archaea [72]). Predicted structure of the archaeal AMO complex supports the integration of new subunits To gain insights into the structural context of the archaeal AMO complex in the light of three additionally proposed subunits, a structural model for the organization of the N. viennensis AMO complex was obtained by employing the multimer-capable The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 595 Table 2. Structural search for missing AMO subunits in N. viennensis. Table 2. Structural search for missing AMO subunits in N. viennensis. Gene Expression & Structural Search Previous Analyses Protein Gene TPM log2a TMb AOA Conservationc BN-PAGE Corr.d AMO Syntenye NVIE_013530 13.82 1 X protein of unknown function NVIE_004540 13.55 1 X X X hypothetical protein NVIE_004550 13.39 1 X X X hypothetical protein amoX 12.14 2 X X X putative ammonia monooxygenase - associated protein coxB 11.87 1 X putative heme-copper oxidase subunit II NVIE_014370 11.75 1 putative Copper binding protein, plastocyanin/ azurin family NVIE_010490 11.75 1 putative phosphoesterase, DHHA1 NVIE_027520 11.40 1 X putative heme/copper-type cytochrome/ quinol oxidase, subunit NVIE_027550 11.33 1 X putative blue (Type 1) copper domain protein atpK 11.30 2 archaeal A1A0-type ATP synthase, subunit K NVIE_021780 10.90 1 exported protein of unknown function NVIE_006540 10.90 1 exported protein of unknown function NVIE_029580 10.20 1 X blue (Type 1) copper domain-containing protein aBased on transcriptomic counts averaged from replete copper conditions from Reyes et al. (2020) [54]. bNumber of predicted transmembrane helices. cAs predicted from Abby et al. (2020) [46]. dRepresents proteins correlated with AmoA, AmoB, and AmoC6 in BN-PAGE bands from N. viennensis in this study. eGenes syntenic with amoA, amoB, and amoC based on the syntenic analysis from this study. tructural search for missing AMO subunits in N. viennensis Table 2. Structural search for missing AMO subunits in N. viennensis. Predicted structure of the archaeal AMO complex supports the integration of new subunits Gene Expression & Structural Search Previous Analyses Protein Gene TPM log2a TMb AOA Conservationc BN-PAGE Corr.d AMO Syntenye NVIE_013530 13.82 1 X protein of unknown function NVIE_004540 13.55 1 X X X hypothetical protein NVIE_004550 13.39 1 X X X hypothetical protein amoX 12.14 2 X X X putative ammonia monooxygenase - associated protein coxB 11.87 1 X putative heme-copper oxidase subunit II NVIE_014370 11.75 1 putative Copper binding protein, plastocyanin/ azurin family NVIE_010490 11.75 1 putative phosphoesterase, DHHA1 NVIE_027520 11.40 1 X putative heme/copper-type cytochrome/ quinol oxidase, subunit NVIE_027550 11.33 1 X putative blue (Type 1) copper domain protein atpK 11.30 2 archaeal A1A0-type ATP synthase, subunit K NVIE_021780 10.90 1 exported protein of unknown function NVIE_006540 10.90 1 exported protein of unknown function NVIE_029580 10.20 1 X blue (Type 1) copper domain-containing protein aBased on transcriptomic counts averaged from replete copper conditions from Reyes et al. (2020) [54]. bNumber of predicted transmembrane helices. cAs predicted from Abby et al. (2020) [46]. dRepresents proteins correlated with AmoA, AmoB, and AmoC6 in BN-PAGE bands from N. viennensis in this study. eGenes syntenic with amoA, amoB, and amoC based on the syntenic analysis from this study. Gene Expression & Structural Search involved all subunit combinations with the exception of AmoZ (Fig. 4F). AmoZ only participated in cross-linking interactions >35 Å, which supports a weaker association with the complex as observed in the BN-PAGE migration patterns. version of AlphaFold 2.1 [64–66]. The resultant models were all similar and represented confident predictions (top model, pLDDT = 71.4 and ptm score = 0.668). All predicted transmem- brane helices from AmoX, AmoY, and AmoZ play a role in anchoring the complex in the membrane along with the transmembrane helices from AmoA, AmoB, and AmoC (Fig. 4A). Additionally, the N-terminal domain of AmoZ was predicted to contain two alpha helices that interact with the N-terminal domain of AmoB, thereby possibly replacing the role of the missing C-terminal soluble domain found in PmoB and offering the final piece of the missing complex in archaea (additional information in Supplementary Discussion). A disulfide bond was also predicted to form within the soluble domain of AmoZ. The overall structure is comparable to a protomer of the pMMO complex (Fig. S6). DISCUSSION Th h l Although there is debate on which subunit harbors the primary active site in CuMMO complexes, there is clear evidence that the metal site(s) in PmoC plays a critical role in the complex of methanotrophs [27, 28, 31, 32]. While the archaeal AmoC lacks a substantial section found in all bacteria that corresponds to two transmembrane helices (Fig. S5), the metal site observed in earlier crystal structures as well as the newly proposed metal site identified via cryo-EM [28], are conserved across all archaeal and bacterial species (Fig. S5). The importance of this subunit is also supported by site directed mutagenesis studies in the genetically tractable Actinobacteria that contain the homologous hydrocar- bon monooxygenase [32]. Regardless of species-specific differences in archaea, the overall predicted archaeal structure, with the new subunits, is reflective of the known bacterial protomer composition (Fig. S6). Definitive proof of the oligomerization and organization of these subunits will not be possible until a definitive structure (i.e., crystal or cryo-EM) of archaeal AMO is realized. Putative protomer interaction sites in the cryo-EM structure of Methylo- coccus capsulatus str Bath (PDB structure 7S4H) [28] appear to be in the section of PmoB that is missing in archaea (Fig. S4). However, the placement of AmoY and AmoZ on the outer regions of the protomer could be facilitating these interactions instead (Fig. S7). This could also explain the high amount of SASD violating interactions between AmoY and AmoZ as the analysis only takes into account a single protomer (Fig. 4F). It is possible that these interactions may instead be between subunits of AmoY and AmoZ in different protomers. Therefore, the predicted protomer models of N. viennensis and N. cavascurensis do not rule out the possibility of a trimeric archaeal AMO complex. With respect to orientation, the present modeling approach is not able to predict exactly how the archaeal AMO sits in the membrane. However, it is likely that the active site as well as the soluble AmoB and AmoZ domains are situated toward the pseudo-periplasmic space. This is supported by previous modeling efforts of nutrient transport in the S-layer of AOA [91] as well as activity-based immunogold labeling of CuMMO complexes in AOB and methanotrophs [92]. The soil model AOA, N. viennensis, like most other soil dwelling AOA from the family Nitrososphaeraceae, encodes multiple homologs of the amoC gene while retaining only single copies of amoA and amoB [15] (Supplementary Dataset 2). DISCUSSION Th h l The varying characteristics within the AMO complex observed in this work further underscore these differences and add to a growing body of evidence that AOA and AOB participate in nitrification under different environmental and/or functional constraints. Data from cross-linking experiments in N. viennensis were mapped to the predicted model and strongly supported the predicted interactions (Fig. 4D) with some exceptions. Out of 67 unique observed cross-links, 27 (40%) satisfied a maximum solvent accessible surface distance (SASD) threshold of ≤35 Å (Fig. 4E) and The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 596 In blue native PAGE protein gels, the AMO complex in both N. viennensis and Nitrosocaldus cavascurensis migrated well above the predicted height of a trimeric protomer complex, even when considering the additional subunits (predicted molecular weight of a homotrimeric complex with six subunits per protomer: 296.9 kDa N. viennensis; 305.1 kDa N. cavascurensis). The archaeal AMO bands are also observed at a higher molecular weight in the gel when compared to the homologous bacterial PMO complex from a Methylomirabilis species that was also extracted using n- dodecyl-β-D-maltoside (DDM) [73]. This could be explained by differences in membrane composition of the strains or potential differences in oligomerization of the protomer. AOA contain unique ether-linked lipids (i.e., crenarchaeols) [37, 74–78] and rely on a proteinaceous S-layer rather than an outer membrane to create a pseudo-periplasmic space [52, 53]. The observation of three distinct peaks of AMO can most likely be explained by the co-migration with other proteins or complexes that it could be physically interacting with, in particular with the S-layer protein. Nitrosocaldus yellowstonensis [86]. This structure confirmed the lack of the C-terminal cupredoxin domain and revealed an extended amino acid region not found in bacteria made up of two helices and two loops. It was proposed that this additional region could help stabilize the existing cupredoxin domain as supportive interactions are lacking due to the absence of the C-terminal domain. However, this amino acid extension is only found within the proposed genus of Nitrosocaldus (Fig. S4). It is more likely that the soluble domain of AmoZ, which is conserved across all AOA, is conferring this stabilizing role. DISCUSSION Th h l , g g In the absence of additional structure-function analyses, it is unclear if the additional subunits in the archaeal complex simply reflect the vast evolutionary distance to all other known protein complexes of the CuMMO family [34], or if this difference in structure also has relevant functional implications. For instance, the bacterial AMO complexes are promiscuous enzymes able to oxidize methane and other compounds [87–90]. Such investiga- tions on alternative substrates have not yet been performed with the archaeal complex but would be important for evaluating the functional role of archaea (and possibly the new subunits) in the environment. Additionally, differences in the AMO complex between AOA species have been identified that have the potential to affect the function of the AMO complex. This includes the extra AmoB loop found within Nitrosocaldus, but is also clearly demonstrated by the newly proposed subunit AmoZ. The genus Nitrososphaera and the family Nitrosocaldaceae (both investigated in this study) are predicted to form a disulfide bond linking the two alpha helices making up the soluble domain of AmoZ (Figs. S7B, D, S10C) via two cysteines that are not conserved in other AOA. Additionally, the genus Nitrosocosmicus is predicted to contain an additional zinc ribbon domain represented by four cysteine residues at the C-terminal end, presumably residing within the cytoplasm (Fig. S10C). The observations of a disulfide bond and zinc ribbon domain within certain AOA lineages could be linked to sensitivity to reactive oxygen species and unique regulation strategies, respectively, and may reflect unique patterns of evolution that complement yet unknown aspects of the metabolism of these specific groups. y y y Previous work on bacteria that rely on CuMMOs have identified other putative proteins involved with the complex. Monocistronic transcripts containing amoABC from the AOB Nitrosococcus oceani ATCC 19707 contained two additional genes assigned as amoR and amoD [79]. The amoR gene was found to be only present in Nitrosococcus and was therefore not thought to be a conserved part of bacterial AMO. A recent study indicated that AmoD/PmoD (and likely the homologous AmoE) play crucial roles in copper homeostasis, but they are not suspected to be a structural part of any CuMMO complex [80]. Rather, crystal and cryo-EM structures of bacterial PMO have consistently confirmed a trimeric protomer structure with one subunit of PmoA, PmoB, and PmoC making up each protomer [22–28]. DISCUSSION Th h l Considering the wide distribution of AOA in virtually all ecosystems [8–14, 34] and their ecological relevance, developing genetic tools for AOA and improving their biomass production will be needed to enable structure-function analysis and to elucidate the full pathway of ammonia oxidation in these archaea. would therefore consist of six subunits instead of three as in other complexes of the CuMMO family. The addition of the new subunits would make the number of transmembrane helices comparable to CuMMO complexes found within bacteria. As the anchoring of pMMO in the membrane has previously been shown to be critical for its activity [26, 28], it seems plausible that the newly identified subunits play an important role for the structural and functional integrity of the archaeal AMO complex. The presence of a soluble domain within AmoZ that could replace the stabilizing function of the missing soluble domain in AmoB also fulfills a potentially crucial missing piece of the AMO complex. Since AmoXYZ appear to have important structural roles, it will be imperative to include all subunits in future expression and structural studies of this environmentally relevant protein complex in archaea. Considering the wide distribution of AOA in virtually all ecosystems [8–14, 34] and their ecological relevance, developing genetic tools for AOA and improving their biomass production will be needed to enable structure-function analysis and to elucidate the full pathway of ammonia oxidation in these archaea. adaptations of archaeal ammonia oxidizers. Proc Natl Acad Sci USA. 2016;113:E7937–46. 16. Walker CB, De La Torre JR, Klotz MG, Urakawa H, Pinel N, Arp DJ, et al. Nitroso- pumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proc Natl Acad Sci USA. 2010;107:8818–23. 17. Berg IA, Kockelkorn D, Buckel W, Fuchs G. A 3-hydroxypropionate 4-hydro- xybutyrate autotrophic carbon dioxide assimilation pathway in archaea. Science. 2007;218:1782–6. 18. Könneke M, Schubert DM, Brown PC, Hügler M, Standfest S, Schwander T, et al. Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation. Proc Natl Acad Sci USA. 2014;111:8239–44. 19. Lancaster KM, Caranto JD, Majer SH, Smith MA. Alternative bioenergy: updates to and challenges in nitrification metalloenzymology. Joule. 2018;2:1–21. 20. Simon J, Klotz MG. Diversity and evolution of bioenergetic systems involved in microbial nitrogen compound transformations. Biochim Biophys Acta - Bioenerg. 2013;1827:114–35. 21. Kozlowski JA, Stieglmeier M, Schleper C, Klotz MG, Stein LY. REFERENCES 1. Wong-Chong GM, Loehr RC. The kinetics of microbial nitrification. Water Res. 1975;9:1099–106. 27. Chang WH, Lin HH, Tsai IK, Huang SH, Chung SC, Tu IP, et al. Copper centers in the cryo-EM structure of particulate methane monooxygenase reveal the catalytic machinery of methane oxidation. J Am Chem Soc. 2021;143:9922–32. 2. Hyman MR, Wood PM. Suicidal inactivation and labelling of ammonia mono- oxygenase by acetylene. Biochem J. 1985;227:719–25. 2. Hyman MR, Wood PM. Suicidal inactivation and labelling of ammonia mono- oxygenase by acetylene. Biochem J. 1985;227:719–25. 28. Koo CW, Tucci FJ, He Y, Rosenzweig AC. Recovery of particulate methane mono oxygenase structure and activity in a lipid bilayer. Science. 2022;375:1287–91. 3. Hollocher TC, Tate ME, Nicholas DJ. Oxidation of ammonia by Nitrosomonas europaea. Definitive 18O-tracer evidence that hydroxylamine formation involves a monooxygenase. J Biol Chem. 1981;256:10834–6. 29. Balasubramanian R, Smith SM, Rawat S, Yatsunyk LA, Stemmler TL, Rosenzweig AC. Oxidation of methane by a biological dicopper centre. Nature. 2010;465:115–9. 4. Winogradsky S. Recherches sur les organismes de la nitrification. Ann Inst Pateur. 1890;4:213–31. 30. Op den Camp HJM, Islam T, Stott MB, Harhangi HR, Hynes A, Schouten S, et al. Environmental, genomic and taxonomic perspectives on methanotrophic Ver- rucomicrobia. Environ Microbiol Rep. 2009;1:293–306. 5. Könneke M, Bernhard AE, De La Torre JR, Walker CB, Waterbury JB, Stahl DA. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature. 2005;437:543–6. 31. Ross MO, MacMillan F, Wang J, Nisthal A, Lawton TJ, Olafson BD, et al. Particulate methane monooxygenase contains only mononuclear copper centers. Science. 2019;364:566–70. 6. Treusch AH, Leininger S, Kietzin A, Schuster SC, Klenk HP, Schleper C. Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environ Microbiol. 2005;7:1985–95. 32. Liew EF, Tong D, Coleman NV, Holmes AJ. Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity. Micro- biology. 2014;160:1267–77. 7. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science. 2004;304:66–74. 7. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science. 2004;304:66–74. 8. Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, et al. Archaea pre- dominate among ammonia-oxidizing prokaryotes in soils. Nature. 2006;442:806–9. 33. DISCUSSION Th h l Pathways and key intermediates required for obligate aerobic ammonia-dependent chemolitho- trophy in bacteria and Thaumarchaeota. ISME J. 2016;10:1836–45. 22. Lieberman RL, Rosenzweig AC. Crystal structure of a membrane-bound metalloen- zyme that catalyses the biological oxidation of methane. Nature. 2005;434:177–82. 23. Hakemian AS, Kondapalli KC, Telser J, Hoffman BM, Stemmler TL, Rosenzweig AC. The metal centres of particulate methane monooxygenase from Methylosinus trichosporium OB3b. Biochemistry. 2008;47:6793–801. DATA AVAILABILITY 24. Smith SM, Rawat S, Telser J, Hoffman BM, Stemmler TL, Rosenzweig AC. Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M. Biochemistry. 2011;50:10231–40. All proteomic data was deposited to the ProteomeXchange Consortium via PRIDE [44] partner repository with identifiers PXD035349, PXD034632, and PXD034475 for BN-Page of N. viennensis, BN-PAGE of N. cavascurensis, and cross-linked samples, respectively. Relevant scripts and code for data analysis can be found at the GitHub repository https://github.com/hodgskiss/Archaeal_AMO. 25. Sirajuddin S, Barupala D, Helling S, Marcus K, Stemmler TL, Rosenzweig AC. Effects of zinc on particulate methane monooxygenase activity and structure. J Biol Chem. 2014;289:21782–94. 26. Ro SY, Ross MO, Deng YW, Batelu S, Lawton TJ, Hurley JD, et al. From micelles to bicelles: effect of the membrane on particulate methane monooxygenase activity. J Biol Chem. 2018;293:10457–65. DISCUSSION Th h l Additional copies of amoC that are spatially disconnected from the AMO operon are encoded by some terrestrial AOB and were implicated in stress response based on transcriptional studies [81, 82]. Within Nitrososphaeraceae, no conserved AMO operons exist (Fig. 3). Duplications of the amoC gene (spatially distant from the other AMO genes) also occur in some species of the AOA marine associated family (Nitrosopumilaceae) and in two MAGs from AOA thermophiles (Nitrosocaldaceae), all discovered in sediments [51, 83, 84]. An amoC duplication is also found in an AOA sponge symbiont and copies of archaeal amoC are even found in marine viruses [85]. These findings together may indicate the metabolic importance of the AmoC subunit for ecophysiological adaptations in ammonia oxidation. While this work identified one particular AmoC (AmoC6; NVIE_028540) to be the primary homolog within the complex of N. viennensis, it is possible that (some of) the other AmoC subunits, which arose by gene duplications at the genus level (Fig. S9), might be incorporated under certain environmental conditions and provide different activity profiles to the enzyme. In conclusion, this study provides evidence through genomic, proteomic, and transcriptomic data for the presence of AmoX and the inclusion of AmoY and AmoZ as subunits within the archaeal AMO complex. A single protomer of the archaeal AMO Beyond comparative genomics, the only confirmed structural information for archaea stems from the crystal structure of a heterologously expressed AmoB originating from Candidatus The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 597 would therefore consist of six subunits instead of three as in other complexes of the CuMMO family. The addition of the new subunits would make the number of transmembrane helices comparable to CuMMO complexes found within bacteria. As the anchoring of pMMO in the membrane has previously been shown to be critical for its activity [26, 28], it seems plausible that the newly identified subunits play an important role for the structural and functional integrity of the archaeal AMO complex. The presence of a soluble domain within AmoZ that could replace the stabilizing function of the missing soluble domain in AmoB also fulfills a potentially crucial missing piece of the AMO complex. Since AmoXYZ appear to have important structural roles, it will be imperative to include all subunits in future expression and structural studies of this environmentally relevant protein complex in archaea. REFERENCES Can- didatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermo- philic archaeon with a highly mobile genome. Front Microbiol. 2018;9:28. 75. Villanueva L, Damsté JSS, Schouten S. A re-evaluation of the archaeal membrane lipid biosynthetic pathway. Nat Rev Microbiol. 2014;12:438–48. 49. Nicol GW, Schleper C. Ammonia-oxidising Crenarchaeota: important players in the nitrogen cycle? Trends Microbiol. 2006;14:207–12. 50. Sheridan PO, Raguideau S, Quince C, Holden J, Zhang L, Gaze WH, et al. Gene duplication drives genome expansion in a major lineage of Thaumarchaeota. Nat Commun. 2020;11:5494. 76. Sinninghe Damsté JS, Schouten S, Hopmans EC, Van Duin ACT, Geenevasen JAJ. Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether mem- brane lipid of cosmopolitan pelagic crenarchaeota. J Lipid Res. 2002;43:1641–51. 51. Luo Z-H, Narsing Rao MP, Chen H, Hua Z-S, Li Q, Hedlund BP, et al. Genomic insights of “Candidatus Nitrosocaldaceae” based on nine new metagenome- assembled genomes, including “Candidatus Nitrosothermus” gen nov. and two new species of “Candidatus Nitrosocaldus”. Front Microbiol. 2021;11:608832. 77. Sinninghe Damsté JS, Rijpstra WIC, Hopmans EC, Jung MY, Kim JG, Rhee SK, et al. Intact polar and core glycerol dibiphytanyl glycerol tetraether lipids of group I.1a 77. Sinninghe Damsté JS, Rijpstra WIC, Hopmans EC, Jung MY, Kim JG, Rhee SK, et al. Intact polar and core glycerol dibiphytanyl glycerol tetraether lipids of group I.1a and I.1b Thaumarchaeota in soil. Appl Environ Microbiol. 2012;78:6866–74. 78. Elling FJ, Könneke M, Mußmann M, Greve A, Hinrichs KU. Influence of tempera- ture, pH, and salinity on membrane lipid composition and TEX86 of marine planktonic thaumarchaeal isolates. Geochim Cosmochim Acta. 2015;171:238–55. 52. Stieglmeier M, Klingl A, Alves RJE, Rittmann SKMR, Melcher M, Leisch N, et al. Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon from soil and a member of the archaeal phylum Thaumarchaeota. Int J Syst Evol Microbiol. 2014;64:2738–52. 79. El Sheikh AF, Poret-Peterson AT, Klotz MG. Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitroso- coccus oceani ATCC 19707. Appl Environ Microbiol. 2008;74:312–8. Thaumarchaeota. Int J Syst Evol Microbiol. 2014;64:2738–52. 80. Fisher OS, Kenney GE, Ross MO, Ro SY, Lemma BE, Batelu S, et al. Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria. Nat Commun. 2018;9:4276. 53. Albers SV, Meyer BH. The archaeal cell envelope. Nat Rev Microbiol. 2011;9:414–26. 54. Reyes C, Hodgskiss LH, Kerou M, Pribasnig T, Abby SS, Bayer B, et al. REFERENCES 598 42. Berger S, Cabrera-orefice A, Jetten MSM, Brandt U, Welte CU. Investigation of central energy metabolism-related protein complexes of ANME-2d methano- trophic archaea by complexome profiling. BBA - Bioenerg. 2021;1862:148308. 67. Klotz MG, Stein LY. Nitrifier genomics and evolution of the nitrogen cycle. FEMS Microbiol Lett. 2008;278:146–56. 68. Yamanaka T, Shinra M. Cytochrome c-552 and cytochrome c-554 derived from Nitrosomonas europaea. J Biochem. 1974;75:1265–73. 43. Hevler JF, Lukassen MV, Cabrera‐Orefice A, Arnold S, Pronker MF, Franc V, et al. Selective cross‐linking of coinciding protein assemblies by in‐gel cross‐linking mass spectrometry. EMBO J. 2021;40:e106174. 69. Arciero DM, Hooper AB, Balny C. Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitroso- monas europaea. Biochemistry. 1991;30:11466–72. 44. Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47:D442–50. 70. Hooper AB, Erickson RH, Terry KR. Electron transport systems of Nitrosomonas: isolation of a membrane-envelope fraction. J Bacteriol. 1972;110:430–8. 71. Whittaker M, Bergmann D, Arciero D, Hooper AB. Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas euro- paea. Biochim Biophys Acta - Bioenerg. 2000;1459:346–55. 45. Melcher M, Hodgskiss LH, Mardini MA, Schleper C, Rittmann SMKR. Analysis of biomass productivity and physiology of Nitrososphaera viennensis grown in continuous culture. Frontiers in Microbiology. (in preparation) 14:206. 72. Elling FJ, Becker KW, Könneke M, Schröder JM, Kellermann MY, Thomm M, et al. Respiratory quinones in Archaea: phylogenetic distribution and application as biomarkers in the marine environment. Environ Microbiol. 2016;18:692–707. 46. Abby SS, Kerou M, Schleper C. Ancestral reconstructions decipher major adap- tations of ammonia-oxidizing archaea upon radiation into moderate terrestrial and marine environments. MBio. 2020;11:e02371–20. 73. Versantvoort W, Guerrero-Castillo S, Wessels HJCT, Van Niftrik L, Jetten MSM, Brandt U, et al. Complexome analysis of the nitrite-dependent methanotroph Methylo- mirabilis lanthanidiphila. Biochim Biophys Acta - Bioenerg. 2019;1860:734–44. 47. Rinke C, Chuvochina M, Mussig AJ, Chaumeil PA, Davín AA, Waite DW, et al. A standardized archaeal taxonomy for the Genome Taxonomy Database. Nat Microbiol. 2021;6:946–59. 74. Pitcher A, Rychlik N, Hopmans EC, Spieck E, Rijpstra WIC, Ossebaar J, et al. Cre- narchaeol dominates the membrane lipids of Candidatus Nitrososphaera gar- gensis, a thermophilic Group I.1b Archaeon. ISME J. 2010;4:542–52. 48. Abby SS, Melcher M, Kerou M, Krupovic M, Stieglmeier M, Rossel C, et al. REFERENCES Musiani F, Broll V, Evangelisti E, Ciurli S. The model structure of the copper- dependent ammonia monooxygenase. JBIC J Biol Inorg Chem. 2020;25:995–1007. 34. Alves RJE, Minh BQ, Urich T, Von Haeseler A, Schleper C. Unifying the global phylogeny and environmental distribution of ammonia-oxidising archaea based on amoA genes. Nat Commun. 2018;9:1517. 9. Nicol GW, Leininger S, Schleper C, Prosser JI. The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria. Environ Microbiol. 2008;10:2966–78. 35. Khadka R, Clothier L, Wang L, Lim CK, Klotz MG, Dunfield PF. Evolutionary history of copper membrane monooxygenases. Front Microbiol. 2018;9:2493. 10. Adair KL, Schwartz E. Evidence that ammonia-oxidizing archaea are more abundant than ammonia-oxidizing bacteria in semiarid soils of northern Arizona, USA. Micro Ecol. 2008;56:420–6. 36. Bartossek R, Spang A, Weidler G, Lanzen A, Schleper C. Metagenomic analysis of ammonia-oxidizing archaea affiliated with the soil group. Front Microbiol. 2012;3:208. 11. Karner MB, Delong EF, Karl DM. Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature. 2001;409:507–10. 37. De La Torre JR, Walker CB, Ingalls AE, Könneke M, Stahl DA. Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ Microbiol. 2008;10:810–8. 12. Shi Y, Tyson GW, Eppley JM, Delong EF. Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean. ISME J. 2011;5:999–1013. 38. Tourna M, Stieglmeier M, Spang A, Könneke M, Schintlmeister A, Urich T. Nitro- sosphaera viennensis, an ammonia oxidizing archaeon from soil. Proc Natl Acad Sci USA. 2011;108:8420–5. 13. Baker BJ, Lesniewski RA, Dick GJ. Genome-enabled transcriptomics reveals archaeal populations that drive nitrification in a deep-sea hydrothermal plume. ISME J. 2012;6:2269–79. 39. Wittig I, Braun H-P, Schägger H. Blue native PAGE. Nat Protoc. 2006;1:418–28. 14. Hollibaugh JT, Gifford S, Sharma S, Bano N, Moran MA. Metatranscriptomic analysis of ammonia-oxidizing organisms in an estuarine bacterioplankton assemblage. ISME J. 2011;5:866–78. 40. Reisinger V, Eichacker LA. Solubilization of membrane protein complexes for blue native PAGE. J Proteom. 2008;71:277–83. 41. De Almeida NM, Wessels HJCT, De Graaf RM, Ferousi C, Jetten MSM, Keltjens JT, et al. Membrane-bound electron transport systems of an anammox bacterium: a complexome analysis. Biochim Biophys Acta - Bioenerg. 2016;1857:1694–704. 15. Kerou M, Offre P, Valledor L, Abby SS, Melcher M, Nagler M, et al. Proteomics and comparative genomics of Nitrososphaera viennensis reveal the core genome and The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. REFERENCES Genome wide transcriptomic analysis of the soil ammonia oxidizing archaeon Nitrososphaera viennensis upon exposure to copper limitation. ISME J. 2020;14:2659–74. 81. Berube PM, Samudrala R, Stahl DA. Transcription of all amoC copies is associated with recovery of Nitrosomonas europaea from ammonia starvation. J Bacteriol. 2007;189:3935–44. 55. Qin W, Amin SA, Lundeen RA, Heal KR, Martens-Habbena W, Turkarslan S, et al. Stress response of a marine ammonia-oxidizing archaeon informs physiological status of environmental populations. ISME J. 2017;12:508–19. 82. Berube PM, Stahl DA. The divergent AmoC3 subunit of ammonia mono- oxygenase functions as part of a stress response system in Nitrosomonas euro- paea. J Bacteriol. 2012;194:3448–56. 56. Carini P, Dupont CL, Santoro AE. Patterns of thaumarchaeal gene expression in culture and diverse marine environments. Environ Microbiol. 2018;20:2112–24. 83. Lebedeva EV, Hatzenpichler R, Pelletier E, Schuster N, Hauzmayer S, Bulaev A, et al. Enrichment and genome sequence of the group I.1a ammonia-oxidizing archaeon ‘Ca. Nitrosotenuis uzonensis’ representing a clade globally distributed in thermal habitats. PLoS One. 2013;8:e80835. 57. Stewart FJ, Ulloa O, Delong EF. Microbial metatranscriptomics in a permanent marine oxygen minimum zone. Environ Microbiol. 2012;14:23–40. 58. Bayer B, Pelikan C, Bittner MJ, Reinthaler T, Könneke M, Herndl GJ, et al. Proteomic response of three marine ammonia-oxidizing archaea to hydrogen peroxide and their metabolic interactions with a heterotrophic alphaproteobacterium. mSystems. 2019;4:e00181–19. 84. Qin W, Heal KR, Ramdasi R, Kobelt JN, Martens-Habbena W, Bertagnolli AD, et al. Nitrosopumilus maritimus gen. nov., sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., four marine ammonia-oxidizing archaea of the phylum Thaumarchaeota. Int J Syst Evol Microbiol. 2017;67:5067–79. 59. Santoro AE, Dupont CL, Richter RA, Craig MT, Carini P, McIlvin MR, et al. Genomic and proteomic characterization of ‘Candidatus Nitrosopelagicus brevis’: an ammonia-oxidizing archaeon from the open ocean. Proc Natl Acad Sci USA. 2015;112:1173–8. 85. Ahlgren NA, Fuchsman CA, Rocap G, Fuhrman JA. Discovery of several novel, widespread, and ecologically distinct marine Thaumarchaeota viruses that encode amoC nitrification genes. ISME J. 2019;13:618–31. 60. Tolar BB, Herrmann J, Bargar JR, van den Bedem H, Wakatsuki S, Francis CA. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea. Environ Microbiol Rep. 2017;9:484–91. 86. Lawton TJ, Ham J, Sun T, Rosenzweig AC. Structural conservation of the B subunit in the ammonia monooxygenase/particulate methane monooxygenase super- family. Proteins. 2014;82:2263–7. 61. AUTHOR CONTRIBUTIONS LHH, MK, SW, MH, and CS conceptualized the research project. Investigation was carried out by LHH, MK, WC, SNS, and RIPT. Biomass production was performed by MM. LHH, MK, and CS wrote the paper with edits and contributions from MM, WC, RIPT, SNS, SW, and MH. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. REFERENCES Diamond S, Lavy A, Crits-Christoph A, Matheus Carnevali PB, Sharrar A, Williams KH, et al. Soils and sediments host Thermoplasmata archaea encoding novel copper membrane monooxygenases (CuMMOs). ISME J. 2022;16:1348–62. 87. Hyman MR, Wood PM. Methane oxidation by Nitrosomonas europaea. Biochem J. 1983;212:31–37. 62. Käll L, Krogh A, Sonnhammer ELL. A combined transmembrane topology and signal peptide prediction method. J Mol Biol. 2004;338:1027–36. 88. Hyman MR, Wood PM. Ethylene oxidation by Nitrosomonas europaea. Arch Microbiol. 1984;137:155–8. 89. Hyman MR, Murton IB, Arp DJ. Interaction of ammonia monooxygenase from Nitrosomonas europaea with alkanes, alkenes, and alkynes. Appl Environ Micro- biol. 1988;54:3187–90. 63. Hakemian AS, Rosenzweig AC. The biochemistry of methane oxidation. Annu Rev Biochem. 2007;76:223–41. 64. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–9. 90. Jones RD, Morita RY. Methane oxidation by Nitrosococcus oceanus and Nitroso- monas europaea. Appl Environ Microbiol. 1983;45:401–10. 65. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, et al. AlphaFold Protein Structure Database: massively expanding the structural cov- erage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50:D439–D444. 91. Li P, Herrmann J, Tolar BB, Poitevin F, Ramdasi R, Bargar JR, et al. Nutrient transport suggests an evolutionary basis for charged archaeal surface layer proteins. ISME J. 2018;12:2389–402. 92. Sakoula D, Smith GJ, Frank J, Mesman RJ, Kop LFM, Blom P, et al. Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria. ISME J. 2022;16:958–71. 66. Evans R, O’Neill M, Pritzel A, Antropova N, Senior A, Green T, et al. Protein complex prediction with AlphaFold-Multimer. bioRxiv. 2022. https://doi.org/ 10.1101/2021.10.04.463034. The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 599 Correspondence and requests for materials should be addressed to Christa Schleper. Correspondence and requests for materials should be addressed to Christa Schleper. Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The ISME Journal (2023) 17:588 – 599 ACKNOWLEDGEMENTS We thank Anas Mohammed Mardini for excellent technical assistance in the cultivation of N. viennensis and Wolfram Weckwerth for valuable input in the initial discussions of the project. We also thank Florian Sikora and Dr. Boris Görke for technical assistance and usage of the OneShot machine for cell lysis and Dr. Stephanie Eichorst for assistance and usage of the ultracentrifuge. We are also appreciative of Dr. Thomas Rattei, Florian Goldenberg, and Johann Dorn of the Division of Computational Systems Biology (CUBE) for providing maintenance and access to the Life Science Computer Cluster (LiSC) at the University of Vienna. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41396-023-01367-3. FUNDING This project was supported by Doktoratskolleg (DK) plus: Microbial nitrogen cycling— from single cells to ecosystems (Austrian Science Fund W1257), ERC Advanced Grant TACKLE (No. 695192), and the European Union’s Horizon 2021–2027 research and innovation programme under grant agreement No 101079299. COMPETING INTERESTS COMPETING INTERESTS COMPETING INTERESTS The authors declare no competing interests. The authors declare no competing interests. © The Author(s) 2023, corrected publication 2023 The ISME Journal (2023) 17:588 – 599
https://openalex.org/W3021957007
https://hal.inria.fr/hal-03188819/document
English
null
Proposed System for a Socio-Technical Design Framework for Improved User Collaborations with Automation Technologies
Lecture notes in computer science
2,020
cc-by
3,832
To cite this version: Parisa Saadati, Jose Abdelnour-Nocera, Torkil Clemmensen. Proposed System for a Socio-Technical Design Framework for Improved User Collaborations with Automation Technologies. 17th IFIP Conference on Human-Computer Interaction (INTERACT), Sep 2019, Paphos, Cyprus. pp.59-67, ￿10.1007/978-3-030-46540-7_7￿. ￿hal-03188819￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03188819 https://inria.hal.science/hal-03188819v1 Submitted on 2 Apr 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Keywords:sociotechnical, human work interaction design, automation, augmentation, Industry 4.0. Proposed System for a Socio-technical Design Framework for Improved User Collaborations with Automation Technologies Parisa Saadati1 [0000-0002-0525-4654], José Abdelnour-Nocera1,2 [0000-0001-7935-7368] andTorkil Clemmensen3 [0000-0002-0934-2336] Parisa Saadati1 [0000-0002-0525-4654], José Abdelnour-Nocera1,2 [0000-0001-7935-7368] andTorkil Clemmensen3 [0000-0002-0934-2336] 1University of West London, UK parisa.saadati@uwl.ac.uk 1University of West London, UK parisa.saadati@uwl.ac.uk 2 ITI/Larsys Portugal abdejos@uwl.ac.uk 1 Introduction During the life cycle of any organisation, a variety of environmental stimuli will in- fluence its operations and decision-making processes. These external factors are de- pendent on economic and social factors, political and legislative changes, and devel- opments in technology and human knowledge. The internal environment may also influence various processes and elements of an organisation such as the staff, infor- mation and monitoring systems or management policies [1]. Complex organisational systems inevitably rely now on large-scale software-intensive systems. In this paper, we hint at a possible sociotechnical HCI framework with customized value proposi- tions and a case presentation for a future investigation of three different scenarios with different levels of automation. Socio-Technical System Design (STSD) developments have identified and ad- dressed several problems in understanding and developing complex systems. Despite many positive outcomes, these methods have not materially changed industrial soft- ware engineering practices. One of the main reasons behind this is involving users only in the testing stage of any new system development instead of the design process [2]. Currently, ‘automation’ is one of the main means for supporting operators using systems that feature high complexity. Automation allows designers to transfer the burden from operators to machine by re-allocating the system tasks that were previ- ously performed by human [3].Researchers have studied different aspects of imple- mentation of advanced interactive technologies employing automation in different platforms [1, 3-7]. Organisations can now improve operations and decision making by implementing cyber-physical systems (CPS) and internet of things (IoT), and potentially linking them to blockchain technology in the future. Rising integration of Internet of Every- thing (IoE) into the industrial value chain is the foundation of “Industry 4.0” technol- ogies [8]. These technologies can improve the end-users’ experience via increasing the self-service options, optimising operations and security processes, and enhancing ground asset management and connectivity. An important point to consider is that implementing new technologies in a complex service-driven work environment (e.g. an airport terminal) does not necessarily and automatically guarantee a positive response from workers and customers [9]. Hence, developments towards future ‘smart workplaces’ need to be carefully designed in order to achieve expected service quality goals for both end-users and employees. The main purpose of this study is to identify all humanistic/social and technological ele- ments in the design of newly automated systems applicable to Industry 4.0 that are affecting the human and machine collaborations. 3Copenhagen Business School tc.digi@cbs.dk 3Copenhagen Business School tc.digi@cbs.dk Abstract.To improve human performance, interactive technologies are going towards more automated systems that involve computers, robots and cyber-physical systems into the deci- sion-making process. While automation can lead to increased performance and reduced impact of human errors, interactive technologies without optimal design can have a negative impact on the experience of operators and end-users, leading to suboptimal performance of the automated systems. In this research, we aim to evaluate and refineHuman Work Interaction Design (HWID) framework to be applicable in various highly-automated settings including Industry 4.0 environments. This will be performed via a thorough literature review as the first step. The list of identified factors playing a potential role in various interactive systems will then be eval- uated and optimised in three case studies. We will try to understand how to maximise collabo- rations between the users and the machine in interactive systems. A practical approach for evaluating both employees’ and end-users' perspectives in three scenarios with different levels of automation will be assessed. We will evaluate the outputs in multiple levels of organisations, employees and end-users. The ultimate output of the study will be a framework or model that will help in designing future research studies for various automation scenarios, especially semi- autonomous systems that involve high levels of interaction between users and the machine. We will provide guidelines for implementation of the proposed framework in different scenari- os. We expect that the framework output of this research will provide a comprehensive guide- line applicable to many Industry 4.0 technologies. Keywords:sociotechnical, human work interaction design, automation, augmentation, Industry 4.0. 2 2 2 Review Automating a process that is embedded into people’s everyday lives will surely im- pact their experience. Automation replaces or rearranges people’s practices and habits that may have been developed over long periods. Therefore, using automation in in- teractive systems requires consideration of potential changes on human activity and the new coordination demands on the human operators. These experiences highly depend on the type and level of automation [7] and to what extent the developer has allowed the machine to make decisions. 1 Introduction This paper is organised as follows. Section 2 introduces the findings of the literature review on different factors affecting the human and machine collaborations and categorising them into three main catego- ries. Section 3 proposes the future research outcome by investigating into these fac- tors from three case studies; university library, research platform and an airport. 3 3 2.1 Technological elements of interactive systems Around 1970s and after a series of technological advances labelled as the third indus- trial revolution (also called “the digital revolution”), the transition towards the fourth industrial revolution (Industry 4.0) is now undergoing that will transform the design, manufacturing, and operation of various products and systems [7]. The increasing integration of the Internet of Everything (IoE) into the industrial value chain has built the foundation for this revolution [8]. The increased connectivity and interaction among systems, humans and machines support the integration of various automated or semi-automated systems, and hence, increasing flexibility and productivity [10]. These automated systems will lead to interconnected manufacturing systems and sup- ply chains with their own challenges. To achieve sufficient autonomous awareness in a system, efficient integration of smart sensors and mobile devices is required alongside industrial communication protocols and standards. Economic impact of this industrial revolution is supposed to be huge [10], as it promises substantial increase in operational effectiveness as well as the development of new business models, services, products and organisational struc- tures and culture [10-12]. Three key components of Industry 4.0 are Internet of Things (IoT), Cyber-Physical Systems (CPS), and smart workplaces. The main objects commonly used in the Indus- try 4.0 are RFID (radio-frequency identifiers), sensors, actuators, and mobile phones that interacts with each other and cooperate with their neighbouring smart compo- nents to reach the common goal. For all these smart objects and people who are going to collaborate with them, there is a need for setting technical standards to enable them to work. Industry 4.0 advancements [7] are categorised into 4 main principles in general: 1. Technical assistance, 2. Interconnections, 3. Decentralised decisions, and 4. Information transparency. The main focus of this research will be on the “Collaborations” sub-principle of the “Interconnections” principle (which includes Collaborations, Standards and Security). 4 4 Three type of collaborations are considered in the context of Industry 4.0: human- human, human-machine and machine-machine collaborations. As a result of recent advances in smart interactive systems, employees’ experience and access to technolo- gy have increased substantially. Recent development of using smart technologies in new domains such as health, education, finance and the impact of Industry 4.0 tech- nologies in manufacturing and logistics have raised new challenges for Human Com- puter Interaction (HCI) researchers and practitioners. Figure 1. The HWID framework [5] Figure 1. The HWID framework [5] For applying HWID models to specific workplaces we need to consider several in- dependent and entangled factors[5]. Considering numerous theories, concepts, tech- niques and methods developed for other work environments is the first step. Envi- ronmental contexts such as national, cultural, geographic, social and organisational factors will have an important role in designing optimal HWID models, as they im- pact interaction between users (i.e. both operators and employees) and smart systems in their work and life. There are more work-related factors including the users’ knowledge/skills, application domain, work contents and goals, as well as the nature of tasks or newly introduced technologies to be considered in the interaction perfor- mance. Developing HWID models requires establishing design goals, evaluation of usability and user experience, engagement of all stakeholders, and provision of trans- parent design processes. 2.3 Smart Workplace “Smart Workplace” is a vision where the organisation is fully connected with all stakeholders via proactive adaptation to the real-time needs of the organisation includ- ing operational necessities and customer requests. As an example, security concerns in airports necessitate more investigations prior to the boarding, which results in long queues and waiting times for passengers. Hence, airports need to be more innovative in operations and handling of stakeholders (passengers and workers) and their needs in real time. 2.2 Human Work Interaction Design Human Work Interaction Design (HWID) is a comprehensive framework that aims to establish relationships between extensive empirical work-domain studies and HCI designs. It builds on the foundation of Cognitive Work Analysis (CWA) [5]. HWID is currently positioned as a modern lightweight version of CWA. HWID studies how to understand, conceptualise, and design for the complex and emergent contexts in which information and communication technologies (ICT) and work are entangled [1]. HWID models are based on the characteristics of humans and work domain contents and the interactions during their tasks and decision making activities (Figure 1). HWID focuses on the integration of work analysis (i.e., CWA methods) and interaction design methods (e.g. goal-oriented design and HCI usabil- ity) for smart workplaces. The ultimate goal of HWID is to empower users by design- ing smarter workplaces in various work domains. 5 5 2.4 Humanistic elements of interactive systems To address human element in designing complex interactive systems, design fiction and design ethnography should be linked[13]. This is in line with considering the impact of anthropology on the design’s future-orientedness by understanding the cul- tural meanings and sensitivity to values and context[14]. Analysis of the allocation of functions is necessary to identify the optimal distribution of both functions and tasks between a partly-autonomous system and the user[3]. Physical support of human workers by robots or machines is an important aspect of new technologies. This is due to involvement of users in conducting a range of tasks that are unpleasant, too exhausting or unsafe [15,16]. For an effective, successful, and safe support of users in physical tasks, it is necessary that robots or machines interact smoothly and intuitively with their human counterparts [15], and that humans are properly trained for this kind of human-machine collaboration[8]. The value of information. In collaborations between human and machine, the value of information is now more recognised given high power of the machine in decision- making in highly-automated systems. For instance, informing users about the sensor’s reading power of Tesla’s automated car can significantly increase their trust [6]. 6 However, other studies show that the number of information items or tasks users re- ceive in an automated process should be personalised and up to the point of their de- sire/tolerability. Not enough functions allocated to a user will lead to underload and boredom and thus decreased performance. [17] Too many allocated functions will lead to cognitive, perceptive or motoric overload and increase negative emotions (e.g. stress, anxiety) [18] and user’s error. [17, 3] Meanwhile, users can cope with emo- tions after spending some time with the autonomous technology and developing some routines. Providing an abundance of information and transparency is an important hypothe- sis in interactive technologies. Trust, transparency and acceptance of losing control (i.e. shared authority between the user and system [8]) can improve the interaction of the user by revealing the ambiguous feelings toward the automation. Other psycho- logical factors under study include worries about practical challenges and security of the technology (e.g. hacking a system) and reliability of the process itself (e.g. flat mobile phone battery for systems that rely on applications). 2.4 Humanistic elements of interactive systems Users may lose their trust in decision-making of an automated system when other humans who will not follow the same process are involved and can impact on the outcome (e.g. if fishermen not using a specific application access to more fish than those using that application). An important situation is when responsibilities are shared between users and the system. Ability to identify responsible party related to a bad outcome (i.e. user error versus system failure) can impact the performance of users [9]. Controllable designed interface and environment of work, as well as feeling safe while using new technolo- gies, are among other factors that can increase the performance of the users. Involving users in the design process. The design process should determine the content and format of information to be shared with users in order to create an experi- ence of certainty and trust. Feedback from the users plays a major role for designing such systems. However, the amount and format of the feedback must be well chosen, otherwise it might question the main advantage of automation itself. Research needs to bridge the gap between the micro-perspective of technology specifications and the macro-perspective of how life will and should change through implementation of that technology. Enacting future systems “in the wild”, as a partic- ular form of prototyping, is certainly an important element of this bridge. Motivating the users to engage with the new technologies is still a challenge due to lack of understanding of the end-users’ individual experience and interaction with such technologies. Users can have different roles or backgrounds that can affect their discovery, collaboration and learning of the interactive system[11].Researchers have tried to recruit users for testing their interaction via use of flyers or instructions ex- plaining the technology (a process known as augmentation) [19]. Furthermore, engaging users in designing the automated or augmented product will change their interaction time. The development teams need to familiarise themselves with space and environment of practices, build trust with the employees and improve design ideas. Studies suggest the relations between modes of discovery, design im- provements, interaction and socio-spatial aspects. These relations can be developed 7 more as an analytical and design tool to redefine the borders of opportunities for so- cial interaction in daily automated spaces. 2.5 Summary of the review We believe that there are unmet needs for evaluation and identification of both tech- nical and humanistic factors involved in partly-autonomous systems[7, 3]. Unlike the extensive technical literature on automation, there is a small research base examining the human capabilities involved in work with automated systems[7]. Several factors such as sociological and psychological exchanges, ergonomic, cul- tural relativity, technology availability and acceptance, etc.,have been proposed to be involved in human-machine collaboration in various settings, especially in higher levels of automation. However, the main problem is that there is no comprehensive list of these factors, and no previous study has tried to develop a model based on these factors. Such a model will be helpful to system designers for developing any new interactive highly-automated system. We therefore see HWID framework as a funnel for socio-technical design, automa- tion technologies, and information system (Figure 2). Figure 2. Main scopes of proposed HWID research on user collaboration with au- tomation in complex settings Figure 2. Main scopes of proposed HWID research on user collaboration with au- tomation in complex settings 3 Proposed further research In the final scenario, current shortcomings and future opportunities will be evaluated by using an HWID model for future smart workplaces using Industry 4.0 framework. Given the variety of environments and different levels of automation, we will poten- tially achieve different lists of factors that affect the performance of both operators and systems. This will help us to update the list for different environments. In the final scenario, current shortcomings and future opportunities will be evaluated by using an HWID model for future smart workplaces using Industry 4.0 framework. 3 Proposed further research For investigating independent and entangled factors related to human and machine collaborations in automated systems, we propose a practical approach for evaluating both end-users’ and employees' (or operators’) perspectives in an automatous envi- ronment. First step (current stage) in this research is to produce a list of relevant factors from different sources including: review of the relevant literature, contact and interview with experts in this domain, and observation of some smart workplaces. This compre- hensive list will then be evaluated and optimised in two scenarios (scenario 1, Univer- 8 8 sity of West London Library, and scenario 2, Indian Research Platform). These sce- narios were selected carefully based on potentially important factors including socio- behavioural (e.g., work pattern), psychological (e.g., trust in system), demographical (e.g., wealth and ethnicity), and geographical characteristics of their user populations. We will analyse previously available (via literature review and expert opinions) and newly-gathered data (via questionnaires and interviews) to produce a model to be validated onscenario 3 settings (i.e.,London based airport). By several iterations in this highly automated environment we will refine and provide the final output of the study, which will be a tool/guideline for designing HWID models for various interac- tive technologies. Figure 3 below depicts the proposed process of research in this study. Figure 3. Research procedure Given the variety of environments and different levels of automation, we will poten- tially achieve different lists of factors that affect the performance of both operators and systems. This will help us to update the list for different environments. In the final scenario, current shortcomings and future opportunities will be evaluated by using an HWID model for future smart workplaces using Industry 4.0 framework. Literature Review& ex- pertopinio n Research Plat- form– Applica- tion data & interview find- ings Refined list of Sociotech- nical factors AirportKi- osks’ data - Interview findings Proposed HWID model UWLLS Kiosks’ data- interview Findings Figure 3. Research procedure Literature Review& ex- pertopinio n Research Plat- form– Applica- tion data & interview find- ings Refined list of Sociotech- nical factors AirportKi- osks’ data - Interview findings Proposed HWID model UWLLS Kiosks’ data- interview Findings Figure 3. Research procedure Figure 3. Research procedure Given the variety of environments and different levels of automation, we will poten- tially achieve different lists of factors that affect the performance of both operators and systems. This will help us to update the list for different environments. Conclusion In summary, the overall objective of this paper was to present a review of the possible theoretical background for a to-be-developed sociotechnical HCI framework, includ- ing customized value propositions for the work domain of choice, and, finally, to present three scenarios to be considered in future research. One of the outcomes that the current stage is a comprehensive list category in main principle and number of sub-principles of the factors impact the machine and human counterpart collaboration from sociotechnical perspective. This is what we hoped to illustrate with this paper as start of a series of papers in different scenarios with various automation level. 9 9 References 1. Barricelli, B.R., Roto, V., Clemmensen, T., Campos, P., Lopes, A., Goncalves, F., Ab- delnour-Nocera, J. (2018) Human Work Interaction Design 2018. Designing Engaging Au- tomation. IFIP AICT 554, pp.215-228.http://doi.org/10.1007/978-3-030-05297-3_15 2. Baxter, G. and Sommerville, I. (2011) 'Socio-technical systems: From design methods to systems engineering', Interacting with Computers, 23(1), pp.4-17. https://doi: 10.1016/j.intcom.2010.07.003 3. Barricelli, B.R., Roto, V., Clemmensen, T., Campos, P., Lopes, A., Goncalves, F., Ab- delnour-Nocera, J. (2018) Human Work Interaction Design 2018. Designing Engaging Au- tomation. IFIP AICT 554, pp.36-56. http://doi.org/10.1007/978-3-030-05297-3_3 4. Abdelnour-Nocera J, Oussena S, Burns C (2015). Human Work Interaction Design of the Smart University. 4th IFIP 13.6 Working Conference on Human Work Interaction Design, AICT-468: pp.127-140 5. Clemmensen T (2011) A Human Work Interaction Design (HWID) Case Study in E- Government and Public Information Systems. International Journal of Public Information Systems 3: pp.105-113 6. Dikmen, M., Burns, C.(2017) Trust in Autonomous Vehicles. 2017 IEEE International Conference on Systems, Man, and Cybernetics: pp.1093–1098. https://doi.org/10.1109/SMC.2017.8122757 7. Parasuraman, R., Sheridan, T.,Wickens, C. (2000) A model for types and level of human interaction with automation. IEEE Transactions on Systems, Man, and Cybernetics - Part A:System and Humans, 30(3), pp.286-297 8. Hermann, M., Pentek, T. and Otto, B. (2016) Design Principles for Industrie 4.0 Scenarios. IEEE, pp. 3928-3937. http://doi.10.1109/HICSS.2016.48 9. World Economic Forum. (2018) The fourth Industrial Revolution, https://www.weforum.org/pages/the-fourth-industrial-revolution-by-klaus-schwab, last ac- cessed 22nd August, 2018 10. Davies, R. (2015) Industry 4.0: Digitalisation for productivity and growth. European Par- liamentary Research Service 11. Lesser, E., Prusak, L. (1999) Communities of Practice, Social Capital and Organizational Knowledge. Information Systems Review 1, pp.3–10. [Online] Available from:http://providersedge.com/docs/km_articles/Cop_-_Social_Capital_-_Org_K.pdf, last accessed 25th Feb, 2019 12. International Airport reviews, [Online] Available from: https://www.internationalairportreview.com/article/25929/personalisation-smart-airport/, last accessed 18th Feb, 2019 13. Lindley, J., Sharma, D., and Potts, R. (2014) Anticipatory Ethnography: Design Fiction as an Input to Design Ethnography. Ethnographic Praxis in Industry Conference Proceedings 2014, pp.237–253. https://doi.org/10.1111/1559-8918.01030 14. Otto, T., Smith, RC., Gunn, W.(2013) Design Anthropology: A Distinct Style of Knowing. Bloomsbury, London, New York, pp.1–29. 15. Awais, M., Henrich, D.(2013) “Human- Robot Interaction in an Unknown Human Inten- tion Scenario”, 11th International Conference on Frontiers of Information Technology, 2013, pp. 89-94.https://doi.org/10.1109/FIT.2013.24 16. Kiesler, S. Hinds, P (2004) “Human-Robot Interaction”, Human-Computer Interaction, Volume 19, 2004 17. Yerkes, RM., Dodson, J.D. (1908) The relation of strength of stimulus to rapidly of habit- formation. Journal of Comparative Neurology and psychology, 18(5), pp.459-482. 10 18. Kirk, A. Brown,D.F. g ( ) pp 19. Wouters, N., Downs, J., Harrop, M., Cox, T., Oliveira, E., Webber, S., Vetere, F., Vande- moere, A. (2016). Uncovering the Honeypot Effect: How Audiences Engage with Public Interactive Systems. Proceedings of the 2016 ACM Conference on Designing Interactive Systems, ACM, pp.5–16. http://doi.org/10.1145/2901790.2901796 References (2003) Employee assistance programs: A review of the management of stress and wellbeing through workplace counselling and consulting, Australian Psy- chologist 38(2), pp.138–143. g ( ) pp 19. Wouters, N., Downs, J., Harrop, M., Cox, T., Oliveira, E., Webber, S., Vetere, F., Vande- moere, A. (2016). Uncovering the Honeypot Effect: How Audiences Engage with Public Interactive Systems. Proceedings of the 2016 ACM Conference on Designing Interactive Systems, ACM, pp.5–16. http://doi.org/10.1145/2901790.2901796
https://openalex.org/W3119473783
https://www.zora.uzh.ch/id/eprint/195855/1/2021_Canepa_Stem_Cell_Res_Ther_Identification_of_ALP%2B_CD73%2B.pdf
English
null
Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry
Stem cell research & therapy
2,021
cc-by
13,422
Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2021 Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry RESEARCH Open Access Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry Canepa, Daisy D ; Casanova, Elisa A ; Arvaniti, Eirini ; Tosevski, Vinko ; Märsmann, Sonja ; Eggerschwiler, Benjamin ; Halvachizadeh, Sascha ; Buschmann, Johanna ; Barth, André A ; Plock, Jan A ; Claassen, Manfred ; Pape, Hans-Christoph ; Cinelli, Paolo DOI: https://doi.org/10.1186/s13287-020-02044-4 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-195855 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International ( Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-195855 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Canepa, Daisy D; Casanova, Elisa A; Arvaniti, Eirini; Tosevski, Vinko; Märsmann, Sonja; Eggerschwiler, Ben- jamin; Halvachizadeh, Sascha; Buschmann, Johanna; Barth, André A; Plock, Jan A; Claassen, Manfred; Pape, Hans-Christoph; Cinelli, Paolo (2021). Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry. Stem Cell Research Therapy, 12:7.t DOI: https://doi.org/10.1186/s13287-020-02044-4 (2021) 12:7 Canepa et al. Stem Cell Research & Therapy https://doi.org/10.1186/s13287-020-02044-4 Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry Daisy D. Canepa1,2†, Elisa A. Casanova1†, Eirini Arvaniti3, Vinko Tosevski4, Sonja Märsmann1, Benjamin Eggerschwiler1,2, Sascha Halvachizadeh1, Johanna Buschmann5, André A. Barth5, Jan A. Plock5, Manfred Claassen3, Hans-Christoph Pape1 and Paolo Cinelli1* Manfred Claassen3, Hans-Christoph Pape1 and Paolo Cinelli1* Abstract Background: The impressive progress in the field of stem cell research in the past decades has provided the ground for the development of cell-based therapy. Mesenchymal stromal cells obtained from adipose tissue (AD- MSCs) represent a viable source for the development of cell-based therapies. However, the heterogeneity and variable differentiation ability of AD-MSCs depend on the cellular composition and represent a strong limitation for their use in therapeutic applications. In order to fully understand the cellular composition of MSC preparations, it would be essential to analyze AD-MSCs at single-cell level. Method: Recent advances in single-cell technologies have opened the way for high-dimensional, high-throughput, and high-resolution measurements of biological systems. We made use of the cytometry by time-of-flight (CyTOF) technology to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. Subcellular composition of the AD-MSCs was investigated in their naïve state as well as during osteogenic commitment, via unsupervised dimensionality reduction as well as supervised representation learning approaches. Result: This study showed a high heterogeneity and variability in the subcellular composition of AD-MSCs upon isolation and prolonged culture. Algorithm-guided identification of emerging subpopulations during osteogenic differentiation of AD-MSCs allowed the identification of an ALP+/CD73+ subpopulation of cells with enhanced osteogenic differentiation potential. We could demonstrate in vitro that the sorted ALP+/CD73+ subpopulation exhibited enhanced osteogenic potential and is moreover fundamental for osteogenic lineage commitment. We finally showed that this subpopulation was present in freshly isolated human adipose-derived stromal vascular fractions (SVFs) and that could ultimately be used for cell therapies. (Continued on next page) * Correspondence: paolo.cinelli@usz.ch †Daisy D. Canepa and Elisa A. Casanova contributed equally to this work. 1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland Full list of author information is available at the end of the article * Correspondence: paolo.cinelli@usz.ch †Daisy D. Canepa and Elisa A. Casanova contributed equally to this work. 1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland Full list of author information is available at the end of the article * Correspondence: paolo.cinelli@usz.ch †Daisy D. Canepa and Elisa A. Casanova contributed equally to this work. 1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland Full list of author information is available at the end of the article Introduction surface markers that could be used to purify cells from tissues to homogeneity. Surgical interventions for bone repair are required for numerous reasons, such as trauma-resulting non-union fractures, or diseases including osteoporosis and osteo- necrosis. Currently, autologous bone grafting is the most commonly used approach, but has a number of short- comings such as the limited amount of harvested spon- giosa and donor site pain [1]. Alternative approaches, including the use of synthetic bone substitutes, are not optimal because they lack the osteoinductive properties which are extremely important for healing large bone defects [2]. Cell therapies based on ex vivo expanded mesenchymal stromal stem cells (MSCs) in combination with appropriate scaffolds may be valuable alternatives to autologous bone grafting [3]. MSCs hold the ability to differentiate into osteoblasts and are available from a wide variety of tissue sources [4]. In particular, human fat tissue has been demonstrated to be a valuable source of MSCs—the so-called adipose-derived stromal cells (AD-MSCs) [3]. An additional advantage of using fat tis- sue is the relatively simple isolation procedure compared to autologous bone isolation [5]. We and others have shown that the combination of AD-MSCs in association with synthetic calcium phosphate bone substitutes may be a good alternative to autologous bone grafting [6–10]. Nevertheless, there are drawbacks linked to the use of MSCs for clinical therapy in humans. In contrast to other stem cell types (e.g., embryonic stem cells), the mechanisms that regulate self-renewal and lineage speci- fication in MSCs are largely unexplored. In particular, MSC heterogeneity exists among donors, tissue sources, and within cell populations [11–14]. The knowledge re- garding how different functional and differentiation attributes of MSCs are specified at the population level is insufficient. This poses significant obstacles in efforts to develop clinical manufacturing protocols that reprodu- cibly generate functionally equivalent MSC populations [15, 16]. Currently, MSCs are defined by cell surface phe- notypes, as well as their functional ability to differentiate into multiple cell lineages including osteoclast, chondro- cyte, adipocyte, or skeletal myocyte lineages [17–19]. With respect to the clinical application of MSCs, much effort has been directed toward the identification of unique cell In 2006, the International Society for Cell Therapy (ISCT) published the minimal criteria for defining MSCs [20]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Page 2 of 16 (Continued from previous page) (Continued from previous page) Conclusion: The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/ CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose tissue for therapeutic applications. Keywords: Adipose-derived mesenchymal stromal cells, Stromal vascular fraction, Osteogenic potential, CyTOF, Multidimensional analysis, Cell subpopulation (Continued from previous page) Conclusion: The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/ CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose tissue for therapeutic applications. Keywords: Adipose-derived mesenchymal stromal cells, Stromal vascular fraction, Osteogenic potential, CyTOF, Multidimensional analysis, Cell subpopulation Classification of osteogenic differentiation ability of 17 human AD-MSCs We have isolated 17 AD-MSCs from the stromal vascular fraction (SVF) of human fat tissue following standard pro- tocols [34]. We further assessed the trilineage potential of the established cell lines by inducing differentiation toward osteogenic, chondrogenic, and adipogenic fate. Expression of lineage-specific markers during the differentiation process was monitored by RTQ-PCR (data not shown) and by classical staining assays (Alizarin Red, Alcian Blue, and Oil Red: Figs. 1 and S1A) at days 14, 17, and 21. Staining in- tensity was quantified using a highly standardized, auto- mated digital image quantification approach [35]. This approach takes into consideration not only the amount of deposited dye in the whole cell culture dish but also the time needed for differentiation [35]. Shortly, for each cell line, the calculated pixels for each differentiation day (days 14, 17, and 21) were summed up to obtain one single value per line (Figs. 1a, b and S1A). Next, the lines were catego- rized into “good,” “intermediate,” and “bad” differentiating cells based on the interquartile range distribution. We cate- gorized lines in the 1st quartile as “bad,” lines in the 2nd and 3rd quartile as “intermediate,” and lines in the 4th quartile as “good” (Figs. 1a, b and S1A). Cells from different donors clearly showed variable differentiation abilities (Figs. 1b, c and S1A). For example, in “good” osteogenic dif- ferentiating lines, calcium deposition was already detected at day 14 whereas “bad” lines did not show differentiation at day 21 but needed in average at least 30 days to fully dif- ferentiate (Fig. 1c). The “intermediate” AD-MSC lines showed Alizarin Red staining around day 17 and classified therefore between the “good” and the “bad” lines (Fig. 1c). A similar trend was also observed for chondrogenic and adipogenic differentiation (Figure S1A). Of interest, “good” lines for one lineage were not necessarily “good” for the other two lineages and the same was true for “bad” lines (Figure S1B). These data suggest either an impairment of the cells to differentiate or the existence of different sub- populations with varying differentiation potential. p ( g ) Visualization of the distribution of the 31 markers in the AD-MSC lines with the dimensionality reduction method Uniform Manifold Approximation and Projec- tion (UMAP) [32] highlighted the intra- and inter-donor heterogeneity (Figs. 2b, c and S2A). Classification of osteogenic differentiation ability of 17 human AD-MSCs Interestingly, all 17 AD-MSC lines formed one compact cloud showing high degree of similarity among cells not only within the cell lines but also among donors (Figs. 2b, c and S2A). Des- pite the high degree of similarity among cells, the ex- pression profiles of the investigated markers were not homogeneously distributed over the cloud but showed a gradient-like distribution all over the 17 AD-MSC lines. Interestingly, this was also the case for the widely ac- cepted MSC markers CD73, CD105, and CD90 [20]. The expression of these key markers mostly co-localized in the same region of the cloud and was overlapping with the expression of other markers described in the litera- ture to be critical for MSCs, such as EGFRα and PDGF Rα (Figs. 2b and S2A). In agreement with the minimal criteria definition [20], the negative markers were indeed not expressed in the AD-MSC lines (Figs. 2b and S2A). Other markers such as CD146, NG2, CD271, and STRO-1 were expressed only by a relatively low number of cells and were heterogeneously distributed over the cloud (Figure S2A). We next generated UMAPs for each individual AD-MSC donor for all 31 markers. Although very small, each marker showed inter-donor variation re- garding not only the amount of positive cells but also the expression intensity of the markers (Fig. 2c). These data clearly highlight in an unprecedented, multiparametric, and multidimensional way the heterogeneous composition of AD-MSC from several donors at single-cell level, sug- gesting the presence of specific subpopulations. Introduction These criteria comprise, besides plastic adherence and trilineage differentiation potential (osteogenic, chon- drogenic, and adipogenic), the expression of CD105, CD73, and CD90, coincident with the lack of the hematopoietic markers CD45, CD34, CD14, CD19b, CD79a, and HLA-DR [20]. Additional markers have been identified over the years and are widely accepted for characterizing MSCs [21–31]. Even though all these markers were identified through functional experiments, in the sense that they correlate with the trilineage potential of the cells, it is not clear how their distribution and expression correlate with the observed dif- ferentiation capacity. Furthermore, questions remain open regarding whether MSCs express any unique surface epi- topes, and more importantly, it is unknown whether the epitopes described to date have value in predicting MSC function. In recent years, it was attempted to identify subpopu- lations of MSCs that show enhanced bone regenerative capability. Of note, most of these studies used a limited number of markers alone or in combinations, thus making comparison and reproducibility of the data difficult. It would be therefore essential to be able to analyze the ex- pression of the identified markers in toto and at single-cell level in order to fully understand which subpopulations are undergoing osteogenic lineage commitment. Recent advances in single-cell technologies have allowed multidimensional, high-throughput, and high-resolution measurements of biological systems. In this study, we ap- plied cytometry by time-of-flight (CyTOF) to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. The goal of this study was to investigate the subcellular composition of AD-MSCs in their naïve state as well as during osteogenic commitment via unsupervised dimensionality reduction [32], as well as by supervised representation learning approaches [33]. The data obtained reveal for the first time, in an unbiased way and at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight the presence of subpopulations of cells with osteogenic lineage commitment properties. Page 3 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Page 3 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy This information is of paramount importance considering the emerging need of MSCs for biomedical applications. This information is of paramount importance considering the emerging need of MSCs for biomedical applications. Introduction unravel cellular heterogeneity in the context of cancer, immune diseases, or cellular differentiation [38–40] as well as for identifying subcellular markers for diseases [41]. However, this technology was never employed to characterize human AD-MSCs. We coupled the high di- mensionality of mass cytometry with advanced cellular barcoding to simultaneously investigate 31 markers in 17 primary human AD-MSC lines to dissect at single- cell level their cellular composition (Fig. 2a). Single-cell, multidimensional analyses reveal high cellular heterogeneity in AD-MSCs We further wondered whether the variable differenti- ation ability of the “good” and “bad” AD-MSCs is due to the presence of specific subpopulations. Since it was pre- viously shown that lineage specification occurs during the first 4 days of differentiation [42], we cultured all 17 AD-MSC lines under osteogenic condition and investi- gated at the single-cell level with CyTOF the population dynamics. Shortly, at five different time points, cells for In order to dissect the differences between the AD- MSCs obtained from different donors, we firstly per- formed single-cell analyses with mass cytometry at their naïve/undifferentiated state. CyTOF allows the simultan- eous analysis at single-cell level of up to 50 different parameters using antibodies conjugated with metal iso- topes [36, 37]. This technique combines flow cytometry and mass spectrometry and has already been used to Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Page 4 of 16 (2021) 12:7 A A Interquartile Osteogenic Distribution Osteogenic Quantification «Good» «Interm.» «Bad» F18 F28 F04 F22 F14 F11 F16 F17 F05 F29 F27 F19 F15 F10 F30 F32 F31 0 1×104 2×104 3×104 4×104 5×104 2×106 4×106 6×106 8×106 1×107 5×107 1×108 1.5×108 2×108 d14 d17 d21 Alizarin Red [a.u.] Alizarin Red [a.u.] 0 5×103 1×104 1.5×104 2×104 1×107 2×107 3×107 4×107 5×107 1×108 1.5×108 2×108 2.5×108 3×108 B C Fig. 1 Classification of in vitro osteogenic differentiation potential of 17 AD-MSC lines. a Strategy used for the quantification of differentiation and AD-MSC classification: (1) Cells were differentiated in vitro into osteogenic lineage, and at three time points (days 14, 17, and 21), they were stained with Alizarin Red staining. (2) For each cell line, images of the whole well were acquired and pixels were counted and summed for the three time points (days 14, 17, and 21). (3) Interquartile distribution was applied, and it was decided that the 4th quartile was representing “good,” the 3rd and the 2nd quartile represented the “intermediate,” and the 1st quartile represented the “bad” differentiating lines. b Sum of the pixels acquired at the three time points (days 14, 17, and 21) for osteogenic differentiation of all 17 AD-MSC lines and interquartile categorization into “good,” “intermediate,” and “bad” AD-MSCs. c In vitro differentiation of one representing “good,” one “intermediate,” and one “bad” AD-MSC after 14, 17, and 21 days under osteogenic condition assessed by Alizarin Red staining. Single-cell, multidimensional analyses reveal high cellular heterogeneity in AD-MSCs Depicted are triplicates of undifferentiated cells (control) and cells cultured under differentiation conditions C Osteogenic Quantification «Good» «Interm.» «Bad» F18 F28 F04 F22 F14 F11 F16 F17 F05 F29 F27 F19 F15 F10 F30 F32 F31 0 1×104 2×104 3×104 4×104 5×104 2×106 4×106 6×106 8×106 1×107 5×107 1×108 1.5×108 2×108 d14 d17 d21 Alizarin Red [a.u.] B C C B Osteogenic Quantification Interquartile Osteogenic Distribution Alizarin Red [a.u.] 0 5×103 1×104 1.5×104 2×104 1×107 2×107 3×107 4×107 5×107 1×108 1.5×108 2×108 2.5×108 3×108 Interquartile Osteogenic Distribution Interquartile Osteogenic Distribution Fig. 1 Classification of in vitro osteogenic differentiation potential of 17 AD-MSC lines. a Strategy used for the quantification of differentiation and AD-MSC classification: (1) Cells were differentiated in vitro into osteogenic lineage, and at three time points (days 14, 17, and 21), they were stained with Alizarin Red staining. (2) For each cell line, images of the whole well were acquired and pixels were counted and summed for the three time points (days 14, 17, and 21). (3) Interquartile distribution was applied, and it was decided that the 4th quartile was representing “good,” the 3rd and the 2nd quartile represented the “intermediate,” and the 1st quartile represented the “bad” differentiating lines. b Sum of the pixels acquired at the three time points (days 14, 17, and 21) for osteogenic differentiation of all 17 AD-MSC lines and interquartile categorization into “good,” “intermediate,” and “bad” AD-MSCs. c In vitro differentiation of one representing “good,” one “intermediate,” and one “bad” AD-MSC after 14, 17, and 21 days under osteogenic condition assessed by Alizarin Red staining. Depicted are triplicates of undifferentiated cells (control) and cells cultured under differentiation conditions each of the 17 AD-MSC lines were collected (day 0: un- differentiated cells, day 1–4: differentiation) and stra- tegically barcoded (Table S2). At day 4, all samples were simultaneously stained and processed for CyTOF acqui- sition (Fig. 3a and Table S1). Cell density plots on the UMAPs of the 17 AD-MSC lines during the initial 4 days of differentiation highlighted an emerging subpopulation, which was very small at day 0 and increased over the differentiation period (Fig. 3b). This subpopulation was clearly visible already at day 0 in Page 5 of 16 Canepa et al. Single-cell, multidimensional analyses reveal high cellular heterogeneity in AD-MSCs Stem Cell Research & Therapy (2 (2021) 12:7 A Human fat tissue isolation Isolation of AD-MSCs Ab Panel development Identification of osteogenic subpopulations tSNE1 tSNE2 31 Ab staining CyTOF BC-strategy C B Fig. 2 Mass cytometry analyses of human AD-MSCs reveal high heterogeneity. a Scheme of mass cytometry analysis on 17 human AD-MSCs from AD-MSC collection to the identification of osteogenic subpopulations. b UMAPs of selected markers in all 17 analyzed AD-MSC lines. c UMAPs of three selected markers (CD73, CD105, PDGFR) in 4 AD-MSC donors. Each dot represents one cell. Blue denotes minimal, green intermediate, and red high expression A Human fat tissue isolation Isolation of AD-MSCs Ab Panel development Identification of osteogenic subpopulations tSNE1 tSNE2 31 Ab staining CyTOF BC-strategy A Human fat tissue isolation BC-strategy Identification of osteogenic subpopulations tSNE1 tSNE2 31 Ab staining CyTOF Identification of osteogenic subpopulations 31 Ab staining C B B C Fig. 2 Mass cytometry analyses of human AD-MSCs reveal high heterogeneity. a Scheme of mass cytometry analysis on 17 human AD-MSCs from AD-MSC collection to the identification of osteogenic subpopulations. b UMAPs of selected markers in all 17 analyzed AD-MSC lines. c UMAPs of three selected markers (CD73, CD105, PDGFR) in 4 AD-MSC donors. Each dot represents one cell. Blue denotes minimal, green intermediate, and red high expression absent in the “bad” cell lines and was confirmed and vali- dated on all later days of the differentiation process (Fig. 3d). Analysis of the percentages of cells positive for ALP and CD73 in each category always confirmed signifi- cant high frequency of ALP+ cells in the “good” lines, moderate frequency in the “intermediate” lines, and very low frequency of ALP+ cells in the “bad” lines over the four osteogenic days (Figure S3A). The percentage of CD73-positive cells was constant during the 4 differenti- ation days in the three categories, but significantly in- creased in the bad lines at day 2 and day 4 compared to “good” lines (Figure S3A). the “good” lines whereas in the “bad” lines it was barely present even at day 4 (Fig. 3b). To further investigate whether it was possible to discriminate the differentiation potential of AD-MSC lines at their undifferentiated state, we applied the CellCNN algorithm [33] to the mass cy- tometry data obtained at day 0 (undifferentiated state). Single-cell, multidimensional analyses reveal high cellular heterogeneity in AD-MSCs Presented with the task of comparing “good” versus “bad” cell lines, CellCNN detected a subpopulation character- ized by high alkaline phosphatase (ALP+) expression and low expression of the MSC marker CD73 (CD73low) (Fig. 3c). This subpopulation was highly frequent in “good, ” moderately present in the “intermediate,” and almost Page 6 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy Population Frequency (%) Day 0 (Training) Day 1 (Validation) Day 2 (Validation) Day 3 (Validation) Day 4 (Validation) Bad Interm Good Bad Interm Good Bad Interm Good Bad Interm Good Bad Interm Good A C B All Cells Selected Population D Fig. 3 Identification of AD-MSC osteogenic subpopulation. a Sample collection and CyTOF approach scheme during 4 days of osteogenic differentiation (d0 = undifferentiated state, d1–d4 = differentiation). b Cell density plots on the UMAPs of the five analyzed days (d0, d1, d2, d3, d4) during osteogenic differentiation. Once the pool of all 17 AD-MSC lines is represented, once only the “good,” the “intermediate” (interm.), and the “bad” AD-MSC lines. Highlighted is the emerging population during osteogenic differentiation. Bright color indicates lower density, and dark color indicates higher cellular density. c Empirical distribution densities of all analyzed 31 marker abundances for the entire cell population (blue) and the cell subset selected by CellCNN (red). The identified subpopulation is characterized by alkaline phosphatase-positive (ALP+) and CD73low expressing cells. d Boxplots indicating the frequencies of the ALP+/CD73low subpopulation selected by CellCNN in all “good,” “intermediate” (interm.), and “bad” osteogenic differentiating lines during the five analyzed days. Error bars represent the mean of the percentage of positive cells present in “good” (n = 6), “intermediate” (n = 4), and “bad” (n = 7) AD-MSCs A A B B Population Frequency (%) Day 0 (Training) Day 1 (Validation) Day 2 (Validation) Day 3 (Validation) Day 4 (Validation) Bad Interm Good Bad Interm Good Bad Interm Good Bad Interm Good Bad Interm Good C B All Cells Selected Population D Fig. 3 Identification of AD-MSC osteogenic subpopulation. a Sample collection and CyTOF approach scheme during 4 days of ost differentiation (d0 = undifferentiated state, d1–d4 = differentiation). b Cell density plots on the UMAPs of the five analyzed days (d d4) during osteogenic differentiation. Once the pool of all 17 AD-MSC lines is represented, once only the “good,” the “intermediat the “bad” AD-MSC lines. Single-cell, multidimensional analyses reveal high cellular heterogeneity in AD-MSCs Highlighted is the emerging population during osteogenic differentiation. Bright color indicates lower de color indicates higher cellular density. c Empirical distribution densities of all analyzed 31 marker abundances for the entire cell po and the cell subset selected by CellCNN (red). The identified subpopulation is characterized by alkaline phosphatase-positive (ALP+ expressing cells. d Boxplots indicating the frequencies of the ALP+/CD73low subpopulation selected by CellCNN in all “good,” “int (interm.), and “bad” osteogenic differentiating lines during the five analyzed days. Error bars represent the mean of the percentage cells present in “good” (n = 6), “intermediate” (n = 4), and “bad” (n = 7) AD-MSCs Population Frequency (%) Day 0 (Training) Day 1 (Validation) Day 2 (Validation) Day 3 (Validation) Day 4 (Validation) Bad Interm Good Bad Interm Good Bad Interm Good Bad Interm Good Bad Interm Good C C Day 3 (Validation) Day 4 (Validation) All Cells Selected Population D D Fig. 3 Identification of AD-MSC osteogenic subpopulation. a Sample collection and CyTOF approach scheme during 4 days of osteogenic differentiation (d0 = undifferentiated state, d1–d4 = differentiation). b Cell density plots on the UMAPs of the five analyzed days (d0, d1, d2, d3, d4) during osteogenic differentiation. Once the pool of all 17 AD-MSC lines is represented, once only the “good,” the “intermediate” (interm.), and the “bad” AD-MSC lines. Highlighted is the emerging population during osteogenic differentiation. Bright color indicates lower density, and dark color indicates higher cellular density. c Empirical distribution densities of all analyzed 31 marker abundances for the entire cell population (blue) and the cell subset selected by CellCNN (red). The identified subpopulation is characterized by alkaline phosphatase-positive (ALP+) and CD73low expressing cells. d Boxplots indicating the frequencies of the ALP+/CD73low subpopulation selected by CellCNN in all “good,” “intermediate” (interm.), and “bad” osteogenic differentiating lines during the five analyzed days. Error bars represent the mean of the percentage of positive cells present in “good” (n = 6), “intermediate” (n = 4), and “bad” (n = 7) AD-MSCs Page 7 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 We further investigated the correlation between ALP+ frequency (measured by CyTOF during the 5 days) and the ability to differentiate into osteocytes (based on the quantification of the staining at days 14, 17, and 21). ALP+/CD73+ cells are present in the SVF of human fat tissue To ultimately prove the clinical utility of the identified ALP and CD73 marker combination, we investigated whether ALP+/CD73+ cells were also present in freshly isolated hu- man adipose stromal vascular fractions (SVFs), and if, upon isolation, they displayed similar properties as the ALP+/ CD73+ cells present in AD-MSC lines. For this purpose, hu- man adipose tissues were collected from 3 healthy donors, and the presence of ALP+/CD73+ cells was investigated. Immunohistochemical staining revealed the presence of ALP+/CD73+ located in fat tissue capillaries (Figs. 5a and S5A). SVFs from the same donors were further processed by FACS sorting, and the fractions (control: unstained cells sorted through FACS; CD45−/ALP+/CD73+, CD45−/ALP −/CD73low, CD45−/ALP−/CD73high) were plated for osteogenic differentiation (Figs. 5b and S5B). Quantification of osteogenic differentiation at d14, d17, and d21 confirmed higher osteogenic differentiation in the ALP+/CD73+ sorted cells compared to the other ones (Figs. 5b and S5C). ALP+CD73+ marker combination can be used for monitoring the osteogenic potential of undifferentiated AD-MSC populations after expansion in vitro p p p A major problem during in vitro expansion of MSCs (and also AD-MSCs) is that they show signs of aging and changes in the subcellular composition, which fi- nally lead to a decrease of the differentiation potential over the passages [14]. To follow the dynamic of the cell composition over prolonged cell culture, we analyzed with our CyTOF antibody panel 3 “good” and 1 “inter- mediate” AD-MSC lines (F28, F14, F22, and F05) from passage 3 (p3) to passage 20 (p20) (Table S4). We could confirm that the median intensity of expression of CD73 was increasing whereas ALP was rapidly diminishing after prolonged culture, mirroring the situation observed in all “bad” lines at p10 (Figs. 4e and S4E). ALP+/CD73+ cells possess enhanced osteogenic differentiation ability In order to further characterize the identified subpopula- tion, we selected four AD-MSC lines (F28, F14, F04, and F22) and sorted three distinct cell subpopulations by FACS: ALP+/CD73+, ALP−/CD73low, and ALP −/CD73high. Although CellCNN analysis on CyTOF data revealed the presence of an ALP+/CD73low popula- tion, this phenotype was not clearly definable by FACS sorting. We could select ALP−/CD73high and ALP −/CD73low, but it was not possible to unambiguously distinguish between ALP+/CD73low and ALP+/ CD73high cells. For this reason, we selected the double positive ALP+ and CD73+ (ALP+/CD73+) population for further experiments (Figure S4A). As a control, we used for each AD-MSC line unstained cells processed through the FACS. After sorting, the different subpopu- lations were directly plated for differentiation into the three lineages followed by lineage-specific staining at days 14, 17, and 21 and quantification according to Eggerschwiler et al. [35]. The sorted ALP+/CD73+ frac- tion showed enhanced osteogenic differentiation when compared to the other sorted populations (Figs. 4a, b and S4B). We next differentiated the cells at p5, p9, and p20, and we observed a decrease in the differentiation capacity over the passages and these changes correlate with the expression of ALP and CD73 (Figs. 4e, f and S4F). In conclusion, we confirmed that ALP+/CD73+ expressing cells possess higher osteogenic differentiation potential and the marker combination of ALP and CD73 can be used to predict the osteogenic differentiation potential of cultured AD-MSCs. Single-cell, multidimensional analyses reveal high cellular heterogeneity in AD-MSCs As expected ALP+ always correlated with the osteogenic differentiation ability (Figure S3B) confirming once more that ALP+ expression correlates with osteogenic lineage commitment. In conclusion, our approach allowed the identification of an osteogenic subpopulation character- ized by the markers ALP+/CD73low that hallmarked ex- clusively the “good” differentiating lines. interquartile categorization of these lines confirmed two predicted “good” lines, one predicted “intermediate” line, and two predicted “bad” lines (Figs. 4c, d and S4C-D). Thus, we could confirm that the presence of the marker combination ALP+/CD73+ is sufficient to predict the osteogenic differentiation ability of a donor AD-MSC line in its undifferentiated state. ALP+CD73+ marker combination is predictive for osteogenic potential in undifferentiated AD-MSC populations We next assessed whether the marker ALP+ (which is also CD73+, see Figure S4A) could be used as a predictor for osteogenic differentiation potential of AD-MSCs in their undifferentiated state. For this purpose, we selected five new AD-MSC lines, which had never been characterized or used in previous experiments. Undifferentiated cells from the new lines, together with 9 already characterized lines (as reference cells), were subjected to CyTOF (Table S3). Quantification of the presence of ALP+ cells in the new lines allowed a predicted categorization based on the 9 AD-MSCs, into “good,” “intermediate,” and “bad” lines (Fig. 4c). We further compared the outcome from the CyTOF data with the differentiation ability observed in vitro (Fig. 4d). Alizarin Red quantification and the Page 8 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy A B Alizarin Red [a.u.] control sorted ALP+ CD73+ ALP- CD73 low ALP- CD73 high d14 d17 d21 A C B Alizarin Red [a.u.] F22 F14 F04 Alizarin Red [a.u.] control sorted ALP+ CD73+ ALP- CD73 low ALP- CD73 high control sorted ALP+ CD73+ ALP- CD73 low ALP- CD73 high d14 d17 d21 d14 d17 d21 d14 d17 d21 d14 d17 d21 D Alizarin Red [a.u.] ALP frequency (%) d14 d17 d21 0 2×103 4×103 6×103 8×103 1×104 2×105 4×105 6×105 8×105 2×107 4×107 6×107 8× 07 p5 p9 p20 E F 0 5 10 15 <Good> <Bad> <Interm.> Fig. 4 ALP+/CD73+ markers possess higher and predictive osteogenic potential. a Alizarin Red staining and quantification of F28 AD-MSC line sorted subpopulations (ALP+/CD73+, ALP−/CD73low, ALP−/CD73high) after 14, 17, and 21 days of osteogenic differentiation. Controls sorted are unstained cells, which were run through the FACS machine. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells cultured under osteogenic differentiation conditions (differentiation). b Quantification of Alizarin Red staining for F04, F14, and F22 AD-MSC lines for the same sorted subpopulations after 14, 17, and 21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. c Predicted categorization based on alkaline phosphatase (ALP) frequency in five new AD-MSC lines (green) and nine already characterized AD-MSC lines (reference) measured by CyTOF. d Alizarin Red staining at day 21 of the five new AD-MSC lines. ALP+CD73+ marker combination is predictive for osteogenic potential in undifferentiated AD-MSC populations Depicted are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation conditions (differentiation). e Histogram for the median intensity of expression of CD73 and ALP of F22 “good” AD-MSC line from passage 3 (p3) till passage 20 (p20). Black is the lowest intensity, and white represents the highest intensity. f Alizarin Red staining and quantification of F22 at passage p5, p9, and p20 after 14, 17, and 21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within h d d f B F22 F14 F04 Alizarin Red [a.u.] control sorted ALP+ CD73+ ALP- CD73 low ALP- CD73 high d14 d17 d21 d14 d17 d21 d14 d17 d21 Alizarin Red [a.u.] d14 d17 d21 0 2×103 4×103 6×103 8×103 1×104 2×105 4×105 6×105 8×105 2×107 4×107 6×107 8× 07 p5 p9 p20 E F C ALP frequency (%) 0 5 10 15 <Good> <Bad> <Interm.> Alizarin Red [a.u.] d14 d17 d21 0 2×103 4×103 6×103 8×103 1×104 2×105 4×105 6×105 8×105 2×107 4×107 6×107 8× 07 p5 p9 p20 F C F F E D D Fig. 4 ALP+/CD73+ markers possess higher and predictive osteogenic potential. a Alizarin Red staining and quantification of F28 AD-MSC line sorted subpopulations (ALP+/CD73+, ALP−/CD73low, ALP−/CD73high) after 14, 17, and 21 days of osteogenic differentiation. Controls sorted are unstained cells, which were run through the FACS machine. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells cultured under osteogenic differentiation conditions (differentiation). b Quantification of Alizarin Red staining for F04, F14, and F22 AD-MSC lines for the same sorted subpopulations after 14, 17, and 21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. c Predicted categorization based on alkaline phosphatase (ALP) frequency in five new AD-MSC lines (green) and nine already characterized AD-MSC lines (reference) measured by CyTOF. d Alizarin Red staining at day 21 of the five new AD-MSC lines. Depicted are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation conditions (differentiation). e Histogram for the median intensity of expression of CD73 and ALP of F22 “good” AD-MSC line from passage 3 (p3) till passage 20 (p20). ALP+CD73+ marker combination is predictive for osteogenic potential in undifferentiated AD-MSC populations Black is the lowest intensity, and white represents the highest intensity. f Alizarin Red staining and quantification of F22 at passage p5, p9, and p20 after 14, 17, and 21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within the same day: *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001. ns, not significant Page 9 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 A A Th d d h ALP /CD 3 ll Di i A B control sorted ALP+CD73+ ALP-CD73low ALP-CD73high Fig. 5 ALP+/CD73+ cells are present in human fat tissue and possess enhanced osteogenic potential. a Immunofluorescence of ALP and CD73 in human fat tissue. Scale 100 μm. BF, bright field. b Alizarin Red staining and quantification of 3 healthy donors’ SVF after 21 days of osteogenic differentiation. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells cultured under osteogenic differentiation conditions. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within the same day: *p ≤ 0.05, **p ≤0.01, and ***p ≤0.001. ns, not significant B B control sorted ALP+CD73+ ALP-CD73low ALP-CD73high Fig. 5 ALP+/CD73+ cells are present in human fat tissue and possess enhanced osteogenic potential. a Immunofluorescence of ALP and CD73 in human fat tissue. Scale 100 μm. BF, bright field. b Alizarin Red staining and quantification of 3 healthy donors’ SVF after 21 days of osteogenic differentiation. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells cultured under osteogenic differentiation conditions. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within the same day: *p ≤ 0.05, **p ≤0.01, and ***p ≤0.001. ns, not significant Discussion However, variability can also be observed when BM-MSCs were autologously isolated over different periods of time or even when isolated bilaterally from the same donor [15], indicating that the cellular composition of MSCs plays an important role and is highly heterogeneous. A possibility to explain this heterogeneity is the variable composition of the tissues used for the establishment of MSC lines (e.g., amount of blood vessels). Dissecting this heterogeneity at single-cell level and identifying subpopula- tions of cells with specific differentiation attributes are ur- gently needed for developing clinical manufacturing protocols that reproducibly generate functionally equivalent MSC populations. In this study, we have aimed at identifying specific AD-MSC subpopulations of cells with higher osteogenic differentiation potential. The novel approach used herein enabled the simultaneous visualization of 31 selected markers in 17 primary AD-MSC lines, thereby offering unprecedented observational dimensionality in a large sample set. This approach allowed circumvention of the classical bulk assays most frequently used for character- izing MSCs and their differentiation potential, which pool signals across entire cell populations, masking cell- to-cell variation. Unexpectedly, the dimensionality re- duction algorithm UMAP revealed a high degree of cel- lular similarity, as observed from the compact clouds that all AD-MSC lines generated. This is in contrast to hematopoietic cells, for example, where UMAPs clearly separate the different cell subpopulations (Bendall et al. [37]). Nevertheless, despite the high degree of similarity among cells, the distribution of the markers within the clouds was highly heterogeneous, forming in some cases gradients (such as CD73, EGFR, PDGFR, SOX9) or small islands (ALP, CD166, STRO-1) (Figs. 2b and S2A). Fur- thermore, each marker showed inter-donor variation re- garding not only the amount of positive cells but also the expression intensity of the markers (Fig. 2c). In vitro selection after prolonged culture represents a major concern for the use of MSCs for therapeutic ap- plications. Expansion on hard tissue culture surfaces may promote cellular divergence and/or reduction in po- tency [57, 58]. Additionally, the culture conditions used are very permissive when compared with the ones employed by other stem cell types, e.g., embryonic stem cells or induced pluripotent stem cells, where specific factors are necessary to maintain the self-renewal cap- acity of the stem cells [59–61]. Discussion These data demonstrate that ALP+/CD73+ cells are present in freshly isolated human fat tissue and possess enhanced osteogenic potential, representing therefore in- teresting cells for therapeutic applications. Even with the most effective protocols, different MSC prep- arations show strong variation in their differentiation per- formance. One possible explanation for this phenomenon Page 10 of 16 Page 10 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 it is an accepted osteoblast marker. CD73 and ALP are GPI (glycophosphatidylinositol)-anchored ectoenzymes with 5′-nucleotidase activity; thus, they share similar functions. CD73 and ALP regulate the extracellular breakdown of ATP to adenosine [51]. Released ATP serves as an autocrine or paracrine regulator of both osteoblast and osteoclast functions [52, 53], and hydroly- zation of pyrophosphate provides inorganic phosphate to promote mineralization. The extracellular nucleotide ATP can be one of the key mediators in bone metabol- ism, not only as a phosphate source, but also as a signal- ing molecule via P2 receptors. In fact, osteoblasts have been reported to release ATP into the extracellular en- vironment constitutively followed by engagement of P2 receptors [54]. Most importantly, ALP+/CD73+ cells are also present and even more abundant in freshly isolated SVFs. The origin of these cells has to be better charac- terized, but it is reasonable to assume that these cells could be of pericytic origin. ALP is a known pericytic marker which was previously described as a marker for the prospective isolation of pericytes from different tis- sues [55, 56]. This is in agreement with our observation that ALP+/CD73+ cells are localized in the capillaries of fat tissue. In this sense, the difference observed between “good” and “bad” AD-MSC lines could be explained with differences in the amount of blood vessels in the isolated fat tissue. Our data suggest the existence of a balanced regulation of ALP and CD73 in human AD-MSCs, which is crucial for the determination of osteogenic lineage commitment. is the high heterogenic cellular composition of MSCs, con- sisting of different cells harboring diverse lineage commit- ment ability [43]. The high donor-to-donor variability observed when MSCs are derived from the same tissue of origin may be due to different factors including donor health [11, 44], age, MSC availability, and/or self-renewal capacity [45–47]. Discussion Our data indicate that progressive loss of ALP+/CD73low cells during passa- ging precludes osteogenic differentiation and constantly monitoring ALP+/CD73low can be used as a quality control procedure to monitor AD-MSC expansion for bone regeneration purposes. Although ALP and CD73 have never been associated together with osteogenic potential, singularly they were previously correlated with osteogenic differentiation. CD73 was shown to regulate bone formation and re- modeling in intramembranous bone repair [48]. In our study, we demonstrated that CD73 expression levels in- versely correlate with the osteogenic differentiation abil- ity of 17 human AD-MSC primary preparations (Figs. 4e, f and S4E-F). Tissue nonspecific ALP has been found in several tissues and cell types, such as activated B cells or pluripotent embryonic stem cells [49, 50], and In conclusion, our study highlights that single-cell and multiparametric analysis identifies gradient expression and co-localization of markers which have not been pre- viously observed. The combination of ALP+/CD73low markers can not only (1) discriminate between “good” and “bad” differentiating lines but can also be used for (2) prospective isolation of selected cells from SVF for bone tissue engineering and (3) to assess the differenti- ation potential of AD-MSC preparation in culture. Page 11 of 16 Page 11 of 16 Page 11 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Osteogenesis Kit or StemPro® Adipogenesis Kit (Gibco/ Life Technologies). For chondrogenic differentiation, cells were cultured at a density of 5 × 103 cells/cm2 in a Nunc™24-well plate (Thermo Fisher Scientific) and dif- ferentiation was induced at the 4th day of culture using the StemPro® Chondrogenesis Kit (Gibco/Life Technolo- gies). All media were changed every 4 days. Osteogenesis Kit or StemPro® Adipogenesis Kit (Gibco/ Life Technologies). For chondrogenic differentiation, cells were cultured at a density of 5 × 103 cells/cm2 in a Nunc™24-well plate (Thermo Fisher Scientific) and dif- ferentiation was induced at the 4th day of culture using the StemPro® Chondrogenesis Kit (Gibco/Life Technolo- gies). All media were changed every 4 days. The use of MSCs in clinical medicine will likely con- tinue to grow rapidly, yet it still is unclear how clinical manufacturing affects MSC biology, particularly regard- ing lineage specification. The development of assays allowing for the monitoring of the production process and assessment of cellular function are urgently needed. Assessment and classification of trilineage differentiation potential Differentiation assessment via specific staining was per- formed for all three differentiation lineages after 14, 17, and 21 days of differentiation. For Alizarin Red S (Sigma) staining, cells were washed with PBS and fixed with 4% (v/v) formaldehyde (Sigma) for 30 min at RT. Upon washing twice with ddH20, Alizarin Red S solution (0.7 g Alizarin Red S diluted in 50 ml ddH2O at pH = 4.2) was added for 20 min at RT. Afterwards, cells were washed four times with ddH2O, dried, and stored in the dark until image acquisition. For Oil Red O (Sigma) staining, cells were washed once with PBS and fixed with 10% (v/ v) formaldehyde (Roth) for 1 h at RT. Afterwards, cells were washed twice with ddH2O, rinsed twice with 60% (v/v) 2-propanole (Sigma) in ddH20, and dried. Oil Red O working solution (0.15 g Oil Red O in 50 ml 60% (v/v) 2-propanole in ddH2O) was added for 10 min at RT. After four ddH2O washing steps, cells were dried and images were directly taken. For Alcian Blue 8GX (Sigma) staining, cells were washed with PBS and then fixed with 4% (v/v) formaldehyde (Sigma) for 20 min at RT. After- wards, cells were washed twice with ddH2O and incu- bated for 3 min with 3% (v/v) acetic acid (Merck Millipore) in ddH20. Alcian Blue solution (0.1 g Alcian Blue 8GX in 100 ml of 3% acetic acid in ddH20 at pH = 2.5) was given for 1 h at RT. Cells were washed four times with ddH2O, dried, and stored in the dark until image acquisition. Images of the entire wells at days 14, 17, and 21 of differentiation were acquired with Cytation 5 imaging reader (BioTek). Quantification of differenti- ation was performed according to [35], and subsequent classification of AD-MSC into “good,” “bad,” and “inter- mediate” differentiating lines was performed applying the interquartile range distribution. We defined cell lines present in the 4th quartile as “good,” lines present in the 2nd and 3rd as “intermediate,” and lines in the 1st quar- tile as “bad.” Discussion The approach chosen in this work might provide a basis for better understanding how different functional attri- butes of MSCs are specified at the population level, and can be used in the development of clinical manufactur- ing protocols that reproducibly generate functionally equivalent MSC populations. Cells and cell culture Twenty-two human adipose tissue samples (100–600 g) were obtained from lipectomies and liposuctions (healthy donors, no diabetic donors) [62]. AD-MSCs were isolated from fat tissue, with the consent of the do- nors according to Swiss (KEK-ZH: StV 7-2009) and international ethical guidelines (ClinicalTrials.gov; Iden- tifier: NCT01218945) [62]. The extraction procedure was performed according to [34]. AD-MSCs were cul- tured in Dulbecco’s modified Eagle’s medium (DMEM) (PAN Biotech) supplemented with 10% of fetal bovine serum (FBS) (Biowest), 1% of antibiotics (100× penicillin, 100× streptomycin) (Biowest), and 1% L-glutamine 200 mM (Sigma) (called AD-MSC medium). Medium was changed every 3 days, and cells were passaged with 1× Trypsin-EDTA (Life Technologies) for 5 min at 37 °C when cells were about 80% confluent. Cells were incu- bated at 37 °C in an atmosphere with 95% humidity and 5% CO2. Material and methods Ethics statement Adipose-derived stromal cells (AD-MSCs) were obtained from lipectomies and liposuctions (healthy donors, no dia- betic donors) upon written informed consent of the do- nors, following the guidelines approved by the Kantonale Ethik Kommission (KEK) Zurich Swiss (KEK-ZH: StV 7- 2009) and international ethical guidelines (ClinicalTrials. gov; Identifier: NCT01218945). The stromal vascular frac- tion (SVF) isolated from human fat tissue was obtained with the consent of the patient according to Swiss ethics (BASEC-Nr.: 2019-01504). Isolation of the stromal vascular fraction For osteogenic differentiation, AD-MSCs were seeded at a density of 1.6 × 104 cells/cm2 in Nunc™24-well plates (Thermo Fisher Scientific) or at a density of 1 × 104 in 96-well plates (TPP). For adipogenic differentiation, cells were cultured at a density of 1.6 × 104 cells/cm2 in Nunc™24-well plates (Thermo Fisher Scientific). Differ- entiation was started 24 h after seeding with StemPro® Stromal vascular fraction (SVF) was isolated from hu- man fat tissue with the consent of the patient according to Swiss ethics (BASEC-Nr.: 2019-01504) and according to [34]. Briefly, lipectomies were cut in small pieces and extensively washed with PBS. Enzymatic digestion was performed with 0.075% collagenase I (Gibco) at 37 °C for Page 12 of 16 Page 12 of 16 Page 12 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Mass cytometry antibody panel and staining procedures The antibody panel consisted of 31 monoclonal anti- human metal-conjugated antibodies, which included cell surface, cytoplasmic, and transcription targets (Table S1). When possible, already metal-conjugated antibodies were purchased from Fluidigm; otherwise, antibodies were con- jugated in-house with isotopically pure lanthanide metals according to the commercially available MaxPar Antibody Labelling Kit (Fluidigm). Labeled antibodies were stored at 4 °C in antibody stabilizer solution (Candor Bioscience). Titration of each antibody was performed on a one-to-one mix of cells consisting of PBMCs (peripheral blood mono- nuclear cells), HEK (human embryonic kidney cells 293), Hela (cervical cancer cells), Jurkat (human T lymphocyte cells), Saos2 (sarcoma cells), Nalm6 (B cell precursor leukemia cells), SHSY5S (neuroblastoma cells), and hu- man AD-MSCs. These different cell lines, which we called MIX, were chosen in order to have for each marker a positive and a negative control cell type. Sample staining was performed as described in the MaxPar Cell Surface, MaxPar Cytoplasmic/Secreted Antigen, and MaxPar Nu- clear Target protocols (Fluidigm) with minor changes. Briefly, cells were first subjected to cell surface antibody staining, followed by cytoplasm staining, and nuclear staining. For the cytoplasmic and intranuclear staining, cell fixation steps were shortened to 10 min. Cells were then resuspended in 4% paraformaldehyde (Electron Microscopy Sciences) and stored at 4 °C until acqui- sition. Fluorescence activating cell sorting (FACS) g g ( ) AD-MSC lines were washed with PBS and stained with ALP-APC (R&D) (1/50) and CD73-FITC (Biolegend) (1/ 160) for 25 min at 4 °C. Upon washing, the cell fractions (controls sorted, ALP+/CD73+, ALP−/CD73high, ALP −/CD73low) were sorted with a FACS BD Aria III 5L and seeded in Nunc™96-well plates (TPP) at a density of 1.2 × 104 cells/cm2 for osteogenic differentiation. Con- trols sorted were unstained cells processed through the FACS and collected without sorting specific subpopula- tions. Differentiation was induced 24 h after seeding. Freshly isolated SVFs were washed with PBS and stained with ALP-APC (R&D) (1/50), CD73-FITC (Biolegend) (1/160), and CD45-PE (Biolegend) (1/160) for 25 min at 4 °C. SVF fractions (controls sorted, CD45−/ALP+/CD73+, CD45−/ALP−/CD73high, CD45−/ALP−/CD73low) were sorted with FACS BD Aria III 5L and plated in vitro at a density of 1 × 104 in 96-well plates (TPP) for osteogenic dif- ferentiation. All media were changed every 4 days. Immunohistochemistry and immunofluorescence Paraffin-embedded samples of human fat tissue were se- lected for immunohistochemical and immunofluores- cence analysis. Samples were deparaffinized with xylene and rehydrated by an increasing ethanol gradient for hematoxylin and eosin (H&E) staining. Target retrieval was performed using the PT Link (DAKO) at pH solu- tion 9.0 (DAKO). Immunohistochemistry staining was performed using a Dako Autostainer Link 48. Primary antibodies used were as follows: rabbit monoclonal ALP (Abcam, 1/200), mouse monoclonal CD73 (Abcam, 1/ 200), mouse monoclonal CD31 (DAKO, 1/200), and the appropriate EnVision HRP secondary antibody (EnVi- sion HRP rabbit or mouse, DAKO, 1/500) according to the manufacturer’s instruction. Immunofluorescence was performed using a Dako Autostainer Link 48 with the following antibodies: rabbit monoclonal ALP (Abcam, 1/ 200), mouse monoclonal CD73 (Abcam, 1/200), Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, 1/200), and Alexa Fluor 546 goat anti-mouse IgG (Thermo Fisher, 1/200) according to the manufacturer’s instruc- tion. Sections were visualized with LEICA DM6600 with a × 20 magnifying objective lens. Isolation of the stromal vascular fraction In the day of CyTOF acquisition, cells were washed with MaxPar Fix and Perm Buffer (Flui- digm) containing Cell-ID Intercalator-IR (Fluidigm) and incubated at RT for 1 h. Cells were washed with ddH2O and then diluted in ddH2O with 10% EQ Calibration Beads (Fluidigm) at 1 million cells/ ml before acquisition with CyTOF 2 mass cyt- ometer (Fluidigm). 45 min in a rotating disk. The reaction was neutralized with AD-MSC medium and centrifuged at 850g for 10 min. For lysis of the red blood cells, the pellet was incu- bated for 10 min at RT in 160 mM NH4Cl and then ex- tensively washed with PBS. The SVF was then filtered through a 100-μm filter nylon mesh and was either dir- ectly processed for FACS sorting followed by osteogenic differentiation, or frozen in AD-MSC medium supple- mented with 10% DMSO (Sigma). Barcoding strategies for the osteogenic differentiation experiments Barcoding strategies for the osteogenic differentiation experiments Mass-tag cellular barcoding g g For all CyTOF experiments, the Cell-ID 20-Plex Pd Bar- coding Kit (Fluidigm) was used following the manufac- tural instructions. In short, 1 million cells per condition and per line were washed with PBS and then incubated with Cell-ID Cisplatin (Fluidigm) for 10 min at RT. Afterwards, cells were fixed with MaxPar Fix Buffer (Fluidigm) for 10 min at RT, washed with MaxPar Barcode Perm Buffer (Fluidigm), and incubated with the appropriate barcode for 30 min at RT. Finally, cells were washed with Cell Staining Buffer (Fluidigm) and com- bined depending on the CyTOF experiment in one or more tubes before antibody staining. Depending on the planned CyTOF experiment, a specific barcoding strat- egy was developed in order to minimize technical bias and highlight biological differences. Page 13 of 16 Page 13 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Barcoding strategy for prediction of differentiation potential in five new AD-MSC lines Mass cytometry measurements were transformed using the inverse hyperbolic sine (arcsinh) function with a co- factor of 5 and subsequently median-centered on a per- marker basis. Five not yet characterized AD-MSC lines (new AD- MSCs) together with 9 already characterized AD-MSC lines (reference) were collected in their undifferentiated state (day 0). Next, together with one MIX, they were all barcoded according to the barcode plan (Table S3) and pooled into one single tube for antibody staining and CyTOF acquisition as described above. Defining the selected cell subpopulation g p p The default CellCNN filter interpretation analysis was performed to define and characterize the selected cell subpopulation. Initially, learned filters were clustered and a single representative filter was retained from each cluster. As a second step, a score was derived for each representative filter, measuring how well this filter alone can classify the validation samples. Only one representa- tive filter achieved a positive score, and this filter was used to define the selected cell subpopulation (i.e., cells with positive score with respect to that filter) in individual mass cytometry samples at d0, d1, d2, d3, and d4. Statistical analyses y Quantification of the staining of the triplicates of undif- ferentiated cells (control) and cells cultured with differ- entiation medium (differentiation) is presented as mean ± s.d. Quantification of the triplicates of the stain- ing of the FACS sorted subpopulations is presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to com- pare the ALP+/CD73+ population with the other sorted fractions within the same day as well as for comparing the percentage of ALP+, CD73+, and CD271+ cells in the “good” category for each day with the same day of the “intermediate” and “bad” ones. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and **** p ≤0.0001. Pearson’s correlation was used to determine the correlation between the ALP frequency measured by CyTOF at days 0, 1, 2, 3, and 4 with the staining intensity measured at days 14, 17, and 21 for the osteogenic differentiation lineage. For this differentiation experiment, we had a total of 102 samples. Thus, having only 20 different barcodes avail- able, we distributed the barcoded samples into 6 tubes (Tables S2). In each tube when possible, there was one “good,” one “bad,” and one “intermediate” line for all the collected time points. The 17 AD-MSC lines cultured under osteogenic condition were collected during the first 5 days (day 0, day 1, day 2, day 3, day 4) of differen- tiation. At each day, the samples were barcoded, pooled into the appropriate tube, and stored at 4 °C until day 4. At day 4, a unique antibody master mix was prepared and distributed into the six tubes. In order to monitor tube-to-tube variations, we added to each of the six tubes twice the MIX (PBMCs, HEK, Hela, Jurkat, Saos2, Nalm6m, SHSY5S, AD-MSCs) for a total of 102 samples (Tables S2). Stability of the barcoded samples stored at 4 °C during the four collection days was extensively proved in preliminary tests (data not shown). Barcoding strategy for the passage experiment Barcoding strategy for the passage experiment AD-MSCs F28, F22, F5, and F14 were cultured in AD- MSC medium in Nunc™6-cm plates (Thermo Fisher Scientific) and passaged when 90% confluence was reached. This was repeated from passage 3 (p3) to pas- sage 20 (p20). At each passage, part of the cells was fro- zen in AD-MSC medium supplemented with 10% DMSO (Sigma). All AD-MSC lines from p3 to p20 were thawed the same day and barcoded according to the bar- code plan (Table S4). All barcoded passages from the same cell line were pooled into one tube. Each tube con- tained also twice a MIX as a control. Cells were stained with the antibody panel following the protocols men- tioned above and then processed in CyTOF2 (Fluidigm). Model training ll CellCNN was trained with the objective to classify “good” versus “bad” AD-MSC lines from their corre- sponding mass cytometry measurements at day 0 (undif- ferentiated state). Training examples (multi-cell inputs) comprised 2000 cells, sampled uniformly at random from the original mass cytometry samples. In total, we sampled 1000 training examples per class (“good” or “bad” cell lines). For the top-k pooling layer, we consid- ered values of k such that the ratio of k over the multi- cell input size would be one of [0.5%, 1%, 3%, 5%]. The remaining CellCNN parameters were set to their default values. Data availability through the FACS sorting machine. Depicted are triplicates of undifferen- tiated cells (control) and cells cultured with osteogenic differentiation medium (differentiation). Error bars indicate the triplicates of the staining and are presented as mean ± s.d. Table S1. Mass cytometry antibody panel. Table S2. Osteogenic differentiation barcoding schema. Table S3. Barcoding plan prediction experiment. Table S4: Barcoding plan for the passage experiment. through the FACS sorting machine. Depicted are triplicates of undifferen- tiated cells (control) and cells cultured with osteogenic differentiation medium (differentiation). Error bars indicate the triplicates of the staining and are presented as mean ± s.d. Table S1. Mass cytometry antibody panel. Table S2. Osteogenic differentiation barcoding schema. Table S3. Barcoding plan prediction experiment. Table S4: Barcoding plan for the passage experiment. through the FACS sorting machine. Depicted are triplicates of undifferen- tiated cells (control) and cells cultured with osteogenic differentiation medium (differentiation). Error bars indicate the triplicates of the staining and are presented as mean ± s.d. Table S1. Mass cytometry antibody panel. Table S2. Osteogenic differentiation barcoding schema. Table S3. Barcoding plan prediction experiment. Table S4: Barcoding plan for the passage experiment. Mass cytometry data that support the findings of this study are available on request from the corresponding author [P.C.]. Supplementary Information h l l The online version contains supplementary material available at https://doi. org/10.1186/s13287-020-02044-4. The online version contains supplementary material available at https://doi. org/10.1186/s13287-020-02044-4. Mass cytometry data analysis Mass cytometry data.fcs files collected from each set of samples were normalized using the executable MATLAB version of the Normalizer tool [63] and concatenated using the .fcs concatenation tool from Cytobank. Indi- vidual samples were debarcoded using the executable MATLAB version of the single-cell debarcoder tool [64]. Page 14 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Authors’ contributions D.D.C., E.A.C., H.C.P., and P.C. conceived the study. D.D.C. and E.A.C. performed all experiments with help from S.M. and B.E. E.A. and M.C. performed the CellCNN analysis. D.D.C., E.A.C., P.C., and V.T. conceived the antibody panel. V.T. assisted with the CyTOF experimental design. J.B., A.A.B., J.A.P., and S.H. provided the adipose tissue samples. D.D.C., E.A.C., and P.C. wrote the manuscript with input from all authors. All authors read and approved the final manuscript. Blue denotes minimal, green intermediate, and red high expression. Fig ure S3. Analyses of the osteogenic subpopulation. A) Means of the per- centage of alkaline phosphatase (ALP) positive cells and CD73 positive cells in the three AD-MSC categories during the five analyzed days of osteogenic differentiation (d0, d1, d2, d3, d4). Error bars represent the mean ± s.d. of the percentage of positive cells present in «good» (n = 6), «intermediate» (n = 4),and «bad» (n = 7) AD-MSCs. B) Pearson correlations of the ALP frequency measured by CyTOF at day 0, 1, 2, 3, 4 with the staining intensities measured at day 14, 17, and 21 for osteogenic differ- entiation. Red dots represent «good», green «intermediate» (interm.), and black «bad» differentiating lines. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to com- pare each day of the “good” AD-MSCs with the same day of “intermedi- ate” and “bad” categories: * p≤0.05, ** p≤0.01, *** p ≤0.001, and **** p Acknowledgements The authors thank Yvonne Neldner for technical assistance, and Ines Kleiber- Schaaf and Andrea Garcete-Bärtschi for paraffin embedding and staining. We acknowledge assistance provided by the Cytometry Facility of the University of Zurich, especially Tess Brodie, Paulina Kulig, and Christoph Schwärzler, and thank Justin Douglas Walter for critically reading the manuscript. This work was supported by the Gottfried and Julia Bangerter Foundation (to P.C.) and the Olga Mayenfisch Foundation (to P.C.). Additional file 1: Figure S1. In vitro chondrogenic and adipogenic categorization of 17 AD-MSCs A) Sum of the pixels acquired at the three time points (day 14, 17, 21) for chondrogenic (left) and adipogenic (right) differentiation of all 17 AD-MSC lines and interquartile categorization into «good», «intermediate», and «bad» AD-MSCs. C) Summary of the categorization of all 17 AD-MSCs for the three differentiation lineages (osteogenic, chondrogenic, and adipogenic). interm. = intermediate. Fig- ure S2. UMAP analyses in the 17 human AD-MSC lines. A) UMAP projec- tions of all 31 markers in 17 AD-MSC lines. Each dot represents one cell. Blue denotes minimal, green intermediate, and red high expression. Fig- ure S3. Analyses of the osteogenic subpopulation. A) Means of the per- centage of alkaline phosphatase (ALP) positive cells and CD73 positive cells in the three AD-MSC categories during the five analyzed days of osteogenic differentiation (d0, d1, d2, d3, d4). Error bars represent the mean ± s.d. of the percentage of positive cells present in «good» (n = 6), «intermediate» (n = 4),and «bad» (n = 7) AD-MSCs. B) Pearson correlations of the ALP frequency measured by CyTOF at day 0, 1, 2, 3, 4 with the staining intensities measured at day 14, 17, and 21 for osteogenic differ- entiation. Red dots represent «good», green «intermediate» (interm.), and black «bad» differentiating lines. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to com- pare each day of the “good” AD-MSCs with the same day of “intermedi- ate” and “bad” categories: * p≤0.05, ** p≤0.01, *** p ≤0.001, and **** p ≤0.0001. ns=not significant. Figure S4. ALP+/CD73+ Sorting analysis and prediction of osteogenic differentiation potential. A) Gating strategy for FACS sorting for the following subpopulations: ALP+/CD73+, ALP-/ CD73low, and ALP-/CD73high. Acknowledgements B) Alizarin Red staining and quantification of the sorted subpopulations in four AD- MSC lines (F04, F14, F22, F28) after 14, 17, and 21 days. Control sorted are unstained cells, which were run through the FACS sorting machine. Depicted are triplicates of undif- ferentiated cells (control) and cells cultured with the differentiation medium (differentiation). Error bars indicate the triplicates of the staining and are presented as mean ± s.d. C) Categorization of the new AD-MSC lines (depicted in green) together with all the 17 already analyzed lines, based on Alizarin Red quantification after 14, 17, and 21 days of osteo- genic differentiation and interquartile distribution of the five new AD- MSCs (depicted in violet). D) Alizarin Red staining and quantification of five new AD-MSCs: two «good» (F08, F26), one «intermediate» (F23), and two «bad» (F20, F24). Depicted are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation conditions (differentiation). Error bars indicate triplicates of the staining and are pre- sented as mean ± s.d. E) Histograms of median intensities of expression of selected markers (CD73 and ALP) in F05, F14, F22 and F28 AD-MSC lines from passage 3 (p3) till passage 20 (p20). Black is the lowest inten- sity and white represents the highest intensity. F) Alizarin Red staining and quantification of F22 at passage p5, p9, and p20 after 14, 17, and 21 days of osteogenic differentiation. Depicted are triplicates of undifferenti- ated cells (control) and cells cultured under osteogenic differentiation medium (differentiation). Error bars indicates the triplicates of the staining and are presented as mean ± s.d. Figure S5. ALP+/CD73+ cells are present in the human fat tissue and stromal vascular fraction A) Hematoxylin/Eosin (H&E) and immunohistochemistry staining of human fat tissue for ALP, CD73, and CD31. Scale 100 μm. B) Gating strategy for sorting the selected subpopulations (CD45- /ALP+/CD73+, CD45-/ALP-/ CD73low, CD45-/ALP-/CD73high) in the SVFs. C) Alizarin Red staining and pixel quantification of sorted SVF fractions (CD45-/ALP+/CD73+, CD45-/ ALP /CD73low CD45 /ALP /CD73high) after 21 days of osteogenic differ Author details 1 B) Gating strategy for sorting the selected subpopulations (CD45- /ALP+/CD73+, CD45-/ALP-/ CD73low, CD45-/ALP-/CD73high) in the SVFs. C) Alizarin Red staining and pixel quantification of sorted SVF fractions (CD45-/ALP+/CD73+, CD45-/ ALP-/CD73low, CD45-/ALP- /CD73high) after 21 days of osteogenic differ- entiation in vitro. Control sorted are unstained SVFs, which were run Received: 7 September 2020 Accepted: 23 November 2020 Author details 1 1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland. 2Life Science Zurich Graduate School, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. 3Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland. 4Mass Cytometry Facility, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. 5Department of Plastic and Hand Surgery, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland. ate and bad categories: p≤0.05, p≤0.01, p ≤0.001, and p ≤0.0001. ns=not significant. Figure S4. ALP+/CD73+ Sorting analysis and prediction of osteogenic differentiation potential. A) Gating strategy for FACS sorting for the following subpopulations: ALP+/CD73+, ALP-/ CD73low, and ALP-/CD73high. B) Alizarin Red staining and quantification of the sorted subpopulations in four AD- MSC lines (F04, F14, F22, F28) after 14, 17, and 21 days. Control sorted are unstained cells, which were run through the FACS sorting machine. Depicted are triplicates of undif- ferentiated cells (control) and cells cultured with the differentiation medium (differentiation). Error bars indicate the triplicates of the staining and are presented as mean ± s.d. C) Categorization of the new AD-MSC lines (depicted in green) together with all the 17 already analyzed lines, based on Alizarin Red quantification after 14, 17, and 21 days of osteo- genic differentiation and interquartile distribution of the five new AD- MSCs (depicted in violet). D) Alizarin Red staining and quantification of five new AD-MSCs: two «good» (F08, F26), one «intermediate» (F23), and two «bad» (F20, F24). Depicted are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation conditions (differentiation). Error bars indicate triplicates of the staining and are pre- sented as mean ± s.d. E) Histograms of median intensities of expression of selected markers (CD73 and ALP) in F05, F14, F22 and F28 AD-MSC lines from passage 3 (p3) till passage 20 (p20). Black is the lowest inten- sity and white represents the highest intensity. F) Alizarin Red staining and quantification of F22 at passage p5, p9, and p20 after 14, 17, and 21 days of osteogenic differentiation. Depicted are triplicates of undifferenti- ated cells (control) and cells cultured under osteogenic differentiation medium (differentiation). Error bars indicates the triplicates of the staining and are presented as mean ± s.d. Figure S5. ALP+/CD73+ cells are present in the human fat tissue and stromal vascular fraction A) Hematoxylin/Eosin (H&E) and immunohistochemistry staining of human fat tissue for ALP, CD73, and CD31. Scale 100 μm. References h van de Peppel J, et al. Identification of three early phases of cell-fate determination during osteogenic and adipogenic differentiation by transcription factor dynamics. Stem Cell Reports. 2017;8:947–60. https://doi. org/10.1016/j.stemcr.2017.02.018. 19. Kobolak J, Dinnyes A, Memic A, Khademhosseini A, Mobasheri A. Mesenchymal stem cells: identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche. Methods. 2016;99:62–8. https://doi.org/10.1016/j.ymeth.2015.09.016. 43. Huang AH, Farrell MJ, Mauck RL. Mechanics and mechanobiology of mesenchymal stem cell-based engineered cartilage. J Biomech. 2010;43: 128–36. https://doi.org/10.1016/j.jbiomech.2009.09.018. 20. Dominici M, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–7. https://doi.org/10.1080/14653240600855905. 44. Wang J, Liao L, Wang S, Tan J. Cell therapy with autologous mesenchymal stem cells-how the disease process impacts clinical considerations. Cytotherapy. 2013;15:893–904. https://doi.org/10.1016/j.jcyt.2013.01.218. 21. Dicker A, et al. Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res. 2005;308:283–90. https://doi.org/ 10.1016/j.yexcr.2005.04.029. 45. D'Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res. 1999;14:1115–22. https://doi.org/ 10.1359/jbmr.1999.14.7.1115. 22. Festy F, et al. Surface protein expression between human adipose tissue- derived stromal cells and mature adipocytes. Histochem Cell Biol. 2005;124: 113–21. https://doi.org/10.1007/s00418-005-0014-z. 46. Katsara O, et al. Effects of donor age, gender, and in vitro cellular aging on the phenotypic, functional, and molecular characteristics of mouse bone marrow-derived mesenchymal stem cells. Stem Cells Dev. 2011;20:1549–61. https://doi.org/10.1089/scd.2010.0280. 23. Gronthos S, et al. Surface protein characterization of human adipose tissue- derived stromal cells. J Cell Physiol. 2001;189:54–63. https://doi.org/10.1002/ jcp.1138. 47. Stenderup K, Justesen J, Clausen C, Kassem M. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone. 2003;33:919–26. 24. Haniffa MA, et al. Adult human fibroblasts are potent immunoregulatory cells and functionally equivalent to mesenchymal stem cells. J Immunol. 2007;179:1595–604. https://doi.org/10.4049/jimmunol.179.3.1595. 25. Mitchell JB, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 2006;24:376–85. https://doi.org/10.1634/stemcells.2005-0234. 48. Bradaschia-Correa V, et al. Ecto-5′-nucleotidase (CD73) regulates bone formation and remodeling during intramembranous bone repair in aging mice. Tissue Cell. 2017;49:545–51. https://doi.org/10.1016/j.tice.2017.07.001. 26. Varma MJ, et al. Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev. 2007;16:91–104. https://doi.org/10.1089/scd.2006.0026. 49. Feldbush TL, Lafrenz D. References h Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211–28. https://doi.org/10.1089/ 107632701300062859. 11. Kuznetsov SA, et al. Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo. J Bone Miner Res. 1997; 12:1335–47. https://doi.org/10.1359/jbmr.1997.12.9.1335. 35. Eggerschwiler B, Canepa DD, Pape HC, Casanova EA, Cinelli P. Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells. Stem Cell Res Ther. 2019;10:69. https://doi.org/10.1186/s13287-019-1170-8. 12. McLeod CM, Mauck RL. On the origin and impact of mesenchymal stem cell heterogeneity: new insights and emerging tools for single cell analysis. Eur Cell Mater. 2017;34:217–31. https://doi.org/10.22203/eCM.v034a14. Ther. 2019;10:69. https://doi.org/10.1186/s13287-019-1170-8. 36. Nassar AF, Ogura H, Wisnewski AV. Impact of recent innovations in the use of mass cytometry in support of drug development. Drug Discov Today. 2015;20:1169–75. https://doi.org/10.1016/j.drudis.2015.06.001. 13. Post S, Abdallah BM, Bentzon JF, Kassem M. Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells. Bone. 2008;43:32–9. https:// doi.org/10.1016/j.bone.2008.03.011. 37. Bendall SC, et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science. 2011; 332:687–96. https://doi.org/10.1126/science.1198704. 14. Selich A, et al. Massive clonal selection and transiently contributing clones during expansion of mesenchymal stem cell cultures revealed by lentiviral RGB-barcode technology. Stem Cells Transl Med. 2016;5:591–601. https://doi. org/10.5966/sctm.2015-0176. 38. Amir el AD, et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol. 2013;31:545–52. https://doi.org/10.1038/nbt.2594. 15. Phinney DG, et al. Donor variation in the growth properties and osteogenic potential of human marrow stromal cells. J Cell Biochem. 1999;75:424–36. 39. Ferrell PB Jr, et al. High-dimensional analysis of acute myeloid leukemia reveals phenotypic changes in persistent cells during induction therapy. PLoS One. 2016;11:e0153207. https://doi.org/10.1371/journal.pone.0153207. 16. Phinney DG. Functional heterogeneity of mesenchymal stem cells: implications for cell therapy. J Cell Biochem. 2012;113:2806–12. https://doi. org/10.1002/jcb.24166. 40. Hamers AAJ, et al. Human monocyte heterogeneity as revealed by high- dimensional mass cytometry. Arterioscler Thromb Vasc Biol. 2019;39:25–36. https://doi.org/10.1161/ATVBAHA.118.311022. 17. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970;3:393–403. 41. Galli E, et al. GM-CSF and CXCR4 define a T helper cell signature in multiple sclerosis. Nat Med. 2019;25:1290–300. https://doi.org/10.1038/s41591-019-0521-4. 18. Pittenger MF, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7. 42. References h 1. Bigham AS, Dehghani SN, Shafiei Z, Torabi Nezhad S. Xenogenic demineralized bone matrix and fresh autogenous cortical bone effects on experimental bone healing: radiological, histopathological and biomechanical evaluation. J Orthop Traumatol. 2008;9:73–80. https://doi.org/ 10.1007/s10195-008-0006-6. 2. Ikada Y. Challenges in tissue engineering. J R Soc Interface. 2006;3:589–601. https://doi.org/10.1098/rsif.2006.0124. 3. Dai R, Wang Z, Samanipour R, Koo KI, Kim K. Adipose-derived stem cells for tissue engineering and regenerative medicine applications. Stem Cells Int. 2016;2016:6737345. https://doi.org/10.1155/2016/6737345. 4. da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci. 2006;119:2204– 13. https://doi.org/10.1242/jcs.02932. 5. Ntege EH, Sunami H, Shimizu Y. Advances in regenerative therapy: a review of the literature and future directions. Regen Ther. 2020;14:136–53. https:// doi.org/10.1016/j.reth.2020.01.004. 6. Gao S, et al. Proliferation of ASC-derived endothelial cells in a 3D electrospun mesh: impact of bone-biomimetic nanocomposite and co- culture with ASC-derived osteoblasts. Injury. 2014;45:974–80. https://doi.org/ 10.1016/j.injury.2014.02.035. 7. Konig MA, et al. Direct transplantation of native pericytes from adipose tissue: a new perspective to stimulate healing in critical size bone defects. Cytotherapy. 2016;18:41–52. https://doi.org/10.1016/j.jcyt.2015.10.002. 7. Konig MA, et al. Direct transplantation of native pericytes from adipose tissue: a new perspective to stimulate healing in critical size bone defects. Cytotherapy. 2016;18:41–52. https://doi.org/10.1016/j.jcyt.2015.10.002. 8. Groninger O, et al. Directing stem cell commitment by amorphous calcium phosphate nanoparticles incorporated in PLGA: relevance of the free calcium ion concentration. Int J Mol Sci. 2020;21. https://doi.org/10.3390/ ijms21072627. Page 15 of 16 Page 15 of 16 Page 15 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 (2021) 12:7 9. Sordi MB, Cabral da Cruz AC, Aragones A, Rodriguez Cordeiro MM, de Souza Magini R. PLGA+HA/betaTCP scaffold incorporating simvastatin: a promising biomaterial for bone tissue engineering. J Oral Implantol. 2020. https://doi. org/10.1563/aaid-joi-D-19-00148. 31. Zhu Y, et al. Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct. 2008;26:664–75. https://doi.org/10.1002/cbf.1488. 32. Becht E, et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol. 2018. https://doi.org/10.1038/nbt.4314. 10. De Luca A, et al. Improvement of osteogenic differentiation of human mesenchymal stem cells on composite poly l-lactic acid/nano- hydroxyapatite scaffolds for bone defect repair. J Biosci Bioeng. 2020;129: 250–7. https://doi.org/10.1016/j.jbiosc.2019.08.001. 33. Arvaniti E, Claassen M. Sensitive detection of rare disease-associated cell subsets via representation learning. Nat Commun. 2017;8:14825. https://doi. org/10.1038/ncomms14825. 34. Zuk PA, et al. References h MicroRNA-21 preserves the fibrotic mechanical memory of mesenchymal stem cells. Nat Mater. 2017;16:379–89. https://doi.org/10.1038/ nmat4780. 58. Li CX, et al. MicroRNA-21 preserves the fibrotic mechanical memory of mesenchymal stem cells. Nat Mater. 2017;16:379–89. https://doi.org/10.1038/ nmat4780. 59. Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev Biol. 1987;121:1–9. https://doi.org/10.1016/0012- 1606(87)90132-1. 59. Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev Biol. 1987;121:1–9. https://doi.org/10.1016/0012- 1606(87)90132-1. 60. Williams RL, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 1988;336:684–7. https://doi.org/10.1038/336684a0. 61. Ying QL, et al. The ground state of embryonic stem cell self-renewal. Nature. 2008;453:519–23. https://doi.org/10.1038/nature06968. 61. Ying QL, et al. The ground state of embryonic stem cell self-renewal. Nature. 2008;453:519–23. https://doi.org/10.1038/nature06968. 62. Buschmann J, et al. Yield and proliferation rate of adipose-derived stromal cells as a function of age, body mass index and harvest site-increasing the yield by use of adherent and supernatant fractions? Cytotherapy. 2013;15: 1098–105. https://doi.org/10.1016/j.jcyt.2013.04.009. 63. Finck R, et al. Normalization of mass cytometry data with bead standards. Cytometry A. 2013;83:483–94. https://doi.org/10.1002/cyto.a.22271. 63. Finck R, et al. Normalization of mass cytometry data with bead standards. Cytometry A. 2013;83:483–94. https://doi.org/10.1002/cyto.a.22271. 64. Zunder ER, et al. Palladium-based mass tag cell barcoding with a doublet- filtering scheme and single-cell deconvolution algorithm. Nat Protoc. 2015; 10:316–33. https://doi.org/10.1038/nprot.2015.020. References h Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor. J Immunol. 1991;147:3690–5. 50. Rohwedel J, Sehlmeyer U, Shan J, Meister A, Wobus AM. Primordial germ cell- derived mouse embryonic germ (EG) cells in vitro resemble undifferentiated stem cells with respect to differentiation capacity and cell cycle distribution. Cell Biol Int. 1996;20:579–87. https://doi.org/10.1006/cbir.1996.0076. 27. Wagner W, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol. 2005;33:1402–16. https://doi.org/10.1016/j.exphem.2005.07.003. 28. Yoshimura K, et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol. 2006;208:64–76. https://doi.org/10.1002/jcp.20636. 51. Yegutkin GG. Nucleotide- and nucleoside-converting ectoenzymes: important modulators of purinergic signalling cascade. Biochim Biophys Acta. 2008;1783:673–94. https://doi.org/10.1016/j.bbamcr.2008.01.024. 29. Yanez R, et al. Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft- versus-host disease. Stem Cells. 2006;24:2582–91. https://doi.org/10.1634/ stemcells.2006-0228. 52. Grol MW, Panupinthu N, Korcok J, Sims SM, Dixon SJ. Expression, signaling, and function of P2X7 receptors in bone. Purinergic Signal. 2009;5:205–21. https://doi.org/10.1007/s11302-009-9139-1. 53. Orriss IR, Burnstock G, Arnett TR. Purinergic signalling and bone remodelling. Curr Opin Pharmacol. 2010;10:322–30. https://doi.org/10.1016/j. coph.2010.01.003. 30. Tapp H, Hanley EN Jr, Patt JC, Gruber HE. Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair. Exp Biol Med (Maywood). 2009;234:1–9. https://doi.org/10.3181/0805/MR-170. Page 16 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 54. Buckley KA, Golding SL, Rice JM, Dillon JP, Gallagher JA. Release and interconversion of P2 receptor agonists by human osteoblast-like cells. FASEB J. 2003;17:1401–10. https://doi.org/10.1096/fj.02-0940com. 55. Dellavalle A, et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol. 2007;9:255–67. https:// doi.org/10.1038/ncb1542. 56. Gerlach JC, et al. Perivascular mesenchymal progenitors in human fetal and adult liver. Stem Cells Dev. 2012;21:3258–69. https://doi.org/10.1089/scd. 2012.0296. 56. Gerlach JC, et al. Perivascular mesenchymal progenitors in human fetal and adult liver. Stem Cells Dev. 2012;21:3258–69. https://doi.org/10.1089/scd. 2012.0296. 57. Yang C, Tibbitt MW, Basta L, Anseth KS. Mechanical memory and dosing influence stem cell fate. Nat Mater. 2014;13:645–52. https://doi.org/10.1038/ nmat3889. 57. Yang C, Tibbitt MW, Basta L, Anseth KS. Mechanical memory and dosing influence stem cell fate. Nat Mater. 2014;13:645–52. https://doi.org/10.1038/ nmat3889. 58. Li CX, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4281722332
https://link.springer.com/content/pdf/10.1007/s11468-022-01649-0.pdf
English
null
The Intensity of the Plasmon–exciton of Three Spherical Metal Nanoparticles On the Semiconductor Quantum Dot Having Three External Fields
Plasmonics
2,022
cc-by
8,530
Abstractl The influence of the plasmon of three spherical metal nanoparticles (MNPs) on the semiconductor quantum dot (SQD) having three external fields is analyzed. The density matrix equations are modified for the description of the optical properties of the SQD-MNPs nanosystem. We study theoretically the role of the plasmon–exciton dipole coupling in the SQD-MNPs nanosys- tem. We investigate the dependence of the plasmon–exciton dipole coupling of the SQD-MNPs nanosystem on the position of three spherical MNPs with respect to SQD as well as on the material parameters of the hybrid nanosystem. The direction and detunings of the three external fields play an important role in the characterization of the SQD-MNPs nanosystem. Keywords  Quantum dot · Plasmon · Dipole–dipole interactions https://doi.org/10.1007/s11468-022-01649-0 Plasmonics (2022) 17:1633–1644 https://doi.org/10.1007/s11468-022-01649-0 Plasmonics (2022) 17:1633–1644 The Intensity of the Plasmon–exciton of Three Spherical Metal Nanoparticles On the Semiconductor Quantum Dot Having Three External Fields Hagar M. Ali1 · Somia Abd‑Elnabi1   · Kariman I. Osman1 Received: 10 January 2022 / Accepted: 25 April 2022 © The Author(s) 2022 / Published online: 27 May 2022 Introduction [17]. Investigating the spatial properties of coherent plas- monic (CP) field and demonstrating how it depends on the collective molecular states of the SQD-MNP system (bright and dark states) are shown in [18, 19] that when the coherent SQD-MNP molecule is in the dark state, i.e., the SQD does not emit light, the CP field is spatially confined around the MNP. It is studied that there were the states of polarization of coherent plasmonic fields of a SQD-MNP system in the environment surrounding the MNP and investigated how the dynamics of these states were evolved with time when this system was interacting with a time-dependent laser field [20–22]. Plasmon-assisted two-photon Rabi oscillations in a semiconductor quantum dot—metal nanoparticle heterodi- mer are investigated in [23]. The demonstration of multipole effect and the intensity of the plasmon–exciton dipole cou- pling in the SQD-MNP nanosystem are shown in [24–27]. The optical properties of complex nanosystems, that when a semiconductor quantum dot (SQD) is in the vicinity of a metallic nanoparticle (MNP), are the area of considerable current interest. So, the optics of the SQD become strongly sensitive to the structural parameters of the nanosystem, the intensity of the plasmon–exciton dipole coupling and the dielectric constant of the environment when combining semiconductor quantum dot (SQD) and plasmonic nano- structures, and hence, it is carried out in the interdiscipli- nary applications of nanoscience. The phenomena that have been studied in these research areas are the phase control of absorption and dispersion as well complete optical trans- parency [1–4], Fano effects in energy absorption [5–8], plasmonic electromagnetically induced transparency (EIT) [9–12], the enhancement of nonlinear Kerr and suscepti- bilities in several quantum systems [13–16]. The terahertz generation enhancement from intraband transition in self- assembled SQD molecules near a (MNP) is discussed in In this paper, we consider a single SQD in close vicinity to three spherical MNPs. The present scheme is based on a coupled SQD-MNPs nanosystem in the presence of the pump, control and probe fields. The SQD is taken as a four-level V-type system in which the distinct excitonic transitions occur. Theoretically, we will study the effect of the plasmon–exciton dipole coupling in the SQD- MNP nanosystem and investigate the dependence of the plasmon–exciton dipole coupling of the SQD-MNP nanosystem on the position of MNPs with a SQD as well as on the material parameters of the hybrid nanosystem. * Somia Abd‑Elnabi somiaabdelnabi@Azhar.edu.eg 1 Department of Mathematics, Faculty of Science and Women’s Section, Al-Azhar University, Cairo, Nasr City, 11754, Egypt Introduction The 1 Department of Mathematics, Faculty of Science and Women’s Section, Al-Azhar University, Cairo, Nasr City, 11754, Egypt (0121 1634 Plasmonics (2022) 17:1633–1644 R1 , R2 and R3 . The schematic diagram of the nanosystem is considered as a V-type four-level SQD structure. It is composed of four states 1⟩ , 2⟩ , 3⟩ and 4⟩ with energies ℏ𝜔1 , ℏ𝜔2 , ℏ𝜔3 and ℏ𝜔4 , respectively, as illustrated in Fig. 1. The nanohybrid structure is subjected to the pump, probe and control fields with amplitudes E2, E3 and E4 and frequencies 휈2 , 휈3 and 휈4 , respectively. The weak probe field derives the excitonic transition 1⟩↔3⟩ with reso- nance frequency 휔13 , where 휔nm = 휔n −휔m and ( n, m = 1, 2, 3, 4) . The pump and control fields derive the excitonic transitions 1⟩↔2⟩ and 3⟩↔4⟩ with reso- nance frequencies 휔12 and 휔34 , respectively. The SQD is situated at center-to-center distance r1, r2 and r3 from the paper is organized as follows: in Section 2, we describe the SQD-MNP nanosystem, derive the density matrix equations describing the dynamics of the system and obtain the form of the plasmon–exciton dipole coupling for the SQD-MNP nanosystem. In Section 3, we discuss our numerical results. Finally, we present our conclusions in Section 4. Theoretical Model and Description In this paper, we theoretically investigate coherent light–matter interaction in a nanohybrid between a small size of SQD and three spherical MNPs of different radii 1 Fig. 1   A schematic diagram of the SQD and three MNPs (hybrid system). SQD have four-level V-type configuration coupling with three fields Fig. 1   A schematic diagram of the SQD and three MNPs (hybrid system). SQD have four-level V-type configurati Fig. 1   A schematic diagram of the SQD and three MNPs (hybrid system). SQD have four-level V-type configuration coupling with three fields Plasmonics (2022) 17:1633–1644 1635 first, second and third spherical MNP, respectively. The distance rj has an angle 휃j ( j = 1, 2, 3 ) with respect to the Z− axis for the first, second and third spherical MNPs as illustrated in Fig. 1, respectively. We consider a first spher- ical MNP is positioned at center-to-center distance r12 and r13 from the second and third spherical MNP, respectively, while the second spherical MNP is at center-to-center dis- tance r23 from the third spherical MNP. The excitonic tran- sitions for the SQD 1⟩↔2⟩ , 1⟩↔3⟩ and 3⟩↔4⟩ are characterized by the transition dipole moments 휇12 , 휇13 and 휇34 , respectively, where the optical excitations in the SQD are excitons and the oscillating external fields give rise to oscillations of conducting electrons in the MNP’s, conventionally called localized surface plasmon (LSP). So, excitons and plasmons are excited in the nanohybrid and interact with each other via the dipole–dipole interaction, which give rise to a renormalization of the field experi- enced by both the SQD and MNPs. The dielectric constant of the SQD is represented by 휖s , and it is surrounded by a material with dielectric constant 휖B , while the three MNPs are treating as classical dielectric particles with dielectric function 휖mj(휔) and mj stands for MNP j where (j = 1, 2, 3) . The dielectric function 휖mj(휔) is obtained for the spherical MNP as [28]: ( 퐄3 ) is along the Y-axis. 퐄ij QD are the fields on SQD from the three MNPs and given by: ( 퐄3 ) is along the Y-axis. Theoretical Model and Description 퐄ij QD are the fields on SQD from the three MNPs and given by: (4) 퐄ij SQD = 1 4휋휖0휖Br3 j [3(퐩ij⋅̂퐫j)̂퐫j −퐩ij ] (4) where the unite vector ̂퐫j along the vector 퐫j is given by: (5) ̂퐫j = cos 휃ĵZ + sin 휃ĵY (j = 1, 2, 3). (5) The vector dipoles 퐩ij originate from the charge induced on the surface of the MNPs and direct in the Z-axis, which is given by: (6) 퐩ij = 훼j퐄ij, 훼j = 4휋휖0휖BR3 j 훾j 휀effmj , 훾j = 휖mj(휔) −휖B 휖mj(휔) + 2휖B (6) where 퐄ij (for i = 2, 3, 4 & j = 1, 2, 3 ) are the fields acting on the three MNPs and given by: (7) 퐄ij = 1 휀effmj [ 퐄i + 퐄SQD ij + 퐄ik ij + 퐄il ij ] (7) mj = 휖mj(휔)+2휖B 3휖B where 휀effmj = 휖mj(휔)+2휖B 3휖B is the screening of the dielectric material of the three spherical MNPs. 퐄SQD ij (for i = 2, 3, 4 & j = 1, 2, 3 ) are the fields from the SQD on the three MNPs and given by: where 휀effmj = 휖mj(휔)+2휖B 3휖B is the screening of the dielectric material of the three spherical MNPs. 퐄SQD ij (for i = 2, 3, 4 & j = 1, 2, 3 ) are the fields from the SQD on the three MNPs and given by: (1) 휖mj(휔) = 1 − 휔2 pj 휔2 + i훾bj휔 (j = 1, 2, 3) (1) 휖mj(휔) = 1 − 휔2 pj 휔2 + i훾bj휔 (j = 1, 2, 3) (1) (8) 퐄SQD ij = 1 4휋휖0휖Br3 j [ 3(퐩i SQD.̂퐫j)̂퐫j −퐩i SQD ] , i = q, 4 (8) where 휔pj is the plasma frequency for spherical MNP and 훾bj is the damping constant. The Hamiltonian of the nanohybrid system can be expressed as: HSQD = ℏ 4 ∑ j=1 𝜔j𝜎jj − [ 훍12.퐄2 SQD𝜎12 + 훍13.퐄3 SQD훔13 + 훍34.퐄4 SQD𝜎34 + H.C. ] (2) (9) pq SQD = 휇1q(휌1q + 휌q1), q = 2, 3 (10) p4 SQD = 휇34(휌34 + 휌34), i = 4. where 휎ij =∣i⟩⟨ j ∣ is the dipole transition operator between∣i⟩ and ⟨ j ∣ of the SQD. 퐄i SQD (i = 2, 3, 4) are the fields felt by the SQD polarized along the ∣1⟩⟺ ∣2⟩ , ∣1⟩⟺ ∣3⟩ and ∣3⟩⟺ ∣4⟩ transitions, respectively. Theoretical Model and Description (13) where: where: (14) Ωeff 2 = [Ω2(Ψ2 + Γ2) + Λ2휌12 ] , (15) Ωeff 3 = [Ω3(Ψ3 + Γ3) + Λ3휌13 ] , (16) Ωeff 4 = [Ω4(Ψ4 + Γ4) + Λ4휌34 ] . (14) For the case (ZYZ), we have the same above equations [14–27], but the subscript (q = 2, 3) in Ψq, Γq and Λq can be exchanged to the subscript (q = 2, 4) and the subscript 4 in Ψ4, Γ4 and Λ4 can be exchanged to the subscript 3 (i.e., the value of Ωeff 3 and Ωeff 4 exchange). For another special case (ZZY) under the condition 훼1 = 훼2 = 훼3 = 훼 and 휃1 = 0, 휃2 = 휋∕2, 휃3 = 휋, we can get the property Ωeff 4 = Ω4 , and this means that the main factor for obtain this result is the direction of the fields. For the case (ZYZ), we have the same above equations [14–27], but the subscript (q = 2, 3) in Ψq, Γq and Λq can be exchanged to the subscript (q = 2, 4) and the subscript 4 in Ψ4, Γ4 and Λ4 can be exchanged to the subscript 3 (i.e., the value of Ωeff 3 and Ωeff 4 exchange). For another special case (ZZY) under the condition 훼1 = 훼2 = 훼3 = 훼 and 휃1 = 0, 휃2 = 휋∕2, 휃3 = 휋, we can get the property Ωeff 4 = Ω4 , and this means that the main factor for obtain this result is the direction of the fields. (15) For the case (ZZY), we have: (17) Ω2 = 𝜇12E2 ℏ𝜀effs , Ω3 = 𝜇13E3 ℏ𝜀effs , Ω4 = 𝜇34E4 ℏ𝜀effs , (18) Ψq = (1 + 3 ∑ j=1 훼jAj), (q = 2, 3) (19) Ψ4 = (1 + 3 ∑ j=1 훼jCj), this means that the main factor for obtain this result direction of the fields. Theoretical Model and Description So, we have : pq SQD = 휇1q(휌1q + 휌q1), q = 2, 3 (9) (10) p4 SQD = 휇34(휌34 + 휌34), i = 4. (10) (3) 퐄i SQD = 1 휀effs [ 퐄i + 3 ∑ j=1 퐄ij SQD ] , (i = 2, 3, 4) Also, the fields 퐄ik ij and 퐄il ij result in the interaction between two polarized MNPs for ( j,  k,  l = 1, 2, 3 and j ≠k ≠l, i = 2, 3, 4) and are given by: (3) where, 휀effs = [ 휖s+2휖B 3휖B ] is the screening of the dielectric mate- rial of SQD. (11) 퐄ik ij = 1 4휋휖0휖Br3 jk [3(퐩ik.̂퐫jk)̂퐫jk −퐩ik ], , (12) 퐄il ij = 1 4휋휖0휖Br3 jl [3(퐩il.̂퐫jl)̂퐫jl −퐩il ] (11) 퐄ik ij = 1 4휋휖0휖Br3 jk [3(퐩ik.̂퐫jk)̂퐫jk −퐩ik ], , (11) Supposing that we have two cases for the direction of the fields ( 퐄i ), the first case (ZZY) that means the direction of the fields ( 퐄2, 퐄3 ) is along the Z-axis and the field ( 퐄4 ) is along the Y −axisand the second case (ZYZ): the direc- tion of the fields ( 퐄2, 퐄4 ) is along the Z −axis and the field (12) 퐄il ij = 1 4휋휖0휖Br3 jl [3(퐩il.̂퐫jl)̂퐫jl −퐩il ] (12) 1 3 3 Plasmonics (2022) 17:1633–1644 1636 (25) S1 = {3[(r2 cos 휃2 −r1 cos 휃1)∕r21]2 −1}∕4휋휖o휖Br3 21 S2 = {3[(r3 cos 휃3 −r2 cos 휃2)∕r32]2 −1}∕4휋휖o휖Br3 32 S3 = {3[(r3 cos 휃3 −r1 cos 휃1)∕r31]2 −1}∕4휋휖o휖Br3 31 (25) S1 = {3[(r2 cos 휃2 −r1 cos 휃1)∕r21]2 −1}∕4휋휖o휖Br3 21 S2 = {3[(r3 cos 휃3 −r2 cos 휃2)∕r32]2 −1}∕4휋휖o휖Br3 32 S3 = {3[(r3 cos 휃3 −r1 cos 휃1)∕r31]2 −1}∕4휋휖o휖Br3 31 where ̂퐫jk = ̂퐫k −̂퐫j and ̂퐫jl = ̂퐫l −̂퐫j . By introducing 퐄i SQD for (i = 2, 3, 4) into Eq. (2), then the total Hamiltonian of the SQD is expressed as: (25) where: (13) HSQD = ℏ 4 ∑ j=1 𝜔j𝜎jj −ℏΩeff 2 𝜎12 −ℏΩeff 3 𝜎13 −ℏΩeff 4 𝜎34 + H.C. (26) T1 = 3[r2 cos 휃2 −r1 cos 휃1][r2 sin 휃2 −r1 sin 휃1]∕4휋휖o휖Br5 21 T2 = 3[r3 cos 휃3 −r2 cos 휃2][r3 sin 휃3 −r2 sin 휃2]∕4휋휖o휖Br5 32 T3 = 3[r3 cos 휃3 −r1 cos 휃1][r3 sin 휃3 −r1 sin 휃1]∕4휋휖o휖Br5 31. Theoretical Model and Description Δ3 = 휈3 −휔31 is the frequency detuning for the i (39) 훽5 = (훾2 + 훾3) −i(Δ2 −Δ3) (40) 훽6 = (훾2 + 훾4) −i(Δ2 −Δ3 −Δ4) (41) 훽7 = (훾3 + 훾4) + iΔ4 with the identity 4∑ n=1 휌nn = 1 and (33) ⋅휌32 = −훽5휌32 −i Ωeff∗ 2 휌31 + i Ωeff 3 휌12 + i Ωeff∗ 4 휌42 (34) ⋅휌42 = −훽6휌42 −i Ωeff∗ 2 휌41 + i Ωeff 4 휌32 (35) ⋅휌43 = −훽7휌43 −i Ωeff∗ 3 휌41 −i Ωeff 4 ( 휌44 −휌33) (36) 훽2 = 훾2 + iΔ2 (37) 훽3 = 훾3 + iΔ3 (38) 훽4 = 훾4 + i(Δ3 + Δ4) where 훾2, 훾3 and 훾4 represent the radiative decay rates of the excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission, respectively. Δ3 = 휈3 −휔31 is the frequency detuning for the weak probe field, and Δ2 = 휈2 −휔21 and Δ4 = 휈4 −휔43 are the frequency detunings for the pump and control fields. In the following section, we present the results of numeri- cal calculations of the plasmonic effects and dipole–dipole interaction of the hybrid MNPs-SQD nanosystem, where the SQD has a V-type four-level structure (39) 훽5 = (훾2 + 훾3) −i(Δ2 −Δ3) (40) 훽6 = (훾2 + 훾4) −i(Δ2 −Δ3 −Δ4) (41) 훽7 = (훾3 + 훾4) + iΔ4 (39) (40) (41) 훽7 = (훾3 + 훾4) + iΔ4 (41) where 훾2, 훾3 and 훾4 represent the radiative decay rates of the excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission, where 훾2, 훾3 and 훾4 represent the radiative decay rates of the it ti t t 2⟩3⟩ d4⟩d t t i i 4 where 훾2, 훾3 and 훾4 represent the radiative decay rates of the it ti t t 2⟩3⟩ d4⟩d t t i i where 훾2, 훾3 and 훾4 represent the radiative decay rates of the excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission, respectively. Δ3 = 휈3 −휔31 is the frequency detuning for the weak probe field, and Δ2 = 휈2 −휔21 and Δ4 = 휈4 −휔43 are the frequency detunings for the pump and control fields. with the identity with the identity respectively. Theoretical Model and Description Under the electric-dipole approximation and the rot wave approximation, we define the equation of moti density matrix elements (the master equation) of the coupled to the three MNPs, as follows: ⋅휌22 = i Ωeff 2 휌12 −i Ω eff∗ 2 휌21 −2훾2휌22 ⋅휌33 = i Ωeff 3 휌13 −i Ωeff∗ 3 휌31 −i Ωeff 4 휌34 + i Ωeff∗ 4 휌43 −2훾3 휌33 + (17) Ω2 = 𝜇12E2 ℏ𝜀effs , Ω3 = 𝜇13E3 ℏ𝜀effs , Ω4 = 𝜇34E4 ℏ𝜀effs , (18) Ψq = (1 + 3 ∑ j=1 훼jAj), (q = 2, 3) (17) Ω2 = 𝜇12E2 ℏ𝜀effs , Ω3 = 𝜇13E3 ℏ𝜀effs , Ω4 = 𝜇34E4 ℏ𝜀effs , (18) Ψq = (1 + 3 ∑ j=1 훼jAj), (q = 2, 3) (19) Ψ (1 3 ∑ C ) (17) Under the electric-dipole approximation and the rotating- wave approximation, we define the equation of motion of density matrix elements (the master equation) of the SQD coupled to the three MNPs, as follows: (18) j=1 (19) Ψ4 = (1 + 3 ∑ j=1 훼jCj), (27) ⋅휌22 = i Ωeff 2 휌12 −i Ω eff∗ 2 휌21 −2훾2휌22 (28) ⋅휌33 = i Ωeff 3 휌13 −i Ωeff∗ 3 휌31 −i Ωeff 4 휌34 + i Ωeff∗ 4 휌43 −2훾3 휌33 + 2훾4 휌44 (27) ⋅휌22 = i Ωeff 2 휌12 −i Ω eff∗ 2 휌21 −2훾2휌22 (27) (19) (19) (28) ⋅휌33 = i Ωeff 3 휌13 −i Ωeff∗ 3 휌31 −i Ωeff 4 휌34 + i Ωeff∗ 4 휌43 −2훾3 휌33 + 2훾4 휌44 (20) (20) Γq = S1훼1훼2(A1 + A2) + S2훼2훼3(A2 + A3) + S3훼3훼1(A3 + A1), (q = 2, 3) (21) Γ4 = T1훼1훼2(C1 + C2) + T2훼2훼3(C2 + C3) + T3훼3훼1(C3 + C1) (29) ⋅휌44 = i Ωeff 4 휌34 −i Ωeff∗ 4 휌43 −2훾4 휌44 (21) Γ4 = T1훼1훼2(C1 + C2) + T2훼2훼3(C2 + C3) + T3훼3훼1(C3 + C1) (29) ⋅휌44 = i Ωeff 4 휌34 −i Ωeff∗ 4 휌43 −2훾4 휌44 (29) (22) Λq = ( 𝜇2 1q ℏ𝜀effs )[ 3 ∑ j=1 𝛼jA2 j + 2(S1𝛼1𝛼2A1A2 + S2𝛼2𝛼3A2A3 + S3𝛼3𝛼1A3A1) ] , (q = 2, 3) (22) Λq = ( 𝜇2 1q ℏ𝜀effs )[ 3 ∑ j=1 𝛼jA2 j + 2(S1𝛼1𝛼2A1A2 + S2𝛼2𝛼3A2A3 + S3𝛼3𝛼1A3A1) ] , (q = 2, 3) (22) (23) Λ4 = ( 𝜇2 34 ℏ𝜀effs )[ 3 ∑ j=1 𝛼jBjCj + T1𝛼1𝛼2(C1B2 + C2B1) + T2𝛼2𝛼3(C2B3 + C3B2) + T3𝛼3𝛼1(C3B1 + C1B3) ] (23) Λ4 = ( 𝜇2 34 ℏ𝜀effs )[ 3 ∑ j=1 𝛼jBjCj + T1𝛼1𝛼2(C1B2 + C2B1) + T2𝛼2𝛼3(C2B3 + C3B2) + T3𝛼3𝛼1(C3B1 + C1B3) ] (23) Λ4 = ( 𝜇2 34 ℏ𝜀effs )[ 3 ∑ j=1 𝛼jBjCj + T1𝛼1𝛼2(C1B2 + C2B1) + T2𝛼2𝛼3(C2B3 + C3B2) + T3𝛼3𝛼1(C3B1 + C1B3) ] (23) (30) ⋅휌21 = −훽2휌21 −i Ωeff 2 ( 휌11 −휌22) −i Ωeff 3 휌23 where where where (24) Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3 j Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3 j Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3 j (30) ⋅휌21 = −훽2휌21 −i Ωeff 2 ( 휌11 −휌22) −i Ωeff 3 휌23 (31) ⋅휌31 = −훽3휌31 −i Ωeff 2 휌32 + i Ωeff 3 ( 휌11 −휌33) + i Ωeff∗ 4 휌41 (32) ⋅휌41 = −훽4휌41 −i Ωeff 2 휌42 −i Ωeff 3 휌43 + i Ωeff 4 휌31 where (24) Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3 j Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3 j Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3 j (30) ⋅휌21 = −훽2휌21 −i Ωeff 2 ( 휌11 −휌22) −i Ωeff 3 휌23 (31) ⋅휌31 = −훽3휌31 −i Ωeff 2 휌32 + i Ωeff 3 ( 휌11 −휌33) + i Ωeff∗ 4 휌41 (32) ⋅휌41 = −훽4휌41 −i Ωeff 2 휌42 −i Ωeff 3 휌43 + i Ωeff 4 휌31 (30) Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3 j Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3 j Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3 j Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3 j Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3 j Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3 j (24) (31) ⋅휌31 = −훽3휌31 −i Ωeff 2 휌32 + i Ωeff 3 ( 휌11 −휌33) + i Ωeff∗ 4 휌41 (32) ⋅휌41 = −훽4휌41 −i Ωeff 2 휌42 −i Ωeff 3 휌43 + i Ωeff 4 휌31 3 Plasmonics (2022) 17:1633–1644 1637 (33) ⋅휌32 = −훽5휌32 −i Ωeff∗ 2 휌31 + i Ωeff 3 휌12 + i Ωeff∗ 4 휌42 (34) ⋅휌42 = −훽6휌42 −i Ωeff∗ 2 휌41 + i Ωeff 4 휌32 (35) ⋅휌43 = −훽7휌43 −i Ωeff∗ 3 휌41 −i Ωeff 4 ( 휌44 −휌33) (39) 훽5 = (훾2 + 훾3) −i(Δ2 −Δ3) (40) 훽6 = (훾2 + 훾4) −i(Δ2 −Δ3 −Δ4) (41) 훽7 = (훾3 + 훾4) + iΔ4 with the identity 4∑ n=1 휌nn = 1 and (33) ⋅휌32 = −훽5휌32 −i Ωeff∗ 2 휌31 + i Ωeff 3 휌12 + i Ωeff∗ 4 휌42 (34) ⋅휌42 = −훽6휌42 −i Ωeff∗ 2 휌41 + i Ωeff 4 휌32 (35) ⋅휌43 = −훽7휌43 −i Ωeff∗ 3 휌41 −i Ωeff 4 ( 휌44 −휌33) where 훾2, 훾3 and 훾4 represent the radiative decay rates of the excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission, respectively. Theoretical Model and Description Δ3 = 휈3 −휔31 is the frequency detuning for the weak probe field, and Δ2 = 휈2 −휔21 and Δ4 = 휈4 −휔43 are the frequency detunings for the pump and control fields. (36) 훽2 = 훾2 + iΔ2 (37) 훽3 = 훾3 + iΔ3 (38) 훽4 = 훾4 + i(Δ3 + Δ4) 훽2 = 훾2 + iΔ2 훽3 = 훾3 + iΔ3 훽4 = 훾4 + i(Δ3 + Δ In the following section, we present the results of numeri- cal calculations of the plasmonic effects and dipole–dipole interaction of the hybrid MNPs-SQD nanosystem, where the SQD has a V-type four-level structure Fig. 2   The spectrum of the plasmon–exciton dipole interaction (Im휂3) of the hybrid MNPs-SQD nanosystem versus probe field detuning ( Δ3 ). 휖s = 2, 휖B = 12, 휃2 = 2휋∕3, 휃3 = 3휋∕2, Ω2 = Ω4 = 6 ns−1and Δ2 = Δ4 = Δ = 2 eV. Figure 2 (a1, a2) for (ZZY) and Fig. 2 (b1, b2) for (ZYZ). Figure 2 (a1, b1) has: ℏ𝜔= 20 eV and Fig. 2 (a2, b2) has: ℏ𝜔= 2.7 eV. 휃1 = 휋∕9 (dashed curve), 휃1 = 휋∕6 (solid curve) and 휃1 = 5휋∕6 (dashed-dotted curve) Fig. 2   The spectrum of the plasmon–exciton dipole interaction (Im휂3) of the hybrid MNPs-SQD nanosystem versus probe field detuning ( Δ3 ). 휖s = 2, 휖B = 12, 휃2 = 2휋∕3, 휃3 = 3휋∕2, Ω2 = Ω4 = 6 ns−1and Δ2 = Δ4 = Δ = 2 eV. Figure 2 (a1, a2) for (ZZY) and Fig. 2 (b1, b2) for (ZYZ). Figure 2 (a1, b1) has: ℏ𝜔= 20 eV and Fig. 2 (a2, b2) has: ℏ𝜔= 2.7 eV. 휃1 = 휋∕9 (dashed curve), 휃1 = 휋∕6 (solid curve) and 휃1 = 5휋∕6 (dashed-dotted curve) 1 3 1638 Plasmonics (2022) 17:1633–1644 Numerical Results and Discussion Figure 3 (a2) displays the effect of damping constant when it is large ( 훾bj = 1.6 eV). For case (ZZY), at 휃1 = 휋∕9 , it is observed a hole in the left side which is converted into small peak at 휃1 = 휋∕6 . But at 휃1 = 휃1 = 5휋∕6 , we find different two peaks with positive values in the left side and another different two peaks with negative values in the right side. We have in this figure zero absorption for all values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 ) at ( Δ3 = −3.2 ) approximately. In Fig. 3 (b2), , we observe the middle peak has top value and another two peaks have in Fig. 2) versus probe field detuning ( Δ3 ). The data are as in Fig. 2, in addition ℏ𝜔= 20 eV. We have Fig. 3 (a1, a2) for case (ZZY) and Fig. 3 (b1, b2) for case (ZYZ), while Fig. 3 (a1, b1) has the data: 훾bj = 0.026 eV and Fig. 3 (a2, b2) has the data: 훾bj = 1.6 eV. At small damping constant ( 훾bj = 0.026 eV), for case (ZZY) as in Fig. 3 (a1) we have an the optical EIT for small 휃1 = 휋∕9 (dashed curve). When increasing 휃1 = 휋∕6 (solid curve), the absorption has three peaks and the optical EIT disappears, but at 휃1 = 5휋∕6 (dashed-dotted curve). It is observed four peaks with negative values. But for case (ZYZ) for all values of 휃1 , we show three peaks only in the negative values with different heights as in Fig. 3 (b1) . Figure 3 (a2) displays the effect of damping constant when it is large ( 훾bj = 1.6 eV). For case (ZZY), at 휃1 = 휋∕9 , it is observed a hole in the left side which is converted into small peak at 휃1 = 휋∕6 . But at 휃1 = 휃1 = 5휋∕6 , we find different two peaks with positive values in the left side and another different two peaks with negative values in the right side. We have in this figure zero absorption for all values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 ) at ( Δ3 = −3.2 ) approximately. In Fig. Numerical Results and Discussion 훾3 = 1 ns−1, 훾4 = 0.01 ns−1, Ω3 = 0.01 ns−1, Ω2 = Ω4 = 6 ns−1 and ( r1, r2, r3) = 10 , 26, 42 nm, respectively. We have Rj = R = 7 nm,  휖s = 2, 휖B = 12, 휃2 = 2휋∕3, 휃3 = 3휋∕2, and Δ2 = Δ4 = Δ = 2 eV. Other parameters are indicated in the figure captions and described in what follows. The numerical calculations for the set of density matrix equations (27-35) at the steady state are done to obtain the coherence 휌13 and 휌34 . We study the influence of the strength of the plasmon–exciton dipole interaction as a function of probe field Im Λ3휌13 = Im 휂3 and control field ImΛ4휌34 = Im휂4 for different parameters of the hybrid MNPs-SQD nanosystem. We consider three spherical gold MNPs with radius aj . The parameters of the MNPs- SQD are taken as ℏ𝜔pj = 9.02 eV,  훾bj = 0.026 eV, and the dipole 휇12 = 휇13 = 휇34 = 0.65 e nm, 훾2 = 0.02 ns−1, Figure 2 shows the spectrum of the plasmon–exciton dipole interaction (Im휂3) of the hybrid MNPs-SQD nano- system for different values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 ) versus probe field detuning ( Δ3 ). Figure 2 (a1, a2) is taken for case (ZZY), and Fig. 2 (b1, b2) is taken for case (ZYZ), while Fig. 2 (a1, b1) has the data: ℏ𝜔= 20 eV and Fig. 2 (a2, b2) has the data: ℏ𝜔= 2.7 eV. Figure 2 (a1) shows different Fig. 3   The spectrum of the plasmon–exciton dipole interaction (Im휂4) of the hybrid MNPs-SQD nanosystem versus probe field detuning ( Δ3 ). The data as in Fig. 2, in addition ℏ𝜔= 20 eV. Figure 3 ( a1, a2 ) for (ZZY) and Fig. 3 (b1, b2) for (ZYZ). Figure  3  (a1, b1) has: 훾bj = 0.026 eV and Fig. 3 (a2, b2) has: 훾bj = 1.6 eV for (ZZY) and Fig. 3 (b1, b2) for (ZYZ). Figure  3  (a1, b1) has: 훾bj = 0.026 eV and Fig. 3 (a2, b2) has: 훾bj = 1.6 eV Fig. 3   The spectrum of the plasmon–exciton dipole interaction (Im휂4) of the hybrid MNPs-SQD nanosystem versus probe field detuning ( Δ3 ). The data as in Fig. 2, in addition ℏ𝜔= 20 eV. Numerical Results and Discussion Figure 3 ( a1, a2 ) 1 3 1639 Plasmonics (2022) 17:1633–1644 impacts and distinctive for the absorption spectra when ℏ𝜔= 20 eV and has negative values for all 휃1 , at small 휃1 = 휋∕9 (dashed curve); the absorption has an optical EIT win- dow at zero detuning ( Δ3 ) besides one peak on each side. With increasing 휃1 = 휋∕6 (solid curve), we have three peaks and notice the optical EIT window disappearing, but at 휃1 = 5휋∕6 (dashed-dotted curve), the peaks are increasing to four peaks. In Fig. 2 (a2) , the peaks are displacing under the effect of angle 휃1 where they have four different peaks with positive values (because of the decrease in ℏ𝜔= 2.7 eV). For case (ZYZ), we notice three peaks only; the peaks for large 휃1 = 5휋∕6 have negative values in Fig. 2 (b1) . All the peaks for all values of 휃1 have negative values for ( ℏ𝜔= 2.7 eV) as in Fig. 2 (b2) , and also the peaks are displacing under the effect of angle 휃1 like Fig. 2 (a2) . We conclude in this figure the absorption spectrum is asymmetric about the vertical axis at ( Δ3 = 0 ), we notice also the resonance frequency ( ℏ𝜔 ) as well the angles, and direction of the fields plays an important role in the plasmon–exciton dipole coupling. in Fig. 2) versus probe field detuning ( Δ3 ). The data are as in Fig. 2, in addition ℏ𝜔= 20 eV. We have Fig. 3 (a1, a2) for case (ZZY) and Fig. 3 (b1, b2) for case (ZYZ), while Fig. 3 (a1, b1) has the data: 훾bj = 0.026 eV and Fig. 3 (a2, b2) has the data: 훾bj = 1.6 eV. At small damping constant ( 훾bj = 0.026 eV), for case (ZZY) as in Fig. 3 (a1) we have an the optical EIT for small 휃1 = 휋∕9 (dashed curve). When increasing 휃1 = 휋∕6 (solid curve), the absorption has three peaks and the optical EIT disappears, but at 휃1 = 5휋∕6 (dashed-dotted curve). It is observed four peaks with negative values. But for case (ZYZ) for all values of 휃1 , we show three peaks only in the negative values with different heights as in Fig. 3 (b1) . Numerical Results and Discussion 3 (b2), , we observe the middle peak has top value and another two peaks have Figure 3 shows the spectrum of the plasmon–exciton dipole interaction (Im휂4) of the hybrid MNPs-SQD nanosystem for different values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 ) (as Fig. 4   The spectrum of the plasmon–exciton dipole interaction (Im휂4) of the hybrid MNPs-SQD nanosystem versus probe field detuning ( Δ3 ) at 휃1 = 0. Figure 4 (a1, a2, a3) for (ZZY) and Fig. 4 (b1, b2, b3) for (ZYZ). Figure 4 (a1, b1)has : ℏ𝜔= 16 eV, Fig. 4 (a2, b2) has: ℏ𝜔= 6 eV and Fig. 4 (a3, b3) has: ℏ𝜔= 2.7 eV. The another data as in Fig. 2 Fig. 4   The spectrum of the plasmon–exciton dipole interaction (Im휂4) of the hybrid MNPs-SQD nanosystem versus probe field detuning ( Δ3 ) at 휃1 = 0. Figure 4 (a1, a2, a3) for (ZZY) and Fig. 4 (b1, b2, b3) for (ZYZ). Figure 4 (a1, b1)has : ℏ𝜔= 16 eV, Fig. 4 (a2, b2) has: ℏ𝜔= 6 eV and Fig. 4 (a3, b3) has: ℏ𝜔= 2.7 eV. The another data as in Fig. 2 1 3 Plasmonics (2022) 17:1633–1644 1640 Fig. 4 (b1, b2, b3) is taken for case (ZYZ). Figure 4 (a1, b1) has the data: ℏ𝜔= 16 eV, Fig. 4 (a2, b2) has: ℏ𝜔= 6 eV and Fig. 4 (a3, b3) has: ℏ𝜔= 2.7 eV. We have different impacts and distinctive for the resonance frequency ( ℏ𝜔 ) on the spectrum (Im휂4) in this figure, when ℏ𝜔= 16 eV. Figure 4 (a1, b1) displays four peaks in the positive values for case (ZZY) and three peaks in the negative values for case (ZYZ), respectively, with different heights. But at ℏ𝜔= 6 eV for case (ZZY), the spectrum exhibits a positive high peak with a Fano-like lineshape in the positive ( Δ3 ) and a negative small peak with a Fano-like lineshape in the negative ( Δ3 ) as in Fig. 4 (a2) . But in Fig. 4 (b2) , the small values for each value of 휃1with different values for peaks. Then, in Fig. 3, the influence of the damping constant on the plasmon–exciton dipole coupling is more obviously for the case (ZZY), and also, the shape of the spectra is asymmetric at ( Δ3 = 0 ). Numerical Results and Discussion 4 (b3) , the spectrum exhibits a trapping at ( Δ3 = 0 ), and we notice also a negative peak at a certain value ( Δ3 = −30 ) and also a positive peak at ( Δ3 = 30 ) approximately. We conclude in this figure the obvious role of the resonance frequency ( ℏ𝜔 ) or the dielectric function 휖mj(휔) for MNPs on the spectrum of the plasmon–exciton dipole interaction (Im휂4) . So, it is clear the influence of the metal nanoparticles is to enhance different phenomena in the regime of exciton–plasmon resonance. spectrum exhibits a negative high peak in the negative ( Δ3 ) and small peak with a Fano-like lineshape in the positive ( Δ3 ). At ℏ𝜔= 2.7 eV for case (ZZY), Fig. 4 (a3) displays a positive peak and a negative peak in the negative ( Δ3 ) and also in the positive ( Δ3 ). For case (ZYZ) in Fig. 4 (b3) , the spectrum exhibits a trapping at ( Δ3 = 0 ), and we notice also a negative peak at a certain value ( Δ3 = −30 ) and also a positive peak at ( Δ3 = 30 ) approximately. We conclude in this figure the obvious role of the resonance frequency ( ℏ𝜔 ) or the dielectric function 휖mj(휔) for MNPs on the spectrum of the plasmon–exciton dipole interaction (Im휂4) . So, it is clear the influence of the metal nanoparticles is to enhance different phenomena in the regime of exciton–plasmon resonance. dielectric constant 휖B is small and equal 휖s ( 휖B = 휖s = 2 ) at ℏ𝜔= 20 eV. The dashed curve is for Ω2 = Ω4 = 6ns−1 and the solid curve is for Ω2 = 4ns−1, Ω4 = 8ns−1 . The another data are as in Fig. 4. Figure  5 (a1, a2, a3) is taken for (Im휂3) , where the spectra have negative val- ues, and Fig. 5 (b1, b2, b3) is taken for ( (Im휂4) , where the spectra have positive values. Figure 5 (a1, b1) has ( Δ = 0 ), Fig. 5 (a2, b2) has ( Δ = 2 ) and Fig. 5 (a3, b3) has ( Δ = 5 ). The optical EIT, when ( Ω2 = Ω4 = 6ns−1 ) (in Fig. 5 (a1, b1) ), is deep and disappears when increasing the ( Δ ) (as in Fig. Numerical Results and Discussion As well, the optical PEIT in the spectrum appears at small damping constant and the hole in the spectrum appears at large damping constant . Figure 4 shows the spectrum of the plasmon–exciton dipole interaction (Im휂4) of the hybrid MNPs-SQD nanosystem for different values of ℏ𝜔 (16, 6,  2.7) versus probe field detuning ( Δ3 ) at 휃1 = 0. The another data are as in Fig. 2. Figure 4 (a1, a2, a3) is taken for case (ZZY), and 1 3 Fig. 5   The spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY) at ( 휖B = 휖s = 2 ), ℏ𝜔= 20 eV. Figure 5 (a1, a2, a3) is for (Im휂3) and Fig. 5 (b1, b2, b3) for ( (Im휂4) . Figure  5 (a1, b1) has ( Δ = 0 ), Fig. 5 (a2, b2) has ( Δ = 2 ) and Fig. 5 (a3, b3) has ( Δ = 5 ). The dashed curve for Ω2 = Ω4 = 6ns−1 and the solid curve for Ω2 = 4ns−1, Ω4 = 8ns−1 . The another data as in Fig. 4 Fig. 5   The spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY) at ( 휖B = 휖s = 2 ), ℏ𝜔= 20 eV. Figure 5 (a1, a2, a3) is for (Im휂3) and Fig. 5 (b1, b2, b3) for ( (Im휂4) . Figure  5 (a1, b1) has ( Δ = 0 ), Fig. 5 (a2, b2) has ( Δ = 2 ) and Fig. 5 (a3, b3) has ( Δ = 5 ). The dashed curve for Ω2 = Ω4 = 6ns−1 and the solid curve for Ω2 = 4ns−1, Ω4 = 8ns−1 . The another data as in Fig. 4 1 3 1641 Plasmonics (2022) 17:1633–1644 spectrum exhibits a negative high peak in the negative ( Δ3 ) and small peak with a Fano-like lineshape in the positive ( Δ3 ). At ℏ𝜔= 2.7 eV for case (ZZY), Fig. 4 (a3) displays a positive peak and a negative peak in the negative ( Δ3 ) and also in the positive ( Δ3 ). For case (ZYZ) in Fig. Numerical Results and Discussion 5 (a3, b3) ) . As well, four peaks appeared for ( Ω2 = 4ns, Ω4 = 8ns−1 ) in Fig. 5 (a1, b1) and decreased to only two peaks when increasing the ( Δ ) (in Fig. 5 (a3, b3) ). Then, the optical EIT is related by the change of ( Δ ), and the value of the ( Δ ) plays an important role in the charac- terization of the spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4). Figure  5 demonstrates the spectrum of the plas- mon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for case (ZZY) when the 1 3 Fig. 6   The spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem at 휖s = 12 , Δ = 0 and 휃1 = 휋∕9 . Figure  6  (a1, a2) for (Im휂3) and Fig. 6  (b1, b2) for ( (Im휂4) . Figure 6 (a1, b1) for (ZZY) and Fig. 6 (a2, b2) for (ZYZ). The dashed-dotted curve for ( 휖B = 2 ), the solid curve for ( 휖B = 6 ) and the (dashed curve) for ( 휖B = 12 ) .The another data as in Fig. 2 Fig. 6   The spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem at 휖s = 12 , Δ = 0 and 휃1 = 휋∕9 . Figure  6  (a1, a2) for (Im휂3) and Fig. 6  (b1, b2) for ( (Im휂4) . Figure 6 (a1, b1) for (ZZY) and Fig. 6 (a2, b2) for (ZYZ). The dashed-dotted curve for ( 휖B = 2 ), the solid curve for ( 휖B = 6 ) and the (dashed curve) for ( 휖B = 12 ) .The another data as in Fig. 2 1 3 1642 Plasmonics (2022) 17:1633–1644 Figure 6 exhibits the spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs- SQD nanosystem when the dielectric constant 휖s is large ( 휖s = 12 ), unlike the above figures, at ( Δ = 0 and 휃1 = 휋∕9 ). Figure 6 (a1, a2) is taken for (Im휂3) and Fig. 6 (b1, b2) is taken for ( (Im휂4) . Figure 6 (a1, b1) is for case (ZZY) and Fig. 6 (a2, b2) is for case (ZYZ). Consent for Publication  Not applicable. Consent for Publication  Not applicable. Conflicts of Interest  We investigated the dependence of the plasmon- exciton dipole coupling of the SQD-MNP nanosystem on the distance between the three metallic nanoparticles (MNPs). Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Conclusion We have derived a compound expression of the effective Rabi frequencies based on the effect of the plasmon–exci- ton dipole coupling in the SQD-MNPs nanosystem which is composed of three sphere metallic nanoparticles (MNPs) and semiconductor quantum dot (SQD) which have three external fields. The strong exciton–plasmon interaction and multipole effects are considered the main focus of this work. The direction and detunings of the three external fields play an important role in the characterization of the SQD-MNPs nanosystem. We investigated the dependence of the plas- mon–exciton dipole coupling of the SQD-MNP nanosystem on the distance between the three MNPs and also the dis- tance between SQD and MNPs. The material parameters of the hybrid nanosystem such as resonance frequency, damp- ing constant and dielectric constant, are demonstrated for many distinct characteristics and phenomena for the spec- tra of the plasmon–exciton dipole interaction (ImΛ3휌13) and (ImΛ4휌34) of the hybrid MNPs-SQD nanosystem. The Numerical Results and Discussion optical experiments on the hybrid SQD-MNPs nanosystem may be analyzed by using the results obtained in this work. Author Contributions  All authors contributed to the study conception, design and preparation. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding Information  Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). The authors declare that no funds, grants or other support was received during the preparation of this manuscript. Data Availability  The datasets generated during and/or analyzed dur- ing the current study are available from the corresponding author on reasonable request. Numerical Results and Discussion The dashed-dotted curve is for ( 휖B = 2 ), the solid curve for ( 휖B = 6 ) and the dashed curve for ( 휖B = 12 ). The another data are as in Fig. 2. We see that when the dielectric constant 휖s is large, the increase in the dielectric constant 휖B can be contributed to the enhancement of the optical EIT for case (ZZY) in the spectrums of the plasmon–exciton dipole interaction ( Im휂3 and Im휂4 ); when 휖s = 휖B = 12 , the optical EIT becomes more deep. The optical EIT for case (ZYZ) in the spec- trums ( Im휂3 and Im휂4 ) is not available, and the three peaks more extend range at increasing the dielectric constant 휖B in Fig. 6 (a2, b2) . So, happening of the optical EIT is related by the value of the dielectric constants 휖B , 휖s and the direction of the fields. Figure  7 demonstrates the influence of the size of the three spherical MNPs on the spectrum of the increase in the dielectric constant 휖B can be contributed to of the three spherical MNPs on the spectrum of the Fig. 7   The spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY), at ( Rj = 4, 6 nm) and ( 휃1 = 0 ). Figure  7  (a1, a2) for (Im휂3) and Fig. 7 (b1, b2) for ( (Im휂4) . Figure  7 (a1, b1) is plotted versus probe field detuning ( Δ3 ) and Fig. 7 (a2, b2) is plotted versus probe field detuning ( Δ4 ). The dashed curve for ( Rj = 4 ) and the solid curve for ( Rj = 6 ). The another data as in Fig. 2 Fig. 7   The spectrum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY), at ( Rj = 4, 6 nm) and ( 휃1 = 0 ). Figure  7  (a1, a2) for (Im휂3) and Fig. 7 (b1, b2) for ( (Im휂4) . Figure  7 (a1, b1) is plotted versus probe field detuning ( Δ3 ) and Fig. 7 (a2, b2) is plotted versus probe field detuning ( Δ4 ). The dashed curve for ( Rj = 4 ) and the solid curve for ( Rj = 6 ). The another data as in Fig. Numerical Results and Discussion 2 1 3 3 Plasmonics (2022) 17:1633–1644 1643 plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for the case (ZZY), and we take the radii with two different values ( R = 4, 6 nm) and ( 휃1 = 0 ), taking into consideration that the center- to-center distances are constant. Figure 7 (a1, a2 ) is taken for (Im휂3) and Fig. 7 (b1, b2) is taken for ( (Im휂4) . Fig- ure 7 (a1, b1) is plotted versus probe field detuning ( Δ3 ) and Fig. 7 (a2, b2) is plotted versus probe field detuning ( Δ4 ). The dashed curve is for ( R = 4 ) and the solid curve is for ( R = 6 ). The another data are as in Fig. 2. In Fig. 7 (a2, b2 ), which is plotted versus probe field detuning ( Δ4 ), it does not show peaks on the two sides of the spectrum as in Fig. 7 (a1, b1) . We notice at ( R = 4 ), the spectrum has small optical EIT; when increasing the radii ( R = 6 ), the optical EIT becomes obvious as in Fig. 7 (a1, b1, a2, b2) , where we have from Eq. (22, 23) that the plasmon–exciton dipole interaction ( Λ3, Λ4 ) depends on 훼j which contains Rj (i.e., the size of spherical MNPs); when we take the another values in these equations’ constant, the dipole interaction for MNP of ( R = 6 ) is more powerful than the dipole interaction for MNP of ( R = 4 ) because the MNP of ( R = 6 ) is the nearest to SQD unlike the MNP of ( R = 4 ) when the center-to-center distances are the same for the two cases ( R = 6, 4 ) [also, see [8, 17, 19, 24, 27]. We notice the ratio of the radius of MNP ( Rj ) to the center-to- center distance ( rj ), which plays an important role in the plasmon–exciton dipole interaction [24]. Then, the spec- trum of the plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem is affected by the size of the three spherical MNPs. optical experiments on the hybrid SQD-MNPs nanosystem may be analyzed by using the results obtained in this work. Ethical Approval  Not applicable. Ethical Approval  Not applicable. Informed Consent  Informed consent was obtained from all individual participants included. References 1. Paspalakis E, Evangelou S, Yannopapas V, Terzis AF (2013) Phase-dependent optical effects in a four-level quantum system near a plasmonic nanostructure. Phys Rev A 88:053832 2. Kosionis S. G, Terzis A. F, Sadeghi S. M, Paspalakis E (2013) Opti- cal response of a quantum dot-metal nanoparticle hybrid interacting with a weak probe field. J. Phys. Condens. Matter 25:045304 3. Yang Yanlian, Guo Kangxian, Yang Tao, Li Keyin, Zhai Wangjian (2019) Enhancement of linear and nonlinear optical absorp- tion coefficients in spherical dome semiconductor nanoshells by surface plasmon resonances. Physica B: Condensed Matter 556:158–162 4. Artuso RD, Bryant GW (2010) Strongly coupled quantum dot- metal nanoparticle systems: Exciton-induced transparency, dis- continuous response, and suppression as driven quantum oscillator effects. Phys. Rev. B 82:195419 4. Artuso RD, Bryant GW (2010) Strongly coupled quantum dot- metal nanoparticle systems: Exciton-induced transparency, dis- continuous response, and suppression as driven quantum oscillator effects. Phys. Rev. B 82:195419 1 3 1644 Plasmonics (2022) 17:1633–1644 5. Zhang W, Govorov AO (2011) Quantum theory of the nonlinear Fano effect in hybrid metal-semiconductor nanostructures: The case of strong nonlinearity Phys Rev B 84:081405fi 17. Carreno F, Anton MA, Melle S, Calderon OG, Cabrera-Granado E, Cox J, Singh MR, Egatz-Gomez A (2014) Plasmon-enhanced terahertz emission in self-assembled quantum dots by femtosec- ond pulses. J Appl Phys 115:64304 6. Cui J, Ji B, Song X, Lin J (2018) Efficient modulation of multipo- lar fano resonances in asymmetric ring-disk/split-ring-disk nano- structure plasmonics 18. Sadeghi SM, Hatef A (2013) Simon Fortin-Deschenes, Michel Meunier, Coherent confinement of plasmonic field in quantum dot-metallic nanoparticle molecules. Nanotechnology 24:205201i p 7. Dongxing Zhao, Wu Jiarui, Gu Ying, Qihuang Gong (2014) Tailoring double Fano profiles with plasmon-assisted quantum interference in hybrid exciton-plasmon system. Appl Phys Lett 105:111112 19. Ryan D (2011) Artuso, Garnett W. Bryant, Using local fields to tailor hybrid quantum-dot/metal nanoparticle systems, Physical Review B 83:235406 20. Sadeghi SM (2010) Gain without inversion in hybrid quantum dot-metallic nanoparticle systems. Nanotechnology 21:455401i 8. Zhang W, Govorov AO, Bryant GW (2006) Semiconductor-Metal Nanoparticle Molecules: Hybrid Excitons and the Nonlinear Fano Effect. Physical Review Letters 97:146804 21. Sadeghi SM (2014) Dynamics of plasmonic field polarization induced by quantum coherence in quantum dot-metallic nanoshell structures. Optics Lett 39 y 9. Hatef A, Sadeghi SM, Singh MR (2012) Plasmonic electromag- netically induced transparency in metallic nanoparticle-quantum dot hybrid systems. Nanotechnology 23:065701 22. References Sadeghi SM (2017) Chuanbin Mao, Quantum sensing using coher- ent control of near-field polarization of quantum dot-metallic nanoparticle molecules. Journal of Applied Physics 121:014309 10. Zamani N, Hatef A, Nadgaran H, Keshavarz A (2017) Control of electromagnetically induced transparency via a hybrid semi- conductor quantum dot-vanadium dioxide nanoparticle system. J Nanophotonics 11(3):036011 23. Nugroho Bintoro S, Iskandar Alexander A, Malyshev Victor A, Knoester Jasper (2019) Plasmon-assisted two-photon Rabi oscil- lations in a semiconductor quantum dot-metal nanoparticle het- erodimer. Phys Rev B 99:075302 11. Behroozian B (2020) Mohammad Reza Rezaie, Hassan Ranjbar Askari, The dispersion behaviors of the tripod-type four-level cylindrical quantum dot under phenomenon of electromagneti- cally induced transparency. Phys. Scr. 95:065506 24. Yan JY, Zhang W, Duan S, Zhao XG, Govorov Alexander O (2008) Optical properties of coupled metal-semiconductor and metal-molecule nanocrystal complexes: Role of multipole effects. Phys Rev B 77:165301 12. Sadeghi SM, Deng L, Li X, Huang W-P (2009) Plasmonic (ther- mal) electromagnetically induced transparency in metallic nano- particle-quantum dot hybrid systems. Nanotechnology 20:365401i 25. Anton MA, Carreno F, Melle S, Calderon O.G, Cabrera-Granado E (2013) Optical pumping of a single hole spin in a p-doped quantum dot coupled to a metallic nanoparticle. Phys Rev B 87:195303 13. Evangelou S, Yannopapas V, Paspalakis E (2014) Modification of Kerr nonlinearity in a four-level quantum system near a plasmonic nanostructure. Journal of Modern Optics 61(18):1458–1464 26. Cox JD, Singh MR, Gumbs G, Anton MA, Carreno Fernando (2012) Dipole-dipole interaction between a quantum dot and a graphene nanodisk. Physical Review B 86:125452 p 14. Terzis AF, Kosionis SG, Boviatsis J, Paspalakis E (2016) Nonlin- ear optical susceptibilities of semiconductor quantum dot - metal nanoparticle hybrids. J Mod Opt 63(5):451–461 15. Lu Zhien, Ka-Di Zhu (2008) Enhancing Kerr nonlinearity of a strongly coupled exciton-plasmon in hybrid nanocrystal mol- ecules. J. Phys. B: At. Mol. Opt. Phys. 41:185503 27. Jiang X, Guo K, Liu G, Yang T, Yang Y (2017) Enhancement of surface plasmon resonances on the nonlinear optical proper- ties in a GaAs quantum dot. Superlattices and Microstructures 105:56–64 y p y 16. Kosionis SG, Terzis AF (2010) Paspalakis, Linear and nonlinear optical properties of a two-subband system in a symmetric semi- conductor quantum well. J Appl Phys 108:034316 28. Stefan A (2007) Maier, Plasmonics: Fundamentals and Applica- tions  Springer Science 1 3 3
https://openalex.org/W2775813442
https://europepmc.org/articles/pmc5723295?pdf=render
English
null
Causes and Consequences of Flavivirus RNA Methylation
Frontiers in microbiology
2,017
cc-by
5,596
Causes and Consequences of Flavivirus RNA Methylation Shelton S. Bradrick* Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States Shelton S. Bradrick* Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States Mosquito-borne flaviviruses are important human pathogens that represent global threats to human health. The genomes of these positive-strand RNA viruses have been shown to be substrates of both viral and cellular methyltransferases. N7-methylation of the 5′ cap structure is essential for infection whereas 2′-O-methylation of the penultimate nucleotide is required for evasion of host innate immunity. N6-methylation of internal adenosine nucleotides has also been shown to impact flavivirus infection. Here, I summarize recent progress made in understanding roles for methylation in the flavivirus life-cycle and discuss relevant emerging hypotheses. Keywords: methylation, flavivirus, Zika virus, dengue virus, hepatitis C virus INTRODUCTION The genomes of mosquito-borne flaviruses are complex, multi-functional RNA molecules that must be translated, replicated and packaged in the face of innate host defenses to accomplish the ultimate viral goal: production of infectious particles to initiate new rounds of infection. Viral genomes must interface with viral proteins and host machinery to accomplish these critical tasks. Such interactions are specified by RNA features within viral genomes, including sequences and secondary/tertiary structures, and trans-acting factors that recognize these cis-acting features (Campos et al., 2017). In addition to RNA sequence and structure, covalent modifications of individual nucleotides represent another layer of cis-acting features that have been shown to impact RNA function (Saletore et al., 2012). An RNA modification fundamental to flavivirus infection is methylation, as evidenced by the existence of virus-encoded RNA methyltransferases (MTase) (Dong et al., 2014). Moreover, a few recent studies implicate flavivirus genomes to be functionally methylated by host enzymes. In this review I summarize the current state of knowledge of flavivirus RNA methylation as well-effects of RNA methylation on flavivirus infection. Edited by: Encarna Martinez-Salas, Centro de Biología Molecular Severo Ochoa (CSIC), Spain Reviewed by: Isabelle Imbert, Aix-Marseille University, France Graham John Belsham, Technical University of Denmark, Denmark *Correspondence: Shelton S. Bradrick ssbradri@utmb.edu Specialty section: This article was submitted to Virology, a section of the journal Frontiers in Microbiology Received: 06 October 2017 Accepted: 16 November 2017 Published: 05 December 2017 Citation: Bradrick SS (2017) Causes and Consequences of Flavivirus RNA Methylation. Front. Microbiol. 8:2374. doi: 10.3389/fmicb.2017.02374 Edited by: Encarna Martinez-Salas, Centro de Biología Molecular Severo Ochoa (CSIC), Spain Reviewed by: Isabelle Imbert, Aix-Marseille University, France Graham John Belsham, Technical University of Denmark, Denmark *Correspondence: Shelton S. Bradrick ssbradri@utmb.edu y y Multiple flaviviruses transmitted by arthropods represent serious human health concerns. These include yellow fever virus (YFV), West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis virus (JEV) and the four serotypes of dengue viruses (DENV) which are the most prevalent, causing nearly 100 million symptomatic infections world-wide (Bhatt et al., 2013). These viruses, comprising part of the flavivirus genus, belong to the Flaviviridae which includes the significant blood-borne human pathogen within the hepacivirus genus, hepatitis C virus (HCV). The genomes of viruses within this family share a similar organization: each contains a single open reading frame flanked by untranslated regions (UTRs) of various sequence, length and structure. The viral UTRs contain functional RNA elements that control viral translation and RNA synthesis (Garcia- Blanco et al., 2016). Unique to members of the flavivirus genus is the presence of a so called “cap” structure at the 5′ end of the genome. As discussed in detail below, methylation of the cap structure and the adjacent penultimate nucleotide of the viral genome critically promotes virus infection by multiple mechanisms. In contrast, HCV, the most prominent member of the hepacivirus genus, is characterized by an uncapped genome that contains an internal ribosome entry site within the 5′ UTR (Tsukiyama-Kohara et al., 1992). Specialty section: This article was submitted to Virology, a section of the journal Frontiers in Microbiology Received: 06 October 2017 Accepted: 16 November 2017 Published: 05 December 2017 Keywords: methylation, flavivirus, Zika virus, dengue virus, hepatitis C virus MINI REVIEW published: 05 December 2017 doi: 10.3389/fmicb.2017.02374 ROLES FOR METHYLATION AT THE 5′ END OF THE FLAVIVIRUS GENOME Cellular mRNAs are modified in the nucleus with a 7- methylguanosine (m7GpppN) cap structure attached to the first base of the transcript via a 5′-5′ triphosphate linker (Figure 1; Shatkin, 1976). This occurs early after the initiation of transcription via recruitment and sequential action of capping enzymes, including an RNA triphosphatase, guanylyltransferase and N7-guanine MTase, to the C-terminal domain of elongating RNA pol II (Phatnani and Greenleaf, 2006). The RNA triphosphatase acts to remove the γ-phosphate from the 5′ nucleotide of the nascent RNA making it available for cap addition by guanylyltransferase. Methylation of the guanosine cap at N7 completes the reaction to generate a so called “type 0” cap structure (Wei C. M. et al., 1975). Importantly, in higher eukaryotic organisms the mRNA is further modified by a separate ribose MTase at the penultimate nucleotide with a 2′-O-methyl group (Figure 1; type 1 cap) and to a lesser extent at the following nucleotide (type 2 cap) (Wei and Moss, 1975). The 5′ cap structure impacts every aspect of mRNA metabolism, including splicing, nuclear export, translation, and decay (Cowling, 2010). In contrast, 2′-O-methylation is a mark that signifies an mRNA as a “self” versus foreign molecule. FIGURE 1 | Depiction of the flavivirus RNA capping and methylation pathway. Nascent flavivirus genomes initiate with a 5′ triphosphorylated adenosine that is dephosphorylated by the RNA triphosphatase (RTPase) activity of NS3. Next, the putative NS5 guanylyltransferase (GTase) attaches guanosine monophosphate (GMP) via a 5′-5′ linkage. NS5 then methylates the guanine N7 position to form the type 0 cap using S-adenosyl methionine (SAM) as a cofactor. Methyl group donation by SAM converts it to S-adenosyl homocysteine (SAH). NS5-mediated 2′-O-methylation of the adenosine nucleotide generates the type I cap structure. Finally, hypothetical m6A methylation of flaviviral RNA at the penultimate adenosine by the METTL3/14 protein complex would result in the formation m6Am. Flaviviruses do not have access to the nuclear m7G-capping machinery and instead have evolved enzymatic activities to carry out all the necessary steps to generate capped genomes. The NS5 protein, in addition to its essential RNA-dependent RNA- polymerase function, harbors guanylyltransferase and MTase enzymes (Egloffet al., 2002; Ray et al., 2006; Issur et al., 2009). NS3 is similarly multifunctional, capable of protease, helicase and RNA triphosphatase activities (Wengler and Wengler, 1993; Bartelma and Padmanabhan, 2002). Citation: Bradrick SS (2017) Causes and Consequences of Flavivirus RNA Methylation. Front. Microbiol. 8:2374. doi: 10.3389/fmicb.2017.02374 December 2017 | Volume 8 | Article 2374 1 Frontiers in Microbiology | www.frontiersin.org Bradrick Flavivirus Methylation At the level of the individual cell all Flaviviridae use a fundamental infection strategy: (i) virus particles attach to various cellular receptors and are internalized via endocytosis, (ii) endosome acidification causes fusion between the viral envelope and endosomal membrane allowing for escape of the viral nucleocapsid into the cytoplasm, (iii) the viral RNA dissociates from capsid and engages the translational machinery to synthesize viral proteins at the cytosolic face of the endoplasmic reticulum (ER), (iv) viral proteins engage the positive-strand genome to synthesize a negative strand intermediate, (v) the negative-strand asymmetrically templates the synthesis of many genomes, (vi) some of which associate with viral structural proteins and bud into the ER to form immature viral particles that (vii) transit through the golgi apparatus where they are modified by host enzymes, and finally, (viii) mature virions are secreted into the extracellular space. Note that multiple phases of the life-cycle, including translation, RNA synthesis and virus assembly, occur concurrently on separate genomes once infection is established although completion of each process is a prerequisite for the following to occur. Frontiers in Microbiology | www.frontiersin.org ROLES FOR METHYLATION AT THE 5′ END OF THE FLAVIVIRUS GENOME The latter of these catalyze the first step in capping: removal of the γ-phosphate from the 5′ adenosine of nascent viral RNAs. NS5 is then believed to cap the RNA and performs sequential methylation reactions to generate (i) m7GpppA (cap-0) and then (ii) m7GpppAm (cap-1) (Ray et al., 2006). The cis- and trans-determinants of putative guanylyltransferase activity have not been well-characterized but the MTase reactions are relatively well-understood. Unlike December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 2 Bradrick Flavivirus Methylation translation initiation may occur via a non-canonical mechanism. First, as noted above cap methylation conferred a relatively small translational advantage to WNV replicon RNAs (Ray et al., 2006). Second, depletion of eIF4E by RNA interference was reported to not affect DENV replication or protein synthesis: whereas DENV translation was reduced by ∼10%, cellular translation was reduced by 60% due to eIF4E knockdown (Edgil et al., 2006). These observations suggest that flaviviruses may use a non-canonical pathway of translation initiation that depends minimally on the presence of a 5′- m7Gppp. cellular MTase which is not believed to discriminate among different RNA substrates, the flavivirus MTase exhibits substrate specificity and will not efficiently methylate the cap of non-viral RNAs (Dong et al., 2007). Cap methylation by NS5 requires the second and third genome nucleotides to be GU and also the presence of a 5′ stem loop which is structurally conserved across all flaviviruses (Brinton and Dispoto, 1988). 2′-O-methylation requires the first two nucleotides (AG) and is enhanced by sequence within the first 20 residues of the genome. Notably, m7GpppA-RNA is strongly preferred over GpppA-RNA as a substrate for 2′-O-methylation, explaining the sequential order of 5′ end methylation reactions (Dong et al., 2008). Finally, it is hypothetically possible that cap methylation protects viral genomes from recognition by factor(s) that sense unmethylated cap structures as invading nucleic acid. No such factor has yet been identified but there are many well-defined “pattern recognition receptors” whose tasks are to detect invading non-self nucleic acids and directly or indirectly, through innate immune pathways, antagonize infection (Wu and Chen, 2014). A pertinent example of these factors is the IFIT family of proteins discussed below. y g What are the functional consequences of cap and 2′-O- methylation? Mutational analyses of the NS5 MTase have identified residues that specifically ablate 2′-O-methylation, cap- methylation, or both. ROLES FOR METHYLATION AT THE 5′ END OF THE FLAVIVIRUS GENOME Interestingly, loss of 2′-O-methylation can be tolerated whereas cap methylation is essential for infection (Zhou et al., 2007; Dong et al., 2010). In considering why cap methylation is critical, it is worthwhile to consider data from Ray et al. (2006) who measured the effects of GpppA, m7GpppA and m7GpppAm caps on translation and RNA synthesis using the WNV replicon system which encodes the Renilla luciferase (RLuc) reporter. Compared to uncapped (pppA) WNV replicon RNA, the addition of GpppA strongly (∼25-fold) enhanced the accumulation of (RLuc) at 2 h post-transfection. Surprisingly, m7GpppA enhanced replicon RNA translation by only twofold compared to unmethylated GpppA cap, and m7GpppAm did not further increase RLuc expression. Unexpectedly, no differences in replicon RNA levels were detected for the differently capped replicons. By 72 h post-transfection, each of the capped replicon RNAs produced similar RLuc, indicating that methylation is not required for initial negative-strand synthesis as this is a prerequisite for synthesis of downstream positive-strand synthesis and consequent production of RLuc. What are the functional consequences of 2′-O-methylation at the penultimate nucleotide? As noted above, loss of this methylation event does not cause virus lethality in contrast to cap methylation. Key insights into this question were made by Diamond and colleagues who observed that a 2′-O-methylation- deficient NS5 mutant (E218A) WNV lacking m7GpppAm was attenuated in immunocompetent mice and primary cells, whereas animals and cells lacking the type I interferon (IFN) receptor (IFNAR-/-) were fully susceptible to infection (Daffis et al., 2010). These authors went on to show that IFN-inducible proteins of the IFIT family (murine IFIT1 and IFIT2) disproportionately restricted WNV lacking 2′-O-methylation compared to WT virus. Some IFIT proteins have been described to inhibit translation via binding and interfering with the function of eIF3 (Hui et al., 2003), a complex of initiation factors that recruit the 40S ribosomal subunit to mRNA via interaction with eIF4G during initiation of translation (Hershey et al., 2012). More recently, several groups have identified human IFIT1 as a protein that binds directly to cap-0 and blocks translation (Kumar et al., 2014; Abbas et al., 2017), presumably by hindering access of eIF4E to the cap structure. This translational suppression coincides with an accelerated innate immune response that compromises infection (Schmid et al., 2015; Chang et al., 2016). ROLES FOR METHYLATION AT THE 5′ END OF THE FLAVIVIRUS GENOME Taken together, these reports strongly suggest that 2′-O-methylation of the cap is an epigenetic RNA modification that allows cells to differentiate self versus non-self RNAs via IFIT proteins. Clearly flaviviruses, and indeed many other types of viruses, have evolved mechanisms to evade IFIT-mediated restriction by encoding their own 2′-O-MTases. There are at least three non-mutually exclusive explanations for a cap methylation requirement by flaviviruses. First, the cap structure itself is known to protect RNA from 5′ to 3′ exonucleases such as Xrn1 (Hsu and Stevens, 1993) and likely plays a significant role in preventing viral RNA decay. However, it is not clear whether cap methylation plays a significant role in stabilizing RNA. Indeed, the human decapping protein Dcp2 cannot act on an capped RNA substrate lacking N7-methylation (Wang et al., 2002), suggesting that methylation actually enables decapping which is a prerequisite for 5′-3′ decay (Wilusz et al., 2001). Nevertheless, it is possible that cap methylation may render flaviviral genomes resistant to cellular RNases by an unknown mechanism. A role in stimulating viral translation initiation is a plausible explanation for a cap methylation requirement by flaviviruses. The canonical mRNA cap-binding protein, eIF4E, is believed to be essential for translation of most cellular mRNAs through indirect recruitment of the 40S ribosomal subunit and associated initiation factors (Hershey et al., 2012). EIF4E strongly discriminates between m7Gppp and Gppp, and early studies by Shatkin and colleagues demonstrated that methylation enhanced cap-dependent translation (Both et al., 1975; Muthukrishnan et al., 1975). There are, however, a few clues that flavivirus Frontiers in Microbiology | www.frontiersin.org ROLES FOR INTERNAL ADENOSINE METHYLATION IN INFECTION BY FLAVIVIRUSES AND HCV It has been recognized for several decades that a prominent modification to cellular mRNA across many diverse organisms is the methylation of adenosine at the N6 position (Desrosiers December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 3 Bradrick Flavivirus Methylation et al., 1974; Perry and Kelley, 1974; Zhao et al., 2016). This occurs at internal mRNA positions (m6A) and also at the penultimate nucleotide of transcripts that initiate with A (Figure 1). The latter is referred to as m6Am as it is also methylated at the 2′- hydroxyl (Wei C. et al., 1975). In the past few years research on m6A has greatly expanded and multiple studies have addressed roles of m6A in virus infection. Several methyltransferase and demethylase enzymes have been identified as well as proteins that can recognize methyl groups in RNA (Zhao et al., 2016; Meyer and Jaffrey, 2017). These factors are referred to as “writers,” “erasers,” and “readers” of m6A. A key recent innovation is the use of m6A-specific antibodies in RNA-immunoprecipitation to allow transcriptome-wide mapping of m6A locations in RNA molecules (Dominissini et al., 2012; Meyer et al., 2012; Linder et al., 2015). This has enabled identification and functional analysis of m6A sites by mutation of the low complexity consensus motif DRACH (D = G/A/U; R = G/A; H = C/A/U). antagonism of ZIKV infection by these reader proteins and YTHDF2 in particular. The authors speculated that YTHDF2 may bind to and destabilize ZIKV RNA. Finally, Lichinchi et al. (2016b) reported that ZIKV infection alters the host m6A methylome, implying that gene expression changes caused by infection may be partly due to altered m6A patterns on cellular mRNA. antagonism of ZIKV infection by these reader proteins and YTHDF2 in particular. The authors speculated that YTHDF2 may bind to and destabilize ZIKV RNA. Finally, Lichinchi et al. (2016b) reported that ZIKV infection alters the host m6A methylome, implying that gene expression changes caused by infection may be partly due to altered m6A patterns on cellular mRNA. Many open questions remain in the nascent field addressing roles for m6A in flavivirus infection. What is the mechanism by which m6A inhibits ZIKV infection? Are similar effects and modes of action operating during infection with other flaviviruses? Does m6A impact cellular innate immune responses? How does infection impact functionality of the machinery that regulates the m6A methylome? Does m6A control flavivirus infection in vivo? ROLES FOR INTERNAL ADENOSINE METHYLATION IN INFECTION BY FLAVIVIRUSES AND HCV Notably, depletion of these enzymes had no effect on HCV translation or RNA synthesis, suggesting a role for m6A in opposing a late stage of infection such as assembly or egress of infectious virus. Consistent with this idea, several known cytosolic reader proteins (YTHDF1-3) suppressed viral titers, co-immunoprecipitated HCV RNA and localized to lipid droplets which are known sites of HCV assembly (Miyanari et al., 2007). Silent mutation of four m6A sites within the envelope coding region enhanced infection, providing further evidence for a restrictive role of m6A in HCV infection. Gokhale et al. (2016) went on to map m6A in the genomes of multiple mosquito-transmitted flaviviruses, including DENV, YFV, WNV, and two divergent strains of ZIKV. Of note, this analysis revealed abundant m6A within the NS5 coding regions of these viruses. ROLES FOR INTERNAL ADENOSINE METHYLATION IN INFECTION BY FLAVIVIRUSES AND HCV In the context of HCV, how do YTHDF proteins suppress late stages of the virus life-cycle? Lichinchi et al. (2016b) observed elevated titer and viral RNA in supernatants of cells with reduced levels of METTL3/METTL14 which implies that m6A modification opposes virus infection. It should be noted that the overall effects on ZIKV production may be considered mild (< ∼2-fold), indicating that m6A acts as a moderate restriction factor for infection in vitro. Nevertheless, the relatively small effects could reflect a viral strategy that is able to counter, in part, otherwise potent restriction by m6A and trans-acting reader proteins. One hypothesis is that the subgenomic flaviviral RNA (sfRNA), a highly stable RNA fragment produced from decay of virus genomes (Pijlman et al., 2008), could act to buffer the negative impact of m6A by sequestering YTHDF reader proteins. It would be of interest, for example, to test whether strains of DENV that produce different amounts of sfRNA would be differentially susceptible to inhibition by m6A (Manokaran et al., 2015). Of course, this is only one hypothesis and there remains much work to be done to gain a thorough understanding of how flavivirus infections are affected by m6A. consensus motif DRACH (D = G/A/U; R = G/A; H = C/A/U). To date m6A mapping and some functional analyses have been performed on multiple viruses including influenza A virus (Courtney et al., 2017), human immunodeficiency virus (Kennedy et al., 2016; Lichinchi et al., 2016a; Tirumuru et al., 2016), HCV (Gokhale et al., 2016), YFV (Gokhale et al., 2016), DENV (Gokhale et al., 2016), WNV (Gokhale et al., 2016), and ZIKV (Gokhale et al., 2016; Lichinchi et al., 2016b). Relevant to this discussion are the studies conducted on Flaviviridae by Gokhale et al. (2016) and Lichinchi et al. (2016b) who characterized functional roles of m6A in HCV and ZIKV infection, respectively. To determine the effects of m6A on infection, Gokhale et al. (2016) depleted the key methylase (METTL3 plus its co-factor METTL14) and demethylases (FTO and ALKBH5) by RNA interference and assayed effects on HCV infection. Intriguingly, knockdown of METTL3/14 enhanced infection while FTO depletion correspondingly reduced infection. These results are consistent with an antiviral role for m6A in the HCV life-cycle. REFERENCES Yeast cells lacking 5′– > 3′ exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5′ cap structure. Mol. Cell. Biol. 13, 4826–4835. doi: 10.1128/MCB.13.8. 4826 Campos, R. K., Garcia-Blanco, M. A., and Bradrick, S. S. (2017). Roles of pro- viral host factors in mosquito-borne flavivirus infections. Curr. Top. Microbiol. Immunol. doi: 10.1007/82_2017_26 [Epub ahead of print]. Hui, D. J., Bhasker, C. R., Merrick, W. C., and Sen, G. C. (2003). Viral stress- inducible protein p56 inhibits translation by blocking the interaction of eIF3 with the ternary complex eIF2·GTP·Met-tRNAi. J. Biol. Chem. 278, 39477– 39482. doi: 10.1074/jbc.M305038200 Chang, D. C., Hoang, L. T., Mohamed Naim, A. N., Dong, H., Schreiber, M. J., Hibberd, M. L., et al. (2016). Evasion of early innate immune response by 2’- O-methylation of dengue genomic RNA. Virology 499, 259–266. doi: 10.1016/j. virol.2016.09.022 Issur, M., Geiss, B. J., Bougie, I., Picard-Jean, F., Despins, S., Mayette, J., et al. (2009). The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure. RNA 15, 2340–2350. doi: 10.1261/rna.1609709 Courtney, D. G., Kennedy, E. M., Dumm, R. E., Bogerd, H. P., Tsai, K., Heaton, N. S., et al. (2017). Epitranscriptomic enhancement of influenza A virus gene expression and replication. Cell Host Microbe 22, 377.e–386.e. doi: 10.1016/j. chom.2017.08.004 Kennedy, E. M., Bogerd, H. P., Kornepati, A. V. R., Kang, D., Ghoshal, D., Marshall, J. B., et al. (2016). Posttranscriptional m6 A editing of HIV-1 mRNAs enhances viral gene expression. Cell Host Microbe 19, 675–685. doi: 10.1016/j.chom.2016. 04.002 Cowling, V. H. (2010). Regulation of mRNA cap methylation. Biochem. J. 425, 295–302. doi: 10.1042/BJ20091352 Daffis, S., Szretter, K. J., Schriewer, J., Li, J., Youn, S., Errett, J., et al. (2010). 2′- O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468, 452–456. doi: 10.1038/nature09489 Kumar, P., Sweeney, T. R., Skabkin, M. A., Skabkina, O. V., Hellen, C. U. T., and Pestova, T. V. (2014). Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5′ppp- mRNAs. Nucleic Acids Res. 42, 3228–3245. doi: 10.1093/nar/gkt1321 Desrosiers, R., Friderici, K., and Rottman, F. (1974). Identification of methylated nucleosides in messenger RNA from Novikoffhepatoma cells. Proc. Natl. Acad. Sci. U. S. A. 71, 3971–3975. REFERENCES Edgil, D., Polacek, C., and Harris, E. (2006). Dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited. J. Virol. 80, 2976–2986. doi: 10.1128/JVI.80.6.2976-2986.2006 Abbas, Y. M., Laudenbach, B. T., Martínez-Montero, S., Cencic, R., Habjan, M., Pichlmair, A., et al. (2017). Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′- O methylations. Proc. Natl. Acad. Sci. U.S.A. 114, E2106–E2115. doi: 10.1073/ pnas.1612444114 Egloff, M.-P., Benarroch, D., Selisko, B., Romette, J.-L., and Canard, B. (2002). An RNA cap (nucleoside-2′-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. EMBO J. 21, 2757–2768. doi: 10.1093/emboj/21.11.2757 O methylations. Proc. Natl. Acad. Sci. U.S.A. 114, E2106–E2115. doi: 10.1073/ pnas.1612444114 Garcia-Blanco, M. A., Vasudevan, S. G., Bradrick, S. S., and Nicchitta, C. (2016). Bartelma, G., and Padmanabhan, R. (2002). Expression, purification, and characterization of the RNA 5′-triphosphatase activity of dengue virus type 2 nonstructural protein 3. Virology 299, 122–132. doi: 10.1006/viro.2002.1504 Garcia-Blanco, M. A., Vasudevan, S. G., Bradrick, S. S., and Nicchitta, C. (2016). Flavivirus RNA transactions from viral entry to genome replication Antiviral Garcia-Blanco, M. A., Vasudevan, S. G., Bradrick, S. S., and Nicchitta, C. (2016). Flavivirus RNA transactions from viral entry to genome replication. Antiviral Res. 134, 244–249. doi: 10.1016/j.antiviral.2016.09.010 onstructural protein 3. Virology 299, 122–132. doi: 10.1006/viro.20 Bhatt, S., Gething, P. W., Brady, O. J., Messina, J. P., Farlow, A. W., Moyes, C. L., et al. (2013). The global distribution and burden of dengue. Nature 496, 504–507. doi: 10.1038/nature12060 Gokhale, N. S., McIntyre, A. B. R., McFadden, M. J., Roder, A. E., Kennedy, E. M., Gandara, J. A., et al. (2016). N6 -methyladenosine in Flaviviridae viral RNA genomes regulates infection. Cell Host Microbe 20, 654–665. doi: 10.1016/j. chom.2016.09.015 Both, G. W., Banerjee, A. K., and Shatkin, A. J. (1975). Methylation-dependent translation of viral messenger RNAs in vitro. Proc. Natl. Acad. Sci. U.S.A. 72, 1189–1193. doi: 10.1073/pnas.72.3.1189 Hershey, J. W. B., Sonenberg, N., and Mathews, M. B. (2012). Principles of translational control: an overview. Cold Spring Harb. Perspect. Biol. 4:a011528. doi: 10.1101/cshperspect.a011528 Brinton, M. A., and Dispoto, J. H. (1988). Sequence and secondary structure analysis of the 5′-terminal region of flavivirus genome RNA. Virology 162, 290–299. doi: 10.1016/0042-6822(88)90468-0 Hsu, C. L., and Stevens, A. (1993). FUNDING infection with virulent viruses (Li et al., 2013; Züst et al., 2013). Thus, understanding the basic molecular mechanisms of flavivirus biology will hopefully lead to measures that reduce the burden caused by these viruses. The author acknowledges Mariano Garcia-Blanco and the University of Texas Medical Branch for support. ACKNOWLEDGMENT The author thanks Gaddiel Galarza-Munoza and Rafael Campos for thoughtful comments. The author confirms being the sole contributor of this work and approved it for publication. CONCLUSION Flavivirus RNA methylation critically impacts infection. Cap methylation (m7GpppA) is essential for infection, at least in part due to its role in stimulating virus translation. Methylation at the 2′-hydroxyl of the penultimate adenosine (m7GpppAm) is inessential for viability but allows the virus to escape the inhibitory actions of IFIT proteins and likely other factor(s) (Szretter et al., 2012). In contrast, internal m6A modifications are somewhat deleterious to ZIKV and HCV infections in vitro although there is much to be learned regarding roles for m6A in infection. Studies addressing viral RNA methylation are informative with respect to basic virus biology but may also allow development of approaches to control infections by pathogenic flaviruses for which there are no currently available therapeutics. Drugs specifically targeting flavivirus MTase enzymes could be potent antivirals for the treatment of patients with acute infections. Moreover, mutant flaviviruses lacking 2′-O-MTase activity have shown promise as candidate vaccine strains because they are attenuated yet induce robust immunity to heterologous In their companion article to the Gokhale et al. (2016) study, Lichinchi et al. (2016b) and colleagues mapped locations m6A on ZIKV RNA and investigated the roles of readers, writers and erasers in infection. Depletion of METTL3 or METTL14 enhanced ZIKV infection in 293T cells whereas ALKBH5 and, to a lesser extent, FTO knockdown reduced infection. Moreover, YTDHF1/2 expression negatively correlated with ZIKV RNA levels released from infected cells, suggesting December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 4 Bradrick Flavivirus Methylation REFERENCES 33, 319–342. doi: 10.1146/annurev-cellbio-100616- 060758 Wang, Z., Jiao, X., Carr-Schmid, A., and Kiledjian, M. (2002). The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc. Natl. Acad. Sci. U.S.A. 99, 12663–12668. doi: 10.1073/pnas.192445599 Meyer, K. D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C. E., and Jaffrey, S. R. (2012). Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646. doi: 10.1016/j.cell.2012. 05.003 Wei, C., Gershowitz, A., and Moss, B. (1975). N6, O2′-dimethyladenosine a novel methylated ribonucleoside next to the 5′ terminal of animal cell and virus mRNAs. Nature 257, 251–253. doi: 10.1038/257251a0 mRNAs. Nature 257, 251–253. doi: 10.1038/257251a0 Miyanari, Y., Atsuzawa, K., Usuda, N., Watashi, K., Hishiki, T., Zayas, M., et al. (2007). The lipid droplet is an important organelle for hepatitis C virus production. Nat. Cell Biol. 9, 1089–1097. doi: 10.1038/ncb1631 Wei, C. M., Gershowitz, A., and Moss, B. (1975). Methylated nucleotides block 5′ terminus of HeLa cell messenger RNA. Cell 4, 379–386. doi: 10.1016/0092- 8674(75)90158-0 Muthukrishnan, S., Both, G. W., Furuichi, Y., and Shatkin, A. J. (1975). 5′-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation. Nature 255, 33–37. doi: 10.1038/255033a0 Wei, C. M., and Moss, B. (1975). Methylated nucleotides block 5′-terminus of vaccinia virus messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 72, 318–322. doi: 10.1073/pnas.72.1.318 Perry, R. P., and Kelley, D. E. (1974). Existence of methylated messenger RNA in mouse L cells. Cell 1, 37–42. doi: 10.1016/0092-8674(74)90153-6 Wengler, G., and Wengler, G. (1993). The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. Virology 197, 265–273. doi: 10.1006/ viro.1993.1587 Phatnani, H. P., and Greenleaf, A. L. (2006). Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 20, 2922–2936. doi: 10.1101/gad.147 7006 Wilusz, C. J., Wormington, M., and Peltz, S. W. (2001). The cap-to-tail guide to mRNA turnover. Nat. Rev. Mol. Cell Biol. 2, 237–246. doi: 10.1038/3506 7025 Pijlman, G. P., Funk, A., Kondratieva, N., Leung, J., Torres, S., van der Aa, L., et al. (2008). A highly structured, nuclease-resistant, noncoding RNA produced by flaviviruses is required for pathogenicity. Cell Host Microbe 4, 579–591. doi: 10.1016/j.chom.2008.10.007 Wu, J., and Chen, Z. J. (2014). Innate immune sensing and signaling of cytosolic nucleic acids. Annu. Rev. Immunol. 32, 461–488. doi: 10.1146/annurev- immunol-032713-120156 Zhao, B. S., Roundtree, I. A., and He, C. (2016). Post-transcriptional gene regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42. REFERENCES doi: 10.1073/pnas.71.10.3971 Dominissini, D., Moshitch-Moshkovitz, S., Schwartz, S., Salmon-Divon, M., Ungar, L., Osenberg, S., et al. (2012). Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206. doi: 10.1038/ nature11112 Li, S.-H., Dong, H., Li, X.-F., Xie, X., Zhao, H., Deng, Y.-Q., et al. (2013). Rational design of a flavivirus vaccine by abolishing viral RNA 2′-O methylation. J. Virol. 87, 5812–5819. doi: 10.1128/JVI.02806-12 Lichinchi, G., Gao, S., Saletore, Y., Gonzalez, G. M., Bansal, V., Wang, Y., et al. (2016a). Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells. Nat. Microbiol. 1, 16011. doi: 10.1038/nmicrobiol. 2016.11 Dong, H., Chang, D. C., Xie, X., Toh, Y. X., Chung, K. Y., Zou, G., et al. (2010). Biochemical and genetic characterization of dengue virus methyltransferase. Virology 405, 568–578. doi: 10.1016/j.virol.2010.06.039 Lichinchi, G., Zhao, B. S., Wu, Y., Lu, Z., Qin, Y., He, C., et al. (2016b). Dynamics of human and viral RNA methylation during Zika virus infection. Cell Host Microbe 20, 666–673. doi: 10.1016/j.chom.2016.10.002 Dong, H., Fink, K., Zust, R., Lim, S. P., Qin, C.-F., and Shi, P.-Y. (2014). Flavivirus RNA methylation. J. Gen. Virol. 95, 763–778. doi: 10.1099/vir.0.062 208-0 Dong, H., Ray, D., Ren, S., Zhang, B., Puig-Basagoiti, F., Takagi, Y., et al. (2007). Distinct RNA elements confer specificity to flavivirus RNA cap methylation events. J. Virol. 81, 4412–4421. doi: 10.1128/JVI.02455-06 Linder, B., Grozhik, A. V., Olarerin-George, A. O., Meydan, C., Mason, C. E., and Jaffrey, S. R. (2015). Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767–772. doi: 10.1038/nmeth. 3453 Dong, H., Ren, S., Li, H., and Shi, P.-Y. (2008). Separate molecules of west nile virus methyltransferase can independently catalyze the N7 and 2′-O methylations of viral RNA cap. Virology 377, 1–6. doi: 10.1016/j.virol.2008.04.026 Manokaran, G., Finol, E., Wang, C., Gunaratne, J., Bahl, J., Ong, E. Z., et al. (2015). Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 5 Bradrick Flavivirus Methylation for epidemiological fitness. Science 350, 217–221. doi: 10.1126/science.aab 3369 for epidemiological fitness. Science 350, 217–221. doi: 10.1126/science.aab 3369 Tsukiyama-Kohara, K., Iizuka, N., Kohara, M., and Nomoto, A. (1992). Internal ribosome entry site within hepatitis C virus RNA. J. Virol. 66, 1476–1483. Meyer, K. D., and Jaffrey, S. R. (2017). Rethinking m6 a readers, writers, and erasers. Annu. Rev. Cell Dev. Biol. REFERENCES doi: 10.1038/nrm.2016.132 Ray, D., Shah, A., Tilgner, M., Guo, Y., Zhao, Y., Dong, H., et al. (2006). West nile virus 5′-cap structure is formed by sequential guanine N-7 and ribose 2′-O methylations by nonstructural protein 5. J. Virol. 80, 8362–8370. doi: 10.1128/JVI.00814-06 Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., et al. (2007). Structure and function of flavivirus NS5 methyltransferase. J. Virol. 81, 3891–3903. doi: 10.1128/JVI.02704-06 Saletore, Y., Meyer, K., Korlach, J., Vilfan, I. D., Jaffrey, S., and Mason, C. E. (2012). The birth of the Epitranscriptome: deciphering the function of RNA modifications. Genome Biol. 13, 175. doi: 10.1186/gb-2012-13- 10-175 Züst, R., Dong, H., Li, X.-F., Chang, D. C., Zhang, B., Balakrishnan, T., et al. (2013). Rational design of a live attenuated dengue vaccine: 2′-O-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques. PLOS Pathog. 9:e1003521. doi: 10.1371/journal.ppat.1003521 Schmid, B., Rinas, M., Ruggieri, A., Acosta, E. G., Bartenschlager, M., Reuter, A., et al. (2015). Live cell analysis and mathematical modeling identify determinants of attenuation of dengue virus 2′-O-methylation mutant. PLOS Pathog. 11:e1005345. doi: 10.1371/journal.ppat.1005345 Conflict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Shatkin, A. J. (1976). Capping of eucaryotic mRNAs. Cell 9, 645–653. doi: 10.1016/ 0092-8674(76)90128-8 Szretter, K. J., Daniels, B. P., Cho, H., Gainey, M. D., Yokoyama, W. M., Gale, M., et al. (2012). 2′-O methylation of the viral mRNA cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo. PLOS Pathog. 8:e1002698. doi: 10.1371/journal.ppat.1002698 Copyright © 2017 Bradrick. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Tirumuru, N., Zhao, B. S., Lu, W., Lu, Z., He, C., and Wu, L. (2016). N6 - methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. Frontiers in Microbiology | www.frontiersin.org REFERENCES Elife 5:e15528. doi: 10.7554/eLife.15528 December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 6
https://openalex.org/W2601523096
https://journal.rniito.org/jour/article/download/488/481
unk
null
CAPABILITIES OF COMPLEX SONOGRAPHIC IMAGING FOR BENIGN SOFT TISSUE TUMORS OF FOOT AND ANKLE
Travmatologiâ i ortopediâ Rossii
2,010
cc-by
4,258
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h  )  %! "    " ($* ( ! ) !%6+689,8 *    > +>79, " $*!" $  ' 2P+PP9, *  !* !% " 2<+<69, *   ! ! "  (      ! ( F+FF9,Q!#"!%  *" #'   g   &  !  !5   ) $!% %)# '  !%(  *"   !   !  4 )"!#" (  "! ' !%#  " *#  2!%  "  "!%  &STUVWXVIYZM[\7;F+V[I]I^N _  ,HW`ab>GGTH+`I^IK\cdcIefK[e 1- , !  (" <6._*  )  ( !*  (" ! $/ * $ &) ('  ! !%  !    1 !)  !% $!!     ! "  " !!% !%     ( (!#("  &! &  !    ) "*( *   !% "*  " !    +g,    ! !  % " ) 23 "   & ( !    h!%  &! "  ( )"" !%!   !;" >   (  . "! " !     !# !   ! "  !%      "!%        !   " (  &!     " (  "!%(! !%(  & ( ?! "    !%  " $*&    ! & &)! ( *      "  ?! " !  & #  "!   !             "!%    ! "#'23 i! !*  $+  !%   !%, + (j  (   (j (, ! !     !  ' %    *   #"  P &      ! ! )')#' +  "     , 4& ! !   )"   4& % &   ! *  ) g *OR+;F <9,Q OP+R9,Q  !% O>+6 ;9,Q"  !+"   '  $  !  ,O;+F9,Q4 &" !%($!  !% *   ! " &! " ( !# !" !     !   !%    !  * !  ! " 2F;+>>9, *  !  $*"! ! "  8!#" &+;89, * !%#  &  &  ( &&"! !#  ! ( &   &" ! !% ! %./00 " ($  "!     '       >7Q<7!    '   )   "!%&    " ( $*      !%  *   " $ * i "!% j!   !  "         "   k    " ! !" :P >=" ($ !" ;; *    "  & !) "!  !  4& ""     !%   +lmG GG;, !*    * "!%    " ($*+;,@"  !'   !($*F +FF9, *  !*   !%   !                     >8 *   /??0@ // " 23A)!   !%*@ @B/CDEFGG> FGG<  &;;+FF9, !    !  " $+! %HIJJIKLMNI,O ;<+P79,Q "  &   (   ! >Q    7Q$ 6Q$! O< +;> F9, ! Q! (!!                          #'& !  &  ($*!%&  &  "   " ! !   ' %! "      # "  "23! "   !*# !        !  !# & (  ) !# &  ) ?  !    ##& ! " &   (  ! "   !%   " *  " !    +g,4   " !    +5, ( * "  ! !    (& (!  _ Q4 %   "    &!  !)   )  ")) (!   *& _!   " ! )    !! &"  "@" )) )#'( "    !    !  %  ! " !   &  ! ( !    & !%"& !% !      "&  &! &:R=                                                                          27 *  " $ +P,  &! &  " !     ! (    !      4&     ""(   ( (    (4&   ) & ! 4& ("& g  (  !  ) ( "!  "!%  % " (A  #'(E"" &!#" &$    +p\YIK,!* ! % !%  ! "  !  F ( "&!"7    ! " )"  0! "  ! $*  *   !* !% > *  +F,2 & *     !  n#4& %  ! ' ! "& (   " ! & "! ""! "   ! 4& (   g        ! )! %  "  ! ! ( !  )  !  & &   ! o& & "   '   !%   '    !  ! " !       !  !  "  %#                      !        " "  # $   "      "                         !        " "  # $   "      "  !        " "  # $   "      "  !        " "  # $   "      "                                            %        &   # '(!   ")    "               &)*   $&+,-.&/0)                          %        &   # '(!   ")    "               &)*   $&+,-.&/0)          1 !% !* !%P *    " 4& (    4&     ) ( ( %#  "   F! & !       !  !% (   #'   !%# +7, k! !" 6  *  F! &!* !%    4&          )#'&  (( !(!  4&  &$& !    (    &! & g (   &   ! @"  '  23Q )% "$$  * % ! "   !% !  Q4 4&   4$$ "   "! !?"  !   " ! #  !   4&  & !# (  !"! "    !   $ !% (! ) !  %  &!%:R= "  )  " )"%   $    !%  ( A !% *E " :; <= !   !#"  4&"  !%($!  !%*  (   " !     !%   !% ! "   " !)  ! "      * !  ! !%   ! "  23? !("!% (  & (!   ! 23  ) (   ! &) ! ()#'&& ( !#  ! " )"  ! "    ! ! "   )   *! +>, q! (!! * !  ! %7 ! 4&      P!  &4!   &!!    !* ! %   *) !  !%   "&!! &)!  _   ! R *    (   @!* ! % 4&   ! !                         4& ( !" (   ! !% (  (                         1          &    &         # "     &$&         1          &    &         # "     &$&    2 3      "                  1          &    &         # "     &$&     1          &    &         # "     &$&    1          &    &         # "     &$&      4         567         2 3      "           2 3      "      F! &!' 4&   !F  & )(    ! <) ( 4& ( !*  !'    !%(! "!  "A   &!!      2    * 373  "   & 3 8       !    ! !" !%  "       & &"! "   & (!         !    !  " +lmG GG;, h   !%%!%   "  )  (!g  ! "  !689  *$ %Q87 R9 "   %Q8P >9    " *! "   ! #' !% ! %   "   "!%        !%   2    * 373  "   & 3 8              !   "#$% &''&"() r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`57:?B3G<H:57@ 1M45"//]"V:3&/ H"F0'"00 ]#H833A YEF58K7GK83MU9KU3:9=434783U37589:U<2 !YE#H833A"F@G38243?@G8$Y394RG45 &''W((]? A /D:3GR477G aCF38<785;GQ5:M87:9G9$U375:9:<:>58?@A 549U379!aCD:3GR477G aJUbG DOU9@45 JCU9K852G< !!682G:3C42"///"V:3/\ HW"F(0"(0(          E  !"    4& (       !;   !   ! %   "  ! "   "  " !  "    ! (   !%" F! &" A &!!  (E !*#'( "&!!  &)!     ! &)!   (+R,      2!%  #"& ! !#"!  >! &?"  ! !  !% 4&     )   4& " ! *(+<,  F! " &&!*  ! &"( (!+ ' (&",   4         567                                              !"#$%&'()*+,-./!0 12  3   .4. .5     0 126 7 8 9:;. dXsZY\eLIt\ XSpMKnNLuMt HvpMKnNLuMt\                   
https://openalex.org/W2214099170
https://www.thno.org/v05p1419.pdf
English
null
High Efficiency Molecular Delivery with Sequential Low-Energy Sonoporation Bursts
Theranostics
2,015
cc-by
6,479
Received: 2015.06.24; Accepted: 2015.08.19; Published: 2015.10.18 Received: 2015.06.24; Accepted: 2015.08.19; Published: 2015.10.18 Abstract Microbubbles interact with ultrasound to induce transient microscopic pores in the cellular plasma membrane in a highly localized thermo-mechanical process called sonoporation. Theranostic ap- plications of in vitro sonoporation include molecular delivery (e.g., transfection, drug loading and cell labeling), as well as molecular extraction for measuring intracellular biomarkers, such as proteins and mRNA. Prior research focusing mainly on the effects of acoustic forcing with poly- disperse microbubbles has identified a “soft limit” of sonoporation efficiency at 50% when including dead and lysed cells. We show here that this limit can be exceeded with the judicious use of monodisperse microbubbles driven by a physiotherapy device (1.0 MHz, 2.0 W/cm2, 10% duty cycle). We first examined the effects of microbubble size and found that small-diameter mi- crobubbles (2 µm) deliver more instantaneous power than larger microbubbles (4 & 6 µm). However, owing to rapid fragmentation and a short half-life (0.7 s for 2 µm; 13.3 s for 6 µm), they also deliver less energy over the sonoporation time. This translates to a higher ratio of FITC-dextran (70 kDa) uptake to cell death/lysis (4:1 for 2 µm; 1:2 for 6 µm) in suspended HeLa cells after a single sonoporation. Sequential sonoporations (up to four) were consequently em- ployed to increase molecular delivery. Peak uptake was found to be 66.1 ± 1.2% (n=3) after two sonoporations when properly accounting for cell lysis (7.0 ± 5.6%) and death (17.9 ± 2.0%), thus overcoming the previously reported soft limit. Substitution of TRITC-dextran (70 kDa) on the second sonoporation confirmed the effects were multiplicative. Overall, this study demonstrates the possibility of utilizing monodisperse small-diameter microbubbles as a means to achieve mul- tiple low-energy sonoporation bursts for efficient in vitro cellular uptake and sequential molecular delivery. Key words: microbubbles, ultrasound contrast agents, drug delivery, drug release, cell uptake, cell viability Ivyspring International Publisher 2015; 5(12): 1419-1427. doi: 10.7150/thno.13033 Theranostics 2015, Vol. 5, Issue 12 Ivyspring International Publisher High Efficiency Molecular Delivery with Sequential Low-Energy Sonoporation Bursts 1. Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309 1. Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309 p g g, y , , 2. Department of Mechanical and Aerospace Engineering, University of Colorado, Colorado Springs, CO 80918 p g g y 2. Department of Mechanical and Aerospace Engineering, University of Colorado, Colorado Springs, CO 80918 g g y 2. Department of Mechanical and Aerospace Engineering, University of Colorado, Colo 3. Department of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO 80045  Corresponding author: Mark A Borden, PhD, Department of Mechanical Engineering, University of Colorado, 1111 Engineering Drive, Boulder, CO 80309-0427. Phone: 303.492.7750; Fax: 303.492.3498; Email: mark.borden@colorado.edu  Corresponding author: Mark A Borden, PhD, Department of Mechanical Engineering, University of Colorado, 1111 CO 80309-0427. Phone: 303.492.7750; Fax: 303.492.3498; Email: mark.borden@colorado.edu © 2015 Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions. © 2015 Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in w See http://ivyspring.com/terms for terms and conditions. 1419 1419 Theranostics 2015, Vol. 5, Issue 12 Materials 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoe- thanolamine-N-[(polyethylene glycol)-2000] (DSPE- PEG2000) lipid powder was obtained from Avanti Polar Lipids (Alabaster, AL, USA) for microbubble prepa- ration. Perfluorobutane (PFB) gas was obtained from FluoroMed (Round Rock, TX, USA). HeLa cells (ATCC, Manassas, VA, USA) were cultured in DMEM solution (Fisher Scientific, Hampton, NH, USA). 70 kDa FITC-dextran (Sigma-Aldrich, St. Louis, MO, USA) was used for sonoporation assays. Plasmid EGFP-C3 (Clontech, Mountain View, CA, USA) was used for transfection assays, and dead cells were stained with ethidium homodimer-1 or lysine-binding dye (Invitrogen, Grand Island, NY, USA). One major challenge for sonoporation has been increasing cell uptake efficiency. A review by Liu et al. revealed that out of 26 in vitro sonoporation studies spanning over a decade, none had demonstrated cel- lular uptake in excess of 50% when accounting for cell lysis and death in their measurements (12). The goal of our study was to surpass this “soft limit” by achieving at least 50% uptake efficiency with mono- disperse microbubbles, while properly accounting for cells that were lysed or otherwise lost during han- dling. Prior in vitro sonoporation studies have focused mainly on optimization of acoustic parameters with commercially available ultrasound contrast agents, which are highly polydisperse in size (13–16). Recent studies, however, have demonstrated microfluidic (17–24) and centrifugal size sorting (25) methods to produce monodisperse microbubbles of select size. In vivo studies have shown dramatic effects of mono- disperse microbubble size on imaging (26) and ther- apeutic (27) performance. This is not surprising: mi- crobubble size is known to affect resonance, oscilla- tion power and stability (28–30). We therefore chose to focus on microbubble size as the key parameter to optimize in vitro sonoporation efficiency, using a new high-throughput cartridge/bracket system with commonly employed ultrasound parameters (1 MHz, 0.53 MPa peak negative pressure) delivered by an inexpensive physiotherapy device. Introduction Sonoporation uses acoustically mediated cavita- tion of microbubbles to porate nearby cells through the induction of micro/nanoscale ruptures in the plasma membrane for intracellular delivery of diverse payloads, such as nucleic acids and nanoparticles (1). Unlike electroporation, which uses strong electric field gradients that act on all structures throughout the sample volume, sonoporation generates localized thermal and mechanical effects that function on cells regardless of cell-media composition. Engineering of the microbubbles allows for more advanced effects, such as targeting of surface proteins and co-imaging with diagnostic ultrasound. One popular application of in vitro sonoporation http://www.thno.org Theranostics 2015, Vol. 5, Issue 12 1420 has been cellular transfection with pDNA (2,3). However, sonoporation offers many more theranostic applications, such as ex vivo transfer of therapeutic and imaging molecules for in vivo transplantation (4–7) (i.e., cell labeling), and transient pore formation for the release and detection of intracellular proteins and mRNA (8,9). Additionally, in vitro sonoporation can serve as a surrogate for drug testing on in vivo disease models (10,11). cells. In the second part, we used this knowledge to engineer a method of sequential sonoporations to overcome the 50% soft limit on uptake efficiency. Microbubble preparation and characterization Lipid-encapsulated perfluorocarbon microbub- bles were generated via sonication of DSPC and DSPE-PEG2000 lipid suspension at a concentration of 2 mg/mL and molar ratio of 9:1 in phosphate buffered saline (PBS) solution, in the presence of PFB gas. Mi- crobubbles with median diameters of 2, 4 and 6 µm were separated based on size using differential cen- trifugation (25,26). Microbubble size and concentra- tion were measured with an Accusizer 780 (PSS Ni- comp, Santa Barbara, CA, USA) and a Multisizer 3® (Beckman Coulter, Brea, CA, USA). Cell culture and handling HeLa cells were procured from ATCC (Cat no. CCL-2) and thawed from 10% DMSO solution. Thawed cells were cultured at 37 oC, 5% CO2 in DMEM with 10% fetal bovine serum supplement (Fisher Scientific, Hampton, NH) and 1% penicil- lin/streptomycin (Sigma-Aldrich, St. Louis, MO) and passaged until healthy growth patterns were ob- served. Cells were trypsinized and harvested at 70% confluence for use in sonoporation studies. Cell con- centrations during sonoporation were held constant at 5x106 ± 2.5x105 for each experimental group. In vitro sonoporation system (a) The in vitro sonoporation apparatus comprised an immersed, sealed treatment cartridge, placed a fixed distance from the ultrasonic transducer. (b) The chamber volume was 0.2 mL, with a total transverse internal width of 2.5 mm. Placement of acoustically transparent polystyrene windows (0.1-mm thick, ~2% reduction in PNP) on the front and back of the chamber allowed ultrasound to travel through with minimal reflections, and a 2-mm magnetic stir bar maintained constant fluid flow in the chamber. each microbubble size were then integrated over time to obtain the energy delivered to each cell suspension, using the following equation: designed for immersion in water up to 40 °C and al- lowed for the exchange of cartridges with minimal variation in cartridge position relative to the trans- ducer. De-ionized and de-gassed water held at a temperature of 37 °C was used as an acoustic medi- um. …(2) ∑ ∆ × = T sono t W E 0 where Δt is the time step (1.0 s) and T is the total ex- posure time of 120 s. In vitro sonoporation system A sonoporation system was constructed to in- corporate a 1-inch diameter ultrasound transducer from a Dynatron® 125 (Dynatronics, Salt Lake City, Utah) applying acoustic pulses to a removable 3-D printed sample holder held at a fixed location by a bracket assembly (34) (Fig. 1). The ultrasonic output from the transducer was characterized by a needle hydrophone (HNC-0200, Onda Corp., Sunnyvale, CA) and shown to output a 1.0 MHz, 0.53 ± 0.03 MPa peak negative pressure sound wave at a setting of 2.0 W/cm2, and 1000 cycles per pulse at 100 Hz pulse repetition frequency at a setting of 10% duty cycle (Fig. S1). The cartridge was designed with two acous- tically transparent polystyrene windows and an inte- rior bevel to minimize cell retention, as well as a 2-mm magnetic stir bar. Window attenuation and waveform distortion were inspected and found to be minimal at a distance of 5 mm from the trailing acoustic window. The polypropylene bracket was Currently, the effect of microbubble size on sonoporation is limited to observations on individual cells. For example, research by Zhou et al. (31,32) demonstrated that larger microbubbles formed larger pores, potentiating the possibility of delivering larger drug molecules than previously possible with com- mercially available small-diameter microbubble for- mulations (33). For cell suspensions, however, the situation is made more complex by the three dimen- sional structure of the cell/microbubble suspension, the transient nature of microbubbles and other effects. We thus structured the first part of our study to ex- amine the influence of microbubble size (2, 4 and 6 µm diameter) on dynamics and stability, and then explore the effects on cell uptake (% treated cells; live, FITC positive), death (% lysine-binding-dye stained cells), lysis (% reduction in total cell count) and unaffected http://www.thno.org http://www.thno.org 1421 Theranostics 2015, Vol. 5, Issue 12 Figure 1. (a) The in vitro sonoporation apparatus comprised an immersed, sealed treatment cartridge, placed a fixed distance from the ultrasonic transducer. (b) The chamber volume was 0.2 mL, with a total transverse internal width of 2.5 mm. Placement of acoustically transparent polystyrene windows (0.1-mm thick, ~2% reduction in PNP) on the front and back of the chamber allowed ultrasound to travel through with minimal reflections, and a 2-mm magnetic stir bar maintained constant fluid flow in the chamber. Figure 1. Microbubble survival during exposure to ul- trasound Microbubble concentration over time was measured under typical sonoporation conditions (37 °C, 1.0 MHz, 2.0 W/cm2, 10% duty cycle, 0-2 min ex- posure time) with experimental groups of 2, 4 and 6-µm diameter microbubbles at an initial concentra- tion of 108/mL. Three 2-μL microbubble samples were taken for each size at 0, 5, 30 and 120 s for a total of 12 samples measured. A mono-exponential function was fit to the resulting concentration-time curves to de- termine half-life and to estimate concentrations be- tween the measured time points. Cellular sonoporation assay Doinikov’s equation for the time-averaged non-dimensional power output was used to estimate the relative power delivered by each microbubble per second under these acoustic forcing conditions (30): FITC-dextran (70 kDa, 0.77 mg/mL, Sig- ma-Aldrich, St. Louis, MO), a long-chain sugar with a fluoroscein isothiocyanate moiety, was chosen as the indicator of cell permeabilization and uptake (33). 20 µL of FITC-dextran (7.7 mg/mL) was added to 180 µL of microbubble/cell suspension (11 μM final concen- tration). The sonoporated volume remained 200 µL for all sonoporation conditions. Microbubble concentra- tion was fixed at 5x107 for each group size (2, 4 and 6 μm diameter). Higher microbubble concentrations were avoided due to the high viscosity exhibited by larger size groups. Cell/microbubble/FITC-dextran suspensions were then subjected to ultrasound (1 MHz, 2.0 W/cm2, 10% duty cycle) in the sonoporation system. Treated cell samples were removed from the …(1) ∫ ×       − ∆ = T dt t C R t R t R W 0 2 0 0 , ) ( 1 ) ( 1 ) ( where Δt is one second (1000 cycles per pulse at 100 Hz pulse repetition frequency), R0 is the initial mi- crobubble radius (1, 2 or 3 µm), C(t) is the dynamic microbubble concentration obtained from persistence data and R(t) is the dynamic radius, which was ob- tained from Marmottant’s model for the experimental acoustic driving conditions (Supplemental: Theoretical radial dynamics calculations). These power values for http://www.thno.org 1422 Theranostics 2015, Vol. 5, Issue 12 homdimer-1) and gating for dead cells (FL4, 630 nm, far-red lysine-binding dye) (Fig. S2, Flow cytometric analysis). cartridges and washed three times at 500 RCF in 15-mL cell media tubes. Anti-fluorescein (Invitrogen, Grand Island, NY) was added at a volume fraction of 2 μL in 1 mL (2 μg/mL) to quench residual fluores- cence on exterior cell surfaces after the third wash to eliminate FITC fluorescence from non-permeabilized cells. Finally, ethidium homodimer-1 was applied at 0.2 M quantity to each sample to identify dead cells and minimize false positives from auto-fluorescence. Characterization of size-isolated microbubbles Each sample of size-isolated microbubbles was shown to maintain a consistent median diameter over the course of experimentation, with average median diameters of 1.7, 4.1 and 6.4 μm, and average mode diameters of 1.7, 4.3 and 6.5 μm (Table 1). These three sizes are referred to as 2, 4 and 6 μm microbubble groups, respectively. Table 1. Size characteristics of microbubbles used in this study. MB size class Mean (μm)* Median (μm) Mode (μm) 2-µm Accusizer 1.53 ± 0.16 1.44 ± 0.06 1.70 ± 0.19 Multisizer 1.80 ± 0.02 1.73 ± 0.03 1.74 ± 0.12 4-µm Accusizer 3.50 ± 0.25 3.67 ± 0.15 4.20 ± 0.20 Multisizer 4.07 ± 0.09 4.13 ± 0.05 4.33 ± 0.07 6-µm Accusizer 5.58 ± 0.28 5.67 ± 0.22 6.36 ± 0.79 Multisizer 6.24 ± 0.11 6.36 ± 0.05 6.46 ± 0.14 *Measurements were made for n ≥ 3 batches, with at least 3 measurements per batch. All size groups were found to be significantly different from each other for both sizing systems (p < 0.01). Table 1. Size characteristics of microbubbles used in this study. *Measurements were made for n ≥ 3 batches, with at least 3 measurements per batch. All size groups were found to be significantly different from each other for both sizing systems (p < 0.01). Sequential sonoporations Separate samples were prepared for each num- ber of sequential sonoporations (n=3); thus, three samples were prepared for one sonoporation and an- alyzed, then three more samples were prepared for two sonoporations and analyzed after the second sonoporation, and so on. Total cell count was obtained before the first sonoporation and after the last sono- poration by removing 50 µL aliquots for flow cy- tometric measurement. For a single sonoporation, the samples were sonicated for ten seconds with 2-µm microbubbles (108/mL in 200 µL). For additional sonoporations, 2×107 microbubbles were added to the 200 µL sonoporation volume (108 microbubbles/mL). The total volume of microbubbles added after the fourth sonoporation (<12 µL) was considered minimal compared to the total sonoporation volume (200 µL). Sonoporations were repeated up to four times, then cells were removed from the cartridge and washed three times at 500 RCF in 1.5-mL tubes to remove ex- cess dye. Lysine-binding dye was applied at 0.2 M quantity to each sample to identify dead cells. Any surviving microbubbles were destroyed by transfer- ring samples to a 12-mL syringe and pressurizing the sample to 10 atm for 5 s. Multi-color sonoporations Sonoporation involves mechanical rupture of the cell membrane owing to the local stresses applied by the oscillating microbubble. In previous studies, this stress was modeled as a function of wall velocity (36–38). We therefore calculated the theoretical radi- us-time curves for the three microbubble sizes using the experimentally validated model by Marmottant et al. for large-amplitude oscillations (39). Under the acoustic driving conditions employed in this study, theory predicts that smaller microbubbles should ex- perience more severe oscillations, i.e., larger relative expansion ratios and wall velocities (Fig. 2). To further characterize the effect of the second sonoporation in a double-sonoporated sample, we used a three-color assay. The first sonoporation was conducted with green FITC-dextran and cells were washed and treated with anti-FITC, and TRITC-dextran (7.5 mg/mL) was then substituted for FITC-dextran in the second sonoporation. Washed cells were treated with anti-tetramethylrhodamine (20 µg/mL) before far-red lysine-binding dye was added to stain dead cells. Data analysis Comparison of sonoporation and transfection results was conducted through unpaired Student’s t-tests between size groups in Prism software (GraphPad, La Jolla, CA, USA). Significant differences were determined for two sample groups if the p-value was found to be smaller than 0.05 (n ≥ 3). Flow cytometric analysis However, the theory does not account for mi- crobubble instabilities, such as dissolution and frag- mentation, which may limit the lifetime. We therefore measured microbubble persistence under these acoustic conditions. Our results showed that larger microbubbles were more stable to sonication than smaller ones (Fig. 3, supplemental Table S1). For 2-μm A flow cytometer (Accuri C5, Ann Arbor, MI) was used to count and analyze populations of fluo- rescent cells. Cells were gated in the forward-vs-side scatter plot and were isolated from the serpentine pattern of microbubbles (35). Once gated in the scatter plot, cells were analyzed for fluorescence by plotting FL1 (520 nm, FITC) vs. FL2 (585 nm, TRITC, ethidium http://www.thno.org http://www.thno.org 1423 Theranostics 2015, Vol. 5, Issue 12 Figure 2. Dimensionless radius-time curves during acoustic forcing for initial microbubble diameters of 2 µm (a), 4 µm (b) and 6 µm (c). See supplemental information for details on the numerical simulations. bubbles, a 97% reduction in concentration was ob- served after five seconds of sonoporation. The 4-μm bubbles demonstrated a 67% reduction in concentra- tion over the same timespan. The 6-μm microbubbles showed the greatest stability, dropping only 7% after five seconds of ultrasonic stimulation. A monoexpo- nential function was fit to the concentration-time data to determine the half-life for each size group and to interpolate concentrations between the experimental measurement times (Fig. 3d). Half-lives of the mi- crobubbles were 0.7, 1.7 and 13.2 s, respectively. These results were consistent with high-speed imaging re- sults of microbubble destruction by Chomas et al. (28), who showed that smaller microbubbles with higher expansion ratios are more likely to experience frag- mentation. bubbles, a 97% reduction in concentration was ob- served after five seconds of sonoporation. The 4-μm bubbles demonstrated a 67% reduction in concentra- tion over the same timespan. The 6-μm microbubbles showed the greatest stability, dropping only 7% after five seconds of ultrasonic stimulation. A monoexpo- nential function was fit to the concentration-time data to determine the half-life for each size group and to interpolate concentrations between the experimental measurement times (Fig. 3d). Half-lives of the mi- crobubbles were 0.7, 1.7 and 13.2 s, respectively. These results were consistent with high-speed imaging re- sults of microbubble destruction by Chomas et al. (28), who showed that smaller microbubbles with higher expansion ratios are more likely to experience frag- mentation. Theoretical power and energy output With knowledge of the experimental lifetimes and theoretical dynamics, we were able to compute theoretical power and energy outputs for each mi- crobubble size from equations 1 and 2, respectively. Our modeling results predicted that increasing mi- crobubble size would reduce the instantaneous power output of each microbubble, but increase the total energy delivered owing to longer persistence (Fig. 4a). This presented an interesting dichotomy: small mi- crobubbles provide high-intensity bursts, whereas large microbubbles produce greater overall exposure at a lower intensity. Our next experiment was de- signed to examine the relative importance of power and energy to sonoporation by measuring the effects on uptake, death and lysis. Figure 2. Dimensionless radius-time curves during acoustic forcing for initial microbubble diameters of 2 µm (a), 4 µm (b) and 6 µm (c). See supplemental information for details on the numerical simulations. Figure 3. Microbubble size distributions and concentrations during ultrasonic stimulation. Electric impedance measurements with the Multisizer III for (a) 2-μm, (b) 4-μm and (c) 6-μm diameter bubbles over the 2-min insonation at 37 °C. The initial concentration for each sample was 108/mL, and concentration measurements were taken at 5, 30 and 120 seconds (n=3). Size distributions were processed using a fourth-order smoothing function. d) The results were aggregated to obtain microbubble concentration over time for each size group. Significant differences were found between all sizes at 5, 30 and 120 s (p < 0.05). The lines are fits to a mono-exponential function. Figure 3. Microbubble size distributions and concentrations during ultrasonic stimulation. Electric impedance measurements with the Multisizer III for (a) 2-μm, (b) 4-μm and (c) 6-μm diameter bubbles over the 2-min insonation at 37 °C. The initial concentration for each sample was 108/mL, and concentration measurements were taken at 5, 30 and 120 seconds (n=3). Size distributions were processed using a fourth-order smoothing function. d) The results were aggregated to obtain microbubble concentration over time for each size group. Significant differences were found between all sizes at 5, 30 and 120 s (p < 0.05). The lines are fits to a mono-exponential function http://www.thno.org Theranostics 2015, Vol. 5, Issue 12 1424 Figure 4. (a) Microbubble size vs. calculated power (dotted line) and energy (solid line), obtained from Doinikov’s non-dimensional power equation (Equa- tion 1) and an energy model incorporating time and observed concentration values (Equation 2), respectively. Sequential sonoporations The second goal of this study was to optimize cellular uptake in context of minimizing cell death and lysis over multiple sonoporations. As imple- mented, sequential sonoporation retains the high-power, low-energy nature of 2-µm bubbles while multiplying the energy output in relatively small in- crements. Figure 5a illustrates the methodology, and Figure 5b shows the effect of sequential sonoporation: that increasing the number of sonoporations resulted in increased number of live fluorescent (“treated”) cells, as well as an increase in cell death and lysis. Importantly, FITC-dextran uptake peaked at 66.1 ± 1.2% after two sonoporations, when including lysis. In this fashion, we were able to exceed the previously reported soft limit of 50% uptake when including both lysed and dead cells in the total count (supplemental Table S2). Omitting lysed cells from the total cell count, sonoporation efficiency was found to be 71.1 ± 1.3%. Corresponding to the decrease in uptake be- tween sonoporation #2 and 3 was a significant in- crease in cell death (17.9 ± 2.0 → 43.8 ± 3.8%). This increase was less pronounced between #3 and 4 (43.8 ± 5.1% → 51.2 ± 6.0%). Instead, cell lysis increased significantly between the third and fourth sono- porations (12.4 ± 3.1% → 29.9 ± 2.6%). Figure 4. (a) Microbubble size vs. calculated power (dotted line) and energy (solid line), obtained from Doinikov’s non-dimensional power equation (Equa- tion 1) and an energy model incorporating time and observed concentration values (Equation 2), respectively. (b) Percentage of sonoporated and dead cells in treated samples comparing 2-, 4- and 6-µm microbubbles. Statistical signifi- cance: * p < 0.05; ** p < 0.05; + p < 0.05; # p < 0.01. Effect of microbubble size on sonoporation Flow cytometry was used to quantify cell up- take, death and lysis (supplemental Fig. S2). Cell up- take of the fluorescence probe was confirmed by flu- orescence microscopy (supplemental Fig. S3). Our results are summarized in Figure 4b. Live cells sono- porated with the largest (6-µm) microbubbles dis- played lower numbers of live sonoporated cells (27.3 ± 5.6%) and the highest cell death (48.7 ± 9.0%) com- pared to the smallest microbubbles. Medium-sized (4-µm) microbubbles produced the lowest numbers of sonoporated cells (25.0 ± 4.4%) and an intermediate number of dead cells (36.9 ± 2.9%). Use of the smallest (2-µm) microbubbles resulted in the highest percent- age of sonoporated cells (38.8 ± 1.3%) with the lowest percentage of dead cells (10.2 ± 2.5%). p ( ) While it is still possible that microbubble and acoustic parameters could be optimized for high-efficiency single sonoporations, there appears to be an intrinsic interaction limit stemming from the transient nature of microbubbles, which restricts the number of cells the microbubble suspension can affect before clearance. It is therefore advantageous to use sequential sonoporations. Our results suggested that sonoporations are multiplicative out to the second sonoporation under these conditions (supplemental Fig. S4), beyond which the cell death and lysis rates increase disproportionately. A second potential ad- vantage of sequential sonoporation is multi-drug de- livery, which we examined next with the use of two different fluorescent probes. These results indicated that the smallest mi- crobubbles produced enough sonication power to induce cell membrane rupture, but the total energy delivered was relatively nonlethal. This suggests a new paradigm for understanding and controlling in vitro sonoporation: that high-intensity bursts are more effective than high-energy exposures. Thus, small-diameter (2 µm) microbubbles are desirable due Theoretical power and energy output (b) Percentage of sonoporated and dead cells in treated samples comparing 2-, 4- and 6-µm microbubbles. Statistical signifi- cance: * p < 0.05; ** p < 0.05; + p < 0.05; # p < 0.01. to their ability to induce high uptake (49.7%) and low cell death (9.9%) after a single sonoporation. Their primary deficiency is the large number of unaffected cells (>39%) remaining after a single sonoporation. In order to overcome this limitation, we next investi- gated the use of sequential sonoporations. Multi-color sonoporations Fractions of live cells were analyzed by mul- ti-color sonoporation in order to separate uptake from the effects of cell death. Fluorescent marker uptake from the first sonoporation indicated that approxi- mately 55% of live cells were treated after each sono- http://www.thno.org 1425 Theranostics 2015, Vol. 5, Issue 12 poration. Changing the fluorescent uptake marker from FITC to TRITC for the second sonoporation re- sulted in three distinct fluorescent populations: FITC-only (23.8 ± 1.3%), TRITC-only (23.3 ± 0.5%), and FITC+TRITC co-fluorescent (26.6 ± 1.6%) cells (Fig. 6, supplemental Table S3). Cell death in multi-color ex- periments were similar to those found in FITC-only trials (22.1 ± 1.4% vs. 17.9 ± 2.0%). Indeed, dual-color sonoporations produced multiplicative trends similar to predicted values (see supplemental information): a single sonoporation induced FITC-dextran uptake in 49.7% of the cells, and the second sonoporation re- sulted in 66% uptake (73.7% when not counting dead or lysed cells). Substituting TRITC for the second sonoporation further supported multiplicative up- take, with 25-30% co-fluorescent (FITC and TRITC-present) cells resulting from two sono- porations. studies done under the microscope (32). In our study, further analyses of cell populations revealed that after two sonoporations, the number of dead co-fluorescent cells was not significantly higher than dead sin- gle-sonoporated cells, suggesting that cell stress thresholds were only exceeded after cells were sono- porated three times. Future studies could investigate the effect of cellular resting periods between sono- porations to further reduce cell stress and improve therapeutic efficiency. Combined with systematic identification of cell stress thresholds, sequential sonoporation allows for predictable and sequential delivery of multiple drugs to cell populations, facilitating the study of interde- pendent drug effects. Further optimization of the se- quential sonoporation methodology could employ microbubbles of various concentrations, diameters and shell compositions in each sonoporation (as well as various ultrasound parameters) to achieve the de- sired percentage, magnitude and specificity of effect for each drug type. As an investigative tool, sequential sonoporation may yield further insight into cellular stress and mortality to supplement single-cell sonoporation Figure 5. (a) A flow diagram of the methodology used in sequential sonoporations. (b) The effect of sequential sonoporations on cell uptake, death and lysis, as measured by flow cytometry. The initial microbubble concentration for each sample was 108/mL, and cell counts were obtained before and after sonoporation to determine cell loss (n=3). Conclusions We conclude that microbubble size is an im- portant parameter for in vitro sonoporation, markedly affecting microbubble persistence and sonoporation mechanics. While acoustic parameters and microbub- ble concentration certainly play significant roles, size appears to provide additional control over the power and energy delivered to cells. Our data indicate that the prolonged persistence of larger microbubbles de- livers more total energy to the cell membrane, in- creasing cell death. In contrast, owing to a drastically larger expansion ratio, smaller microbubbles deliver high power and a short half-life, porating cells with minimal loss of viability. By extending this efficiency gain through multiple bursts of low-energy sono- poration, we were able to further augment cellular uptake – above the putative 50% limit – without ex- cessive cell death and lysis. Indeed, two sequential sonoporations with 2-μm bubbles induced the highest FITC-dextran uptake. Our multi-color sonoporation assay utilizing FITC- and TRITC-dextran confirmed that this gain proceeds in a multiplicative fashion. Further treatments resulted in diminished viability, indicating a finite energy threshold for cell death. This general strategy to first explore the effects of power and energy on cell uptake and viability, followed by optimization of multiple treatments, can be applied to diagnostic and therapeutic treatment of other cell types in vitro and in vivo to further enhance the utility of sonoporation for future theranostic applications. 5. Mo R, Lin S, Wang G, Wang Y, Wu EX. Preliminary in vitro study of ultra- sound sonoporation cell labeling with superparamagnetic iron oxide particles for MRI cell tracking. Conf Proc Annu Int Conf IEEE Eng Med Biol Soc IEEE Eng Med Biol Soc Annu Conf. 2008;2008:367–70. g 6. Skachkov I, Luan Y, van Tiel S, van der Steen T, Kooiman K, Bernsen M, et al. Ultrasound contrast agents mediated cell labeling for MRI tracking. New York: Ieee; 2012. 7. Kolarova M, Polakova K, Tomankova K, Havrdova M, Markova Z, Zboril R. Rapid Cellular Uptake of Superparamagnetic Iron Oxide Nanoparticles by Using Low-Intensity Ultrasound. Nanocon 2013 5th Int Conf. 2014;578–83. 8. D’Souza AL, Tseng JR, Pauly KB, Guccione S, Rosenberg J, Gambhir SS, et al. A strategy for blood biomarker amplification and localization using ultra- sound. Proc Natl Acad Sci. 2009;106(40):17152–7. ( ) 9. Forbrich A, Paproski R, Hitt M, Zemp R. Microbubble-Enhanced Ultrasound Liberation of mRNA Biomarkers In Vitro. Ultrasound Med Biol. 2013 Jun;39(6):1087–93. 10. Li YS, Davidson E, Reid CN, McHale AP. Multi-color sonoporations FITC-Dextran uptake peaked at the second sonoporation (p<0.05), with cell death and lysis increasing disproportionately over subsequent sonoporations. Figure 5. (a) A flow diagram of the methodology used in sequential sonoporations. (b) The effect of sequential sonoporations on cell uptake, death and lysis, as measured by flow cytometry. The initial microbubble concentration for each sample was 108/mL, and cell counts were obtained before and after sonoporation to determine cell loss (n=3). FITC-Dextran uptake peaked at the second sonoporation (p<0.05), with cell death and lysis increasing disproportionately over subsequent sonoporations. Figure 5. (a) A flow diagram of the methodology used in sequential sonoporations. (b) The effect of sequential sonoporations on cell uptake, death and lysis, as measured by flow cytometry. The initial microbubble concentration for each sample was 108/mL, and cell counts were obtained before and after sonoporation to determine cell loss (n=3). FITC-Dextran uptake peaked at the second sonoporation (p<0.05), with cell death and lysis increasing disproportionately over subsequent sonoporations. http://www.thno.org 1426 Theranostics 2015, Vol. 5, Issue 12 Figure 6. FITC- and TRITC-dextran uptake over two sonoporations. FITC-dextran was used as an uptake marker in the first sonoporation, and cells were washed and processed before a second sonoporation in the presence of TRITC-dextran (n=3). References 1. Husseini GA, Pitt WG. Micelles and nanoparticles for ultrasonic drug and gene delivery. Adv Drug Deliv Rev. 2008 Jun;60(10):1137–52. 1. Husseini GA, Pitt WG. Micelles and nanoparticles for ultrasonic drug and gene delivery. Adv Drug Deliv Rev. 2008 Jun;60(10):1137–52. Figure 6. FITC- and TRITC-dextran uptake over two sonoporations. FITC-dextran was used as an uptake marker in the first sonoporation, and cells were washed and processed before a second sonoporation in the presence of TRITC-dextran (n=3). g y g J ; ( ) 2. Miller DL, Bao S, Morris JE. Sonoporation of cultured cells in the rotating tube exposure system. Ultrasound Med Biol. 1999;25(1):143–9. g y g 2. Miller DL, Bao S, Morris JE. Sonoporation of cultured cells in the rotating tube exposure system. Ultrasound Med Biol. 1999;25(1):143–9. y 3. Christiansen JP, French BA, Klibanov AL, Kaul S, Lindner JR. Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic mi- crobubbles. Ultrasound Med Biol. 2003;29(12):1759–67. 3. Christiansen JP, French BA, Klibanov AL, Kaul S, Lindner JR. Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic mi- crobubbles. Ultrasound Med Biol. 2003;29(12):1759–67. ( ) 4. Partlow KC, Brant JA, Marsh JN, Nolta JA, Hughes MS, Lanza GM, et al. Ultrasound energy rapidly labels stem/progenitor cells with nanoparticle beacons without disrupting membrane integrity. 2007 Ieee Ultrasonics Sym- posium Proceedings, Vols 1-6. New York: Ieee; 2007: 1768–71. 4. Partlow KC, Brant JA, Marsh JN, Nolta JA, Hughes MS, Lanza GM, et al. Ultrasound energy rapidly labels stem/progenitor cells with nanoparticle beacons without disrupting membrane integrity. 2007 Ieee Ultrasonics Sym- posium Proceedings, Vols 1-6. New York: Ieee; 2007: 1768–71. Acknowledgments The authors would like to thank Dr. Shashank Sirsi for help with experimental methodology and insightful discussions. Funding was provided by NSF grant DMR 1409972 to M.A.B., Colorado Clinical and Translational Sciences Institute (CCTSI) grant to M.A.B. and A.G.H., and the UCCS Center for the Bio- frontiers Institute to M.L.C. Competing Interests The authors have declared that no competing interest exists. Conclusions Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: Potential applications for gene therapy of cancer. Cancer Lett. 2009 Jan;273(1):62–9. 11. Iwanaga K, Tominaga K, Yamamoto K, Habu M, Maeda H, Akifusa S, et al. Local delivery system of cytotoxic agents to tumors by focused sonoporation. Cancer Gene Ther. 2007;14(4):354–63. ( ) 12. Liu Y, Yan J, Prausnitz MR. Can Ultrasound Enable Efficient Intracellular Uptake of Molecules? A Retrospective Literature Review and Analysis. Ul- trasound Med Biol. 2012; 13. Hallow DM, Mahajan AD, Prausnitz MR. Ultrasonically targeted delivery into endothelial and smooth muscle cells in< i> ex vivo</i> arteries. J Controlled Release. 2007;118(3):285–93. ( ) 14. Reslan L, Mestas J-L, Herveau S, Béra J-C, Dumontet C. Transfection of cells in suspension by ultrasound cavitation. J Controlled Release. 2010;142(2):251–8. 15. Qiu Y, Zhang C, Tu J, Zhang D. Microbubble-induced sonoporation involved in ultrasound-mediated DNA transfection in vitro at low acoustic pressures. J Biomech. 2012 May;45(8):1339–45. 16. Escoffre J, Novell A, Piron J, Zeghimi A, Doinikov A, Bouakaz A. Microbubble attenuation and destruction: are they involved in sonoporation efficiency? IEEE Trans Ultrason Ferroelectr Freq Control [Internet]. 2013;60(1). http://ieeexplore.ieee.org/lpdocs/epic03/wrapper.htm?arnumber=6396485 17. Hettiarachchi K, Talu E, Longo ML, Dayton PA, Lee AP. On-chip generation of microbubbles as a practical technology for manufacturing contrast agents for ultrasonic imaging. Lab Chip. 2007;7(4):463–8. g g p ( ) 18. Talu E, Hettiarachchi K, Powell RL, Lee AP, Dayton PA, Longo ML. Main- taining Monodispersity in a Microbubble Population Formed by Flow-Focusing. Langmuir. 2008 Mar;24(5):1745–9. g g ( ) 19. Talu E, Hettiarachchi K, Zhao S, Powell RL, Lee AP, Longo ML, et al. Tailoring the size distribution of ultrasound contrast agents: possible method for im- proving sensitivity in molecular imaging. Mol Imaging. 2007;6(6):384. Supplementary Material Theranostics 2015, Vol. 5, Issue 12 21. Van Hoeve W, Dollet B, Versluis M, Lohse D. Microbubble formation and pinch-off scaling exponent in flow-focusing devices. Phys Fluids 1994-Present. 2011;23(9):092001. 21. Van Hoeve W, Dollet B, Versluis M, Lohse D. Microbubble formation and pinch-off scaling exponent in flow-focusing devices. Phys Fluids 1994-Present. 2011;23(9):092001. ; ( ) 22. Chen C, Zhu Y, Leech PW, Manasseh R. Production of monodispersed mi- cron-sized bubbles at high rates in a microfluidic device. Appl Phys Lett. 2009;95(14):144101. 23. Parhizkar M, Edirisinghe M, Stride E. Effect of operating conditions and liquid physical properties on the size of monodisperse microbubbles produced in a capillary embedded T-junction device. Microfluid Nanofluidics. 2013 May;14(5):797–808. y ( ) 24. Parrales MA, Fernandez JM, Perez-Saborid M, Kopechek JA, Porter TM. Acoustic characterization of monodisperse lipid-coated microbubbles: Rela- tionship between size and shell viscoelastic properties. J Acoust Soc Am. 2014;136(3):1077–84. ( ) 25. Feshitan JA, Chen CC, Kwan JJ, Borden MA. Microbubble size isolation by differential centrifugation. J Colloid Interface Sci. 2009;329(2):316–24. g 26. Sirsi S, Feshitan J, Kwan J, Homma S, Borden M. Effect of microbubble size on fundamental mode high frequency ultrasound imaging in mice. Ultrasound Med Biol. 2010;36(6):935–48. ( ) 27. Choi JJ, Feshitan JA, Baseri B, Shougang Wang, Yao-Sheng Tung, Borden MA, et al. Microbubble-Size Dependence of Focused Ultrasound-Induced Blood-Brain Barrier Opening in Mice In Vivo. IEEE Trans Biomed Eng. 2010 Jan;57(1):145–54. J ( ) 28. Chomas JE, Dayton P, May D, Ferrara K. Threshold of fragmentation for ultrasonic contrast agents. J Biomed Opt. 2001;6:141. 29. Postema M, van Wamel A, Lancée CT, de Jong N. Ultrasound-induced en- capsulated microbubble phenomena. Ultrasound Med Biol. 2004 Jun;30(6):827–40. J ( ) 30. Doinikov AA, Haac JF, Dayton PA. Resonance frequencies of lipid-shelled microbubbles in the regime of nonlinear oscillations. Ultrasonics. 2009;49(2):263–8. ( ) 31. Zhou Y, Kumon RE, Cui J, Deng CX. The size of sonoporation pores on the cell membrane. Ultrasound Med Biol. 2009;35(10):1756–60. 32. Zhou Y, Yang K, Cui J, Ye JY, Deng CX. Controlled permeation of cell mem- brane by single bubble acoustic cavitation. J Controlled Release [Internet]. 2011 [cited 2012 Aug 21]; Available from: http://www.sciencedirect.com/science/article/pii/S0168365911008534 33. Karshafian R, Bevan PD, Williams R, Samac S, Burns PN. Sonoporation by ultrasound-activated microbubble contrast agents: effect of acoustic exposure parameters on cell membrane permeability and cell viability. Ultrasound Med Biol. 2009;35(5):847–60. ( ) 34. Song KH, Fan A, Brlansky J, et al. Supplementary Material 20. Dollet B, van Hoeve W, Raven J-P, Marmottant P, Versluis M. Role of the Channel Geometry on the Bubble Pinch-Off in Flow-Focusing Devices. Phys Rev Lett [Internet]. 2008;100(3). http://link.aps.org/doi/10.1103/ PhysRevLett.100.034504 Figures S1-S5. http://www.thno.org/v05p1419s1.pdf Figures S1-S5. http://www.thno.org/v05p1419s1.pdf http://www.thno.org 1427 Theranostics 2015, Vol. 5, Issue 12 Efficient microbubble-assisted sonoporation for in vitro applications. Manuscript. 35. Satinover SJ, Dove JD, Borden MA. Single-Particle Optical Sizing of Mi- crobubbles. Ultrasound Med Biol. 2014 Jan;40(1):138–47. 36. Lewin PA, Bjørnø L. Acoustically induced shear stresses in the vicinity of microbubbles in tissue. J Acoust Soc Am. 1982;71:728. 37. Wu J. Theoretical study on shear stress generated by microstreaming sur- rounding contrast agents attached to living cells. Ultrasound Med Biol. 2002;28(1):125–9. ( ) 38. Doinikov AA, Bouakaz A. Theoretical investigation of shear stress generated by a contrast microbubble on the cell membrane as a mechanism for sono- poration. J Acoust Soc Am. 2010;128(1):11. p ( ) 39. Marmottant P, Meer S van der, Emmer M, Versluis M, Jong N de, Hilgenfeldt S, et al. A model for large amplitude oscillations of coated bubbles accounting for buckling and rupture. J Acoust Soc Am. 2005 Dec 1;118(6):3499–505. http://www.thno.org
https://openalex.org/W3119174025
https://digibug.ugr.es/bitstream/10481/65362/1/19-Article%20Text-245-2-10-20201231.pdf
English
null
Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships
International journal of racket sports science
2,020
cc-by
7,381
Int. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-4508 Int. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-4508 Original Investigation Correspondence author: Tamás Laczkó, joola.hu@gmail.com Cite this article as: Laczkó, T., Ács, P., Stocker, M., Paár D. (2020). Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships. International Journal of Racket Sports Science, 2(2), 37-46. Cite this article as: Laczkó, T., Ács, P., Stocker, M., Paár D. (2020). Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships. International Journal of Racket Sports Science, 2(2), 37-46. Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships Tamás Laczkó 1; Pongrác Ács 1; Miklós Stocker 2; Dávid Paár 1 1 University of Pécs, Faculty of Health Sciences, Pécs, Hungary 2 Corvinus University of Budapest, Institute of Business Economics, Budapest, Hungary Tamás Laczkó 1; Pongrác Ács 1; Miklós Stocker 2; Dávid Paár 1 1 University of Pécs, Faculty of Health Sciences, Pécs, Hungary 2 Corvinus University of Budapest, Institute of Business Economics, Budapest, Hungary INTRODUCTION Tourism areas for sports and physical activity have been constantly growing since the turn of the millennium. Tourism and economic professionals predict a dynamic expansion in active and passive sports tourism for the next decade (Dreyer, 2002; Bánhidi, 2015; Borbély & Müller, 2015). Sports events tourism is one of the highlights of sports tourism and it has been growing significantly in recent decades. Sports events and especially international sports events are nowadays characterized by fast paced evolution. Organisers focus on intangible values i.e. experience and entertainment (sometimes even at the expense on sport’s professional aspects), which generate the emergence of new competition series and forms (e.g. new multisport events such as the European Games or the new World Table Tennis Series). At the same time, the number of viewers of worldwide sports events and the amount of its revenues are growing, and we can clearly see the trend of globalisation regarding the international sports events (Stocker, 2013; Bánhidi, 2018; Laflin, 2018; Laczkó & Stocker, 2020). One of the characteristics of sports events tourism is that they have two main target groups that differ in their motivation and characteristics. The first is the participants’ group (such as athletes, delegates, organisers, etc.) whose travels are classified as professional tourism based on their characteristics and motivations, while the second is the spectators’ group (local, domestic and foreign spectators) whose touristic consumption is interpreted as a part of leisure tourism (Standeven & De Knopp, 1999; Lasztovicza & Béki, 2016). The size of the global spectators’ market and its rapid expansion have placed the study of spectators’ characteristics and consumption habits into the focus of international sports and economics researches (Bánhidi, 2018). Based on this research, it can be said that nowadays spectators attend sports events primarily to gain experiences and let themselves to be entertained, therefore, they are looking for non-sports related attractions and services, and can be characterized by increased demands for these attractions compared to the average tourist (Albers, 2004; Chen & Funk, 2010; Stocker & Laczkó, 2020). Hungary has a very successful past in the sport of table tennis and the Hungarian Table Tennis Association has considerable experience in organising international sports competitions. Hungary is the second most successful table tennis nation with 202 medals at the World Table Tennis Championships. These results came from a series of successes between the two world wars and the 1950s and 1970s. Abstract Organising international sports events became one of the most important elements of the prioritized sports sector in Hungary. The main goal of this study was to examine the economic impact of Liebherr 2019 ITTF World Table Tennis Championships (WTTC) for the Hungarian GDP. The impact is based on consumption habits of stakeholders and total budget of organisers so it was necessary to analyse the characteristics of passive sports tourists and all other stakeholders and their spending too. Consumption of stakeholders were surveyed with questionnaire (n=1097) and the budget of the organisers were presented by the Hungarian Table Tennis Association. We have used the secondary data of Hungarian Statistical Office and Eurostat for input-output modelling. Expenditures and spending behaviour of stakeholders were calculated by inferential statistics, differences were tested by independent-samples T tests, ANOVA and Chi-square tests. Input-output modelling method was used for estimating the direct and indirect macroeconomic impacts. Daily spending of domestic (47.9 EUR/day) and foreign (102.8 EUR/day) passive sports tourists coming to the WTTC were calculated. Foreign passive sports tourists spent an average 3.85 days in the country. Examining the macroeconomic effects it can be stated that every EUR of government support increased the country’s GDP by 1.21 EUR and generated a tax of 1.01 EUR in 2019. Expenditures of spectators and participants contributed 24% of the generated GDP. Passive sports tourists of WTTC spent more money than the general domestic and foreign tourists (20.6 EUR/day and 51.8 EUR/day respectively) or even the domestic or foreign sports tourists (24.7 EUR/day and 54.7 EUR/day respectively). They spent more time than general foreign tourists or foreign sports tourists as well (2.26 days and 2.29 days respectively) in Hungary. The Hungarian government provided almost 4 million euros support to the organisers and this amount is exceeded by the total contribution of WTTC to the Hungarian GDP (4.7 million EUR). World Table Tennis Championships, consumption habits, economic impact, input-output mode Correspondence author: Tamás Laczkó, joola.hu@gmail.com This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 37 Int. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-4508 Laczkó, T. et. al. government’s decision to handle sport as a strategic sector since 2010, as well as the fact that the area also appears as a priority in tourism development strategies at various levels. Abstract Organising international sports events became one of the most important elements of the prioritised sports sector (Stocker & Szabó, 2017) and its over-spilling effects are in focus of scientific researches nowadays (Laczkó & Paár, 2018; Paár & Laczkó, 2018). As a result the number of international sports events applied for and held in the country increased significantly from 2010 and also included events with higher prestige and professional quality. More than 120 international sports competitions were held in Hungary in 2019. The Formula One race, the World Table Tennis Championships (WTTC), the Canoe Sprint World Championships, the Fencing World Championships and the Modern Pentathlon World Championships were held among others this year. According to the data of Sportcal Global Communications, Budapest was the 3rd, while Hungary was the 18th in the world ranking of settlements and countries organising international sports events in 2019 (Sportcal Global Communications, 2019a). Design & Procedures g We created slightly different questionnaires for different stakeholder groups in international sports events to obtain primary research data, and we selected the sample with quota sampling. Interviewers were sent to the event and they recorded the answers of the spectators. The questionnaires were created with different types of questions: open-ended questions (some financial and some textual), closed- ended questions and Likert-scale questions about the basic statistical data of the respondents, the different types of expenditures spent and their amounts, the length of stay, the different tourist appeals, the quality of the event organisation, the willingness to return to the country and the attitude about how spectators rated Hungarian people’s hospitality. Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships Table 1. Sample sizes and number of participants at Liebherr 2019 ITTF World Table Tennis Championships (Budapest, HU)   Domestic spectators Foreign spectators Professionals Total Sample size (n) 355 461 281 1,097 Participants (N) 5,577 3,810 2,199 11,586 Proportion (%) 6.4% 12.1% 12.8% 9.5% Sources: Authors’ compilation MATERIALS AND METHODOLOGY Information about the consumer behaviour of spectators and participants was obtained by the survey methodology and information about the budget of the event was provided by the local organising committee. Analysis y The direct and indirect impacts of the World Championships were calculated by the economic input-output modelling method, which is widely used by impact calculation studies in event tourism (European Commission, 2012; Kóródi, 2011; Kundi, 2012; KPMG, 2015; Laczkó-Stocker, 2018; Stocker-Laczkó, 2020). Economic impact is mostly connected to the organisation of the event and the consumption of the participants as the consumption of the spectators is usually responsible for a smaller amount in table tennis. Further primary research data was provided by the local organising committee about the budget of the events and the numbers of different participants. According to secondary sources, we used public data from the national sports associations, the Hungarian Central Statistical Office (HCSO), the Statistical Office of the European Union (Eurostat) and other sports professional fora. The gathered primary and secondary data were used as inputs for the input- output modelling. The economic impact of the international sports event was calculated from the direct expenditure of the event and its participants and the indirect impact they caused in the national economy. From the spectators’ expenditure the expenditure of entry tickets was ignored as these were included in the budget of organisers. Travel costs of foreign spectators were also excluded as these occur usually in their home country which means it does not have impact in the Hungarian economy. Those spectators’ expenditures were excluded from the impact analysis who reported that their primary travel reason was different than the World Championships, as their economic impact should have been calculated in the impact analysis of the primary reason of travel. According to Hungarian spectators related calculations in the impact analysis we decreased the number of Hungarian spectators with 1.7% as their primary motivation was not the WTTC and we also decreased the expenditures of the remaining Hungarian spectators by 16.7% as they would have been spent money in Hungary if the WTTC would not take place in the country. Exact measures are the benchmarks taken from another Olympic sport’s World Championships in Hungary in 2019. Expenditures and spending behaviour of spectators, travel and other tourist behaviour were calculated by the primary data with inferential statistics. The differences of the given variables were calculated with independent-sample T tests, ANOVA tests and Chi-square tests depending on the operationalization of the given variable. The significance level was set at p < 0.05 (Ács, 2007; Ács, 2014). INTRODUCTION Hungary has paid special attention to the application and organisation of international table tennis competitions in the recent years. In addition to the annual World Tour series in the country, the European Championships (EC) was held in 2016, while the individual WTTC was held in April 2019 and the European Veteran Championships (EVC) was held one month later. Budapest hosted the WTTC for the fourth time in its history in 2019 (after 1929, 1931 and 1950). Budapest is the second placed settlement in the world after London regarding the number of organised World Table Tennis Championships. Budapest wanted to become the centre of international table tennis life with these events in 2019. Although the main focus of the Hungarian tourism is not sports tourism, it can be said that more and more attention has been paid to the development of sports-related tourism trends in recent years (Sulyok & Magyar, 2014; Hungarian Tourist Agency, 2017). The expansion in the sector is explained and supported by several social and economic trends as well as government decisions. Among the social trends, the rising participation rate of the Hungarian population in recreational sports for health purposes and the increasing sports consumption must be highlighted (Kovács, Paár, Elbert, Welker, Stocker, & Ács, 2015; European Commission, 2018). An important driver of the development of Hungarian sports tourism is the The main research question of this paper is the economic impact of the Liebherr 2019 ITTF World Table Tennis Championships. This total impact can only be calculated including the consumer habits of different stakeholders of the event, therefore the consumer habits will be demonstrated first, then the economic impact calculations will come. The hypothesis of the study is that the Liebherr 2019 ITTF World Table Tennis Championships contributed more to the Gross Domestic Product of Hungary than the subsidy it got from the government. 38 RESULTS Figure 1. Distribution of Hungarian Spectators’ Expenditure (%) Consumption and touristic characteristics of the WTTC The characteristics and opinions of domestic spectators are based on a sample of 355 people, which is 6.4% of the Hungarian spectators. A total of 75.2% of Hungarian spectators were men, 24.8% were women at the WTTC in 2019. In terms of age distribution 22.0% were under 20 years of age, 14.4% were 20-29 years of age, 10.7% were 30-39 years of age, 24.8% were 40-49 years of age, 16.1% were 51-60 years of age and 12.0% were over 60 years. Figure 1. Distribution of Hungarian Spectators’ Expenditure (%) A total of 30.7% of the respondents came from Budapest to the competition. 18.5% of domestic spectators travelled for up to 1 hour and a further 25.1% travelled between 1 and 2 hours to watch the competition in addition to the this. 25.7% of the domestic spectators came from a distance of more than 2 hours. It can be said that more than half of the domestic spectators (50.8%) travelled at least 1 hour to watch the competition. Domestic spectators came to the WTTC from all counties of the country. Domestic spectators rated the entertainment value of the competition as good, as 62.5% of the respondents rated the WTTC as five on a scale of 1 to 5. The mean of the ratings was 4.48 (SD = 0.87). Only 5.9% of the respondents rated the entertainment value of the competition as 3 or lower. The followings were considered important among the entertaining elements of the competition: the players’ playing level, the quality of the matches (4.71, SD = 0.07), the information announcements (4.44, SD = 0.09), the work of the organising staff (4.41, SD = 0.09) and the presence of domestic players (4.26, SD = 0.1). The presence of show elements (3.46, SD = 0.14) and the competition’s unpredictability factor (3.89, SD = 0.15) were considered significantly less important. According to modes of travel 71.2% of the spectators arrived by car (or minibus), 18.6% by public transport, 8.8% by train/coach, and 1.4% used other vehicles (such as bicycles etc.). More than a third of the respondents (33.8%) watched the competition with their families, 22.1% of them came alone, 19.8% came with friends and acquaintances, while 24.3% came with sports mates. The quality of the competition’s organisation was rated as slightly weaker, averaging 4.30 (SD = 1.11). Participants This meant a total of 3,569 guest nights in the capital during the competition period. Most of the used accommodations were cheaper ones, such as sleeping with friends or lower- rated commercial accommodations. Only 38.0% of those who spent guest nights related to the event slept in 3-stars or higher rated hotels (12.7% in 4-stars hotels and 25.3% in 3-stars hotels), while 40.1% of them slept by friends and acquaintances and 19.7% of them spent their nights in rented apartments or other non-commercial accommodation. Figure 1 shows the distribution of Hungarian spectators’ expenditure. Domestic spectators spent an average of 47.9 EUR (SD = 49.5) per day of the competition. This amount is significantly higher than the average 20.6 EUR (t = 10.4, p = 0.000) daily spending by the average Hungarian tourists on multi-day trips in Hungary in 2019 and the average spending of 24.7 EUR on cultural and sports events (t = 8.8, p = 0.000) (HCSO, 2020). All statistical calculations were made by IBM SPSS Statistics 25 and Microsoft Excel (from Microsoft Office 365 ProPlus). Participants Our interviewers recorded 1139 survey entries from the spectators’ and the participants’ questionnaires together at the Liebherr 2019 ITTF World Table Tennis Championships. We had to exclude 42 entries because of interviewer data entry problems or because of the unfinished status of the questionnaires. The final sample consists of n=1,097 survey entries from which n=355 are from domestic spectators, n=641 are from foreign spectators and n=281 are from professionals (athletes and sports professionals) as it can be seen in table 1. Altogether n=1,097 person was included in the sample from the participating 11.5 thousand which means 9.5% sample rate. Financial contribution of sponsors was included in the budget of the organisers. Non-financial sponsorships were included in the research too. These were products, services and sporting goods. Sponsors also contributed to the event with letting their employees work in the event, their fair value was also included in the research. As the organisation of the event also included imported goods but these imported goods created value for their home economy, therefore they were excluded from the calculations. 39 Laczkó, T. et. al. nt. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-450 in and around Budapest. This meant a total of 3,569 guest nights in the capital during the competition period. Most of the used accommodations were cheaper ones, such as sleeping with friends or lower- rated commercial accommodations. Only 38.0% of those who spent guest nights related to the event slept in 3-stars or higher rated hotels (12.7% in 4-stars hotels and 25.3% in 3-stars hotels), while 40.1% of them slept by friends and acquaintances and 19.7% of them spent their nights in rented apartments or other non-commercial accommodation. The created input-output model used the previously described data. We used multipliers obtained from the input-output analysis of Hungarian sectors to estimate the indirect impact as well (Stocker & Boda, 2018). The different expenditures spent in accordance with the World Championships were spent in the following sectors: sports activities and amusement and recreation activities; accommodation and food service activities; transportation and storage; manufacture of food products, beverages; manufacture of wearing apparel; human health activities; services to buildings and landscape activities; and education. Sectoral multipliers were used for the expenditure to the respective sectors and we could estimate the impact of foreign spectators’ expenditures on the Hungarian GDP with these calculated multiplier effects. in and around Budapest. Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships years of age, 13.8% were 51-60 years of age and 8.5% were over 60 years (see Figure 2). years of age, 13.8% were 51-60 years of age and 8.5% were over 60 years (see Figure 2). years of age, 13.8% were 51-60 years of age and 8.5% were over 60 years (see Figure 2). Figure 3. Distribution of foreign spectators’ expenditure (%) Figure 2. Age Distribution of Domestic and Foreign Spectators at the WTTC Figure 3. Distribution of foreign spectators’ expenditure (%) Examining the tourism related consumption of foreign spectators, it can be said that almost two thirds of the respondents (61.0%) were curious about the tourist attractions of Budapest and its surroundings. Several tourist destinations were visited by 37.2% of foreign spectators on several occasions in addition to the WTTC. Foreign spectators viewed mostly the main tourist attractions of Budapest similarly like general tourists. The city centre (Parliament, Danube riverbank) and the UNESCO World Heritage’s sites (Buda Castle, Heroes’ Square, Andrassy Street) were visited by 88.3% of the foreign spectators, while 10% of foreign spectators were looking for entertainment opportunities, but cultural (1%) and shopping tourism (0.4%) motives were negligible. Only 2% of spectators were involved in programs organised by tourism providers. Figure 2. Age Distribution of Domestic and Foreign Spectators at the WTTC The sample included citizens of 34 countries. 14.1% of respondents came from Asia (China, Japan, India, Kazakhstan, Lebanon, Philippines), 0.4% came from the Americas (USA, Brazil) while the vast majority came from European countries (85.5%). Most Europeans came from Germany (24.7%), Slovakia (17.1%), Romania (6.1%), Sweden (5.4%) and England (4.3%). More than a third (34.7%) of foreign spectators arrived by road by their own car (or minibus) and a significant number of them also arrived by plane (45.4%) to Hungary. Due to the favourable geographical location of Budapest, a significant number of foreign spectators also arrived by train (19.2%). Foreigners rated the hospitality of Hungarians averagely at 4.6 (SD = 0.61) based on the experiences of their trip to Hungary related to the WTTC. The foreign spectators of the WTTC spent an average of 3.85 nights (SD = 2.08) in Hungary. 53.7% of them spent their nights in at least 3-stars hotels (38.4% in 4- and 5-stars hotels), while 35.4% slept in rented apartments. Foreign spectators spent a total of 13,488 nights related to the WTTC. RESULTS The characteristics and the opinions of the foreign spectators are based on a sample of 461 people, which is 12.1% of the foreign spectators. The Hungarian spectators watched an average of 2.56 (SD = 2.15) competition days. According to accommodation 20.0% of the spectators booked accommodation related to the competition and they spent an average of 3.18 (SD = 2.56) nights. Taking all domestic viewers into account, they spent an average of 0.64 (SD = 1.71) guest nights A total of 75.1% of foreign spectators were men and 24.9% were women. In terms of age distribution 8.1% were under 20 years of age, 26.5% were 20-29 years of age, 21.0% were 30-39 years of age, 22.1% were 40-49 40 Table 2. Net direct expenditure by stakeholders in LIEBHERR 2019 WTTC (EUR) Table 2. Net direct expenditure by stakeholders in LIEBHERR 2019 WTTC (EUR) Net direct expenditure by stakeholders in LIEBHERR 2019 WTTC (EUR) Organisers Sponsors Extra ex­ penditure of Participants Foreign spectators Domestic spectators Total 5,106,235 EUR 59,090 EUR 461,854 EUR 1,259,134 EUR 264,742 EUR 7,151,055 EUR Source: Authors’ compilation Figure 4. Distribution of the participants’ local expenditure (%) Direct revenue to the central budget from the WTTC was 2.32 million EUR, which consists of VAT, employee related taxes and insurance. Local government will get tourist tax and local business tax and indirect tax revenues which are also created by the multiplying effect of the demand of WTTC. Figure 4. Distribution of the participants’ local expenditure (%) Average import ratio of the segments from where the WTTC procured products and services is 4%, therefore the demand side of the organisation and the other stakeholders expenditure had to be decreased by this amount in the economic impact analysis. Estimated import of the event was 294 thousand EUR, therefore direct national impact of WTTC was 6.86 million EUR. The hospitality of the participants (in terms of accommodation, meals and transfers) was part of the accreditation, so we wanted to know more about their additional expenses. The participants spent an average of 265.3 EUR (SD = 386.1) during their stay in Budapest, which was 44.9 EUR (SD = 65.3) per day in addition to their basic hospitality. Players spent significantly the least (29.2 EUR/day; SD = 37.7), while other participants (ITTF members, media professionals, umpires and referees, technical staff) spent the most (61.6 EUR/day; SD = 87.4) among participants (coaches and delegates spent 44.1 EUR/day; SD = 39.1) (p = 0.034). The overall structure of their spending is shown by Figure 4. For the suppliers be able to deliver products and services indirect products and services were needed, therefore the given segments’ economic multiplier effect had to be taken into account. According to the structure of the expenditure the weighted average multiplier became 1.71, which means that the 7.15 million EUR demand of the WTTC needed 11.75 million EUR output from the Hungarian supply chain, which means the WTTC generated 11.75 million EUR output in the Hungarian economy. The quality of organisation was rated averagely 4.24 (SD = 0.85) by the participants. Int. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-4508 Laczkó, T. et. al. Economic impact analysis of the 2019 World Table Tennis Championships Economic impact analysis of the 2019 World Table Tennis Championships Foreign participants came to Budapest from 99 countries of all five continents. The foreign participants spent an average of 6.0 (SD = 2.1) and a total of 13,003 guest nights in the event’s 9 official hotels (two 3-stars, five 4-stars and one 5-stars) in Budapest. Direct expenditure related to the 2019 World Table Tennis Championships was 7.15 EUR million as it can be seen in table 2. 82% of the participants arrived into the Hungarian capital by plane, while 16% arrived by road (car, minibus). Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships The vast majority of foreign spectators would like to return into Hungary in the future. More than half of them (57.7%) would like to return within 5 years based on their previous experiences, while the proportion of those who refuse to return is only 3.5%. Foreign spectators spent an average of 102.8 EUR (SD = 64.3) per day without travel costs during the competition, while this amount was 128.2 EUR (SD = 95.2) per day if travel costs were included. This meant an average of 624 EUR (SD = 645) including travel costs for the entire stay in Budapest. The daily spending of foreign spectators of the WTTC was significantly higher than the general foreign tourists’ 51.8 EUR/day (t = 17.0, p = 0.000) and the average spending of tourists arriving especially at sports events which was 54.7 EUR/day (t = 16.0; p = 0.000) in Hungary in 2019 (HCSO, 2020). Distribution of foreign spectators’ expenditure can be seen in Figure 3. There is a significant relationship between the perception of hospitality and the willingness to return (χ2 = 37.19; p = 0.000). It can be said that those who rated hospitality more positively plan to return to Budapest in the future (64% of those who rated hospitality as 5 plan to return to Budapest within 5 years). The willingness to return was also significantly influenced by the assessment of the organisation of the competition (χ2 = 28.9; p = 0.004). Those who considered the quality of the organisation to be more favourable would return to the Hungarian capital in a larger proportion in the future. Almost two-thirds (63.4%) of the foreign spectators who rated the organisation as the best possible (as 5) would like to return in the next 5 years. Foreign spectators rated the quality of the WTTC as favourable, as more than four-fifths (85.1%) of the respondents rated the organisation of the competition as at least four. 11.4%, of them rated it as three, and only a very small number (3.5%) gave a less favourable rating. The average rating of the foreign spectators was 4.26 (SD = 0.80). The characteristics and the opinions of the professional participants are based on a sample of 281 people, which is 12.8% of the professional participants. 41 Int. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-4508 Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships onsumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships Fi 5 E i i t f Li bh 2019 WTTC Figure 5. Economic impact of Liebherr 2019 WTTC Figure 5. Economic impact of Liebherr 2019 WTTC Figure 5. Economic impact of Liebherr 2019 WTTC Gross financial subsidy of the Hungarian government was 3.95 million EUR. The hypothesis of the study was verified, the Liebherr 2019 ITTF World Table Tennis Championships contributed more to the Gross Domestic Product of Hungary (4.78 million EUR) than the subsidy it has got from the government. Even the tax revenues the Hungarian Central Budget realized related to event were higher (4.02 million EUR) than the subsidies. in WTTC whereas 233 EUR in World Tour, 307 EUR in U13 Hungarian Open and 307 EUR in Badminton U18 Hungarian Open. This shows that those who came to the Table Tennis World Championships were willing to spend significantly more than those who came to smaller international events. This is supporting expectations however as if someone comes to a major event then he/she is willing to pay more. We can also understand that the spending in U13 Hungarian Open was higher than in the other two remaining events as with young children parents also travel, who are eager to spend more. Tax revenues per participant is very interesting as these averages shows that at least how much money is a participant worth in government subsidy. As in most cases significant immaterial value is created besides the economic value (Stocker, 2013), therefore if an event’s tax contribution can break even on the government subsidy all immaterial value would be a net benefit. WTTC contributed 347 EUR per participant tax revenues to the Hungarian Central Budget, whereas World Tour contributed 102 EUR, U13 Hungarian Open 133 EUR and Badminton U18 Hungarian Open 132 EUR per participant. Table 2. Net direct expenditure by stakeholders in LIEBHERR 2019 WTTC (EUR) The weighted average added value multiplier of the segments of the expenditure structure of WTTC is 0.36, which means that WTTC generated 4.28 million EUR added value in the Hungarian economy. A total of 47% of the participants visited the city centre because of non-competitive motivations in addition to their duties related to the sports event. The range of tourist attractions visited was almost exclusively concentrated in the downtown and World Heritage sites (96.9%), only 2% visited spas and other activities. Other taxes can be also calculated from input- output analysis, the weighted average multiplier of the respective economic segments was 0.04, which means other taxes generated by WTTC were 502 thousand EUR. WTTC created 4.78 million EUR GDP contribution altogether. The hospitality of the Hungarians was judged by the participants very favourably similarly to the foreign spectators, averaging 4.6 (SD = 0.69). The willingness to return among the participants was also reported very similarly as by the spectators (although probably with different goals) at the same time, 55% of foreign participants plan to return to Budapest within 5 years, while only 2.6% do not want to come to Hungary again. Income taxes and social security paid to the central budget from the generated GDP of WTTC was 1.12 million EUR on top of what 2.9 million EUR VAT was also paid into the budget. 42 CONCLUSIONS The organisation of the competition was rated on average 4.27 by all respondents (SD = 0.92), which can be considered a favourable rating mainly in view of the fact that 86.6% of the respondents rated the event as 4 or 5. However if we compare the participants (mean = 4.24; SD = 0.85), the foreign spectators (mean = 4.26; SD = 0.80) and the Hungarian spectators (mean = 4.30; SD = 1.11), it can be stated that Hungarian spectators rated the organisation significantly better than foreign spectators and participants (χ2 = 10.648; p = 0.005). Foreign passive sports tourists coming to the WTTC spent more time at the event and other tourist attractions than foreign general tourists or sports tourists. Average spending of both foreign and domestic passive sports tourists was higher than foreign or domestic general or sports tourists respectively. The full experience package was important for the spectators in addition to the attractiveness of the event, but almost half of the participants also had additional experiences in Hungary beyond the competition. The proportion of foreigners was high (41%) among the spectators, which may be explained by Hungary’s favourable transport and geographical situation, relative low prices, the popularity of Budapest among Chinese tourists and the fact that an event of similar importance has not been held in the CEE region for more than a decade. It can be said that there is a justification of organising world sports events in Hungary for Olympic sports based on the responses of the spectators and participants and their consumer behaviour. Both the Hungarian organisation and hospitality are so high level that the respondents would like to return to the country in the near future, when they will spend again to fuel the Hungarian economy. The groups participating in the WTTC spent a total of more than 30,000 guest nights (30,060 nights) in Budapest, of which 71.8% were spent in a hotel with at least a three-stars rating. The Hungarian government provided almost 4 million euros support to the organisers of the WTTC. Examining the macroeconomic effects of the competition, it can be stated that every EUR of government support increased the country’s GDP by 1.21 EUR and generated a tax of 1.01 EUR in 2019. Expenditures of spectators and participants contributed 24% of the generated GDP, which were not part of the WTTC budget. DISCUSSION Laczkó and Stocker (2018) calculated economic impact of international racket sports events organised in Hungary with which the WTTC can be compared with as it can be seen in table 3. Table 3. Economic impact per participant of international sports events in racket sports in Hungary  Impact per participant (person) Net spending Total National Output GDP contribution Tax revenues LIEBHERR 2019 ITTF World Championship 617 EUR 1,015 EUR 413 EUR 347 EUR Table Tennis World Tour 2017 233 EUR 374 EUR 158 EUR 102 EUR Table Tennis U13 Hungarian Open 2017 307 EUR 490 EUR 203 EUR 133 EUR Badminton U18 Hungarian Open 2017 265 EUR 437 EUR 184 EUR 132 EUR Source: Authors’ compilation based on Laczkó & Stocker (2018) p. 72 Table 3. Economic impact per participant of international sports events in racket sports in Hungary According to empirical results domestic spectators are elemental in international sports events organised in the European Union as their proportion is from 70% to 99% of all spectators depending on the given sports (Schwark, 2005; Sportcal Global Communications, 2017; Laflin, 2018; Sportcal Global Communications, 2019a; Sportcal Global Communications, 2019b). However the proportion of domestic spectators was only 59% is at WTTC. This difference is due to the favourable geographic and economic situation of Hungary in this aspect, namely that Budapest is very easy to reach from other European countries with around 1-2 hour flight. The purchasing power of most European spectators are very strong if they spend in Hungary. These make a world championship in Budapest easy to reach and affordable. The different sizes of the events are clearly shown in the table, but average spending and contribution also vary. Net spending per participant was 617 EUR 43 nt. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-450 Laczkó, T. et. al. According to Sportcal Global Communications (2018) the daily spectator expenditure was 116.8 EUR in the World Games 2017 which was organised in Wroclaw. The average total expenditure was 138 EUR per spectator at the IFF Men’s World Floorball Championships 2018 in Prague which was 37.5 EUR per day for domestic (Czech) spectators and 96.25 EUR per foreign visitors. In contrast daily expenditure of foreign visitors was 128.2 EUR and 624 EUR for the entire stay while 47.9 EUR per day and 122.6 EUR for the entire competition at WTTC (Sportcal Global Communications, 2017; Sportcal Global Communications, 2019b). DISCUSSION reaching 665 million viewers (of which 558 million people were in China, where the number of unique viewers reached almost 150 million). Internet streaming broadcasts were followed over 8 days in 148 countries, for which 4.8 million searches were registered. In the case of social media interfaces viewers watched 34.7 million minutes on the event’s YouTube channel, while 27.56 million impressions were registered on Facebook (Péli, 2019). CONCLUSIONS It is important to emphasize from tourism’s perspective that foreigners (spectators and participants together) coming to Budapest due to the WTTC considered the hospitality of the Hungarian people very favourable (an average of 4.61 on a scale of 1-5; SD = 0.64), which was significantly associated with a favourable trend in the willingness to return. 57.1% of foreigners plan to return to Hungary within 5 years, but among those who rated hospitality as 5, this proportion was already 62.2%. More than half (55.8%) of the WTTC’s foreign spectators and participants took time to visit one or more sights of the capital. Regarding the macroeconomic effects of the WTTC, it can be said that it contributed more than 4.7 million EUR to Hungary’s GDP which is more than the subsidy given by the government because of the event. WTTC generated 2.9 million EUR in VAT revenue for the budget and further 1.1 million EUR additional tax revenue for the government’s budget. The WTTC’s economic impact generated extremely low amount of imports and most of the WTTC related expenditures were spent within the country. A higher willingness to spend compared to the general tourists and the general sports tourists was clearly identifiable for both domestic and foreign spectators. This can be explained by the location of the competition, which is the settlement with the highest price level and the widest range of tourist offers in Hungary. The peculiarity of the WTTC is that the most important table tennis manufacturers and distributors in the world came to the event venue with special offers which encourages spectator groups to buy table tennis related goods. There were also positive sports policy effects in addition to the economic and tourism related effects and Budapest became a focal point in the international media during the competition. There are also some limitations on the use of input- output modelling in the international literature when estimating the complex economic impacts of sports The 2019 WTTC set a record in terms of media. TV channels broadcasted in 145 countries in 1176 hours, 44 Consumption habits and economic impact of Liebherr 2019 ITTF World Table Tennis Championships sociological and health aspects of sports tourism]. Wiesbaden, Germany: Universitatsverlag. competitions (Vörös & Koppány, 2019). These include supply-side income leakage, one-sided interpretation of demand growth, crowding-out and substitution effects. CONCLUSIONS When quantifying the macroeconomic effects generated by the WTTC some of the mentioned methodological shortcomings do not appear (due to the size of the event) and we tried to handle the constraint related to domestic viewers (substitution effect). European Commission (Eds) (2012). Study on the Contribution of Sport to Economic Growth and Employment in the EU. Retrieved from https:// ec.europa.eu/assets/eac/sport/library/studies/ study-contribution-spors-economic-growth-final- rpt.pdf European Commission (2018). Eurobarometer - Sport and Physical Activity. Brussels, Belgium. Retrieved from http://ec.europa.eu/commfrontoffice/ publicopinion/index.cfm/survey/getsurveydetail/ instruments/special/surveyky/2164 We consider it important to monitor the macroeconomic impact of other world table tennis events in the future, taking into account the local specificities of the host countries, which can contribute to a well-founded tendering process for the organisation of these events. Particular emphasis may also be placed on the ITTF’s current efforts to transform adult and youth competition series into a new, even more business-based type. Hungarian Central Statistical Office (2020). Information database, Main Indicators of foreign tourists in Hungary [Data Set]. http://statinfo.ksh.hu/ Statinfo/themeSelector.jsp?page=2&szst=OGT Hungarian Central Statistical Office (2020). Information database, Characteristics of multi-day domestic trips [Data Set]. http://statinfo.ksh.hu/Statinfo/ themeSelector.jsp?page=2&szst=OGT REFERENCES com/Impact/EventStudies Laczkó, T., & Paár, D. (2018). Társadalmi hatások vizsgálata a 2018. évi hazai rendezésű nemzetközi sportesemények kapcsán [Research of social effects of domestic organised sport events in 201]. In T. Szabó, M. Bánhidi, & G. Szőts (Eds.), A sportturizmus gazdasági és társadalmi kérdései Magyarországon (pp. 77–102). Budapest, Hungary: Hungarian Society of Sport Science. Sportcal Global Communications Ltd. (2019a). Global Sports Impact Report 2019. Retrieved from https:// drive.google.com/file/d/1xp768UG8LsouOvI0N7EPL ixR6SkjO3s5/view Sportcal Global Communications Ltd. (2019b). IFF Men’s World Floorball Championships 2018. Retrieved from http://dltaw1vhj9zy5.cloudfront.net/2019/05/ Sportcal-GSI-Event-Study-IFF-Mens-World- Championships-2018-FINAL-Public-version.pdf Laczkó, T., & Stocker, M. (2018). 2018. évi hazai rendezésű nemzetközi sportesemények gazdasági és turisztikai hatásainak vizsgálata [Analysis of socio-economic and tourism impacts of domestic organized sport events in 2018]. In T. Szabó, M. Bánhidi, & G. Szőts (Eds.), A sportturizmus gazdasági és társadalmi kérdései Magyarországon (pp. 37-76). Budapest, Hungary: Hungarian Society of Sport Science. Standeven, J., & De Knop, P. (1999). Sport tourism. Leeds, United Kingdom: Human Kinetics Europe. Stocker, M. (2013). Dematerializálódás szerepe az értékteremtésben [Demate value creation]. Vezetéstudomány, 14(12), 44-53. Laczkó, T., & Stocker, M. (2020). A 2017. évi hazai rendezésű nemzetközi sporteseményekre érkező külföldi turisták fogyasztásának nemzetgazdasági hatásai [The economic impact of consumption by foreign visitors to major international sporting events in Hungary in 2017]. Turizmus Bulletin, 20(2), 22-33. https://doi.org/10.14267/TURBULL.2020v20n2.3 Stocker, M. & Boda, Gy. (2018). A sportszektor gazdaságdinamizáló hatása [The Economic Dynamising Effect of Sports Sector]. Magyar Sporttudományi Szemle, 19(75), 78. Stocker, M., & Laczkó, T. (2020). Expenditures of CEE and Non-CEE spectators in major international sport events in Hungary. Problemy zarządzania - Management issues, 2020(87):81-98. DOI: 10.7172/1644-9584.87.4 Laflin, M. (2018). The Explosion of International Sport Events. Retrieved from https://drive.google.com/ file/d/1GbwBRjfV9kB5Y6cbfi2v2PYebDe7a5Lc/view Lasztovicza, D., & Béki, P. (2016). A sport és a turizmus kapcsolata, a sportturizmus fogalmi rendszere [The Connection between Sport and Tourism, the Conceptual System of Sport Tourism]. Testnevelés, Sport, Tudomány, 1(1), 39-50. https://doi. org/10.21846/TST.2016.1.4 Stocker, M. & Szabó, T. (2017). A hazai sportirányítás szerepe és tevékenysége a kiemelt hazai sportesemények esetében [Role and activity of Hungarian national sport governance in international sport events]. In M. Szmodis, G. Szőts (Eds.), A sportirányítás gazdasági kérdései – 2017, Magyar Sporttudományi Füzetek XVI. (pp. 56–77). Budapest, Hungary: Hungarian Society of Sport Science. Paár, D., & Laczkó, T. (2018). 2018. évi hazai rendezésű nemzetközi sportesemények sportszakmai, sportpolitikai és sporttechnológiai hatásainak vizsgálata [Research of domestic organised sport events’ effects from the point of view sport performance, sport politics and sport technology in 2018]. REFERENCES Ács, P. (2007). Bevezetés a sportstatisztikába [Introduction to Sports Statistics]. Budapest-Pécs, Hungary: Dialóg Campus. Hungarian Tourist Agency (2017). Magyar Turizmusfejlesztési Stratégia 2030 [Hungarian Tourism Development Strategy]. Budapest: Hungarian Tourist Agency. Ács, P. (2015). Research Methodology in Sport Sciences. Pécs, Hungary: University of Pécs, Faculty of Health Sciences. Kóródi, M. (2011). Turizmus kutatások módszertana [Methodology of Tourism Research]. Pécs, Hungary: University of Pécs. Albers, A. (2004). Sport als Imageträgen im Tourismus: Auswirkungen und Chancen von Sportevents für Destinationen [Sport as image carriers in tourism: Effects and opportunities of sporting events for destinations]. Paderborn, Germany: Verlag des Faches Geographie, Universität Paderborn. Kovács, A., Paár, D., Elbert, G., Welker, Zs., Stocker, M., & Ács, P. (2015). A magyar háztartások sportfogyasztási szokásainak felmérése [Survey of Sports Consumption Habits of Hungarian Households]. Pécs, Hungary: University of Pécs, Faculty of Health Sciences. Bánhidi, M. (2015). Sportturisztikai termékek. In: T. Laczkó & M. Bánhidi (Eds.), Sport és egészségturizmus alapjai [Basics of Sport and Health Tourism]. (pp. 47-75). Pécs, Hungary: University of Pécs, Faculty of Health Sciences. KPMG (2015). Futóturizmus Budapesten. A Budapest Futóiroda 2015 őszi budapesti futóversenyeinek nemzetgazdasági hatásvizsgálata [Running Tourism in Budapest. National Economic Impact Assessment of the Budapest Running Office’s Running Competitions in Budapest in the Autumn 2015]. Retrieved from http://www.futanet.hu/kep0/ PR/gazdasagi_sajttaj/KPMG_BSI_2015-Gazdasagi_ hatastanulm%C3%A1ny_Vezetoi_osszefoglalo.pdf Bánhidi M. (2018). A nemzetközi sportturizmus területén végzett kutatások elemzése sporttudományi megközelítésben [International sport tourism literature analysis from sport science perspectives]. In T. Szabó, M. Bánhidi & G. Szőts (Eds.), A sportturizmus gazdasági és társadalmi kérdései Magyarországon (pp. 9-36). Budapest, Hungary: Hungarian Society of Sport Science. Kundi, V. (2012). Fesztiválok városokra gyakorolt gazdasági- és társadalmi-kulturális hatásainak elemzése. A Győri Magyar Táncfesztivál és a Miskolci Operafesztivál példáján keresztül [Analysis of the Economic and Socio-cultural Impacts of Festivals on Cities. The Example of Hungarian Dance Festival in Győr and Miskolc Opera Festival]. [Doctoral dissertation, Széchenyi István University]. Retrieved from https://rgdi.sze.hu/images/RGDI/ honlapelemei/fokozatszerzesi_anyagok/kundi_ viktoria_vegleges_disszertacio.pdf Borbély A., & Müller A. (2015). Sport és turizmus [Sport and Tourism]. Debrecen, Hungary: Campus. Chen, N., & Funk, D.C. (2010). Exploring destination image, experience and revisit intention: a comparison of sport and non-sport tourist perceptions. Journal of Sport Tourism, 15(3), 239-259. https://doi.org/10.108 0/14775085.2010.513148 Dreyer, A. (2002). Sport und Tourismus. Wirtschaftliche, soziologische und gesundheitliche Aspekte des Sport-Tourismus [Sport and tourism. Economic, 45 Int. j. racket sports sci. vol. 2(2), 2020, 37-46. eISSN: 2695-4508 Laczkó, T. et. al. Sportcal Global Communications Ltd. (2018). The World Games 2017. Retrieved from https://www.sportcal. REFERENCES In T. Szabó, M. Bánhidi, & G. Szőts (Eds.), A sportturizmus gazdasági és társadalmi kérdései Magyarországon (pp. 103–130). Budapest, Hungary: Hungarian Society of Sport Science. Sulyok, J., & Magyar, Zs. (2014). Az aktív turizmus helyzete Magyarországon. Fókuszban a kerékpározás és a természetjárás [The Situation of Active Tourism in Hungary. Cycling and Hiking in Focus]. Turizmus Bulletin, 16(3-4), 15-26. Vörös, T., & Koppány, K. (2019). Sportlétesítmény- beruházások társadalmi-gazdasági hatásainak értékelési lehetőségei [Methodological approaches to assess social and economic impacts of sport facility investments]. In T. Gyömörei (Ed.), Egészség, Sport, Gazdaság (pp. 18-31). Győr, Hungary: Széchenyi István University of Győr. Péli, L. (2019). Asztalitenisz-vb tévés és host broadcast tapasztalatok [TV and Host Broadcast Experiences of World Table Tennis Championship]. Retrieved from https://pelicom.hu/portfolio/asztalitenisz- vb-teves-es-host-broadcast-tapasztalatok/ Schwark, J. (2005). Wirtschaftliche Bedeutung des 3. KarstadtRuhrMarathons 2005. Bocholt, Germany: FH Gelsenkirchen http://www.sport-tourismus.de/ pdf/2005_karstadt_ruhr_marathon_zuschauer.pdf Sportcal Global Communications Ltd. (2017). GSI Event Study / FIS Nordic World Ski Championships 2017. Retrieved from https://www.sportcal.com/ Reports/Search/44 46
https://openalex.org/W3021577374
https://europepmc.org/articles/pmc7204209?pdf=render
English
null
Recurrent costs in primary health care in Ethiopia: facility and disease specific unit costs and their components in government primary hospitals and health centers
BMC health services research
2,020
cc-by
9,793
Agarwal et al. BMC Health Services Research (2020) 20:389 https://doi.org/10.1186/s12913-020-05218-1 Agarwal et al. BMC Health Services Research (2020) 20:389 https://doi.org/10.1186/s12913-020-05218-1 (2020) 20:389 Open Access Recurrent costs in primary health care in Ethiopia: facility and disease specific unit costs and their components in government primary hospitals and health centers Anubhav Agarwal1,2, Carlyn Mann1,3*, Engida Abdella4, Workie Mitiku4, Abebe Alebachew4 and Peter Berman1,5 Abstract Background: Continued investment, especially from domestic financing, is needed for Ethiopia to achieve universal health coverage and a sustainable health system over time. Understanding costs of providing health services will assist the government to mobilize adequate resources for health, and to understand future costs of changes in quality of care, service provision scope, and potential decline in external resources. This study assessed costs per unit of service output, “unit costs”, for government primary hospitals and health centers, and disease-specific services within each facility. Methods: Quantitative and qualitative data were collected from 25 primary hospitals and 47 health centers across eight of the eleven regions of Ethiopia for 2013/14, and 2014/15 and 2015/16 but only for primary hospitals, and supplemented by other related health and financial institutions records. A top-down costing approach was used to estimate unit costs for each facility by department – inpatient, outpatient, maternal and child health, and delivery. A mixed-method approach was used for the disease-specific unit costs exempt from fees. Results: Health center median unit cost was 146 Ethiopian birr (ETB) (17 PPP$, 2012), the Delivery department had the highest median unit cost (647 ETB; 76 PPP$, 2012) and Outpatient department (OPD) had the lowest (124 ETB; 14 PPP$, 2012). Primary hospital median unit cost was 339 ETB (40 PPP$, 2012), with Inpatient department having the highest median unit cost (1288 ETB; 151 PPP$, 2012), while OPD was the lowest (252 ETB; 29 PPP$, 2012). Drugs and pharmaceutical supplies accounted for most of the costs for both facilities. Among the exempted services offered, tuberculosis and antiretroviral treatment are the costliest with median unit costs from 1091 to 1536 ETB (128–180 PPP$, 2012), with drugs and supplies accounting for almost 90% of the costs. (Continued on next page) * Correspondence: cmann@hsph.harvard.edu 1Global Health and Population Department, Harvard T.H. Chan School of Public Health, 677 Huntington Ave, Boston, MA 02115, USA 3Present Address: 500 D St SW, Washington DC 20024, USA Full list of author information is available at the end of the article * Correspondence: cmann@hsph.harvard.edu 1Global Health and Population Department, Harvard T.H. Chan School of Public Health, 677 Huntington Ave, Boston, MA 02115, USA 3Present Address: 500 D St SW, Washington DC 20024, USA Full list of author information is available at the end of the article 1Somali region was initially included to add to the representation of the developing regions; however, data was very limited and aggregated at the woreda level rather than facility level. Therefore, facilities from this region could not be included in the study. Study setting y g Ethiopia’s government primary health care delivery organization is comprised of health posts, health centers, and primary hospitals. A health post, the lowest level of the primary health care system, mainly provides promo- tive and preventive health care services; serving 3000– 5000 people in a woreda. A health center is a referral center for health posts, and provide promotive, prevent- ive, curative and rehabilitative outpatient care including basic laboratory and pharmacy services; serving 15,000– 25,000 people in a woreda. A primary hospital is the highest level of primary health care and provides in- patient and ambulatory services. This includes all of the same services offered at health centers, as well as add- itional emergency surgical services, including caesarian sections and blood transfusions, and serves as a referral center for health centers that reside within the primary hospital’s catchment area. Primary hospitals serve 60, 000–100,000 people in a woreda. Investment in primary hospitals is still ongoing so not all health centers are linked with primary hospitals and some primary hospitals may serve several woredas [11, 12]. Ethiopia is focusing on providing universal access to quality health care without financial burden and ensuring an adaptive health system to meet the population’s chan- ging health needs [4, 5]. Continued investment is needed to meet this goal and a substantial increase in domestic fi- nancing for health will allow for a more sustainable health system [6]. Understanding the costs of providing the current health service envelope is first needed to deter- mine the resources that the government needs to mobilize for health, and to begin understanding the potential future costs of changes in quality of care, scope of service provision, and possible decline in external funding. Few representative data on the costs or resources used to provide services at government-funded health care facilities exist for Ethiopia. One previous costing exercise in Ethiopia consisted of a very small sample of health facilities for pricing the potential social health insurance benefits package [7]. The results of that study do not reflect the significant changes to the health system since 2007. Other work includes normative costing exercises for the 5-year health sector plans and the essential health service package [8–10]. This type of costing is based on standards and norms to provide health care services but might not reflect real costs of service provision under field conditions. Background Ethiopia has made great strides in improving the health of its population over the last decade. The maternal mortality ratio dropped to 412 maternal deaths per 100, 000 live births (LBs) in 2016, from 676 maternal deaths in 2011 [1, 2]. Infant mortality rate also halved from 97 deaths per 1000 LBs in 2000 to 48 deaths in 2016, and antenatal care by a skilled birth attendant increased from 27% in 2000 to 62% in 2016 [2]. This is partially due to sustained economic growth, a rapid increase of financial investments into the health system and moving away from an urban-centric model with greater emphasis on primary health care [3]. Nonetheless, more progress is needed if Ethiopia is to continue to improve health out- comes and sustain the progress made during the Millennium Development Goal era. (Continued from previous page) (Continued from previous page) p p g Conclusions: High unit costs of service provision could be indicative of underutilization of the primary health care system, coupled with inefficiencies associated with organization and delivery of health services. Data from this study are being used to assess efficiency and productivity among primary care facilities, facilitate premium setting for health insurance, and improve budgeting and allocating health resources for a more sustainable and effective primary health care system. Keywords: Unit costs, Disease-specific costs, Health care services, Public facilities, Ethiopia inputs in estimating service output efficiency, explaining causes of variations in cost/output ratios, identifying the right strategies to improve efficiency and quality, and sup- port efforts to mobilize more domestic resources for health. We partnered with Ethiopia’s Ministry of Health (MoH) to conduct a primary health care service costing study in seven of the eleven regions in Ethiopia. The measurement of this type of data, and subsequent analyses that it can be used for, will contribute to Ethiopia’s health transformation agendas of equitable and quality of health care; improving data quality and use for effective decision- making; and woreda (district) transformation [4]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Agarwal et al. BMC Health Services Research (2020) 20:389 Page 2 of 12 Costing methodology Data collected during this phase was 2013/14 (the Ethi- opian fiscal year (EFY) 2006). In the second phase of the study additional primary hospitals were purposively se- lected, using a proportional to population size sampling method among the four major regions – Tigray, Am- hara, Oromia and Southern Nations, Nationalities, and Peoples’ Region (SNNPR) – where an adequate number of functioning primary hospitals were in existence for at least 1 year at the time of this supplemental data collec- tion. Data collected during this phase was for 2014/15 (EFY 2007), with the exception of primary hospitals in Oromia which were not fully operational during this year. Data from primary hospitals in this region was col- lected from the last quarter of 2014/15 along with the first three quarters of 2015/16. No statistical difference was found among primary hospital facility characteristics and service statistics from 1 year to the next [11]. A total of 40 health centers and 24 primary hospitals had data of sufficient quality for analysis. The costing framework and analysis followed the major steps and recommendations outlined in guidelines by Hanson and Gilson, and Creese and Parker [13, 14] for conducting a cost accounting analysis in primary health care and hospitals (see Supplementary file 3). “Cost” is defined as the monetary value of non-capital, recurrent expenditures incurred, and resources used to produce a defined set of health service outputs or to operate spe- cific health facilities. The recurrent costs include drugs and supplies, salaries, and other operational costs (e.g., electricity, running water, maintenance, etc.) incurred on a regular basis that were allocated as direct or indirect costs. Direct costs are costs directly attributable to a spe- cific service output and consists of drugs and supplies and salaries. Indirect costs are other operational costs (e.g., electricity, running water, etc.) not attributed directly to a specific output. Both direct and indirect costs were included. Costs were estimated irrespective of where the money to cover such costs came from; such sources include revenue obtained through user fees, funds provided by federal block grants, and resources provided by development partners. Some primary care facilities rely on technical support from partners and nongovernmental organizations to administer certain exempted services. This study does not cost such tech- nical assistance because this information is not collected and reported in the standard administrative records of the individual health facilities. Sample selection The study was conducted in two phases in seven1 of the eleven regions of Ethiopia (Table 1). The first phase used Health service costing data have a variety of uses, such as health sector budgeting and planning, pricing of ser- vices, and reimbursement modalities for public sector ser- vices as part of insurance schemes. They can be valuable Agarwal et al. BMC Health Services Research (2020) 20:389 Page 3 of 12 Table 1 Final sample for analysis by geographic location Major Regions Developing Regions City Administration Health Facility Type Amhara Oromia SNNPR Tigray Benishangul-Gumuz Addis Ababa Dire Dawaa Total for Analysis Primary hospitals 8 8 5 3 0 0 0 24 Health Center 9 9 0 0 5 9 8 40 Total 17 17 5 3 5 9 8 64 Note: Data was collected from 94 PHC facilities (25 primary hospitals, 47 health centers, and 22 health posts). Poor data quality led to a reduction in the sample size from 94 to 64 PHC facilities during data analysis, where 1 primary hospital, 7 health centers, and 22 health posts had incomplete or poor data quality records beyond ‘fixing’ using extrapolation aDire Dawa sample selection process based on primary hospitals and health centers with data availability and accessibility (not based on woreda selection first since woredas do not exist in this city administration) Table 1 Final sample for analysis by geographic location Note: Data was collected from 94 PHC facilities (25 primary hospitals, 47 health centers, and 22 health posts). Poor data quality led to a reduction in the sample size from 94 to 64 PHC facilities during data analysis, where 1 primary hospital, 7 health centers, and 22 health posts had incomplete or poor data quality records beyond ‘fixing’ using extrapolation aDire Dawa sample selection process based on primary hospitals and health centers with data availability and accessibility (not based on woreda selection first since woredas do not exist in this city administration) Costing methodology a multi-stage cluster sampling to account for variation among regions and woredas with regard to major deter- minants that affect health service demand and utilization ensuring that units with particular characteristics (urban, rural, etc.) were included in the sample. Two regions, or city administrations, were selected within each stratified group. Three woredas were selected within each identi- fied region, and in each selected woreda one primary hospital, three health centers (under the primary hospital catchment), and two health posts (under at least one of the health centers catchment) were selected for data col- lection. However, the inclusion of health posts for the analysis was not possible due to incomplete data records. Data collected during this phase was 2013/14 (the Ethi- opian fiscal year (EFY) 2006). In the second phase of the study additional primary hospitals were purposively se- lected, using a proportional to population size sampling method among the four major regions – Tigray, Am- hara, Oromia and Southern Nations, Nationalities, and Peoples’ Region (SNNPR) – where an adequate number of functioning primary hospitals were in existence for at least 1 year at the time of this supplemental data collec- tion. Data collected during this phase was for 2014/15 (EFY 2007), with the exception of primary hospitals in Oromia which were not fully operational during this year. Data from primary hospitals in this region was col- lected from the last quarter of 2014/15 along with the first three quarters of 2015/16. No statistical difference was found among primary hospital facility characteristics and service statistics from 1 year to the next [11]. A total of 40 health centers and 24 primary hospitals had data of sufficient quality for analysis. a multi-stage cluster sampling to account for variation among regions and woredas with regard to major deter- minants that affect health service demand and utilization ensuring that units with particular characteristics (urban, rural, etc.) were included in the sample. Two regions, or city administrations, were selected within each stratified group. Three woredas were selected within each identi- fied region, and in each selected woreda one primary hospital, three health centers (under the primary hospital catchment), and two health posts (under at least one of the health centers catchment) were selected for data col- lection. However, the inclusion of health posts for the analysis was not possible due to incomplete data records. Costing methodology The health facilities also do not record if the patients procure drugs and supplies from a private pharmacy outside the health facility, or the costs associated with patient referrals from one health facility to another. Thus, such costs were excluded from this analysis. Furthermore, capital costs were not included in this study due to study feasibility and other data limitations. Capital costs may not vary much by type of facility when amortized over long periods of time. Secondary data including financial, administrative and health utilization (drug consumption, utilization rates, etc.) was extracted from the administrative records at the health facilities and other related health and financial institutions. Additionally, interviews with the health facility personnel were used to supplement data collection. A key informant interview guide was developed and used (Supplementary file 1). A paper-based survey instrument (Supplementary file 2) was used to extract the necessary data from the health facilities and other related institutions for this study. Direct and indirect costs were allocated across defined departments or cost centers for primary hospitals and health centers. The existing institutional arrangement for primary care services in Ethiopia was used to identify Agarwal et al. BMC Health Services Research (2020) 20:389 Page 4 of 12 Page 4 of 12 Page 4 of 12 the four departments – Inpatient department (IPD), Outpatient department (OPD), Maternal and Child Health department (MCH), and Delivery department. Notably, the MCH department provides non-emergency maternal and child health services (such as immuniza- tions, family planning, antenatal care, and post-natal care), excluding deliveries. On the other hand, the deliv- ery department focuses on basic obstetric care (health centers) and comprehensive obstetric care (primary hos- pitals). Staff cost allocation was based on their formal work assignments because a detailed time-motion based allocation was not feasible. Key informant interviews at the facility were used to adjust any allocations of human resource (HR) costs where staff work in more than one department. A two-step approach was used to allocate the drugs and supplies costs by department. The first step consisted of focusing on program drugs and sup- plies that are used for specific treatments that would be offered under one of the four departments. The second step was to allocate drugs and supplies that could not be clearly allocated by department, consisting of non-pro- gram drugs and supplies for IPD, OPD, and Delivery de- partment. 2Capital costs may not vary much by type of facility when amortized over long periods of time. These costs could be added later from other data sources on inputs and their costs. Limitations d h Readers should be cautious of the limitations of the study while interpreting the results. Unit cost estimates are affected by the quality of the available data. For ex- ample, number of patient contacts, the denominator for the unit cost estimates, was based on health manage- ment information systems data, which were at times found to be incomplete for the whole year and data im- putations were used to fill data gaps. Also, problems were encountered during the data collection process, leading to a higher attrition of primary health care facil- ities in the study than expected and thus reducing our sample size from the originally planned 96 primary health care facilities to the 76 used in the costing ana- lysis. See reference [11, 16] for more details of these issues. All operating costs for a primary health care facil- ity could not be included in this study. It excludes cap- ital costs,2 potential costs that are provided by technical assistance from development partners, and unforeseen additional costs to either a health provider or patient such as procuring drugs and supplies from a private pharmacy when stock-outs occur. Our analysis also does not reflect possible combined costs resulting from pa- tient referrals from one health facility to another. Lastly, data issues to estimate exempted services limited disag- gregating family planning services by specific types, de- termining the unit cost of treatment of leprosy, and led Unit cost output Mi f E l Microsoft Excel 2010 and STATA 14 was used to ana- lyse the data. Median estimates, instead of averages, are reported for unit and annual costs to minimize the effect of outliers. The health facility unit cost is the ratio of the total re- current costs relative to the total number of patient con- tacts for a given health facility. The department-wise unit cost is the ratio of total recurrent costs estimated for that department relative to the total patient contacts of that department. Cost estimates are reported in both current Ethiopian birr (ETB) and purchasing power parity (PPP) with 2012 as the base year. Costing methodology The MCH department was not included because services offered at the MCH department are only considered as program-related services, such as family planning and vaccinations. For non-program drugs and supplies a ratio of 1:4 was used to allocate these costs across inpatient (IPD and Delivery) and out- patient departments [11]. This is a similar ratio found in paper by Özaltın and Cashin [15]. To allocate the non- program costs across the three departments, one-ninth of the costs went to OPD, four-ninths went to IPD, and the remaining four-ninths went to the Delivery department. for all patients across the 4 defined departments. The unit costs for ANC and PNC were estimated using a cost-mix approach with normative and field-based costs because the drugs and supplies consumed for these two services could not be attributed directly to those particu- lar services, unlike other exempted services such as malaria treatment or family planning. Costing of exempted services Exempted services are defined as the services that are of- fered for free to everyone among government-provided health facilities regardless of their income level [10]. Exempted services include care for tuberculosis, mater- nal and childcare (prenatal, delivery, postnatal, immuni- zations), family planning, antiretroviral treatment (ART) for HIV/AIDS, leprosy, and fistula and epidemics. A large portion of these services are financed by external funding, especially reproductive and maternal health [3]. Costing exempted services thus deserves special atten- tion as the sustainability of these services is uncertain. The costing framework for exempted services uses a similar approach as the primary hospitals and health centers departmental unit costs. Exempted services were classified under their respective departments (see Table 2). Some health centers do not offer all exempted services and thus omitted from such estimates. The HR and indirect costs were based on average per visit cost Agarwal et al. BMC Health Services Research (2020) 20:389 Page 5 of 12 Table 2 Costed exempted services by department Outpatient department (OPD) Maternal and child health department (MCH) Delivery department Tuberculosis Expanded program for immunizations (all vaccines) Delivery (natural and complicated) Anti-retroviral treatment Family planning Malaria Antenatal care Postnatal care Table 2 Costed exempted services by department Outpatient department (OPD) Maternal and child health department (MCH) Delivery department Tuberculosis Expanded program for immunizations (all vaccines) Delivery (natural and complicated) Anti-retroviral treatment Family planning Malaria Antenatal care Postnatal care Delivery department Delivery department Delivery (natural and complicated) (Fig. 1). Drugs and supplies accounted for a substantial proportion of facility costs at 52% for health centers and 42% for primary hospitals. to a normative-based costing for ANC and PNC services rather than field representation. All of these limitations with this costing analysis either lead to overestimating (e.g., using normative costing instead of field-based cost- ing for ANC and PNC) or underestimating (e.g., not in- cluding annual capital costs) costs for primary hospitals and health centers. y The main cost driver were drugs and supplies for health centers with the exception for the Delivery de- partment and OPD (Fig. 2). Both of these departments typically require more HR time for service provision, but the high HR costs might also be attributed to inefficien- cies in human resource allocation based on patient load. Exploring this further, what “drives costs” is a combin- ation of the costs and availability of drugs and supplies combined with the productivity of human resources. Unit costs bl l Table 5 lists unit costs per service output per year for provision of various health services at the mentioned health facilities. The overall facility median unit cost for health centers was 146 ETB (17 PPP $, 2012), with the Delivery department having the highest median unit cost at 647 ETB (76 PPP $, 2012) and OPD with the lowest unit cost at 124 ETB (14 PPP $, 2012). The median health facility unit cost for primary hospitals was 339 ETB (40 PPP $, 2012), more than double compared to health centers. IPD had the highest median unit cost (1288 ETB (151 PPP $, 2012)), while OPD was the low- est (252 ETB (29 PPP $, 2012)) among primary hospitals. Costing of exempted services Unpacking this further requires additional analysis that is forthcoming. Indirect costs (e.g., electricity, running water, etc.) only accounted for a small portion of the total costs. We see a slightly different picture for primary hospitals where HR was the main cost driver, accounting for at least 50% of total costs with the exception of the MCH department (35%). Profile of study centers The median catchment population for a health center and primary hospital in the study sample was 28,342 people and 352,805 people, respectively (Table 3). Most primary hospitals have a catchment population substan- tially higher than the standard (with a median almost 3.5 times more than the standard) since rollout of this rela- tively new health facility is still underway and typically serve multiple woredas. Primary hospitals have about 3 times the clinical staff (doctors, nurses, health officers, and midwives) com- pared to health centers. The median per capita contact rate for health centers (0.805) is more than primary hos- pitals (0.660). This rate simply uses catchment popula- tion as the denominator and doesn’t account for possible spillover effects facilities may experience from other woredas. Annual costs Referring to Table 4, the median annual recurrent costs for health centers and primary hospitals were ETB 3.9 million (0.46 million PPP $, 2012) and ETB 12 million (1.4 million PPP $, 2012), respectively. HR costs were comparable across the two health facilities, accounting for 39% for health centers and 40% for primary hospitals Table 3 Health facility summary statistics (medians) Characteristic Health centers (N = 40) Primary Hospitals (N = 24) Catchment population 28,342 352,805 Annual per capita contact rate for catchment area population 0.805 0.660 Total staff count 47 154 Total clinical staff counta 20 63 Outpatient attendance per year 13,141 35,554 Total deliveries per year 239 744 Total IPD discharges per year 43 1104 aClinical staff includes doctors, nurses, health officers, and midwives Table 3 Health facility summary statistics (medians) Catchment population Agarwal et al. BMC Health Services Research (2020) 20:389 Page 6 of 12 Table 4 Annual costs of delivering healthcare services by health facility (median costs) Health Centers (n = 40) Primary Hopitals (n = 24) Annual Cost ETB PPP $, 2012 ETB PPP $, 2012 Drugs and pharmaceutical supplies 1,690,499 197,951 4,726,543 553,459 Human resources 1,241,826 145,413 4,483,344 524,982 Indirect expenditures 287,367 33,650 1,968,421 230,494 Total expenditure 3,924,119 459,499 12,012,302 1,405,152 PPP Purchasing power parity conversion factor = 8.54 Ethiopian birr to international dollar [17] Table 4 Annual costs of delivering healthcare services by health facility (median costs) comprised of HR (45%), followed by drugs and supplies (39%), and indirect costs (17%). Looking more closely at the individual vaccines under this program, the median unit costs vary. For the health centers, the measles vac- cine had the lowest median cost to administer (63 ETB (7 PPP $, 2012) per vaccination), while the pentavalent vaccine had the highest median cost to administer (123 ETB (14 PPP $, 2012) per vaccination). Among these primary hospitals, the Pneumococcal vaccine had the highest median cost to administer (233 ETB (27 PPP $, 2012) per vaccination), while the BCG and measles vaccine had the lowest median cost to administer (161 ETB (19 PPP $, 2012) per vaccination each). Unit cost for family planning services Unit cost for family planning services An “acceptor” for family planning is a patient of repro- ductive age (15–49 years) receiving a modern contracep- tive method. Contraceptive services include provision of contraceptive supplies as well as routine check-ups for ongoing use of a long-term method such as Norplant, intrauterine device (IUD), etc. The median recurrent unit cost for a family planning service was moderately high for both health centers and primary hospitals, at Regional distribution of unit costs The distribution of median unit costs varied significantly by region across the studied health centers, ranging from 116 to 171 ETB (14–20 PPP $, 2012), and primary hos- pitals, ranging from 71 to 457 ETB (8–53 PPP$, 2012) (Table 6). Across all the regions, OPD and MCH had substantially lower median unit costs compared to both IPD and Delivery department (Figs 3 and 4). Unit cost for EPI The median recurrent unit cost for the EPI vaccines ad- ministered at health centers and primary hospitals was 84 ETB (10 PPP $, 2012) and 189 ETB (22 PPP $, 2012) per vaccination, respectively. Among primary hospitals, the largest share of this cost comes from HR (42%), followed by indirect costs (37%), and drugs and supplies (21%). A majority of the EPI costs for health centers is Fig. 1 Percent distribution of annual median costs in ETB for health centers and primary hospitals. This figure presents the proportion of the cost components – human resources, drugs and pharmaceutical supplies, and indirect costs – from the total annual median costs for primary hospitals and health centers. The proportional break down of the median costs for health centers was: 52% for drugs and supplies, 39% for human resources, and the remaining 9% for indirect costs. The proportional break down of the median costs for primary hospitals was: 42% for drugs and supplies, 40% for human resources, and the remaining 18% for indirect costs. The sample size (n) for each health facility is presented – 40 health centers and 24 primary hospitals were used in the cost study Fig. 1 Percent distribution of annual median costs in ETB for health centers and primary hospitals. This figure presents the proportion of the cost components – human resources, drugs and pharmaceutical supplies, and indirect costs – from the total annual median costs for primary hospitals and health centers. The proportional break down of the median costs for health centers was: 52% for drugs and supplies, 39% for human resources, and the remaining 9% for indirect costs. The proportional break down of the median costs for primary hospitals was: 42% for drugs and supplies, 40% for human resources, and the remaining 18% for indirect costs. The sample size (n) for each health facility is presented – 40 health centers and 24 primary hospitals were used in the cost study Fig. 1 Percent distribution of annual median costs in ETB for health centers and primary hospitals. This figure presents the proportion of the cost components – human resources, drugs and pharmaceutical supplies, and indirect costs – from the total annual median costs for primary hospitals and health centers. The proportional break down of the median costs for health centers was: 52% for drugs and supplies, 39% for human resources, and the remaining 9% for indirect costs. Unit cost of exempted services Table 7 shows the estimated median unit costs for exempted services (health services that are free to all regardless of welfare) in both ETB and PPP $ (2012) for the studied primary hospitals and health centers. Unit cost for tuberculosis, ART and Malaria treatment Unit cost for tuberculosis, ART and Malaria treatment The median recurrent unit cost per tuberculosis case per year was 1259 ETB (147 PPP $, 2012) for a health center and 1179 ETB (138 PPP $, 2012) for primary hospitals. Drugs and supplies were the main cost drivers for this treatment for both primary hospitals (81%) and health centers (87%). ART median unit cost estimates were based on the number of people living with HIV/AIDS currently receiving ART for EFY 2006, and was esti- mated at 1091ETB (128 PPP $, 2012) and 1536 ETB (180 PPP $, 2012) per case per year for health centers and primary hospitals, respectively. A majority of ART costs were for drugs and supplies, consisting of 84% of the median unit costs for primary hospitals and 88% for health centers. Malaria treatment included both compli- cated or severe cases (infections complicated by organ failure or abnormalities in patient’s blood or aSample for IPD unit costs for the health centers was reduced from 40 to 30 because only 30 health centers had IPDs Unit cost for EPI The proportional break down of the median costs for primary hospitals was: 42% for drugs and supplies, 40% for human resources, and the remaining 18% for indirect costs. The sample size (n) for each health facility is presented – 40 health centers and 24 primary hospitals were used in the cost study Agarwal et al. BMC Health Services Research (2020) 20:389 Page 7 of 12 Fig. 2 Percent share of total department expenditure in ETB for health centers and primary hospitals. This figure presents the proportion of the cost components for each health facility department – IPD, OPD, MCH, and Delivery. The main cost driver are drugs and supplies for health centers with the exception for the Delivery department and OPD. Indirect costs (e.g., electricity, running water, etc.) only account for a small portion of the total costs. We see a slightly different picture for primary hospitals where human resources are the main cost driver, accounting for at least 50% of total costs with the exception of the MCH department (35%) Fig. 2 Percent share of total department expenditure in ETB for health centers and primary hospitals. This figure presents the proportion of the cost components for each health facility department – IPD, OPD, MCH, and Delivery. The main cost driver are drugs and supplies for health centers with the exception for the Delivery department and OPD. Indirect costs (e.g., electricity, running water, etc.) only account for a small portion of the total costs. We see a slightly different picture for primary hospitals where human resources are the main cost driver, accounting for at least 50% of total costs with the exception of the MCH department (35%) 359 ETB (42 PPP $, 2012) and 641 ETB 75 PPP $, 2012) per acceptor per year, respectively. Drugs and supplies, which constitute of birth control commodities (pills, injectables, and IUD), condoms, and emergency contra- ception, were the major cost drivers for family planning services at both primary hospitals (67%) and health centers (78%). median unit costs were based on a normal vaginal deliv- ery conducted at the health facility and were estimated at 647 ETB (76 PPP $, 2012) for health centers and 945 ETB (111 PPP $, 2012) for primary hospitals. The major cost driver for a delivery was HR costs at both health centers (59%) and primary hospitals (62%). Unit cost for ANC, delivery, and PNC High unit costs might be due to a high number of staff relative to de- mand or inefficiencies in resource use driving up the costs. The former could be reasonable in a rapidly grow- ing system, which anticipates significant future increases in demand and is taking proactive measures to be pre- pared for that to happen [23]. Also, possible stock-outs may lead primary care facilities to procure drugs and supplies from costlier private pharmacies. Moreover, an estimated 73% of government primary health care spending is from external funding [6]. Under the current circumstances of uncertainty in the flow of external funding, it will be difficult for Ethiopia to main- tain business as usual in domestic health spending and sustain these high unit costs of government-funded service provision [6]. metabolism) and uncomplicated cases (malaria attack that lasts 6–10 h) [9, 18]. Uncomplicated cases would have a lower unit cost estimate compared to complicated ones, however the distinction between these two types of malaria cases was not possible for this analysis. The median unit cost for malaria treatment was 126 ETB (15 PPP $, 2012) per case per year for health centers and 302 ETB (35 PPP $, 2012) for the primary hospitals. HR were the main cost driver for malaria treatment at 43 and 68% in primary hospitals and health centers, respectively. metabolism) and uncomplicated cases (malaria attack that lasts 6–10 h) [9, 18]. Uncomplicated cases would have a lower unit cost estimate compared to complicated ones, however the distinction between these two types of malaria cases was not possible for this analysis. The median unit cost for malaria treatment was 126 ETB (15 PPP $, 2012) per case per year for health centers and 302 ETB (35 PPP $, 2012) for the primary hospitals. HR were the main cost driver for malaria treatment at 43 and 68% in primary hospitals and health centers, respectively. Drugs and pharmaceutical supplies are the highest cost component for health centers (52%) and primary hospi- tals (42%), which is substantially higher compared to other low-and-middle income countries’ (LMICs’) pri- mary care facilities where the share of personnel costs was over 50% [24–26]. A systematic review found that the availability of quality drugs is an indicator of the dif- ference in quality of private versus public ambulatory health care in LMICs [27]. Unit cost for ANC, delivery, and PNC ANC unit costs were based on at least 4 ANC visits per year, while PNC unit costs were based on a typical post- partum visit within the first 24 h of a newborn’s birth during the study period. ANC services typically included regular check-ups, tetanus toxoid shots, and/or syphilis detection and treatment. The median unit cost for 4 ANC visits among health centers was 231 ETB (27 PPP $, 2012) and 645 ETB (76 PPP $, 2012) for primary hos- pitals. PNC services consisted of treatments for newborn sepsis, treatment of postpartum hemorrhage, and pre- scribing Chlorhexidine for treatment of the umbilical cord among newborns, among others. The median unit cost of an average PNC service among health centers and primary hospitals was 68 ETB (8 PPP $, 2012) and 171 ETB (20 PPP $, 2012), respectively. The delivery Table 5 Unit costs of delivering health care services by health facility (median cost) Table 5 Unit costs of delivering health care services by health facility (median cost) Health Center Unit Costs (N = 40) Primary Hospitals Unit Costs (N = 24) Facility/Department Type ETB PPP $, 2012 ETB PPP $, 2012 Health facility 146 17 339 40 OPD 124 14 252 29 IPDa 340 40 1288 151 MCH 145 17 310 36 Delivery 647 76 945 111 aSample for IPD unit costs for the health centers was reduced from 40 to 30 because only 30 health centers had IPDs Agarwal et al. BMC Health Services Research (2020) 20:389 Page 8 of 12 Page 8 of 12 Table 6 Regional distribution of median unit costs by department for each health facility type Health Centers Primary Hospitals Regions ETB PPP$, 2012 ETB PPP$, 2012 Amhara 116 14 336 39 Addis Ababa 145 17 na na Benishangul-Gumuz 161 19 na na Dire Dawa 158 19 na na Oromia 171 20 353 41 SNNPR na na 457 53 Tigray na na 71 8 na not applicable; data was not collected in the region for that health facility Table 6 Regional distribution of median unit costs by department for each health facility type PPP $, 2012) (2013/14) excluding external sources [19]. These are also high compared to other Sub-Saharan Af- rican countries [20, 21], and given that Ethiopia is still a relatively low-income country [22]. Unit cost for ANC, delivery, and PNC Thus, spending more on drugs and supplies might be encouraging as it may re- flect adequate spending on supplies needed to deliver quality health services at publicly funded facilities. How- ever, it might also indicate inefficiencies with possible losses due to improper storage and expiration or even Discussion This paper provides an estimate of the total annual and unit costs for Ethiopian government-funded primary health care facilities, departments, and exempted ser- vices. Spending on primary health care services are rela- tively high when considering them within the overall low total government per capita expenditure of ETB 169 (20 Fig. 3 Regional distribution of median unit costs in ETB by department for health centers. This figure shows the regional distribution of the median unit costs (in Ethiopian birr) across the 4 departments for health centers. OPD and MCH had substantially lower median unit costs compared to IPD and Delivery department across all regions. The lower median unit costs in certain regions could be due to cost-efficient use of resources, high utilization rates, or even inefficiencies not captured by this analysis (such as low expenditures due to high stock-out rates). The highest median unit costs were for inpatient services provided in Addis Ababa and delivery services provided in Dire Dawa. Both Addis Ababa and Dire Dawa are urban areas Fig. 3 Regional distribution of median unit costs in ETB by department for health centers. This figure shows the regional distribution of the median unit costs (in Ethiopian birr) across the 4 departments for health centers. OPD and MCH had substantially lower median unit costs compared to IPD and Delivery department across all regions. The lower median unit costs in certain regions could be due to cost-efficient use of resources, high utilization rates, or even inefficiencies not captured by this analysis (such as low expenditures due to high stock-out rates). The highest median unit costs were for inpatient services provided in Addis Ababa and delivery services provided in Dire Dawa. Both Addis Ababa and Dire Dawa are urban areas Agarwal et al. BMC Health Services Research (2020) 20:389 Page 9 of 12 Fig. 4 Regional distribution of median unit costs in ETB by department for primary hospitals. This figure shows the regional distribution of the median unit costs (in Ethiopian birr) across the 4 departments for primary hospitals. Overall, the lowest median unit costs for almost all regions were for outpatient and maternal and child health services. The lower median unit costs in certain regions could be due to cost-efficient use of resources, high utilization rates, or even inefficiencies not captured by this analysis (such as low expenditures due to high stock-out rates). Discussion The highest median unit costs were inpatient services provided in Oromia primary hospitals and delivery services provided in SNNPR primary hospitals Fig. 4 Regional distribution of median unit costs in ETB by department for primary hospitals. This figure shows the regional distribution of the median unit costs (in Ethiopian birr) across the 4 departments for primary hospitals. Overall, the lowest median unit costs for almost all regions were for outpatient and maternal and child health services. The lower median unit costs in certain regions could be due to cost-efficient use of resources, high utilization rates, or even inefficiencies not captured by this analysis (such as low expenditures due to high stock-out rates). The highest median unit costs were inpatient services provided in Oromia primary hospitals and delivery services provided in SNNPR primary hospitals Fig. 4 Regional distribution of median unit costs in ETB by department for primary hospitals. This figure shows the regional distribution of the median unit costs (in Ethiopian birr) across the 4 departments for primary hospitals. Overall, the lowest median unit costs for almost all regions were for outpatient and maternal and child health services. The lower median unit costs in certain regions could be due to cost-efficient use of resources, high utilization rates, or even inefficiencies not captured by this analysis (such as low expenditures due to high stock-out rates). The highest median unit costs were inpatient services provided in Oromia primary hospitals and delivery services provided in SNNPR primary hospitals low quality of care with limited higher-paid health staff (e.g., doctors). utilization rates, or even inefficiencies not captured by this analysis (such as low expenditures due to high stock-out rates). Funding for exempted services is provided by either external resources or from the government, as they are to be exempt from individuals paying user fees for these services. External support for these services is mostly in- kind, providing a majority of the drugs and supplies. Drugs and pharmaceutical supplies are the main cost driver, meaning the domestic health budget would have to increase substantially to at least maintain the current exempted service package, should external support de- cline due to continued rapid economic development, changing global financial landscape, and additional com- peting priorities with the Sustainable Development Goals (SDGs). One of the most expensive exempted services was ART and almost 90% of the cost is from drugs and supplies. Discussion With the relatively recent cuts in the United States President’s Emergency Plan for AIDS Relief (PEP- FAR) funding for Ethiopia [6], the government has to cover these high costs to ensure that such services continue for free. The Ethiopian health system is undergoing a transition in order to be more responsive to the changing popula- tion health needs. This includes expanding primary hos- pital coverage, increasing funding of exempted services from domestic resources, and revising the essential health services package list. The cost estimates in this study provide critical data for policy makers and health sector managers to support their financial planning for such changes. Addressing major data limitations of this study would further refine future unit cost estimates for primary care service provision. Data quality and availability were the largest hurdles faced during data collection and analysis. Although the MoH has in place a good system of report- ing and recording of input- and output-related data, we often found these data to be incomplete or not available at facility level. For instance, the estimation of program drugs consumption was done using the monthly Report and Requisition Forms (RRFs). Many of the health facil- ities did not have a completed set of RRFs pertaining to the study period. In such cases, extrapolations were car- ried out using the available RRFs. This can potentially affect the validity of drug consumption estimates. Simi- larly, service volume statistics were obtained from the health management information system (HMIS). Data were extrapolated to estimate the service volume for those health facilities where completed HMIS reports were not available for the entire duration of the study period. To strengthen health system performance for Unit cost results show some patterns of variation be- tween facility types and location. For example, health centers in rural areas have lower department-level unit costs, as compared to the urban, except for the MCH departments. A variety of factors, including quality of care, cost implications due to stock-outs, or additional costs incurred because of remoteness (e.g., additional transportation costs to delivery drugs and supplies to more remote health facility) may contribute to such dif- ferences. The lower median unit costs in certain regions could be due to cost-efficient use of resources, high Agarwal et al. Discussion BMC Health Services Research (2020) 20:389 Page 10 of 12 Table 7 Median unit cost and cost components of exempted services by health facility ource: Berman et al. 2016. HR: Human resources D + S: Drugs and pharmaceutical supplies I: Indirect An acceptor consists of new and repeat patients, from the previous fiscal year, of reproductive age (15–49 years) that receives a modern contraceptive service in he calendar year At least four ANC visits during the EFY A PNC visit within 24 h of a newborn’s birth during the EFY Tuberculosis case (all forms) that was registered at each facility for the EFY. A person living with HIV/AIDS (adult or child) that received ART for the EFY, with assumption that an individual did not stop treatment or miss regimens hroughout the year New malaria case (complicated or severe) registered at the health center during the EFY Table 7 Median unit cost and cost components of exempted services by health facility coupled with inefficiencies associated with organization and delivery of health services. The data from this study could be further used to assess efficiency and productiv- ity among the primary care facilities, facilitate premium setting for health insurance, and improve budgeting and allocating health resources for a more sustainable and effective primary health care system. Based on this study’s findings more work is needed to understand con- tributing factors of potential resource input inefficien- cies, causes of regional variations in unit costs, and explanations for the low level of personnel costs relative to drugs and supplies. quality and efficiency, more effort will be needed to as- sure that these essential data are maintained and used. This is one of the key goals of Ethiopia’s Health Sector Transformation Plan (HSTP, 2015/16–2019/20) in its transformational agenda for an “information revolution” [4]. The relatively recent roll-out of the District Health Information Software 2 across Ethiopia is seen as a pathway to improve data quality and increase use. Additionally, better record keeping and regular audits of primary health care facilities could help in minimizing po- tential estimation biases resulting from extrapolated data. Consent for publication Not applicable. Consent for publication Not applicable. Funding ll f All costs for the writing and publication of this paper were provided through a grant from the Bill & Melinda Gates Foundation, the RTM project, which was awarded to the Harvard T.H. Chan School of Public Health in partnership with Breakthrough International Consultancy, PLC. 10. Federal Democratic Republic of Ethiopia, Ministry of Health. Essential health service package for Ethiopia. Addis Ababa: Federal Ministry of Health; 2005. 11. Berman P, Alebachew A, Mann C, Agarwal A, Abdella E. Costs of publicly funded primary care facilities, departments, and exempted Services in Ethiopia. Boston and Addis Ababa: Harvard T.H. Chan School of Public Health; Breakthrough International Consultancy, PLC; 2016. Competing interests h h d l h The authors declare that they have no competing interests. The authors alone are responsible for the views expressed in this article and they do not necessarily represent the views of the organizations listed. Additional file 2: Supplementary file 2. Cost study instruments. This paper-based survey instrument was used to extract the necessary data from the health facilities and other related institutions to capture relevant data for this study such as service statistics, drugs and supplies con- sumed, human resource data including salaries, etc. Acknowledgements 4. Federal Democratic Republic of Ethiopia, Ministry of Health. Health sector transformation plan 2008–12. Addis Ababa: Federal Democratic Republic of Ethiopia; 2015. We would like to thank the Ethiopia’s Ministry of Health for their support and inputs during the process of conducting the costing study. We would also like to thank our partner on the Resource Tracking and Management (RTM) project, Breakthrough International Consultancy PLC, who contributed to the data collection process and data entry. The success of the data collection process for this study would not have happened without the cooperation and time committed by the staff from the woreda health offices, woreda offices of finance and economic cooperation, and health facilities. 5. Federal Democratic Republic of Ethiopia, Ministry of Health. Health care financing strategy 2017–25. Addis Ababa: Federal Democratic Republic of Ethiopia; 2017. 6. Berman P, Mann C, Riculli ML. Can Ethiopia finance the continued development of its primary health care system if external resources decline? Health Syst Reform. 2018:1–12. https://doi.org/10.1080/23288604.2018.1448240. 6. Berman P, Mann C, Riculli ML. Can Ethiopia finance the continued development of its primary health care system if external resources decline? Health Syst Reform. 2018:1–12. https://doi.org/10.1080/23288604.2018.1448240. 7. Federal Democratic Republic of Ethiopia, Ministry of Health. The baseline assessment of unit cost of health Services at Different Levels of care: background study for introducing of social health insurance. Addis Ababa: Federal Democratic Republic of Ethiopia; 2007. Availability of data and materials The instruments used to collect the data at the primary health care facilities to conduct the costing study presented in this article are included in this article (Supplementary files 1 and 2). The data used and analysed for this study are available from the corresponding author on reasonable request. 12. Alebachew A, Hatt L, Kukla M, Nakhimovsky S. Universal health coverage measurement in a low--income context: an Ethiopian case study. Bethesda: Health finance & governance project, Abt Associates Inc; 2014. 13. Creese AL, Parker D. Cost analysis in primary health care: a training manual for programme managers. Geneva: World Health Organization; 1994. 13. Creese AL, Parker D. Cost analysis in primary health care: a training manual for programme managers. Geneva: World Health Organization; 1994. Abbreviations C l ANC: Antenatal care; ART: Antiretroviral treatment; BCG: Bacille Calmette- Guerin; EPI: Expanded program for immunizations; ETB: Ethiopian birr; EFY: Ethiopian fiscal year; MCH: Maternal and child health; HR: Human resources; NGO: Non-governmental organization; LB: Live births; SNNPR: Southern Nations, Nationalities, and Peoples' Region; IPD: Inpatient department; MoH: Ministry of Health; PEPFAR: The United States President’s Plan for AIDS Relief; PNC: Postnatal care; OPD: Outpatient department; PPP: Purchasing power parity; HSTP: Health Sector Transformation Plan; LMIC: Low-and-middle income countries; SDGs: Sustainable development goals; RRF: Report and requisition form References C l 1. Central Statistical Agency [Ethiopia] and ICF International. Ethiopia demographic and health survey 2011. Addis Ababa: Central Statistical Agency and ICF International; 2012. 1. Central Statistical Agency [Ethiopia] and ICF International. Ethiopia demographic and health survey 2011. Addis Ababa: Central Statistical Agency and ICF International; 2012. 1. Central Statistical Agency [Ethiopia] and ICF International. Ethiopia demographic and health survey 2011. Addis Ababa: Central Statistical Agency and ICF International; 2012. 2. Central Statistical Agency (CSA) [Ethiopia] and ICF International. Ethiopia demographic and health survey 2016. Addis Ababa: CSA and ICF; 2016. 3. Ruducha J, Mann C, Singh N, et al. How Ethiopia achieved millennium development goal 4 through multisectoral interventions: a countdown to 2015 case study. Lancet Glob Health. 2017;5(11):e1142–51. https://doi.org/ 10.1016/S2214-109X(17)30331-5. Authors’ contributions AA1, PB, AA2, CM designed the study. AA1, EA, WM conducted the field work. AA1 performed the analysis with oversight from CM. AA1 is the lead author and CM, PB, and AA2 made significant contributions to the writing of the manuscript. All named authors read and approved the final manuscript. 8. Federal Democratic Republic of Ethiopia, Ministry of Health. Health sector development plan (HSDP-III) 2005/6–2009/10. Addis Ababa: Federal Democratic Republic of Ethiopia; 2005. 9. Federal Democratic Republic of Ethiopia, Ministry of Health. Health sector development plan IV 2010/11–2014/15. Addis Ababa: Federal Democratic Republic of Ethiopia; 2010. Supplementary information in-depth interviews as per the IRB protocol. Letters were written by the Ethi- opian Ministry of Health requesting cooperation from the sub-national gov- ernment, following standard procedure of approvals for research in Ethiopia. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-05218-1. Additional file 1: Supplementary file 1. Key informant interview guide. This is the key informant guide that was used to conduct interviews with health facility heads or top management at health facilities in the study sample. This interview was to capture additional information on resource allocation and use challenges, identified solutions, and best practices to supplement data collection at the health facilities. Author details 1 1Global Health and Population Department, Harvard T.H. Chan School of Public Health, 677 Huntington Ave, Boston, MA 02115, USA. 2Present address: School of Epidemiology and Public Health, University of Ottawa, 600 Peter Morand Crescent, Ottawa K1G 5Z3, ON, Canada. 3Present Address: 500 D St SW, Washington DC 20024, USA. 4Breakthrough International Consultancy, PLC, Alem Birhan Plaza 4th Floor, Room No. 401 Kirkos Sub City, Kebele 17/ 18, House Number 005 (Near St. Urael Church), Addis Ababa, Ethiopia. 5Present address: School of Population and Public Health, University of British Columbia, Vancouver, BC, Canada. Additional file 3: Supplementary file 3. Cost accounting steps. This document lays out the specific cost accounting steps used in this costing study. This consists of description of each of the six steps taken: 1) Define the final product; 2) Define the cost centers; 3) Identify and allocate direct costs; 4) Identify and allocate indirect costs; 5) Allocate all costs to cost center; and 6) Compute total and average costs for each final cost center. 5Present address: School of Population and Public Health, University of British Columbia, Vancouver, BC, Canada. Received: 25 March 2019 Accepted: 13 April 2020 Conclusion The data intensive nature of costing exercises also points to the importance of maintaining records and data systems at the facility level to enable accuracy and replicability of future studies, for which our study could serve as a baseline. This study reports empirical cost data for Ethiopia’s government-provided primary health care system. High unit costs of service provision could be indicative of underutilization of the primary health care system, Page 11 of 12 Page 11 of 12 Agarwal et al. BMC Health Services Research (2020) 20:389 Ethics approval and consent to participate Cost of delivering health Care Services in Public Sector Primary and Community Health Centres in North India. PLoS One. 2016;11(8):e0160986. https://doi. org/10.1371/journal.pone.0160986. 26. Ensor T, Firdaus H, Dunlop D, et al. Budgeting based on need: a model to determine sub-national allocation of resources for health services in Indonesia. Cost effectiveness and resource allocation. Cost Eff Resource Alloc. 2012;10:11. https://doi.org/10.1186/1478-7547-10-11. 27. Berendes S, Heywood P, Oliver S, Garner P. Quality of private and public ambulatory health care in low and middle income countries: systematic review of comparative studies. PLoS Med. 2011;8(4):e1000433. https://doi. org/10.1371/journal.pmed.1000433. Ethics approval and consent to participate The study was determined to be not human research by the Internal Review Board (IRB) from the Harvard T.H. Chan School of Public Health (IRB15–0824). Written consent was provided by health facility staff who participated in the 14. Hanson K, Gilson L. Cost, resource use and financing methodology for district health services: a practical manual. Bamako initiative technical report, no. 34. 2nd ed. New York: UNICEF; 1996. 14. Hanson K, Gilson L. Cost, resource use and financing methodology for district health services: a practical manual. Bamako initiative technical report, no. 34. 2nd ed. New York: UNICEF; 1996. Page 12 of 12 Agarwal et al. BMC Health Services Research (2020) 20:389 15. Ozaltin A, Cashin C. Costing of health Services for Provider Payment. Washington, DC: Joint Learning Network (JLN) for Universal Health Coverage; 2014. 16. Berman P, Alebachew A, Mann C, Agarwal A, Abdella E. Costs of publicly funded primary hospitals, departments, and exempted Services in Ethiopia Supplement to paper 1 with expanded sample of primary hospitals. Boston and Addis Ababa: Harvard T.H. Chan School of Public Health; breakthrough international consultancy, PLC; 2016. 17. United Nations Statistical Division. 2016. Available at: http://data.un.org/ Data.aspx?d=MDG&f=seriesRowID%3a699. (Accessed April 6, 2016). 18. Center for Disease Control (CDC). About malaria: disease; 2015. Retrieved from: http://www.cdc.gov/malaria/about/disease.html (Accessed 20 April 2016). 19. Federal Democratic Republic of Ethiopia, Ministry of Health. Ethiopia health accounts, 2013/14. Addis Ababa: Federal Democratic Republic of Ethiopia; 2017. 20. Flessa S, et al. Basing care reforms on evidence: the Kenya health sector costing model. BMC Health Serv Res. 2011;11:128. https://doi.org/10.1186/ 1472-6963-11-128. 21. Institute for Health Metrics and Evaluation (IHME). Health service provision in Ghana: assessing facility capacity and costs of care. Seattle: IHME; 2015. 22. World Bank. World development indicators database; 2016. https://data. worldbank.org/indicator/ (Accessed 20 April 2016). 23. Mann C, Dessie E, Adugna M, Berman P. Measuring efficiency of public health centers in Ethiopia. Boston and Addis Ababa: Harvard T.H. Chan School of Public Health and Federal Democratic Republic of Ethiopia Ministry of Health; 2016. 24. Aboagye AQQ, Degboe ANK, Obuobi AAD. Estimating the cost of healthcare delivery in three hospitals in southern Ghana. Ghana Med J. 2010;44(3):83–92. https://doi.org/10.4314/gmj.v44i3.68890. 24. Aboagye AQQ, Degboe ANK, Obuobi AAD. Estimating the cost of healthcare delivery in three hospitals in southern Ghana. Ghana Med J. 2010;44(3):83–92. https://doi.org/10.4314/gmj.v44i3.68890. 25. Prinja S, Gupta A, Verma R, Bahuguna P, Kumar D, Kaur M, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3002434014
https://hal.inria.fr/hal-03759111/document
English
null
ATMF: A Student-Centered Framework for the Effective Implementation of Alternative Teaching Methods for CSEd
IFIP advances in information and communication technology
2,020
cc-by
5,308
1 Cambridge Online Dictionary: http://dictionary.cambridge.org/dictionary/english/alternative, last accessed June 2019 1 University of Peloponnese, Greece 1 University of Peloponnese, Greece Abstract. The dynamic development of Informatics introduces new educational and pedagogical challenges, including the instructional design of teaching and learning. How can we teach our students better in such a growing and demanding field? Moreover, how can we motivate them and together have better learning results? This paper aims to give answers to these questions, through a student- centered framework for effectively encompassing alternative teaching methods within CS. The proposed framework (ATMF) summarizes the benefits of alter- native teaching methods in contrast with known issues of traditional teaching. In addition, ATMF is built upon empirical evidence and concludes that alternative practices, if used under the right conditions, can provide increased motivation for learning and better learning outcomes. Keywords: Computer Science Education, Teaching Framework, Alternative Teaching Methods. Keywords: Computer Science Education, Teaching Framework, Alternative Teaching Methods. This document is the original author manuscript of a paper submitted to an IFIP conference proceedings or other IFIP publication by Springer Nature. As such, there may be some differences in the official published version of the paper. Such differences, if any, are usually due to reformatting during preparation for publication or minor corrections made by the author(s) during final proofreading of the publication manuscript. This document is the original author manuscript of a paper submitted to an IFIP conference proceedings or other IFIP publication by Springer Nature. As such, there may be some differences in the official published version of the paper. Such differences, if any, are usually due to reformatting during preparation for publication or minor corrections made by the author(s) during final proofreading of the publication manuscript. This document is the original author manuscript of a paper submitted to an IFIP conference proceedings or other IFIP publication by Springer Nature. As such, there may be some differences in the official published version of the paper. Such differences, if any, are usually due to reformatting during preparation for publication or minor corrections made by the author(s) during final proofreading of the publication manuscript. Keywords: Computer Science Education, Teaching Framework, Alternative Teaching Methods. ATMF: A student-centered framework for the effective implementation of alternative teaching methods for CSEd Anastasios Theodoropoulos1[0000-0002-0143-4228], Costas Vassilakis1, Angeliki Antoniou1, Manolis Wallace1 and George Lepouras1 1 Introduction Informatics -also termed as “Computer Science” (CS)- is on the brink of an enormous possible growth and therefore it draws great interest from governments, businesses and other organizations as a top educational priority. Moreover, the dynamic development of the field introduces new educational and pedagogical challenges, including the in- structional design of teaching and learning. How can we teach our students better in such a growing and demanding field? Furthermore, how can we motivate them and together consume better learning results? Alternative Teaching Methods (ATM) that support students’ active involvement in the learning process, according to the constructivism and constructionism principles, could be very helpful [1]. Alternative is considered everything that is relating to activ- ities that depart from or challenge traditional norms1. Therefore, ATM refers to provid- ing students with different approaches/strategies to learning the same information. There are several instructional methodologies used by educators and researchers in or- der to deal with the complexity and the needs of various cases. Nevertheless, using them could be adapted in more approaches with similar features. As Morrison, Ross [2] 2 state, instructors at any level of education should implement various pedagogical ap- proaches and teaching methods when the discipline and learning tasks vary. However, most instructors have little practice or experience with teaching methods other than traditional lecturing [3]. Some have a particular teaching style that they use in every teaching context, regardless of the type or level of student learning they expect. Others, because of the CS discipline, may already use various technology applications, but their decisions may not be based on student-centered pedagogy [4, 5]. Finally, a number of instructors are concerned that students will respond negatively to teaching and learning activities that are new to them [6]. Thus, any of these factors can hinder the reframing of teaching to a student-centered perspective. It becomes apparent that there is a need for a clear and easy-to-adopt, student-centered framework encompassing alternative teaching concepts. One may argue that several models exist, standards, curriculum guidelines or frame- works that can be used for teaching and learning purposes. However, most of them refer to teaching in a general scope [7] or teaching in a specific discipline different from CS, like frameworks from math, science, and technology education. Therefore, the existing frameworks neither consider the identity and particularities of CS Education (CSE),nor prescribe suitable alternative teaching strategies. 2.1 The CS discipline CSE is learning about CS and focuses on teaching the fundamental concepts of the discipline, just as core Mathematics and Physics courses do. Nevertheless, confusion arises when trying to distinguish between the most common areas of computing educa- tion offered in schools like CS, IT and Educational Technology [11]. There are many different aspects of CSE. To the same reason, there is an ignorance about the core con- cepts and characteristics that students are taught through CSE. This reflects to the wider ignorance by the general public about the nature, methodologies, and contributions of the field in the modern world. However, through CS learners can acquire many valuable skills and characteristics, beyond the narrow scope of programming. Coding is an indispensable tool for CS, en- abling the creation of software, but CS is a broader field covering many different con- cepts that go well beyond coding. Through CS, one can develop logical reasoning and gain awareness of the resources required to implement, test, and deploy a solution, and how to deal with real-world constraints. All these skills are applicable in many contexts, from science and engineering to the humanities and business, and they have enabled deeper understanding in these and other areas. 1 Introduction For example, within the CS field, the “K–12 Computer Science Framework”, describes the foundational literacy in CS, aim- ing to show that CS is essential for all students [8]. This framework includes standards, curriculum, course pathways and even professional development suggestions for all K– 12 grade levels, but it does not include any specific teaching guidelines. Other national frameworks, describe CS concepts and practices that students should know, but do not focus on alternative teaching methods. Finally, other well established frameworks in higher education, like the Advanced Placement CS Principles curriculum framework and the ACM’s curriculum guidelines for undergraduate CS programs [9], provide standards for students and curricula, but not for teachers who want to teach students. Additionally, although a teaching framework could be based on existing research and well-established practices, it should also be evolving, taking into account new empirical studies. The lack of empirical research on best practices in CSE has led researchers to repetitively ask similar basic questions without clear progress toward resolving them, although current practical research shows promise [10]. Given the above-mentioned research motivations, it is clear that there is potential for learning achievement to be improved in CSE through the effective design and use of new methods, strategies and approaches. Thus, the aim of this study is the development of a student-centered framework for teaching that can provide shared understandings, which can help improve the quality of instructional design, course and lesson planning, learning and assessment. The next section maps a territory of topics encountered in CSE. The third section presents the proposed framework and the fourth section presents briefly the empirical studies that were conducted during its formulation. Finally, we summarize this article in the fifth section. 3 3 2.3 Alternative Teaching Methods In addition to traditional teaching, educators and curriculum designers must consider learning activities and instructional techniques that aim to student motivation and learn- ing. There are many parameters that need to be considered, like the students’ age and experience on technology matters or the learning goals. There are also several methods used by educators and researchers in order to deal with the complexity and the needs of various cases. Table 1gives a summary of the ATMs used in CSE by the time this article was written. Table 1. Alternative Teaching Methods used in CSE. 1 Peer Learning 11 Non-Textual Programming 2 Problem-Based Learning 12 Contextualized Learning 3 Project-Based Learning 13 CS Unplugged 4 Studio-Based Learning 14 Subgoal Learning 5 Inquiry-Based Learning 15 Programming Puzzles 6 Process Oriented Guided Inquiry Learning 16 Extreme Programming 7 Team Learning 17 Program Visualization 8 Game-Based Learning 18 Competency-Based Learning 9 Educational Robotics 19 Social Networks Learning 10 Physical Computing 20 Emerging Technologies Table 1. Alternative Teaching Methods used in CSE. 2.2 Traditional Strategies Issues Probably the main argument posed against traditional classroom styles involves how little it truly engages students [12]. In addition, traditional, lecture-based structures serve particularly adroit conduits for rote learning and memorization. Another issue with traditional strategies involves teacher bias. Most subjects are not objective, and since the instructor stands as the highest authority in the room, the more strict, rote structure only presents some perspectives on the matters at hand [13]. The most effec- tive educational settings are more active and allow students to consider content from multiple angles and form multiple opinions, rather than adopting what teachers transfer [12]. Moreover, not every teacher is good at public speaking. Poor communicators and speaking anxiety can seriously screw over different learners, even if they typically ben- efit from traditional lecture structures [14]. It is crucial that teachers understand where their public speaking limitations lie and alter their styles accordingly for maximum ed- ucational achievement [14]. Furthermore, traditional strategies may suit better some students than others and strengthen a limited number of skills. The highly dynamic field of CS demands that learners must be empowered with reflective lifelong learning skills in order to be suc- cessful. They need to develop skills such as CT, problem solving, teamwork, commu- nication, critical thinking, and creativity. The traditional teacher-centric pedagogy is focused on the course content and only on transferring knowledge to the students whereas a learner-centric view is focused on assisting students to develop or build knowledge [15]. In CSE a move to a learner-centered design is recommended [16], because active elements of a 21st century education receive little attention. Therefore, 4 reliance on the traditional lecture as the main mode of student learning has been criti- cized and new alternative strategies are needed. 3 The ATM Framework Learning different CS concepts more effectively, requires the development of a multi- level framework that could be applied in different contexts using different teaching ap- proaches to a multitude of application domains. This article proposes a framework for the effective implementation of ATM in CS. 3.1 A new conceptual framework 3.1 A new conceptual framework This new framework is called the ATMF (Alternative Teaching Methods Framework). ATMF is a broad description of the context, characteristics, content and sequence of learning expected of all learners - but not at the level of detail of grade-by-grade stand- ards or, at the course description and standards. Figure 1 illustrates the five phases of the methodology that was adapted in order to develop the ATMF. The current literature on ATM, has provided the theoretical framework for the studies reported in this paper. The proposed framework is intended as a guide to instructors (formal or informal set- tings) as well as for curriculum designers, assessment developers, researchers and pro- fessionals responsible for CSE. Thus, it describes the major concepts, and disciplinary core ideas that all instructors should be familiar with in order to enhance their teaching with alternative strategies, and providing an outline of how these practices, concepts, 5 and ideas should be developed across different settings. ATMF was based both on pre- vious theoretical frameworks and models as well as empirical guidelines. Fig. 1.The five-phased methodology adapted for developing the ATM framework. Phase 1: We researched alternative teaching methods within CS other frameworks Quantitative and Qualitative case studies Traditional teaching issues ATM benefits Framework of ATM Quantitative Study with DGBL Relate with long-lasting motivations Qualitative Study with Robotics Relate with teachers’ perceptions/perspective Quantitative Study with DGBL Relate with personalization in programming Phase 1 Researching Phase 3 Testing/Refining Phase 4 Testing/Refining Phase 5 Final Framework Phase 2 Analysis & Concepting Literature Problem identification Phase 1: We researched alternative teaching methods within CS, other frameworks and contacted two initial empirical studies with quantitative and qualitative data. Back- ground expert work from well know educational models and frameworks was reused. The process on one hand revealed the teaching issues that the traditional teaching meth- ods have and on the other hand highlighted the key advantages of ATM. 3.1 A new conceptual framework In this phase the framework was tested once more, enhanced with additional elements and finalized. 3.1 A new conceptual framework Quantitative and Qualitative case studies Traditional teaching issues ATM benefits Framework of ATM Quantitative Study with DGBL Relate with long-lasting motivations Qualitative Study with Robotics Relate with teachers’ perceptions/perspective Quantitative Study with DGBL Relate with personalization in programming Phase 1 Researching Phase 3 Testing/Refining Phase 4 Testing/Refining Phase 5 Final Framework Phase 2 Analysis & Concepting Literature Problem identification Phase 4 Testing/Refining Phase 1: We researched alternative teaching methods within CS, other frameworks and contacted two initial empirical studies with quantitative and qualitative data. Back- ground expert work from well know educational models and frameworks was reused. The process on one hand revealed the teaching issues that the traditional teaching meth- ods have and on the other hand highlighted the key advantages of ATM. Phase 1: We researched alternative teaching methods within CS, other frameworks and contacted two initial empirical studies with quantitative and qualitative data. Back- ground expert work from well know educational models and frameworks was reused. The process on one hand revealed the teaching issues that the traditional teaching meth- ods have and on the other hand highlighted the key advantages of ATM. 6 Phase 2: We analyzed the concepts used at the ATMF. In this phase the framework was initially shaped through literature review and the initial empirical research. Phase 3: We tested and refined the ATMF. In this phase the framework was exam- ined and enhanced with additional elements through another empirical research study with Digital Game-Based Learning (DGBL). Phase 4: We tested and refined the ATMF. In this phase the framework was tested again and enhanced with additional elements, though another empirical research study. Phase 5: We tested and shaped the final ATMF. In this phase the framework was d h d i h ddi i l l d fi li d Phase 4: We tested and refined the ATMF. In this phase the framework was tested again and enhanced with additional elements, though another empirical research study. Phase 4: We tested and refined the ATMF. In this phase the framework was tested again and enhanced with additional elements, though another empirical research study. Phase 5: We tested and shaped the final ATMF. In this phase the framework was tested once more, enhanced with additional elements and finalized. again and enhanced with additional elements, though another empirical research study. Phase 5: We tested and shaped the final ATMF. 3.2 Analysis - Concepting The ATMF comprises the need to consider context, content, pedagogy, instructor and the learner as part of the design process, so-called dimensions. Dimensions contain components, where each component defines a distinct aspect of a dimension and com- ponents are consisted of elements that describe specific features. Table 2. Components end Elements of Dimension 1. Dimension 1: Context Component 1a Education Level Element 1 Formal Education / Type of School / Level Element 2 Informal Education Element 3 Non-formal Education Component 1b Environment (physical) Element 1 Infrastructure (availability) Element 2 Supporting Resources Component 1c Educational System Element 1 Policies Element 2 Compulsory Curriculum Element 3 CS standards Element 4 General public attitudes Element 5 Time restrictions In particular, in advance of planning a lesson with the use of ATMF, it is suggested that a comprehensive learning analysis be produced that sufficiently covers the following standpoints: Table 2. Components end Elements of Dimension 1. In particular, in advance of planning a lesson with the use of ATMF, it is suggested that a comprehensive learning analysis be produced that sufficiently covers the following standpoints: Dimension 1. Context: The components in this dimension describe the surrounding environment around the learning process (Table 2). This dimension deals with the need to consider the educational level and the place where learning is taking place, the re- sources available (e.g. access to laptops/computers, mobiles, technical support), and the disciplinary context (e.g. in school, in ICT lab, in a university, at home, in the work- place). The components establish the environment that supports the learning and are not directly associated with the learning of any particular content, instead, they set the stage for all learning processes. The specific elements of the learning environment are captured in three categories: Teaching Level, Classroom Level and Educational Sys- tem. 7 Dimension 2. Participants: This dimension describes the participants’ characteris- tics in two main components, learners and instructors (Table 3). The environment in- cluding the relationship between instructor and learners and the cultural norms (char- acteristics) play a significant role in what can and does occur during the teaching and learning. Both learner and instructor characteristics are important for instructional de- signers as they allow them to design and create tailored instructions for a target group. Table 3. Components end Elements of Dimension 2. Table 3. Components end Elements of Dimension 2. 3.2 Analysis - Concepting Dimension 2: Participants’ Characteristics Component2a Learner-related Element 1 Age Element 2 Gender Element 3 Cultural background Element 4 Socioeconomic status Element 5 ICT use Element 6 ICT experience Element 7 Attitudes Element 8 Personality traits Component2b Instructor-related Element 1 Age Element 2 Gender Element 3 Teaching experience Element 4 Academic characteristics Element 5 Skills - Abilities Element 6 Attitudes Element 7 Personality Traits Component 2b also contains instructors’ attitudes (element 6) about alternative ways of teaching. For instance, teachers that prefer traditional teaching believe that it gives bet- ter learning results and hesitate to include active-learning methods in their teaching, although they see some advantages in them. Fig. 2. The ATM Framework for CSE. Fig. 2. The ATM Framework for CSE. Fig. 2. The ATM Framework for CSE. Finally, personality traits (element 7) relate to such things as actions, attitudes and be- haviors that determine different personality styles like cognitive style and intelligence 8 type. Being positive and upbeat can influence learners around an instructor, and so can negativity. The personalities of both instructor and learner interacting with one another and with the content create a unique environment. It is expected that by taking into account the participants’ characteristics can be designed and developed more efficient, effective and motivating instructional materials. Dimension 3. Content: The components of this dimension reflect process work- flows from teaching and learning theories and from the ATMs. Those components de- scribe how an instructor organizes the content that the students are to learn and how he/she designs instruction (Table 4). Component 3a comprises of five elements, related to what is the content to be taught. Elements 1 to 3, refer to lesson planning and effort to answer queries like: What is the subject to be taught? What are the objectives and goals that instructor aims to achieve? Does the lesson planning refer to short or long- term features? etc. Table 4. Components end Elements of Dimension 3. Dimension 3: Content Component3a Learning Element 1 Subject Element 2 Objectives Element 3 Short-term & Long-term Planning Element 4 Expectations for learning Element 5 Relations to other subjects / disciplines Component3b Teaching Element 1 Lecture-based Element 2 ATMs Table 4. Components end Elements of Dimension 3. 3.3 Shaping the ATMF Fig. 2 (above) provides the visual description that was used to for the ATMF. This visual recognized the importance of multiple dimensions of: context (describing the setting, curriculum, policies and infrastructure), participants (describing the instructors and learners’ characteristics), content (describing the subject matter, purposes and val- ues, pedagogy and strategies) and evaluation (describing the outcomes for both learners and instructors). Those four dimensions are presented as being a complex and intercon- nected whole with ATM at the hub connecting all the dimensions. ATM is in the center of all the process in order to emphasize that instructors have to think about and reflect upon the multiple dimensions as they investigated each topic or assignment of imple- menting ATM. The overall goal is to guide instructors in developing an integrated, in- terconnected knowledge for implementing effectively ATM in their teaching. 3.2 Analysis - Concepting Dimension 3: Content Component3a Learning Element 1 Subject Element 2 Objectives Element 3 Short-term & Long-term Planning Element 4 Expectations for learning Element 5 Relations to other subjects / disciplines Component3b Teaching Element 1 Lecture-based Element 2 ATMs Table 4. Components end Elements of Dimension 3. Table 4. Components end Elements of Dimension 3. Dimension 4. Evaluation: This dimension addresses the outcomes of the teaching and learning processes, which deal with assessment and evaluation. The outcomes (di- rect or indirect) are examined in two main components as shown at Table 5. Table 5. Components end Elements of Dimension 4. Table 5. Components end Elements of Dimension 4. Dimension 4: Evaluation Component4a Learner-related Element 1 Knowledge / Performance Element 2 Motivation Element 3 Skills Element 4 Metacognition Element 5 Beliefs Component4b Instructor-related Element 1 Assessment Criteria / Method Element 2 Monitoring of Learning (Learning Analytics) Element 3 Feedback to Learners Element 4 Long-term Evaluation Element 5 Teaching Evaluation 9 4 Experience with the ATMF Fig. 3 provides a graphical overview of the empirical studies conducted in several Greek educational settings to help formulate the proposed framework. This model con- sists of three interrelated levels: Initially the key characteristics that were researched for every empirical study are reported. Next, is shown the alternative teaching approach that was implemented for each study. Finally, at the bottom of this visualization, are presented the proposed framework’s stages corresponded with the previous approaches and studies. Fig. 3. A map of the empirical studies within ATMF. Fig. 3. A map of the empirical studies within ATMF. All the studies described in this section, are all previously reported elsewhere in peer- reviewed publications and therefore are presented briefly. 4.2 Research study 2: Social Networks in Education Secondly, we researched Social Networks (SN) for assisted learning, through an obser- vational study with undergraduate students [18]. Facebook was used as a teaching tool in higher education and ways that SNs can be used in teaching and learning were in- vestigated. Both qualitative and quantitative data were gathered (n=66 students) through one academic term. The results reported that students were highly motivated to participate in the lesson through the Facebook page although there seem to exist some personalities, cultural and gender differences about the usage. This study was also used as an input for the ATMF while it confirmed the positive effects of SNs in pro- moting learning and motivation. 4.1 Research study 1: Peer Learning Initially, we conducted an experiment using Peer Learning and Collaboration tech- niques comparing them to traditional teaching, to secondary education students [17]. The learning from these techniques was assessed and students’ attitudes towards the alternative teaching were researched through quantitative data (n=57 students). In ad- dition, teachers’ opinions about that way of teaching and learning were explored through means of qualitative data. The empirical findings of this study confirmed the 10 positive effects of student-centered learning techniques at CSE. The study was used as an input during the design of the ATMF and provided evidence that the specific ATM had better learn results and increased motivation by learners. 4.3 Research study 3: Game-Based Learning Next, we examined the Game-Based Learning method (GBL), in order to teach basic programming concepts to primary school pupils [19]. The empirical findings of this study (quantitative data from n=94 students gathered) confirmed the positive effects of GBL in promoting basic programming principles to young children. Empirical findings derived from this analysis provided valuable information games and pupils’ satisfaction and willingness to use them for acquiring programming knowledge. In addition, Pair Programming method was students’ choice for learning programming. However, the short activity of this study did not have long term results and motivation at children and thus the element of long-term planning was added to the ATMF. 4.4 Research study 4: Physical Computing The empirical findings of this study demonstrated the importance of referring to alter- native learning through the instructor’s views. In particular Educational Robotics through a national competition was researched [20]. This observational study investi- gated the benefits of students’ involvement with robotics about skills, motivation and learning through the teachers’ eyes (a qualitative methodology was used n=18). The results showed that there are numerous benefits for students: they increased their col- laboration, problem solving and creativity skills; understand STEM concepts about CS and engineering and especially gaining programming knowledge. Therefore, the ATMF was enhanced with the instructors’ perspective about the learners’ knowledge and mo- tivations. 11 4.5 Research study 5: Personal Learning Characteristics Finally, the empirical findings of this research identified different personality traits and especially cognitive style as an important factor in programming learning through seri- ous games. This study investigated students’ attitudes (quantitative data gathered from n=77 students) from gaming activities to reveal the quality of their learning experience and correlated it with their cognitive profile to reveal potential differences [21]. In ad- dition, through an empirical way it was revealed the GBL effectiveness and next it was compared to students’ cognitive styles. Cognitive style was found to be a significant learning characteristic that should be taken into consideration when using digital games to learn programming. This study was used to finalize the ATMF and provided evidence that personality traits may affect both teaching and learning. 5 Conclusion In this paper, we present ATMF, a conceptual framework for CSE that allows teachers to continuously experiment with and improve their teaching. The proposed framework is about student-centered teaching pedagogy trying to address issues around learning and teaching of CS concepts. The ATMF is implemented based on the perspective that learning is a socially embedded cognitive process and knowledge is socially con- structed through interaction and activity with others. The main objective of the ATMF is to promote motivation and enhance learning. It proposes that this model can be used in examining instructors’ CS lessons of any level and any context and in designing experiences for teachers on the integration of student-centered practices in CS teaching. The proposed framework for teaching with the use of alternative methods can make teaching both more effective and more efficient, by helping create the conditions that support student learning and minimize the need for revising materials, content, and pol- icies. While implementing these principles requires a commitment in time and effort, it often saves time and energy later on. However, more work needs to be done. We con- sider that the framework itself is robust and therefore it will not change. Likewise, we expect that for ATM some practices may change and new ones may be added. In this paper, we present ATMF, a conceptual framework for CSE that allows teachers to continuously experiment with and improve their teaching. The proposed framework is about student-centered teaching pedagogy trying to address issues around learning and teaching of CS concepts. The ATMF is implemented based on the perspective that learning is a socially embedded cognitive process and knowledge is socially con- structed through interaction and activity with others. The main objective of the ATMF is to promote motivation and enhance learning. It proposes that this model can be used in examining instructors’ CS lessons of any level and any context and in designing experiences for teachers on the integration of student-centered practices in CS teaching. h d f k f hi i h h f l i h d k The proposed framework for teaching with the use of alternative methods can make teaching both more effective and more efficient, by helping create the conditions that support student learning and minimize the need for revising materials, content, and pol- icies. 5 Conclusion While implementing these principles requires a commitment in time and effort, it often saves time and energy later on. However, more work needs to be done. We con- sider that the framework itself is robust and therefore it will not change. Likewise, we expect that for ATM some practices may change and new ones may be added. 2. Morrison, G.R., et al., Designing effective instruction. 2010: John Wiley & Sons. 3. Yadav, A., et al., Expanding computer science education in schools: understanding teacher experiences and challenges. Computer Science Education, 2016: p. 1-20. 4. Cuny, J., Transforming high school computing: a call to action. ACM Inroads, 2012. 3(2): p. 32-36. References 1. Theodoropoulos, A., A. Antoniou, and G. Lepouras, Students teach students: Alternative teaching in Greek secondary education. Education and Information Technologies, 2014: p. 1-27. 2. Morrison, G.R., et al., Designing effective instruction. 2010: John Wiley & Sons. 3. Yadav, A., et al., Expanding computer science education in schools: understanding teacher experiences and challenges. Computer Science Education, 2016: p. 1-20. 4. Cuny, J., Transforming high school computing: a call to action. ACM Inroads, 2012. 3(2): p. 32-36. 12 5. Yadav, A., H. Hong, and C. Stephenson, Computational Thinking for All: Pedagogical Approaches to Embedding 21st Century Problem Solving in K-12 Classrooms. TechTrends, 2016: p. 1-4. 6. Stes, A., D. Gijbels, and P. Van Petegem, Student-focused approaches to teaching in relation to context and teacher characteristics. Higher Education, 2008. 55(3): p. 255- 267. 7. Danielson, C., Enhancing professional practice: A framework for teaching. 2011: ASCD. 8. Committee, K.-C.S.F.S., K-12 Computer Science Framework. 2016, ACM. p. 307. 9. Joint Task Force on Computing Curricula, A.f.C.M. and I.C. Society, Computer Science Curricula 2013: Curriculum Guidelines for Undergraduate Degree Programs in Computer Science. 2013: ACM. 518. 10. Porter, L. and B. Simon. Retaining nearly one-third more majors with a trio of instructional best practices in CS1. in Proceeding of the 44th ACM technical symposium on Computer science education. 2013. ACM. 11. CSTA, K., Computer science standards. Computer Science Teachers Association. 12. Felder, R.M. and R. Brent, Teaching and learning STEM: A practical guide. 2016: John Wiley & Sons. 13. Ary, D., et al., Introduction to research in education. 2018: Cengage Learning. 14. King, P.E. and A.N. Finn, A test of attention control theory in public speaking: Cognitive load influences the relationship between state anxiety and verbal production. Communication Education, 2017. 66(2): p. 168-182. 15. Wright, G.B., Student-centered learning in higher education. International Journal of Teaching and Learning in Higher Education, 2011. 23(1): p. 92-97. 16. Guzdial, M., Learner-centered design of computing education: Research on computing for everyone. Synthesis Lectures on Human-Centered Informatics, 2015. 8(6): p. 1- 165. 17. Theodoropoulos, A., A. Antoniou, and G. Lepouras, Students teach students: Alternative teaching in Greek secondary education. Education and Information Technologies, 2016. 21(2): p. 373-399. 18. Antoniou, A., et al., Facebook as teaching tool in higher education: A case study. International Journal of Advances in Social Science and Humanities, 2014. 2(3): p. 43- 56. 19. Theodoropoulos, A., A. Antoniou, and G. References Lepouras, The Little ones, the Big ones and the Code: Utilization of digital educational games in primary school pupils, in 7th Conference on Informatics in Education (CIE2015). 2015, Greek Computer Society (GCS): Piraeus, Greece. p. 40-49. 20. Theodoropoulos, A., A. Antoniou, and G. Lepouras. Educational Robotics in the service of CSE: A study based on the PanHellenic competition. in Proceedings of the 11th Workshop in Primary and Secondary Computing Education. 2016. ACM. 21. Theodoropoulos, A., A. Antoniou, and G. Lepouras, How Do Different Cognitive Styles Affect Learning Programming? Insights from a Game-Based Approach in Greek Schools. Trans. Comput. Educ., 2016. 17(1): p. 1-25.
https://openalex.org/W3033141616
https://iimmun.ru/iimm/article/download/1254/1198
Russian
null
A search for new molecular targets for optimizing plague preventive vaccination and therapy
Infekciâ i immunitet
2,020
cc-by
16,096
Reviews Russian Journal of Infection and Immunity = Infektsiya i immunitet 2021, vol. 11, no. 2, pp. 265–282 Инфекция и иммунитет 2021, Т. 11, № 2, с. 265–282 ПОДБОР НОВЫХ МОЛЕКУЛЯРНЫХ МИШЕНЕЙ ДЛЯ ОПТИМИЗАЦИИ ВАКЦИНОПРОФИЛАКТИКИ И ТЕРАПИИ ЧУМЫ Е.А. Красильникова, А.С. Трунякова, А.С. Вагайская, Т.Э. Светоч, Р.З. Шайхутдинова, С.В. Дентовская рикладной микробиологии и биотехнологии Роспотребнадзора, п. Оболенск, Московская область, Россия Резюме. Возбудитель чумы, Yersinia pestis, — особо опасный бактериальный патоген и потенциальный агент биотерроризма. Преобладающими клиническими формами инфекции являются бубонная и легочная чума, отличающиеся путем инфицирования. При отсутствии лечения смертность при бубонной форме чумы со- ставляет 40–60%, в то время как легочная чума без лечения летальна всегда. Развитие инфекционного процес- са в организме восприимчивого хозяина обеспечивается наличием у чумного микроба комплекса факторов патогенности различной функциональной направленности, экспрессирующихся в зависимости от стадии инфекционного процесса. Нокаутные мутации генов, кодирующих эти факторы, могут не влиять на виру- лентность микроба или приводить к его аттенуации. Поиск новых факторов патогенности Y. pestis и разработ- ка на их основе высокоэффективных субъединичных и живых чумных вакцин, индуцирующих развитие вы- раженных клеточных и гуморальных иммунных реакций, и/или оценка потенциального использования этих факторов в качестве молекулярных мишеней для химиотерапии чумы остаются по-прежнему актуальны- ми, так как обе лицензированные на настоящий день чумные вакцины не соответствуют требованиям ВОЗ, а на Мадагаскаре выделены штаммы чумного микроба, устойчивые ко всем лекарственным препаратам, реко- мендованным для антибактериальной терапии чумы. В настоящей статье обобщены сведения относительно вклада известных и вновь открытых факторов патогенности в вирулентность штаммов Y. pestis и протектив- ную активность в отношении чумы. Описано также влияние утраты генов регуляторных белков и мутаций в генах различных транспортных систем Y. pestis на аттенуацию вирулентных штаммов. Проведена оценка перспективности включения охарактеризованных антигенов в состав прототипов субъединичных вакцин и тех или иных полученных мутантов в состав прототипов живых аттенуированных вакцин. Использование антибиотиков постулируется комитетом экспертов Всемирной организации здравоохранения по чуме как «золотой стандарт» лечения чумы. Однако появление устойчивых к антибиотикам штаммов Y. pestis привело к необходимости поиска альтернативных способов терапии. В обзоре обобщены предварительные результаты попыток лечения чумы ингибиторами фактора патогенности. Препараты, нацеленные на ключевые факторы патогенности, предоставляют новые перспективные возможности в борьбе с устойчивыми к антибиотикам бактериями. Ключевые слова: чума, Yersinia pestis, фактор патогенности, молекулярная мишень, вакцинопрофилактика. вые слова: чума, Yersinia pestis, фактор патогенности, м Адрес для переписки: Дентовская Светлана Владимировна 142279, Россия, Московская область, Серпуховский р-н, п. Оболенск, ФБУН ГНЦ ПМБ. Тел.: 8 (4967) 36-01-17. E-mail: dentovskaya@obolensk.org Адрес для переписки: Дентовская Светлана Владимировна 142279, Россия, Московская область, Серпуховский р-н, п. Оболенск, ФБУН ГНЦ ПМБ. Тел.: 8 (4967) 36-01-17. E-mail: dentovskaya@obolensk.org Contacts: Svetlana V. Dentovskaya 142279, Russian Federation, Moscow Region, Serpukhov District, Obolensk, State Research Center for Applied Microbiology and Biotechnology. ПОДБОР НОВЫХ МОЛЕКУЛЯРНЫХ МИШЕНЕЙ ДЛЯ ОПТИМИЗАЦИИ ВАКЦИНОПРОФИЛАКТИКИ И ТЕРАПИИ ЧУМЫ Phone: +7 (4967) 36-01-17. E-mail: dentovskaya@obolensk.org Обзоры Обзоры Reviews Для цитирования: Для цитирования: Красильникова Е.А., Трунякова А.С., Вагайская А.С., Светоч Т.Э., Шайхутдинова Р.З., Дентовская С.В. Подбор новых молекулярных мишеней для оптимизации вакцинопрофилактики и терапии чумы // Инфекция и иммунитет. 2021. Т. 11, № 2. С. 265–282. doi: 10.15789/2220- 7619-SNM-1254 Для цитирования: Красильникова Е.А., Трунякова А.С., Вагайская А.С., Светоч Т.Э., Шайхутдинова Р.З., Дентовская С.В. Подбор новых молекулярных мишеней для оптимизации вакцинопрофилактики и терапии чумы // Инфекция и иммунитет. 2021. Т. 11, № 2. С. 265–282. doi: 10.15789/2220- 7619-SNM-1254 Krasil’nikova E.A., Trunyakova A.S., Vagaiskaya A.S., Svetoch T.E., Shaikhutdinova R.Z., Dentovskaya S.V. A search for new molecular targets for optimizing plague preventive vaccination and therapy // Russian Journal of Infection and Immunity = Infektsiya i immunitet, 2021, vol. 11, no. 2, pp. 265–282. doi: 10.15789/2220-7619-SNM-1254 © Красильникова Е.А. и соавт., 2021 DOI: http://dx.doi.org/10.15789/2220-7619-SNM-1254 265 Е.А. Красильникова и др. Инфекция и иммунитет A SEARCH FOR NEW MOLECULAR TARGETS FOR OPTIMIZING PLAGUE PREVENTIVE VACCINATION AND THERAPY Krasil’nikova E.A., Trunyakova A.S., Vagaiskaya A.S., Svetoch T.E., Shaikhutdinova R.Z., Dentovskaya S.V. State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russian Federation Abstract. The causative agent of plague, Yersinia pestis, is a highly virulent bacterial pathogen and a potential bioweapon. Depending on the route of infection, two prevalent forms of the disease — bubonic and pneumonic, are known. The latter is featured by a high fatality rate. Mortality in untreated bubonic plague patients reaches up to 40–60%, whereas untreated pneumonic plague is always lethal. The development of the infectious process in susceptible host is accounted for by a whole set of pathogenicity factors in plague pathogen displaying various functional modalities being expressed depending on stage of infectious process, providing their coordinated expression. Knocking out any of such factors, in turn, may not either affect microbe virulence or lead to its attenuation. A search for new Yersinia pestis pathogenicity factors and subsequent development of highly effective subunit and live attenuated plague vaccines inducing development of pronounced cellular and humoral immune reactions, and/or assessment of their potential use as molecular targets for plague therapy still remain a pressing issue, as both currently licensed plague vaccines do not meet the WHO requirements, whereas strains of plague microbe isolated in Madagascar are resistant to all drugs recommended for plague antibacterial therapy. Here we summa- rize an impact of described and newly discovered pathogenicity factors into the virulence of Y. pestis strains and their protec- tive anti-plague activity. An effect of loss of genes encoding regulatory proteins as well as mutations in the genes for various transport systems of Y. pestis on attenuation of virulent strains is described as well. Perspectives for introducing character- ized antigens into prototype su bunit vaccine as well as some other obtained mutants into prototypes of living attenuating vaccines were assessed. The use of antibiotics for plague treatment has been embraced by the World Health Organization Expert Committee on Plague as the “gold standard” treatment. However, concerns regarding development of antibiotic- resistant Y. pestis strains accounted for further exploring alternatives to plague therapy. Several research groups continue to seek for other alternative approaches, e. g. treatment with inhibitors of pathogenicity factors. Preliminary data attempting to treat plague patients with pathogenicity factor inhibitors are summarized. Аntivirulence drugs targeting key microbial factors represent new promising therapeutic options in the fight against antibiotic-resistant bacteria. A SEARCH FOR NEW MOLECULAR TARGETS FOR OPTIMIZING PLAGUE PREVENTIVE VACCINATION AND THERAPY 11, № 2 пуринов, уже не рассматривают как факторы па- тогенности [1, 14, 41], но выявлен целый ряд но- вых кандидатов на роль факторов патогеннос ти чумного микроба. кое распространение этого белка среди грамо- трицательных патогенов делает перспективным его использование для разработки ингибиторов с широким спектром действия. Липопротеин Брауна (Lpp) локализован в на- ружной мембране бактерий семейства Entero- bacteriaceae, связывает слой пептидогликанов с наружной бактериальной мембраной [63]. Структурно Lpp выступает в качестве спейсе- ра между внутренней и наружной мембраной, поддерживая открытость периплазматического пространства и целостность наружной мембра- ны [159]. В процессе созревания липопротеина Брауна происходит модификация липидно- го домена, катализируемая глицеролтранс- феразой, О-ацилтрансферазой, сигнальной транс феразой II и N-ацилтрансферазой [64]. Показано, что вместе с липополисахаридом Lpp индуцирует развитие септического шока, продукцию цитокинов TNFα, IFNα, IL-1β, IL-6 [179], активирует комплемент и систему свертывания крови [71, 110]. Установлено, что сигнал от Lpp, приводящий к индукции ци- токинов, идет через рецептор TLR-2 (Toll-Like Receptor — Toll-подобный рецептор) [7]. У воз- будителя чумы Lpp имеет размер 8,3 kDa. Sha J. и соавт. [145, 146] получили одинарный делеци- онный мутант по гену lpp и двойной делецион- ный мутант по генам lpp и msbB на модели ви- рулентного штамма Y. pestis CO92 и аттенуиро- ванного штамма KIMD27(Pgm–). Иммунизация полученными мутантными штаммами защи- щала от гибели мышей и крыс при заражении высоковирулентными штаммами дикого типа в модельных экспериментах бубонной и легоч- ной чумы. Van Lier C.J. и соавт. [170] показали, что штамм Y. pestis CO92ΔlppΔpla, авирулентный для мышей при подкожном и аэрогенном вве- дении, индуцирует гуморальный и клеточно- опосредованный иммунный ответ, обеспечивая защиту животных от заражения летальными дозами Y. pestis CO92. Исследователи впервые продемонстрировали, что полученный ими двойной мутант не способен эффективно пере- живать в макрофагах мыши и человека в отли- чие от штамма дикого типа. Galindo C.L. и со- авт. [58] исследовали влияние мутации по гену lpp на экспрессию ряда генов стресс-ответа и вирулентности при температурах культивиро- вания чумного микроба 26°С и 37°С. Если при температуре 26°С у мутанта в основном обнару- живали изменение экспрессии генов, отвечаю- щих за метаболизм, то при 37°С уменьшалась экспрессия генов стресс-ответа и вирулентнос- ти (htpN, degQ, htrA, lcrQ/yscM, iscR, nifS и др.). Результаты эксперимента подтвердили роль Lpp в адаптации Y. pestis к условиям организма хозяи- на. Таким образом, Lpp обладает еще и регулятор- ной функцией. A SEARCH FOR NEW MOLECULAR TARGETS FOR OPTIMIZING PLAGUE PREVENTIVE VACCINATION AND THERAPY До настоящего времени поиск новых анти- генов для разработки субъединичных и живых аттенуированных чумных вакцин, а также со- вершенствование препаратов для лаборатор- ной диагностики и лечения чумы сохраняют свою актуальность, так как в природных оча- гах ежегодно заболевает до 3000 человек [57], что, с учетом высокой контагиозности легоч- ной формы заболевания, представляет серьез- ную опасность возникновения эпидемических осложнений. Отбор потенциальных генов-мишеней для аттенуации вирулентных штаммов проводят с помощью произвольного мутагенеза индиви- дуально меченными транспозонами, используя методики обратной (реверсивной) вакциноло- гии или исследуя аналоги генов других бакте- риальных патогенов, мутации в которых ведут к снижению вирулентности [41]. Аналоги генов других бактериальных патогенов, мутации в которых ведут к снижению вирулентности A SEARCH FOR NEW MOLECULAR TARGETS FOR OPTIMIZING PLAGUE PREVENTIVE VACCINATION AND THERAPY Key words: plague, Yersinia pestis, pathogenicity factor, molecular target, vaccine prevention. Чума — острое инфекционное природно- очаговое трансмиссивное заболевание, сопро- вождающееся лихорадкой, тяжелой интокси- кацией, серозно-геморрагическим воспале- нием в лимфатических узлах, легких и других органах, а также сепсисом. Высокая леталь- ность необходима для переноса возбудителя чумы — Yersinia pestis — инфицированными блохами, вынужденными покидать мертво- го грызуна в поисках нового хозяина-про- кормителя. Два других представителя рода Yersinia — энтеропатогенные Y. pseudotuberculosis и Y. enterocolitica — вызывают у людей чаще все- го склонные к затяжному течению заболевания желудочно-кишечного тракта средней тяжести, обеспечивающие длительное выделение па- тогенов в окружающую среду и последующее алиментарное заражение новых хозяев. Одного из них — Y. pseudotuberculosis — принято считать прародителем Y. pestis. Дивергенция этих бакте- рий произошла 5000–20 000 лет назад [5]. щиты макроорганизма. Развитие инфекцион- ного процесса в организме восприимчивого хо- зяина требует присутствия у возбудителя чумы целого набора факторов патогенности различ- ной функциональной направленности и систем регуляции, обеспечивающих их координиро- ванную экспрессию. «Выключение» любого из этих факторов, в свою очередь, может либо не влиять на вирулентность микроба, либо при- водить к его аттенуации. щиты макроорганизма. Развитие инфекцион- ного процесса в организме восприимчивого хо- зяина требует присутствия у возбудителя чумы целого набора факторов патогенности различ- ной функциональной направленности и систем регуляции, обеспечивающих их координиро- ванную экспрессию. «Выключение» любого из этих факторов, в свою очередь, может либо не влиять на вирулентность микроба, либо при- водить к его аттенуации. В середине прошлого века Burrows T.W. [33] определил набор признаков, присутствующий у всех изученных им вирулентных штаммов Y. pestis, — классические «детерминанты виру- лентности». К их числу он относил способность клеток сорбировать экзогенные красители и ге- мин (Pgm+), зависимость роста при температуре 37°С от наличия в среде ионов Са2+, синтез V- (а позднее — LcrV) и W-антигенов, «мышиного» токсина (Tox+), капсульного антигена FI (Fra+ или F1+, а позднее — Cаf1+), сочетанный синтез пестицина (Pst+), фибринолизина (Fb+) и плаз- мокоагулазы (Cg+), пуринонезависимость, или способность синтезировать эндогенные пури- ны (Pur+). После шести десятилетий исследо- ваний и дискуссий, прошедших с момента пос- тулирования детерминант вирулентности, не- которые из них, такие как синтез W-антигена, пестицина и способность к синтезу эндогенных Особенности патогенеза инфекционных за- болеваний и вирулентность вызывающих их микробов определяются имеющимися у по- следних вируломами — «наборами» факторов патогенности. При этом каждая из биомоле- кул (факторов патогенности) может обладать несколькими активностями, направленными на преодоление различных звеньев системы за- 266 Молекулярные мишени для профилактики чумы 2021, Т. Полифункциональные белки наружной мембраны, кодируемые хромосомой Наружная мембрана грамотрицательных бак- терий содержит множество белков, помогаю- щих поддерживать структурную целостность клеточной оболочки бактерий. Белок SurA (Sur- vival protein A) впервые идентифицировали как фактор, играющий важную роль в выживании клеток Escherichia coli в стационарной фазе ро- ста [163]. Показано, что парвулин-подобный до- мен белка обладает пептидил-пролил изомераз- ной активностью [90, 138], а N-концевой домен — шаперонной активностью [23]. Уста новлено, что именно шаперонная функция связана с ролью SurA в патогенезе инфекций, вызываемых неко- торыми видами бактерий [22]. Например, деле- ция гена surA оказывает плейотропный эффект на бактериальную клетку Y. pseudotuberculosis, ведущий к аттенуации штамма при мыши- ной модели инфекции [112, 113], что отчасти может быть связано с ролью белка в биогене- зе наружной мембраны, а именно утратой Inv, необходимого возбудителю псевдотуберкуле- за для процесса инвазии [112]. Делеционный мутант вирулентного штамма Y. pestis оказался более чувствителен к мембранопроникающим агентам и не вызывал гибели мышей при под- кожном введении 2,1 × 105 КОЕ культуры [153]. Таким образом, SurA можно рассматривать как мишень для антимикробных агентов, а широ- 267 Е.А. Красильникова и др. Инфекция и иммунитет NlpD (novel lipoprotein D) — липопротеин на- ружной мембраны чумного микроба, содержа- щий 379 аминокислотных остатков и типичную для липидов консенсусную последовательность на N-конце. Ген nlpD вместе с генами surE, pcm, rpoS входит в состав хромосомного pcm-локуса, обнаруживаемого у микроорганизмов различ- ных видов семейства Enterobacteriaceae. Lange R. и соавт. [70, 85] и Tidhar A. и соавт. [161] пока- зали, что гены nlpD и rpoS образуют оперон. Используя набор изогенных мутантов штам- ма Y. pestis Kimberly 53, Tidhar A. и соавт. [161] изучили экспрессию и роль продуктов генов pcm-локуса в патогенезе чумы. Авторы обнару- жили, что только NlpD существенен для виру- лентности чумного микроба. Мутация по гену nlpD приводила к полной аттенуации Y. pestis, а сконструированный nlpD-мутант превосхо- дил вакцинный штамм EV76 по защите мы- шей от гибели при их последующем заражении бубонной или легочной формой чумы [161]. Дентовская С.В. и соавт. [42] сконструировали ΔnlpD-мутанты на основе других родительских штаммов Y. pestis, включая непатогенные для морских свинок и человека штаммы подвида microti. Сравнительная оценка подтвердила, что при подкожной иммунизации мышей ΔnlpD- мутанты индуцировали иммунитет, превосхо- дящий по напряженности в 105 раз таковой в от- вет на введение вакцинного штамма EV. В то же время NlpD– варианты бактерий практически не защищали морских свинок при подкожном заражении возбудителем чумы, уступая штам- му EV по напряженности формируемого имму- нитета в 106 раз. клеточном переживании патогена [56, 102, 128]. Полифункциональные белки наружной мембраны, кодируемые хромосомой Chen Y. и соавт. подтвердили высокую степень гомологии нуклеотидной и аминокислотной последовательностей OmpA у всех трех патоген- ных видов Yersinia: Y. pestis, Y. pseudotuberculosis и Y. enterocolitica. Анализ последовательнос- тей OmpA 262 штаммов Y. pestis, 134 штаммов Y. pseudotuberculosis и 219 штаммов Y. enterocolitica выявил 100, 98,8 и 97,7%-ную гомологию [38]. Для идентификации генов, продукты которых участвуют в реализации функции пережива- ния Y. pestis в макрофагах, Bartra S.S. и соавт. [17] использовали подход, основанный на гибри- дизации по местам встраивания транспозонов (TraSH — transposon site hybridization). Авторы провели скрининг пула транспозоновых мутан- тов на модели культуры клеток. Результаты ис- следования подтвердили необходимость OmpA для переживания Y. pestis и родственного патоге- на Y. pseudotuberculosis в макрофагах. Таким обра- зом, порины как белки, играющие важную роль во взаимоотношениях патогена с организмом хозяина, способные индуцировать образование протективных антител, привлекательны с точ- ки зрения использования при конструировании вакцинных и диагностических препаратов. По данным Erova T. и соавт. [46], введение рекомбинантного OmpA Y. pestis мышам вызы- вало развитие протективного иммунного от- вета против бубонной, но не легочной формы чумы при заражении Cаf1– мутантом штамма CO92. Однако титры протективных антител в сыворотке вакцинированных мышей оказа- лись намного ниже титров анти-Ail и анти-F1- V-антител. Тем не менее полученные экспери- ментальные данные позволяют рассматривать рекомбинантный OmpA, а также Pla и Ail в ка- честве дополнительных антигенов в составе субъединичных вакцин к традиционно исполь- зуемым Cаf1 и LcrV для повышения протектив- ности последних [46]. OmpA (Outer membrane protein A — белок на- ружной мембраны А) является представителем порообразующих белков наружной мембраны грамотрицательных бактерий, или поринов. Неспецифические порины доминируют среди белков наружной мембраны бактерий и предна- значены для пассивной диффузии гидрофиль- ных молекул с молекулярной массой не более 600 Da [40]. Порины чрезвычайно устойчивы к действию протеаз, повышенной температуре и другим денатурирующим факторам, что обу- словлено особенностью их структуры. По физи- ко-химическим свойствам порины — это слабо- кислые белки, содержащие необычно большое для интегральных белков количество полярных аминокислотных остатков [4]. Регуляция транс- порта различных молекул через наружную мем- брану, поддержание структурной целостности клетки, связывание различных веществ и ад- гезия — основные функции поринов. Было по- казано, что OmpA обладает пороформирующей активностью [155], опосредует резистентность к комплементу и антибактериальным пепти- дам, играет важную роль в инвазии и внутри- Факторы адгезии и колонизации В процесс адгезии и колонизации вовлечены различные поверхностные молекулы Y. pestis. Кроме ранее идентифицированных фимбрий с известными или потенциальными функция- ми адгезинов [47, 99, 139], адгезивные и инва- зивные свойства обнаружены у некоторых бел- ков наружной мембраны возбудителя чумы не- фимбриальной природы. Белок Ail/OmpX (Attachment invasion locus — Ail) принадлежит к Ail/Lom семейству белков наружной мембраны и обеспечивает защиту Y. pestis от комплемент-опосредованного кил- линга [18]. Более того, Ail является доминант- ной молекулой адгезии и играет важную роль в доставке эффекторов системы секреции III типа к клеткам-мишеням хозяина в ходе ин- 268 Молекулярные мишени для профилактики чумы 2021, Т. 11, № 2 фекционного процесса [48]. Белок Ail Y. pestis идентифицировали в качестве одного из глав- ных адгезинов Y. pestis [18, 48, 50, 83]. Мутанты Y. pestis с делецией Δail обладают сниженной способностью к системному распростране- нию и той или иной степенью аттенуации, бо- лее выраженной на модели крыс, чем мышей. Однако показано, что адаптивный иммунный ответ у крыс формировался после введения вы- соких доз мутантного штамма (3 × 104 КОЕ), о чем свидетельствовали высокие титры анти- тел к Y. pestis Cаf1-антигену [68]. Более того, Kolodziejek A.M. и соавт. сообщили, что делеция гена ail у штамма Y. pestis CO92 приводила к вы- сокой степени аттенуации на крысиной модели легочной чумы [82]. На модели штамма Y. pestis KIM5 с делецией pgm-локуса показано, что Ail более важен для связывания клеток хозяи- на и индукции цитотоксичности и для виру- лентности на мышиной модели инфекции при внутривенном введении, чем Pla, и что опос- редованная Pla секреция Yop не зависит от его протеазной активности [50]. Ail связывает фиб- ронектин, белок внеклеточного матрикса, ко- торый, возможно, выступает в качестве связую- щего рецептора между Ail и хозяйскими клетка- ми [169]. Антитела к фибронектину подавляют KIM5-опосредованную цитотоксичность кле- ток хозяина в Ail-зависимой манере, показывая необходимость этого взаимодействия для до- ставки Yop [169]. белком GST-YadC137-409, содержащим аминокис- лотные остатки YadC с 137 по 409, присоеди- ненные к С-концу глутатион-S-трансферазы (glutathione S-transferase — GST), обеспечивала частичную защиту против заражения Cаf1– штаммом Y. pestis и стимулировала смешанный Th1/Th2 иммунный ответ [108]. Данные наход- ки выводят YadC в число перспективных ком- понентов кандидатных вакцин. Факторы инвазии В конце прошлого века большинство ис- следователей рассматривали возбудителя чумы как внеклеточный патоген вследствие обнару- жения мутаций в двух генах, кодирующих глав- ные гомологи инвазина (Inv) и адгезина (YadA) Y. pseudotuberculosis [119, 148]. Однако позднее было установлено, что Y. pestis может проникать внутрь нефагоцитирующих эукарио тических клеток, и данная способность опосредуется рядом факторов патогенности, а именно ак- тиватором плазминогена (Pla), белком наруж- ной мембраны OmpX (Ail) и pH6-антигеном (Psa) [12, 35, 83, 86]. Причем ни один из данных факторов в одиночку не может полностью обе- спечить процесс инвазии. Seo J. и соавт. [144] идентифицировали один из добавочных по- верхностных белков Y. pestis, интимин/ин- вазин-подобный белок (intimin/invasin-like protein — Ilp), кодируемый хромосомным геном YPO3944 и сходный с поверхностными белка- ми семейства интиминов/инвазинов грамотри- цательных бактерий. Авторы показали, что Ilp не участвует в колонизации блох, но необходим для адгезии к клеткам млекопитающего и регу- лирует системное распространение и вирулент- ность возбудителя чумы на мышиной модели инфекции, о чем свидетельствует 55-кратное увеличение LD50 для Δilp-мутанта при интра- назальном введении. Учитывая, что Ilp игра- ет важную роль в развитии легочной формы чумы, он может быть перспективен в качестве кандидата для конструирования вакцинных препаратов. Кроме того, адгезивные свойства описа- ны для нескольких белков автотранспортеров чумного микроба [176], таких как YapC [49], YapE [88, 89] и YadA-подобных олигомерных белков-автотранспортеров [55, 108]. К настоя- щему времени показана уникальная роль YapE, YapJ и YapK в патогенезе чумы. YapE необхо- дим для полного проявления вирулентности штаммов при бубонной, но не легочной фор- ме инфекции [88]. Продукты генов yapK и yapJ играют важную роль при развитии системной (не бубонной) чумной инфекции, при этом мутанты Y. pestis, лишенные обоих генов yapK и yapJ, проявляют более аттенуированный фе- нотип, чем индивидуальные мутанты [93]. YadB и YadC Y. pestis, два новых белка семейства оли- гомерных адгезинов [6, 137], способные к фор- мированию тримеров, наличие которых корре- лирует с инвазивностью Y. pestis для эпителио- идных клеток [49, 55]. Установлено, что YadC при экспрессии в E. coli опосредует агрегацию бактерий и прилипание к мышиным макрофа- гоподобным клеткам и эпителиальным клет- кам человека. Утрата yadBC ведет к незначи- тельному снижению инвазивности для эпите- лиоидных клеток и понижению вирулентности мутантов при бубонной, но не легочной чуме у мышей [55]. Иммунизация мышей слитым Эффекторные Yop-белки Для секреции и транслокации эффекторных Yop-белков в клетки млекопитающих Y. pestis ис- пользует систему секреции белков третьего типа (type III secretion system — T3SS). Белок YopR (19 kDa) — фактор патогенности, секретируе- мый Y. pestis на ранних стадиях развития инфек- ции, причем транслоцируется он не в цитозоль клеток хозяина, а во внеклеточное простран- ство. На мышиной модели инфекции показано, что делеция гена yopR приводит к 10–30-крат- ному снижению вирулентности Y. pestis [92]. Schubot F.D. и соавт. [142] исследовали кристал- лическую структуру резистентного к протеа- 269 Е.А. Красильникова и др. Инфекция и иммунитет прерыванию образования стресс-волокон и фа- гоцитоза макрофагов. VASP играет основную роль в регуляции актинового цитоскелета, ко- торый, как было показано, является мишенью при развитии инфекций, вызванных Helico- bacter pylori и Listeria monocytogenes [16, 19, 25]. Киназный и GDI домены YpkA совместно ре- моделируют цитоскелет клеток хозяина и пре- рывают хозяйские клеточные процессы, вызы- вающие перестройки цитоскелета, такие как фагоцитоз, подвижность и слияние клеток. Помимо эффектов, направленных на цитоске- лет, YpkA инициирует гибель клеток хозяина, так как показано, что N-концевые аминокис- лотные остатки 133–262 ответственны за ин- дукцию апоптоза [117]. На мышиной модели инфекции установлено значительное сниже- ние вирулетности YpkA-дефицитного мутанта Y. pseudotuberculosis [174]. Сведения о протектив- ности YpkA Y. pestis ограничены. Установлено, что вакцинация YpkA Y. pestis стимулирует у мышей образование значительных уровней антител, достоверно удлиняет среднюю про- должительность жизни, но не влияет на вы- живаемость при подкожном заражении бес- капсульным вариантом вирулентного штамма возбудителя чумы [10]. Wang S. и соавт. [171] попытались использовать ДНК-иммунизацию как подход для определения протективной эффективности YpkA при интразальном за- ражении. Однако даже после модификаций генетической последовательности ypkA авто- рам удалось добиться лишь частичной защиты при заражении вирулентным штаммом Y. pestis KIM1001. зам домена кора YopR (38–149 аминокислотные остатки) и обнаружили в этой области пять аль- фа-спиралей. Структура рассматриваемой обла- сти оказалась подобна структуре одного из до- менов центрального регулятора T3SS Y. pestis YopN. По мнению авторов, YopR, возможно, также участвует в регуляции T3SS. У ΔyopR- мутантов Y. pestis нарушался процесс сборки «иглы» аппарата секреции T3SS. При этом YscF не поступал к месту сборки, а секретировался в среду. Предположительно, YopR способен ре- гулировать секрецию YscF. Альтернативным ва- риантом может являться непосредственное его участие в полимеризации белка YscF, что менее вероятно, так как YopR не обнаруживали в ассо- циации с очищенными «иглами» [26]. Эффекторные Yop-белки YpkA — один из эффекторных Yop-белков возбудителя чумы, доставляемых в клетки хо- зяина посредством системы секреции белков третьего типа, локализующийся на внутрен- ней поверхности плазматической мембраны. YpkA — мультидоменный эффектор, состоящий из N-концевого секреторного сигнального пеп- тида, который узнается T3SS-аппаратом, подоб- ного эукариотическому серин/треонин- (Ser/ Thr) киназного домена, GDI-домена (Guanosine Dissociation Inhibitors — GDI) и С-концевого ак- тин-связывающего домена [109, 129]. YpkA у бак- терий является неактивной киназой, актива- ция фермента проходит внутри цитозоля клет- ки хозяина путем связывания с коактиватором актином, которым богаты эукариотические клетки [168]. После активации YpkA подверга- ется автофосфорилированию и фосфорилиру- ет целый набор внутриклеточных субстратов, включая Gαq (α-субъединица гетеротример- ных G-белков), фосфопротеин, стимулиро- ванный вазодилятатором (VASP — vasodilator- stimulated phosphoprotein), убиквитин тио- эстеразу (otubain-1) и актин. Цитотоксические эффекты YpkA проявляются преимущественно в виде нарушения актинового цитоскелета, ис- чезновения стрессовых волокон и округления клеток, которое, по-видимому, зависит от ки- назной активности и GDI-домена [45, 76, 94, 109, 129]. Показано, что мембранно-локализо- ванный домен, расположенный в N-концевом регионе, направляет YpkA к внутренней по- верхности клеточной мембраны, где ГТФазы выполняют их функции [60]. GDI-домен YpkA связывается ГТФ- или ГДФ-связывающими ГТФазами, мимикрируя под белки GDI хо- зяина, для подавления обмена нуклеотидами при участии RhoA и Rac1 [129]. YpkA преры- вает Gαq-опосредованный сигнальный путь, фосфорилируя Gαq в положении Ser47 [109]. VASP фосфорилируется YpkA преимуществен- но в положении Ser157, что ведет к подавлению VASP-опосредованной полимеризации актина, Регуляторные белки Рецептор циклического аденозинмонофос- фата (Cyclic AMP receptor protein — Crp) явля- ется регулятором вирулентности многих пато- генов [114, 124, 133, 149, 177]. Crp контролирует экспрессию множества генов бактерий, ассо- циированных с вирулентностью и отвечающих за приспособление к изменяющимся услови- ям окружающей среды [34]. Crp активен толь- ко в присутствии малой молекулы индуктора cAMP. Образовавшийся комплекс cAMP-Crp связывается с промоторными областями целе- вых генов, чтобы активировать или репресси- ровать их транскрипцию [34]. Комплекс cAMP- Crp модулирует экспрессию более 6% генов Y. pestis, продукты которых обладают большим разнообразием функций [177]. Crp необхо- дим для развития бубонной и легочной чумы, в основе которых лежит Crp-зависимая экс- прессия pla и оперона sycO-ypkA-yopJ системы секреции III типа Yop-Ysc Y. pestis [80, 99, 178]. В то же время экспрессия Crp положительно регулируется PhoP на уровне транскрипции и Hfq (главный посттранскрипционный ре- гулятор, который контролирует образование Факторы, обеспечивающие репликацию в макрофагах Y. pestis — факультативный внутриклеточ- ный патоген, способный реплицироваться на ранней стадии инфицирования макрофа- гов [36, 37, 72, 154]. Pujol C. и соавт. [130] пока- зали, что локус pgm, локализованный на хро- мосоме возбудителя чумы и ранее ассоцииро- ванный с системой транспорта железа, важен и для репликации клеток Y. pestis в макрофагах. Репликация Pgm+ бактерий Y. pestis в активиро- ванных макрофагах коррелирует с уменьшени- ем содержания в них NO, при этом репликация Δpgm бактерий Y. pestis возможна только в iNOS- макрофагах (iNOS — индуцибельная NO синта- за), в отсутствии NO. Было показано, что в со- ставе pgm-локуса находится оперон, названный rip (required for intracellular proliferation), состоя- щий из трех генов ripA, ripB, ripC и необходи- мый для пролиферации бактериальных клеток внутри макрофагов. Показано, что мутантные штаммы с делецией любого из генов ripA и ripB неспособны снижать уровень NO и реплици- 270 Молекулярные мишени для профилактики чумы 2021, Т. 11, № 2 биопленки и вирулентность у штаммов Y. pestis) на посттранскрипционном уровне [87, 180], что указывает на значимость роли Crp в регуля- ции генов, кодирующих факторы патогеннос- ти Y. pestis. Crp или его гомологи регулируют контроль образования биопленок у многих па- тогенов: Listeria monocytogenes [140], Xanthomonas campestris pv. campestris [104], Aggregatibacter actinomycetemcomitans [167], Vibrio cholerae [53] и Serratia marcescens [77]. Установлено, что по- теря Crp ведет к значительному снижению об- разования биопленки возбудителем чумы [175]. Причем показано, что Crp оказывает прямое влияние на транскрипцию gmhA и опосредован- но активирует транскрипцию waaAE-coaD пу- тем прямого влияния на phoPQ-YPO1632, но не оказывает регуляторного влияния на гены hms на уровне транскрипции [100]. роваться в активированных макрофагах [61, 130, 141, 147]. Белок RipA, кодируемый геном ripA, принадлежит семейству структурно гомо- логичных CoA трансфераз I, ответственных за перенос CoAS– аниона от донора CoA тио- эфира к акцептору — свободной кислоте [166]. RipA, представляющий собой тетрамер, функ- ционирует как бутирил-CoA-трансфераза [164]. Предполагаемая функция белка RipB — ено- ил-гидролаза. Анализ последовательности гена ripC и соответствующего ему белка, аннотиро- ванного ранее как субъединица белка цитрат- лиазы, а также отсутствие в геноме Y. pestis генов, кодирующих цитратлиазу, позволили Torres R. и соавт. [165] выдвинуть предположение о при- надлежности белка RipC к производному CoA. Таким образом, возможный сценарий действия оперона — синтез ацетил-CoA, являющегося метаболическим предшественником в биосин- тезе жирных кислот, необходимых для строи- тельства клеточной стенки и, возможно, являю- щихся причиной падения содержания NO [107, 135, 136]. Ингибиторы лизоцима Возбудитель чумы обладает гомологами пе- риплазматического ингибитора лизоцима Ivy и мембраносвязанного ингибитора лизоцима MliC. Показано, что MliC не требуется чумному микробу для устойчивости к лизоциму и разви- тия инфекции. Делеция гена ivy снижала спо- собность вирулентного штамма Y. pestis СО92 противостоять лизоциму и полиморфноядер- ным нейтрофилам, но не влияла на способность бактерии к росту в сыворотке или устойчивость к макрофагам. Штамм Y. pestis, лишенный Ivy, обладал сниженной вирулентностью для мы- шей при подкожном или интраназальном вве- дении. При введении лизоцим-M-дефицитным мышам линии FVB, неспособным продуци- ровать лизоцим LysM, «дикий» штамм Y. pestis и его Δivy-мутант обладали одинаковой виру- лентностью [43]. Факторы, обеспечивающие репликацию в макрофагах Авторы допустили, что конечным про- дуктом rip-оперона является бутират, который косвенно обладает способностью подавлять IFN-опосредованную индукцию макрофагами продукции NO, предотвращая гибель бактери- альных клеток [81, 118]. BLAST анализ доступ- ных геномов прокариот показал, что rip-оперон присутствует у мик роорганизмов различных видов, в том числе Salmonella и Burkholderia, что свидетельствует о его роли в эволюции виру- лентных микроорганизмов. Белки, участвующие в формировании биопленок Важным процессом, обеспечивающим пе- редачу возбудителя чумы новым теплокров- ным хозяевам, является размножение Y. pestis в организме блохи в виде бактериальных био- пленок, заполняющих ее преджелудок и бло- кирующих прохождение пищи [1, 11, 32, 66, 67, 74, 75, 103, 122, 123]. Формирование биопленок у Y. pestis активируется циклическим дигуани- латмонофосфатом (ц-ди-ГМФ, с-di-GMP) [29] и подав ляется двухкомпонентной регуля- торной системой Rcs (regulation of capsular synthesis). Rcs состоит из двух мембранных белков — RcsD и RcsC, ДНК-связывающего ре- гулятора — RcsB и вспомогательного белка — RcsA. После стимуляции RcsC сенсор автофос- форилируется и переносит фосфатную группу к RcsD и затем к RcsB. Фосфорилированный RcsB формирует RcsB гомополимер или RcsB/ 271 Инфекция и иммунитет Е.А. Красильникова и др. необходимы высокоаффинные системы полу- чения Zn2+ в ходе инфекционного процесса. Для преодоления низкой концентрации Zn2+, наблюдаемой внутри эукариотических кле- ток, многие патогенные бактерии используют транспортер цинка ZnuABC [59, 62, 78, 125]. Bobrov A.G. и соавт. [27] показали, что помимо транспортера ZnuABC возбудитель чумы об- ладает вторичным транспортером Zn2+, вклю- чающим компоненты иерсиниабактина (Ybt), сидерофорозависимой транспортной системы железа. Авторы установили, что критичным для функционирования вторичной транспорт- ной системы Zn2+ возбудителя чумы являет- ся синтез сидерофора Ybt и белка внутренней мембраны YbtX. Наконец, авторы показали, что система ZnuABC и Ybt-синтетаза HMWP2, предположительно через синтез Ybt, способ- ствуют развитию летальной инфекции на мо- дели септической чумы у мышей. В дальней- шем Bobrov A.G. и соавт. [28] продемонстриро- вали в эксперименте на модели бубонной и ле- гочной чумы, что двойной мутант ΔybtXΔznu аттенуирован для мышей, тогда как одинар- ный мутант ΔybtX сохранил вирулентность на уровне исходного штамма. Избыток Zn2+ обладает антимикробной активностью, сни- жая внутриклеточное выживание бактерий. Установлено, что экспортер Zn2+ ZntA способ- ствует устойчивости к токсическим эффектам Zn2+ in vitro, однако двойной мутант ΔzntAΔzur сохраняет высокую вирулентность на моделях легочной и бубонной чумы и высокую выжи- ваемость в макрофагах. Данные авторов по- казывают, что Ybt или модифицированный Ybt принимают участие в цинк-связывающей активности или способствуют таковой в су- пернатантах культур и вовлечены в получение Zn2+ чумным микробом. RcsA гетеродимер. Гетерополимер RcsB/RcsA подавляет экспрессию HmsT, фермента ди- гуанилатциклазы, которая синтезирует c-di- GMP, что в итоге приводит к ингибированию продукции биопленок [156]. Наличие регуля- тора RcsB/RcsA в клетках Y. pseudotuberculosis приводит к невозможности формирования бактериями данного вида биопленок в орга- низме насекомых [157, 158]. В штаммах Y. pestis ген rcsA инактивирован, замена данного гена на функциональный ген из возбудителя псев- дотуберкулеза ведет к подавлению способнос- ти штаммов Y. Факторы нутриционной вирулентности Утрата вирулентности и/или снижение вну- триклеточной выживаемости, вызванные мута- циями в транспортных системах марганца (Mn), были продемонстрированы для различных патогенов, включая Borrelia burgdorferi, Brucella abortus, Neisseria gonorrhoeae, Porphyromonas gingi- valis, Salmonella spp., разные виды стрептококков и Yersinia pseudotuberculosis [8, 15, 24, 30, 44, 65, 73, 79, 96, 105, 115, 116, 151]. У грам отрицательных бактерий известны две системы транспорта марганца: Yfe/Sit и/или MntH. Установлено, что система Yfe Y. pestis участвует в транспорте Fe и Mn [20, 21, 120]. Позднее было показано, что мутация в yfe или mntH существенно не влия- ла на рост чумного микроба в аэробных усло- виях in vitro при дефиците Mn. Двойной мутант ΔyfeΔmntH показал умеренный дефект роста, который компенсировался добавлением Mn. Наконец, двойной мутант Y. pestis ΔyfeΔmntH имел ~133-кратную потерю вирулентности на модели бубонной чумы у мышей, но сохра- нял вирулентность на модели легочной чумы. Это говорит о том, что доступность Mn, потреб- ность бактерии в Mn или транспортеры Mn, ис- пользуемые Y. pestis, отличаются в легких (ле- гочная чума) по сравнению с другими органами и системами [121]. Белки, участвующие в формировании биопленок pestis образовывать биопленки в преджелудке блох, что свидетельствует о не- обходимости этой стадии в эволюционном из- менении механизма распространения микро- организма. необходимы высокоаффинные системы полу- чения Zn2+ в ходе инфекционного процесса. Для преодоления низкой концентрации Zn2+, наблюдаемой внутри эукариотических кле- ток, многие патогенные бактерии используют транспортер цинка ZnuABC [59, 62, 78, 125]. Bobrov A.G. и соавт. [27] показали, что помимо транспортера ZnuABC возбудитель чумы об- ладает вторичным транспортером Zn2+, вклю- чающим компоненты иерсиниабактина (Ybt), сидерофорозависимой транспортной системы железа. Авторы установили, что критичным для функционирования вторичной транспорт- ной системы Zn2+ возбудителя чумы являет- ся синтез сидерофора Ybt и белка внутренней мембраны YbtX. Наконец, авторы показали, что система ZnuABC и Ybt-синтетаза HMWP2, предположительно через синтез Ybt, способ- ствуют развитию летальной инфекции на мо- дели септической чумы у мышей. В дальней- шем Bobrov A.G. и соавт. [28] продемонстриро- вали в эксперименте на модели бубонной и ле- гочной чумы, что двойной мутант ΔybtXΔznu аттенуирован для мышей, тогда как одинар- ный мутант ΔybtX сохранил вирулентность на уровне исходного штамма. Избыток Zn2+ обладает антимикробной активностью, сни- жая внутриклеточное выживание бактерий. Установлено, что экспортер Zn2+ ZntA способ- ствует устойчивости к токсическим эффектам Zn2+ in vitro, однако двойной мутант ΔzntAΔzur сохраняет высокую вирулентность на моделях легочной и бубонной чумы и высокую выжи- ваемость в макрофагах. Данные авторов по- казывают, что Ybt или модифицированный Ybt принимают участие в цинк-связывающей активности или способствуют таковой в су- пернатантах культур и вовлечены в получение Zn2+ чумным микробом. Использование методик обратной (реверсивной) вакцинологии Клоны с мутациями внутри открытых рамок считывания гена rbsA, кодирующего предпола- гаемый АТФ-связывающий белок транспортной системы рибозы, и гена vasK, кодирующего ком- понент системы секреции VI типа, продемон- стрировали аттенуацию при введении 11 или 12 LD50 на модели легочной чумы у мышей. Cреди остальных 18 мутантов с сигнатурными метка- ми 9 также были аттенуированы (от 40 до 100%) на модели легочной чумы у мышей при введении 12 LD50. Ранее авторы обнаружили, что делеции в генах, кодирующих липопротеин Брауна (Lpp) и ацилтрансферазу (MsbB), снижают вирулент- ность штамма Y. pestis CO92 на моделях бубонной и легочной чумы [145, 146]. Удаление генов rbsA и vasK в штаммах, несущих одиночную Δlpp или двойную ΔlppΔmsbB мутации, дополняло аттену- ированный фенотип и обеспечивало выживание от 90 до 100% мышей на модели легочной чумы при заражающей дозе от 20 до 50 LD50. После вве- дения тройного мутанта ΔlppΔmsbBΔrbsA в дозе 50 LD50 до 90% мышей были защищены от гибе- ли при последующем заражении 12 LD50 штамма «дикого» типа Y. pestis CO92, подтверждая тем самым, по мнению авторов, что данный мутант или другие, несущие комбинацию делеций ге- нов, могут быть дополнительно испытаны при создании кандидата в живую аттенуированную вакцину против чумы. Позднее авторы созда- ли и охарактеризовали еще три штамма с му- тациями внутри открытых рамок считывания ypo0815 (кодирует белок GspE системы секреции II типа), ypo2884 (продукт обладает гомологией к суперсемейству BG crystallin), ypo3614-3168 (cyoABCDE — оперон цитохром c-оксидазы) и ypo1119-1120 (кодируют систему Tol-Pal) [9]. Созданные делеционные мутантные штаммы демонстрировали различные уровни аттенуации (до 100%) на моделях бубонной или легочной инфекции у мышей. Снижение вирулентности мутантов могло быть дополнительно усилено за счет создания сочетанных делеций, а имен- ру р у В дальнейшем Zvi A. и соавт. [181] использо- вали комбинацию высокопроизводительного компьютерного и экспериментального под- ходов для идентификации CD8+ Т-клеточных (CD8+ T-cell — CTL) эпитопов у белков Y. pestis. С помощью современных алгоритмов прогно- зирования авторы проанализировали потенци- альную способность продуктов 4067 открытых рамок считывания Y. pestis связываться с моле- кулами класса I главного комплекса гистосов- местимости (ГКГС). После тестирования про- дукции IFNγ спленоцитами вакцинированных мышей, индуцированной введением 1532 син- тезированных пептидов, впервые отобрали 178 (11,8%) эпитопов, охватывающих 113 белков Y. pestis, способных стимулировать специфи- ческий Т-клеточный ответ. Ни один из этих белков не совпал с 34 белками-индукторами Т-клеточного ответа, обнаруженными в пре- дыдущем исследовании [95]. Использование методик обратной (реверсивной) вакцинологии Выбор среди вновь выявленных пептидов наиболее иммуногенных, способных обеспечивать эффективную защи- ту животных от гибели при заражении чумой, может быть проведен с помощью различных подходов, в числе которых доставка нескольких повторяющихся эпитопов в виде ДНК-вакцин и/или иммунизация препаратами, состоящими из смесей синтетических пептидов, усиленных добавлением различных адъювантов, возмож- но, вместе с известными иммунодоминантными антигенами чумного микроба. Использование методик обратной (реверсивной) вакцинологии Одной из главных задач при создании субъ- единичных вакцинных препаратов против чумы является идентификация антигенов Y. pestis, способных стимулировать протектив- ный Т-клеточный ответ, уникальную возмож- ность обнаружения которых предоставляют полногеномное секвенирование, биоинформа- тика и протеомный анализ. Для поиска антиге- нов, способных стимулировать протективный Т-клеточный ответ, Li B. и соавт. [95] на осно- ве биоинформационного анализа и предвари- тельно опубликованных результатов выбра- ли 261 ген Y. pestis для экспрессии в Escherichia coli BL21(DE3). После выделения и очистки 101 белок использовали для оценки способности Организм млекопитающего с помощью целого ряда механизмов ограничивает для внедрившихся патогенов доступность Zn2+. Уровень Zn2+ в сыворотке крови представля- ет собой микромолярные количества, металл часто хелатирован белками трансферрином, альбумином или α2-макро гло булином [54, 132, 134, 173]. В ходе инфекции организм млекопи- тающего снижает концентрацию Zn2+ на си- стемном уровне и локально, пытаясь огра- ничить доступность этого необходимого для патогенов микроэлемента [39, 54, 69, 78, 101, 132, 134, 152, 173]. Поэтому микроорганизмам 272 Молекулярные мишени для профилактики чумы 2021, Т. 11, № 2 индуцировать продукцию IFNγ у мышей, вак- цинированных Y. pestis EV76 методом ELispot. Авторы обнаружили, что 34 белка обладают способностью стимулировать T-клеточное звено иммунитета. Протективная эффектив- ность 24 из них была предварительно оцене- на на модели бубонной чумы у мышей линии BALB/c. Кроме известного LcrV внутримышеч- ная иммунизация девятью белками (YPO0606, YPO1914, YPO0612, YPO3119, YPO3047, YPO1377, YPCD1.05c, YPO0420 и YPO3720) предоставля- ла частичную защиту от гибели при заражении малыми дозами Y. pestis (20 × LD50), но только YPO0606 был способен частично защитить мы- шей при заражении высокой дозой Y. pestis (200 × LD50). По мнению авторов, данный белок может быть рекомендован в качестве компонента при конструировании субъединичной вакцины. и провели скрининг на модели легочной чумы у мышей. На основании гибридизации входной и выходной ДНК из мутантных пулов с 53 уни- кальными сигнатурными тегами авторы отобра- ли 118 клонов, неспособных к распространению в селезенку при заражающей дозе, эквивалент- ной 5 × LD50 штамма Y. pestis CO92. Остаточная вирулентность 20 из выявленных мутантов была снижена и на модели бубонной чумы у мышей при подкожном введении 8 × LD50, причем за- ражение десятью из них привело к 40%-ной или более высокой выживаемости при инфицирую- щей дозе 40 × LD50. При проведении фрагмент- ного секвенирования авторы обнаружили, что шесть аттенуированных мутантов несут нару- шения в генах, кодирующих гипотетические белки или белки с предполагае мыми функция- ми. Произвольный мутагенез индивидуально меченными транспозонами Используя высокопроизводительный мута- генез индивидуально меченными транспозона- ми, Ponnusamy D. и соавт. [127] создали библио- теку из 5088 мутантов штамма Y. pestis CO92 273 Е.А. Красильникова и др. Инфекция и иммунитет но ΔlppΔypo0815, ΔlppΔypo2884, ΔlppΔcyoABCDE, ΔvasKΔhcp6 и Δypo2720-2733Δhcp3. На модели ле- гочной чумы мыши, пережившие заражение му- тантами ΔlppΔcyoABCDE, ΔvasKΔhcp6 и Δypo2720- 2733Δhcp3, были на 55–100% защищены от гибели при последующем введении штаммов «дикого» типа CO92. Кроме того, оценка in vitro аттенуи- рованных штаммов с мутациями в генах T6SS выявила значительные изменения в фагоцитозе, внутриклеточной выживаемости в макрофагах мышей и способности оказывать цитотоксичес- кие эффекты на макрофаги. Представленные в работе результаты служат дополнительным доказательством целесо образности STM-скри- нинга для идентификации новых факторов патогенности. Еще одной привлекательной мишенью яв- ляется активатор плазминогена Pla [31]. Бак- териальные протеазы — важные факторы патогенности, инактивирующие защитные белки хозяина и способствующие разруше- нию тканей и распространению бактерий. Протеазы наружной мембраны семейства ом- птинов, примером которых является акти- ватор плазминогена Pla Y. pestis, обнаружены в некоторых грамотрицательных бактериях. Омптины расщепляют множество субстратов на границе раздела «хозяин–патоген», вклю- чая плазминоген и антимикробные пептиды. Было выявлено несколько субстратов омпти- на, имеющих отношение к инфекции; тем не менее эффективный ингибитор омптинина еще предстоит найти. К настоящему моменту показано, что ингибитор сериновой протеа- зы апротинин действует как конкурентный ингибитор активности Pla и препятствует расщеплению мышиного кателицидин-род- ственного антимикробного пептида, причем активность проявляется в микромолярном диапазоне. Понимание механизмов, которые бактерия использует для адаптации к организму мле- копитающего при температуре 37°С, является центральным звеном при разработке вакцин и лекарственных препаратов для профилак- тики или лечения чумы. Используя библио- теку, содержащую около 1 млн случайных Tn5-мутантов штамма Y. pestis CO92, для сек- венирования сайтов встраивания транспозо- на, Senior N.J. и соавт. [143] идентифицировали 530 генов, необходимых бактерии для роста при температуре 28°С, и 19 генов — при температу- ре 37°С, но не 28°С, в том числе принимаю- щих участие в функционировании системы секреции III типа, в репликации ДНК и т. д. Комбинированный компьютерно-эксперимен- тальный подход позволил выявить 54 гена, про- дукты которых необходимы для роста микроба при температуре тела млекопитающего и могут быть потенциальными мишенями для разра- ботки препаратов для профилактики и лече- ния чумы. Y. pestis обладает системой активного погло- щения железа на основе сидерофоров — секре- тируемых, хелатирующих железо соединений с чрезвычайно высоким сродством к нему [52]. Произвольный мутагенез индивидуально меченными транспозонами Сидерофоры играют основную роль в усвоении бактериями железа в организме человека, где концентрация свободного железа значительно ниже той, которая требуется для роста бактерий и проявления вирулентности. Последнее делает биосинтез сидерофора привлекательной ми- шенью при разработке новых антимикробных препаратов. р р Известно, что метилирование аденина в ДНК регулирует многочисленные метабо- лические процессы в бактериях, а изменение экспрессии ДНК-аденин-метилтрансферазы (Dam) аттенуирует целый ряд патогенов, вклю- чая Y. pestis. Отсутствие же у человека функ- ционально схожего фермента делает Dam подходящей молекулярной мишенью для разработки новых терапевтических средств против чумы [106]. Кандидатные препара- ты оценивали на их способность ингибиро- вать активность Dam в скрининговом анали- зе. Идентифицировали серию арилстибоно- вых кислот, которые ингибируют Dam in vitro. Наиболее активная 4-стибонбензолсульфо- кислота демонстрирует конкурентный способ ингибирования в отношении ДНК и Ki, рав- ный 6,46 нМ. Обработка бактериальных кле- точных культур этими препаратами приводила к снижению метилирования ДНК. Высказано предположение о том, что эти ингибиторы мо- гут ослаблять бактериальную инфекционность и со временем заменить антибиотики. Заключение Аналогичная ситуация складывается и с вы- бором генов-мишеней для аттенуации вирулент- ных штаммов чумного микроба. Продолжается поиск мишеней, нокаут которых не только обес- печивает отсутствие остаточной вирулентнос- ти, но и сохраняет высокую иммуногенность штамма для всех видов лабораторных живот- ных, используемых для оценки протективности чумных вакцин. В последние три десятилетия достигнут зна- чительный прогресс в понимании патогенеза чумной инфекции. Но несмотря на значитель- ное количество публикаций, посвященных оценке возможности использования вновь вы- являемых факторов патогенности в качестве молекулярных мишеней для совершенство- вания вакцинопрофилактики и/или терапии чумы, «идеальная» вакцина или «идеальное» антибактериальное средство пока еще не соз- даны. Наибольшие усилия исследователей при создании чумных субъединичных вакцин скон- центрированы на разработке препаратов, со- держащих два основных иммунодоминантных антигена Y. pestis, Cаf1 и LcrV, выявленных еще до середины прошлого века [51, 131, 150, 162]. Что же касается других антигенов/факторов патогенности чумного микроба, то все не так однозначно. В целом ряде случаев различными группами были получены противоречивые дан- ные о значимости отдельных факторов пато- генности в пато- и иммуногенезе чумы [41], что может быть связано с использованием разных пород/линий лабораторных животных [172]. Однако целый ряд исследователей считает, что сосредотачиваться исключительно на Cаf1 и LcrV как на единственных протективных белках, подходящих для создания кандидат- ных чумных вакцин, было бы недальновидным по причине обнаружения структурного и се- рологического полиморфизма LcrV Y. pestis [13] и Caf1 [84]. Определенное беспокойство вызы- вают и высоковирулентные природные штам- мы возбудителя чумы, лишенные способности продуцировать Cаf1 и благодаря этому преодо- левающие иммунитет, индуцированный вак- цинами на основе данного антигена [150, 162]. Кроме того, несмотря на то что субъединичные вакцины на основе Cаf1 и LcrV обеспечива- ют выраженный протективный эффект на не- скольких животных моделях, данные белки не являются доминантными антигенами, сти- мулирующими Т-клеточное звено иммунитета для продолжительной защиты человека. В не- скольких исследованиях продемонстрировано, что введение живых аттенуированных штаммов Y. pestis праймирует CD4+ и CD8+ T-лимфоциты, что помогает макроорганизму распознать набор антигенов чумного микроба, отличных от Cаf1 и LcrV, которые стимулируют гуморальное зве- но иммунитета [97, 126]. Следовательно, долж- ны существовать другие белки, имеющие зна- чение для стимуляции клеточного иммунного ответа. Найти такие антигены поможет исполь- зование омиксного подхода, включающего ге- номику, транскриптомику и метаболомику. Каждый из рассмотренных в обзоре канди- датов на роль факторов патогенности чумного микроба обладает и достоинствами, и недостат- ками. Использование молекулярных мишеней для терапии чумы Появление штаммов Y. pestis, устойчивых к антибиотикам, обусловливает необходи- мость разработки альтернативных антимик- робных препаратов, эффективных против чумного микроба. В последнее время популяр- ным стало исследование «ингибиторов виру- лентности», или «генов домашнего хозяйства». Наибольшее количество работ связано с си- стемой секреции III типа, обеспечивающей доставку в цитоплазму клеток иммунной си- стемы хозяина ряда цитотоксинов. В качестве молекулярной мишени чаще всего исследуют тирозинфосфатазу YopH, которая парализует лимфоциты и макрофаги, дефосфорилируя критические тирозинкиназы и молекулы сиг- нальной трансдукции [91, 111, 160]. 274 2021, Т. 11, № 2 Молекулярные мишени для профилактики чумы Заключение В настоящее время уже не оспаривается тот факт, что живые аттенуированные штаммы патогенных микроорганизмов, по сравнению с химическими и субъединичными вакцина- ми, способны стимулировать гораздо более эффективный иммунитет, по напряженнос- ти приближающийся к постинфекционному и защищающий от заражения различными по антигенному составу вариантами патоге- на. Однако ревакцинацию живыми чумными вакцинами можно проводить не ранее чем че- рез 12 месяцев [3]. В более ранние сроки сохра- няется достаточно напряженный иммунитет, не позволяющий клеткам вакцинного штамма размножиться и персистировать в иммунизи- руемом организме ограниченное время, доста- точное для ревакцинации. Очевидно, что для экстренной ревакцинации в ранние сроки по- сле вакцинации живой вакциной оптимальной является схема иммунизации, предложенная Дальвадянцем С.М. [2], включающая иммуни- зацию живой вакциной, а затем ревакцинацию субъединичной. Поиски оптимального способа аттенуации при конструировании вакцинного штамма или определение антигенного/эпитопного и адъю- вантного составов молекулярной вакцины против чумы, а также способов ее презента- ции продолжаются. На наш взгляд, наиболее перспективными мишенями для иммунопро- филактики и иммунотерапии чумы являются поверхностно расположенные белки внешней мембраны и/или адгезины Y. pestis, а в качестве мишеней для антимикробной терапии лучше выбирать факторы нутриционной вирулент- ности чумного микроба, что позволит повысить эффективность разнонаправленного бактери- цидного и/или бактериостатического действия иммунобиологических и/или лекарственных препаратов. Работа выполнена в рамках отраслевой научно- исследовательской программы Роспотреб надзора на 2016–2020 гг. «Проблемно-ориентированные научные исследования в области эпидемиологичес- кого надзора за инфекционными и паразитарными болезнями». 275 Е.А. Красильникова и др. Инфекция и иммунитет Список литературы/References 1. Анисимов А.П. Факторы Yersinia pestis, обеспечивающие циркуляцию и сохранение возбудителя чумы в экоси- стемах природных очагов. Сообщение 2 // Молекулярная генетика, микробиология и вирусология. 2002. № 3. С. 3–23. [Anisimov A.P. Yersinia pestis factors ensuring circulation and persistence of the plague pathogen in ecosystems of natu- ral foci. Communication 2. Molekulyarnaya genetika, mikrobiologiya i virusologiya = Molecular Genetics, Microbiology and Virology, 2002, no. 3, pp. 3–23. (In Russ.)] 2. Дальвадянц С.М., Дятлов И.А., Еремин С.А., Щуковская Т.Н., Саяпина Л.В., Сергеева Г.М., Кутырев В.В. Исследования по иммунизации против чумы. Сообщение 4. Опыт ревакцинации волонтеров «химической» и живой чумной вакцинами // Проблемы особо опасных инфекций. 2006. № 91. C. 57–61 [Dalvadyants S.M., Dyatlov I.A., Yeremin S.A., Schukovskaya T.N., Sayapina L.V., Sergheyeva G.M., Kutyrev V.V. Plague immunization studies. Communication 4. An expe- rience of volunteer revaccination with the “chemical” and live plague vaccines. Problemy osobo opasnykh infektsiy = Problems of Particularly Dangerous Infections, 2006, vol. 91, pp. 57–61. (In Russ.)] icularly Dangerous Infections, 2006, vol. 91, pp. 57–61. (In R 3. Наумов А.В., Ледванов М.Ю., Дроздов И.Г. Иммунология чумы. Саратов, 1992. 172 с. [Naumov A.V., Ledvanov M.Yu., Drozdov I.G. Immunology of plague. Saratov, 1992. 172 p. (In Russ.)] p ( ) 4. Achouak W., Heulin T., Pagès J.M. Multiple facets of bacterial porins. Protein Sci., 2001, vol. 199, no. 1, 7 p. doi: 10.1111/j.1574- 6968.2001.tb10642.x 5. Achtman M., Zurth K., Morelli G., Torrea G., Guiyoule A., Carniel E. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc. Natl. Acad. Sci. USA, 1999, vol. 96, pp. 14043–14048. doi: 10.1073/pnas.96.24.14043 6. Ackermann N., Tiller M., Anding G., Roggenkamp A., Heesemann J. Contribution of trimeric autotransporter C-terminal do- mains of oligomeric coiled-coil adhesin (Oca) family members YadA, UspA1, EibA, and Hia to translocation of the YadA pas- senger domain and virulence of Yersinia enterocolitica. J. Bacteriol., 2008, vol. 190, pp. 5031–5043. doi: 10.1128/JB.00161-08 7. Aliprantis A.O., Yang R.B., Mark M.R., Suggett S., Devaux B., Radolf J.D., Klimpel G.R., Godowski P., Zychlinsky A. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science, 1999, vol. 285, pp. 736–739. doi: 10.1126/ science.285.5428.736 8. Anderson E.S., Paulley J.T., Gaines J.M., Valderas M.W., Martin D.W., Menscher E., Brown T.D., Burns C.S., Roop R.M. The manganese transporter MntH is a critical virulence determinant for Brucella abortus 2308 in experimentally infected mice. Infect. Immun., 2009, vol. 77, pp. 3466–3474. doi: 10.1128/IAI.00444-09 9. Список литературы/References Andersson J.A., Sha J., Erova T.E., Fitts E.C., Ponnusamy D., Kozlova E.V., Kirtley M.L., Chopra A.K. Identification of new virulence factors and vaccine candidates for Yersinia pestis. Front. Cell. Infect. Microbiol., 2017, vol. 7: 448. doi: 10.3389/ fcimb.2017.00448 f 10. Andrews G.P., Strachan S.T., Benner G.E. Protective efficacy of recombinant Yersinia outer proteins against bubonic plague caused by encapsulated and non-encapsulated Yersinia pestis. Infect. Immun., 1999, vol. 67, pp. 1533–1537. 11. Anisimov A.P. Factors providing the blocking activity of Yersinia pestis. Mol. Gen. Mikrobiol. Virusol., 1999, vol. 4, pp. 11–15. 12. Anisimov A.P., Amoako K.K. Treatment of plague: promising alternatives to antibiotics. J. Med. Microbiol., 2006, vol. 55, pp. 1461–1475. doi: 10.1099/jmm.0.46697-0 p g g y p , , , pp 12. Anisimov A.P., Amoako K.K. Treatment of plague: promising alternatives to antibiotics. J. Med. Microbiol., 2006, vol. 55, pp. 1461–1475. doi: 10.1099/jmm.0.46697-0 13. Anisimov A.P., Dentovskaya S.V., Panfertsev E.A., Svetoch T.E., Kopylov P.Kh., Segelke B.W., Zemla A., Telepnev M.V., Motin V.L. Amino acid and structural variability of Yersinia pestis LcrV protein. Infect. Genet. Evol., 2010, vol. 10, pp. 137–145. doi: 10.1016/j.meegid.2009.10.003 /j g 14. Anisimov A.P., Lindler L.E., Pier G.B. Intraspecific diversity of Yersinia pestis. Clin. Microbiol. Rev., 2004, vol. 17, no. 2, pp. 434– 464. doi: 10.1128/CMR.17.2.434-464.2004 15. Arirachakaran P., Benjavongkulchai E., Luengpailin S., Ajdić D., Banas J.A. Manganese affects Streptococcus mutans virulence gene expression. Caries Res., 2007, vol. 41 (6), pp. 503–11. doi: 10.1159/000110883 16. Auerbuch V., Loureiro J.J., Gertler F.B., Theriot J.A., Portnoy D.A. Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility. Mol. Microbiol., 2003, vol. 49, no. 5, pp. 1361–1375. 17. Bartra S.S., Gong X., Lorica C.D., Jain C., Nair M.K., Schifferli D., Qian L., Li Z., Plano G.V., Schesser K. The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microb. Pathog., 2012, vol. 52, no. 1, pp. 41–46. doi: 10.1016/j.micpath.2011.09.009 18. Bartra S.S., Styer K.L., O’Bryant D.M., Nilles M.L., Hinnebusch B.J., Aballay A., Plano G.V. Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein. Infect. Immun., 2008, vol. 76, pp. 612–622. doi: 10.1128/IAI.01125-07 / 19. Bear J.E., Gertler F.B. Ena/VASP: towards resolving a pointed controversy at the barbed end. J. Cell Sci., 2009, vol. 122, pp. 1947– 1953. doi: 10.1242/jcs.038125 /j 20. Bearden S.W., Staggs T.M., Perry R.D. 5. Bierne H., Miki H., Innocenti M., Scita G., Gertler F.B., Takenawa T. WASP-related proteins, Abi1 and Ena/VA for Listeria invasion induced by the Met receptor. J. Cell Sci., 2005, vol. 118, pp. 1537–1547. doi: 10.1242/jcs.0228 Список литературы/References An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11. J. Bacteriol., 1998, vol. 180, pp. 1135–1147. doi: 10.1128/JB.180.5.1135-1147.1998 21. Bearden S.W., Perry R.D. The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague. Mol. Microbiol., 1999, vol. 32, pp. 403–414. doi: 10.1046/j.1365-2958.1999.01360.x 22. Behrens S., Kneip S. The role of SurA factor in outer membrane protein transport and virulence. Int. J. Me vol. 300, pp. 421–428. doi: 10.1016/j.ijmm.2010.04.012 23. Behrens S., Maier R., de Cock H., Schmid F.X., Gross C.A. The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO J., 2001, vol. 15, pp. 285–294. doi: 10.1093/emboj/20.1.285 p y , , , pp / j/ 24. Berry A.M., Paton J.C. Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumonia. Infect. Immun., 1996, vol. 64, pp. 5255–5262. doi: 10.1128/IAI.64.12.5255-5262.1996. 25. Bierne H., Miki H., Innocenti M., Scita G., Gertler F.B., Takenawa T. WASP-related proteins, Abi1 and Ena/VASP are required for Listeria invasion induced by the Met receptor. J. Cell Sci., 2005, vol. 118, pp. 1537–1547. doi: 10.1242/jcs.02285 276 2021, Т. 11, № 2 Молекулярные мишени для профилактики чумы 26. Blaylock B., Berube B.J., Schneewind O. YopR impacts type III needle polymerization in Yersinia species. Mol. Microbiol., 2010, vol. 75, pp. 221–229. doi: 10.1111/j.1365-2958.2009.06988.x 27. Bobrov A.G., Kirillina O., Fetherston J.D., Miller M.C., Burlison J.A., Perry R.D. The Yersinia pestis siderophore, yersiniabac- tin, and the ZnuABC system both contribute to Zinc acquisition and the development of lethal septicemic plague in mice. Mol. Microbiol., 2014, vol. 93, pp. 759–775. doi: 10.1111/mmi.12693 , , , pp / 28. Bobrov A.G., Kirillina O., Fosso M.Y., Fetherston J.D., Miller M.C., van Cleave T.T. Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice. Metallomics, 2017, vol. 21, pp. 757–772. doi: 10.1039/c7mt00126f 29. Bobrov A.G., Kirillina O., Ryjenkov D.A., Waters C.M., Price P.A., Fetherston J.D. Systematic analysis of cyclic di-GMP signal- ling enzymes and their role in biofilm formation and virulence in Yersinia pestis. Mol. Microbiol., 2011, vol. 79, no. 2, pp. 533–551. doi: 10.1111/j.1365-2958.2010.07470.x 30. Boyer E., Bergevin I., Malo D., Gros P., Cellier M.F.M. Acquisition of Mn(II) in addition to Fe(II) is requir of Salmonella enteric serovar Typhimurium. Infect. Immun., 2002, vol. 70, pp. 6032–6042. Список литературы/References doi: 10.1128/IAI.70 of Salmonella enteric serovar Typhimurium. Infect. Immun., 2002, vol. 70, pp. 6032 6042. doi: 10.1128/IAI.70.11.6032 6042.2002 31. Brannon J.R., Burk D.L., Leclerc J.M., Thomassin J.L., Portt A., Berghuis A.M., Gruenheid S., Le Moual H. Inhibition of out- er membrane proteases of the omptin family by aprotinin. Infect. Immun., 2015, vol. 83, no. 6, pp. 2300–2311. doi: 10.1128/ IAI.00136-15 32. Brubaker R.R. Factors promoting acute and chronic diseases caused by Yersinia. Clin. Microbiol. Rev., 1991, vol. 4, pp. 309–324. doi: 10.1128/CMR.4.3.309 / 33. Burrows T.W. Virulence of Pasteurella pestis. Nature, 1957, vol. 179, pp. 1246–1247. / 33. Burrows T.W. Virulence of Pasteurella pestis. Nature, 1957, vol. 179, pp. 1246–1247. Burrows T.W. Virulence of Pasteurella pestis. Nature, 1957, 34. Busby S., Ebright R.H. Transcription activation by catabolite activator protein (CAP). J. Mol. Biol., 1999, vol. 2 doi: 10.1006/jmbi.1999.3161 35. Butler T., Fu Y.S., Furman L., Almeida C., Almeida A. Experimental Yersinia pestis infection in rodents after intragastric inocu- lation and ingestion of bacteria. Infect. Immun., 1982, vol. 36, pp. 1160–1167. g f , , , pp 36. Cavanaugh D.C., Randall R. The role of multiplication of Pasteurella pestis in mononuclear phagocytes in the pathogenesis of fleaborne plague. J. Immunol., 1959, vol. 83, pp. 348–371. 37. Charnetzky W.T., Shuford W.W. Survival and growth of Yersinia pestis within macrophages and an effect of the loss of the 47-megadalton plasmid on growth in macrophages. Infect. Immun., 1985, vol. 47, pp. 234–241. 38. Chen Y., Duan R., Li X., Li K., Liang J., Liu C., Qiu H., Xiao Y., Jing H., Wang X. Homology analysis and cross-immunogenicity of OmpA from pathogenic Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. Mol. Immunol., 2015, vol. 68, pp. 290–299. doi: 10.1016/j.molimm.2015.09.016 pp /j 39. Corbin B.D., Seeley E.H., Raab A., Feldmann J., Miller M.R., Torres V.J. Metal chelation and inhibition of bacterial growth in tissue abscesses. Science, 2008, vol. 319, pp. 962–965. doi: 10.1126/science.1152449 40. Delcour A. Functon and modulation of bacterial porins: insight from electrophysiology. FEMS Microbiol. Lett pp. 115–125. doi: 10.3390/ijms20030674 pp / j 41. Dentovskaia S.V., Kopylov P.Kh., Ivanov S.A., Ageev S.A., Anisimov A.P. A molecular basis of the plague vaccine development. Mol. Gen. Mikrobiol. Virusol., 2013, vol. 3, pp. 3–12. 42. Dentovskaya S.V., Ivanov S.A., Kopylov P.Kh., Shaikhutdinova R.Z., Platonov M.E., Kombarova T.I., Gapelchenkova T.V., Balakhonov S.V., Anisimov A.P. Selective protective potency of Yersinia pestis ΔnlpD mutants. Acta Naturae, 2015, vol. 7, no. Список литературы/References 1, pp. 102–108. pp 43. Derbise A., Pierre F., Merchez M. Inheritance of the lysozyme inhibitor Ivy was an important evolutionary step by Yersinia pestis to avoid the host innate immune response. J. Infect. Dis., 2013, vol. 207, no. 10, pp. 1535–1543. doi: 10.1093/infdis/jit057 44 Di ilh A All i G G d l C Cl J P C d i l f S i Ad d P A 44. Dintilhac A., Alloing G., Granadel C., Claverys J.P. Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases. Mol. Microbiol., 1997, vol. 25, pp. 727–739. doi: 10.1046/j.1365-2958.1997.5111879.x , pp /j 45. Dukuzumuremyi J.M., Rosqvist R., Hallberg B., Akerstrom B., Wolf-Watz H., Schesse K. The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor. J Biol. Chem., 2000, vol. 275, no. 45, pp. 35281–35290. doi: 10.1074/jbc. M003009200 46. Erova T.E., Rosenzweig J.A., Sha J., Suarez G., Sierra J.C., Kirtley M.L., van Lier C.J., Telepnev M.V., Motin V.L., Chopra A.K. Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine. Clin. Vaccine Immunol., 2013, vol. 20, no. 2, pp. 227–238. doi: 10.1128/CVI.00597-12 47. Felek S., Jeong J.J., Runco L.M., Murray S., Thanassi D.G., Krukonis E.S. Contributions of chaperone/u binding, biofilm formation and Yersinia pestis virulence. Microbiology, 2011, vol. 157, pp. 805–818. doi: 10.10 48. Felek S., Krukonis E.S. The Yersinia pestis Ail protein mediates binding and Yop delivery to host cells required for plague viru- lence. Infect. Immun., 2009, vol.77, pp. 825–836. doi: 10.1128/IAI.00913-08 f , , , pp / 49. Felek S., Lawrenz M.B., Krukonis E.S. The Yersinia pestis autotransporter YapC mediates host cell binding, autoaggregation and biofilm formation. Microbiology, 2008, vol. 154, pp. 1802–1812. doi: 10.1099/mic.0.2007/010918-0 gy pp / / 50. Felek S., Tsang T.M., Krukonis E.S. Three Yersinia pestis adhesins facilitate Yop delivery to eukaryotic cells and contribute to plague virulence. Infect. Immun., 2010, vol. 78, pp. 4134–4150. doi: 10.1128/IAI.00167-10 51. Feodorova V.A., Corbel M.J. Prospects for new plague vaccines. Expert Rev. Vaccines, 2009, vol. 8, pp. 1721–173 erv.09.129 52. Ferreras J.A., Ryu J.S., Di Lello F., Tan D.S., Quadri L.E. Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis. Nat. Chem. Biol., 2005, vol. 1, no. 1, pp. 29–32. doi: 10.1038/nchembio706 p pp / 53. 54. Foote J.W., Delves H.T. Albumin bound and a2-macroglobulin bound zinc concentrations in the sera of health Pathol., 1984, vol. 37, pp. 1050–1054. doi: 10.1136/jcp.37.9.1050 p , , , , pp / 53. Fong J.C., Yildiz F.H. Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling rae biofilm formation. J. Bacteriol., 2008, vol. 190, pp. 6646–6659. doi: 10.1128/JB.00466-08 Список литературы/References Med. Biol., 2003, vol. 529, pp. 55–62. doi: 10.1007/0-306-48416-1_11 68. Hinnebusch B.J., Jarrett C.O., Callison J.A., Gardner D., Buchanan S.K., Plano G.V. Role of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte response during bubonic plague. Infect. Immun., 2011, vol. 79, pp. 4984– 4989. doi: 10.1128/IAI.05307-11 69. Hood M.I., Skaar E.P. Nutritional immunity: transition metals at the pathogen-host interface. Nat. Rev. Micro pp. 525–537. doi: 10.1038/nrmicro2836 70. Ichikawa J.K., Li C., Fu J., Clarke S. A gene at 59 minutes on the Escherichia coli chromosome encodes a lipoprotein with unusual amino acid repeat sequences. J. Bacteriol., 1994, vol. 176, pp. 1630–1638. doi: 10.1128/jb.176.6.1630-1638.1994 p q , , , pp /j 71. Jacob A., Hensley L.K., Safratowich B.D., Quigg R.J., Alexander J.J. The role of the complement cascade in endotoxin-induced septic encephalopathy. Lab. Invest., 2007, vol. 87, pp. 1186–1194. doi: 10.1038/labinvest.3700686 72. Janssen W.A., Surgalla M.J. Plague bacillus: survival within host phagocytes. Science, 1969, vol. 163, no. 3870, pp. 950–952. doi: 10.1126/science.163.3870.950 73. Janulczyk R., Ricci S., Björck L. MtsABC is important for manganese and iron transport, oxidative stress re lence of Streptococcus pyogenes. Infect. Immun., 2003, vol. 71, pp. 2656–2664. doi: 10.1128/IAI.71.5.2656-26 73. Janulczyk R., Ricci S., Björck L. MtsABC is important for manganese and iron transport, oxidative stress resistance, and viru- lence of Streptococcus pyogenes. Infect. Immun., 2003, vol. 71, pp. 2656–2664. doi: 10.1128/IAI.71.5.2656-2664.2003 74. Jarrett C.O., Deak E., Isherwood K.E., Oyston P.C., Fischer E.R., Whitney A.R. Transmission of Yersinia pestis from an infec- 74. Jarrett C.O., Deak E., Isherwood K.E., Oyston P.C., Fischer E.R., Whitney A.R. Transmission of Yersinia p tious biofilm in the flea vector. J. Infect. Dis., 2004, vol. 190, no. 4, pp. 783–792. doi: 10.1086/422695 f , , , , pp / 75. Jarrett C.O., Sebbane F., Adamovicz J.J., Andrews G.P., Hinnebusch B.J. Flea-borne transmission model to evaluate vaccine efficacy against naturally acquired bubonic plague. Infect. Immun., 2004, vol. 72, no. 4, pp. 2052–2056. doi: 10.1128/iai.72.4.2052-2056.2004 76. Juris S.J., Rudolph A.E., Huddler D., Orth K., Dixon J.E. A distinctive role for the Yersinia protein kinase: actin binding, kinase activation, and cytoskeleton disruption. Proc. Natl. Acad. Sci., 2000, vol. 97, no. 17, pp. 9431–9436. doi: 10.1073/pnas.170281997 77. Kalivoda E.J., Stella N.A., O’Dee D.M., Nau G.J., Shanks R.M. The cyclic AMP-dependent catabolite repression system of Serratia marcescens mediates biofilm formation through regulation of type 1 fimbriae. Appl. Environ. Microbiol., 2008, vol. 74, pp. 3461–3470. Список литературы/References Fong J.C., Yildiz F.H. Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio chole- rae biofilm formation. J. Bacteriol., 2008, vol. 190, pp. 6646–6659. doi: 10.1128/JB.00466-08 54. Foote J.W., Delves H.T. Albumin bound and a2-macroglobulin bound zinc concentrations in the sera of healthy adults. J. Clin. Pathol., 1984, vol. 37, pp. 1050–1054. doi: 10.1136/jcp.37.9.1050 54. Foote J.W., Delves H.T. Albumin bound and a2-macroglobulin bound zinc concentrations in the sera of healthy adults. J. Clin. Pathol., 1984, vol. 37, pp. 1050–1054. doi: 10.1136/jcp.37.9.1050 277 Е.А. Красильникова и др. Инфекция и иммунитет 55. Forman S., Wulff C.R., Myers-Morales T., Cowan C., Perry R.D., Straley S.C. yadBC of Yersinia pestis, a new virulence deter- minant for bubonic plague. Infect. Immun., 2008, vol. 76, pp. 578–587. doi: 10.1128/IAI.00219-07 56. Fu H., Belaaouaj A.A., Dahlgren C., Bylund J. Outer membrane protein A-deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides. Microbes Infect., 2003, vol. 5, pp. 781–788. doi: 10.1016/s1286-4579(03)00145-x / ( ) 57. Gage K.L., Kosoy M.Y. Natural history of plague: perspectives from more than a century of research. Annu. Rev. Entomol., 2005, vol. 50, pp. 505–528. doi: 10.1146/annurev.ento.50.071803.130337 58. Galindo C.L., Sha J., Moen S.T., Agar S.L., Kirtley M.L., Foltz S.M., McIver L.J., Kozlova E.V., Garner H.R., Chopra A.K. Comparative global gene expression profiles of wild-type Yersinia pestis CO92 and its braun lipoprotein mutant at flea and human body temperatures. Comp. Funct Genomics., 2010. doi: 10.1155/2010/342168 59. Graham A.I., Hunt S., Stokes S.L., Bramall N., Bunch J., Cox A.G. Severe zinc depletion of Escherichia coli: roles for high- affinity zinc binding by ZinT, zinc transport and zinc-independent proteins. J. Biol. Chem., 2009, vol. 284, pp. 18377–18389. doi: 10.1074/jbc.M109.001503 /j 60. Hakansson S., Galyov E.E., Rosqvist R., Wolf-Watz H. The Yersinia YpkA Ser/Thr kinase is translocated and subsequently target- ed to the inner surface of the HeLa cell plasma membrane. Mol. Microbiol., 1996, vol. 20, no. 3, pp. 593–603. doi: 10.1046/j.1365- 2958.1996.5251051.x T., Ishii Y., Danbara H., Okada N. Genome-wide identification of novel genomic islands that contribute to Salmon e in mouse systemic infection. FEMS Microbiol. Lett., 2009, vol. 297, pp. 241–249. doi: 10.1111/j.1574-6968.2009.0168 61. Haneda T., Ishii Y., Danbara H., Okada N. Genome-wide identification of novel genomic islands that contribute to Salmonella virulence in mouse systemic infection. FEMS Microbiol. Lett., 2009, vol. 297, pp. 241–249. doi: 10.1111/j.1574-6968.2009.01686.x 62. Hantke K. Список литературы/References Bacterial zinc uptake and regulators. Curr. Opin. Microbiol., 2005, vol. 8, pp. 196–202. doi: 10.1016/j.mib.2005.02.001 63. Hantke K., Braun V. Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the mureinlipoprotein of the Escherichia coli outer membrane. Eur. J. Biochem., 1973, vol. 34, pp. 284–296. virulence in mouse systemic infection. FEMS Microbiol. Lett., 2009, vol. 297, pp. 241 249. doi: 10.1111/j.1574 6968.2009.01686.x 62. Hantke K. Bacterial zinc uptake and regulators. Curr. Opin. Microbiol., 2005, vol. 8, pp. 196–202. doi: 10.1016/j.mib.2005.02.001 63 H k K B V C l bi di f li id i Di l id d id li k d f id h N i l virulence in mouse systemic infection. FEMS Microbiol. Lett., 2009, vol. 297, pp. 241 249. doi: 10.1111/j.1574 6968.2009.01686.x 62. Hantke K. Bacterial zinc uptake and regulators. Curr. Opin. Microbiol., 2005, vol. 8, pp. 196–202. doi: 10.1016/j.mib.2005.02.001 63. Hantke K., Braun V. Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the mureinlipoprotein of the Escherichia coli outer membrane. Eur. J. Biochem., 1973, vol. 34, pp. 284–296. doi: 10.1111/j.14321033.1973.tb02757.x rial zinc uptake and regulators. Curr. Opin. Microbiol., 200 62. Hantke K. Bacterial zinc uptake and regulators. Curr. Opin. Microbiol., 2005, vol. 8, pp. 196–202. doi: 10.1016/j.mib.2005.02.0 2. Hantke K. Bacterial zinc uptake and regulators. Curr. O 63. Hantke K., Braun V. Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the mureinlipoprotein of the Escherichia coli outer membrane. Eur. J. Biochem., 1973, vol. 34, pp. 284–296. doi: 10.1111/j.14321033.1973.tb02757.x /j 64. Hayashi S., Wu H.C. Lipoproteins in bacteria. J. Bioenerg. Biomembr., 1990, vol. 22, no. 3, pp. 451–471. doi: 10.1007/BF00763177 65. He J., Miyazaki H., Anaya C., Yu F., Yeudall W.A., Lewis J.P. Role of Porphyromonas gingivalis FeoB2 in metal uptake and oxi- dative stress protection. Infect. Immun., 2006, vol. 74, pp. 4214–4223. doi: 10.1128/IAI.00014-06 65. He J., Miyazaki H., Anaya C., Yu F., Yeudall W.A., Lewis J.P. Role of Porphyromonas gingivalis FeoB2 in metal uptake and oxi- dative stress protection. Infect. Immun., 2006, vol. 74, pp. 4214–4223. doi: 10.1128/IAI.00014-06 / 66. Hinnebusch B.J. Biofilm-dependent and biofilm-independent mechanisms of transmission of Yersinia pestis by fleas. Adv. Exp. Med. Biol., 2012, vol. 954, pp. 237–243. doi: 10.1007/978-1-4614-3561-7_30 67. Hinnebusch B.J. Transmission factors: Yersinia pestis genes required to infect the flea vector of plague. Adv. Exp. Список литературы/References Transcriptional profiling of a mice plague model: insights into interaction be nia pestis and its host. J. Basic Microbiol., 2009, vol. 49, no. 1, pp. 4992–4999. doi: 10.1002/jobm.200800027 99. Liu H., Wang H., Qiu J., Wang X., Guo Z., Qiu Y. Transcriptional profiling of a mice plague model: insights tween Yersinia pestis and its host. J. Basic Microbiol., 2009, vol. 49, no. 1, pp. 4992–4999. doi: 10.1002/jobm.2 100. Liu L., Fang H., Yang H., Zhang Y., Han Y., Zhou D., Yang R. CRP is an activator of Yersinia pestis biofilm formation that oper- ates via a mechanism involving gmhA and waaAE-coaD. Front. Microbiol., 2016, vol. 7. doi: 10.3389/fmicb.2016.00295 101. Liuzzi J.P., Lichten L.A., Rivera S., Blanchard R.K., Aydemir T.B., Knutson M.D. Interleukin-6 regulates the zinc transport- er Zip14 in liver and contributes to the hypozincemia of the acute-phase response. Proc. Natl. Acad. Sci. USA, 2005, vol. 102, pp. 6843–6848. doi: 10.1073/pnas.0502257102 pp /p 102. Llobet E., March C., Giménez P., Bengoechea J.A. Klebsiella pneumoniae OmpA confers resistance to antimicrobial peptides. Antimicrob. Agents Chemother., 2009, vol. 53, pp. 298–302. doi: 10.1128/AAC.00657-08 103. Lorange E.A., Race B.L., Sebbane F., Hinnebusch B.J. Poor vector competence of fleas and the evolution of hypervirulence in Yersinia pestis. J. Infect. Dis., 2005, vol. 191, no. 11, pp. 1907–1912. doi: 10.1086/429931 104. Lu X.H., An S.Q., Tang D.J., McCarthy Y., Tang J.L., Dowetal J.M. RsmA regulates biofilm formation in Xanthomonas cam- pestris through a regulatory network involving cyclicdi-GMP and the Clp transcription factor. PLoS One, 2012, vol. 7: e52646. doi: 10.1371/journal.pone.0052646 /j p 105. Marra A., Lawson S., Asundi J.S., Brigham D., Hromockyj A.E. In vivo characterization of the psa genes from Streptococcus pneumoniae in multiple models of infection. Microbiology, 2002, vol. 148, pp. 1483–1491. doi: 10.1099/00221287-148-5-1483 106. McKelvie J.C., Richards M., Harmer J.E., Milne T.S., Roach P.L., Oyston P.C. Inhibition of Yersinia pestis DNA adenine methyl- transferase in vitro by a stibonic acid compound: identification of a potential novel class of antimicrobial agents. Br. J. Pharmacol., 2013, vol. 168, no. 1, pp 172–188. doi: 10.1111/j.1476-5381.2012.02134.x , , , pp /j 107. Melching L., Vas S.I. Effects of serum components on gram-negative bacteria during bactericidal reactions. Infect. Immun., 1971, vol. 3, pp. 107–115. 108. Murphy B.S., Wulff C.R., Garvy B.A., Straley S.C. Yersinia pestis YadC: a novel vaccine candidate against pla Biol., 2007, vol. 603, pp. 400–414. doi: 10.1007/978-0-387-72124-8_37 / 109. Список литературы/References 9: e0162308. doi: 10.1371/journal.pone.0162308 85. Lange R., Hengge-Aronis R. The nlpD gene is located in an operon with rpoS on the Escherichia coli chromosome and encodes a novel lipoprotein with a potential function in cell wall formation. Mol. Microbiol., 1994, vol. 13, no. 4, pp. 733–743. doi: 10.1111/ j.1365-2958.1994.tb00466.x 84. Kopylov P.Kh., Platonov M.E., Ablamunits V.G., Kombarova T.I., Ivanov S.A., Kadnikova L.A., Somov A.N., Dentovskaya S.V., Uversky V.N., Anisimov A.P. Yersinia pestis Caf1 protein: effect of sequence polymorphism on intrinsic disorder propensity, sero- logical cross-reactivity and cross-protectivity of isoforms. PLoS One, 2016, vol. 11, no. 9: e0162308. doi: 10.1371/journal.pone.0162308 85. Lange R., Hengge-Aronis R. The nlpD gene is located in an operon with rpoS on the Escherichia coli chromosome and encodes a novel lipoprotein with a potential function in cell wall formation. Mol. Microbiol., 1994, vol. 13, no. 4, pp. 733–743. doi: 10.1111/ j.1365-2958.1994.tb00466.x 86. Lathem W.W., Price P.A., Miller V.L., Goldman W.E. A plasminogen-activating protease specifically controls t of primary pneumonic plague. Science, 2007, vol. 15, pp. 509–513. doi: 10.1126/science.1137195 87. Lathem W.W., Schroeder J.A., Bellows L.E., Ritzert J.T., Koo J.T., Price P.A. Posttranscriptional regulation of the Yersinia pestis cyclic AMP receptor protein Crp and impacton virulence. M. Bio., 2014, vol. 5: e01038-13. doi: 10.1128/mBio.01038-13 88. Lawrenz M.B., Lenz J.D., Miller V.L. A novel autotransporter adhesin is required for ef plague. Infect. Immun., 2009, vol. 77, pp. 317–326. doi: 10.1128/IAI.01206-08 88. Lawrenz M.B., Lenz J.D., Miller V.L. A novel autotransporter adhesin is required for efficient colonization during bubonic plague. Infect. Immun., 2009, vol. 77, pp. 317–326. doi: 10.1128/IAI.01206-08 89. Lawrenz M.B., Pennington J., Miller V.L. Acquisition of OmpT in reveals cryptic virulence function of autotransporter YapE in Yersinia pestis. Mol. Microbiol., 2013, vol. 89, pp. 276–287. doi: 10.1111/mmi.12273 90. Lazar S.W., Kolter R. SurA assists the folding of Escherichia coli outer membrane proteins. J. Bacteriol., 1996, vol. 178, pp. 1770– 1773. doi: 10.1128/jb.178.6.1770-1773.1996 90. Lazar S.W., Kolter R. SurA assists the folding of Escherichia coli outer membrane proteins. J. Bacteriol., 1996, vol. 178, pp. 1770– 1773. doi: 10.1128/jb.178.6.1770-1773.1996 91 Lee K Gao Y Yao Z J Phan J Wu L Liang J Waugh D S Zhang Z Y Burke T R Jr Tripeptide inhibitors of Yersinia pro 91. Lee K., Gao Y., Yao Z.J., Phan J., Wu L., Liang J., Waugh D.S., Zhang Z.Y., Burke T.R. Jr. Tripeptide inhibitors tein-tyrosine phosphatase. Bioorg. Med. Chem. Lett., 2003, vol. 13, no. Список литературы/References doi: 10.1128/AEM.02733-07 78. Kehl-Fie T.E., Skaar E.P. Nutritional immunity beyond iron: a role for manganese and zinc. Curr. Opin. Chem. Biol., 2010, vol. 14, pp. 218–224. doi: 10.1016/j.cbpa.2009.11.008 79. Kehres D.G., Janakiraman A., Slauch J.M., Maguire M.E. SitABCD is the alkaline Mn2+ transporter of Salmonella enterica serovar Typhimurium. J. Bacteriol., 2002, vol. 184, pp. 3159–3166. doi: 10.1128/JB.184.12.3159-3166.2002 80 Kim T J Chauhan S Motin V L Goh E B Igo M M Young G M Direct transcriptional control of the plasminogen activator 79. Kehres D.G., Janakiraman A., Slauch J.M., Maguire M.E. SitABCD is the alkaline Mn2+ transporter of Salmonella enterica serovar Typhimurium. J. Bacteriol., 2002, vol. 184, pp. 3159–3166. doi: 10.1128/JB.184.12.3159-3166.2002 80. Kim T.J., Chauhan S., Motin V.L., Goh E.B., Igo M.M., Young G.M. Direct transcriptional control of the plasminogen activator gene of Yersinia pestis by the cyclic AMP receptor protein J Bacteriol 2007 vol 189 pp 8890 8900 doi: 10 1128/JB 00972 07 serovar Typhimurium. J. Bacteriol., 2002, vol. 184, pp. 3159–3166. doi: 10.1128/JB.184.12.3159-3166.2002 80. Kim T.J., Chauhan S., Motin V.L., Goh E.B., Igo M.M., Young G.M. Direct transcriptional control of the plasminogen activator gene of Yersinia pestis by the cyclic AMP receptor protein. J. Bacteriol., 2007, vol. 189, pp. 8890–8900. doi: 10.1128/JB.00972-07 81. Kim W.K., Jang P.G., Woo M.S., Han I.O., Piao H.Z., Lee K., Lee H., Joh T.H., Kim H.S. A new anti-inflammatory agent KL-1037 represses proinflammatory cytokine and inducible nitric oxide synthase (iNOS) gene expression in activated microglia. Neuropharmacology, 2004, vol. 47, no. 2, pp. 243–252. doi: 10.1016/j.neuropharm.2004.03.019 82. Kolodziejek A.M., Schnider D.R., Rohde H.N., Wojtowicz A.J., Bohach G.A., Minnich S.A., Hovde C.J. Outer membrane pro- tein X (Ail) contributes to Yersinia pestis virulence in pneumonic plague and its activity is dependent on the lipopolysaccharide core length. Infect. Immun., 2010, vol. 78, pp. 5233–5243. doi: 10.1128/IAI.00783-10 83. Kolodziejek A.M., Sinclair D.J., Seo K.S., Schnider D.R., Deobald C.F., Rohde H.N., Viall A.K., Minnich S.S., Hovde C.J., Minnich S.A., Bohach G.A. Phenotypic characterization of OmpX, an Ail homologue of Yersinia pestis KIM. Microbiology, 2007, vol. 153, pp. 2941–2951. doi: 10.1099/mic.0.2006/005694-0 278 Молекулярные мишени для профилактики чумы 2021, Т. 11, № 2 84. Kopylov P.Kh., Platonov M.E., Ablamunits V.G., Kombarova T.I., Ivanov S.A., Kadnikova L.A., Somov A.N., Dentovskaya S.V., Uversky V.N., Anisimov A.P. Yersinia pestis Caf1 protein: effect of sequence polymorphism on intrinsic disorder propensity, sero- logical cross-reactivity and cross-protectivity of isoforms. PLoS One, 2016, vol. 11, no. Список литературы/References 15, pp. 2577–2581. doi: 10.1016/S0960-89 92. Lee V.T., Schneewind O. Type III machines of pathogenic yersiniae secrete virulence factors into the extracellular milieu. Mol. Microbiol., 1999, vol. 31, pp. 1619–1629. doi: 10.1046/j.1365-2958.1999.01270.x 93. Lenz J.D., Temple B.R., Miller V.L. Evolution and virulence contributions of the autotransporter proteins YapJ and YapK of Yersinia pestis CO92 and their homologs in Y. pseudotuberculosis IP32953. Infect. Immun., 2012, vol. 80, pp. 3693–3705. doi: 10.1128/IAI.00529-12 / 94. Letzelter M., Sorg I., Mota L.J., Meyer S., Stalder J., Feldman M. The discovery of SycO highlights a new function for type III secretion effector chaperones. EMBO J., 2006, vol. 25, no. 13, pp. 3223–3233. doi: 10.1038/sj.emboj.7601202 94. Letzelter M., Sorg I., Mota L.J., Meyer S., Stalder J., Feldman M. The discovery of SycO highlights a new function for type III secretion effector chaperones. EMBO J., 2006, vol. 25, no. 13, pp. 3223–3233. doi: 10.1038/sj.emboj.7601202 95. Li B., Guo J., Wang X., Ni B., Ke Y., Zhu Z., Guo Z., Yang R. High-throughput identification of new protective antigens f Y i i i li i b li k d i I f I 2009 l 77 10 4356 4361 95. Li B., Guo J., Wang X., Ni B., Ke Y., Zhu Z., Guo Z., Yang R. High-throughput identification of new protective antigens from a Yersinia pestis live vaccine by enzyme-linked immunospot assay. Infect. Immun., 2009, vol. 77, no. 10, pp. 4356–4361. doi: 10.1128/IAI.00242-09 96. Lim K.H.L., Jones C.E., vanden Hoven R.N., Edwards J.L., Falsetta M.L., Apicella M.A., Jennings M.P., McEwan A.G. Metal binding specificity of the MntABC permease of Neisseria gonorrhoeae and its influence on bacterial growth and interaction with cervical epithelial cells. Infect. Immun., 2008, vol. 76, pp. 3569–3576. doi: 10.1128/IAI.01725-07 p f , , , pp / 97. Lin J.S., Szaba F.M., Kummer L.W., Chromy B.A., Smiley S.T. Yersinia pestis YopE contains a dominant CD8 T cell epitope that confers protection in a mouse model of pneumonic plague. J. Immunol., 2011, vol. 187, no. 2, pp. 897–904. doi: 10.4049/jim- munol.1100174 98. Liu F., Chen H., Galván E.M., Lasaro M.A., Schifferli D.M. Effects of Psa and F1 on the adhesive and invasive interactions of Yersinia pestis with human respiratory tract epithelial cells. Infect Immun., 2006, vol. 74, pp. 5636–5644. doi: 10.1128/ IAI.00612-06 ng H., Qiu J., Wang X., Guo Z., Qiu Y. Список литературы/References Navarro L., Koller A., Nordfelth R., Wolf-Watz H., Taylor S., Dixon J.E. Identification of a molecular target for the Yersinia pro- tein kinase A. Mol. Cell., 2007, vol. 26, pp. 465–477. doi: 10.1016/j.molcel.2007.04.025 , , , pp /j 110. Neilsen P.O., Zimmerman G.A., McIntyre T.M. Escherichia coli Braun lipoprotein induces a lipopolysaccharide-like endotoxic response fromprimary human endothelial cells. J. Immunol., 2001, vol. 167, pp. 5231–5239. doi: 10.4049/jimmunol.167.9.5231 111. Nordfelth R., Kauppi A.M., Norberg H.A., Wolf-Watz H., Elofsson M. Small-molecule inhibitors specifically targeting type III secretion. Infect. Immun., 2005, vol. 73, no. 5, pp. 3104–3114. doi: 10.1128/IAI.73.5.3104-3114.2005 / 110. Neilsen P.O., Zimmerman G.A., McIntyre T.M. Escherichia coli Braun lipoprotein induces a lipopolysaccharide-like endotoxic response fromprimary human endothelial cells. J. Immunol., 2001, vol. 167, pp. 5231–5239. doi: 10.4049/jimmunol.167.9.5231 111. Nordfelth R., Kauppi A.M., Norberg H.A., Wolf-Watz H., Elofsson M. Small-molecule inhibitors specifically targeting type III secretion. Infect. Immun., 2005, vol. 73, no. 5, pp. 3104–3114. doi: 10.1128/IAI.73.5.3104-3114.2005 279 Е.А. Красильникова и др. Инфекция и иммунитет 112. Obi I.R., Francis M.S. Demarcating SurA activities required for outer membrane targeting of Yersinia pseudotuberculosis adhe- sions. Infect. Immun., 2013, vol. 81, pp. 2296–2308. doi: 10.1128/IAI.01208-12 f , , , pp / 113. Obi I.R., Nordfelth R., Francis M.S. Varying dependency of periplasmic peptidylprolyl cis-transisomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity. Biochem. J., 2011, vol. 439, pp. 321–332. doi: 10.1042/BJ20110767 114. Oh M.H., Lee S.M., Lee D.H., Choi S.H. Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence. Infect. Immun., 2009, vol. 77, pp. 1208–1215. doi: 10.1128/IAI.01006-08 15. Ouyang Z., He M., Oman T., Yang X.F., Norgard M.V. A manganese transporter, BB0219 (BmtA), is required for virule disease spirochete, Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA, 2009, vol. 106, pp. 3449–3454. doi: 10.1073/pnas.0 116. Paik S., Brown A., Munro C.L., Cornelissen C.N., Kitten T. The sloABCR operon of Streptococcus mutans encodes an Mn and Fe transport system required for endocarditis virulence and its Mn-dependent repressor. J. Bacteriol., 2003, vol. 185, pp. 5967– 5975. doi: 10.1128/JB.185.20.5967-5975.2003 / 117. Park H., Teja K., O’Shea J.J., Siegel R.M. The Yersinia effector protein YpkA induces apoptosis independen merization. J. Immunol., 2007, vol. 178, no. 10, pp. 6426–6434. doi: 10.4049/jimmunol.178.10.6426 118. Park J.S., Lee E.J., Lee J.C., Kim W.K., Kim H.S. Список литературы/References 64, pp. 146–153. doi: 10.1128/IAI.64.1.146-153.1996 129. Prehna G., Ivanov M.I., Bliska J.B., Stebbins C.E. Yersinia virulence depends on mimicry of host Rho-family nucleotide disso- ciation inhibitors. Cell, 2006, vol. 126, no. 5, pp. 869–880. doi: 10.1016/j.cell.2006.06.056 /j 130. Pujol C., Grabenstein J.P., Perry R.D., Bliska J.B. Replication of Yersinia pestis in interferon-(-activated macrophages requires ripA, a gene encoded in the pigmentation locus. PNAS, 2005, vol. 102, no. 36, pp. 12909–12914. doi: 10.1073/pnas.0502849102 131 Quenee L E Schneewind O Plague vaccines and the molecular basis of immunity against Yersinia pestis Hum Vaccin 2009 / 130. Pujol C., Grabenstein J.P., Perry R.D., Bliska J.B. Replication of Yersinia pestis in interferon-(-activated macrophages requires ripA, a gene encoded in the pigmentation locus. PNAS, 2005, vol. 102, no. 36, pp. 12909–12914. doi: 10.1073/pnas.0502849102 131. Quenee L.E., Schneewind O. Plague vaccines and the molecular basis of immunity against Yersinia pestis. Hum. Vaccin., 2009, vol. 5, pp. 817–823. doi: 10.4161/hv.9866 p , g p g , , , , pp /p 131. Quenee L.E., Schneewind O. Plague vaccines and the molecular basis of immunity against Yersinia pestis. Hum. Vaccin., 2009, vol. 5, pp. 817–823. doi: 10.4161/hv.9866 132. Rahuel-Claremont S., Dunn M.F. The biological chemistry of zinc. In: Rainsford K.D., Milanino R., Sorenson R.J., Velo G.P. Copper and zinc in inflammatory and degenerative diseases. Kluwer Academic Publisher, 1998, pp. 47–59. 133. Rickman L., Scott C., Hunt D.M., Hutchinson T., Menendez M.C., Whalan R. A member of the cAMP receptor protein family of transcription regulatorsin Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. Mol. Microbiol., 2005, vol. 56, pp. 1274–1286. doi: 10.1111/j.1365-2958.2005.04609.x 134. Rink L., Haase H. Zinc homeostasis and immunity. Trends Immunol., 2007, vol. 28, pp. 1–4. doi: 10.1016/j.it. / 135. Roediger W.E. Nitric oxide-dependent nitrosation of cellular CoA: a proposal for tissue responses. Nitric. Oxide, 2001, vol. 5, no. 2, pp. 83–87. doi: 10.1006/niox.2001.0336 136. Roediger W.E., Babidge W.J. Nitric oxide effect on colonocyte metabolism: co-action of sulfides and peroxide. Mol. Cell Biochem., 2000, vol. 206, no. 1–2, pp. 159–167. doi: 10.1023/a:1007034417320 137. Roggenkamp A., Ackermann N., Jacobi C.A., Truelzsch K., Hoffmann H., Heesemann J. Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA. J. Bacteriol., 2003, vol. 185, pp. 3735–3744. doi: 10.1128/ JB.185.13.3735-3744.2003 138. Rouviere P.E., Gross C.A. Список литературы/References Anti-inflammatory effects of short chain fatty acids in IFN-gamma-stimulated RAW 264.7 murine macrophage cells: involvement of NF-kappaB and ERK signaling pathways. Int. Immunopharmacol., 2007, vol. 7, pp. 70–77. doi: 10.1016/j.intimp.2006.08.015 n B.W., Thomson N.R. Genome sequence of Yersinia pestis, the causative agent of plague. Nature, 2001, vol. 413, i: 10.1038/35097083 119. Parkhill J., Wren B.W., Thomson N.R. Genome sequence of Yersinia pestis, the causative agent of plague. Na pp. 523–527. doi: 10.1038/35097083 pp / 120. Perry R.D., Abney J., Mier I. Jr., Lee Y., Bearden S.W., Fetherston J.D. Regulation of the Yersinia pestis Yfe and Ybt iron transport systems. Adv. Exp. Med. Biol., 2003, vol. 529, pp. 275–283. doi: 10.1007/0-306-48416-1_53 121. Perry R.D., Craig S.K., Abney J., Bobrov A.G., Kirillina O., Mier I. Jr., Truszczynska H., Fetherston J.D. Manganese transporters Yfe and MntH are Fur-regulated and important for the virulence of Yersinia pestis. Fur. Microbiol., 2012, vol. 158, pp. 804–815. doi: 10.1099/mic.0.053710-0 / 122. Perry R.D., Fetherston J.D. Yersinia pestis — etiologic agent of plague. Clin. Microbiol. Rev., 1997, vol. 10, no. 1, pp. 35–66. doi: 10.1128/CMR.10.1.35 123. Perry R.D., Shah J., Bearden S.W., Thompson J.M., Fetherston J.D. Yersinia pestis TonB: role in iron, heme, and hemoprotein utilization. Infect. Immun., 2003, vol. 71, no. 7, pp. 4159–4162. doi: 10.1128/iai.71.7.4159-4162.2003 124. Petersen S., Young G.M. Essential role for cyclic AMP and its receptor protein in Yersinia enterocolitica virulence. Infect. Immun., 2002, vol. 70, pp. 3665–3672. doi: 10.1128/IAI.70.7.3665-3672.2002 125. Petrarca P., Ammendola S., Pasquali P., Battistoni A. The Zur-regulated ZinT protein is an auxiliary component of the high- affinity ZnuABC zinc transporter that facilitates metal recruitment during severe zinc shortage. J. Bacteriol., 2010, vol. 192, pp. 1553–1564. doi: 10.1128/JB.01310-09 126. Philipovskiy A.V., Smiley S.T. Vaccination with live Yersinia pestis primes CD4 and CD8 T cells that synergistically protect against lethal pulmonary Y. pestis infection. Infect. Immun., 2007, vol. 75, no. 2, pp. 878–885. doi:10.1128/IAI.01529-06 127. Ponnusamy D., Fitts E.C., Sha J., Erova T.E., Kozlova E.V., Kirtley M.L. High-throughput, signature-tagged mutagenic ap- proach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection. Infect. Immun., 2015, vol. 83, pp. 2065–2081. doi: 10.1128/IAI.02913-14 28. Prasadarao N.V., Wass C.A., Weiser J.N., Stins M.F., Huang S.H., Kim K.S Outer membrane protein A of Escherichia to invasion of brain microvascular endothelial cells. Infect. Immun., 1996, vol. 64, pp. 146–153. doi: 10.1128/IAI.64.1.1 to invasion of brain microvascular endothelial cells. Infect. Immun., 1996, vol. Список литературы/References SurA, a periplasmic protein with peptidyl-prolylisomerase activity, participates in the assembly of outer membrane porins. Genes Dev., 1996, vol. 10, pp. 3170–3182. doi: 10.1101/gad.10.24.3170 39. Runco L.M., Myrczek S., Bliska J.B., Thanassi D.G. Biogenesis of the fraction 1 capsule and analysis of the of Yersinia pestis. J. Bacteriol., 2008, vol. 190, pp. 3381–3385. doi: 10.1128/JB.01840-07 140. Salazar J.K., Wu Z., Yang W., Freitag N.E., Tortorello M.L., Wang H. Roles of a novel Crp/Fnr family transcription factor Lmo0753 in soil survival, biofilm production and surface attachment to fresh produce of Listeria monocytogenes. PLoS One, 2013, vol. 8: e75736. doi: 10.1371/journal.pone.0075736 280 2021, Т. 11, № 2 Молекулярные мишени для профилактики чумы 141. Santiviago C.A., Reynolds M.M., Porwollik S., Choi S.H., Long F. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice. PLoS Pathog., 2009, vol. 5: e1000477. doi: 10.1371/ journal.ppat.1000477 D., Cherry S., Austin B.P., Tropea J.E., Waugh D.S. Crystal structure of the protease-resistant core domain of Yersinia ence factor YopR. Protein Sci., 2005, vol. 14, pp. 1679–1683. doi: 10.1110/ps.051446405 142. Schubot F.D., Cherry S., Austin B.P., Tropea J.E., Waugh D.S. Crystal structure of the protease-resistant core pestis virulence factor YopR. Protein Sci., 2005, vol. 14, pp. 1679–1683. doi: 10.1110/ps.051446405 143. Senior N.J., Sasidharan K., Saint R.J., Scott A.E., Sarkar-Tyson M., Ireland P.M., Bullifent H.L., Rong Yang Z., Moore K., Oyston P.C.F., Atkins T.P., Atkins H.S., Soyer O.S., Titball R.W. An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions. BMC Microbiol., 2017, vol. 17, no. 1: 163. doi: 10.1186/s12866-017-1073-8 144. Seo K.S., Kim J.W., Park J.Y., Viall A.K., Minnich S.S., Rohde H.N. Role of a new intimin/invasin-like protein in Yersinia pestis virulence. Infect. Immun., 2012, vol. 80, pp. 3559–3569. doi: 10.1128/IAI.00294-12 145. Sha J., Agar L.S., Baze W.B., Olano J.P., Fad A.A., Erova T.E., Wang S., Foltz S.M., Suarez G., Motin V.L., Chauhan S., Klimpel G.R., Peterson J.W., Chopra A.K. Braun lipoprotein (Lpp) contributes to virulence of Yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague. Infect. Immun., 2008, vol. 76, no. 4, pp. 1390–1409. doi: 10.1128/IAI.01529-07 146. Sha J., Kirtley L.M., van Lier C.J., Wang S., Erova T.E., Kozlova E.V., Cao A., Cong Y., Fitts E.C., Rosenzweig J.A., Chopra A.K. Deletion of the braun lipoprotein-encoding gene and altering the function of lipopolysaccharide attenuate the plague bacterium. Infect. Список литературы/References Sun Y.C., Guo X.P., Hinnebusch B.J., Darby C. The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT. J. Bacteriol., 2012, vol. 194, no. 8, pp. 2020–2026. doi: 10.1128/JB.06243-11 157. Sun Y.C., Hinnebusch B.J., Darby C. Experimental evidence for negative selection in the evolution of Yersinia pestis pseudogene. Proc. Nat. Acad. Sci., 2008, vol. 105, no. 23, pp. 8097–8101. doi: 10.1073/pnas.0803525105 155. Sugawara E., Nikaido H. Pore forming activity of OmpA protein of Escherichia coli. J. Biol. Chem., 1992, vol. 267, pp. 2507 2511. 156. Sun Y.C., Guo X.P., Hinnebusch B.J., Darby C. The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT. J. Bacteriol., 2012, vol. 194, no. 8, pp. 2020–2026. doi: 10.1128/JB.06243-11 157 S Y C Hi b h B J D b C E i l id f i l i i h l i f Y i i i d 156. Sun Y.C., Guo X.P., Hinnebusch B.J., Darby C. The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT. J. Bacteriol., 2012, vol. 194, no. 8, pp. 2020–2026. doi: 10.1128/JB.06243-11 157. Sun Y.C., Hinnebusch B.J., Darby C. Experimental evidence for negative selection in the evolution of Yersinia pestis pseudogene. Proc. Nat. Acad. Sci., 2008, vol. 105, no. 23, pp. 8097–8101. doi: 10.1073/pnas.0803525105 157. Sun Y.C., Hinnebusch B.J., Darby C. Experimental evidence for negative selection in the evolution of Yersinia pestis pseudogene. Proc. Nat. Acad. Sci., 2008, vol. 105, no. 23, pp. 8097–8101. doi: 10.1073/pnas.0803525105 157. Sun Y.C., Hinnebusch B.J., Darby C. Experimental evidence for negative selection in the evol Proc. Nat. Acad. Sci., 2008, vol. 105, no. 23, pp. 8097–8101. doi: 10.1073/pnas.0803525105 / 158. Sun Y.C., Jarrett C.O., Bosio C.F., Hinnebusch B.J. Retracing the evolutionary path that led to flea-borne transmission of Yersinia pestis. Cell Host Microbe, 2014, vol. 15, no. 5, pp. 578–586. doi: 10.1016/j.chom.2014.04.003 159. Suzuki H., Nishimura Y., Yasuda S., Nishimura A., Yamada M., Hirota Y. Murein-lipoprotein of Escherichi involved in the stabilization of bacterial cell envelope. Mol. Gen. Genet., 1978, vol. 167, no. 1, pp. 1–9. doi: 10.100 160. Tautz L., Bruckner S., Sareth S., Alonso A., Bogetz J., Bottini N., Pellecchia M., Mustelin T. Inhibition of Yersinia tyrosine phosphatase by furanyl salicylate compounds. J. Biol. Chem., 2005, vol. 280, no. 10, pp. 9400–9408. doi: 10.1074/jbc.M413122200 161. Список литературы/References Immun., 2013, vol. 81, pp. 815–828. doi: 10.1128/IAI.01067-12 147. Shi L., Adkins J.N., Coleman J.R., Schepmoes A.A., Dohnkova A. Proteomic analysis of Salmonella enterica serovar typhimu- rium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar typhi- murium inside macrophages. J. Biol. Chem., 2006, vol. 281, pp. 29131–29140. doi: 10.1074/jbc.M604640200 p g , , , pp /j 148. Simonet M., Riot B., Fortineau N., Berche P. Invasin production by Yersinia pestis is abolished by insertion of an IS200-like ele- ment within the inv gene. Infect. Immun, 1996, vol. 64, pp. 375–379. 149. Skorupski K., Taylor R.K. Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin-coregulated pilusin Vibrio cholerae. Proc. Natl. Acad. Sci., 1997, vol. 94, pp. 265–270. doi: 10.1073/pnas.94.1.265 150 S il S T I d f i t i l I l R 2008 l 225 256 271 d i 10 1111/j 1600 149. Skorupski K., Taylor R.K. Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin-coregulated pilusin Vibrio cholerae. Proc. Natl. Acad. Sci., 1997, vol. 94, pp. 265–270. doi: 10.1073/pnas.94.1.265 150. Smiley S.T. Immune defense against pneumonic plague. Immunol. Rev., 2008, vol. 225, pp. 256–271. doi: 10.1111/j.1600- 065X.2008.00674.x 150. Smiley S.T. Immune defense against pneumonic plague. Immunol. Rev., 2008, vol. 225, pp. 256–271. 065X.2008.00674.x 151. Smith A.J., Ward P.N., Field T.R., Jones C.L., Lincoln R.A., Leigh J.A. MtuA, a lipoprotein receptor antigen from Streptococcus uberis, is responsible for acquisition of manganese during growth in milk and is essential for infection of the lactating bovine mammary gland. Infect. Immun., 2003, vol. 71, pp. 4842–4849. doi: 10.1128/IAI.71.9.4842-4849.2003 152. Sohnle P.G., Hunter Michael J., Hahn B., Chazin Walter J. Zinc reversible antimicrobial activity of recom (migration inhibitory factor-related proteins 8 and 14). J. Infect. Dis., 2000, vol. 182, pp. 1272–1275. doi: 10.1 153. Southern S.J., Scott A.E., Jenner D.C., Ireland P.M., NorvilleI H., Sarkar-Tyson M. Survival protein A is essential for virulence in Yersinia pestis. Microb. Pathog., 2016, vol. 92, pp. 50–53. doi: 10.1016/j.micpath.2015.12.013 154. Straley S.C., Harmon P.A. Growth in mouse peritoneal macrophages of Yersinia pestis lacking established virulence determi- nants. Infect. Immun., 1984, vol. 45, no. 3, pp. 649–654. 155. Sugawara E., Nikaido H. Pore-forming activity of OmpA protein of Escherichia coli. J. Biol. Chem., 1992, vol. 267, pp. 2507–2511. 156. 168. Trasak C., Zenner G., Vogel A., Yuksekdag G., Rost R., Haase I. Yersinia protein kinase YopO is activated by a novel G-actin binding process. J. Biol. Chem., 2007, vol. 282, no. 4, pp. 2268–2277. doi: 10.1074/jbc.M610071200 Список литературы/References Tidhar A., Flashner Y., Cohen S., Levi Y., Zauberman A., Gur D., Aftallon M., Elhanany E., Zvi A., Shafferman A., Mamroud E. The NlpD lipoprotein is a novel Yersinia pestis virulence factor essential for the development of plague. PLoS One, 2009, vol. 4: e7023. doi: 10.1371/journal.pone.0007023 /j p 162. Titball R.W., Williamson E.D. Yersinia pestis (plague) vaccines. Expert. Opin. Biol. Ther., 2004, vol. 4, pp. 965–973. doi: 10.1517/14712598.4.6.965 163. Tormo A., Almiron M., Kolter R. SurA, an Escherichia coli gene essential for survival in stationary phase. J. Bacteriol., 1990, vol. 172, pp. 4339–4347. doi: 10.1128/jb.172.8.4339-4347.1990 pp /j 164. Torres R., Chim N., Sankaran B., Pujol C., Bliska J., Gouldinga C.W. Structural insights into RipC, a putative citrate lyase b subunit from a Yersinia pestis virulence operon. Acta. Cryst., 2012, vol. 68, pp. 2–7. doi: 10.1107/S1744309111048056 165. Torres R., Lan B., Latif Y., Chima N., Goulding C.W. Structural snapshots along the reaction pathway of Yersi a putative butyryl-CoA transferase. Acta. Cryst., 2014, vol. 70, pp. 1074–1085. doi: 10.1107/S1399004714000911 166. Torres R., Swift R.V., Chim N., Wheatley N., Lan B., Atwood B.R., Pujol C., Sankaran B., Bliska J.B., Amaro R.E., Goulding C.W. Biochemical, structural and molecular dynamics analyses of the potential virulence factor RipA from Yersinia pestis. PLoS One, 2011, vol. 6, iss. 9: e25084. doi: 10.1371/journal.pone.0025084 167. Torres-Escobar A., Juarez-Rodriguez M.D., Lamont R.J., Demuth D.R. Transcriptional regulation of Aggregatibacter actino- mycetemcomitans lsrACDBFG and lsrRK operons and their role in biofilm formation. J. Bacteriol., 2013, vol. 195, pp. 56–65. doi: 10.1128/JB.01476-12 168. Trasak C., Zenner G., Vogel A., Yuksekdag G., Rost R., Haase I. Yersinia protein kinase YopO is activated by a novel G-actin binding process. J. Biol. Chem., 2007, vol. 282, no. 4, pp. 2268–2277. doi: 10.1074/jbc.M610071200 281 Е.А. Красильникова и др. Инфекция и иммунитет 169. Tsang T.M., Felek S., Krukonis E.S. Ail binding to fibronectin facilitates Yersinia pestis binding to host cells and Yop delivery. Infect. Immun., 2010, vol. 78, pp. 3358–3368. doi: 10.1128/IAI.00238-10 170. Van Lier С.J., Tiner B.L., Chauhan S., Motin V.L., Fitts E.C., Huante M.B., Endsley J.J., Ponnusamy D., Sha J., Chopra A.K. Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages. Microb. Pathog., 2015, vol. 80, pp. 27–38. doi: 10.1016/j.micpath.2015.02.005 171. Wang S., Joshi S., Mboudjeka I., Liu F., Ling T., Goguen J.D., Lu S. Список литературы/References Relative immunogenicity and protection potential of can- didate Yersinia pestis antigens against lethal mucosal plague challenge in Balb/C mice. Vaccine, 2008, vol. 26, pp. 1664–1674. doi: 10.1016/j.vaccine.2008.01.024 /j 172. Weening E.H., Cathelyn J.S., Kaufman G., Lawrenz M.B., Price P., Goldman W.E., Miller V.L. The dependence of the Yersinia pestis capsule on pathogenesis is influenced by the mouse background. Infect. Immun., 2011, vol. 79, no. 2, pp. 644–652. doi: 10.1128/IAI.00981-10 / 173. Weinberg E.D. Infectious diseases influenced by trace element environment. Ann. NY Acad. Sci., 1972, vol. 199, pp. 274–284. doi: 10.1111/j.1749-6632.1972.tb54344.x 174. Wiley D.J., Nordfeldth R., Rosenzweig J., DaFonseca C.J., Gustin R., Wolf-Watz H. The Ser/Thr kinase activity of the Yersinia protein kinase A (YpkA) is necessary for full virulence in the mouse, mollifying phagocytes, and disrupting the eukaryotic cy- toskeleton. Microb. Pathog., 2006, vol. 40, no. 5, pp. 234–243. doi: 10.1016/j.micpath.2006.02.001 175. Willias S.P., Chauhan S., Lo C.C., Chain P.S., Motin V.L. CRP-mediated carbon catabolite regulation of Yersinia pestis biofilm formation is enhanced by the carbon storage regulator protein, CsrA. PLoS One, 2015, vol. 10: e0135481. doi: 10.1371/journal. pone.0135481 176. Yen Y.T., Karkal A., Bhattacharya M., Fernandez R.C., Stathopoulos C. Identification and characterization of autotransporter proteins of Yersinia pestis KIM. Mol. Membr. Biol., 2007, vol. 24, pp. 28–40. doi: 10.1080/09687860600927626 177. Zhan L., Han Y., Yang L., Geng J., Li Y., Gao H. The cyclic AMP receptor protein, CRP, is required for both virulence and expres- sion of the minimal CRP regulon in Yersinia pestis biovar Microtus. Infect. Immun., 2008, vol. 76, pp. 5028–5037. doi: 10.1128/ IAI.00370-08 178. Zhan L., Yang L., Zhou L., Li Y., Gao H., Guo Z. Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis. BMC Microbiol., 2009, vol. 9: 178. doi: 10.1186/1471-2180-9-178 179. Zhang H.J., Peterson J.W., Niesel D.W., Klimpel G.R. Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production. J. Immunol., 1997, vol. 159, pp. 4868–4878. 180. Zhang Y., Wang L., Han Y., Yan Y., Tan Y., Zhou L. Autoregulation of PhoP/PhoQ and positive regulation of the tor protein-cyclic AMP complex by PhoP in Yersinia pestis. J. Bacteriol., 2013, vol. 195, pp. 1022–1030. doi: 10 180. Zhang Y., Wang L., Han Y., Yan Y., Tan Y., Zhou L. Autoregulation of PhoP/PhoQ and positive regulation of the cyclic AMP recep- tor protein-cyclic AMP complex by PhoP in Yersinia pestis. J. Поступила в редакцию 19.07.2019 Отправлена на доработку 11.11.2019 Принята к печати 26.11.2019 Список литературы/References Bacteriol., 2013, vol. 195, pp. 1022–1030. doi: 10.1128/JB.01530-12 181. Zvi A., Rotem Sh., Zauberman A., Elia U., Aftalion M., Bar-Haim E., Mamroud E., Cohen O. Novel CTL epitopes identified through a Y. pestis proteome-wide analysis in the search for vaccine candidates against plague. Vaccine, 2017, vol. 35, no. 44, pp. 5995–6006. doi: 10.1016/j.vaccine.2017.05.092 181. Zvi A., Rotem Sh., Zauberman A., Elia U., Aftalion M., Bar-Haim E., Mamroud E., Cohen O. Novel CTL epitopes identified through a Y. pestis proteome-wide analysis in the search for vaccine candidates against plague. Vaccine, 2017, vol. 35, no. 44, pp. 5995–6006. doi: 10.1016/j.vaccine.2017.05.092 Авторы: Авторы: Krasil’nikova E.A., Junior Researcher, Laboratory for Plague Microbiology, Department of Infectious Diseases, State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russian Federation; Красильникова Е.А., младший научный сотрудник лаборатории микробиологии чумы отдела особо опасных инфекций ГНЦ прикладной микробиологии и биотехнологии, Красильникова Е.А., младший научный сотрудник лаборатории микробиологии чумы отдела особо опасных инфекций ГНЦ прикладной микробиологии и биотехнологии, Krasil’nikova E.A., Junior Researcher, Laboratory for Plague Microbiology, Department of Infectious Diseases, State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russian Federation; п. Оболенск, Московская область, Россия; Трунякова А.С., стажер-исследователь лаборатории микробиологии чумы отдела особо опасных инфекций ГНЦ прикладной микробиологии и биотехнологии, п. Оболенск, Московская область Россия; Вагайская А.С., младший научный сотрудник лаборатории микробиологии чумы отдела особо опасных инфекций ГНЦ прикладной микробиологии и биотехнологии, п. Оболенск, Московская область, Россия; Светоч Т.Э., научный сотрудник лаборатории микробиологии чумы отдела особо опасных инфекций ГНЦ прикладной микробиологии и биотехнологии, п. Оболенск, Московская область, Россия; Shaikhutdinova R.Z., Senior Researcher, Laboratory for Plague Microbiology, Department of Infectious Diseases, State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russian Federation; Шайхутдинова Р.З., старший научный сотрудник лаборатории микробиологии чумы отдела особо опасных инфекций ГНЦ прикладной микробиологии и биотехнологии, п. Оболенск, Московская область, Россия; Дентовская С.В., д.м.н., главный научный сотрудник лаборатории микробиологии чумы отдела особо опасных инфекций ГНЦ прикладной микробиологии и биотехнологии, п. Оболенск, Московская область, Россия. Dentovskaya S.V., PhD, MD (Medicine), Head Researcher, Laboratory for Plague Microbiology, Department of Infectious Diseases, State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russian Federation. Поступила в редакцию 19.07.2019 Отправлена на доработку 11.11.2019 Принята к печати 26.11.2019 Received 19.07.2019 Revision received 11.11.2019 Accepted 26.11.2019 282
https://openalex.org/W4294832850
https://www.frontiersin.org/articles/10.3389/fcteg.2022.953768/pdf
English
null
Control of dead-time process: From the Smith predictor to general multi-input multi-output dead-time compensators
Frontiers in control engineering
2,022
cc-by
14,024
TYPE Review PUBLISHED 06 September 2022 DOI 10.3389/fcteg.2022.953768 TYPE Review PUBLISHED 06 September 2022 DOI 10.3389/fcteg.2022.953768 TYPE Review PUBLISHED 06 September 2022 DOI 10.3389/fcteg.2022.953768 OPEN ACCESS This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS time delay, MIMO processes, dead-time compensators, robustness, discrete-time models KEYWORDS time delay, MIMO processes, dead-time compensators, robustness, discrete-time models CITATION Normey-Rico JE, Santos TLM, Flesch RCC and Torrico BC (2022), Control of dead-time process: From the Smith predictor to general multi-input multi-output dead-time compensators. Front. Control. Eng. 3:953768. doi: 10.3389/fcteg.2022.953768 OPEN ACCESS OPEN ACCESS EDITED BY Carlos Renato Vazquez, Monterrey Institute of Technology and Higher Education (ITESM), Mexico REVIEWED BY Stefan Palis, International University of Applied Sciences, Germany Adrián Navarro Díaz, Monterrey Institute of Technology and Higher Education (ITESM), Mexico *CORRESPONDENCE Julio E. Normey-Rico, julio.normey@ufsc.br SPECIALTY SECTION This article was submitted to Control and Automation Systems, a section of the journal Frontiers in Control Engineering RECEIVED 26 May 2022 ACCEPTED 18 July 2022 PUBLISHED 06 September 2022 CITATION Normey-Rico JE, Santos TLM, Flesch RCC and Torrico BC (2022), Control of dead-time process: From the Smith predictor to general multi-input multi-output dead-time compensators. Front. Control. Eng. 3:953768. doi: 10.3389/fcteg.2022.953768 EDITED BY Carlos Renato Vazquez, Monterrey Institute of Technology and Higher Education (ITESM), Mexico Julio E. Normey-Rico1*, Tito L. M. Santos2, Rodolfo C. C. Flesch1 and Bismark C. Torrico3 1Departamento de Automação e Sistemas (DAS), Universidade Federal de Santa Catarina, Florianópolis, Brazil, 2Departamento de Engenharia Elétrica e de Computação (DEEC), Universidade Federal da Bahia, Salvador, Brazil, 3Departamento de Engenharia Elétrica (DEE), Universidade Federal do Ceará, Fortaleza, Brazil This review paper deals with the analysis, design, and tuning of dead-time compensators for stable and unstable multi-input multi-output (MIMO) processes with multiple time delays. It is well known that, even in the single-input single-output case, processes with significant dead times are difficult to control using standard feedback controllers. For MIMO systems, the study of processes with dead time is more involved, particularly when the process behavior exhibits different dead times in the different input-output relationships. Because of this, much research has been conducted in the last 50 years on this subject, with different approaches and proposals of controllers for covering a variety of objectives. Thus, this paper gives an overview of this important topic, focusing on the solutions derived from the Smith Predictor. First, a historical perspective of the different controllers proposed in the literature is presented. Then, the general solution of the problem is developed, paying particular attention to robustness and disturbance rejection properties, because of their importance and usefulness in industrial processes. All the development is done in the discrete-time case, which allows direct digital implementation. Two simulation case studies are presented to illustrate some of the ideas discussed in the paper, and an experimental case study is used to discuss aspects of practical implementation. © 2022 Normey-Rico, Santos, Flesch and Torrico. © 2022 Normey-Rico, Santos, Flesch and Torrico. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 2 Historical perspective—from the original idea of Smith to the general multi-input multi-output-dead-time compensators solutions The feedback control design is even more complicated in a general multiple-input and multiple-output (MIMO) system, because, in this case, the effect of the delay is added to the coupling effects between the inputs and outputs typically observed in MIMO plants. Moreover, in the MIMO case, it is difficult to define a “process delay,” because each signal path between inputs and outputs may have a different delay (Skogestad and Postlethwaite, 2005). As already mentioned, most of the DTCs proposed in the literature were derived from the original idea of Smith, who proposed in 1957 one of the most popular dead-time compensating methods for SISO processes, called the Smith predictor (SP) (Smith, 1957). The SP is a model-based controller which separates the process model into two parts, the fast model (delay-free) and the delay model, and considers in its structure also two parts, the predictor itself and the so-called primary controller, which uses the output of the predictor to compute the control action. With this idea, the SP allows to create a virtual signal that anticipates the process output behaviour, which is used to eliminate the delay from the closed-loop characteristic equation, at least in the nominal case, in which there is no plant-model mismatch. As a consequence, the design of the closed-loop controller becomes much easier than in the original case, which contains the delay in the characteristic equation. Nowadays, the SP is the best known and most widely used algorithm for SISO dead-time compensation in academy and industry. As it was proposed to control only open-loop stable plants, several works were published after 1957 to allow its use with integrating or unstable processes. Moreover, many works studied tuning rules of the primary controller, how to improve robustness or disturbance rejection of the closed loop, and also its analysis and design in the discrete- time domain. A review of several of these proposed methods for the SISO case can be found, for example, in the book chapter (Palmor, 1996) and in the books (Normey-Rico and Camacho, 2007) and (Visioli and Zhong, 2011). One of the most used control strategies for improving the closed-loop performance of SISO and MIMO dead-time processes consists of using a predictor structure plus a primary controller. and Camacho, 2007). Even for the case of single-input and single-output (SISO) systems, the dead time complicates the feedback control design mainly because: 1) the effects of the control action and the load disturbances take some time to be felt in the controlled variable; 2) the control action that is applied based on the actual error tries to correct a situation that originated some time before. These conditions normally cause two undesirable effects on the closed- loop performance of simple loops: oscillations in the controlled and manipulated variables when the designer tries to reduce the closed-loop settling time, or very sluggish transients when the tuning avoids these oscillations (Aström and Hägglund, 1995). In the frequency domain, it is easy to understand the negative effect of dead time by simply computing the extra decrease in the system phase introduced by the delay (Normey-Rico and Camacho, 2008). The rest of the paper is organized as follows: Section 2 presents a historical perspective of the research in the topic, from the original idea of Smith in 1957 to the general MIMO- DTC solutions studied in the last years. In Section 3 the MIMO- DTC solutions for stable processes are presented. Section 4 presents MIMO-DTC solutions that can cope with integrating and unstable processes, obtaining internally stable structures. The most important aspects related to performance, robustness, and disturbance rejection are discussed in Section 5. Section 6 presents simulation and experimental case studies, followed by conclusions in Section 7. 2 Historical perspective—from the original idea of Smith to the general multi-input multi-output-dead-time compensators solutions With the predictor in the loop, the primary controller can “see” an equivalent system composed by the process and the predictor, and the equivalent system exhibits no dead time or a smaller dead time than the one of the process (Normey-Rico and Camacho, 2007). These structures, which are based on the original idea of Smith proposed in 1957 (Smith, 1957), are known in the literature as dead-time compensators (DTCs), and they have been applied with success to many engineering fields, mainly in industry (Takatsu et al., 1998). 1 Introduction In industry, as well as in other areas, many processes exhibit dead times (or delays) in their dynamic behavior. In most of the cases, the causes of dead time are energy, mass or information transportation phenomena, processing time of sensors and reaction time of actuators. Moreover, apparent dead-time can be perceived in many processes because of Frontiers in Control Engineering Frontiers in Control Engineering 01 frontiersin.org Normey-Rico et al. 10.3389/fcteg.2022.953768 the dynamic effect of the accumulation of time lags in a number of simple dynamic systems connected in series (Normey-Rico and Camacho, 2007). This work deals with the analysis, design, and tuning of MIMO DTCs derived from the Smith Predictor when controlling stable and unstable MIMO processes with multiple delays. et al., 1998). As expected, MIMO dead-time compensator structures are more difficult to analyze and tune to obtain efficient solutions. Note that MIMO-DTC strategies have to cope with nonsquare systems, different types of disturbances in each one of the controlled variables, and coupling effects. Moreover, as in the SISO case, they have to cope with unstable modes and achieve a desired robustness level. Because of this, many researchers have been working on dead-time compensators for MIMO plants during the last years, proposing particular solutions to some of the cited problems (see for example Ogunnaike and Ray (1979), Chen et al. (2011); Garrido et al. (2016)), or general structures capable to achieve a robust stable feedback control system considering a complete set of MIMO closed-loop specifications, as for instance, the works of García and Albertos (2010) and Santos et al. (2014). Considering MIMO processes, the first works that extended the ideas of the SP considered only the case of stable open-loop processes. In the work of Alevisakis and Seborg (1973), the authors studied MIMO stable processes with a single delay, Frontiers in Control Engineering frontiersin.org 02 Normey-Rico et al. 10.3389/fcteg.2022.953768 in this work; however, the procedure is valid only for stable processes. Sánchez-Peña et al. (2009) present a robust performance analysis of the MIMO-SP, but the study is limited to models in which the dead times can be factorized into input and output delays. Therefore, plants with internal coupling delays cannot be considered. A study of two inverted decoupling techniques for stable MIMO delayed processes with non-minimum-phase zeros is presented in Chen et al. (2011), and PI/PID controllers are designed for decoupled processes. Moreover, a robustness analysis is performed in the paper and low bounds of the control parameters are derived to guarantee closed-loop robust stability. In Mirkin et al. (2011), the authors convert the dead-time control problem into an equivalent delay- free problem via loop-shifting arguments, concluding that the structure of the dead-time compensator should rely upon the structure of the regulated output and/or the way in which exogenous signals affect the measurement. In Zheng et al. (2017), a discrete-time dynamic output feedback control is designed for systems with multiple dead times, considering uncertainties. A Lyapunov-Krasovskii function approach is used together with a system decomposition into two subsystems, thus facilitating the feedback controller design only based on the system output. In Bezerra-Correia et al. et al., 1998). (2017), the authors combine the best properties of both DTC and optimal control for MIMO processes with delay. Using a state-space optimal approach, they analyze an explicit dead-time compensation structure used in model predictive control to impose desired performance and robustness properties to the closed loop. In Shaqarin et al. (2019), a robust H∞controller is designed using the mixed sensitivity loop shaping design. that is, all the input-output channels of the processes are assumed to have the same dead time. For this simple case, the main properties of the SP are maintained, thus, the virtual output computed in the predictor anticipates the process output and a delay-free closed-loop characteristic equation is obtained. However, as this simple case is in general not very useful in practice, some years after this work, improved versions of this MIMO-DTC were presented, extending the results for the case of multiple delays (Ogunnaike and Ray, 1979; Jerome and Ray, 1986). In the first paper the proposed solution tries to maintain the delay-free characteristic equation of the closed-loop system, using a MIMO delay-free model with no delays, however losing the prediction property, as the obtained virtual signal is no more an anticipation of the process output. This causes a more involved tuning procedure of the controller to achieve a desired performance. The second work proposes a different fast model, in order to maintain the prediction property, however, as a consequence, obtaining a closed-loop characteristic equation with delays. Although this last property can be seen as a drawback, it is possible to show that with this choice the closed-loop performance can be improved, primarily for MIMO processes with bigger delays. Moreover, this approach gives more flexibility to the control engineer to adequate the solution to the specific characteristics of the plant, as for example, to prioritize some controlled variables against the others. Although this last solution was proposed only for square MIMO plants, the paper contains a very interesting discussion of the advantages and drawbacks of the MIMO dead-time compensation schemes. It is interesting to note that in all these approaches, the primary controller of the MIMO-DTC is in general composed by a set of PI or PID controllers. Some years later, Rao and Chidambaram (2006) and Zhang and Lin (2006) extended the ideas of the MIMO-SP to non-square stable MIMO systems with multiple time delays. et al., 1998). In Rao and Chidambaram (2006) the authors considered the case where the process has more inputs than outputs, and, in the primary controller, a set of centralized MIMO PI controllers were tuned using a pseudo inverse of the steady-state gain matrix. Decoupling strategies have been extensively studied in the last years to improve the control performance of controllers for MIMO delayed systems, not limited to extensions of the MIMO- SP. In Zhang et al. (2016), an H2 analytical decoupling control scheme with a MIMO disturbance observer is proposed. The controller can be used for both stable and unstable MIMO processes with multiple dead times. The proposed control scheme can improve the closed-loop behaviour of the system even under model mismatches and strong external disturbances. Other advantages are: 1) analytical forms of the controller and observer can be obtained, which have low orders, 2) the design procedure is simple, and 3) performance and robustness can be adjusted easily by tuning the parameters in the designed controller and observer. In Tang et al. (2018), a strategy based on a decoupling block plus an SP controller is proposed in order to reduce the negative effects of network-induced delays in the stability in MIMO networked control systems. A modified fuzzy immune feedback control algorithm is used to tune, online, the PID used in the primary controller, to improve both robustness and performance. The advantages of the algorithm proposed in Tang et al. (2018) are that it does not need to measure, estimate, or identify the network delay. In Chuong et al. (2019), a simplified decoupling strategy is proposed coupled with a All the previously cited works focused mainly on the predictor structure. However, other works were oriented to tuning strategies for the primary controller or robustness analysis, sometimes for the general case and in other works for particular models. For example, different decoupling strategies have been analyzed along the time, optimal controllers have been proposed, first-order plus dead-time non-square systems have been analyzed, etc. In the work of Zhang and Lin (2006), a decoupled MIMO-SP is designed by factorizing the model, to separate the delay-free model into minimum phase and non-minimum phase parts, in order to analytically derive an optimal controller. In (Liu et al., 2007), the authors present an analytical decoupling scheme for square MIMO systems, based on an H2 optimal performance specification. et al., 1998). Stability and robustness analysis are also studied Frontiers in Control Engineering frontiersin.org 03 Normey-Rico et al. 10.3389/fcteg.2022.953768 10.3389/fcteg.2022.953768 work, the use of two different dead-time free models is analyzed in order to enable more flexibility in the controller design. Moreover, for open-loop stable processes, a tuning procedure is proposed to speed-up the closed-loop disturbance rejection response, which is an important point that was not studied in many of the previous approaches. A simplified tuning strategy of the MIMO-FSP was presented in Santos et al. (2016). In this approach, offset-free control for step references and disturbances is achieved without explicitly using integral action in the primary controller, then reducing the number of tuning parameters. MIMO-SP structure. The idea is to solve decoupling realizability by using a modified particle swarm optimization algorithm. The controller improves the system performance in terms of the servomechanism problem. work, the use of two different dead-time free models is analyzed in order to enable more flexibility in the controller design. Moreover, for open-loop stable processes, a tuning procedure is proposed to speed-up the closed-loop disturbance rejection response, which is an important point that was not studied in many of the previous approaches. A simplified tuning strategy of the MIMO-FSP was presented in Santos et al. (2016). In this approach, offset-free control for step references and disturbances is achieved without explicitly using integral action in the primary controller, then reducing the number of tuning parameters. The SP can be analyzed using the more general structure of the internal model control (IMC) (Morari and Zafiriou, 1989) which enables a complete analysis of performance and robustness of the controller. Because of this, many researchers developed DTC control strategies and tuning procedures for MIMO delayed plants using the IMC approach. One of the first works was (Maciejowski, 1994), where the author analyzes the stability and robust stability of MIMO-SP considering an IMC approach. More recently, in Guo and Peng (2011), a MIMO-SP is proposed for process with right-half-plane zeros. The proposed strategy uses a decoupling compensation control method, and the IMC technique is applied to design the SP. To reduce the order of the obtained controller a sub-optimal reduction algorithm is proposed, such that the design process of the controller is simplified. In Chen et al. et al., 1998). (2011), the authors presented an IMC approach of the MIMO-SP for a particular case, considering the elements of the MIMO model as first-order- plus-time-delay transfer functions. Later, in the work of Jin et al. (2013), the IMC is applied to an equivalent model of the plant, obtained using the concept of effective open-loop transfer function. The idea in this paper is to ease the primary PID control design, thus avoiding using decoupling strategies as in previous solutions. In Garrido et al. (2014), a new tuning method of the main controller of an IMC strategy is proposed based on the centralized inverted decoupling structure, considering square MIMO plants. The advantages of the approach are that very simple general expressions for the controller elements are obtained and filters can be used to improve the disturbance rejection without modifying the nominal set-point response. An important point to be highlighted is that these new works present results is the discrete-time domain, with control laws that are implementable without any polynomial approximation of the dead time. Moreover, in the particular case of the MIMO-FSP, any control strategy can be used in the primary controller, thus allowing to combine the predictor properties with some optimal or decoupling strategies, or even to introduce some practical aspects such as constraints in the manipulated variables or varying delays. For example, the case of time-varying dead time is studied in Normey-Rico et al. (2012) for the MIMO-FSP, using a delay-dependent Linear Matrix Inequality-based condition to compute a maximum delay interval and tolerance to model uncertainties such that the closed-loop system remains stable. In Garrido et al. (2016), the idea of the MIMO-FSP is used together with a new method to design a centralised inverted decoupling structure. The primary controller, which is applied to square stable or unstable plants, can be obtained with simple general expressions and also a simple tuning of the filters allows improving the disturbance rejection responses. In Lima et al. (2016), the idea of the FSP is combined with a model predictive control strategy to improve robustness or disturbance rejection properties of the closed loop. The controller, named filtered dynamic matrix control (FDMC), is a modification of the well-known Dynamic Matrix Control widely used in industry. In Santos et al. (2017), the MIMO- FSP is extended to reject sinusoidal disturbances in steady state. et al., 1998). Using the same transfer matrix description of the plant, the prediction error filter is re-tuned to deal with this new type of disturbance. As in the case of the FDMC controller, the filter design can be used in model predictive controllers with inactive constraints in steady state. In Giraldo et al. (2018), a method to design a MIMO-FSP for square processes with multiple time delays is presented based on the decentralized direct decoupling structure. Under certain realizability conditions, an easy tuning of the primary controller is presented in the paper, based on the simplification of the MIMO problem to multiple single loops. More recently, in Santos et al. (2021), the authors proposed an anti-windup strategy to be used with the MIMO-FSP predictor for the case of square plants when the control input saturates. The approach is based on a causal modified implementation of the primary controller, and interestingly, the strategy does not add any tuning parameters. Stability analysis is also presented in the paper using linear matrix inequality conditions. More general strategies which allow to control unstable processes and also non-square plants have been proposed in the last years. An approach for square unstable plants was presented in García and Albertos (2010). The idea of the paper is to compute a stable predictor which copes with multiple delays and to design a controller in two parts, first using a stabilizing strategy for the delay-free model and then including extra elements to achieve some output tracking and regulation requirements. In Flesch et al. (2011), a unified MIMO- DTC for square processes with multiple delays was presented, and it can be used to control stable, integrating, and unstable dead-time MIMO processes. The proposed strategy generalizes the filtered Smith predictor (FSP) controller originally proposed in Normey-Rico and Camacho (2009) for SISO plants. An interesting feature of this approach, named MIMO-FSP, is that it compensates the minimal output dead time of each output and allows a tuning procedure considering performance and robustness specifications. An improved version of the MIMO-FSP was presented in Santos et al. (2014), that can also be used for non-square plants. In this Frontiers in Control Engineering frontiersin.org 04 10.3389/fcteg.2022.953768 Normey-Rico et al. FIGURE 1 Block diagram representation of general SISO SP for stable plants. implemented in digital processors with no loss of generality. Anyway, continuous-time counterparts can be found in related works (Normey-Rico and Camacho, 2007, 2009). et al., 1998). The Smith predictor for SISO systems provides three key properties (Jerome and Ray, 1986), (Normey-Rico and Camacho, 2007, Chapter 5) in the absence of modeling error (Pn(z) = P(z)): 1) dead-time compensation, 2) output prediction, and 3) ideal dynamic compensation. As can be seen from the previous analysis there is a historical and conceptual line going from the original SP used to control SISO stable plants and arriving to the MIMO-FSP which allows to control even unstable processes with multiple delays. Thus, in the following sections a review of the main ideas of these developments are presented, directly in the discrete-time domain. The dead-time compensation property, indicated as Property 1, comes from the fact that the delay is removed from the characteristic equation (1 + Gn(z)C(z) = 0). Hence, C(z) can be designed to define the closed-loop poles from the fast model given by Gn(z). In summary, despite the fact that the nominal process is represented by Pn(z)  Gn(z)z−dn, the closed-loop poles are defined by the roots of 1 + Gn(z)C(z). This property is useful as the phase margin is not reduced by z−dn, for instance. et al., 1998). The SISO SP is illustrated in Figure 1, where P(z) = G(z)z−d represents the process with dead time, d is the discrete-time dead time, Pn(z)  Gn(z)z−dn describes a nominal model for P(z), Gn(z) is a model without delay, also known as fast model, and C(z) is a linear controller. r(k), q(k), and n(k) are, respectively, the reference, the load disturbance and the noise signals. If the modeling error is neglected (Pn(z) = P(z)), the closed-loop transfer functions from the external signals to the output are given by FIGURE 1 Block diagram representation of general SISO SP for stable plants. Hyr z ( )  Pn z ( )C z ( ) 1 + Gn z ( )C z ( ), (1) Hyq z ( )  Pn z ( ) 1 − Pn z ( )C z ( ) 1 + Gn z ( )C z ( )  , (2) Hyn z ( )  1 − Pn z ( )C z ( ) 1 + Gn z ( )C z ( ), (3) (1) (2) Applications of the predictor ideas of the SP and FSP to nonlinear systems also appear in literature. The main idea in these works is to separate the delay from the nonlinear model of the process and combine an SP or an FSP with a nonlinear controller, in such a way that the predictor compensates the delay and the nonlinear controller is only designed for the delay-free model. For example, in Gálvez-Carrillo et al., (2007), the authors present the application of a Smith-predictor-based nonlinear predictive controller to a solar power plant. Chapter 13 of Normey-Rico and Camacho (2007) is dedicated to analyze the use of the predictor structure in the nonlinear case, and in Lima et al. (2015), a robust nonlinear FSP which extends the robustness properties of the prediction filter to the nonlinear case is presented. (3) where Hyr(z)  Z{y(k)}/Z{r(k)}, Hyq(z)  Z{y(k)}/Z{q(k)}, and Hyn(z)  Z{y(k)}/Z{n(k)}. The closed-loop requirements such as robustness margins, noise attenuation, and disturbance rejection performance are commonly defined with respect to the Sensitivity Function, namely S(z), and the Complementary Sensitivity Function, given by C(z)  1 −S(z). For linear closed-loop systems, the Sensitivity Function can be obtained from S(z)  Hyn(z). Hence, notice that Hyq(z)  Pn(z)S(z), and Hyr(z)  C(z). 3 Simple multi-input multi-output- dead-time compensators for stable plants Figure 1 can be used to illustrate the output prediction property of the SP, identified as Property 2 in this work. Consider the nominal case without external disturbance, then Yp(z) = P(z)U(z) + Gn(z)U(z) −Pn(z)U(z), Y(z) = P(z)U(z) with Gn(z) = G(z), and z−d  z−dn. Hence, Y(z)  Gn(z)z−dnU(z), and Yp(z) = Gn(z)U(z), such that yp(k) = y(k + d). Once y(k) = yp(k −d), the nominal set-point tracking responses can be defined for yp(k) because y(k−d) is a direct delayed version of yp(k). That is, the property of output prediction states that the output signal of the predictor is an anticipation of the process output. Notice that, because of this property, the nominal set- point tracking response can be designed for the delay-free This section presents the fundamental properties of the DTC for MIMO stable processes. The original Smith predictor for SISO systems is briefly revisited to point out some of the MIMO dead-time compensation challenges. 3.2 Multi-input multi-output-Smith predictor—single delay case The extension of the SP to the MIMO case with a single delay is somehow straightforward because the key ingredients are preserved (Alevisakis and Seborg, 1973). A process with single delay can be written as P z ( )  G11z−d / G1mz−d ... 1 ... Gn1z−d / Gnmz−d ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ G11 / G1m .. . 1 .. . Gn1 / Gnm ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦z−d  G z ( )z−d, (4) (4) where Gij(z)z−d is the transfer function from the j-th input to the i-th output, being Gij(z) a delay-free transfer function and z−d the single time delay of the system. The matrix G(z) is known as the delay-free matrix function or fast model. The basic idea of GMDC is to write the model of the plant as the effective time delays for each output and the remaining dynamics. GMDC is able to compensate for the effective output delays and the primary controller is responsible for guaranteeing the desired performance for the remaining dynamics. Notice that the remaining dynamics may also contain some time delays in this case, so the tuning of the primary controller may have to consider a model which still contains some delays. Consider an extension of the plant model in (Eq. 4) to consider different delays in each input-output pair given by A model for the delay-free process is defined by Gn(z) and, as in the SISO case, dn is a nominal value for d. It is simple to derive the nominal closed-loop relationship from the external signals to the output as Hyr z ( )  z−dnGn z ( )C z ( ) I + Gn z ( )C z ( ) [ ]−1, (5) Hyq z ( )  z−dn I −z−dnGn z ( )C z ( ) I + Gn z ( )C z ( ) [ ]−1 Gn z ( ), (6) Hyn z ( )  I −z−dnGn z ( )C z ( ) I + Gn z ( )C z ( ) [ ]−1. (7) (5) (6) P z ( )  G11z−d11 / G1mz−d1m ... 1 ... 3.1 A brief review of the single-input and single-output-Smith predictor In this work, the DTC analysis and properties are directly discussed in a discrete-time paradigm as the predictors are Frontiers in Control Engineering frontiersin.org 05 Normey-Rico et al. 10.3389/fcteg.2022.953768 predicted system from u(k) to yp(k), which is also observed in closed loop from u(k) to y(k + d). Therefore, the relationships are analogous to the ones obtained in the SISO case, and the three key properties are verified for this case. The Property 3 is the ideal dynamic compensation, that has been used to highlight the achievable performance bounds for input disturbance rejection. Firstly notice that the output response is given by Y(z) = Hyr(z)R(z) + Hyq(z)Q(z) + Hyn(z) N(z). If a theoretical “ideal” controller is defined with an infinity gain, then Hyr(z) →z−d, Hyq(z) →Pn(z)[1 −z−d], Hyn(z) →1 −z−d, so that Y(z) →z−dR(z) + Pn(z)[1 −z−d]Q(z) + [1 −z−d]N(z). Hence, the delayed response cannot be avoided for reference changes and disturbance rejection response, which is expected due to the typical causality constraints. In summary, the achievable performance bounds depend on the delay length. Hence, despite the fact that C(z) should be designed to deal with the MIMO closed-loop poles defined by det(I + Gn(z)C(z)) = 0, the dead-time compensation challenge is similar to the one faced in the SISO problem in the case of a single value for the input delay. The main conclusion of this initial extension comes from the fact that the MIMO design challenge for C(z) depends on the coupling structure of Gn(z). On the other hand, the MIMO dead-time compensation challenge for the SP is similar to the SISO case if a single delay is considered. However, from a practical point of view, the single delay assumption is quite restrictive. It should be highlighted that the SP cannot be used to control open-loop unstable processes because Hyq(z)  Pn(z)S(z) is not stable if Pn(z) has poles outside the unit circle. In order to achieve internal stability, modified SP versions are used, but this discussion is presented directly for the MIMO case due to the generality of the MIMO result. 3.3 Multi-input multi-output-Smith predictor—multiple delay case The first extension of SP for MIMO plants with different delays in each of the input-output pairs was proposed in Ogunnaike and Ray (1979). The idea of the method was to remove all the delays from the model of the plant to obtain a prediction structure which does not contain any delay. This approach makes the tuning of the primary controller simple with respect to the definition of the nominal closed-loop poles, since all the delays are removed from the model which is considered for the controller design. However, the prediction property of the SP (Property 2) does not hold for this case, so it is quite challenging to tune the controller to satisfy performance criteria. This idea was latter improved in Jerome and Ray (1986) to keep the prediction property and is known as generalized multidelay compensator (GMDC). Jerome and Ray (1986) ha L z ( )  diag z−d1, . . . , z−dn Jerome and Ray (1986) have also used the concept of rearrangement test to define whether it is possible or not to find a primary controller which at the same time provides a decoupled and best performance response. A system passes the rearrangement test if it is possible to place the minimum delay of each process variable in the main diagonal by only interchanging rows or columns. In this case, it is possible to compensate for the delays in all the loops and tune a primary controller which is able to provide a perfectly decoupled response. Based on this idea, they show that the control system performance can often be improved (or at least not degraded) by considering extra time delays in specific inputs so that the resulting system passes the rearrangement test. as the MIMO delay of the plant model P(z), and G(z) as the model without the common delays (also known as fast model), it is possible to write the dynamics of the plant model as P z ( )  L z ( )G z ( ). The primary controller can then be tuned to control G(z), which is given by The primary controller can then be tuned to control G(z), which is given by G z ( )  G11z−d11+d1 / G1mz−d1m+d1 .. . 1 .. . Gn1z−dn1+dn / Gnmz−dnm+dn ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦, As happens in the SISO case of the Smith predictor, GMDC is not able to properly deal with open-loop processes which are not strictly stable. For integrating plants, the structure is not able to reject step-like load disturbances in steady state, while for unstable systems the whole compensation structure becomes internally unstable. The extension of the ideas of GMDC for unstable plants is discussed in Section 4. if a control structure as the one provided in Figure 2 is used. In Figure 2, Gn(z) is the nominal fast model, Ln(z) is the nominal delay matrix, C(z) is the primary controller, and F(z) is a reference filter. In addition, r(k) is the reference vector, y(k) is the process variable vector, yp(k) is the vector of model outputs, q(k) is a vector of load disturbances, and n(k) is a vector of measurement noise and output disturbances. Jerome and Ray (1986) ha For this particular structure, in the nominal case (perfect model), the transfer function matrices which define the closed-loop response are given by 3.2 Multi-input multi-output-Smith predictor—single delay case Gn1z−dn1 / Gnmz−dnm ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦, (8) Here Hyr(z) defines the matrix function between the reference vector and process variable vector, Hyq(z) defines the matrix function between the load disturbance vector and the process variable vector, and Hyn(z) defines the matrix function between the vector of measurement noise and output disturbances and the process variable vector. (8) where the definitions are the same as in Eq. 4, except for dij, which is the discrete-time delay associated with the transfer function from the j-th input to the i-th output. frontiersin.org Frontiers in Control Engineering 06 Frontiers in Control Engineering 06 frontiersin.org 10.3389/fcteg.2022.953768 Normey-Rico et al. Gn z ( )  Gn,11z−dn,11+dn,1 / Gn,1mz−dn,1m+dn,1 ... 1 ... Gn,n1z−dn,n1+dn,n / Gn,nmz−dn,nm+dn,n ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦. FIGURE 2 Block diagram representation of GMDC. FIGURE 2 Block diagram representation of GMDC. Hence, nominal prediction property holds in the absence of disturbances as Yp(z) = Gn(z)U(z) and Y(z) = Ln(z)Gn(z)U(z). In other words, yp(k)  [y(k + dn,1) y(k + dn,2) /y(k + dn,n)]′. However, Property 1 holds partially as the characteristic equation is defined by det([I + Gn(z)C(z)])  0, but Gn(z) is not a delay-free model. Clearly, the GMDC removes the effective output delay defined by Ln(z) as the controller design without DTC must consider det([I + Ln(z)Gn(z)C(z)])  0. However, the advantage of the GDMC depends on the input-output dead- time for each output. The ideal compensation property (Property 3) is also preserved by using a theoretical controller with infinity gain as Y(z) →Ln(z)F(z)R(z) + Pn(z)[I −Ln(z)]Q(z) + [I −Ln(z)]N(z). The effective time delay of each output i is di, which is obtained as the minimum value of the time delays associated with that particular output, i.e. di  minj1...m(dij). Thus, defining 4 Multi-input multi-output-dead- time compensators for unstable plants The nominal closed-loop relationships from the external signal to the output are given by Hyr z ( )  Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1F z ( ), (12) Hyq z ( )  I −Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1Fr z ( ) Pn z ( ), (13) Hyn z ( )  I −Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1Fr z ( ). (14) The internal stability of the predictors is an important problem for unstable systems as IMC-based and SP-based strategies can be interpreted as cancellation-based control strategies. This problem is widely discussed in the DTC literature for SISO systems, but the MIMO problem is commonly neglected. This section presents the analysis of a unified MIMO-FSP for unstable processes that can be used to achieve prediction properties and to ensure internal stability. If G•(z) = Gf(z) is employed in this general structure, then det([I + Gf(z)C(z)])  0 is a delay-free characteristic equation. However, the prediction property does not hold in the nominal case because Yp(z) = Gf(z)U(z), Y(z) = Pn(z)U(z), and Pn(z) ≠ L(z)Gf(z) if dn,ij ≠dn,i for any j. The ideal compensation property is lost as Pn(z)C(z) ≠L(z)Gf(z)C(z). The main consequence of this modified fast model comes from the difficulty to define C(z) with respect to performance specification as y(k) is not a direct delayed version of yp(k). Hence, this full dead-time compensation preserves SP Property 1, but the Property 2 and Property 3 are lost. The general MIMO-FSP can be modified to achieve internal stability by using the implementation structure presented in Figure 4, where S• z ( )  G• z ( ) −Fr z ( )Pn z ( ) (15) (15) is a stable filter. For analysis purposes, Figure 3 and Figure 4 are equivalent, but Fr(z) should be designed such that the minimal representation of S•(z) has no pole outside the unit circle. The output feedback property and the ideal compensation properties are directly ensured if G•(z) = Go(z) because Pn(z) = L(z)Go(z). Then, the GMDC properties are also verified in this case, such that det([I + Go(z)C(z)])  0 is not a delay-free characteristic equation, as previously discussed. 3.4 General solution for stable plants Hyr z ( )  Pn z ( )C z ( ) I + Gn z ( )C z ( ) [ ]−1F z ( ), (9) Hyq z ( )  I −Pn z ( )C z ( ) I + Gn z ( )C z ( ) [ ]−1 Pn z ( ), (10) Hyn z ( )  I −Pn z ( )C z ( ) I + Gn z ( )C z ( ) [ ]−1. (11) (9) The filtered Smith predictor for MIMO stable processes (Normey-Rico and Camacho, 2007; Flesch et al., 2011; Santos et al., 2014) is presented as a general solution for stable systems due to the design flexibility provided by the robustness filter Fr(z). The dead-time compensation properties achieved by the methods described in Ogunnaike and Ray (1979) and Jerome and Ray (1986) can be recovered from the implementation structure in Figure 3, where G•(z) can be either a delay-free model Gf(z) or a model without the effective output delay, given by Go(z). In this general solution, the models are given by In order to define closed-loop design requirements and for comparison with other controllers define, respectively, the MIMO Sensitive Function and MIMO Complementary Sensitive Function, as, S(z)  I −Pn(z)C(z)[I + Gn(z)C(z)]−1, and C(z)  Pn(z)C(z)[I + Gn(z)C(z)]−1. In this problem, it should be remarked that the fast model is given by Frontiers in Control Engineering Frontiers in Control Engineering frontiersin.org 07 10.3389/fcteg.2022.953768 Normey-Rico et al. FIGURE 3 Block diagram representation of general MIMO-FSP for stable plants. FIGURE 4 Block diagram representation of general unified MIMO-FSP for unstable systems. FIGURE 4 Block diagram representation of general unified MIMO-FSP for unstable systems. FIGURE 4 Block diagram representation of general unified MIMO-FSP for unstable systems. FIGURE 3 Block diagram representation of general MIMO-FSP for stable plants. FIGURE 3 Block diagram representation of general MIMO-FSP for stable plants. FIGURE 4 Block diagram representation of general unified MIMO-FSP for unstable systems. FIGURE 4 Block diagram representation of general unified MIMO-FSP for unstable systems. FIGURE 3 Block diagram representation of general MIMO-FSP for stable plants. C(z)  Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z). Indeed, for open- loop stable processes, the only internal stability requirement for Fr(z) is to be stable if the roots of det([I + G•(z)C(z)])  0 are inside the unit circle. This property provides a significant flexibility with respect to the definition of Fr(z). 3.4 General solution for stable plants As the Complementary Sensitivity Function is directly related to the robustness filter, it becomes a powerful ingredient to deal with loop-requirement trade-offs. Conditions to achieve robust stability and deterministic disturbance rejection are discussed in Section 5. Go z ( )  Gn,11z−dn,11+dn,1 / Gn,1mz−dn,1m+dn,1 ... 1 ... Gn,n1z−dn,n1+dn,n / Gn,nmz−dn,nm+dn,n ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦, r Gf z ( )  Gn,11 / Gn,1m .. . 1 .. . Gn,n1 / Gn,nm ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦. or or Gf z ( )  Gn,11 / Gn,1m .. . 1 .. . Gn,n1 / Gn,nm ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦. 4 Multi-input multi-output-dead- time compensators for unstable plants ( (16) An interesting interpretation of this solution based on the integral action of the primary controller C(z) comes from the fact that if the stability of S•(z) is ensured and the filter static gain is such that ∥S•(1)∥= 0, then yp(k) →y(k) in steady state and the integral action of C(z) ensures the constant disturbance rejection. This result shows that the stability of S•(z) is required to ensure the internal stability in the MIMO problem, which is a direct extension of the SISO FSP result. Details of the complete proof can be found in Santos et al. (2014). The effects of Fr(z) on the disturbance rejection and robustness properties are discussed in Section 5. 4 Multi-input multi-output-dead- time compensators for unstable plants The transfer function from the input-disturbance to the output is similar to the one derived in the stable case, but it requires some additional manipulation as follows Hyqg z ( )  I −Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1Fr z ( ) Pq z ( ). (17) As the delays do not modify the static gain, then limz→1∥Pn(z) −G•(z)∥= 0 and limz→1∥Pn(z)C(z) −G•(z) C(z)∥= 0. Hence, a typical solution is to impose that Fr(1) = I and to include a multivariable integral action in C(z) such that Pn(1)C(1)[I + G•(1)C(1)]−1  I and ∥Hyqg(1)∥= ∥(I −I) Pq(1)∥= 0, which ensures constant disturbance rejection for a stable Pq(z) due to the final value theorem. It should be remarked that if Pq(z) is not Bounded-Input Bounded-Output (BIBO) stable, as verified with Pq(z) = Pn(z) if Pn(z) has poles outside the unit circle, then Fr(z) has to be tuned to eliminate these poles as previously discussed based on the unified dead- time compensation structure. Therefore, S•(z) should be stable to guarantee internal stability and ∥Hyqg(1)∥= 0 to ensure constant disturbance rejection. Both conditions can be handled by the design of Fr(z). The general case can be solved by defining a filter Fr(z) that guarantees the elimination of the undesired poles of Pq(z) from a minimal realization for Hyqg(z). Hyq z ( )  Pn z ( ) −Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1Fr z ( )Pn z ( )  Pn z ( ) I + C z ( )G• z ( ) [ ]−1 I + C z ( )G• z ( ) [ ] −Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1Fr z ( )Pn z ( )  Pn z ( ) I + C z ( )G• z ( ) [ ]−1 + Pn z ( )C z ( ) × I + G• z ( )C z ( ) [ ]−1 G• z ( ) −Fr z ( )Pn z ( ) [ ]   H1 z ( ) + H2 z ( )S• z ( ). 5.1.1 Modified condition to ensure constant disturbance rejection Inspired by the ideas presented in Torrico et al. (2013) for SISO systems, a relaxed condition for constant disturbance rejection has been proposed for MIMO processes in Santos et al. (2016). Actually, the integral action may not be directly imposed by C(z), but by means of a modified steady-state condition for Fr(1). The new condition is given by: 1) S•(1) C(1) = I for constant disturbance rejection and 2) F(1) = Fr(1) for constant set-point tracking. Moreover, if Pn(z) is stable, then S•(1)C(1) = I can be achieved by imposing Fr(1)  {Pn(1)C(1)[I + G•(1)C(1)]−1}−1. In these relaxed conditions, the disturbance rejection problem has been simplified because the integral action of C(z) is not required anymore. Notice that the previous approach with integral action is defined such that {Pn(1)C(1)[I + G•(1)C(1)]−1}−1  I and Fr(1) = I, which characterizes a particular solution. Disturbance rejection performance and robust condition define a relevant trade-off for practical control problems. The design conditions to achieve closed-loop stability in the presence plant-model mismatch and to reject persistent disturbances are even more challenging in the presence of multiple input-output delays. This section presents some of the key conditions to achieve disturbance rejection and robust stability driven to the MIMO-FSP for either stable or unstable processes. 4 Multi-input multi-output-dead- time compensators for unstable plants The design of Fr(z) in order to achieve internal stability is not a difficult task once it can be interpreted as a combination of SISO internal stability conditions. Notice that the elements of S•(z) are defined by S•,ij(z) = G•,ij(z) −Fr,ii(z)Pn,ij(z), i = 1, .., n, j = 1, .., m if Fr(z) is defined as a diagonal filter. This problem can be expressed as a determined system of linear equations where the number of decision variables is defined by the coefficient of the numerator of Fr,ii(z). In this case, the robustness filter cannot be freely defined. This stability condition can be interpreted as a The proposed general structure is a filtered Smith predictor for MIMO systems where Fr(z) is a robustness filter that can be designed to deal with loop-requirement trade-offs, preserving nominal set-point tracking response. Notice that Hyr(z) does not depend on Fr(z), but S(z)  I − Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z) and Frontiers in Control Engineering frontiersin.org 08 Normey-Rico et al. 10.3389/fcteg.2022.953768 applications. For presentation generality, assume that n(k)  Z−1{Pq(z)Z{qg(k)}} is a generalized disturbance signal and that its effect depends on a certain disturbance model Pq(z). For instance, if Pq(z) = P(z), then qg(k) can be used to represent q(k), but if Pq(z) = I, then qg(k) is equivalent to n(k). In this case, the nominal transfer matrix from qg(k) to y(k) is given by loop constraint because a minimum gain is necessary to stabilize any open-loop unstable systems. The poles of Fr(z) are typically used as design parameters, but the numerators of the partial elements of the robustness filter, namely the numerators of Fr,ii(z), are used to ensure internal stability. The stabilizing properties of S•(z) can be explained by comparing the transfer functions of the unified solution for unstable processes with the general solution for stable systems. Assume that C(z) is a stabilizing controller such that H1(z)  Pn(z)[I + C(z)G•(z)]−1 and H2(z)  Pn(z)C(z)[I + G•(z)C(z)]−1 are stable transfer matrices. Hence, Hyr(z) = H1(z)F(z) is stable if F(z) is a stable reference filter. As previously discussed, Hyn(z) = I −H1(z)Fr(z) is also stable if Fr(z) is defined as a stable robustness filter. 5.1 Conditions to achieve disturbance rejection Firstly, the rejection of constant disturbances is considered due to the relevance of this loop requirement for several Frontiers in Control Engineering frontiersin.org 09 Normey-Rico et al. 10.3389/fcteg.2022.953768 FIGURE 5 Δ −M structure for robustness analysis. Once more, if Pq(z) is stable, then ∥Hyqg(1)∥= ∥(I −I) Pq(1)∥= 0, which ensures the constant disturbance rejection due to the final value theorem. On the other hand, if Pn(z) = Pq(z) and Pn(z) has integral action, the internal stability requires that S•(z) should be a BIBO stable matrix and the modified condition may be expressed as S•(1)C(1) = I. The BIBO stability of S•(z) imposes the elimination of the integrating poles of Pq(z) from the disturbance rejection response. I −Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1Fr z ( ) z±e−jωTs  0. (18) (21) In case of poles with multiplicity greater than one, such as qq(k) = kA sin(ωTsk + ϕ)I or ramp disturbances, then the condition is imposed to Hyqg and to its derivatives, where the derivative order is one degree lower than the multiplicity of the disturbance poles. In the Eq. 21, the functions W1(ejΩ) and W2(ejΩ) give the spatial and frequency structure of the uncertainty. Now, define Y(z) = Pn(z)U(z) + ΔP(z)U(z), Q(z) = ΔP(z)U(z) such that: 1) Q(z) = ΔP(z)U(z) and 2) U(z) = Huq(z)Q(z). The transfer matrix Huq(z) is given by U(z) = −C(z)Fr(z)Q(z) −C(z) G•(z)U(z). Then, M•(z) = Huq(z) is given by 1 The notation σ(X) represents the maximum singular value of matrix X. 5.2 Robustness Persistent deterministic disturbances such as sinusoidal signals and ramp-like disturbances can be handled in the same final value theorem paradigm. For instance, assume that qg(k) = A sin(ωTsk + ϕ)I. In order to apply the final value theorem, then Hyqg(z)Qg(z) should be stable. As a consequence, the zeros of Hyqg(z) should be placed in the same position of the poles of Qg(z) over the unit circle. Hence, a new design condition is imposed to Fr(z) as follows (Hyqg(z)|z±e−jωTs  0 ) The robustness analysis can be performed using the standard unstructured description (Normey-Rico and Camacho, 2007). Assume that the model uncertainty can be described by P z ( )  Pn z ( ) + ΔP z ( ), (20) (20) where ΔP(z) is a stable unknown transfer matrix that represents plant-model mismatch (Skogestad and Postlethwaite, 2005). Moreover, the uncertainty model can be rewritten as follows1 ΔP ejΩ    W2 ejΩ  Δ ejΩ  W1 ejΩ  , σ Δ ejΩ    ≤1, ∀Ω ∈0,π [ ]. (21) (18) I −Pn z ( )C z ( ) I + G• z ( )C z ( ) [ ]−1Fr z ( ) z±e−jωTs  0. (18) 5.1.3 Disturbance rejection performance (22) M• z ( )  −I + C z ( )G• z ( ) [ ]C z ( )Fr z ( ). (22) The unified structure of the filtered Smith predictor has been defined such that the open-loop unstable poles of Pn(z) are avoided in the transfer matrix from q(k) to y(k). This idea, that has been used to stabilize the predictor for open-loop unstable processes, can be also applied to avoid slow open- loop poles to appear in the disturbance rejection response. This solution can be implemented by defining Fr(z) such that the undesired poles of the disturbance model are eliminated from Hyqg(z). The robust stability condition can be derived from the Δ −M structure as depicted in Figure 5. Hence ∥Δ∥∞∥M(z)∥∞< 1, where M(z) = W1(z)M•(z)W2(z). Alternatively, the condition may be given by σ M ejΩ     < 1, ∀Ω ∈0, π [ ], (23) (23) σ M ejΩ     < 1, ∀Ω ∈0, π [ ], for block diagonal perturbations. Notice that M(z) = −W1(z)[I + C(z)G•(z)]C(z)Fr(z)W2(z), such that a low-pass frequency response for Fr(z) may be directly used to reduce the loop gain at a given frequency range in order to achieve the robust stability criterion. Once more, the transfer function from the generalized disturbance to the output is given by Hyqg z ( )  S z ( )Pq z ( ), (19) (19) If the system is open-loop unstable, the internal stability condition implicitly imposes design constraints for Fr(z) as a minimum gain is required to stabilize the prediction. Moreover, low-pass robustness filters slow down disturbance rejection performance even for open-loop stable processes. Hence, the where S(z)  I −Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z) and C(z)  Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z). Hence, the where S(z)  I −Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z) and C(z)  Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z). Hence, the Complementary Sensitivity Function and the Sensitivity Function, as a consequence, can be directly shaped by Fr(z). As a result, the disturbance rejection response can be improved, but the cost to pay comes from the typical trade-off between disturbance rejection performance and robustness. where S(z)  I −Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z) and C(z)  Pn(z)C(z)[I + G•(z)C(z)]−1Fr(z). Hence, the Complementary Sensitivity Function and the Sensitivity Function, as a consequence, can be directly shaped by Fr(z). 6.1 single-input and single-output unstable process In this study the FSP is tuned to control an unstable process with transfer function given by Fr z ( )  z −a z −b 1 −b 1 −a, P z ( )  0.2214z−10 z −1.221 , is enough to achieve both these conditions, where b is the free parameter to define the settling time of the responses and a is computed for the condition 1 −z−dFr(z) = 0 for z = 1.221 and the nominal delay d = 10. Note that this condition is directly derived from the SISO expression of S• in (15), given by S(z) = Gn(z)(1 − z−dFr(z)), which must be stable. Thus, all the poles of Gn(z) outside the unit circle must be cancelled with the roots of the numerator of 1 −z−dFr(z). In this case, tuning b = 0.915 to obtain a closed-loop time response with settling time of approximately 7 s gives a = 0.9914. Thus, the resulting prediction filter is and model Y(z) = P(z)[U(z) + Q(z)], where U(z) is the manipulated variable, Q(z) is the disturbance, and Y(z) is the process output. The sampling time is Ts = 0.2 s. To achieve zero steady-state error for step references, a PI controller C(z) and a reference filter F(z) are used in the primary controller of the FSP. The tuning of the primary controller is not the focus of this case study, so in order to obtain a stable closed-loop system Y(z)/R(z), being R(z) the reference signal, C(z) and F(z) can be tuned as and model Y(z) = P(z)[U(z) + Q(z)], where U(z) is the manipulated variable, Q(z) is the disturbance, and Y(z) is the process output. The sampling time is Ts = 0.2 s. To achieve zero steady-state error for step references, a PI controller C(z) and a reference filter F(z) are used in the primary controller of the FSP. The tuning of the primary controller is not the focus of this case study, so in order to obtain a stable closed-loop system Y(z)/R(z), being R(z) the reference signal, C(z) and F(z) can be tuned as Fr z ( )  9.843z −9.758 z −0.915 . C z ( )  2.657z −2.496 z −1 , F z ( )  0.0606z z −0.9394. C z ( )  2.657z −2.496 z −1 , F z ( )  0.0606z z −0.9394. 6 Case studies Y z ( ) R z ( )  0.0359z z2 −1.633z + 0.6689z−10, This section presents three case studies. In the first one, a SISO simulated unstable process is considered, to illustrate some details of the filter implementation. In the second simulation study, a MIMO plant is used in order to illustrate some of the discussed points related to the predictor structure and disturbance rejection response. Finally, in the third one, experimental results are presented to show the applicability of the dead-time compensation strategy in practice. with two poles: z = 0.8166 + 0.0449j and z = 0.8166 −0.0449j, plus the poles which characterize the delay. The step response does not present overshoot and has a setting time of about 7 s after the delay is elapsed. In this case, the prediction filter Fr(z) has to be tuned to obtain a closed-loop disturbance rejection transfer function Y(z)/ Q(z) without the open-loop pole of the model at z = 1.221, which is outside the unit circle. Moreover, the condition Fr(1) = 1 must be met to achieve the rejection of step-like disturbances in steady state. A simple first-order filter with unity steady-state gain given by 5.1.3 Disturbance rejection performance As a result, the disturbance rejection response can be improved, but the cost to pay comes from the typical trade-off between disturbance rejection performance and robustness. Frontiers in Control Engineering Frontiers in Control Engineering frontiersin.org 10 Normey-Rico et al. 10.3389/fcteg.2022.953768 FIGURE 6 Unstable SISO system: disturbance response in closed-loop. Control action in dotted line and process output in solid line. trade-off between disturbance rejection and robustness should be taken into account. For presentation simplicity, the additive uncertainty description has been used in order to illustrate the robustness filter impact with respect to a robust stability criterion, but this type of simplified uncertainty description is not suitable to deal with unstable additive uncertainty terms. For the general problem, other types of uncertainty descriptions such as the multiplicative input, the multiplicative output and their inverse counterparts can be derived from the definitions presented by Skogestad and Postlethwaite (2005, Chapter 8). The proposed criterion is conservative because it spreads the uncertainty over the whole transfer matrix before defining a magnitude bound, but it is widely used in the robustness analysis of MIMO plants (Skogestad and Postlethwaite, 2005) and the robustness filter effect can be directly highlighted. FIGURE 6 Unstable SISO system: disturbance response in closed-loop. Control action in dotted line and process output in solid line. Frontiers in Control Engineering 6.2 Multi-input multi-output integrating plant Particularly, this case study considers the simplified MIMO- FSP. For this purpose, a challenging example of a three-stage evaporator system, already studied by several authors (Normey- Rico and Camacho 2007); (Santos et al. 2017), is chosen. A MIMO model can represent this process with two inputs, two outputs, and multiple dead times as Fr1 z ( )  1 + 12.143 z −1 ( ) z 0 −14.286 z −1 ( ) z 6 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ , Fr2 z ( )  1 + 0.6071 z −1 ( ) z −0.95 ( ) 0 −0.7143 z −1 ( ) z −0.95 ( ) 6 0.05z z −0.95 ( ) ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ . (27) Pn s ( )  3.5 e−s s −e−5s 2s + 1 2 e−7s 1.5s + 1 −e−5s 3.2s + 1 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ , Pq s ( )  3.5 e−3s s −4.5 e−2s 2s + 1 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦, (24) (27) (24) where the time is in minutes. The inputs are the juice and steam flows, while the outputs are the level and temperature in the storage tank. For the control design, a discrete-time model is given by It is worth mentioning that for this particular case of constant disturbance, is possible to use the same robustness filter to satisfy the disturbance rejection properties for both the full and output DTC fast models. This is possible because the two models have the same transfer function in the element (1, 1) of the fast model, which is the one with the integrative mode. And, as the filter is block diagonal, the internal stability condition is achieved tuning Fr,11(z). It is worth mentioning that for this particular case of constant disturbance, is possible to use the same robustness filter to satisfy the disturbance rejection properties for both the full and output DTC fast models. This is possible because the two models have the same transfer function in the element (1, 1) of the fast model, which is the one with the integrative mode. And, as the filter is block diagonal, the internal stability condition is achieved tuning Fr,11(z). Pn z ( )  0.7 z−5 z −1 −0.0952 z−25 z −0.9048 0.2497 z−35 z −0.8752 −0.0606 z−25 z −0.9394 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ , (25) (25) Figure 7 shows the simulation results. 6.2.1 Constant disturbance This subsection presents comparative results of the simplified MIMO-FSP considering both full DTC and output DTC fast models and also different tuning of the robustness filters. In the first case, the filter Fr1(z) was used in the two DTCs to accelerate the disturbance rejection response; and in the second case, again for the two DTCs, a slow pole was used in Fr2(z) in order to achieve a more robust solution (Santos et al., 2014). Note that in general, the filter tuning has to satisfy a trade-off between robustness and disturbance rejection response (Santos et al., 2014), thus it is expected a better robustness in case 2. In this particular case, the used filters are 6.1 single-input and single-output unstable process For the controller implementation, the output prediction yp(z) is computed using: Thus, the closed-loop transfer function in the nominal case is: Yp z ( )  S z ( )U z ( ) + Fr z ( )Y z ( ), Frontiers in Control Engineering 11 Frontiers in Control Engineering frontiersin.org 11 Normey-Rico et al. 10.3389/fcteg.2022.953768 models have, for this case study, the same elements in the diagonal, and different delays in the non-diagonal elements. where S(z) = Gn(z)(1 −z−dFr(z)) = Gn(z)z−d(zd −Fr(z)) is stable thanks to the imposed conditions in Fr(z). Note that S(z) must be implemented eliminating the polo-zero cancellation at z = 1.221, as this value is a root of both the numerator and denominator of S(z). Thus, S(z) can be written as S(z)  Ns(z) (z−0.915)z10, being Ns(z) = [z10 + 0.3064z9 + 0.3743z8 + 0.4572z7 + 0.5584z6 + 0.6820z5 + 0.8330z4 + 1.0174z3 + 1.2427z2 + 1.5178z −7.9893] the quotient of the polynomial division when eliminating the root at z = 1.221. The reference filter F(z) is also a static gain that is tuned to obtain Hyr(1) = 1, then , C  0.2 0 0 −0.2 ⎡⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎦, F  1 0 0 6 ⎡⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎦. (26) (26) The design of the robustness filter depends on the characteristic of the disturbance to be attenuated. In this study, constant and sinusoidal plus constant disturbances are considered in two different subsections. The objective is to illustrate the tuning in these two cases and to analyse different aspects of the closed-loop behavior. The closed-loop response of the system is show in Figure 6 for a step disturbance of amplitude 1 applied at t = 0. As can be seen, the closed-loop system is stable, the disturbance is rejected in steady state, and the settling time is, as expected, about 7 s. Note that as the system has a delay of 2 s, the response is not affected by the disturbance in the first 2 s and then the system is in open loop from t = 2 s to t = 4 s, since a control action taken at t = 2 s only affects the system response after t = 4 s. Thus, the settling time of about 7 s is measured from t = 4 s to t = 11 s (for 95% of attenuation). 6.2 Multi-input multi-output integrating plant Two step changes were applied to the references r1 and r2 at t = 0 min and t = 20 min, respectively. Then, a constant disturbance qg(t) = −0.02 was applied at the input of Pq(s) at t = 50 min. It can be seen that both controllers have similar performance for the first setpoint change. In the second setpoint, the process coupling affected the FSP with the full DTC fast model more severely, originating an undershoot of 37% on y1(t). In comparison, the output DTC presented an overshoot of 18%. As expected, the robustness filters do not affect the nominal setpoint response. Regarding where Ts = 0.2 min is used. Note that the output disturbance in this case is n(z) = Pq(z)qg(z), where Pq(z) is the discrete-time model obtained from Pq(s) considering step type disturbances. As already mentioned, one of the advantages of the simplified MIMO-FSP is that a simple primary controller C(z) can be used. In this case, for simplicity, this controller is chosen as a diagonal static gain. It is tuned to stabilize both the nominal full DTC and output DTC fast models (Gf(z) and Go(z)). Note that these two Frontiers in Control Engineering frontiersin.org 12 Normey-Rico et al. 10.3389/fcteg.2022.953768 FIGURE 7 Three-stage evaporator system: constant disturbance. (A) Results for the robustness filter Fr1(z); (B) Results for the robustness filter Fr2(z); URE 7 ee-stage evaporator system: constant disturbance. (A) Results for the robustness filter Fr1(z); (B) Results for the robustness filter Fr2(z Note that the robustness filter has the same static gain as F(z), which is necessary to obtain an integral action. In addition, for a fair comparison, all the poles of the filter Fr(z) are set to be equal to the MPC proposed in Santos et al. (2017). In order to perform the simulations, step changes were applied in the reference r1 at t = 10 min and in r2 at t = 50 min; a constant disturbance of amplitude −0.2 was applied at t = 120 min, and a sinusoidal disturbance defined by 0.2 sin(0.7t) was introduced at t = 180 min. disturbance rejection, in the case of the more aggressive filter Fr1(z) (Figure 7A), the output y1(t) of the output DTC presented a better response with no overshoot. However, the performance of the full DTC, for the case of Fr2(z) (see Figure 7B), was better for the same output. 6.2.2 Constant plus sinusoidal disturbances In this subsection, the simplified FSP with output DTC fast model is compared to a model-based predictive control (MPC) implementation, which is a common choice for dealing with MIMO processes with coupling. The robustness filter is computed to obtain the desired disturbance rejection characteristics. In this case, the process is supposed to be subject to a disturbance signal which is composed of a constant part plus a sinusoidal part with a 0.7 rad/min frequency. In this case, the obtained filter Fr(z) for internal stability and sinusoidal disturbance rejection is computed using the method proposed in (Santos et al., 2017), giving 6.2 Multi-input multi-output integrating plant Finally, the disturbance rejection of the second output y2(t) is quite similar for both controllers. This example shows that the process coupling and different dead times can deteriorate the setpoint response of the controller with the full DTC fast model. Thus, a reference filter may be necessary to improve the setpoint response. Furthermore, there is no general rule for disturbance attenuation because it is difficult to specify the effect of the uncompensated delays of the fast models in the responses. Thus, specific tuning rules of the MIMO-DTC is an open research topic. As shown in Figure 8, for this tuning, the MPC has a slightly better setpoint response because it attenuates better the process coupling using a stronger control action. In this case, the disturbance rejection response is slightly better in the simplified FSP for both constant and sinusoidal disturbances, despite the simplified FSP having filters with lower order than the adaptive MPC. It is fair to mention that the MPC uses an adaptive filter that estimates the frequency of the sinusoidal disturbance online, which causes an additional transient in the disturbance rejection responses. This last topic is out of the scope of this paper, but further details about the adaptive approach can be found in (Santos et al., 2017). Furthermore, this example shows that despite the MPC having a great ability to deal with complex MIMO time-delayed processes, the MIMO-FSP might give a similar performance. 6.3 Control of a neonatal intensive care unit Fr z ( )  1+ z−1 ( ) −2.7288z+2.8503 ( ) z−0.9 ( )2 −7.0075 z−1 ( ) z−0.9 z−1 ( ) −6.0903z+5.9474 ( ) z−0.9 ( )2 6+ −14.9620 z−1 ( ) z−0.9 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ . (28) This section presents a practical implementation of a MIMO-DTC for controlling the temperature (T) and relative humidity (RH) of a neonatal intensive care unit (NICU) prototype. The objective here is to illustrate, in a real case, (28) Frontiers in Control Engineering Frontiers in Control Engineering frontiersin.org 13 Normey-Rico et al. 10.3389/fcteg.2022.953768 FIGURE 8 Three-stage evaporator system: constant plus sinusoidal disturbances. FIGURE 8 Three-stage evaporator system: constant plus sinusoidal disturbances. Fr z ( )  0.3883z2 −0.7503z+0.3625 z−0.98 ( )2 0 0 0.01516z2 −0.0217z+0.0069 z−0.98 ( )2 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ . (31) Fr z ( )  0.3883z2 −0.7503z+0.3625 z−0.98 ( )2 0 0 0.01516z2 −0.0217z+0.0069 z−0.98 ( )2 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ . (31) the implementation of the MIMO-FSP, using the fast model Go(z) in the predictor structure and considering tuning aspects for a robust behavior. A picture of the NICU prototype and its schematic representation are shown in Figure 9. The manipulated variables of the NICU are the power of a heating resistor and a humidifier, which are controlled through a driving circuit. A fan circulates the heated and humidified air through an acrylic canopy to obtain uniform humidity and temperature in the NICU dome. The linear model of the process with a sampling time Ts = 12 s is given by Y(z) = Pn(z)U(z), where (31) In this case, as the model is a simple representation of the real process, a robust tuning is used. Thus, the PI controllers do not accelerate the closed-loop responses and low-pass filters with two poles are used in the diagonal of the MIMO filter to obtain a robust closed-loop system. Moreover, this low-pass characteristic of the filters is important to attenuate the effect of the noise in the control signal. Pn z ( )  0.6065 z−4 z −0.9754 −0.1678 z−17 z −0.9879 −0.05795 z−15 z −0.9742 0.08129 z−14 z −0.9821 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ . (29) Figure 10 shows an experiment with the proposed controller. Figure 10 shows an experiment with the proposed controller. Two setpoint changes were applied in the relative humidity and temperature setpoints to test the performance of the controller. 6.3 Control of a neonatal intensive care unit In addition, it is worth mentioning that the unmodeled dynamics and the environmental conditions act as unknown disturbances. In Figure 10, the relative humidity (in blue) and temperature (in red) signals are at the top, while the controls signals uH (in blue) and uR (in red) are at the bottom. Note that the controller has a good performance, since the process outputs track the step-like references, even in the presence of unknown environmental disturbances and the coupling between the process variables. In addition, the relative humidity and temperature measurement noises are attenuated by the prediction filter and they do not affect the control signals. Furthermore, as linear controllers are used, an undesired overshoot occurs in the temperature during the first setpoint change. This effect, called windup, originated because the control signal u2 saturates. This effect can be eliminated using an anti-windup technique, such as the one used in Santos et al. (2021), which generalizes the ideas in Flesch et al. (2017). However, that is not in the scope of this case study. (29) The output vector is given by Y  [RH T]′ and the input vector is U  [uH uR]′, being uH the control signal that manipulates the humidifier and uR the one that manipulates the heating resistor. For this case study, a diagonal PI controller C(z) is proposed, which is tuned based on the fast model, obtained by extracting the effective delay z−4 in the first output and the effective delay z−14 in the second output. The obtained primary controller is C z ( )  0.0421 z −0.9691 ( ) z −1 0 0 0.5934 z −0.985 ( ) z −1 ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ , (30) (30) while the robustness filter is defined by Frontiers in Control Engineering 14 Frontiers in Control Engineering 14 frontiersin.org 14 10.3389/fcteg.2022.953768 Normey-Rico et al. FIGURE 9 NICU prototype picture and scheme. FIGURE 9 NICU prototype picture and scheme. FIGURE 9 NICU prototype picture and scheme. NICU prototype picture and scheme. the flexibility of the predictor filter. However, from the practical control point of view, tuning aspects can be further studied. The better choice of the fast model is an important subject for research. 6.3 Control of a neonatal intensive care unit Although, in general, the use of Go(z) in the predictor gives good closed-loop results, there are some particular type of MIMO process, with very different delays in their elements, that can be controlled with better performance using Gf(z) as fast model. This point can be analyzed in a more general form, in order to evaluate the advantages and drawbacks of the two options and to determine which process characteristics should be considered in the decision. Another important research point is the optimal tuning of the primary controller and filters, considering a set of specifications. FIGURE 10 Experimental results of relative humidity and temperature control in a NICU. This paper is based on Smith-predictor concepts for processes with multiple delays that are described by input- output transfer matrices. Input-output models are widely used in practical problems due to the identification simplicity. An interesting topic for further investigation is the extension of the DTC properties for nonlinear input-output descriptions such as Hammerstein, nonlinear autoregressive moving average with exogenous input (NARMAX), and Volterra models. The networked control systems have introduced some interesting control challenges, such as networked induced delay, packet disorder, and data dropout. The multivariable problem with dead-time compensation in the presence of a single-input time-varying delay has already been investigated in a related work (Normey-Rico et al., 2012). However, the study of the conservativeness of the robust stability criteria for systems with time-varying delay deserves further investigation. Moreover, adaptive control strategies for system with networked induced delay and the case of multiple time- varying delays may also be interesting topics for future works. 7 Future research topics As it has been analyzed in previous sections, the MIMO-FSP gives a control structure capable of dealing with stable and unstable MIMO plants with multiple delays, considering, simultaneously, several closed-loop specifications, such as disturbance rejection, robustness, and performance. All these advantages are mainly related to the predictor structure used in the controller, and to Frontiers in Control Engineering frontiersin.org 15 Normey-Rico et al. 10.3389/fcteg.2022.953768 Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions All authors contributed to conception of the work and the first draft of the manuscript. JN-R organized the historical 8 Conclusion perspective. TS and RF performed the solutions for MIMO plants. BT prepared the simulations. All authors contributed to manuscript revision, read, and approved the submitted version. This work presented a historical and technical overview of the design of dead-time compensators for MIMO processes with multiple dead times. It was shown how the original idea of the Smith Predictor, presented in 1957, was used in a modified form, to propose a controller capable to control MIMO stable and unstable processes satisfying a set of closed- loop specifications. It was also shown that the key point in the analysis is the correct design of the predictor structure, that has to be defined to guaranty internal stability of the closed- loop system, and to offer enough degree of freedom for tuning, looking for a trade-off between robustness and performance. This overview emphasized that the MIMO-DTC challenge depends on the internal delay structure, as different output delays may increase significantly the compensation difficulties. A unified discussion was provided such that disturbance rejection and robustness requirements can be directly handled by using any of the MIMO-FSP alternatives. The main conclusion is that the MIMO filtered Smith predictor-based controller is a generalized solution that can be used to deal with multiple loop requirements due to great flexibility provided by the robustness filter. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This research received funding from CNPq/Brazil—Conselho Nacional de Desenvolvimento Científico e Tecnológico—under grants 304032/2019–0, 308741/2021-8, 315546/2021-2, and 313000/2021-2. References Garrido, J., Vazquez, F., and Morilla, F. (2014). Inverted decoupling internal model control for square stable multivariable time delay systems. J. Process Control 24, 1710–1719. doi:10.1016/j.jprocont.2014.09.003 Alevisakis, G., and Seborg, D. (1973). An extension of the Smith Predictor method to multivariable linear systems containing time delays. Int. J. Control 3, 541–551. doi:10.1080/00207177308932401 Garrido, J., Vázquez, F., Morilla, F., and Normey-Rico, J. E. (2016). Smith predictor with inverted decoupling for square multivariable time delay systems. Int. J. Syst. Sci. 47, 374–388. doi:10.1080/00207721.2015.1067338 Aström, K., and Hägglund, T. (1995). PID controllers: Theory, design and tuning. Reseach Tirangle Park, NC: Instrument Society of America. Aström, K., and Hägglund, T. (1995). PID controllers: Theory, design and tuning. Reseach Tirangle Park, NC: Instrument Society of America. Bezerra-Correia, W., Claure-Torrico, B., and Olímpio-Pereira, R. D. (2017). Optimal control of MIMO dead-time linear systems with dead- time compensation structure. Dyna 84, 62–71. doi:10.15446/dyna. v84n200.54348 Giraldo, S. A. C., Flesch, R. C. C., Normey-Rico, J. E., and Sejas, M. Z. P. (2018). A method for designing decoupled filtered Smith predictor for square MIMO systems with multiple time delays. IEEE Trans. Ind. Appl. 54, 6439–6449. doi:10.1109/tia. 2018.2849365 Chen, J., He, Z.-F., and Qi, X. (2011). A new control method for MIMO first order time delay non-square systems. J. Process Control 21, 538–546. doi:10.1016/j. jprocont.2011.01.007 Guo, M., and Peng, Y. (2011). The control method of multivariable time-delay square system containing right half plane zeros. Procedia Eng. 15, 1004–1009. doi:10.1016/j.proeng.2011.08.186 Chuong, V. L., Vu, T. N. L., Truong, N. T. N., and Jung, J. H. (2019). An analytical design of simplified decoupling Smith predictors for multivariable processes. Appl. Sci. 9, 2487. doi:10.3390/app9122487 Jerome, N., and Ray, W. (1986). High performance multivariable control strategies for systems having time delays. AIChE J. 32, 914–931. doi:10.1002/aic.690320603 Flesch, R. C., Normey-Rico, J. E., and Flesch, C. A. (2017). A unified anti-windup strategy for SISO discrete dead-time compensators. Control Eng. Pract. 69, 50–60. doi:10.1016/j.conengprac.2017.09.002 Jin, Q., Hao, F., and Wang, Q. (2013). A multivariable IMC-PID method for non- square large time delay systems using NPSO algorithm. J. Process Control 23, 649–663. doi:10.1016/j.jprocont.2013.02.007 Flesch, R. C., Torrico, B. C., Normey-Rico, J. E., and Cavalcante, M. U. (2011). Unified approach for minimal output dead time compensation in MIMO processes. J. Process Control 21, 1080–1091. doi:10.1016/j.jprocont.2011.05.005 Lima, D. M., Maia Santos, T. L., and Normey-Rico, J. E. (2015). References “A robust predictor for nonlinear systems with dead time,” in 2015 54th IEEE Conference on Decision and Control (CDC), 7548–7553. doi:10.1109/CDC.2015.7403411 Lima, D. M., Normey-Rico, J. E., and Santos, T. L. M. (2016). Temperature control in a solar collector field using filtered dynamic matrix control. ISA Trans. 62, 39–49. doi:10.1016/j.isatra.2015.09.016 Gálvez-Carrillo, M., De Keyser, R., and Ionescu, C. (2007). Application of a smith predictor based nonlinear predictive controller to a solar power plant. IFAC Proc. Vol. 40, 414–419. doi:10.3182/20070822-3-ZA-2920.00068 Gálvez-Carrillo, M., De Keyser, R., and Ionescu, C. (2007). Application of a smith predictor based nonlinear predictive controller to a solar power plant. IFAC Proc. Vol. 40, 414–419. doi:10.3182/20070822-3-ZA-2920.00068 García, P., and Albertos, P. (2010). Dead-time-compensator for unstable MIMO systems with multiple time delays. J. Process Control 20, 877–884. doi:10.1016/j. jprocont.2010.05.009 Liu, T., Zhang, W., and Gao, F. (2007). Analytical decoupling control strategy using a unity feedback control structure for MIMO processes with time delays. J. Process Control 17, 173–186. doi:10.1016/j.jprocont.2006.08.010 Frontiers in Control Engineering frontiersin.org 16 Normey-Rico et al. 10.3389/fcteg.2022.953768 Maciejowski, J. (1994). Robustness of multivariable Smith predictors. J. Process Control 4, 29–32. doi:10.1016/0959-1524(94)80021-9 Santos, T. L., Silva, B. P., and Uzêda, L. (2017). Multivariable filtered Smith predictor for systems with sinusoidal disturbances. Int. J. Syst. Sci. 48, 2182–2194. doi:10.1080/00207721.2017.1309591 Mirkin, L., Palmor, Z. J., and Shneiderman, D. (2011). Dead-time compensation for systems with multiple i/o delays: A loop-shifting approach. IEEE Trans. Autom. Contr. 56, 2542–2554. doi:10.1109/tac.2011.2108530 Santos, T. L., Torrico, B. C., and Normey-Rico, J. E. (2016). Simplified filtered Smith predictor for MIMO processes with multiple time delays. ISA Trans. 65, 339–349. doi:10.1016/j.isatra.2016.08.023 Morari, M., and Zafiriou, E. (1989). Robust process control. Englewood Cliffs, NJ: Prentice-Hall. Shaqarin, T., Al-Rawajfeh, A., Hajaya, M., Alshabatat, N., and Noack, B. R. (2019). Model-based robust H∞control of a granulation process using Smith predictor with reference updating. J. Process Control 77, 38–47. doi:10.1016/j. jprocont.2019.03.003 Normey-Rico, J., and Camacho, E. (2007). Control of dead-time processes. London: Springer. Normey-Rico, J., and Camacho, E. (2008). Dead-time compensators: A survey. Control Eng. Pract. 16, 407–428. doi:10.1016/j.conengprac.2007.05.006 Skogestad, S., and Postlethwaite, I. (2005). Multivariable feedback control. Analysis and design. New York: Wiley. Normey-Rico, J. E., and Camacho, E. F. (2009). Unified approach for robust dead- time compensator design. J. Process Control 19, 38–47. doi:10.1016/j.jprocont.2008. 02.003 Smith, O. (1957). The biosynthesis of some connective tissue components. Prog. Biophys. Biophys. Chem. 53, 217–240. doi:10.1016/s0096-4174(18)30149-5 Takatsu, H., Itoh, T., and Araki, M. References (1998). Future needs for the control theory in industries—Report and topics of the control technology survey in Japanese industry. J. Process Control 8, 369–374. doi:10.1016/s0959-1524(98)00019-5 Normey-Rico, J. E., Garcia, P., and Gonzalez, A. (2012). Robust stability analysis of filtered Smith predictor for time-varying delay processes. J. Process Control 22, 1975–1984. doi:10.1016/j.jprocont.2012.08.012 Tang, Y., Du, F., Cui, Y., and Zhang, Y. (2018). New Smith predictive fuzzy immune pid control algorithm for MIMO networked control systems. EURASIP J. Wirel. Commun. Netw. 1, 212–215. doi:10.1186/s13638-018-1229-8 Ogunnaike, B., and Ray, W. (1979). Multivariable controller design for linear systems having multiple time delays. AIChE J. 25, 1043–1057. doi:10.1002/aic. 690250616 Torrico, B. C., Cavalcante, M. U., Braga, A. P., Normey-Rico, J. E., and Albuquerque, A. A. (2013). Simple tuning rules for dead-time compensation of stable, integrative, and unstable first-order dead-time processes. Ind. Eng. Chem. Res. 52, 11646–11654. doi:10.1021/ie401395x Palmor, Z. (1996). The control handbook. Time delay compensation: Smith predictor and its modifications. Mumbai: CRC Press and IEEE Press. chap. 10.8. 224–238. Rao, A., and Chidambaram, M. (2006). Smith delay compensator for multivariable non-square systems with multiple time delays. Comput. Chem. Eng. 30, 1243–1255. doi:10.1016/j.compchemeng.2006.02.017 Visioli, A., and Zhong, Q.-C. (2011). Control of integral processes with dead time, 54. London: Springer. Zhang, W., and Lin, C. (2006). Multivariable Smith predictors design for nonsquare plants. IEEE Trans. Control Syst. Technol. 14, 1145–1149. doi:10. 1109/tcst.2006.880219 Sánchez-Peña, R. S., Bolea, Y., and Puig, V. (2009). MIMO Smith predictor: Global and structured robust performance analysis. J. Process Control 19, 163–177. doi:10.1016/j.jprocont.2007.12.004 Zhang, W., Wang, Y., Liu, Y., and Zhang, W. (2016). Multivariable disturbance observer-based h2 analytical decoupling control design for multivariable systems. Int. J. Syst. Sci. 47, 179–193. doi:10.1080/00207721.2015.1036479 Santos, T. L., Flesch, R. C., and Normey-Rico, J. E. (2014). On the filtered Smith predictor for MIMO processes with multiple time delays. J. Process Control 24, 383–400. doi:10.1016/j.jprocont.2014.02.011 Zheng, W., Wang, H.-b., and Zhang, Z.-m. (2017). Dynamic output feedback compensation control for discrete closed-loop nonlinear system with multiple time- delays. Math. Problems Eng. 2017, 1–11. doi:10.1155/2017/6427807 Santos, T. L., Franklin, T. S., and Torrico, B. C. (2021). Anti-windup strategy for processes with multiple delays: A predictor-based approach. J. Frankl. Inst. 358, 1812–1838. doi:10.1016/j.jfranklin.2020.12.022 Frontiers in Control Engineering Frontiers in Control Engineering 17 frontiersin.org
W4387524638.txt
https://www.ritpu.ca/fr/../files/numeros/119/ritpu-v20n2-06.pdf
fr
Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes
Revue internationale des technologies en pédagogie universitaire
2,023
cc-by
7,770
RITPU|IJTHE ritpu.org | ijthe.org Revue internationale des technologies en pédagogie universitaire International Journal of Technologies in Higher Education Volume 20, n°2, p. 53-67 ISSN 1708-7570 2023 Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Benefits and Limitations of Distant Learning for Pre-Service Teacher Education https://doi.org/10.18162/ritpu-2023-v20n2-06 Chantal TREMBLAY a Université du Québec à Montréal, Canada Mis en ligne : 11 octobre 2023 Résumé Bien que la pandémie ait montré la faisabilité d’offrir des cours de formation initiale à l’enseignement dans une modalité à distance, il semble important de s’interroger sur les apports et les limites de cette modalité pour soutenir l’apprentissage. S’appuyant sur le concept de la valeur ajoutée pédagogique (VAP), cette réflexion compare les apports et limites des modalités à distance et en présence à l’aide d’écrits scientifiques pertinents. Trois facteurs qui peuvent influencer la VAP seront ainsi discutés : l’alignement pédagogique, l’accessibilité des études et l’intégration des étudiants et étudiantes à leur profession future. Il sera alors recommandé d’analyser la VAP pour faire des choix de modalité adéquats, menant à conclure que les formations hybrides seraient préférables pour bénéficier des apports de chacune d’elles. Mots-clés Formation à distance, formation hybride, valeur ajoutée pédagogique, développement de compétences, technologies d’apprentissage, enseignement supérieur Abstract Although the pandemic has shown the feasibility of offering pre-service teacher education courses in a distance learning mode, it seems important to weigh the benefits and limitations of this mode in terms of how it supports learning. Based on the concept of pedagogical added value (PAV), this reflection draws on relevant scientific literature to compare the benefits and limitations of distance and face-to-face learning modes. Three factors that can influence PAV will be discussed: pedagogical alignment, accessibility of studies, and the integration of students into their future profession. This will be followed by a recommendation to analyze the PAV to make appropriate learning mode choices, leading to the conclusion that hybrid training would be preferable so as to offer the benefits of each mode. (a) Département de didactique. © Autrice. Cette œuvre, disponible à https://doi.org/10.18162/ritpu-2023-v20n2-06, est distribuée sous licence Creative Commons Attribution 4.0 International http://creativecommons.org/licences/by/4.0/deed.fr 53 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Keywords Distance learning, hybrid learning, pedagogical added value, skills development, learning technologies, higher education Introduction La fermeture des campus d’universités en 2020 a engendré un transfert massif de cours en présentiel vers une modalité à distance, synchrone ou asynchrone, et ce, pendant plus d’une année universitaire. Une très grande majorité de cours de programmes universitaires, incluant ceux visant la formation initiale à l’enseignement, ont été assurés à distance à au moins une reprise. Cet évènement a montré la faisabilité d’offrir ces programmes dans une modalité majoritairement, voire, dans certains cas, entièrement à distance. Depuis, on assiste à une rupture avec le modèle traditionnel d’enseignement universitaire en présentiel, qui s’observe par une augmentation de l’offre de cours et de formations assurés dans une modalité à distance depuis la réouverture des campus (Johnson, 2021a; Parent et al., 2021; Pelletier et al., 2021, 2022). Par exemple, la TÉLUQ a récemment lancé un programme de 2e cycle en éducation (D.E.S.S. de 30 crédits) qui se donnera entièrement à distance et qui mènera au brevet d’enseignement, nécessaire pour devenir une personne enseignante légalement qualifiée au Québec (Université TÉLUQ, 2023). Devant cette hausse marquée de la formation à distance (FAD) en période postpandémique, il semble nécessaire de se questionner sur ses apports, ses enjeux et ses limites. Plus globalement, quelle ou quelles modalités sont préférables pour mieux soutenir les apprentissages et faciliter le développement de compétences? Pour y répondre, ce texte de réflexion pédagogique s’appuie sur le concept de la valeur ajoutée pédagogique (Beaudoin et al., 2022; Docq et al., 2010) et présente des enjeux et des apports potentiels de la FAD comparativement à la formation en présentiel pour soutenir l’apprentissage et le développement des compétences des futurs enseignants et enseignantes au Québec. La littérature actuelle suggère que les définitions des termes liés à la FAD et à la formation hybride ne font pas consensus au sein de la communauté (Freiman et al., 2021; Forget-Dubois, 2020; Johnson, 2021b; Joseph. et Dallaire, 2015; Parent et al., 2021; Pelletier et al., 2021), surtout depuis les transformations numériques engendrées par la pandémie. Ainsi, pour clarifier cette réflexion, les définitions des termes utilisés dans ce texte qui réfèrent à la FAD sont fournies à la section suivante, puis le référentiel de compétences de la profession enseignante (Ministère de l’Éducation du Québec [MEQ], 2020) est brièvement exposé pour présenter le contexte. La question qui anime cette réflexion est ensuite soumise, menant à expliquer le concept de valeur ajoutée pédagogique (Beaudoin et al., 2022; Docq et al., 2010) et sa pertinence pour choisir une modalité (à distance, en présence, hybride) adéquate. La réflexion s’articulera alors autour de trois éléments à considérer pour analyser la valeur ajoutée pédagogique de chaque modalité dans le contexte de cours de formation initiale à l’enseignement au Québec : l’alignement pédagogique (Biggs, 1999), l’accessibilité des études et l’intégration des étudiants et étudiantes à leur profession. La conclusion propose alors un cadre d’analyse pour faire des choix de modalités éclairés, soulignant ainsi que les programmes de formations hybrides sont potentiellement les plus prometteurs pour mieux soutenir l’apprentissage de ces étudiants et étudiantes. 1. Une définition des termes relatifs à la formation à distance La formation à distance (FAD) est un terme employé abondamment dans la littérature qui ne revêt pas systématiquement le même sens (Johnson, 2021b). Il importe donc de préciser les définitions 2023 - Revue internationale des technologies en pédagogie universitaire, 20(2) ritpu.org 54 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes retenues des modalités présentées dans cette réflexion. En s’appuyant sur plusieurs écrits issus de la littérature scientifique et d’organismes publics qui les définissent (Bates, 2022; Forget-Dubois, 2020; MEQ, 2022; Freiman et al., 2021; Johnson, 2021b; Parr, 2019; Pelletier et al., 2021), les paragraphes suivants expliquent ce que nous entendons par modalités de formation à distance, en présence et hybride. Il est à noter que pour des raisons de concision, la variante comodale de la formation hybride, qui est composée de séances synchrones où des étudiants ou étudiantes sont en présence et d’autres à distance (McGee et Reis, 2012), ne sera pas abordée dans ce texte. En cohérence avec la suite de cette réflexion, qui s’appuie sur le concept de la valeur ajoutée pédagogique, ces définitions de modalités sont adaptées à une séquence d’enseignementapprentissage (aussi nommée séquence pédagogique ou didactique dans la littérature) qui correspond à un « regroupement logique d’éléments ciblés par les enseignants et que les élèves doivent apprendre dans une matière scolaire donnée » (Centre de transfert pour la réussite éducative du Québec, 2018). En enseignement supérieur, un cours est donc composé de plusieurs séquences pédagogiques, chacune visant l’atteinte d’un ou plusieurs objectifs d’apprentissage. Ainsi, la modalité d’une séquence d’enseignement-apprentissage sera qualifiée de FAD si elle comporte les trois caractéristiques suivantes, qui sont les plus fréquemment observées dans la littérature pour définir cette modalité selon le Conseil supérieur de l’éducation (CSE; ForgetDubois, 2020). Premièrement, en se référant à la définition des modalités « remote learning » de Johnson (2021b), « fully online (distance) » de Bates (2022), de la FAD du Campus numérique (2022), ainsi que des éléments des définitions scientifiques de la FAD recensées par le CSE (Forget-Dubois, 2020), on peut conclure que la FAD implique une distance physique entre l’étudiant ou l’étudiante et le campus universitaire. Cette distance signifie que toutes les activités pédagogiques, synchrones et asynchrones, peuvent être effectuées à distance : la présence physique étudiante sur le campus n’est donc jamais exigée. Deuxièmement, cette absence de présence physique amène à une « distance temporelle » (Forget-Dubois, 2020, p. 21) dans les échanges entre l’enseignant et l’étudiant et entre les étudiants causée par l’utilisation d’outils numériques de communication asynchrones (ex. courriel, forum, messagerie). Précisons que cette deuxième caractéristique est cohérente avec la définition de l’apprentissage en ligne de Freiman et al. (2021), qui utilisent ce terme pour définir une catégorie de FAD où les interactions sociales sont importantes et très présentes pour soutenir l’apprentissage. La troisième caractéristique de cette définition de la FAD vise à préciser le contexte actuel de médiatisation. Ainsi, cette modalité implique minimalement l’usage d’un environnement numérique d’apprentissage (ex. Moodle) ou d’une classe virtuelle (ex. Teams). Autrement dit, le contexte actuel postpandémique amène à exclure des modalités de FAD qualifiées de « offline distance learning » par Johnson (2021b), où étudiants et étudiantes n’ont accès qu’à des ressources pédagogiques tangibles qui peuvent leur être envoyées par la poste, car elles ne reflètent plus des pratiques courantes en FAD. En enseignement supérieur, l’usage d’environnements numériques d’apprentissage ou de classes virtuelles et le recours à différents outils et ressources numériques pour l’enseignement en présentiel sont très fréquents (Bates, 2022; Johnson, 2021b; Pelletier et al., 2021, 2022). Les étudiants et étudiantes peuvent, entre les séances de cours, consulter des ressources ou exploiter des outils numériques leur permettant de réaliser des travaux et d’autres activités d’apprentissage (Forget-Dubois, 2020). Les usages de classes virtuelles, d’environnements numériques d’apprentissage et de leurs outils pédagogiques ont connu une hausse fulgurante depuis la pandémie, qui devrait se maintenir à long terme (Pelletier et al., 2021, 2022). Ainsi, pour distinguer une séquence offerte dans une modalité « en présence » d’une séquence « hybride », un seul critère sera utilisé dans ce texte, soit l’importance des activités en présentiel qui exigent la présence 2023 – International Journal of Technologies in Higher Education, 20(2) ijthe.org 55 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes étudiante sur le campus. Une séquence en présence comporte un nombre important d’activités qui se déroulent sur le campus et qui exigent une telle présence dans ces lieux. Par exemple, un cours en présence de trois crédits universitaires au Québec est généralement composé d’une séance hebdomadaire de trois heures qui se tient dans une salle de classe, un laboratoire, un gymnase ou tout autre espace physique de l’établissement d’enseignement. Suivant cette logique et en cohérence avec la définition de la modalité « in-person learning » de Johnson (2021b), une séquence en présence d’une durée de quatre semaines inclurait alors quatre rencontres dans une salle de classe physique, où il y aurait au moins une enseignante ou un enseignant et plusieurs étudiantes et étudiants présents qui réaliseraient des activités d’apprentissage. La modalité hybride se distingue donc de la modalité en présentiel par le nombre limité d’activités en présentiel. Cette définition fait référence à celle de la modalité hybride ou flexible de Bates (2022), qui explique que les activités en présentiel de cette modalité sont peu fréquentes et qu’elles visent surtout à permettre aux étudiants et étudiantes de réaliser des tâches importantes pour leur apprentissage qui peuvent difficilement être effectuées à distance. L’auteur précise que la modalité hybride implique de revoir l’ensemble des activités d’apprentissage du cours (ou de la séquence) pour tirer profit de chaque modalité (en présence et à distance). Par exemple, une séquence d’une durée de quatre semaines serait qualifiée d’hybride si elle ne contenait qu’une seule séance sur le campus, qui serait consacrée à la réalisation d’une activité pédagogique qui ne peut s’effectuer à distance, comme une simulation d’enseignement. Bien que cette distinction entre la modalité « en présence » et la modalité « hybride » ne soit pas observée dans toutes les définitions consultées pour cette réflexion, elle apparaît pertinente pour effectuer l’analyse de la valeur ajoutée pédagogique des modalités et faire des choix éclairés. En d’autres termes, cette distinction amène à réfléchir à la pertinence de planifier des rencontres en présence de façon régulière (modalité en présence) ou ponctuelle (modalité hybride) pour soutenir judicieusement les apprentissages dans le contexte de la formation à l’enseignement. Cette notion de pertinence est à la base de la question qui guide cette réflexion, présentée à la section suivante. 2. Le contexte et la question de cette réflexion Rappelons que cette réflexion pédagogique se situe dans le contexte de la formation initiale à l’enseignement au Québec. En 2020, le ministère de l’Éducation du Québec a mis à jour le référentiel de compétences de la profession enseignante (MEQ, 2020) qui sert de base aux programmes de formation à l’enseignement menant à l’obtention d’un brevet d’enseignement, nécessaire pour acquérir une qualification légale d’enseignement aux niveaux primaire et secondaire. Bien qu’il n’y ait ni brevet ni formation obligatoire pour enseigner aux niveaux postsecondaires au Québec (collégial et universitaire), plusieurs universités offrent des programmes de formation à la pédagogie de l’enseignement supérieur, dont les compétences visées sont apparentées à celles du référentiel ou basées sur celles-ci. Le référentiel est composé de treize compétences qui sont indiquées au tableau 1 et qui couvrent toutes les activités liées à la profession enseignante, incluant les compétences directement liées à l’enseignement (compétences 1 à 8), mais aussi des compétences associées à la collaboration (compétences 9 et 10), au développement professionnel (compétence 11), à l’utilisation du numérique à des fins pédagogiques (compétence 12) ainsi qu’à l’agir éthique (compétence 13). Avant la pandémie, la majorité de ces programmes de formation étaient offerts en présence. Or, avec la fermeture des campus en 2020-2021, de nombreux cours de ces programmes ont été convertis pour être offerts à distance. Depuis la réouverture des campus, plusieurs se questionnent sur la pertinence de revenir en présence ou de maintenir l’enseignement à distance. Bien qu’il 2023 - Revue internationale des technologies en pédagogie universitaire, 20(2) ritpu.org 56 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes existe de nombreuses études sur l’efficacité, les retombées ou les avantages des modalités à distance ou hybrides (ex. Castro et Tumibay, 2021; McGee et Reis, 2012; Raes et al., 2020), la diversité des contextes et des contenus disciplinaires ne permet pas de conclure à un consensus sur la supériorité d’une modalité relativement à une autre pour soutenir l’apprentissage. De plus, d’autres études montrent des résultats mitigés quant à l’appréciation des enseignants et enseignantes et des étudiants et étudiantes relativement aux modalités à distance (Beaudoin et al., 2022; CSE, 2021; Johnson, 2021a; Legault et Fichten, 2022; Parent et al., 2021; Pelletier et al., 2021). Face à ces constats, on peut se poser la question suivante : Quelle ou quelles modalités choisir pour mieux soutenir les apprentissages et faciliter le développement des compétences professionnelles des futurs enseignants et enseignantes? Autrement dit, sur quoi s’appuyer pour faire des choix de modalités éclairés? Tableau 1 Treize compétences du référentiel de compétences de la profession enseignante. Source : Ministère de l’Éducation du Québec (2020, p. 43). no Compétence 1 Agir en tant que médiatrice ou médiateur de culture 2 Maîtriser la langue d’enseignement 3 Planifier les situations d’enseignement-apprentissage 4 Mettre en œuvre les situations d’enseignement-apprentissage 5 Évaluer les apprentissages 6 Gérer le fonctionnement du groupe-classe 7 Tenir compte de l’hétérogénéité des élèves 8 Soutenir le plaisir d’apprendre 9 S’impliquer activement au sein de l’équipe-école 10 Collaborer avec la famille et les partenaires de la communauté 11 S’engager dans un développement professionnel continu et dans la vie de la profession 12 Mobiliser le numérique 13 Agir en accord avec les principes éthiques de la profession Nous proposons donc d’explorer le concept de la valeur ajoutée pédagogique du numérique (Beaudoin et al., 2022; Docq et al., 2010) pour y répondre. Cette proposition est cohérente avec le CSE qui, dans l’édition 2021 de son rapport sur l’état et les besoins de l’éducation, rappelle l’importance de planifier la FAD en s’appuyant sur des principes pédagogiques et en considérant les enjeux qu’elle soulève. Ce concept est défini à la section suivante, puis nous poursuivons en présentant trois éléments importants à considérer pour analyser la valeur ajoutée de la FAD, relativement à la formation en présence. 3. La valeur ajoutée pédagogique (VAP) du numérique et de la FAD Selon Beaudoin et al. (2022), la valeur ajoutée pédagogique (VAP) du numérique correspond à une utilisation qui permet d’améliorer, de bonifier ou d’enrichir les apprentissages et le développement des compétences des étudiants et étudiantes. Cette définition peut se transposer à la FAD, ainsi celle-ci possède une VAP si elle mène à des gains d’apprentissage, comparativement à la modalité en présence. Docq et al. (2010), s’appuyant sur Lebrun et al. (2010), proposent trois catégories de VAP pour analyser des dispositifs de formation hybrides, qui peuvent également être 2023 – International Journal of Technologies in Higher Education, 20(2) ijthe.org 57 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes adaptées pour le contexte de la FAD. Premièrement, la modalité doit permettre une meilleure centration sur l’apprentissage, par exemple par l’intégration d’activités d’apprentissage actif. Docq et al. (2010) proposent cinq facteurs qui décrivent comment l’utilisation du numérique peut mener à une meilleure centration sur l’apprentissage. Le premier correspond à une amélioration de la disponibilité des ressources informationnelles, dont celles qui permettent une ouverture sur la société. Le deuxième facteur est lié à la motivation : la modalité doit mener à une augmentation de l’implication des étudiantes et étudiants dans les activités d’apprentissage ou elle doit leur permettre de se familiariser avec des outils professionnels qu’ils devront maîtriser lors de leur entrée sur le marché du travail. Le troisième facteur est directement lié aux activités d’apprentissage. Il y a donc une valeur ajoutée pédagogique de la modalité si les activités qui y sont associées permettent de stimuler l’apprentissage, de développer des habiletés cognitives de haut niveau ou de soutenir l’apprentissage autorégulé. Le quatrième facteur porte sur le potentiel de la modalité pour susciter des interactions, lorsque celle-ci rend possible la réalisation d’activités collaboratives ou améliore les interactions entre enseignants et étudiants. Enfin, le dernier facteur de VAP de la modalité associée à une centration sur l’apprentissage est son potentiel pour amener les étudiants et étudiantes à réaliser des productions visibles qui témoignent de leurs apprentissages. La deuxième catégorie de VAP de la modalité correspond à l’amélioration de la prise en considération du contexte actuel et des besoins des étudiants et étudiantes. Autrement dit, la modalité apporte une VAP si elle mène à des gains en matière de rétention étudiante, qui se manifesteront ensuite par des gains d’apprentissage. Enfin, la troisième catégorie porte sur l’influence de la modalité pour stimuler le développement professionnel des enseignants et enseignantes. Si l’adoption d’une modalité implique une amélioration des pratiques pédagogiques, alors cela devrait éventuellement se traduire par des gains d’apprentissage. Ainsi, ce concept amène à réfléchir aux apports de la FAD sous l’angle des gains d’apprentissage qu’elle peut engendrer. Ses limites concernent alors les difficultés d’apprentissage, les échecs ou les abandons qu’elle peut générer. Bien que plusieurs facteurs puissent être considérés pour déterminer la valeur ajoutée pédagogique de la FAD, nous proposons d’en analyser trois qui nous semblent essentiels, car ils sont applicables à de nombreuses séquences d’enseignementapprentissage associées à la formation à l’enseignement au Québec. 4. Trois facteurs qui influencent la valeur ajoutée pédagogique d’une modalité Dans cette section, nous présentons trois facteurs qui peuvent influencer la valeur ajoutée pédagogique (VAP) de la FAD. D’abord, nous analysons les limites des apports potentiels de la modalité pour améliorer l’alignement pédagogique de la séquence d’enseignement-apprentissage. Ensuite, nous examinons l’influence de la modalité sur l’accessibilité aux études, puis nous terminons par un facteur particulièrement important dans le contexte de la formation à l’enseignement, soit l’intégration des étudiants et étudiantes à leur profession future. 4.1 L’alignement pédagogique Un premier facteur qui permet d’analyser la VAP de la FAD correspond à son potentiel pour améliorer l’alignement pédagogique de la séquence d’enseignement-apprentissage, ce qui signifierait que la modalité permet une meilleure centration sur l’apprentissage. L’alignement pédagogique, au sens de Biggs (1999), consiste à maintenir une cohérence entre les objectifs d’apprentissage, les activités pédagogiques et les modalités d’évaluation d’un cours (figure 1). Pour l’enseignant ou l’enseignante, il s’agit donc de concevoir des activités d’apprentissage qui 2023 - Revue internationale des technologies en pédagogie universitaire, 20(2) ritpu.org 58 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes sont efficaces et pertinentes pour que les étudiantes et étudiants puissent atteindre les objectifs. Ensuite, il ou elle doit construire des évaluations qui contiennent des tâches représentatives de celles qui ont été effectuées lors des activités d’apprentissage pour assurer leur cohérence avec les objectifs d’apprentissage visés. Figure 1 L’alignement pédagogique de Biggs (1999) L’alignement pédagogique repose principalement sur la taxonomie du domaine cognitif de Bloom (1956) révisée par Anderson et al. (2001). Cette taxonomie présente six niveaux cognitifs, dont les deux premiers (mémoriser, comprendre) portent sur l’acquisition de connaissances et les suivants décrivent des habiletés intellectuelles qui permettent leur mobilisation (appliquer, analyser, évaluer, créer). La taxonomie est représentée sous la forme d’une pyramide (figure 2) pour illustrer la progression des apprentissages : une personne doit d’abord comprendre un concept avant d’être en mesure de le mobiliser pour analyser une situation, par exemple. Cette pyramide montre également un degré d’abstraction qui augmente à chaque niveau. En général, les trois premiers niveaux sont qualifiés de « bas niveaux taxonomiques cognitifs », car ils font référence à des apprentissages plus concrets que ceux des trois derniers niveaux, qualifiés de « hauts niveaux taxonomiques cognitifs ». Figure 2 Les niveaux de la taxonomie du domaine cognitif de Bloom (1956) révisée par Anderson et al. (2001) Par exemple, une séquence d’enseignement-apprentissage dont l’objectif d’apprentissage correspond à créer un plan de leçon (niveau créer de la taxonomie) devrait d’abord inclure des activités pédagogiques qui permettent de comprendre ce qu’est un plan de leçon (niveau 2023 – International Journal of Technologies in Higher Education, 20(2) ijthe.org 59 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes comprendre) et poursuivre par des activités où l’étudiant ou l’étudiante crée certaines parties d’un plan en suivant un modèle très structuré (niveau appliquer). Dans un troisième temps, la séquence devrait permettre aux étudiants et étudiantes d’analyser différents plans et d’évaluer leurs forces, limites et améliorations possibles (niveaux analyser et évaluer), puis elle se terminerait par des activités les amenant à créer un plan de leçon complet et applicable dans un contexte réel (niveau créer). Ainsi, cette séquence inclurait des activités d’apprentissage cohérentes avec le niveau taxonomique cognitif de l’objectif, donc il y aurait un alignement pédagogique entre ces deux éléments. Pour poursuivre l’alignement avec les évaluations, il serait donc nécessaire d’en concevoir en cohérence avec les niveaux taxonomiques cognitifs de chaque activité d’apprentissage de la séquence et de l’objectif visé. Au moins une évaluation devrait alors permettre aux étudiants et étudiantes de montrer leur capacité à créer un plan de leçon complet. L’alignement pédagogique permet donc une meilleure centration des pratiques sur l’apprentissage par son influence sur le choix des activités et des tâches qu’effectuent les étudiants et étudiantes en classe. Ainsi, un facteur à considérer pour analyser la VAP de la FAD consiste à déterminer si cette modalité permet un meilleur alignement pédagogique de la séquence d’enseignementapprentissage que la formation en présence. À ce sujet, Grenon (2020) recommande de s’assurer de l’alignement pédagogique lors de la conception de cours en ligne ou hybride, car cela peut influencer les apprentissages. Dans un même ordre d’idées, Lafleur et al. (2021) soutiennent l’importance d’une « cohérence pédagonumérique » (p. 29), soit l’intégration des modalités et les potentialités du numérique au concept d’alignement pédagogique. En FAD, cela suppose d’évaluer la nature des activités d’apprentissage qui peuvent être effectuées grâce à l’utilisation d’outils et de ressources numériques, pour s’assurer qu’elles sont cohérentes avec les objectifs visés. Dans les paragraphes qui suivent, nous présentons donc trois enjeux et trois apports potentiels de la FAD en lien avec l’alignement pédagogique. Comme chaque contexte est différent et qu’il peut mener à une évaluation différente de la valeur ajoutée de la FAD, il importe de préciser que ces enjeux et ces apports potentiels sont présentés à titre d’exemples, pour guider la réflexion d’une personne qui souhaite utiliser la VAP pour choisir une modalité pertinente pour une séquence d’enseignement-apprentissage donnée. 4.1.1 Trois enjeux de la FAD liés à l’alignement pédagogique Un premier enjeu de la FAD concerne la réalisation de tâches de haut niveau taxonomique cognitif par les étudiants et étudiantes. À ce sujet, Grenon (2020) s’appuie sur une recension d’écrits sur la formation hybride pour soutenir que certaines ressources numériques couramment utilisées en FAD (vidéos, balados, sites Web) seraient inefficaces pour développer des connaissances de haut niveau taxonomique cognitif. De plus, l’étude de Guyet (2021) témoigne de difficultés rencontrées par des étudiants et étudiantes universitaires ayant eu à accomplir des tâches liées à l’analyse ou à l’évaluation lors de situations de travail coopératif à distance. Dans cette étude, des simulations virtuelles ont été employées pour développer la capacité de raisonnement clinique d’étudiants et étudiantes en kinésiologie, qui devaient collaborer pour effectuer l’analyse de dossiers de patients et patientes. Les résultats montrent qu’une majorité des personnes participantes ont éprouvé des difficultés à communiquer avec les membres de leur équipe, ce qui a eu pour effet de réduire leur perception du développement de compétence. Bref, comme le soutient Grenon (2020), il est essentiel de porter une attention particulière aux tâches que les étudiants et étudiantes effectuent à distance pour s’assurer de l’alignement pédagogique et, conséquemment, de l’atteinte des objectifs d’apprentissage. 2023 - Revue internationale des technologies en pédagogie universitaire, 20(2) ritpu.org 60 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Un second enjeu lié à l’alignement pédagogique et exprimé par le CSE (2021) concerne la formation pratique dans des programmes professionnels. En effet, plusieurs études présentent des difficultés vécues par des étudiants et étudiantes lors de séances d’apprentissage expérientiel à distance, notamment lors d’activités de modelage (Kidd et Murray, 2020), pour la mise en œuvre d’une activité de laboratoire en sciences (Legault et Fichten, 2022), lors d’ateliers pratiques ou d’activités d’apprentissage qui impliquent l’utilisation de matériel spécialisé (la Velle et al., 2020). Les résultats de l’étude de Latorre-Cosculluela et al. (2021) suggèrent que la réalisation d’activités d’apprentissage actif dans un contexte de classe inversée à distance mène à des apprentissages moindres que lorsque celles-ci sont réalisées en présence. Par ailleurs, la Velle et al. (2020) soutiennent qu’une réduction de temps de classe en présence accordée à des ateliers pratiques pourrait mener à une préparation inadéquate des futurs enseignants et enseignantes. En somme, ces études soulignent que lorsque les étudiantes et étudiants sont à distance, ils peuvent éprouver des difficultés à accomplir des tâches liées à des activités d’apprentissage expérientiel qui s’apparentent à celles qu’ils auront à effectuer dans leur futur contexte professionnel. Par conséquent, cela les empêche d’atteindre le niveau taxonomique cognitif visé et la FAD peut donc causer une rupture d’alignement pédagogique. Leurs apprentissages pourraient alors se limiter au niveau taxonomique cognitif de la compréhension, à cause de leur incapacité à réaliser ces tâches adéquatement. Par ailleurs, un troisième enjeu de la FAD en lien avec l’alignement pédagogique porte sur la cohérence entre les objectifs et les évaluations des apprentissages. Rappelons que pendant la fermeture des campus, les enseignantes et enseignants ont dû adapter leurs évaluations afin qu’elles puissent être réalisées en ligne et à distance, ce qui a constitué un défi pour plusieurs d’entre eux, tant au Québec (Parent et al., 2021) que dans le reste du Canada (Johnson, 2021a). Ces évaluations numériques ont fait l’objet de plusieurs analyses, notamment concernant des types à privilégier pour éviter le plagiat (Hébert et Fontaine, 2022; Kozanitis, 2021). Pour analyser la VAP de la FAD, il semble donc essentiel de déterminer s’il est possible de concevoir des évaluations numériques dont les tâches sont cohérentes avec les niveaux taxonomiques cognitifs des objectifs d’apprentissage. 4.1.2 Trois apports potentiels de la FAD en lien avec l’alignement pédagogique Certaines technologies qui peuvent être employées en FAD pourraient améliorer l’alignement pédagogique d’une séquence d’enseignement-apprentissage. La réalité virtuelle peut améliorer l’authenticité des activités d’apprentissage et permettre aux étudiants et étudiantes de s’expérimenter dans un environnement simulé (Dalgarno et Lee, 2010). Son utilisation pourrait alors améliorer l’alignement pédagogique de la séquence, si les objectifs d’apprentissage font référence à des tâches qui se réalisent uniquement dans un contexte de classe. Aussi, la Velle et al. (2020) proposent d’explorer le potentiel de la réalité virtuelle pour concevoir des activités d’apprentissage qui visent l’atteinte d’objectifs de haut niveau taxonomique cognitifs. Un second apport potentiel de la FAD pour améliorer l’alignement pédagogique d’une séquence concerne l’ajout d’activités d’évaluations formatives intégrées à des outils numériques (ex. questionnaire en ligne) qui permettent d’offrir une rétroaction rapide et adaptée à l’étudiant ou à l’étudiante (Paterson et al., 2020). Combinées à des activités de pratique réflexive et à une rétroaction de l’enseignant ou de l’enseignante, ces activités pourraient mieux soutenir le développement des compétences, comme celles liées à l’enseignement (Kidd et Murray, 2020; la Velle et al., 2020; Legault et Fichten, 2022). 2023 – International Journal of Technologies in Higher Education, 20(2) ijthe.org 61 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Enfin, le troisième apport de la FAD concerne la douzième dimension du référentiel de compétences de la profession enseignante (tableau 1), qui consiste à être capable de mobiliser le numérique à des fins pédagogiques. Autrement dit, pour préparer les futurs enseignants et enseignantes à former à distance, il serait judicieux d’inclure des activités d’apprentissage qui sont basées sur cette modalité, ce qui reflète une valeur ajoutée pédagogique de la FAD. 4.2 L’accessibilité des études Un deuxième facteur qui influence la VAP de la FAD consiste à évaluer son potentiel pour améliorer l’accessibilité des études. Il s’agit de déterminer si la FAD permet une meilleure prise en considération du contexte actuel ou une réponse plus adéquate aux besoins des étudiants et étudiantes (2e catégorie de VAP du modèle de Docq et al.). Pour orienter la réflexion sur ce facteur, deux enjeux importants à considérer sont présentés dans cette section, soit le niveau de compétence numérique des étudiants et étudiantes et leur accès à un environnement propice aux apprentissages. La section se poursuit par une description de trois apports potentiels de la FAD : flexibilité spatio-temporelle, conciliation travail-famille-études et réduction du temps et des coûts de déplacement. Tout d’abord, Lafleur et al. (2021) insistent sur l’importance de « placer l’apprenant au cœur de l’espace pédagonumérique » (p. 29), ce qui signifie prendre en considération non seulement les apprentissages qu’il doit réaliser, mais aussi son accessibilité aux outils et ressources numériques, de même que son niveau de compétence numérique. Dans certains cas, la multiplication des plateformes et le recours à un trop grand nombre d’outils numériques complexifieraient l’apprentissage des étudiants et étudiantes (Parent et al., 2021). Il faudrait donc considérer leur niveau de compétence numérique pour déterminer s’il influence la VAP de la FAD. Autrement dit, il serait judicieux de s’interroger sur la capacité des étudiantes et étudiants à exploiter efficacement les outils et ressources numériques à leur disposition, considérant qu’ils devront apprendre à les utiliser à distance sans disposer du même niveau d’accompagnement qu’il est possible d’offrir en présence. L’accessibilité au numérique et à un environnement propice aux apprentissages en FAD est aussi un enjeu exprimé par les étudiants et étudiantes (CSE, 2021; Legault et Fichten, 2022) et qui préoccupe plusieurs membres de la communauté universitaire québécoise et canadienne (Johnson, 2021b; Parent et al., 2021). Certains étudiants et étudiantes préféreraient la modalité en présence, justement pour bénéficier d’un environnement adéquat (Pelletier et al., 2021). À noter que leur niveau de compétence numérique pourrait influencer cette préférence (CSE, 2021). Le rapport de Pelletier et al. (2022) souligne des préoccupations en matière d’équité qui peuvent émerger lorsque les cours sont transférés dans une modalité à distance. De façon analogue, Beaudoin et al. (2022) font ressortir l’importance de considérer les inégalités liées au numérique dans la décision de l’intégrer dans les activités d’apprentissage. Pour surmonter ces enjeux, Pelletier et al. (2022) soutiennent que les établissements d’enseignement devraient fournir aux étudiantes et étudiants le soutien, l’environnement et les ressources nécessaires pour qu’ils puissent accéder et participer aux cours offerts à distance. Ils ajoutent que ceux-ci devraient avoir l’occasion de s’exprimer et d’être entendus quant aux décisions prises relativement aux modalités de leur formation. Autrement dit, ils suggèrent de faire des choix de modalités qui susciteront l’adhésion et l’approbation étudiantes, donc qui amélioreront l’accessibilité des études, ce qui est cohérent avec le concept de VAP. En somme, il semble important de s’assurer que la FAD ne réduise pas l’accessibilité des études, notamment en évaluant le soutien, l’accompagnement ou les ressources dont peuvent bénéficier les étudiants et étudiantes, pour éviter que cela puisse nuire à la VAP. 2023 - Revue internationale des technologies en pédagogie universitaire, 20(2) ritpu.org 62 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Cependant, la FAD pourrait aussi améliorer l’accessibilité des études de certaines catégories étudiantes, permettant alors d’augmenter sa valeur ajoutée pédagogique. Premièrement, plusieurs études montrent que des étudiants et étudiantes préfèrent la FAD parce qu’elle permet une flexibilité spatio-temporelle, c’est-à-dire qu’elle leur permet de réaliser leurs activités d’apprentissage dans le lieu qui leur convient, voire à l’extérieur du pays, et au moment de leur choix (CSE, 2021; Grenon, 2020; Parent et al., 2021; Pelletier et al., 2021). La FAD favoriserait aussi la conciliation travail-études-famille (CSE, 2021; Pelletier et al., 2021) et une meilleure gestion des horaires (Grenon, 2020). Elle pourrait également réduire le temps et les coûts de déplacement (CSE, 2021; Pelletier et al., 2021). En résumé, l’accessibilité des études est un facteur qui peut restreindre ou améliorer la valeur ajoutée pédagogique de la FAD. L’influence négative ou positive de ce facteur dépend principalement du contexte de la formation et des besoins des étudiantes et étudiants qui y sont inscrits, ainsi que des mesures de soutien et d’accompagnement mises en place pour les accompagner. 4.3 L’intégration des étudiantes et étudiants à leur future profession Le dernier facteur présenté dans ce texte pour analyser la VAP de la FAD concerne l’intégration des étudiantes et étudiants à leur future profession enseignante. Le référentiel de compétences professionnelles indique que la collaboration et la communication sont des compétences fondamentales pour exercer la profession enseignante (MEQ, 2020) et qu’elles contribuent à l’identité professionnelle : « Former des enseignantes et des enseignants capables de travailler en équipe et qui comprennent que le travail collaboratif est au cœur de l’enseignement appelle le développement d’une identité professionnelle forte et de compétences particulières » (p. 24). On ajoute aussi que la collaboration est une pratique essentielle, qui favorise le développement d’une « culture d’apprentissage collectif » (p. 24) où le partage des connaissances pour soutenir les apprentissages des élèves est encouragé. Les compétences 9 (s’impliquer activement au sein de l’équipe-école) et 10 (collaborer avec la famille et les partenaires de la communauté) visent précisément des habiletés relationnelles et de communication, qui se développent généralement en côtoyant des membres de sa communauté. Plus loin dans ce référentiel, on indique que le développement professionnel des enseignantes et enseignantes s’effectue, entre autres, par la participation à des communautés d’apprentissage plus ou moins formelles, selon les scénarios. Bref, les relations professionnelles, les compétences de collaboration et de communication, et le développement de l’identité professionnelle et du sentiment d’appartenance à la communauté enseignante sont des éléments essentiels de la formation à l’enseignement. Il faut donc les considérer pour évaluer la VAP de la FAD afin de déterminer si elle favorise leur développement, ce qui constituerait une meilleure centration sur l’apprentissage (1re catégorie du modèle de Docq et al.). De façon plus globale, ces éléments font aussi référence à l’isolement et à la distance transactionnelle (Garrison, 2016; Moore et Marty, 2015) que peut entraîner la FAD, ainsi qu’à l’importance de créer un sentiment de présence. À noter que le niveau d’autonomie de l’étudiant ou de l’étudiante est un facteur qui peut influencer cette perception de distance transactionnelle (Forget-Dubois, 2020). Le développement du sentiment de présence et d’appartenance à une communauté peut être facilité par la création de communautés de personnes apprenantes (Freiman et al., 2021; Garrison, 2016) et par une présence soutenue de l’enseignante ou de l’enseignant (Grenon, 2020) observable par 2023 – International Journal of Technologies in Higher Education, 20(2) ijthe.org 63 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes un nombre important d’interactions avec l’étudiante ou l’étudiant (Forget-Dubois, 2020). L’ajout d’outils numériques de collaboration et de communication ainsi que de séances synchrones peut aussi contribuer à développer ce sentiment. Néanmoins, Legault et Fichten (2022) suggèrent de privilégier une modalité de cours hybride dont les premières séances seraient données en présence. Ainsi, cela pourrait aider les étudiantes et étudiants nouvellement admis à se construire un sentiment d’appartenance, à établir une relation de confiance avec leurs enseignants et enseignantes et à avoir la possibilité de s’impliquer dans des activités sociales, parascolaires ou sportives de leur établissement d’enseignement. Ils pourraient alors se créer un réseau qui serait ensuite maintenu à distance grâce aux outils numériques de communication et de collaboration. Ainsi, l’intégration des étudiantes et étudiants à leur future profession ou à leur programme d’études est un facteur qui influence la valeur ajoutée pédagogique de la FAD. Il faut donc évaluer le potentiel de la modalité pour améliorer le développement des habiletés de collaboration essentielles à la profession enseignante et la construction de l’identité professionnelle. Conclusion : quelques repères pour faire des choix éclairés de modalités Ce texte de réflexion pédagogique visait à approfondir des enjeux associés à la formation à distance dans les programmes d’études de formation initiale à l’enseignement au Québec. Considérant que des gestionnaires estiment que la FAD peut être exploitée pour soutenir la croissance de leur établissement d’enseignement ou stabiliser leurs revenus (CSE, 2021), il nous semblait essentiel d’approfondir la réflexion sur cette modalité pour éviter que des décisions institutionnelles soient prises sans égard à leurs répercussions sur les apprentissages des étudiants et étudiantes. Nous avons donc soutenu que le choix de planifier une séquence d’enseignement-apprentissage en FAD devrait s’appuyer sur la valeur ajoutée pédagogique (VAP) que cette modalité peut procurer, comparativement à la modalité traditionnelle en présence. Trois facteurs d’influence de la VAP ont été présentés, soit l’alignement pédagogique, l’accessibilité des études et l’intégration des étudiants et étudiantes à leur profession future. Néanmoins, cette réflexion n’a pas permis de discuter de la VAP de la formation en présence ni de celle de la formation hybride (incluant le comodal), bien que cela soit aussi essentiel pour faire des choix de modalités éclairés. Ainsi, nous proposons que le choix d’une modalité s’effectue après l’évaluation de la valeur ajoutée pédagogique de chacune de ces formations. Celle qui apporte la plus grande valeur ajoutée pédagogique serait à privilégier. Ensuite, il serait judicieux de considérer les répercussions de la modalité choisie pour limiter leurs potentiels effets néfastes sur l’apprentissage. Au besoin, des mesures d’accompagnement pourraient alors être offertes pour les étudiants et étudiantes dont les apprentissages risquent d’être limités par ce choix. En terminant, il apparaît donc que les cours hybrides sont potentiellement les plus prometteurs pour mieux soutenir les apprentissages. En effet, il est fort probable qu’à l’intérieur d’un cours, il y ait des séquences pour lesquelles la distance est préférable et d’autres pour lesquelles la présence ou l’hybridité est à privilégier. Néanmoins, il sera nécessaire de conduire de nouvelles recherches pour s’en assurer. Celles-ci viseraient à comparer la VAP de différentes modalités pour une même séquence d’enseignement-apprentissage, afin de déterminer celles qui sont préférables selon différents contextes. 2023 - Revue internationale des technologies en pédagogie universitaire, 20(2) ritpu.org 64 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Références Anderson, L. W. et Krathwohl, D. R. (dir.). (2001). A taxonomy for learning, teaching, and assessing: A revision of Bloom’s taxonomy of educational objectives (éd. abrégée). Longman. Bates, A. W. (Tony). (2022). Teaching in a digital age (3e éd.). Pressbooks. http://pressbooks.bccampus.ca/... Beaudoin, J., Laferrière, T., Collin, S. et Voyer, S. (2022). Rapport ÉVA – Équité et valeur ajoutée dans les usages du numérique pour l’enseignement et l’apprentissage. CTREQ. http://ctreq.qc.ca/... Biggs, J. (1999). What the student does: Teaching for enhanced learning. Higher Education Research & Development, 18(1), 57-75. https://doi.org/drgphk Bloom, B. S. (1956). Taxonomy of educational objectives: The classification of educational goals. David McKay. Campus numérique. (2022). Typologie des modes de formation. Ministère de l’Éducation du Québec. http://education.gouv.qc.ca/... Castro, M. D. B. et Tumibay, G. M. (2021). A literature review: Efficacy of online learning courses for higher education institution using meta-analysis. Education and Information Technologies, 26(2), 1367-1385. https://doi.org/jk44 Centre de transfert pour la réussite éducative du Québec. (2018). Apprentissages essentiels. Collaborer pour apprendre. http://projetcar.ctreq.qc.ca/... Conseil supérieur de l’éducation. (2021). Revenir à la normale? Surmonter les vulnérabilités du système éducatif face à la pandémie de COVID-19 – Rapport sur l’état et les besoins de l’éducation 2020-2021. Gouvernement du Québec. https://cse.gouv.qc.ca/... Dalgarno, B. et Lee, M. J. W. (2010). What are the learning affordances of 3-D virtual environments? British Journal of Educational Technology, 41(1), 10-32. https://doi.org/cmm6xk Docq, F., Lebrun, M. et Smidts, D. (2010). Analyse des effets de l’enseignement hybride à l’université : détermination de critères et d’indicateurs de valeurs ajoutées. Revue internationale des technologies en pédagogie universitaire, 7(3), 48-59. https://doi.org/10.18162/ritpu.2010.187 Forget-Dubois, N. (2020). Définitions et modalités de la formation à distance. Gouvernement du Québec, Conseil supérieur de l’éducation. https://cse.gouv.qc.ca/... Freiman, V., Giroux, P., Kamba, J. et Cyr, P.-P. (2021). Guide sur le développement de compétences non techniques (CNT) dans la formation à distance (FAD) pour les institutions d’enseignement francophones canadiennes. Réseau d’enseignement francophone à distance du Canada (REFAD). https://constellation.uqac.ca/... Garrison, D. R. (2016). E-learning in the 21st century: A community of inquiry framework for research and practice. Taylor & Francis. https://doi.org/hxpt 2023 – International Journal of Technologies in Higher Education, 20(2) ijthe.org 65 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Grenon, V. (2020). Une recension nord-américaine des conditions à mettre en place pour favoriser le développement de cours hybrides dans un programme de formation en enseignement supérieur. Dans F. Lafleur et G. Samson (dir.), État de situation sur l’hybridité de la formation à distance en contexte postsecondaire, tome 2 : ce qu’en disent les recherches (p. 11-26). Presses de l’Université du Québec. https://doi.org/10.2307/j.ctv18b5czs.8 Guyet, D. (2021). Innovation pédagogique à distance pendant la pandémie de COVID-19 : l’exemple d’une séquence pédagogique de e-coopération induite par simulation en formation professionnelle. Formation et profession, 29(2). https://doi.org/10.18162/fp.2021.629 Hébert, M.-H. et Fontaine, S. (2022). Et si les cas de plagiat et de tricherie étaient liés à la nature des activités d’évaluation? Médiations et médiatisations, (9), 148-151. https://doi.org/10.52358/mm.vi9.265 Johnson, N. (2021a). Leçons tirées de la pandémie de COVID-19 – Rapport national 2021. Association canadienne de recherche sur la formation en ligne (ACRFL). http://cdlra-acrfl.ca/... Johnson, N. (2021b). Evolving definitions in digital learning: A national framework for categorizing commonly used terms. Canadian Digital Learning Research Association (CDLRA). http://cdlra-acrfl.ca/... Joseph, G. et Dallaire, F. (2015). Guide sur l’apprentissage mobile et son impact sur la formation à distance dans la francophonie canadienne. Réseau d’enseignement francophone à distance du Canada (REFAD). https://eduq.info/... Kidd, W. et Murray, J. (2020). The Covid-19 pandemic and its effects on teacher education in England: How teacher educators moved practicum learning online. European Journal of Teacher Education, 43(4), 542-558. https://doi.org/gp66pf Kozanitis, A. (2021). Évaluation des apprentissages en formation à distance : les situations authentiques à la rescousse. Revue internationale des technologies en pédagogie universitaire, 18(1), 276-290. https://doi.org/10.18162/ritpu-2021-v18n1-24 Lafleur, F., Nolla, J.-M. et Samson, G. (dir.). (2021). Évaluation des apprentissages en formation à distance : enjeux, modalités et opportunités de formation en enseignement supérieur. Presses de l’Université du Québec. Latorre-Cosculluela, C., Suárez, C., Quiroga, S., Sobradiel-Sierra, N., Lozano-Blasco, R. et Rodríguez-Martínez, A. (2021). Flipped classroom model before and during COVID-19: Using technology to develop 21st century skills. Interactive Technology and Smart Education, 18(2), 189-204. https://doi.org/10.1108/ITSE-08-2020-0137 la Velle, L., Newman, S., Montgomery, C. et Hyatt, D. (2020). Initial teacher education in England and the Covid-19 pandemic: Challenges and opportunities. Journal of Education for Teaching, 46(4), 596-608. https://doi.org/gg7t3b Lebrun, M., Docq, F. et Smidts, D. (2010). Une plateforme Internet pour former les enseignants. Éducation et formation, (e-294), 95-103. http://revueeducationformation.be/... 2023 - Revue internationale des technologies en pédagogie universitaire, 20(2) ritpu.org 66 C. Tremblay Apports et limites de la formation à distance pour la formation initiale des futurs enseignants et enseignantes Legault, A. et Fichten, C. S. (2022). Enseignement et apprentissage dans l’enseignement supérieur à l’ère de la COVID-19 : une synthèse des connaissances [rapport de recherche]. Fonds de recherche du Québec – Société et culture. http://frq.gouv.qc.ca/... McGee, P. et Reis, A. (2012). Blended course design: A synthesis of best practices. Online Learning, 16(4). https://doi.org/10.24059/olj.v16i4.239 Ministère de l’Éducation du Québec. (2020). Référentiel de compétences professionnelles – Profession enseignante. Gouvernement du Québec. http://cdn-contenu.quebec.ca/... Ministère de l’Éducation du Québec. (2022). Typologie des modes de formation. Campus numérique. http://education.gouv.qc.ca/... Moore, M. G. (2015). La théorie de la distance transactionnelle (O. Marty, trad.). HAL SHS. (Texte original paru en 2007 sous le titre The theory of transactional distance, dans M. G. Moore (dir.), Handbook of distance education [2e éd., p. 89-105], Lawrence Erlbaum.) http://halshs.archives-ouvertes.fr/... Parent, S., Poellhuber, B., Johnson, N. et Seaman, J. (2021). L’apprentissage numérique dans les établissements postsecondaires canadiens – Rapport du Québec 2021. Association canadienne de recherche sur la formation en ligne (ACRFL). http://cdlra-acrfl.ca/... Parr, M. (2019). Pour apprivoiser la distance – Guide de formation et de soutien aux acteurs de la formation à distance. Réseau d’enseignement francophone à distance du Canada (REFAD). http://refad.ca/... Paterson, C., Paterson, N., Jackson, W. et Work, F. (2020). What are students’ needs and preferences for academic feedback in higher education? A systematic review. Nurse Education Today, 85, article 104236. https://doi.org/10.1016/j.nedt.2019.104236 Pelletier, K., Brown, M., Brooks, D. C., McCormack, M., Reeves, J., Arbino, N., Bozkurt, A., Crawford, S., Czerniewicz, L., Gibson, R., Linder, K., Mason, J. et Mondelli, V. (2021). 2021 EDUCAUSE Horizon Report – Teaching and learning edition. http://library.educause.edu/... Pelletier, K., McCormack, M., Reeves, J., Robert, J. et Arbino, N., Al-Freih, M., Dickson-Deane, C., Guevara, C., Koster, L., Sánchez-Mendiola, M., Bessette, L. S. et Stine, J. (2022). 2022 EDUCAUSE Horizon Report – Teaching and learning edition. http://library.educause.edu/... Raes, A., Detienne, L., Windey, I. et Depaepe, F. (2020). A systematic literature review on synchronous hybrid learning: Gaps identified. Learning Environments Research, 23(3), 269-290. https://doi.org/gg84hk Université TÉLUQ. (2023). DESS en éducation préscolaire et en enseignement primaire. Récupéré le 16 mai 2023 de http://teluq.ca/... 2023 – International Journal of Technologies in Higher Education, 20(2) ijthe.org 67
https://openalex.org/W2800060814
https://www.mdpi.com/2306-5354/5/3/53/pdf?version=1531208314
English
null
Gel Dosimetry with Radio-Fluorogenic Coumarin
null
2,018
cc-by
7,421
bioengineering bioengineering bioengineering Article Received: 8 May 2018; Accepted: 5 July 2018; Published: 10 July 2018 Abstract: Gel dosimeters are attractive detectors for radiation therapy, with properties similar to biological tissue and the potential to visualize volumetric dose distributions. Radio-fluorogenesis is the yield of fluorescent chemical products in response to energy deposition from ionizing radiation. This report shares the development of a novel radio-fluorogenic gel (RFG) dosimeter, gelatin infused with coumarin-3-carboxlyic acid (C3CA), for the quantification of imparted energy. Aqueous solutions exposed to ionizing radiation result in the production of hydroxyl free radicals through water radiolysis. Interactions between hydroxyl free radicals and coumarin-3-carboxylic acid produce a fluorescent product. 7-hydroxy-coumarin-3-carboxylic acid has a blue (445 nm) emission following ultra-violet (UV) to near UV (365–405 nm) excitation. Effects of C3CA concentration and pH buffers were investigated. The response of the RFG was explored with respect to strength, type, and exposure rate of high-energy radiation. Results show a linear dose response relationship independent of energy and type, with a dose-rate dependency. This report demonstrates increased photo-yield with high pH and the utility of gelatin-RFG for phantom studies of radiation dosimetry. Keywords: gel dosimetry; radiation dosimetry; radio-fluorogenic gel; luminescent dosimetry Bioengineering 2018, 5, 53; doi:10.3390/bioengineering5030053 www.mdpi.com/journal/bioengineering Peter A. Sandwall 1,*, Brandt P. Bastow 2, Henry B. Spitz 3, Howard R. Elson 4, Michael Lamba 4, William B. Connick 2 and Henry Fenichel 5 Department of Radiation Oncology, OhioHealth, 330 Glessner Ave., Mansfield, OH 44903, USA f Ch C ll f A d S f C C O 1 Department of Radiation Oncology, OhioHealth, 330 Glessner Ave., Mansfield, OH 44903, USA 2 Department of Chemistry, College of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45221, 1 Department of Radiation Oncology, OhioHealth, 330 Glessner Ave., Mansfield, OH 44903, U 1 Department of Radiation Oncology, OhioHealth, 330 Glessner Ave., Mansfield, OH 44903, USA 2 Department of Chemistry, College of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45221, USA; brandtbastow@gmail.com (B.P.B.); connicwb@ucmail.uc.edu (W.B.C.) 2 Department of Chemistry, College of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45221, USA; brandtbastow@gmail.com (B.P.B.); connicwb@ucmail.uc.edu (W.B.C.) 2 Department of Chemistry, College of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45221 USA; brandtbastow@gmail.com (B.P.B.); connicwb@ucmail.uc.edu (W.B.C.) g 3 Department of Nuclear Engineering, College of Engineering, University of Cincinnati, Cincinnati, OH 45221, USA; spitzh@ucmail.uc.edu 4 3 Department of Nuclear Engineering, College of Engineering, University of Cincinnati, Cincinnati, OH 45221, USA; spitzh@ucmail.uc.edu 4 4 Department of Radiation Oncology, College of Medicine, University of Cincinnati, Cincinnati, OH 4522 USA; elsonhr@ucmail.uc.edu (H.R.E.); lambama@ucmail.uc.edu (M.L.) 5 Department of Physics, College of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45221, USA; fenichel@ucmail.uc.edu 5 Department of Physics, College of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45221, USA; fenichel@ucmail.uc.edu * Correspondence: pasandwall@gmail.com; Tel.: +1-513-307-8156   Received: 8 May 2018; Accepted: 5 July 2018; Published: 10 July 2018 Radio-Fluorogenic Gel Dosimetry with Coumarin Peter A. Sandwall 1,*, Brandt P. Bastow 2, Henry B. Spitz 3, Howard R. Elson 4, Michael Lamba 4 William B. Connick 2 and Henry Fenichel 5 1. Introduction Advancements in radiation therapy technology have supported study of tissue-equivalent gels containing active chemical sensors for the measurement of absorbed dose of radiation. Gel dosimeters have radiological properties similar to biological tissue, and are suitable substitutes with the potential to resolve three-dimensional dose distributions. The development of gel dosimeters was dormant for many years, but has recently been developing at a rapid pace. The first reported use of a gel dosimeter was in 1950 by Day and Stein, using the colorimetric dye methylene blue [1]. Andrews, et al. explored chloral hydrate and trichloroethylene in agar [2]. Stein and Tomkiewicz later investigated gelatin with ferricyanide; the radiation induced oxidation of ferricyanide was well characterized and known to produce colorimetric changes [3,4]. Fricke-type gel dosimeters, named after the discoverer of the radiation induced ferric to ferrous reaction, gained significant interest following study by Gore and King using magnetic resonance (MR) imaging [5]. Investigations in 1990s introduced polymer systems which, at a basic level, consist of monomers in solution that polymerize in response to radiation [6]. Studies in the 21st century have yielded plastic leuco-dye Bioengineering 2018, 5, 53; doi:10.3390/bioengineering5030053 www.mdpi.com/journal/bioengineering 2 of 9 Bioengineering 2018, 5, 53 dosimeters containing a colorless dye that reacts with free-radicals to produce color in proportion to energy deposition [7], and most recently, a polymer radio-fluorogenic gel (RFG) dosimeter, where a radio-fluorogenic monomer is activated as it is taken up into a polymer chain [8]. The above-referenced gel dosimeters all have features, often related to fabrication, limiting widespread application in radiotherapy [9]. With Fricke-type systems, the mobility of the ferrous ions allows rapid diffusion of the radiation product and subsequent loss of spatial information. Early polymer system required an evacuated glove box. Current formulations utilize an oxygen scavenger, but still suffer from monomer toxicity, although efforts have been made to minimize this [10]. Leuco-dyes are formed by mixing part-A (pre-polymer solution) and part-B (dye, catalyst, and radical initiator) solutions in a mold. Proper formation requires control of temperature and pressure. Polymer-RFG production involves a nitrogen-flushed glove box and pre-irradiation prior to use as a dosimeter. With strict fabrication methods and sometimes toxic constituents, the hunt for the ideal sensor element and gel substrate continues. g Two natural gel substrates are agarose and gelatin. Gelatin is a derivative of bovine or porcine collagen: the primary element of skin, bone, and connective tissue. 1. Introduction Agarose is a polysaccharide isolated from agar with highest gelling potential: agar is derived from seaweed. Gelatin and agarose are both capable of creating hydrogels with low percentages of gelling agent. However, agarose is opaque and induces light scattering, while gelatin is relatively translucent. The opacity of agar makes it less than ideal for optical analysis. The clarity and transparency of gelatin are strongly dependent on raw material history, purity, and preparation. Commercial gelatin consists of tropocollagen rods in the order of 300 nm in length with 1.5 nm diameter [11]. Raw collagen is processed with acid or base solutions yielding “Type A” (hydrogen chloride) or “Type B” (sodium hydroxide). Type A is denser than type B, with a greater intrinsic viscosity [12]. Gelation speed also affects rigidity, with structure being a function of formation temperature. Slow gelation yields increased organization and orientation of chain elements with greater lateral bonding, resulting in the formation of fine well-ordered lattices [13]. Additionally, gelation is not susceptible to ionic effects [11]. Originating from mammalian tissue with well-understood mechanisms of gelation, gelatin presents as an attractive substrate for exploration of optically active sensors. Radio-fluorogenic sensors are chemical elements that allow for dosimetry and quantification of energy deposition from of ionizing radiation through measurement of molecular fluorescence. Fluorescent detection methods are particularly promising because of their ability to form selective high-resolution images. Initially reported by Day and Stein in 1949, fluorescence spectroscopy can be used to determine absorbed dose in aqueous solutions of aromatic compounds [14–16]. Ionizing radiation initiates radiolysis of water yielding hydroxyl free radicals that hydroxylate aromatic compounds via electrophilic substitution. Numerous aromatic compounds are recognized as radio-fluorogenic, with hydroxylation producing fluorescent products. The first fluorescent sensor investigated for radiation dosimetry was aqueous benzoic acid [16]. Other potential sensors are terephthalic, trimesic, and pyromellitic acid [17–19]. Each improved the yield of fluorescent products by restraining positions on the aromatic compounds for substitution by hydroxyl radicals. However, each of the single-ring aromatic compounds possesses excitation wavelengths in the range of ultra-violet (UV) light, with a limited depth of penetration in a gel. This is the result of Rayleigh scattering of light, proportional to 1/λ4, resulting in rapid reduction of transmission for shorter wavelengths. Because of their macromolecular nature, organic gels are naturally turbid; thus, it is preferable to use longer excitation wavelengths with a greater range of penetration. 2.1. Chemical Preparation 2.1. Chemical Preparation p Reagents were all purchased from Fisher Scientific (Baltimore, MD, USA): 98% C3CA (Acros Organics, Baltimore, MD, USA) and 99% umbelliferone (Infodine Chemical Company; Hillsborough, NJ, USA), sodium bicarbonate, sodium hydroxide, phosphate buffered saline, and food grade porcine type A gelatin (bloom strength 260, pH 5, and viscosity 40). All solutions were prepared with water from an EASYpure water purification system (Barnstead International; Boston, MA, USA). Large volumes of RFGs were prepared and aliquots separated, irradiated, and analyzed. Individual aliquots were stored at low temperature (5 °C) between processing steps to inhibit Reagents were all purchased from Fisher Scientific (Baltimore, MD, USA): 98% C3CA (Acros Organics, Baltimore, MD, USA) and 99% umbelliferone (Infodine Chemical Company; Hillsborough, NJ, USA), sodium bicarbonate, sodium hydroxide, phosphate buffered saline, and food grade porcine type A gelatin (bloom strength 260, pH 5, and viscosity 40). All solutions were prepared with water from an EASYpure water purification system (Barnstead International; Boston, MA, USA). Large volumes of RFGs were prepared and aliquots separated, irradiated, and analyzed. Individual aliquots were stored at low temperature (5 ◦C) between processing steps to inhibit microbial growth. Individual aliquots were stored at low temperature (5 °C) between processing steps to inhibit microbial growth. Preparation was as follows: Gelatin was “wet” to allow for effective dispersion. Seven percent by weight gelatin was placed into a beaker to soak for 20 min, with half the total volume of water. In a separate beaker, C3CA was brought into solution by bringing a small volume of water containing C3CA to boil. The C3CA solution was then added, along with the remaining portion of water, to the “wet” gelatin. The temperature of the RFG solution was then raised to 35 °C. It is important for the temperature to remain below 40 °C to prevent denaturation of the gelatin. The RFG was maintained at 35 °C for approximately 90 min, or until it presented as optically clear and free of visible colloidal structures The RFG was then removed from heat and pipetted into poly methyl methacrylate Preparation was as follows: Gelatin was “wet” to allow for effective dispersion. Seven percent by weight gelatin was placed into a beaker to soak for 20 min, with half the total volume of water. In a separate beaker, C3CA was brought into solution by bringing a small volume of water containing C3CA to boil. 1. Introduction Fluorescence of aromatic compounds is due to their conjugated system of alternating single and double-bonds; pi-orbital overlap allows for delocalization of electrons. Larger conjugated systems require less energy for delocalization or excitation [20]. Therefore, selection of a multi-cyclic radio-fluorogenic sensor would provide the most attractive fluorescent product, ideally with excitation from visible light. Multi-cyclic coumarin-3-carboxylic acid (C3CA) is a promising sensor candidate. 3 of 9 Bioengineering 2018, 5, 53 Aqueous C3CA has been identified as a chemical dosimeter with potential for application in radiotherapy; it demonstrates favorable traits, including linear dose response, reproducibility, and long-term stability [21]. The radio-fluorogenic mechanism of C3CA has been studied in aqueous solution [22]. Positive features of C3CA include high solubility in aqueous solutions, simple organic composition, and favorable excitation and emission spectra. C3CA reacts with hydroxyl radicals to yield the fluorescent product, 7-hydroxycoumarin-3-carboxylic acid (umbelliferone), Figure 1. Bioengineering 2018, 5, x FOR PEER REVIEW 3 of 9 long-term stability [21]. The radio-fluorogenic mechanism of C3CA has been studied in aqueous solution [22]. Positive features of C3CA include high solubility in aqueous solutions, simple organic composition, and favorable excitation and emission spectra. C3CA reacts with hydroxyl radicals to yield the fluorescent product, 7-hydroxycoumarin-3-carboxylic acid (umbelliferone), Figure 1. Figure 1. Hydroxyl radicals react with C3CA to yield umbelliferone through hydrogen abstraction, transfer, and substitution. Figure 1. Hydroxyl radicals react with C3CA to yield umbelliferone through hydrogen abstraction, transfer, and substitution. Figure 1. Hydroxyl radicals react with C3CA to yield umbelliferone through hydrogen abstraction, transfer and substitution Figure 1. Hydroxyl radicals react with C3CA to yield umbelliferone through hydrogen abstraction, transfer, and substitution. Optical imaging of biomarkers is an active area of study with several investigators exploring the use of C3CA labels for radiometric assessment [23–25]. The present investigation explored C3CA in gelatin as a potential RFG dosimeter. Concentration effects of C3CA were studied, and the influence of pH buffers was investigated with respect to fluorescent yield. Radiation response was examined subject to dose, rate, energy, and type for megavoltage electron and photon energies. Optical imaging of biomarkers is an active area of study with several investigators exploring the use of C3CA labels for radiometric assessment [23–25]. The present investigation explored C3CA in gelatin as a potential RFG dosimeter. Concentration effects of C3CA were studied, and the influence of pH buffers was investigated with respect to fluorescent yield. 1. Introduction Radiation response was examined subject to dose, rate, energy, and type for megavoltage electron and photon energies. 2.2. Irradiation and Analysis 2.2. Irradiation and Analysis Irradiations were conducted with a high-energy medical linear accelerator (Varian Medical Systems; Palo Alto, CA, USA). Samples were irradiated with two nominal megavoltage (MV) photon energies (6 & 23 MV) and one electron energy (9 MeV). Irradiations were conducted with samples placed in a polystyrene phantom containing a void for 4 cuvettes. The phantom was designed to provide favorable geometry for the establishment of electronic equilibrium, providing an even distribution of imparted energy. A computed tomography (CT) scan was performed on the phantom, and images were imported into the Eclipse treatment planning system (Varian Medical Systems; Palo Alto, CA, USA) for calculation of dose, Figure 2. Irradiations were conducted with a high-energy medical linear accelerator (Varian Medical Systems; Palo Alto, CA, USA). Samples were irradiated with two nominal megavoltage (MV) photon energies (6 & 23 MV) and one electron energy (9 MeV). Irradiations were conducted with samples placed in a polystyrene phantom containing a void for 4 cuvettes. The phantom was designed to provide favorable geometry for the establishment of electronic equilibrium, providing an even distribution of imparted energy. A computed tomography (CT) scan was performed on the phantom, and images were imported into the Eclipse treatment planning system (Varian Medical Systems; Palo Alto, CA, USA) for calculation of dose, Figure 2. A C B Figure 2. (A) Polystyrene phantom plate with void for irradiation of cuvettes; (B) dose calculation of phantom (heat map represents relative dose); (C) medical linear accelerator used for irradiations. Figure 2. (A) Polystyrene phantom plate with void for irradiation of cuvettes; (B) dose calculation of phantom (heat map represents relative dose); (C) medical linear accelerator used for irradiations. A C A B Figure 2. (A) Polystyrene phantom plate with void for irradiation of cuvettes; (B) dose calculation of phantom (heat map represents relative dose); (C) medical linear accelerator used for irradiations. Figure 2. (A) Polystyrene phantom plate with void for irradiation of cuvettes; (B) dose calculation of phantom (heat map represents relative dose); (C) medical linear accelerator used for irradiations. Instrumental analysis was conducted with a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.; Pal Alto, CA, USA). Excitation and emission slit widths were set to 5 nm, emission scans were performed, and peak emission values recorded and plotted. Dose response was measured by observing the intensity of 445 nm emissions. Instrumental analysis was conducted with a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.; Pal Alto, CA, USA). 2.1. Chemical Preparation 2.1. Chemical Preparation The C3CA solution was then added, along with the remaining portion of water, to the “wet” gelatin. The temperature of the RFG solution was then raised to 35 ◦C. It is important for the temperature to remain below 40 ◦C to prevent denaturation of the gelatin. The RFG was maintained at 35 ◦C for approximately 90 min, or until it presented as optically clear and free of visible colloidal structures. The RFG was then removed from heat and pipetted into poly-methyl-methacrylate (PMMA) cuvettes. Cuvettes were left to cool overnight at ambient temperature. structures. The RFG was then removed from heat and pipetted into poly-methyl-methacrylate (PMMA) cuvettes. Cuvettes were left to cool overnight at ambient temperature. For studies with different pHs, umbelliferone was used to mimic the radio-fluorogenic product. Seven percent gelatin by weight and 0.9 mM C3CA and 0.1 mM umbelliferone solutions were prepared with deionized water (pH 6 0) 0 05 mM sodium bicarbonate and 0 1 mM sodium For studies with different pHs, umbelliferone was used to mimic the radio-fluorogenic product. Seven percent gelatin by weight and 0.9 mM C3CA and 0.1 mM umbelliferone solutions were prepared with deionized water (pH 6.0), 0.05 mM sodium bicarbonate and 0.1 mM sodium hydroxide (basic reagents, pH 10), and stock phosphate buffered saline (PBS, pH 7.4). For dose response 4 of 9 Bioengineering 2018, 5, 53 investigations, RFGs with various coumarin concentrations (1 mM, 5 mM, 10 mM, and 20 mM) were prepared using basic reagents. Bioengineering 2018, 5, x FOR PEER REVIEW 4 of 9 2.2. Irradiation and Analysis 2.2. Irradiation and Analysis Excitation and emission slit widths were set to 5 nm, emission scans were performed, and peak emission values recorded and plotted. Dose response was measured by observing the intensity of 445 nm emissions. 3.1. pH Response 3.1. pH Response The influence of pH buffers on fluorescent response was examined. Results showed a doubling of fluorescent yield in basic solution (pH 10). A spectral shift of the excitation maxima was also observed. Specifically, peak excitation shifted from 365 nm in solutions prepared with water The influence of pH buffers on fluorescent response was examined. Results showed a doubling of fluorescent yield in basic solution (pH 10). A spectral shift of the excitation maxima was also observed. 5 of 9 Bioengineering 2018, 5, 53 Specifically, peak excitation shifted from 365 nm in solutions prepared with water compared to 405 nm in solutions prepared with basic reagents, Figure 3. Bioengineering 2018, 5, x FOR PEER REVIEW 5 of 9 Bioengineering 2018, 5, x FOR PEER REVIEW 5 of 9 Specifically, peak excitation shifted from 365 nm in solutions prepared with water compared to 405 nm in solutions prepared with basic reagents, Figure 3. Bioengineering 2018, 5, x FOR PEER REVIEW 5 of 9 Bioengineering 2018, 5, x FOR PEER REVIEW 5 of 9 Figure 3. Fluorescence properties of coumarin-gelatin RFG dosimeter, excitation (dashed-lines) and emission (solid-lines) spectra for solutions of 7% gelatin with 0.9 mM C3CA and 0.1 mM umbelliferone. Curves are labeled in the graph. X-axis represents wavelength of emitted and collected light, Y-axis is not shown but represents an arbitrary unit of light intensity. 320 340 360 380 400 420 440 460 480 water solution PBS solution basic solution PBS solution water solution basic solution Figure 3. Fluorescence properties of coumarin-gelatin RFG dosimeter, excitation (dashed-lines) and emission (solid-lines) spectra for solutions of 7% gelatin with 0.9 mM C3CA and 0.1 mM umbelliferone. Curves are labeled in the graph. X-axis represents wavelength of emitted and collected light, Y-axis is not shown but represents an arbitrary unit of light intensity. Figure 3. Fluorescence properties of coumarin-gelatin RFG dosimeter, excitation (dashed-lines) and emission (solid-lines) spectra for solutions of 7% gelatin with 0.9 mM C3CA and 0.1 mM umbelliferone. Curves are labeled in the graph. X-axis represents wavelength of emitted and collected light, Y-axis is not shown but represents an arbitrary unit of light intensity. 320 340 360 380 400 420 440 460 480 water solution PBS solution basic solution PBS solution water solution basic solution basic solution basic solution basic solution basic solution Figure 3. 3.1. pH Response 3.1. pH Response Fluorescence properties of coumarin-gelatin RFG dosimeter, excitation (dashed-lines) and emission (solid-lines) spectra for solutions of 7% gelatin with 0.9 mM C3CA and 0.1 mM umbelliferone. Curves are labeled in the graph. X-axis represents wavelength of emitted and collected light, Y-axis is not shown but represents an arbitrary unit of light intensity. Figure 3. Fluorescence properties of coumarin-gelatin RFG dosimeter, excitation (dashed-lines) and emission (solid-lines) spectra for solutions of 7% gelatin with 0.9 mM C3CA and 0.1 mM umbelliferone. Curves are labeled in the graph. X-axis represents wavelength of emitted and collected light, Y-axis is not shown but represents an arbitrary unit of light intensity. Figure 3. Fluorescence properties of coumarin-gelatin RFG dosimeter, excitation (dashed-lines) and emission (solid-lines) spectra for solutions of 7% gelatin with 0.9 mM C3CA and 0.1 mM umbelliferone. Curves are labeled in the graph. X-axis represents wavelength of emitted and collected light, Y-axis is not shown but represents an arbitrary unit of light intensity. 3.2. C3CA Concentration 3.2. C3CA Concentration 3.2. C3CA Concentration Varying the concentration of C3CA in solutions prepared with basic reagents (pH 10) demonstrated a significantly increased response with concentrations 5 mM and greater, Figure 4. Repeated measures, using four samples for each data point, demonstrated standard deviations of less than 1%. Varying the concentration of C3CA in solutions prepared with basic reagents (pH 10) demonstrated a significantly increased response with concentrations 5 mM and greater, Figure 4. Repeated measures, using four samples for each data point, demonstrated standard deviations of less than 1%. Varying the concentration of C3CA in solutions prepared with basic reagents (pH 10) demonstrated a significantly increased response with concentrations 5 mM and greater, Figure 4. Repeated measures, using four samples for each data point, demonstrated standard deviations of less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. 3.2. C3CA Concentration 3.2. C3CA Concentration 3.2. C3CA Concentration Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. Figure 4. Dose plotted against intensity for concentrations of C3CA in 7% gelatin. X-axis represents central axis dose for the depth of cuvette, Y-axis, represents an arbitrary unit of light intensity. Error bars represent a 5% variation from the mean; calculated standard deviation from n = 4 samples per data point, less than 1%. 6 of 9 6 of 9 6 of 9 Bioengineering 2018, 5, 53 Bioengineering 2018, 5, x FO Bioengineering 2018, 5, x FO 3.3. Dose Response p Dose response 3.3. Dose Response D Dose response was determined by plotting 445 nm emissions versus dose, Figure 5. A linear response was observed in the range investigated, independent of type (photon or electron) and energy, Figure 6. Using a definition of three times the standard deviation of the background, the minimum detectable amount (MDA) was extrapolated from 9 MeV electron data, and found to be 1.5 Gy, Figure 7. Dose response was determined by plotting 445 nm emissions versus dose, Figure 5. A linear response was observed in the range investigated, independent of type (photon or electron) and energy, Figure 6. Using a definition of three times the standard deviation of the background, the minimum detectable amount (MDA) was extrapolated from 9 MeV electron data, and found to be 1.5 Gy, Figure 7. Dose response was determined by plotting 445 nm emissions versus dose, Figure 5. A linear response was observed in the range investigated, independent of type (photon or electron) and energy, Figure 6. Using a definition of three times the standard deviation of the background, the minimum detectable amount (MDA) was extrapolated from 9 MeV electron data, and found to be 1.5 Gy, Figure 7. Figure 5. Nominal dose plotted against intensity. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized fluorescent intensity. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 5. Nominal dose plotted against intensity. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized fluorescent intensity. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 5. Nominal dose plotted against intensity. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized fluorescent intensity. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 5. Nominal dose plotted against intensity. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized fluorescent intensity. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 5. Nominal dose plotted against intensity. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized fluorescent intensity. 3.3. Dose Response p Dose response 3.3. Dose Response D Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 5. Nominal dose plotted against intensity. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized fluorescent intensity. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 6 Normalized response plotted against nominal dose for 23 MV, 6 MV, and 9 MeV beams. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized dose response. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 6 Normalized response plotted against nominal dose for 23 MV, 6 MV, and 9 MeV beams. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized dose response. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 6. Normalized response plotted against nominal dose for 23 MV, 6 MV, and 9 MeV beams. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized dose response. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 6 Normalized response plotted against nominal dose for 23 MV, 6 MV, and 9 MeV beams. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized dose response. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 6 Normalized response plotted against nominal dose for 23 MV, 6 MV, and 9 MeV beams. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized dose response. Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. Figure 6. Normalized response plotted against nominal dose for 23 MV, 6 MV, and 9 MeV beams. X-axis represents central axis dose for the depth of cuvette, and Y-axis the normalized dose response. 4. Discussion 4. Discussion Basic solutions (pH 10) of the gelatin-coumarin dosimeter were observed to double emission intensity and shift the peak excitation wavelength from 365 nm to 405 nm. Transition between excited and ground states, i.e., the energy gap, is known to be influenced by the micro-environment through molecular motion, collision, rotational and translational diffusion, and the formation of complexes. Smaller quantum yields are observed with large energy gaps due to the availability of alternative relaxation pathways. The observed increase in quantum yield is consistent with previous studies in aqueous solution; however, the wavelength shift was greater than previously observed (385 nm) [26]. The increased spectral shift may be an unexplored result of interactions with gelatin. Basic solutions (pH 10) of the gelatin-coumarin dosimeter were observed to double emission intensity and shift the peak excitation wavelength from 365 nm to 405 nm. Transition between excited and ground states, i.e., the energy gap, is known to be influenced by the micro-environment through molecular motion, collision, rotational and translational diffusion, and the formation of complexes. Smaller quantum yields are observed with large energy gaps due to the availability of alternative relaxation pathways. The observed increase in quantum yield is consistent with previous studies in aqueous solution; however, the wavelength shift was greater than previously observed (385 nm) [26]. The increased spectral shift may be an unexplored result of interactions with gelatin. (385 nm) [26]. The increased spectral shift may be an unexplored result of interactions with gelatin. Dose response was notably more pronounced for concentrations of C3CA above 5 mM. A concentration of 10 mM was selected for further study, but 5 mM may have been a better selection; 10 mM values appear to demonstrate decreased intensities, most likely due to the inner filter effect. Observations show an independent linear response with respect to dose, energy, and type of ionizing radiation (electron and photon). With respect to type, an independent response is expected, since photon dose is predominately deposited by delta rays, secondary electrons. A dose rate dependency was observed, which was consistent with other findings [21]. This dose rate dependency has previously been suggested to be the result of metallic impurities in C3CA. Future work should explore successive distillations to remove impurities and an expanded dose range. Dose response was notably more pronounced for concentrations of C3CA above 5 mM. 4. Discussion 4. Discussion A concentration of 10 mM was selected for further study, but 5 mM may have been a better selection; 10 mM values appear to demonstrate decreased intensities, most likely due to the inner filter effect. Observations show an independent linear response with respect to dose, energy, and type of ionizing radiation (electron and photon). With respect to type, an independent response is expected, since photon dose is predominately deposited by delta rays, secondary electrons. A dose rate dependency was observed, which was consistent with other findings [21]. This dose rate dependency has previously been suggested to be the result of metallic impurities in C3CA. Future work should explore successive distillations to remove impurities and an expanded dose range. work should explore successive distillations to remove impurities and an expanded dose range. The potential of RFGs for determination of spatial dose distributions has been previously demonstrated [27,28], and the use of planar laser-induced fluorescence (PLIF) has been suggested and demonstrated to yield high-resolution images [29,30]. Independent investigators have recognized PLIF as a valid method, and are currently developing a reader system [31]. However, it is our belief that the finest imaging resolution for RFG gel dosimetry will be achieved by applying methods of two-photon excitation microscopy, allowing the use of longer wavelength light possessing a greater depth of penetration. With recent reports of an RFG using a nanoclay substrate and advances in the fabrication of 3D printable phantom materials, further study is encouraged The potential of RFGs for determination of spatial dose distributions has been previously demonstrated [27,28], and the use of planar laser-induced fluorescence (PLIF) has been suggested and demonstrated to yield high-resolution images [29,30]. Independent investigators have recognized PLIF as a valid method, and are currently developing a reader system [31]. However, it is our belief that the finest imaging resolution for RFG gel dosimetry will be achieved by applying methods of two-photon excitation microscopy, allowing the use of longer wavelength light possessing a greater depth of penetration. With recent reports of an RFG using a nanoclay substrate and advances in the fabrication of 3D printable phantom materials, further study is encouraged [32,33]. 3.3. Dose Response p Dose response 3.3. Dose Response D Error bars represent a 5% variation from the mean; calculated standard deviations from n = 4 samples per data point, less than 1%. ioengineering 2018, 5, 53 7 o oengineering 2018, 5, x FOR PEER REVIEW 7 of Figure 7. Plot of 9 MeV dose response plotted with extrapolated MDA. X-axis represents central axis dose for the depth of cuvettes. Y-axis is the normalized fluorescent intensity. y = 1.5382x + 305.67 R² = 0.9995 306 316 326 336 346 356 366 376 386 0 10 20 30 40 50 Intensity (AU) Nominal Dose (Gy) 9MeV Extrapolated MDA Figure 7. Plot of 9 MeV dose response plotted with extrapolated MDA. X-axis represents central axis dose for the depth of cuvettes. Y-axis is the normalized fluorescent intensity. 7 of 9 7 of 9 Bioengineering 2018, 5, 53 Bioengineering 2018 5 x FO y = 1.5382x + 305.67 R² = 0.9995 Figure 7. Plot of 9 MeV dose response plotted with extrapolated MDA. X-axis represents central axis dose for the depth of cuvettes. Y-axis is the normalized fluorescent intensity. Figure 7. Plot of 9 MeV dose response plotted with extrapolated MDA. X-axis represents central axis dose for the depth of cuvettes. Y-axis is the normalized fluorescent intensity. References 1. Day, M.J.; Stein, G. Chemical effects of ionizing radiation in some gels. Nature 1950, 166, 146–147. [CrossRef] [PubMed] 2. Andrews, H.L.; Murphy, R.E.; LeBrun, E.J. Gel dosimeter for depth-dose measurements. Rev. Sci. Instrum. 1957, 28, 329–332. [CrossRef] 3. Stein, G.; Tomkiewicz, M. Radiation chemistry of gelatin gels containing ferricyanide, radiation research. Radiat. Res. 1970, 43, 25–33. [CrossRef] [PubMed] 4. Fricke, H.; Hart, E.J. The oxidation of the ferrocyanide, arsenite and selenite ions by the irradiation of their aqueous solutions with X-rays. J. Chem. Phys. 1935, 3, 596. [CrossRef] 4. Fricke, H.; Hart, E.J. The oxidation of the ferrocyanide, arsenite and selenite ions by the irradiation of their aqueous solutions with X-rays. J. Chem. Phys. 1935, 3, 596. [CrossRef] 5. Gore, J.C.; Kang, Y.S. Measurement of radiation dose distributions by nuclear magnetic resonance (NMR) imaging. Phys. Med. Biol. 1984, 29, 1189–1197. [CrossRef] [PubMed] Maryanski, M.J.; Ibbott, G.S.; Eastman, P.; Schulz, R.J.; Gore, J.C. Radiation therapy dosimetry using magnetic resonance imaging of polymer gels. Med. Phys. 1996, 23, 699–705. [CrossRef] [PubMed] 7. Adamovics, J.; Maryanski, M.J. Characterisation of PRESAGE™: A new 3-D radiochromic solid p dosemeter for ionising radiation. Radiat. Prot. Dosim. 2006, 120, 107–112. [CrossRef] [PubMed] 7. Adamovics, J.; Maryanski, M.J. Characterisation of PRESAGE™: A new 3-D radiochromic solid polymer dosemeter for ionising radiation. Radiat. Prot. Dosim. 2006, 120, 107–112. [CrossRef] [PubMed] 8. Warman, J.M.; De Haas, M.P.; Luthjens, L.H. High-energy radiation monitoring based on radio-fluorogenic co-polymerization. I: Small volume in situ probe. Phys. Med. Biol. 2009, 54, 3185–3200. [CrossRef] [PubMed] 8. Warman, J.M.; De Haas, M.P.; Luthjens, L.H. High-energy radiation monitoring based on radio-fluorogenic co-polymerization. I: Small volume in situ probe. Phys. Med. Biol. 2009, 54, 3185–3200. [CrossRef] [PubMed] 8. Warman, J.M.; De Haas, M.P.; Luthjens, L.H. High-energy radiation monitoring based on radio-fluorogenic co-polymerization. I: Small volume in situ probe. Phys. Med. Biol. 2009, 54, 3185–3200. [CrossRef] [PubMed] 9. Doran, S.J. The history and principles of chemical dosimetry for 3-D radiation fields: Gels, polymers and plastics. Appl. Radiat. Isotopes 2009, 67, 393–398. [CrossRef] [PubMed] p y p y 9. Doran, S.J. The history and principles of chemical dosimetry for 3-D radiation fields: Gels, polymers and plastics. Appl. Radiat. Isotopes 2009, 67, 393–398. [CrossRef] [PubMed] p pp p 10. Senden, R.J.; De Jean, P.; McAuley, K.B.; Schreiner, L.J. Polymer gel dosimeters with reduced toxicity: A preliminary investigation of the NMR and optical dose–response using different monomers. Phys. Med. Biol. [32,33]. 5. Conclusions 5. Conclusions Our report shares some studies of a novel coumarin-gelatin RFG dosimeter. We found that a significant increase in quantum yield can be achieved with a coumarin-gelatin RFG in basic solution. Gels had a linear dose response with potential for application in the therapeutic dose range. RFG dosimeters warrant further study due to the selective high-resolution images that can be obtained Our report shares some studies of a novel coumarin-gelatin RFG dosimeter. We found that a significant increase in quantum yield can be achieved with a coumarin-gelatin RFG in basic solution. Gels had a linear dose response with potential for application in the therapeutic dose range. RFG dosimeters warrant further study due to the selective high-resolution images that can be obtained with fluorescent analysis. 8 of 9 Bioengineering 2018, 5, 53 Author Contributions: P.A.S. conceived and designed the experiments; B.P.B. supported the performance the experiments; H.B.S., H.R.E., M.L., W.B.C., and H.F. supported the analysis of data and contributed reagents/materials/analysis tools; P.A.S. wrote the paper. Author Contributions: P.A.S. conceived and designed the experiments; B.P.B. supported the performance the experiments; H.B.S., H.R.E., M.L., W.B.C., and H.F. supported the analysis of data and contributed reagents/materials/analysis tools; P.A.S. wrote the paper. Funding: P.A.S. was supported by funds from the National Institute of Occupational Safety and Health (NIOSH); through the University of Cincinnati, NIOSH Education and Research Center. Funding: P.A.S. was supported by funds from the National Institute of Occupational Safety and Health (NIOSH); through the University of Cincinnati, NIOSH Education and Research Center. Acknowledgments: P.A.S. would like to honor the luminous lives of co-authors; H.R. Elson and W.B. Connick, both of whom made the world a better place due to their compassion, integrity, and love of learning. Acknowledgments: P.A.S. would like to honor the luminous lives of co-authors; H.R. Elson and W.B. Connick, both of whom made the world a better place due to their compassion, integrity, and love of learning. Acknowledgments: P.A.S. would like to honor the luminous lives of co-authors; H.R. Elson and W.B. Connick, both of whom made the world a better place due to their compassion, integrity, and love of learning. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 2006, 51, 3301–3314. [CrossRef] [PubMed] 11. Veis, A. The Macromolecular Chemistry of Gelatin; Academic Press: New York, NY, USA, 1964. 12. Djagny, K.B.; Wang, Z.; Xu, S. Gelatin: A valuable protein for food and pharmaceutical industries: Review. Crit. Rev. Food Sci. Nutr. 2001, 41, 481–492. [CrossRef] [PubMed] 13. Roussenova, M.; Enrione, J.; Diaz-Calderon, P.; Taylor, A.J.; Ubbink, J.; Alam, M.A. A nanostructural investigation of glassy gelatin oligomers: Molecular organization and interactions with low molecular weight diluents. New J. Phys. 2012, 14, 035016. [CrossRef] 14. Day, M.J.; Stein, G. Chemical Measurement of Ionizing Radiations. Nature 1949, 164, 671–672. [CrossRef] [PubMed] 15. Stein, G.; Weiss, J. Detection of Free Hydroxyl Radicals by Hydroxylation of Aromatic Compounds. Nature 1950, 166, 1104–1105. [CrossRef] [PubMed] 16. Armstrong, W.A.; Grant, D.W. A highly sensitive chemical dosimeter for ionizing radiation. Nature 1958, 182, 747. [CrossRef] [PubMed] 17. Barreto, J.C.; Smith, G.S.; Strobel, N.H.; McQuillin, P.A.; Miller, T.A. Terephthalic acid: A dosimeter for the detection of hydroxyl radicals in vitro. Life Sci. 1994, 56, PL89–PL96. [CrossRef] 8. Matthews, R.W. Aqueous chemical dosimetry. Int. J. Appl. Radiat. Isotopes 1982, 33, 1159–1170. [CrossR 18. Matthews, R.W. Aqueous chemical dosimetry. Int. J. Appl. Radiat. Isotopes 1982, 33, 1159–1170. [CrossRef] 19. Nadrowitz, R.; Coray, A.; Boehringer, T.; Dunst, J.; Rades, D. A liquid fluorescence dosimeter for proton dosimetry. Phys. Med. Biol. 2012, 57, 1325–1333. [CrossRef] [PubMed] 9. Nadrowitz, R.; Coray, A.; Boehringer, T.; Dunst, J.; Rades, D. A liquid fluorescence dosimeter for pro dosimetry. Phys. Med. Biol. 2012, 57, 1325–1333. [CrossRef] [PubMed] 20. Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, NY, USA, 2006. 21. Collins, A.K.; Makrigiorgos, G.M.; Svensson, G.K. Coumarin chemical dosimeter for radiation therapy. Med. Phys. 1994, 21, 1741–1747. [CrossRef] [PubMed] 9 of 9 Bioengineering 2018, 5, 53 22. Yamashita, S.; Baldacchino, G.; Maeyama, T.; Taguchi, M.; Muroya, Y.; Lin, M.; Kimura, A.; Murakami, T.; Katsumura, Y. Mechanism of radiation-induced reactions in aqueous solution of coumarin-3-carboxylic acid: Effects of concentration, gas and additive on fluorescent product yield. Free Radic. Res. 2012, 46, 861–871. [CrossRef] [PubMed] 23. Perry, C.C.; Tang, V.J.; Konigsfeld, K.M.; Aguilera, J.A.; Milligan, J.R. Use of a coumarin-labeled hexa-arginine peptide as a fluorescent hydroxyl radical probe in a nanoparticulate plasmid DNA condensate. J. Phys. Chem. B 2011, 115, 9889–9897. [CrossRef] [PubMed] 24. Gallina, M.E.; Kim, T.J.; Vasquez, J.; Tuerkcan, S.; Abbyad, P.; Pratx, G. References Single-cell analysis of radiotracers’ uptake by fluorescence microscopy: Direct and droplet approach. In Proceedings of the Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV, San Francisco, CA, USA, 16 February 2017; International Society for Optics and Photonics: Bellingham, WA, USA, 2017; Volume 10068, p. 100680Y. uptake by fluorescence microscopy: Direct and droplet approach. In Proceedings of the Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV, San Francisco, CA, USA, 16 February 2017; International Society for Optics and Photonics: Bellingham, WA, USA, 2017; Volume 10068, p. 100680Y. 25. Gallina, M.E.; Kim, T.J.; Shelor, M.; Vasquez, J.; Mongersun, A.; Kim, M.; Tang, S.K.; Abbyad, P.; Pratx, G. Toward a Droplet-Based Single-Cell Radiometric Assay. Anal. Chem. 2017, 89, 6472–6481. [CrossRef] [PubMed] 25. Gallina, M.E.; Kim, T.J.; Shelor, M.; Vasquez, J.; Mongersun, A.; Kim, M.; Tang, S.K.; Abbyad, P.; Pratx, G. Toward a Droplet-Based Single-Cell Radiometric Assay. Anal. Chem. 2017, 89, 6472–6481. [CrossRef] [PubMed] 26. Dai, X.; Rollin, E.; Bellerive, A.; Hargrove, C.; Sinclair, D.; Mifflin, C.; Zhang, F. Wavelength shifters for water Cherenkov detectors. Nucl. Instrum. Methods Phys. Res. Sect. A Accel. Spectrom. Detect. Assoc. Equip. 2008, 589, 290–295. [CrossRef] 27. Warman, J.M.; de Haas, M.; Luthjens, L.; Murrer, L. A Radio-Fluorogenic Organic Gel for Real-Time, 3D Radiation Dosimetry. Adv. Mater. 2011, 23, 4953–4955. [CrossRef] [PubMed] 28. Sandwall, P.A.; Spitz, H.B.; Elson, H.R.; Lamba, M.A.; Connick, W.B.; Fenichel, H. Measuring the photon depth dose distribution produced by a medical linear accelerator in a water-equivalent radio-fluorogenic gel. J. Radioanal. Nucl. Chem. 2016, 307, 2505–2508. [CrossRef] 29. Sandwall, P.A. Spatial Dosimetry with Violet Diode Laser-Induced Fluorescence of Water-Equivalent Radio-Fluorogenic Gels. Ph.D. Thesis, University of Cincinnati, Cincinnati, OH, USA, 2014. 30. Sandwall, P.; Spitz, H.; Elson, H.; Lamba, M.; Connick, W.; Fenichel, H. Radio-fluorogenic dosimetry with violet diode laser-induced fluorescence. In Proceedings of the Medical Imaging: Physics of Medical Imaging, San Diego, CA, USA, 19 March 2014; International Society for Optics and Photonics: Bellingham, WA, USA, 2014; Volume 9033, p. 90333Y. 31. Yao, T.; Gasparini, A.; De Haas, M.P.; Luthjens, L.H.; Denkova, A.G.; Warman, J.M. A tomographic UV-sheet scanning technique for producing 3D fluorescence images of X-ray beams in a radio-fluorogenic gel. Biomed. Phys. Eng. Express 2017, 3. [CrossRef] 32. Maeyama, T.; Shinnosuke, H. Nanoclay gel-based radio-fluorogenic gel dosimeters using various fluorescence probes. Radiat. Phys. Chem. 2018, 151, 42–46. [CrossRef] 33. References Dahal, E.; Badal, A.; Zidan, A.; Alayoubi, A.; Hagio, T.; Glick, S.J.; Badano, A.; Ghammraoui, B. Stable gelatin-based phantom materials with tunable X-ray attenuation properties and 3D printability for X-ray imaging. Phys. Med. Biol. 2018, 63. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3093701922
https://dugi-doc.udg.edu/bitstream/10256/20611/1/031934.pdf
English
null
Maternity leave and female labor force participation: evidence from 159 countries
Journal of population economics
2,020
public-domain
11,463
∗Elena Del Rey, Campus de Montilivi, 17071 Girona, email: elena.delrey@udg.edu; Andreas Kyriacou, Campus de Montilivi, 17071 Girona, email: andreas.kyriacou@udg.edu; Jos´e I. Silva, Campus de Montilivi, 17071 Girona, email:jose.silva@udg.edu, Tel.: (+34) 972418779, corresponding author. Maternity Leave and Female Labor Force Participation: Evidence from 159 Countries∗ 1Universitat de Girona 2Universitat de Girona and University of Kent September 8, 2020 September 8, 2020 September 8, 2020 Keywords: female labor force participation, fertility, maternity leave, social norms. JEL Classification: D13, J13, J16, J22. 1Studies have also considered the effect of leave duration on outcomes in the short and long-term. In general, longer leaves reduce female employment in the time immediately after birth as recent mothers take advantage of the eligibility, but have a limited or negligible impact on female participation rates in the long run (see, for example, Hanratty and Trzcinski (2009) for the case of Canada, Lalive and Zweim¨uller (2009) for Austria, Sch¨onberg and Ludsteck (2014) for Germany and Dahl et al. (2016) for Norway). We do not consider the short- versus long-run effect of leave duration in this article. Our results should be interpreted as average effects across time and households. Abstract In this paper, we account for the direct and indirect effects of maternity leave entitlements on female labor force participation. We first explore theoretically the impact of maternity leave dura- tion on female labor supply in the presence of fertility decisions. We assume that maternity leave duration affects female labor supply through two main channels: reducing the time cost of female market work, and reducing women’s earnings. Our theoretical model allows for non-monotonic effects of leave duration on female labor supply. We test the predictions of our model using an unbalanced panel of 159 countries for the years 1994, 2004 and 2011. We confirm the existence of an inverted U-shaped relationship between maternity leave duration and female participation, and find a maternity leave threshold of around 30 weeks above which female participation falls. Below this threshold, increasing maternity leave increases female labor force participation because the positive effect due to the reduction of work-time cost of employed mothers strongly dominates the negative wage penalty effect. Beyond this threshold, the opposite happens. Our analysis also confirms the relevance of social norms for female labor supply throughout the world. Keywords: female labor force participation, fertility, maternity leave, social norms. JEL Classification: D13, J13, J16, J22. 1 Introduction It is generally acknowledged that the impact of maternity leave on labor market outcomes depends, in part, on the duration of leave. Using data for nine European countries over the period 1969- 1993, Ruhm (1998) shows that a parental leave of around three months is associated with increases in the employment of women, especially those of child-bearing age, while lengthier leaves (around nine months) have no impact on employment but are associated with lower female hourly earnings. Th´evenon and Solaz (2012) exploit a sample of 30 OECD countries between 1970 and 2010 and find that parental leave below two years has a positive effect on female employment, while longer leaves reduce employment. For the U.S., Rossin-Slater (2017) reports non-monotonic effects of family leave policies on women’s employment rates: while leave entitlements less than one year in length improve their employment rates, longer leaves can have negative effects. Focusing on Canada, Baker and Milligan (2008) find that leave entitlements of 17-18 weeks don’t change the amount of time women spend away from work, but longer leaves of up to 70 weeks lead them to spend more time at home. Olivetti and Petrongolo (2017) review this extensive literature and add to cross-country evidence with data from 30 OECD countries from 1970 to 2014. They confirm the existence of a non-monotonic effect such that the employment rate rises with leaves up to 50 weeks and declines thereafter.1 Job-protected maternity leave can also impact fertility. Olivetti and Petrongolo (2017) report that the effect of parental leave on fertility is non-monotonic (increasing more than proportionally as the length of the leave increases), but quantitatively negligible in OECD countries. Still, Averett and Whittington (2001) find positive effects of parental leave on fertility in the U.S., as does Bj¨orklund (2006) in Sweden. Lalive and Zweim¨uller (2009) find strong positive effects on fertility due to an extension of paid protected leave in Austria in the 90s, and Raute (2019) also reports evidence from Germany suggesting that leave policies can affect fertility. Importantly, leave policies can also have an indirect effect on female labor force via fertility. In relation to this, Aaronson et al. (2018) and Bloom et al. (2009) report that fertility may lower female labor participation rates. To account for both the direct and indirect effects of maternity leave duration on female participa- 2 tion rates, we extend previous work in two ways. 2Job absence due to maternity leave can generate direct costs for firms in the guise of replacement and overtime costs, and indirect ones in the form of productivity losses. Survey information provided by SHRM and Kronos (2014) shows that the direct costs and the productivity loss of a planned absence related to vacations, sick time and parental leave are equal to 29.4% and 15.2% of total payroll in Europe, respectively (see Tables 11 and 12 in the report). The report also calculates similar costs in the U.S., Australia, China, India and Mexico. Additionally, as mentioned by Th´evenon and Solaz (2012), if employees take up long leave entitlements, they may become detached from the labor market as their skills depreciate. For these reasons, we assume that the wage penalty associated with the productivity loss depends on the length of the leave awarded to mothers and that its effect is quadratic. 1 Introduction We first explore theoretically the impact of maternity leave duration on female labor supply in the presence of fertility decisions. We take as our starting point Bloom et al.’s (2009) unitary model, where households choose both women’s labor supply and the number of children. We extend this model by including maternity leave entitlements that reduce the time cost of female market work, may reduce the wage received during their absence, and are costly to the firm.2 Overall, maternity leave affects decisions through two main channels: reducing the time cost of female market work, and reducing women’s earnings. The first channel has a positive direct effect on female labor supply but, indirectly, also reduces labor supply because a lower time cost of female market work raises fertility. The second channel reduces labor supply when the substitution effect of lower wages dominates. Thus, our theoretical model allows for non-monotonic effects of leave duration on female labor supply, including its indirect effect through fertility decisions. To the best of our knowledge, the existing theoretical literature has not explored the relationship between maternity leave duration and labor supply in the presence of fertility decisions. Our theoretical model also extends Bloom et al. (2009) by incorporating the role of social norms in women’s labor supply and fertility decisions. We consider social norms concerning the extent to which it is appropriate for women to work, and assume that they affect each household’s utility in two ways. First, there is a specific cost for women of finding a job that decreases as views towards working women are more favorable. Second, the social norm affects the costs of avoiding fertility, as suggested in Goldin and Katz (2002). Thus, we assume that where views towards working women are more favorable, the cost of avoiding fertility is lower. Interestingly, our model shows that, once fertility decisions are internalized, social norms have an ambiguous effect in labor supply. Specifically, if social views towards working women become more favorable, there is a direct positive effect on female labor supply because the time cost of finding a job decreases. However, an ambiguous indirect effect via fertility appears: on the one hand, lower search costs can increase fertility and thus decrease female labor supply; on the other hand, the cost of reducing fertility decreases, resulting in fewer children 3 3 and thereby increasing female labor supply. and thereby increasing female labor supply. 1 Introduction Our second contribution is empirical. Specifically, unlike previous cross-country evidence that has focused on samples of European or OECD countries, we test for the existence of a non-linear relationship between maternity leave duration and female labor market participation rates in a wide sample of 159 countries. We test the theoretical predictions of our model using an unbalanced panel covering the years 1994, 2004 and 2011. We estimate a set of ordinary least square (OLS) regressions and, consistent with our theoretical priors, we report the existence of an inverted U-shaped relationship between female labor supply and maternity leave across countries. Specifically, we find a maternity leave threshold of around 30 weeks above which female participation falls. According to the theoretical model, to the left of this threshold, increasing maternity leave increases female labor force participation because the positive effect due to the reduction of work-time cost of employed mothers strongly dominates the negative wage penalty effect. Beyond 30 weeks, the wage penalty effect is stronger, thus reducing female labor supply. Finally, the regressions confirm the relevance of social norms for female labor force participation (e.g.: Akerlof and Kranton; 2010; Bertrand, 2011; Fernandez, 2013; Bertrand et al., 2015; Bertrand et al., 2016; Codazzi et al., 2018; Petrongolo, 2019; Casarico and Profeta, 2020). In the words of Bertrand (2011, page 1571): “as long as there is a strong behavioral prescription indicating that men work in the labor force and women work in the home, norms regarding gender identity could explain why women have been slow at increasing their labor force participation”. Our estimates indicate that an increase of 10 percentage points (about one standard deviation) in the prevalence of positive attitudes towards working women increases the female labor force participation by almost 6.8 percentage points. As suggested by our theoretical model, the reduction in both the time cost of finding a job and the cost of reducing fertility may be behind this positive effect. The rest of the paper is organized as follows. In Section 2 we review the theoretical work that has linked fertility and labor supply decisions. We then present the theoretical model linking maternity leave to female labor supply in Section 3. In Section 4 we explain how we measure our key variables, and in Section 5 we present and discuss our main empirical results as well as related robustness analysis. We conclude the article with Section 6. 4 4 2 Theoretical literature Female labor market outcomes and fertility are clearly interconnected. The modeling of women’s labor supply and fertility as conflicting simultaneous decisions has a long history in the theoretical literature. Following the tradition of Becker’s unitary model, Cigno (1986) considers households as single decision units characterized by a utility function of joint adult consumption, number and quality of children. This generates important insights into the design of family policies. It points, for instance, to a potential trade-offbetween the number and quality of children in the absence of externalities. Cigno (1998) extends this work to allow for fertility adjustments accounting for infant mortality. Galor and Weil (1996) consider an overlapping generations general equilibrium model where men supply inelastically one unit of mental labor and one unit of physical labor and women, endowed with less physical labor, choose how much mental labor to supply. There is no leisure: households decide only how many children to have and labor supply adjusts automatically. This paper predicts a negative relationship between income, positively associated with women’s labor force participation, and fertility. However, in poor countries, households are constrained in the number of live births. When income increases in these countries, more children survive and women spend more time childrearing and less in labor-force participation. This replicates the U shape relationship between female labor supply and income in Goldin (1994): when output is low, an increase in income is related to less labor-force participation (and more children), when output is high more income is related to more labor force participation (and less children). Apps and Rees (2004) note that the traditional negative relationship between female labor supply and fertility becomes positive in the 90s. They propose a very simple model where men supply one unit of labor inelastically and women allocate their time to market work and childcare, and use it to attribute the change in tendency to a change to public policy from joint to individual taxation, and from child-related (cash) transfers to child-care (in-kind) transfers. Bloom et al. (2009) extend this simple model to account for women’s leisure, child mortality, fertility control and the role of urbanization, and identify the (positive) causal effect of fertility reductions on female labor force participation. In spite of these achievements, the unitary model has clear limitations. 2 Theoretical literature For example, because all incomes are pooled together, it fails to account for empirical regularities concerning the adjustment 5 5 of both fertility and labor supplies to changes in incomes allocated to one or the other member of the household (e.g. Schultz, 1990). This limitations are still more patent when it comes to conducting normative welfare analysis, leaving no room to determine the optimal allocation of consumption, labor supply, housework, in sum, welfare, between the members of the household. Thus, another strand of the literature has diverged from the unitary model by recognizing that individual preferences within the household may differ. Decision-making within this (game theoretical) framework can be assumed to follow a non-cooperative (Cigno, 2012) or a cooperative (Chiappori, 1997) pattern. In the former case, each member of the household maximizes her utility given the choices of the other member. Because this can lead to Pareto inefficient intra-household allocations, the use of cooperative models based on bargaining the- ory (the collective approach) has received more support (see Vermeulen (2002) and Chiappori et al. (2019)). In these models, members of the household have distinctive preferences and cooperate in, or bargain over the production of a common household good or public good. Although the public good can certainly be interpreted as children like in Blundell et al. (2005), fertility decisions are generally not explicit in these models. Eswaran (2002) considers fertility but not labor supply decisions. As we have mentioned before, our model goes back to the unitary tradition and extends Bloom et al. (2009) in several directions. Despite the aforementioned limitations of this unitary model, our predictions concerning the relationship between maternity leave duration and labor supply are strongly supported by the empirical evidence. We present the model in detail next. 3 The Model The model is based on Bloom et. al (2009). A household consists of a man, who supplies inelastically one unit of efficient labor, and a woman that divides her time among work, childcare and leisure. The utility function of a representative household is defined over household consumption c, female leisure d, and fertility n, and is given by U(c, d, n) = log (c −c0) + α log (d) + η(1 −m)n −k (Σ, Λ) (N −n) (1) (1) (1) For simplicity it is assumed that utility is logarithmic in consumption and leisure and linear in the number of surviving children (1−m)n where m is the infant mortality rate. The weight on consumption in utility is normalized to 1, the relative weight of leisure is α > 0, and the relative weight given For simplicity it is assumed that utility is logarithmic in consumption and leisure and linear in the number of surviving children (1−m)n where m is the infant mortality rate. The weight on consumption in utility is normalized to 1, the relative weight of leisure is α > 0, and the relative weight given 6 to surviving children is η > 0. Parameter c0 represents subsistence consumption. The potential reproductive capacity (usually 15 pregnancies) can be reduced at a cost k that represents the cost of actions such as abstinence, using contraceptives, or interrupting pregnancies. While Bloom et al. (2009) take this cost as given, we allow it to be a function of the social norm parameter Σ and other, potentially exogenous, factors Λ, k(Σ, Λ).3 Parameter Σ measures the general attitude towards working women, with 0 meaning least and 1 most favorable towards working women. Our assumption is that the more favorable are views towards working women in a country the less costly it is to avoid fertility therein (kΣ < 0). Parameter Λ can collect diverse elements with positive or negative effects on the costs of reducing fertility but as in Bloom et al. (2009) we will consider the extent of legal grounds permitting abortion that will have a negative effect on the costs of reducing fertility (kΛ < 0).4 Total time available to a woman during the year is normalized to 1. This is divided between working time lf, leisure time d, and childcare. 3Goldin and Katz (2002) point to the role of social norms in maintaining laws that prohibited the sale of contraceptives in the U.S. in the middle of the 20th century. Other things, such as country imitation effects or pressure from international organizations, could have an independent effect on the costs of reducing fertility k. 3 The Model Time allocated to childcare is linear in the number of surviving children n(1 −m) where n stands for fertility and m for infant mortality rates, with a time cost per child of b. There is a work-time-cost per unit of labor supplied, φu, where φ > 0 and u is an indicator of urbanization. This time cost is meant to account for commuting time, which is larger the larger the size of the city. Having to commute for a longer time implies more time devoted to supplying the same amount of labor. Going beyond Bloom et al. (2009), we assume that urbanization has a positive effect on income and wages, as will be made clear below. In addition, we introduce a time cost of finding a job ψ that is normalized to zero in the case of men and depends negatively on the social norm parameter Σ in the case of women. Thus, the specific cost of finding a job for a woman ψ(Σ) decreases as views towards working women are more favorable: ψ′(Σ) < 0. Finally, we model parental maternity leave mandates λ < 1 as reducing the work-time-cost of employed mothers.5 In other words, a maternity leave of duration λ represents λlf more time available for mothers on a contract for lf hours. Summing up, the yearly leisure constraint of a typical woman is: d = 1 −(1 −λ + φu + ψ(Σ))lf −bn(1 −m) (2) (2) 3Goldin and Katz (2002) point to the role of social norms in maintaining laws that prohibited the sale of contraceptives in the U.S. in the middle of the 20th century. Other things, such as country imitation effects or pressure from international organizations, could have an independent effect on the costs of reducing fertility k. 3Goldin and Katz (2002) point to the role of social norms in maintaining laws that prohibited the sale of contraceptives in the U.S. in the middle of the 20th century. Other things, such as country imitation effects or pressure from international organizations, could have an independent effect on the costs of reducing fertility k. 4Although it can be argued that this too affects social norms, we will allow for Λ to affect household decisions through an additional independent channel. 5Parental leave rights can only be used once a year and because of this, we do not multiply this term by the number of surviving children. 4Although it can be argued that this too affects social norms, we will allow for Λ to affect household decisions through an additional independent channel. 5 3 The Model 4Although it can be argued that this too affects social norms, we will allow for Λ to affect household decisions through an additional independent channel. 5Pa e tal lea e ights ca o l be sed o ce a ea a d beca se of this e do ot lti l this te b the be l leave rights can only be used once a year and because of this, we do not multiply this term by the numb children. 7 As in Bloom et al. (2009), men supply inelastically one unit of labor and receive the male wage wm in exchange. Women supply lf hours of work and earn the wage wf while not on leave. We assume that, while on leave, they earn a proportion ρ of their wage. Household consumption possibilities are therefore given by As in Bloom et al. (2009), men supply inelastically one unit of labor and receive the male wage wm in exchange. Women supply lf hours of work and earn the wage wf while not on leave. We assume that, while on leave, they earn a proportion ρ of their wage. Household consumption possibilities are therefore given by c = wm + wflf (1 −λ (1 −ρ)) (3) (3) Substituting (2) and (3) into (1) we obtain the indirect utility function: (2) and (3) into (1) we obtain the indirect utility function: Substituting (2) and (3) into (1) we obtain the indirect utility function: V (lf) = log [(wflf (1 −λ (1 −ρ)) + wm) −c0] + α log [1 −(1 −λ + φu + ψ(Σ))lf −bn(1 −m)] +η(1 −m)n −k(Σ, Λ)(N −n) (4) (4) We first assume that fertility decisions are exogenous and study the choice of female labor supply for n given. Then, in section 3.1 we account for the endogeneity of fertility decisions. 6Congestion of local public goods can also arise when urbanization increases, resulting in a negative effect on A (Combes and Gobillon, 2015). 3.1 Female labor supply with exogenous fertility If ρ = 1, the duration of the leave will have a positive linear effect on female labor supply.8 The negative effect of the square of leave duration λ2 suggests and inverted U shape relationship between leave duration and female labor force participation. More children n (or lower child mortality m) reduce available time, and the social norm that reduces the cost of finding a job for a woman Σ, has a positive effect on the female labor supply when fertility is given. Finally, living in a larger city (represented by the urbanization parameter u) increases commuting costs, which reduces female labor supply, and generates higher income through agglomeration effects, which also reduce female labor supply. The reason for this is that the male wage increases, with an income effect that reduces female labor supply, and although the female wage also increases, with an income and a substitution effect, income effects can be shown to dominate (see Appendix). In the presence of congestion costs due to urbanization (A′(u) < 0, see lf = β + βyy + βhmhm + βhfhf + βρρ + βλλ + βλ2λ2 + βnn + βmm + βΣΣ + βuu (8) and we can show that: βy < 0, βhm < 0, βhf > 0, βρ > 0, βλ ≶0, βλ2 < 0, βn < 0, βm > 0, βΣ > 0, βu < 0 (see Appendix). In words, income y, and the human capital of men hm, both have a negative effect on female labor supply through an income effect. In contrast, the human capital of women hf, and the wage replacement rate while on leave ρ, can increase female labor supply if the substitution effect of the higher wage dominates.7 The linear effect of leave duration on the labor supply of women βλ could be positive or negative when the number of children is given. This is due to the fact that the leave endows women with more time and this has a positive effect on labor supply, but may reduce earnings when working if ρ < 1, and this has a negative effect on labor supply. If ρ = 1, the duration of the leave will have a positive linear effect on female labor supply.8 The negative effect of the square of leave duration λ2 suggests and inverted U shape relationship between leave duration and female labor force participation. 3 The Model To estimate wages from human capital achievement (years of schooling), we use a Cobb-Douglas production function: Y = A(u)KσE1−σ where A(u) is an increasing function of the urbanization indicator u, accounting for positive agglom- eration effects, K stands for capital, and E stands for efficiency units of labor:6 where A(u) is an increasing function of the urbanization indicator u, accounting for positive agglom- eration effects, K stands for capital, and E stands for efficiency units of labor:6 E = Emhm + Efhf(1 −θλ2) The term θλ2 captures administrative costs and potential losses of productivity experienced by the firm when a worker is on temporary leave. Accounting for previous evidence mentioned in the Introduction, this effect is assumed to be convex on the duration of the leave, with θ > 0. Firms are competitive and wages equal their marginal products: wm = (1 −σ)yhm (5) wf = (1 −σ)yhf 1 −θλ2 (6) wm = (1 −σ)yhm (5) wf = (1 −σ)yhf 1 −θλ2 (6) (5) (6) with y = Y/E = A(u)(K/E)σ. We now derive the equations to estimate and provide their linearized form together with a discus- sion of the predicted signs of the coefficients (see the Appendix for further details). 6Congestion of local public goods can also arise when urbanization increases, resulting in a negative effect on A (Combes and Gobillon, 2015). 8 3.1 Female labor supply with exogenous fertility 7As in Bloom et al. (2009) the substitution effect will dominate provided that wm > c0. 8 8The sign of the marginal effect of leave duration on female labor supply will however not only depen so on βλ2 being βλ + 2βλ2. 3.1 Female labor supply with exogenous fertility From (4), and using (5)-(6), we obtain the first order condition determining female labor supply: From (4), and using (5)-(6), we obtain the first order condition determining female labor supply: l∗ f = (1 −bn(1 −m)) (1 + α) (1 −λ + φu + ψ(Σ)) − α ((1 −σ)yhm −c0) (1 + α) (1 −λ (1 −ρ)) (1 −σ)yhf (1 −θλ2) (7) where y = A(u)(K/E)σ. Thus, in addition to the negative effect of urbanization on female labor supply through the reduction in available time in Bloom et al. (2009), there is a positive effect on wages due to agglomeration effects. l∗ f = (1 −bn(1 −m)) (1 + α) (1 −λ + φu + ψ(Σ)) − α ((1 −σ)yhm −c0) (1 + α) (1 −λ (1 −ρ)) (1 −σ)yhf (1 −θλ2) (7) where y = A(u)(K/E)σ. Thus, in addition to the negative effect of urbanization on female labor supply through the reduction in available time in Bloom et al. (2009), there is a positive effect on wages due to agglomeration effects. (7) Linearizing (7) we can write: Linearizing (7) we can write: lf = β + βyy + βhmhm + βhfhf + βρρ + βλλ + βλ2λ2 + βnn + βmm + βΣΣ + βuu (8) and we can show that: βy < 0, βhm < 0, βhf > 0, βρ > 0, βλ ≶0, βλ2 < 0, βn < 0, βm > 0, βΣ > 0, βu < 0 (see Appendix). In words, income y, and the human capital of men hm, both have a negative effect on female labor supply through an income effect. In contrast, the human capital of women hf, and the wage replacement rate while on leave ρ, can increase female labor supply if the substitution effect of the higher wage dominates.7 The linear effect of leave duration on the labor supply of women βλ could be positive or negative when the number of children is given. This is due to the fact that the leave endows women with more time and this has a positive effect on labor supply, but may reduce earnings when working if ρ < 1, and this has a negative effect on labor supply. loom et al. (2009) the substitution effect will dominate provided that wm > c0. 3.1 Female labor supply with exogenous fertility More children n (or lower child mortality m) reduce available time, and the social norm that reduces the cost of finding a job for a woman Σ, has a positive effect on the female labor supply when fertility is given. Finally, living in a larger city (represented by the urbanization parameter u) increases commuting costs, which reduces female labor supply, and generates higher income through agglomeration effects, which also reduce female labor supply. The reason for this is that the male wage increases, with an income effect that reduces female labor supply, and although the female wage also increases, with an income and a substitution effect, income effects can be shown to dominate (see Appendix). In the presence of congestion costs due to urbanization (A′(u) < 0, see (8) 9 footnote 6), reduced income would imply a positive effect of u on female labor supply. Then the sign of βu would be ambiguous. We now consider the endogenous choice of fertility n. 3.2 Female labor supply with endogenous fertility 3.2 Female labor supply with endogenous fertility mal fertility choice will be given by: From (4), the optimal fertility choice will be given by: n∗= (1 −(1 −λ + φu + ψ(Σ))lf) b(1 −m) − α (η(1 −m) + k (Σ, Λ)) (9) (9) Since fertility n depends on labor supply, and to account for this endogeneity, we first linearize (9) and then substitute the outcome in (8). The linear version of (9) is: (10) n = γ + γλλ + γlflf + γmm + γuu + γΣΣ + γΛΛ (10) where γλ > 0, γlf < 0, γm ≶0, γu < 0, γΣ ≷0, and γΛ < 0 (see Appendix). A larger duration of the maternity leave has a positive effect on fertility as expected. Also as expected, a higher labor supply reduces fertility. Higher child mortality has an ambiguous effect (it increases available time but requires higher fertility to attain the desired number of children). Urbanization u increases the time cost of finding a job, reducing available time and hence fertility of working mothers and having no effect on women who do not work. More favorable views towards working mothers Σ decrease the time cost of finding a job for those women who want to work, thus increasing available time and, hence, fertility. However, such favorable views also decrease the cost of reducing fertility, and this has a negative effect on n. Thus the final effect of Σ on fertility is ambiguous in sign, but negative for non-working women. Finally, more permissive abortion laws Λ make abortion easier and hence reduce fertility. We now substitute (10) into (8) and, collecting terms, obtain the equation that we estimate in the next section: lf = µ + µyy + µhmhm + µhf hf + µρρ + µλλ + µλ2λ2 + µmm + µΣΣ + µΛΛ + µuu (11) The signs of these parameters are derived in the Appendix. We obtain: µy < 0, µhm < 0, µhf > 0, µρ > 0, µλ ≷0, µλ2 < 0, µm ≷0, µu < 0, µΣ ≷0, and µΛ > 0. In the model, income y, the human capital of males and females hm and hf, the term λ2, and ρ, do not affect the optimal choice of fertility. For this reason, the sign of the effect of these variables on female labor supply is the 10 same as the one obtained when fertility is assumed exogenous. 3.2 Female labor supply with endogenous fertility The linear effect of the duration of the leave λ on female labor supply, µλ, continues to be ambiguous but, now, it is so even if ρ = 1: a higher duration reduces the time cost of working, may reduce earnings (or not, if ρ = 1) and, now, also induces higher fertility which has a negative effect on female labor supply. Infant mortality m which had an unambiguous positive effect on labor supply when fertility was exogenous, now has an indeterminate influence because of the ambiguous effect of child mortality on fertility. Urbanization u has a direct negative effect on female labor supply and also affects the fertility choice negatively. For this reason, the final effect of u on female labor supply may be ambiguous. Still, the calculations displayed in the Appendix show that the global effect is still negative when we account for endogenous fertility. Finally, the effect of the social norm Σ, clearly positive when fertility is taken as exogenous, is now ambiguous: µΣ ≷0. If social views towards working women become more favorable, there is a direct positive effect on female labor supply because the time cost of finding a job decreases. But positive views towards female employment also have an ambiguous effect on female labor supply via fertility because, on the one hand, working women have more time to have children and, on the other hand, the cost of reducing fertility decreases resulting in less children. In contrast, the permissiveness of abortion laws Λ has an unambiguous positive effect on female labor supply (µΛ > 0). The rest of the paper will empirically test these theoretical predictions. 4 Data As already stated in the Introduction, our data takes the form of an unbalanced panel covering the periods 1994, 2004 and 2011 and 159 countries. Table 1 provides summary statistics for all the variables employed in the article, including the overall-O, between-B (variation between countries) and within-W (variation within countries) standard deviations (SD). The dependent variable is the female labor force participation rate lf taken from the International Labor Organization (ILO). The female labor force participation rate is the proportion of women aged between 15 and 64 years old who are economically active. It ranges from 8.8% to 89.8% with an average of 50.8%. Countries on the lower end of the distribution include Iran and Algeria while those with the highest rates are Iceland and Mozambique, among others. Most of the variation in this variable is between countries as attested by the overall SD of 17.5, a between SD of 17.3 and a within variation of 3.2. 11 We are interested in the effect of maternity leave, λ on female labor supply. We measure leave length by using the total duration (in weeks) of maternity leave whether paid or unpaid for singleton and uncomplicated childbirths taken from the ILO Working Conditions Laws Database. Leave duration λ has an average value of 14.1 weeks and ranges from 4 in Tunisia in 1994 and 2004, to 58.6 weeks in Croatia in 2004 and 2011. As was the case for female force participation rates, within country variation in the maternity leave indicator is rather limited: within countries SD 2.1 compared to between countries SD of 7.3 in the context of an overall SD of 6.8. 4 Data The explanatory variables in equation (8) are: the fertility rate n (births per woman) taken from the World Population Policies Database; social norms Σ based on the percentage of the population agreeing with the assessment that It is perfectly acceptable for any woman in your family to have a paid job outside the home if she wants one taken from Gallup and ILO (2017); the replacement maternity leave rate ρ refers to the percentage of previous earnings replaced by the maternity benefit over the length of the paid leave entitlement taken from the ILO Working Conditions Laws Database; the infant mortality rate m per 1000 live births taken from the World Bank database; the male and female years of schooling (hm and hf) from the Human Development Report; and the GDP per capita y based on purchasing power parity (PPP) in constant 2011 international dollars and applying logs. When estimating Eq. (11), we replace fertility with a proxy of the cost of avoiding childbirth. Following Bloom et al. (2009), we include the abortion index from the World Population Policies Database, which measures the total number of legal grounds on which abortion is permitted (from 0 to 7). This index reflects legal provisions under which the Government permits induced abortion in the country and includes seven legal reasons for an abortion: to save the life of the woman; to preserve her physical health; to preserve her mental health; consequent on rape or incest; fetal impairment; economic or social reasons; and available on request. A value index of 0(7) means than none(all) of these laws apply to the country. The higher the number of legal grounds permitting abortion Λ, the lower the expected cost of reducing fertility k. This will reduce the number of children and, therefore, increase female labor force participation. As explained more fully below, for robustness we also employ a range of additional control variables namely, general government consumption as a share of GDP from the World Bank, legal traditions (La Porta el al. 2008) and religious affiliations (North et al. 2013). 4 Data 12 Table 1: Model variables: Summary statistics Variable N Mean O(SD) B(SD) W(SD) Min Max Female labor force participation, lf (%) 477 50.8 17.5 17.3 3.2 8.8 89.8 Maternity leave, λ (weeks) 429 14.1 6.8 7.3 2.1 4 58.6 Positive attitudes towards working women , Σ (%) 378 85.25 12.13 12.13 12.13 35 100 Replacement maternity leave rate, ρ (%) 419 87.1 21.3 20.3 7.8 0 100 Fertility rate (births per woman), n 476 3.3 1.7 1.7 0.5 1.2 7.8 Abortion index, Λ 466 3.9 2.5 2.4 0.6 0 7 Infant mortality rate, m (per 1000 live births) 477 36.0 33.3 31.3 11.5 2 168 Urban population rate, u (%) 477 54.9 23.2 23.0 3.4 7.0 100 Years of schooling male, hm 431 7.8 3.0 2.9 0.8 1.1 13.9 Years of schooling female, hf 432 7.0 3.5 3.5 0.9 0.2 13.8 GDP per capita, y (constant dollars 2011 PPS) 457 15,520 18,948 19,292 3,687 477 124,024 General government consumption (% of GDP) 424 15.8 6.0 5.4 2.8 1.8 36.6 Legal origins French code 471 0.456 0.499 0.499 0 0 1 Legal origins German code 471 0.032 0.176 0.176 0 0 1 Legal origins Scandinavian code 471 0.032 0.176 0.176 0 0 1 Legal origins socialist communist laws 471 0.178 0.383 0.383 0 0 1 Religious affiliation Protestants 465 0.185 0.226 0.226 0 0 0.93 Religious affiliation Catholic 468 0.297 0.383 0.383 0 0 0.94 Religious affiliation Eastern Orthodox 468 0.052 0.162 0.162 0 0 0.91 Religious affiliation Muslim 468 0.231 0.346 0.346 0 0 0.99 Note: Variables as defined in the main text. The database is available from the authors on request. 5 Empirical Results In Table 2, we report the OLS estimates when regressing female labor supply on maternity leave and the variables included in the theoretical model. In column 1 we regress female participation on maternity leave without considering any additional variables. Columns 2 and 3 correspond to Equation (8) of the theoretical section 3 that assumes exogenous fertility. Column 3 augments the regression in column 2 by including a squared of (log) GDP per capita term to account for the possibility of a quadratic relationship between GDP per capita and female participation (Goldin 1994; Mammen and Paxson 2000; Bloom et al. 2009). Controlling for fertility allows us to consider the direct effect of maternity leave on female participation or, in other words, allows us to separate out the indirect effect of leave on participation passing through fertility. In column 4 we replace fertility with the abortion index, which Bloom et al. (2009) employ as an exogenous determinant of fertility rates. This corresponds to Equation (11) of the theoretical section, capturing the total effect of maternity leave on female participation rates, including its effect via fertility. Consistent with our theoretical priors, the results clearly indicate that maternity leave λ is asso- 13 Table 2: Determinants of female participation rates estimation Regressors: OLS(1) OLS(2) OLS(3) OLS(4) Maternity leave (λ) 1.908*** 0.548* 0.634** 0.637** (0.378) (0.341) (0.313) (0.296) Maternity leave squared(λ2) -0.028*** -0.011** -0.011** -0.011** (0.006) (0.005) (0.005) (0.004) Maternity leave threshold (weeks) 34.3 24.9 28.8 29.0 Fertility rate (n) - 0.695 -0.833 - - (1.065) (1.067) - Abortion index (Λ) - - - 1.629*** - - - (0.289) Attitudes working women (Σ) - 0.907*** 0.814*** 0.767*** - (0.068) (0.068) (0.065) Maternity replacement rate (ρ) - 0.017 0.020 0.025 - (0.030) (0.028) (0.028) Infant mortality rate (m) - 0.182*** 0.121* 0.122** - (0.064) (0.062) (0.051) Urban population rate (u) - -0.367*** -0.285*** -0.193*** - (0.061) (0.062) (0.059) Years of schooling male (hm) - 0.121 -0.411 -1.681** - (0.929) (0.866) (0.832) Years of schooling female (hf) - 3.477*** 3.615*** 4.146*** - (0.889) (0.829) (0.806) log GDP per capita (log(y)) - -3.033** -61.560*** -56.871*** - (1.412) (9.853) (8.756) log GDP per capita squared (log(y)2) - - 3.093*** 2.813*** - - (0.520) (0.455) Number of observations 429 330 330 322 Adjusted R-squared 0.058 0.587 0.640 0.672 Note: *, **, *** measures statistical significance at the 10, 5 and 1 percent levels respectively. 5 Empirical Results All regressions include a constant and report robust errors. Note: *, **, *** measures statistical significance at the 10, 5 and 1 percent levels respectively. All regressions include a constant and report robust errors. ciated with female participation rates but that the association is not linear. In particular, we confirm the existence of an inverted U-shaped relationship and, more specifically, we find a maternity leave threshold of between 25 and 34 weeks above which female participation falls. According to the the- oretical model, if one is on the left of this threshold, increasing maternity leave will increase female labor force participation because the positive effect due to the reduction of work-time cost of employed mothers strongly dominates the negative wage penalty effect. Alternatively, beyond this threshold, 14 the latter effect will overwhelm that due to the reduction in the work-time cost. In addition, we find that labor supply is positively associated mortality rate m. In turn, participa- tion rates are higher in societies that are favorable to female participation in the labor market. This is in line with the theoretical expectation that social or cultural norms can reduce the search cost experienced by women as well as the cost of avoiding fertility. Thus, our estimates indicate that an increase of 10 percentage points (near to one standard deviation) in the prevalence of positive attitudes towards working women Σ, increases labor force participation by around 8 percentage points. We also find a positive effect of female education hf on labor supply due to its positive effects on female wages (thus implying a dominant substitution effect according to the model). Specifically, an additional year of schooling increases labor force by more than 4 percentage points (see column 4). Additionally, urbanization reduces female labor supply, suggesting the presence of a work-time-cost effect that increases with the level of urbanization. Finally, columns 3 and 4 confirm the quadratic relationship between economic development and female participation in the labor market. As previously stated, the results reported in column 4, show the total effect of maternity leave on female labor participation rates, including its effect via fertility (Equation (11)). 5 Empirical Results They suggest that incorporating the indirect effect on female labor supply going through fertility, has a negligible effect on the maternity leave threshold (28.8 in OLS(3) to 29.0 in OLS(4)), indicating that the positive direct effect of maternity leave duration on female participation dominates the negative indirect effect running through fertility rates. 6 Robustness Analysis The limited within variation in female participation rates and maternity leave duration, leads us to eschew country fixed-effects in the empirical analysis. However, because we are aware that omitted variable bias may affect our estimates, we pursue the robustness of our results in several directions. First, we account for the impact of time varying variables common to our cross-section units through the application of period fixed effects, as well as the effect of time constant factors common to countries in specific world continents via regional fixed effects for Africa, America, Europe, Asia and Oceania (all regressions in Table 3). We, moreover, control for the size of the public sector based on general government final con- sumption expenditure as percentage of GDP (regressions 6 to 9 in Table 3). 6 Robustness Analysis This allows us to account 15 Regressors: OLS(5) OLS(6) OLS(7) OLS(8) OLS(9) Maternity leave (λ) 0.563* 0.599* 0.819*** 1.010*** 1.037*** (0.348) (0.354) (0.321) (0.366) (0.370) Maternity leave squared(λ2) -0.011** -0.011** -0.015*** -0.017*** -0.016*** (0.005) (0.005) (0.005) (0.005) (0.005) λ × low GDP per capita (Dy) - - - - 0.008 - - - - (0.207) λ2 × low GDP per capita (Dy) - - - - -0.004 - - - - (0.004) Maternity leave threshold (weeks) 25.6 27.2 27.3 29.8 32.4 Abortion index (Λ) 1.766*** 1.788*** 1.439*** 1.635*** 1.648*** (0.294) (0.301) (0.318) (0.339) (0.340) Attitudes working women (Σ) 0.785*** 0.816*** 0.828*** 0.677*** 0.685*** (0.078) (0.082) (0.084) (0.100) (0.100) Maternity replacement rate (ρ) 0.022 0.025 0.040 0.037 0.036 (0.029) (0.031) (0.031) (0.032) (0.032) Infant mortality rate (m) 0.164*** 0.145** 0.120** 0.119** 0.114** (0.054) (0.059) (0.057) (0.058) (0.058) Urban population rate (u) -0.201*** -0.199*** -0.143** -0.117* -0.108* (0.062) (0.063) (0.058) (0.060) (0.061) Years of schooling male (hm) -1.675* -1.918** -1.733* -2.242** -2.331** (0.885) (0.928) (0.972) (1.007) (1.017) Years of schooling female (hf) 3.882*** 3.971*** 2.986*** 3.328*** 3.348*** (0.872) (0.916) (0.961) (0.949) (0.950) log GDP per capita (log(y)) -54.409*** -56.625*** -55.886*** -52.621*** -51.523*** (9.412) (10.037) (10.373) (10.847) (10.849) log GDP per capita squared (log(y)2) 2.722*** 2.853*** 2.818*** 2.641*** 2.561*** (0.494) (0.530) (0.562) (0.587) (0.591) Government consumption (% of GDP) - 0.127 -0.146 -0.180 -0.200 - (0.157) (0.163) (0.171) (0.174) Time fixed effects yes yes yes yes yes Continental fixed effects yes yes yes yes yes Legal origins no no yes yes yes Religion no no no yes yes Number of observations 322 309 306 303 303 Adjusted R-squared 0.680 0.671 0.700 0.710 0.711 Note: *, **, *** measures statistical significance at the 10, 5 and 1 percent levels respectively. All regressions include a constant, fixed time trends and regional dummies for Asia, Africa, Europe and America and report robust errors. Final consumption of the general government appears in regressions (6)-(9). Regressions (7)-(9) include Legal origins, which are four dummy variables that identify the legal origin of the company law or commercial code of each country: (i) French commercial code; (ii) German commercial code; (iii) Scandinavian commercial code; (iv) socialist communist laws. Regression (9) also includes four dummies for religious affiliation (Protestants, Catholic, Eastern Orthodox, Muslim) as a percentage of population in 2000. 6 Robustness Analysis Regression (9) includes the interaction between maternity leave and its square with a dummy variable Dy for countries with a GDP per capita lower than 15,500 constant PPS dollars. 16 somewhat for differences in the welfare states across countries and over time; differences that are likely to be associated with a range of model variables including female labor supply, maternity leave, fertil- ity and social norms. In addition, (in regressions 7 to 9) we control for a country’s legal tradition on the premise that this captures the preference for public intervention in the private sphere ranging from a relatively laissez-fair approach in common law systems (originating in the UK), greater intervention in civil law regimes (radiating from France, Germany and Scandinavia) and greatest intervention in countries with a socialist-communist legal heritage (La Porta et al., 2008). We also control for the percentage of the population belonging to the main monotheistic religions (Protestants, Catholics, Eastern Orthodox or Muslim) in the year 2000 (North et al. 2013). This allows us to consider the confounding effects of cultural norms beyond those that we focus on this article. As can be seen in regressions 7 and 8 of Table 3, the estimated effect of positive attitudes towards working women is reduced from 0.828 to 0.677. Although not shown, this is mostly due to the fact that we are now controlling for the percentage of the population that identify themselves as muslims. This religion seems to have a wider impact on female labor supply, above and beyond the social norms we focus on this article. In addition, regression (9) allows for different slopes in maternity leave in low compared to high income countries by multiplying λ and λ2 with a dummy variable Dy that takes the value of one when the country has a GDP per capita lower than the average value observed in the data (15,500 PPS dollars). Thus, the coefficients of λ × Dy and λ2 × Dy measure the difference in the effect of maternity leave and its square between low and high income countries. These coefficients are not statistically different from zero, meaning that maternity leave duration has a similar effect in both types of countries. Finally, figure 1 shows the predicted relationship between maternity leave and the female labor force participation rate obtained from the most saturated estimated equation (OLS(8) in Table 3). 6 Robustness Analysis It clearly maps the inverted U-shape relationship between these two variables. The predicted female labor force participation rate first increases from 46.0% to a maximum of 60.3% when maternity leave goes from 1 week (the lowest observed value of maternity leave) to 30 weeks, and then decreases until it reaches a minimum of 45.7% when maternity leave is equal to 60 weeks (around the highest value of maternity leave in the data). 17 17 40 45 50 55 60 65 Prediction of female labor force participation (%) 0 20 40 60 Maternity leave duration (weeks) Predictive Margins with level(90)% CIs Figure 1: Predicted relationship between maternity leave and female labor force rate Note: Predicted female labor force rate using regression OLS(8) in Table 3 with confidence intervals of 90%. Predictive Margins with level(90)% CIs Figure 1: Predicted relationship between maternity leave and female labor force rate Note: Predicted female labor force rate using regression OLS(8) in Table 3 with confidence intervals of 90% Note: Predicted female labor force rate using regression OLS(8) in Table 3 with confidence intervals 90% Before concluding, it is necessary to address the issue of reverse causality. As mentioned by Olivetti and Petrongolo (2017), changes in female labor supply may create political support for parental leave rights and lead to extended rights including additional weeks of maternity leave. Unaccounted for, reverse causality may potentially introduce upward bias in the estimated impact of maternity leave on female labor force participation (since stronger political support may reasonably be expected to increase leave duration). One way to address reverse causality would be by way of instrumental vari- ables and 2SLS regressions. Unfortunately, valid instrumental variables are difficult to find, especially for the square of maternity leave duration. On the other hand, the impact of reverse causality is likely to be reduced by the inclusion of public sector size, legal origins and social norms in the re- gressions. Public sector size and legal origins help account for differences in the way political support for parental leave may lead to changes in public policies. Female labor market participation may also impact on maternity leave policies by changing social norms regarding female employment (Olivetti and Pentrongolo, 2017), thus highlighting the usefulness of including social norms in the regressions. 7 Conclusion It is generally acknowledged that the impact of maternity leave on labor market outcomes depends, in part, on the duration of maternity leave. Moreover, female labor market outcomes and fertility are clearly interconnected. We account both theoretically and empirically for the effects of paid and unpaid maternity leave duration on female labor force participation in the presence of fertility decisions. From a theoretical perspective, we extend Bloom et al.’s (2009) unitary model by including ma- ternity leave duration through two main channels: (i) the time cost of female market work and; (ii) women’s earnings. The first channel increases female labor supply if the leave is fully paid. The wage penalty effect reduces labor supply because the absence from work, due to the birth of a child, does not only reduce a mother’s earnings if the leave is not fully paid but may also have a negative effect on the marginal product of female labor. Due to the presence of on-leave related costs to the firm, we assume that the wage penalty associated with the productivity loss depends on the length of the leave awarded to mothers and that its effect is quadratic. Thus, our theoretical model considers non-monotonic effects of leave duration on labor supply including its indirect effect through fertility decisions. This is important because maternity leave duration induces higher fertility which has a negative effect on female labor supply. As a result, our model shows that the total effect of leave duration on female labor supply is ambiguous and it is thus entirely an empirical question. We test the theoretical predictions of our model using an unbalanced panel covering the periods 1994, 2004 and 2011 and 159 countries. Our findings confirm the existence of an inverted U-shaped relationship between maternity leave duration and female labor force participation, and identify a ma- ternity leave duration threshold of around 30 weeks above which female participation falls. According to the theoretical model, if one is on the left of this threshold, increasing maternity leave will increase female labor force participation because the positive effect due to the reduction of work-time cost of employed mothers strongly dominates the negative wage penalty effect. Alternatively, beyond this threshold, the latter effect will overwhelm the effect due to the reduction in the work-time cost. Previ- ous studies have found a similar relationship but only in OECD economies. 6 Robustness Analysis Notwithstanding this discussion, the possibility persists for upward bias in the estimated effect of parental leave and therefore, the threshold maternity leave duration of 30 weeks reported should be taken as an upper bound. 18 18 7 Conclusion Thus, our study confirms the existence of an inverse U-shape effect of maternity leave duration on labor market outcomes for a much wider cross-section of countries. The 30 weeks threshold, however, should be taken as an upper bound due to potential presence of reverse causality from labor force participation to maternity leave 19 entitlements. We also study the effects of social norms on female labor force participation. Our estimates indicate that an increase of 10 percentage points (near to one standard deviation) in the prevalence of positive attitudes towards working women increases the female labor force participation by 6.8 percentage points. According to our theoretical model, the reduction in both the time cost of finding a job and the cost of reducing fertility may be behind this positive effect. Finally, future research could explore the extent to which the non-linear effect of maternity leave duration may impact on other labor market outcomes, most notably, wages and gender gaps. Compliance with Ethical Standards: Funding: This study was founded by Ministerio de Econom´ıa y Competitividad, Spain -ECO2016- 76255P (Del Rey) and ECO2017-82350-R (Silva); Ministerio de Ciencia e Innovaci´on, Spain -PID2019- 106642GB-I00 (Del Rey) and PID2019-104723RB-I00 (Kyriacou); Generalitat de Catalunya -2017SGR- 558 (Kyriacou and Silva). Conflict of Interest: The authors declare that they have no conflict of interest. Acknowledgement: We would like to thank the editor, two anonymous referees, Oriana Silva for her assistant with the database, Dirk Foremny and Pedro Trivin for their comments and suggestions. Availability of data and code: The database and the Stata regressions code are available from the authors on request. References Aaronson, D., R. Dehejia, A. Jordan, C. Pop-Eleches, C. Samii and K. Schulze (2018): The effect of fertility on mother’s labor supply over the last two centuries. NBER Working paper 23717, National Bureau of Economic Research, Inc. Akerlof, G., and R. Kranton (2000): Economics and identity. Quarterly Journal of Economics, 115, 715–753. Apps, P. and R. Rees (2004): Fertility, Taxation and Family Policy, The Scandinavian Journal of Economics, 106 (4), 745-763. Apps, P. and R. Rees (2004): Fertility, Taxation and Family Policy, The Scandinavian Journal of Economics, 106 (4), 745-763. 20 Averett, S.L and L. A. Whittington (2001): Does Maternity Leave Induce Births? Southern Economic Journal, 68(2), 403-417. Baker, M. and K. Milligan (2008): How Does Job-Protected Maternity Leave Affect Mothers’ Em- ployment? Journal of Labor Economics, 26 (4), 655-691. Bertrand, M. (2011): New Perspectives on Gender. Handbook of Labor Economics, vol. 4B, Chapter 17, 1543-1590. Bertrand, M., E. Kamenica, and J. Pan (2015): Gender Identity and Relative Income within House- holds. Quarterly Journal of Economics, 130, 571-614. Bertrand, M., P. Cort´es, C. Olivetti, and J. Pan (2016): Social Norms, Labor Market Opportunities, and the Marriage Gap for Skilled Women. NBER Working Paper No. 22015, National Bureau of Economic Research, Inc. Bloom, D. E., D. Canning, G. Fink and J. E. Finlay (2009): Fertility, female labor force participation, and the demographic dividend. Journal of Economic Growth, 14: 79-101. Blundell, R., L. Dearden, and B. Sianesi (2005): Evaluating the effect of education on earnings: models, methods and results from the National Child Development Survey, Journal of the Royal Statistical Society, 163 (3), 473-512. Bj¨orklund, A. (2006): Does family policy affect fertility? Lessons from Sweden. Journal of Population Economics, 19, 3-24. Casarico, A. and P. Profeta (2020): Introduction to the Special Issue on Gender Perspectives in Public Economics. Hacienda P´ublica Espa˜nola-Review of Public Economics, 235, forthcoming. Economics. Hacienda P´ublica Espa˜nola-Review of Public Economics, 235, forthcoming. Chiappori, P.-A. (1997): Introducing Household Production in Collective Models of Labor Supply, Journal of Political Economy, 105 (1), 191-209. Chiappori, P.-A., J. A. Molina, J. I. Gimenez-Nadal, and J. Velilla (2019): Intertemporal Labor Supply and Intra-Household Commitment. IZA DP No. 12353. Cigno, A. (1986): Fertility and the Tax-Benefit System: A Reconsideration of the Theory of Family Taxation, Economic Journal, 96 (384), 1035-1051. 21 Cigno, A. (1998): Fertility decisions when infant survival is endogenous. Journal of Population Eco- nomics, 11, 21– 28. Cigno, A. References (2012): Marriage as a commitment device. Review of Economics of the Household, 10. 10.1007/s11150-012-9141-1. Codazzi, K., V. Pero and A. Alburquerque Sant’Anna (2018): Social norms and labor force partici- pation in Brazil. Review of Development Economics, 22(4), 1513-1535. Combes, P.-P. and L. Gobillon (2015): The empirics of agglomeration economies. Handbook of Regional and Urban Economics, 5, 247-348. Dahl, G., K. Løken, M. Mogstad and K. V. Salvanes (2016): What Is the Case for Paid Maternity Leave?, The Review of Economics and Statistics, 98(4), 655-670. Eswaran, M. (2002): The empowerment of women, fertility, and child mortality: Towards a theoretical analysis. Journal of Population Economics, 15, 433-454. Galor, O. and D. N. Weil (1996):The Gender Gap, Fertility, and Growth, American Economic Review, 86 (3), 374-387. Gallup and ILO (2017): Towards a better future for women and work: Voices of women and men. Gallup, Inc and International Labour Organization. English edition. Goldin, C. (1994): The U-Shaped Female Labor Force Function in Economic Development and Eco- nomic History, NBER Working Papers 4707, National Bureau of Economic Research, Inc. Goldin, C. and L. F. Katz (2002): The Power of the Pill: Oral Contraceptives and Women’s Career and Marriage Decisions. Journal of Political Economy, 110(4), 730-770. Fernandez, R. (2013): Cultural change as learning: The evolution and female labor force participation over a century. American Economic Review, 103(1), 472-500. Hanratty, M. and & E. Trzcinski (2009): Who benefits from paid family leave? Impact of expansions in Canadian paid family leave on maternal employment and transfer income,Journal of Population Economics, 22(3), 693-711. Lalive, R. and J. Zweim¨uller (2009): How does parental leave affect fertility and return to work? Evidence from two natural experiments. Quarterly Journal of Economics, 124(3), 1363- 1402. 22 La Porta, R., F. Lopez-de-Silanes, and A. Shleifer (2008): The Economic Consequences of Legal Origins, Journal of Economic Literature, 46(2), 285-332. Mammen, K., and C. Paxson (2000): Women’s Work and Economic Development, Journal of Eco- nomic Perspectives, 14(4), 141-164. North, C., O. Hakim, and R. Gwin (2013): Religion, Corruption, and the Rule of Law, Journal of Money, Credit and Banking, 45(5), 757-779. Olivetti C. and B. Petrongolo (2017): The Economic Consequences of Family Policies: Lessons from a Century of Legislation in High-Income Countries. Journal of Economic Perspectives 31, 205-230. Petrongolo, B. (2019): The gender gap in employment and wages. Nature Human Behaviour, 3, 316-318. Raute, A. (2019): Can financial incentives reduce the baby gap? References Evidence from a reform in maternity leave benefits. Journal of Public Economics, 169, 203-222. Rossin-Slater. M. (2017): Maternity and family leave policy. NBER Working Paper 23069, National Bureau of Economic Research, Inc. Ruhm, C. J. (1998): The Economic Consequences of Parental Leave Mandates: Lessons from Europe. Quarterly Journal of Economics, 113(1): 285-317. Schultz, T. P. (1990): Testing the Neoclassical Model of Family Labor Supply and Fertility, The Journal of Human Resources, 25(4), 599-634 SHRM and Kronos (2014): Total Financial Impact of Employee Absences Across the United States, China, Australia, Europe, India and Mexico, A Research Report by the Society for Human Resource SHRM and Kronos (2014): Total Financial Impact of Employee Absences Across the United States, China, Australia, Europe, India and Mexico, A Research Report by the Society for Human Resource Management (SHRM). Sch¨onberg, U. and & J. Ludsteck (2014): Expansions in Maternity Leave Coverage and Mothers’ Labor Market Outcomes after Childbirth, Journal of Labor Economics, 32(3), 469-505. Th´evenon, O. and A. Solaz. (2012): Labour Market Effects of Parental Leave Policies in OECD Countries. OECD Social, Employment and Migration Working Papers 141. Vermeulen, F. (2002): Collective Household Models: Principles and Main Results, Journal of Eco- nomic Surveys, 16 (4), 533-564. 23 Appendix Following Bloom et al. (2009), in order to derive an equation to estimate, we linearize Eq. (7) around the point (¯y, ¯hf, ¯hm, ¯λ, ¯λ2, ¯ρ, ¯n, ¯m, ¯u, ¯Σ) and obtain: Following Bloom et al. (2009), in order to derive an equation to estimate, we linearize Eq. (7) around the point (¯y, ¯hf, ¯hm, ¯λ, ¯λ2, ¯ρ, ¯n, ¯m, ¯u, ¯Σ) and obtain: lf ≈ lf(¯y, ¯hf, ¯hm, ¯λ, ¯λ2, ¯ρ, ¯n, ¯m, ¯u, ¯Σ) + − c0α (1 + α) (1 −σ)¯hf  1 −θ¯λ2 (1 + α) 1 −¯λ (1 −¯ρ)  (1 −σ)¯y¯hf 1 −θ¯λ22 ! ∗y + −α (1 + α) 1 −¯λ (1 −¯ρ)  ¯hf 1 −θ¯λ2 ! ∗hm + α (1 −σ)¯y¯hm −c0  (1 + α) 1 −¯λ (1 −¯ρ)  (1 −σ)¯y 1 −θ¯λ2 ¯hf 2 ! ∗hf + λα (1 −σ)¯y¯hm −c0  (1 + α) (1 −σ)¯y¯hf  1 −θ¯λ2 1 −¯λ (1 −¯ρ) 2 ! ∗ρ + (1 −b¯n(1 −¯m)) (1 + α) 1 −¯λ + φ¯u + ψ(¯Σ) 2 − (1 −¯ρ) α (1 −σ)y¯hm −c0  (1 + α) (1 −σ)¯y¯hf 1 −¯λ (1 −¯ρ) 2 1 −θ¯λ22 ! ∗λ + − θα (1 −σ)y¯hm −c0  (1 + α) 1 −¯λ (1 −¯ρ)  (1 −σ)¯y¯hf 1 −θ¯λ22 ! ∗λ2 + −b(1 −¯m) (1 + α)  1 −¯λ + φ¯u + ψ(¯Σ)  ! ∗n + b¯n (1 + α)  1 −¯λ + φ¯u + ψ(¯Σ)  ! ∗m + −(1 + α) ψ′(¯Σ) (1 + α) (1 −¯λ + φ¯u + ψ(¯Σ)) 2 ! ∗Σ + − φ (1 −b¯n(1 −¯m)) (1 + α)  1 −¯λ + φ¯u + ψ(¯Σ) 2 − α (1 + α) 1 −¯λ (1 −¯ρ)  c0 (1 + α) 1 −¯λ (1 −¯ρ)  wf(¯u) 2 dwf du ! ∗u (A.1) (A.1) where lf(¯y, ¯hf, ¯hm, ¯λ, ¯λ2, ¯n, ¯m, ¯u, σ, ¯ρ) collects all constant terms. Let βx denote each term in brackets multiplying each variable x = {y, hf, hm, λ, λ2, ρ, n, m, u, Σ} in (A.1). Then, we can write the linearized equation (A.1) as (8). Appendix The predicted sign of each β can be readily seen in (A.1): βy < 0, βhm < 0, βhf and βρ positive provided that (1 −σ)y¯hm −c0 > 0 (the substitution effect dominates, see footnote 7), βλ ≶0, βλ2 < 0, βn < 0, βm > 0, βΣ > 0, βu < 0. where lf(¯y, ¯hf, ¯hm, ¯λ, ¯λ2, ¯n, ¯m, ¯u, σ, ¯ρ) collects all constant terms. Let βx denote each term in brackets multiplying each variable x = {y, hf, hm, λ, λ2, ρ, n, m, u, Σ} in (A.1). Then, we can write the linearized equation (A.1) as (8). The predicted sign of each β can be readily seen in (A.1): βy < 0, βhm < 0, βhf and βρ positive provided that (1 −σ)y¯hm −c0 > 0 (the substitution effect dominates, see footnote 7), βλ ≶0, βλ2 < 0, βn < 0, βm > 0, βΣ > 0, βu < 0. We also linearize the first order condition determining optimal fertility given female la We also linearize the first order condition determining optimal fertility given female labor supply 24 (9) around the point (¯lf, ¯λ, ¯m, ¯u, ¯Σ, ¯Λ) and obtain, collecting all constant terms in n(¯lf, ¯λ, ¯m, ¯u, ¯Σ, ¯Λ): n ≈ n(¯lf, ¯λ, ¯m, ¯u, ¯Σ, ¯Λ) + −(1 −¯λ + φ¯u + ψ(¯Σ)) b(1 −¯m)  ∗lf +  ¯lf b(1 −¯m)  ∗λ + −  1 −(1 −¯λ + φ¯u + ψ(¯Σ)¯lf  b (1 −¯m)2 + ηα η(1 −¯m) + k ¯Σ, ¯Λ 2 ! ∗m +  −φ¯lf b(1 −¯m)  ∗u + −ψ′(¯Σ)¯lf b(1 −¯m) + αkΣ η(1 −¯m) + k ¯Σ, ¯Λ 2 ! ∗Σ + αkΛ η(1 −¯m) + k ¯Σ, ¯Λ 2 ! ∗Λ + αkΛ η(1 −¯m) + k ¯Σ, ¯Λ 2 ! ∗Λ (A.2) (A.2) that we can write as (10) letting γy denote each term in brackets multiplying each variable y = {lf, λ, m, u, Σ, Λ}. The sign of each γy is clear from (A.2). Appendix Substituting (10) into (8) and collecting terms we obtain Substituting (10) into (8) and collecting terms we obtain Substituting (10) into (8) and collecting terms we obtain (1 −βnγlf) lf = (β + βnγ) + βyy + βhmhm + βhfhf + βρρ + (βλ + βnγλ) λ + βλ2λ2 + (βm + βnγm) m + (βu + βnγu) u + (βΣ + βnγΣ) Σ + βnγΛ (A.3) (A.3) that we write as (11) letting µz denote each term in brackets multiplying variable z = {y, hf, hm, λ, λ2, m, u, Σ, Λ}. From (A.3): µy = βy 1 −βnγlf , µhm = βhm 1 −βnγlf , µhf = βhf 1 −βnγlf , µρ = βρ 1 −βnγlf , µλ = βλ + βnγλ 1 −βnγlf , µλ2 = βλ2 1 −βnγlf , µm = βm + βnγm 1 −βnγlf , µu = βu + βnγu 1 −βnγlf , µΣ = βΣ + βnγΣ 1 −βnγlf , µΛ = βnγΛ 1 −βnγlf . To determine the predicted signs of each µz, note first that the denominator is always positive: To determine the predicted signs of each µz, note first that the denominator is always positive: 1 −βnγlf = 1 − 1 (1 + α) > 0 1 −βnγlf = 1 − 1 (1 + α) > 0 Therefore, sign µy = sign βy < 0 sign µy = sign βy < 0 and sign µhm = sign βhm < 0. sign µhm = sign βhm < 0. sign µhm = sign βhm < 0. The signs of µhf and µρ are, respectively equal to the signs of βhf and βρ, and positive in both cases if (1 −σ)y¯hm −c0 > 0. In turn, sign µλ = sign (βλ + βnγλ) ≷0, 25 sign µλ2 = sign βλ2 < 0, sign µλ2 = sign βλ2 < 0, sign µλ2 = sign βλ2 < 0, and and sign µm = sign (βm + βnγm) ≷0. sign µm = sign (βm + βnγm) ≷0. Appendix Also, Also, sign µu = sign (βu + βnγu) < 0 sign µu = sign (βu + βnγu) < 0 since, after some manipulation, and using d = 1 −b¯n(1 −¯m) −¯lf 1 −¯λ + φ¯u + ψ(Σ)  , and since, after some manipulation, and using d = 1 −b¯n(1 −¯m) −¯lf 1 −¯λ + φ¯u + ψ(Σ)  , and since, after some manipulation, and using d = 1 −b¯n(1 −¯m) −¯lf 1 −¯λ + φ¯u + ψ(Σ)  , and dwf du = (1 −σ)hf 1 −θλ2 A′(u)(K/E)σ > 0, e can write, we can write, βu + βnγu = − φd (1 + α) 1 −¯λ + φ¯u + ψ(¯Σ) 2 − α (1 + α) 1 −¯λ (1 −¯ρ)  c0 (1 + α) 1 −¯λ (1 −¯ρ)  wf(¯u) 2 dwf du < 0. (1 + α) 1 λ + φu + ψ(Σ)  (1 + α) 1 λ (1 ρ)  wf(u)  Finally, Finally, Finally, sign µΣ = sign (βΣ + βnγΣ) ≶0 sign µΣ = sign (βΣ + βnγΣ) ≶0 sign µΣ = sign (βΣ + βnγΣ) ≶0 and, and, sign µΛ = sign (βnγΛ) > 0 sign µΛ = sign (βnγΛ) > 0 since βn < 0, and γΛ < 0. since βn < 0, and γΛ < 0. 26
https://openalex.org/W4308332976
https://boa.unimib.it/bitstream/10281/400963/1/Staurenghi-2022-Antioxidants-VoR.pdf
English
null
ApoE3 vs. ApoE4 Astrocytes: A Detailed Analysis Provides New Insights into Differences in Cholesterol Homeostasis
Antioxidants
2,022
cc-by
15,700
Citation: Staurenghi, E.; Leoni, V.; Lo Iacono, M.; Sottero, B.; Testa, G.; Giannelli, S.; Leonarduzzi, G.; Gamba, P. ApoE3 vs. ApoE4 Astrocytes: A Detailed Analysis Provides New Insights into Differences in Cholesterol Homeostasis. Antioxidants 2022, 11, 2168. https://doi.org/10.3390/ antiox11112168 Keywords: astrocytes; alzheimer’s disease; apolipoprotein E; ApoE4; cholesterol metabolism; lipids; oxysterols antioxidants antioxidants antioxidants ApoE3 vs. ApoE4 Astrocytes: A Detailed Analysis Provides New Insights into Differences in Cholesterol Homeostasis Erica Staurenghi 1 , Valerio Leoni 2,3 , Marco Lo Iacono 1 , Barbara Sottero 1, Gabriella Testa 1 , Serena Giannelli 1 , Gabriella Leonarduzzi 1,† and Paola Gamba 1,*,† urenghi 1 , Valerio Leoni 2,3 , Marco Lo Iacono 1 , Barbara Sottero 1, Gabriella Testa 1 , annelli 1 , Gabriella Leonarduzzi 1,† and Paola Gamba 1,*,† 1 Department of Clinical and Biological Sciences, University of Turin, Orbassano, 10043 Turin, Italy 1 Department of Clinical and Biological Sciences, University of Turin, Orbassano, 10043 Turin, Italy 2 Laboratory of Clinical Biochemistry, Hospital Pius XI of Desio, ASST-Brianza, University of Milano-Bicocca, 20126 Monza, Italy 2 Laboratory of Clinical Biochemistry, Hospital Pius XI of Desio, ASST-Brianza, University of Milano-Bicocca, 20126 Monza, Italy y 3 Department of Medicine and Surgery, University of Milano-Bicocca, 20126 Monza, Italy y 3 Department of Medicine and Surgery, University of Milano-Bicocca, 20126 Monza, Italy * Correspondence: paola.gamba@unito.it † These authors equally contributed to this work. Abstract: The strongest genetic risk factor for sporadic Alzheimer’s disease (AD) is the presence of the ε4 allele of the apolipoprotein E (ApoE) gene, the major apolipoprotein involved in brain cholesterol homeostasis. Being astrocytes the main producers of cholesterol and ApoE in the brain, we investi- gated the impact of the ApoE genotype on astrocyte cholesterol homeostasis. Two mouse astrocytic cell lines expressing the human ApoE3 or ApoE4 isoform were employed. Gas chromatography–mass spectrometry (GC-MS) analysis pointed out that the levels of total cholesterol, cholesterol precursors, and various oxysterols are altered in ApoE4 astrocytes. Moreover, the gene expression analysis of more than 40 lipid-related genes by qRT-PCR showed that certain genes are up-regulated (e.g., CYP27A1) and others down-regulated (e.g., PPARγ, LXRα) in ApoE4, compared to ApoE3 astro- cytes. Beyond confirming the significant reduction in the levels of PPARγ, a key transcription factor involved in the maintenance of lipid homeostasis, Western blotting showed that both intracellular and secreted ApoE levels are altered in ApoE4 astrocytes, as well as the levels of receptors and transporters involved in lipid uptake/efflux (ABCA1, LDLR, LRP1, and ApoER2). Data showed that the ApoE genotype clearly affects astrocytic cholesterol homeostasis; however, further investigation is needed to clarify the mechanisms underlying these differences and the consequences on neighboring cells. Indeed, drug development aimed at restoring cholesterol homeostasis could be a potential strategy to counteract AD. antioxidants antioxidants 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Alzheimer’s disease (AD) is a highly complex neurodegenerative condition that affects millions of people around the world, and the number of AD patients is increasing due to aging of the global population. Two features are the primary hallmarks in the AD brain: intracellular neurofibrillary tangles (NFTs) made of hyperphosphorylated tau protein, and extracellular senile plaques caused by amyloid β (Aβ) accumulation [1]. Most AD cases are sporadic, due to the complex combination of genetic and environ- mental risk factors. The strongest genetic risk factor for sporadic AD is the presence of the ε4 allele of the apolipoprotein E (ApoE) gene, the major apolipoprotein involved in brain cholesterol transport [2,3]. Indeed, changes in lipid metabolism, in particular that of choles- terol, predispose to AD playing a substantial role in the different phases of the disease [4]. In contrast to the most common isoform ApoE3, present in 78% of the population, ApoE4 increases the risk of developing AD by three to twelve times depending on whether the ε4 allele is present in heterozygosis or homozygosis [3]. Indeed, the ApoE4 isoform has been shown to affect lipid and glucose metabolism, but also many other processes involved Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/antioxidants Antioxidants 2022, 11, 2168. https://doi.org/10.3390/antiox11112168 Antioxidants 2022, 11, 2168 2 of 21 in AD pathology including Aβ clearance, neuroinflammation, and synaptic function [5]; however, the molecular mechanisms underlying ApoE4-induced pathogenic processes are not yet clear. In addition to AD, ApoE4 has been associated with the progression of related dementia, such as frontotemporal dementia and Lewy body dementia [6,7]. In humans, the brain is the organ with the highest level of cholesterol, approximately 20% of the whole-body cholesterol pool [8]. Cholesterol homeostasis in the normal brain is tightly regulated in order to keep cholesterol steady-state levels in the brain stable, es- sential for neuronal functioning and brain development. In the brain most cholesterol is produced de novo by astrocytes, and then transported to neurons and other cells by com- bining with ApoE, mainly synthesized by astrocytes, to form lipoproteins that are secreted through the ATP-binding cassette (ABC) transporters [9,10]. 2.1. Cell Cultures Immortalized mouse astrocytes, obtained from ApoE-TR mouse primary astrocytes ex- pressing human ApoE3 or ApoE4 under the control of endogenous mouse ApoE promoter, were kindly gifted by Prof. David Holtzman [18]. Astrocyte cultures were maintained at 37 ◦C with 5% CO2 in DMEM high glucose medium (Corning, Corning, NY, USA) sup- plemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 4 mM L-Alanyl-L-Glutamine (Glutagro™Supplement, Corning), 100 U/mL penicillin, and 100 µg/mL streptomycin (Corning). The growing medium was replaced with serum-free medium 24 h before collecting astrocyte-conditioned media (ACM) and cell pellets for protein or RNA extraction. 1. Introduction The association between choles- terol homeostasis alterations and AD onset and progression has been evident for many years [11–13]. For instance, cholesterol oxidation products (i.e., oxysterols) that accumulate in the brain are key actors in AD pathogenesis because they lead to neuron dysfunction and degeneration, mainly by enhancing oxidative stress and inflammation [11,14]. Since astrocytes are the main producers of both cholesterol and ApoE in the brain and brain cholesterol homeostasis alterations are closely linked to AD [10], understanding how the ApoE4 genotype affects lipid homeostasis in astrocytes is of primary importance. In this regard, transcriptomic studies of ApoE4 astrocytes deriving from human-induced pluripotent stem cells (hiPSCs) of AD patients demonstrated that lipid metabolism is one of main altered processes [15,16]. Moreover, alterations of lipid metabolism were shown to distinguish ApoE4 astrocytes deriving from human ApoE-targeted replacement (ApoE-TR) mice [17]. These studies clearly highlight the key role of the ApoE4 genotype in affecting lipid homeostasis in astrocytes, but further analyses are needed to clarify its specific impact on cholesterol metabolism and to elucidate the molecular mechanisms involved in the induced metabolic alterations. To fill this gap, this paper aimed at deepening the impact of ApoE4 genotype on astrocytic cholesterol homeostasis, providing a detailed analysis of the main molecules involved in cholesterol metabolism and transport in ApoE3 and ApoE4 astrocytic cell lines obtained from ApoE-TR mouse primary astrocytes [18]. For the first time, we demon- strated substantial differences between ApoE3 and ApoE4 astrocytes at the level of various molecules involved in cholesterol metabolism and transport. Interestingly, the obtained re- sults pointed out that ApoE4 astrocytes contain altered levels of total cholesterol, cholesterol precursors, and of the main oxysterols. Of note, the trend of the oxysterol concentrations in ApoE4 astrocytes compared to ApoE3 astrocytes was similar to that observed in human AD brain cortices compared to healthy brains [19], suggesting their potential implication in ApoE4 deleterious effects on AD risk. 2.2. Sterol Quantification by GC-MS Cholesterol, cholesterol precursors (lathosterol, lanosterol and desmosterol, mark- ers of cholesterol synthesis), and the oxysterols 25-hydroxycholesterol (25-OHC) and 27- hydroxycholesterol (27-OHC) (markers of reverse cholesterol transport with immunomod- Antioxidants 2022, 11, 2168 3 of 21 3 of 21 ulatory and metabolic actions), 24(S)-hydroxycholesterol (24-OHC) (cerebrosterol, brain- specific cholesterol elimination product and marker of brain cholesterol turnover), 7- ketocholesterol (7-KC), 7α-hydroxycholesterol (7α-OHC), and 7β-hydroxycholesterol (7β- OHC) (markers of oxidative stress) were measured by isotope dilution gas chromatography- mass spectrometry (GC-MS), as described elsewhere [20–22]. The cell number of each sample (about 206 cells) was evaluated to normalize the corresponding sterol amounts. Cellular homogenates, prepared from pellets suspended in water (250 µL) and sonicated for 15 min, were transferred to a screw-capped vial sealed with a Teflon septum together with 50 µg epicoprostanol (Sigma-Aldrich, Merck, Darmstadt, DE, Germany), 500 ng of lathosterol-25, 26, 26, 26, 27, 27, 27-d7, 50 ng of lanosterol-26, 26, 26, 27, 27, 27-d6, 50 ng of 7β-OHC-25, 26, 26, 26, 27, 27, 27-d7, 50 ng of 7-KC-25, 26, 26, 26, 27, 27, 27-d7, 50 ng of 24(R/S)-OHC-25, 26, 26, 26, 27, 27, 27-d7, 50 ng of 25-OHC-26, 26, 26, 27, 27, 27-d6, 50 ng of 27-OHC-25, 26, 26, 26, 27, 27-d6 (Avanti Polar Lipids Inc., Birmingham, AL, USA) as internal standards, as well as 50 µL of butylated hydroxytoluene (BHT) (5 g/L, Sigma-Aldrich) and 50 µL of K3-ethylenediamine tetra acetic acid (EDTA) (10 g/L, Sigma-Aldrich) to prevent autooxidation. Each vial was flushed with argon for 10 min to remove air. Alkaline hydrolysis was carried out at room temperature for 1 h in the presence of ethanolic KOH 1M. Sterols and oxysterols were extracted twice with 5 mL of cyclohexane followed by centrifugation (3500× g for 10 min at 4 ◦C). The organic solvents were evapo- rated under a gentle stream of argon and converted into trimethylsilyl ethers with 100 µL N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Sigma-Aldrich) (70 ◦C for 60 min). Isotope dilution GC-MS analysis was performed by a 6890N Network GC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP 7687 series autosampler and a HP 7683 series injector (Agilent Technologies) and coupled to a quadruple mass selective detector HP5975B Inert MSD (Agilent Technologies). For GC separation a B-XLB column (30 m × 0.25 mm i.d. 2.2. Sterol Quantification by GC-MS × 0.25 µm film thickness; J&W Scientific Alltech, Folsom, CA, USA) was used and the following oven temperature program: an initial temperature of 180 ◦C held for 1 min, followed by a linear ramp of 20 ◦C/min to 270 ◦C, and then a linear ramp of 5 ◦C/min until the final temperature of 290 ◦C held for 11 min. Helium was used as a carrier gas at a flow rate of 1 mL/min. Injection of the sample (1 µL) was carried out in splitless mode, at 250 ◦C with a flow rate of 20 mL/min. The MS temperature parameters were 290 ◦C for the transfer line, 150 ◦C for the filament, and 220 ◦C for the quadrupole, according with the manufacturer indication. Mass spectrometric data were acquired in selected ion monitoring mode (OTMSi- ethers) at m/z = 465 for lathosterol-d7, m/z = 458 for lathosterol, m/z = 343 for desmosterol, m/z = 399 for lanosterol-d6, m/z = 393 for lanosterol, m/z = 463 for 7α-OHC-d7, m/z = 456 for 7α-OHC, m/z = 463 for 7β-OHC-d7, m/z = 456 for 7β-OHC, m/z = 479 for 7-KC-d7, m/z = 472 for 7-KC, m/z = 137 for 25-OHC-d6, m/z = 131 for 25-OHC, m/z = 420 for 24- OHC-d7, m/z = 413 for 24(S)-OHC, m/z = 462 for 27-OHC-d6, and m/z = 456 for 27-OHC (Avanti Polar Lipids Inc.). Peak integration was performed manually, and the analytes were quantified from SIM analysis against internal standards using standard curves for the listed compounds. (Avanti Polar Lipids Inc.). Peak integration was performed manually, and the analytes were quantified from SIM analysis against internal standards using standard curves for the listed compounds. 2.4. Gel Electrophoresis and Western Blotting After incubating astrocytes with serum-free medium for 24 h, ACM were collected, centrifuged at 3000 rpm for 5 min, and a protease inhibitor cocktail (P8340, Sigma-Aldrich) was added before freezing them. To detect secreted ApoE, 3 mL of ACM were concentrated by using centrifugal filters (Amicon Ultra, Millipore, Merck). Astrocytes were washed with phosphate-buffered saline (PBS), pelleted, and lysed with ice-cold PBS containing 10 µL/mL Triton-X100, 10 µL/mL SDS 10%, protease inhibitor (Complete Mini EDTA-Free Protease Inhibitor Cocktail, Roche, Basel, CH) and phosphatase inhibitor (P0044, Sigma-Aldrich) cocktails. Total protein concentration of culture media and cell lysates was determined by Bradford assay (Bio-Rad Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein samples (40–100 µg) were mixed with sample buffer (Nu- PAGE LDS Sample Buffer 4X, Invitrogen) and reducing agent (NuPAGE Sample Reducing Agent 10X, Invitrogen) before boiling them. Then, protein samples were separated by electrophoresis by using 10% acrylamide gels (TGX Stain-Free FastCast Acrylamide kit, Bio- Rad Laboratories) or 4–12% precast gels (Bolt Bis-Tris Plus gels, Invitrogen) and transferred to nitrocellulose membranes (Amersham Protran, GE Healthcare, Chicago, IL, USA). After blocking with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 ◦C. The following primary antibodies (1:1000 dilution) were used: anti-ApoE (D7I9N, Cell Signaling Technol- ogy, Danvers, MA, USA), anti-PPARγ (C26H12, Cell Signaling Technology), anti-ABCA1 (MA5-16026, Invitrogen), anti-ApoER2 (LRP8, PA5-109269, Invitrogen), anti-LDLR (MA5- 32075, Invitrogen), anti-LRP1 (37-7600, Invitrogen), and anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with TBS1X-Tween20 0.05% to remove the unbound antibody, the appropriate HRP-linked secondary antibody (1:3000, Cell Signaling Technology) was added for 1 h at room temperature. Membranes were washed with TBS1X- Tween20 0.05%, incubated with Clarity Western ECL Substrate (Bio-Rad Laboratories) and scanned by using a ChemiDoc Imaging System (Bio-Rad Laboratories). Band intensities were quantified by using the Image Lab Software (Bio-Rad Laboratories) and normalized to the corresponding β-actin or, in the case of concentrated media samples, to the total protein content of the sample through the Stain-Free imaging technology (Bio-Rad Laboratories). 2.3. RNA Extraction and Real-Time RT-PCR Total RNA was extracted by using TRIzol™Reagent (Invitrogen, Thermo Fisher Scientific) following the manufacturers’ instructions. Possible traces of DNA were removed by using the TURBO DNA-free™Kit (Invitrogen) and RNA was dissolved in RNase-free water with RNase inhibitors (SUPERase-In RNase Inhibitor, Invitrogen). The amount and purity of the extracted RNA were assessed spectrophotometrically. The cDNA was synthesized by reverse transcription of 4 µg of RNA by using a commercial kit and random primers (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Thermo Fisher Scientific). 4 of 21 Antioxidants 2022, 11, 2168 Real-time RT-PCR was performed on 50 ng of cDNA by using a 7500 Fast Real- Time PCR System and the TaqMan Array Mouse Lipid Regulated Genes 96-well Plate or TaqMan Gene Expression Assays for mouse CYP7A1 (Mm00484150_m1), CYP27A1 (Mm00470430_m1), CYP46A1 (Mm00487306_m1), CH25H (Mm00515486_s1), DHCR24 (Mm00519071_m1), HMGCR (Mm01282499_m1), and GAPDH (Mm99999915_g1) (Applied Biosystems). The PCR cycling parameters were set up as previously described [19]. The fractional cycle number (Ct) was determined for each considered gene. Gene expression analysed by using the TaqMan Array Mouse Lipid Regulated Genes 96-well Plate was normalized to the average expression levels of three endogenous control genes included in the plate: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phospho- ribosyltransferase 1 (HPRT1), and β-glucuronidase (GUSB). The expression levels of two of the 43 genes included in the plate were not calculated because they were undetectable in both ApoE3 and ApoE4 astrocytic cell lines. Gene expression results obtained by us- ing single TaqMan Gene Expression Assays were normalized to the GAPDH expression levels. Relative quantification of target gene expression was achieved by a mathematical method [23]. 2.5. Immunocytochemistry Astrocytes were plated on coverslips (16 mm diameter, thickness No. 1) in 12-well plates. After 24 h in serum-free medium, cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, and then washed again twice with PBS. Cells were permeabilized and blocked (2% goat serum, 0.1% Triton in PBS) for 5 of 21 Antioxidants 2022, 11, 2168 1 h at room temperature before being incubated with a mix of anti-ApoE (1:100, D7I9N, Cell Signaling Technology) and anti-β-actin (1:50, sc-47778, Santa Cruz Biotechnology) primary antibodies for 2 h at room temperature. After two washes in PBS, astrocytes were incu- bated with a mix of Alexa Fluor 594 (1:250, ab150080, Abcam, Cambridge, UK) and Alexa Fluor 488 (1:250, A11001, Invitrogen) secondary antibodies for 1 h at room temperature; then, nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (2 µg/mL in PBS, Sigma-Aldrich) for 30 min at room temperature. Cells were imaged by using an LSM800 confocal microscope (Carl Zeiss, Oberkochen, DE, Germany). 2.6. Statistical Analysis Data were analysed by using Student’s t-test with Welch correction (GraphPad Prism 7 Software, Graphpad Software, La Jolla, CA, USA). Results were considered statistically significant when p < 0.05. Data are represented as means ± standard deviation (SD). 3.1. ApoE Intra- and Extracellular Levels Are Altered in ApoE4 Astrocytes 3.1. ApoE Intra- and Extracellular Levels Are Altered in ApoE4 Astrocytes 3.1. ApoE Intra- and Extracellular Levels Are Altered in ApoE4 Astrocytes Alterations of ApoE synthesis and release have been shown to characterize hiPSCs- derived ApoE4 astrocytes [15,16,24]. Results confirmed that the ApoE intracellular content in immortalized ApoE4 astrocytes is less than halved compared to the ApoE amount present in ApoE3 astrocytes, as shown by both immunocytochemistry (Figure 1A) and Western blotting (p < 0.0001; Figure 1B). On the other hand, the levels of ApoE released in ACM by the ApoE4 cell line resulted to be more than doubled compared to that secreted by the ApoE3 cell line (p < 0.01; Figure 1B). EVIEW 6 of 22 Figure 1. Cont. Figure 1. Cont. 6 of 21 Antioxidants 2022, 11, 2168 Figure 1. ApoE synthesis and release is altered in ApoE4 astrocytes. (A) ApoE protein levels were examined by immunocytochemistry. Antibodies detecting ApoE (red), and -actin (green), were used and nuclei were stained with DAPI (blue). Representative im- ages are shown. Cells were imaged by using an LSM800 confocal microscope (Zeiss, 63X oil objective; scale bar: 50 m). (B) ApoE protein levels were analysed by Western blot- ting in both cell lysates and concentrated media from ApoE3 and ApoE4 immortalized mouse astrocytes. Concerning cell lysates, ApoE levels were normalized to the corre- sponding -actin levels and data are represented as percentages of average ApoE3 val- ues. Data are expressed as mean values ± SD of four different experiments (n = 14, Figure 1. ApoE synthesis and release is altered in ApoE4 astrocytes. (A) ApoE protein levels were examined by immunocytochemistry. Antibodies detecting ApoE (red), and β-actin (green), were used and nuclei were stained with DAPI (blue). Representative images are shown. Cells were imaged by using an LSM800 confocal microscope (Zeiss, 63X oil objective; scale bar: 50 µm). (B) ApoE protein levels were analysed by Western blotting in both cell lysates and concentrated media from ApoE3 and ApoE4 immortalized mouse astrocytes. Concerning cell lysates, ApoE levels were normalized to the corresponding β-actin levels and data are represented as percentages of average ApoE3 values. Data are expressed as mean values ± SD of four different experiments (n = 14, Student’s t-test). Concerning concentrated media, ApoE levels were normalized to total proteins loaded into the corresponding well (Stain-free method) and data are represented as percentage of average ApoE3 values. 3.1. ApoE Intra- and Extracellular Levels Are Altered in ApoE4 Astrocytes Data are expressed as mean values ± SD of four different experiments (n = 11, Student’s t-test). **** p < 0.0001, ** p < 0.01 vs. ApoE3 astrocytes. Figure 1. ApoE synthesis and release is altered in ApoE4 astrocytes. (A) ApoE protein levels were examined by immunocytochemistry. Antibodies detecting ApoE (red), and -actin (green), were used and nuclei were stained with DAPI (blue). Representative im- ages are shown. Cells were imaged by using an LSM800 confocal microscope (Zeiss, 63X oil objective; scale bar: 50 m). (B) ApoE protein levels were analysed by Western blot- ting in both cell lysates and concentrated media from ApoE3 and ApoE4 immortalized mouse astrocytes. Concerning cell lysates, ApoE levels were normalized to the corre- sponding -actin levels and data are represented as percentages of average ApoE3 val- ues. Data are expressed as mean values ± SD of four different experiments (n = 14, Figure 1. ApoE synthesis and release is altered in ApoE4 astrocytes. (A) ApoE protein levels were examined by immunocytochemistry. Antibodies detecting ApoE (red), and β-actin (green), were used and nuclei were stained with DAPI (blue). Representative images are shown. Cells were imaged by using an LSM800 confocal microscope (Zeiss, 63X oil objective; scale bar: 50 µm). (B) ApoE protein levels were analysed by Western blotting in both cell lysates and concentrated media from ApoE3 and ApoE4 immortalized mouse astrocytes. Concerning cell lysates, ApoE levels were normalized to the corresponding β-actin levels and data are represented as percentages of average ApoE3 values. Data are expressed as mean values ± SD of four different experiments (n = 14, Student’s t-test). Concerning concentrated media, ApoE levels were normalized to total proteins loaded into the corresponding well (Stain-free method) and data are represented as percentage of average ApoE3 values. Data are expressed as mean values ± SD of four different experiments (n = 11, Student’s t-test). **** p < 0.0001, ** p < 0.01 vs. ApoE3 astrocytes. 3.2. ApoE4 Astrocytes Show Different Expression Levels of Several Lipid-Related Genes Transcriptomic studies showed that lipid metabolism is one of the main altered pro- cesses in ApoE4 astrocytes [15,16]. In order to investigate this aspect, the expression levels of 43 lipid-related genes were analysed in both ApoE3 and ApoE4 astrocytes and sev- eral genes resulted to be up- or down-regulated in ApoE4 compared to ApoE3 astrocytes (Figure 2). Antioxidants 2022, 11, 2168 Antioxidan 7 of 21 Figure 2. Gene expression analysis of lipid-related genes. Gene expression levels of 43 genes were analysed in ApoE3 and ApoE4 immortalized mouse astrocytes by using Array Mouse Lipid Regulated Genes 96-well Plate. The Figure shows the expression le gene in ApoE4 astrocytes (up-regulated genes: red; down-regulated genes: blue) c ApoE3 astrocytes. Gene expression results were normalized to the average expression le endogenous control genes included in the plate: glyceraldehyde-3-phosphate deh (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and -glucuronidase ( chinodate 5-lipoxygenase (ALOX5) and lipoprotein lipase (LPL) expression levels wer ble in ApoE4 astrocytes, so it was not possible to calculate the Log2(FC) of these gen expressed as mean values of three different experiments (n = 6, Student’s t-test). **** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes; ns: not significant. 3 3 ApoE4 Astrocytes Are Characterized by A Lower Cholesterol Synthesis Figure 2. Gene expression analysis of lipid-related genes. Gene expression levels of 43 lipid-related genes were analysed in ApoE3 and ApoE4 immortalized mouse astrocytes by using the TaqMan Array Mouse Lipid Regulated Genes 96-well Plate. The Figure shows the expression levels of each gene in ApoE4 astrocytes (up-regulated genes: red; down-regulated genes: blue) compared to ApoE3 astrocytes. Gene expression results were normalized to the average expression levels of three endoge- nous control genes included in the plate: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and β-glucuronidase (GUSB). Arachinodate 5-lipoxygenase (ALOX5) and lipoprotein lipase (LPL) expression levels were undetectable in ApoE4 astrocytes, so it was not possible to calculate the Log2(FC) of these genes. Data are expressed as mean values of three different experiments (n = 6, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes; ns: not significant. Figure 2. Gene expression analysis of lipid-related genes. Gene expression levels of 43 genes were analysed in ApoE3 and ApoE4 immortalized mouse astrocytes by using Array Mouse Lipid Regulated Genes 96-well Plate. 3.2. ApoE4 Astrocytes Show Different Expression Levels of Several Lipid-Related Genes The Figure shows the expression le gene in ApoE4 astrocytes (up-regulated genes: red; down-regulated genes: blue) c ApoE3 astrocytes. Gene expression results were normalized to the average expression le endogenous control genes included in the plate: glyceraldehyde-3-phosphate deh (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and -glucuronidase ( chinodate 5-lipoxygenase (ALOX5) and lipoprotein lipase (LPL) expression levels wer ble in ApoE4 astrocytes, so it was not possible to calculate the Log2(FC) of these gen expressed as mean values of three different experiments (n = 6, Student’s t-test). **** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes; ns: not significant. 3 3 ApoE4 Astrocytes Are Characterized by A Lower Cholesterol Synthesis Figure 2. Gene expression analysis of lipid-related genes. Gene expression levels of 43 lipid-related genes were analysed in ApoE3 and ApoE4 immortalized mouse astrocytes by using the TaqMan Array Mouse Lipid Regulated Genes 96-well Plate. The Figure shows the expression levels of each gene in ApoE4 astrocytes (up-regulated genes: red; down-regulated genes: blue) compared to ApoE3 astrocytes. Gene expression results were normalized to the average expression levels of three endoge- nous control genes included in the plate: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and β-glucuronidase (GUSB). Arachinodate 5-lipoxygenase (ALOX5) and lipoprotein lipase (LPL) expression levels were undetectable in ApoE4 astrocytes, so it was not possible to calculate the Log2(FC) of these genes. Data are expressed as mean values of three different experiments (n = 6, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes; ns: not significant. Antioxidants 2022, 11, 2168 8 of 21 8 of 21 Of the genes that were found significantly more expressed in ApoE4 astrocytes, starting from the most up-regulated one, are those coding for cholesterol 27-hydroxylase (CYP27A1; p < 0.0001), arachidonate 12-lipoxygenase (ALOX12; p < 0.05), arachidonate 15-lipoxygenase (ALOX15; p < 0.0001), arachidonate 5-lipoxygenase activating protein (ALOX5AP; p < 0.01), solute carrier family 16 member 6 (SLC16A6; p < 0.0001), phospholipase A2 (PLA2G4A; p < 0.01), sterol regulatory element binding transcription factor 1 (SREBF1; p < 0.01), hydroxy-methylglutaryl-CoA synthase 1 (HMGCS1; p < 0.0001), leukotriene A4 hydrolase (LTA4H; p < 0.01), stearoyl-CoA desaturase (SCD1; p < 0.05), fatty acid desaturase (FADS1; p < 0.05), and acetyl-CoA acetyltransferase 1 (ACAT1; p < 0.05). 3.2. ApoE4 Astrocytes Show Different Expression Levels of Several Lipid-Related Genes p ) y y ( p ) On the other hand, the most down-regulated genes in ApoE4 astrocytes are peroxisome proliferator activated receptor γ (PPARγ; p < 0.0001), mitochondrial uncoupling protein 2 (UCP2; p < 0.05), nuclear receptor subfamily 1 group H member 3 (NR1H3, also known as LXRα; p < 0.0001), solute carrier family 27 member 3 (SLC27A3; p < 0.05), insulin-induced gene 1 protein (INSIG1; p < 0.05), prostaglandin-endoperoxide synthase 2 (PTGS2, also known as COX-2; p < 0.05), ABC subfamily A member 1 (ABCA1; p < 0.05), and glycerol kinase (GYK; p < 0.01). Notably, the expression levels of lipoprotein lipase (LPL) and arachidonate 5-lipoxygenase (ALOX5) were found to be down-regulated but undetectable in ApoE4 astrocytes, indicating the almost total absence of expression of these genes. Thus, it was not possible to calculate the exact extent and significance of these down-regulations. The expression levels of ABCG1 and interleukin 1β (IL1β) are not reported because they were undetectable in both cell lines. 3.3. ApoE4 Astrocytes Are Characterized by a Lower Cholesterol Synthesis Quantification of cholesterol and its precursors, and analysis of key enzymes expression. Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coenzyme A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydroxylase; CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7-hydroxylase; DE: desmosterol; DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7-OHC: 7- hydroxycholesterol; 7-OHC; 7-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25-OHC: 25- hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. (B) Total cholesterol and cholesterol precur- sors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. (C) Gene expression levels of the enzymes HMGCR and DHCR24 were analysed in Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coenzyme A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydroxylase; CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7α-hydroxylase; DE: desmosterol; DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7α-OHC: 7α-hydroxycholesterol; 7β-OHC; 7β-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25-OHC: 25-hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. (B) Total cholesterol and cholesterol pre- cursors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. (C) Gene expression levels of the enzymes HMGCR and DHCR24 were analysed in ApoE3 and ApoE4 immortalized mouse astrocytes. Gene expression results were normalized to GAPDH expression levels. Data are expressed as mean values of three different experiments (n = 8, Student’s t-test). ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. g Q p , y y y p (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coenzyme A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydroxylase; CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7-hydroxylase; DE: desmosterol; DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7-OHC: 7- hydroxycholesterol; 7-OHC; 7-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25-OHC: 25- hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. 3.3. ApoE4 Astrocytes Are Characterized by a Lower Cholesterol Synthesis It has previously been shown that cholesterol homeostasis is impaired in ApoE4 astro- cytes; however, results are still controversial, and it seems that they depend on the specific experimental model [15,16,24]. Thus, we quantified total cholesterol and some biosynthetic cholesterol precursors by GC-MS to have a broader view of the lipid profile of the ApoE3 and ApoE4 cell lines. A simplified representation of cholesterol synthesis and oxidation is depicted in Figure 3A. The analysis pointed out that the levels of the cholesterol precursors lanosterol (+54%; p < 0.0001) and lathosterol (+142%; p < 0.0001) are higher in ApoE4 astrocytes, whereas desmosterol levels are lower (−32%; p < 0.01) compared to ApoE3 astrocytes. Overall, total cholesterol levels are slightly but significantly reduced (−14%; p < 0.05) (Figure 3B). Moreover, we analysed the gene expression levels of two key enzymes involved in cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and 24-dehydrocholesterol reductase (DHCR24). As shown in Figure 3C, HMGCR (p < 0.05) and DHCR24 (p < 0.01) expression levels are significantly reduced in ApoE4 astrocytes. 9 of 21 Antioxidants 2022, 11, 2168 Antioxidants 2021, 10, x FOR Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coenzyme A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydroxylase; CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7-hydroxylase; DE: desmosterol; DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7-OHC: 7- hydroxycholesterol; 7-OHC; 7-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25-OHC: 25- hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. (B) Total cholesterol and cholesterol precur- sors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. (C) Gene expression levels of the enzymes HMGCR and DHCR24 were analysed in Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coenzyme A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydroxylase; CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7α-hydroxylase; DE: desmosterol; DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7α-OHC: 7α-hydroxycholesterol; 7β-OHC; 7β-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25-OHC: 25-hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. 3.3. ApoE4 Astrocytes Are Characterized by a Lower Cholesterol Synthesis (B) Total cholesterol and cholesterol pre- cursors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coenzyme A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydroxylase; CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7-hydroxylase; DE: desmosterol; DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7-OHC: 7- hydroxycholesterol; 7-OHC; 7-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25-OHC: 25- hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. (B) Total cholesterol and cholesterol precur- sors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. (C) Gene expression levels of the enzymes HMGCR and DHCR24 were analysed in Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expre (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coen A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydrox CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7α-hydroxylase; DE: desmo DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA redu LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7α- 7α-hydroxycholesterol; 7β-OHC; 7β-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25- 25-hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. (B) Total cholesterol and cholestero cursors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-M ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. A astrocytes. (C) Gene expression levels of the enzymes HMGCR and DHCR24 were analysed in A Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. Figure 3 Quantification of cholesterol and its precursors and analysis of key enzymes ex Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. (A) Schematic representation of cholesterol synthesis and oxidation Acetyl-CoA: Acetyl coenzyme Figure 3. 3.3. ApoE4 Astrocytes Are Characterized by a Lower Cholesterol Synthesis (B) Total cholesterol and cholesterol precur- sors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. (C) Gene expression levels of the enzymes HMGCR and DHCR24 were analysed in Figure 3. Quantification of cholesterol and its precursors, and analysis of key enzymes expression. (A) Schematic representation of cholesterol synthesis and oxidation. Acetyl-CoA: Acetyl coenzyme A; Chol: cholesterol; CH25H: cholesterol 25-hydroxylase; CYP46A1: cholesterol 24-hydroxylase; CYP27A1: cholesterol 27-hydroxylase; CYP7A1: cholesterol 7α-hydroxylase; DE: desmosterol; DHCR24: 24-dehydrocholesterol reductase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LA: lanosterol; LT: lathosterol; ROS: reactive oxygen species; 7-KC: 7-ketocholesterol; 7α-OHC: 7α-hydroxycholesterol; 7β-OHC; 7β-hydroxycholesterol; 24-OHC: 24-hydroxycholesterol; 25-OHC: 25-hydroxycholesterol; 27-OHC: 27-hydroxycholesterol. (B) Total cholesterol and cholesterol pre- cursors (LA, LT, and DE) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. (C) Gene expression levels of the enzymes HMGCR and DHCR24 were analysed in ApoE3 and ApoE4 immortalized mouse astrocytes. Gene expression results were normalized to GAPDH expression levels. Data are expressed as mean values of three different experiments (n = 8, Student’s t-test). ** p < 0.01, and * p < 0.05 vs. ApoE3 astrocytes. 10 of 21 10 of 21 Antioxidants 2022, 11, 2168 3.4. Oxysterol Profile Is Different in ApoE4 Astrocytes 3.4. Oxysterol Profile Is Different in ApoE4 Astrocytes Since AD is associated with abnormalities not only in cholesterol biosynthesis but also in its catabolism by oxidation [11,19,25], we also analysed oxysterol levels in ApoE3 and ApoE4 astrocytes by GC-MS. Concerning the oxysterols of enzymatic origin, ApoE4 astrocytes have been shown to produce higher levels of 25-OHC (+282%; p < 0.0001) and 27-OHC (+72%; p < 0.001), whereas cholesterol oxidation to 24-OHC (−36%; p < 0.0001) and 7α-OHC (−51%; p < 0.0001) resulted in being lower. Among the oxysterols of non- enzymatic origin, 7β-OHC levels were found to be lower (−52%; p < 0.0001) and 7-KC levels did not change significantly in ApoE4 astrocytes (Figure 4A). In order to better understand the obtained results concerning oxysterol levels, we analysed the gene expression levels of the enzymes responsible for the synthesis of the quantified enzymatically produced oxysterols. In particular, the latter are the enzymes cholesterol 24-hydroxylase (CYP46A1), cholesterol 25-hydroxylase (CH25H), CYP27A1, and cholesterol 7α-hydroxylase (CYP7A1), respectively, responsible for the synthesis of 24-OHC, 25-OHC, 27-OHC, and 7α-OHC, as outlined in Figure 3A. Specifically, their expression levels resulted in reflecting the corresponding oxysterol trend; indeed, CH25H (p < 0.0001) and CYP27A1 (p < 0.0001) gene expression levels markedly increased in ApoE4 astrocytes, whereas those of CYP46A1 (p < 0.05) resulted to be reduced compared to ApoE3 astrocytes. Instead, CYP7A1 expression did not significantly change (Figure 4B). EVIEW 12 of 22 Figure 4. Cont. Figure 4. Cont. Figure 4. Cont. 11 of 21 Antioxidants 2022, 11, 2168 Figure 4. Quantification of oxysterols and gene expression analysis of the enzymes involved in cho- lesterol oxidation. (A) The amount of the oxysterols 24-hydroxycholesterol (24-OHC), 25-hydroxy- cholesterol (25-OHC), 27-hydroxycholesterol (27-OHC), 7-hydroxycholesterol (7-OHC), 7-hy- droxycholesterol (7-OHC), and 7-ketocholesterol (7-KC) were quantified by gas chromatography- mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are ex- pressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, *** p < 0.001 vs. ApoE3 astrocytes. (B) Gene expression levels of the enzymes cholesterol 24-hydrox- ylase (CYP46A1), cholesterol 25-hydroxylase (CH25H), cholesterol 27-hydroxylase (CYP27A1), and cholesterol 7-hydroxylase (CYP7A1) were analysed in ApoE3 and ApoE4 immortalized mouse as- trocytes. Gene expression results were normalized to GAPDH expression levels. Data are shown either as Log2(gene/GAPDH) or as Log2(FC). 3.4. Oxysterol Profile Is Different in ApoE4 Astrocytes Data are expressed as mean values ± SD of three dif- ferent experiments (n = 8, Student’s t-test). **** p < 0.0001, *p < 0.05 vs. ApoE3 astrocytes. Figure 4. Quantification of oxysterols and gene expression analysis of the enzymes involved in cholesterol oxidation. (A) The amount of the oxysterols 24-hydroxycholesterol (24-OHC), 25-hydroxycholesterol (25-OHC), 27-hydroxycholesterol (27-OHC), 7α-hydroxycholesterol (7α- OHC), 7β-hydroxycholesterol (7β-OHC), and 7-ketocholesterol (7-KC) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astro- cytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, *** p < 0.001 vs. ApoE3 astrocytes. (B) Gene expression levels of the enzymes choles- terol 24-hydroxylase (CYP46A1), cholesterol 25-hydroxylase (CH25H), cholesterol 27-hydroxylase (CYP27A1), and cholesterol 7α-hydroxylase (CYP7A1) were analysed in ApoE3 and ApoE4 immortal- ized mouse astrocytes. Gene expression results were normalized to GAPDH expression levels. Data are shown either as Log2(gene/GAPDH) or as Log2(FC). Data are expressed as mean values ± SD of three different experiments (n = 8, Student’s t-test). **** p < 0.0001, * p < 0.05 vs. ApoE3 astrocytes. 3 5 A E4 A t t S th ti L A t f KEY P t i I l d i Figure 4. Quantification of oxysterols and gene expression analysis of the enzymes involved in cho- lesterol oxidation. (A) The amount of the oxysterols 24-hydroxycholesterol (24-OHC), 25-hydroxy- cholesterol (25-OHC), 27-hydroxycholesterol (27-OHC), 7-hydroxycholesterol (7-OHC), 7-hy- droxycholesterol (7-OHC), and 7-ketocholesterol (7-KC) were quantified by gas chromatography- mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astrocytes. Data are ex- pressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, *** p < 0.001 vs. ApoE3 astrocytes. (B) Gene expression levels of the enzymes cholesterol 24-hydrox- ylase (CYP46A1), cholesterol 25-hydroxylase (CH25H), cholesterol 27-hydroxylase (CYP27A1), and cholesterol 7-hydroxylase (CYP7A1) were analysed in ApoE3 and ApoE4 immortalized mouse as- trocytes. Gene expression results were normalized to GAPDH expression levels. Data are shown either as Log2(gene/GAPDH) or as Log2(FC). Data are expressed as mean values ± SD of three dif- ferent experiments (n = 8, Student’s t-test). **** p < 0.0001, *p < 0.05 vs. ApoE3 astrocytes. Figure 4. Quantification of oxysterols and gene expression analysis of the enzymes involved in cholesterol oxidation. 3.4. Oxysterol Profile Is Different in ApoE4 Astrocytes (A) The amount of the oxysterols 24-hydroxycholesterol (24-OHC), 25-hydroxycholesterol (25-OHC), 27-hydroxycholesterol (27-OHC), 7α-hydroxycholesterol (7α- OHC), 7β-hydroxycholesterol (7β-OHC), and 7-ketocholesterol (7-KC) were quantified by gas chromatography-mass spectrometry (GC-MS) in ApoE3 and ApoE4 immortalized mouse astro- cytes. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001, *** p < 0.001 vs. ApoE3 astrocytes. (B) Gene expression levels of the enzymes choles- terol 24-hydroxylase (CYP46A1), cholesterol 25-hydroxylase (CH25H), cholesterol 27-hydroxylase (CYP27A1), and cholesterol 7α-hydroxylase (CYP7A1) were analysed in ApoE3 and ApoE4 immortal- ized mouse astrocytes. Gene expression results were normalized to GAPDH expression levels. Data are shown either as Log2(gene/GAPDH) or as Log2(FC). Data are expressed as mean values ± SD of three different experiments (n = 8, Student’s t-test). **** p < 0.0001, * p < 0.05 vs. ApoE3 astrocytes. 3.5. ApoE4 Astrocytes Synthetize Lower Amounts of KEY Proteins Involved in Cholesterol Homeostasis In order to better characterize the ApoE4 astrocytic cell line, we analysed the levels of key proteins involved in the regulation of cholesterol homeostasis in both ApoE3 and ApoE4 cell lines. We observed that ApoE4 astrocytes are characterized by substantial lower levels of proteins involved in cholesterol efflux and uptake, i.e., the transporter ABCA1 (p < 0.0001) and the receptors low-density lipoprotein receptor (LDLR; p < 0.0001), LDLR- related protein 1 (LRP1; p < 0.0001), and ApoE receptor 2 (ApoER2; p < 0.001) (Figure 5). In addition, data confirmed that PPARγ protein levels are markedly decreased in ApoE4 compared to ApoE3 astrocytes (p < 0.0001) (Figure 5), as suggested by its very low gene expression levels (Figure 2). 12 of 21 ase i ery low 12 of 21 ase i ery low Antioxidants 2022, 11, 2168 Figure 5. Analysis of the levels of key proteins involved in lipid homeostasis. The protein levels of the ATP binding cassette subfamily A member 1 (ABCA1), ApoE receptor 2 (ApoER2), low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), and peroxisome proliferator activated receptor  (PPAR) were determined by Western blotting in ApoE3 and ApoE4 immortalized mouse astrocytes. Data were normalized to the corresponding -actin levels and are shown as percentage of average ApoE3 values. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001 and *** p < 0.001 vs. ApoE3 astrocytes. Figure 5. 3.4. Oxysterol Profile Is Different in ApoE4 Astrocytes p p p ( ), p ( ), p p receptor γ (PPARγ) were determined by Western blotting in ApoE3 and ApoE4 immortalized mouse astrocytes. Data were normalized to the corresponding β-actin levels and are shown as percentage of average ApoE3 values. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001 and *** p < 0.001 vs. ApoE3 astrocytes. 3.4. Oxysterol Profile Is Different in ApoE4 Astrocytes Analysis of the levels of key proteins involved in lipid homeostasis. The protein levels of the ATP binding cassette subfamily A member 1 (ABCA1), ApoE receptor 2 (ApoER2), low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), and peroxisome proliferator activated receptor γ (PPARγ) were determined by Western blotting in ApoE3 and ApoE4 immortalized mouse astrocytes. Data were normalized to the corresponding β-actin levels and are shown as percentage of average ApoE3 values. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001 and *** p < 0.001 vs. ApoE3 astrocytes. Figure 5. Analysis of the levels of key proteins involved in lipid homeostasis. The protein levels of the ATP binding cassette subfamily A member 1 (ABCA1), ApoE receptor 2 (ApoER2), low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), and peroxisome proliferator activated receptor  (PPAR) were determined by Western blotting in ApoE3 and ApoE4 immortalized mouse astrocytes. Data were normalized to the corresponding -actin levels and are shown as percentage of average ApoE3 values. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001 and *** p < 0.001 vs. ApoE3 astrocytes. Figure 5. Analysis of the levels of key proteins involved in lipid homeostasis. The protein levels of the ATP binding cassette subfamily A member 1 (ABCA1), ApoE receptor 2 (ApoER2), low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), and peroxisome proliferator activated receptor γ (PPARγ) were determined by Western blotting in ApoE3 and ApoE4 immortalized mouse astrocytes. Data were normalized to the corresponding β-actin levels and are shown as percentage of average ApoE3 values. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001 and *** p < 0.001 vs. ApoE3 astrocytes. lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), and peroxisome proliferator activated receptor  (PPAR) were determined by Western blotting in ApoE3 and ApoE4 immortalized mouse astrocytes. Data were normalized to the corresponding -actin levels and are shown as percentage of average ApoE3 values. Data are expressed as mean values ± SD of three different experiments (n = 9, Student’s t-test). **** p < 0.0001 and *** p < 0.001 vs. ApoE3 astrocytes. 4. Discussion 4. Discussion ApoE is the primary apolipoprotein responsible for the transport of cholesterol in the brain, where astrocytes are its main producers [26]. After ApoE secretion and lipidation, ApoE-containing lipoproteins are taken up by neurons by binding to different types of receptors, playing a key function in neuronal maintenance and repair (e.g., synaptic ho- meostasis and axonal regeneration) [27,28]. Since ApoE4 has been identified as the strong- est genetic risk factor for sporadic AD, more and more studies have been carried out in order to understand the structural and functional consequences due to the single amino ApoE is the primary apolipoprotein responsible for the transport of cholesterol in the brain, where astrocytes are its main producers [26]. After ApoE secretion and lipida- tion, ApoE-containing lipoproteins are taken up by neurons by binding to different types of receptors, playing a key function in neuronal maintenance and repair (e.g., synaptic homeostasis and axonal regeneration) [27,28]. Since ApoE4 has been identified as the strongest genetic risk factor for sporadic AD, more and more studies have been carried out in order to understand the structural and functional consequences due to the single amino acid change that characterizes ApoE4 (Arg112) compared to the most common ApoE3 isoform (Cys112) [29]. Even if Arg112 is not part of the LDLR binding site, studies showed that ApoE4 has different specificities in binding lipids, and the lipidation status is assumed to affect its ability to bind receptors [30,31]. However, the current knowledge about ApoE role in AD pathology is still limited because native and lipidated ApoE forms present in vivo have not yet been analysed, due to the complexity of the structures and to the insufficient methods currently available [29]. Concerning ApoE4 brain implications, ApoE4 carriers showed an increased Aβ deposition [32,33], a greater hippocampal and cortical atrophy [34], and a higher risk of progression from mild cognitive impairment to AD [35]. Moreover, many of these features also characterize ApoE4-TR mice, which showed cognitive impairment [36,37], Aβ and hyperphosphorylated tau accumulation [38], and deficits in synaptic transmission [39]. Antioxidants 2022, 11, 2168 13 of 21 13 of 21 Although the role of ApoE4 in AD onset and progression remains unclear, many studies pointed out that ApoE4 may contribute to AD pathogenesis affecting neuronal structure and function, Aβ production and clearance, tau pathology, and neuroinflamma- tion [40]. 4. Discussion 4. Discussion In particular, new mouse models, in vitro models (e.g., derived from hiPSC), and genome editing technics allowed to a deeper understanding of the impact of ApoE4 in different brain cell types, including astrocytes [41]. ApoE4 has been shown to impact on many physiological functions played by astrocytes, affecting their ability to clear Aβ, to uptake glutamate, to promote neuronal synaptogenesis, and to maintain lipid homeosta- sis [15,16,42]. Among all these aspects, being astrocytes the main producers of cholesterol and ApoE in the brain, we decided to deepen the impact of ApoE4 genotype on astrocytic cholesterol homeostasis in order to better characterize the induced metabolic and molecular alterations. For this purpose, ApoE3 and ApoE4 astrocytic cell lines obtained from ApoE-TR mouse primary astrocytes were used as experimental models [18]. As shown in Figure 1A, the two cell lines have a different morphology, with ApoE4 astrocytes resulting as smaller compared to ApoE3 astrocytes. Morphological differences have been also observed by other researchers in hiPSC-derived ApoE4 astrocytes, which showed a reduced expression of genes involved in the regulation of actin cytoskeleton and focal adhesions, as well as a reduced attached cell surface area [15]. Concerning ApoE levels, we observed that ApoE intracellular levels in ApoE4 astrocytes are less than half compared to the levels detected in ApoE3 astrocytes (Figure 1A,B). In agreement with our data, significantly reduced ApoE levels were also observed in hiPSC-derived ApoE4 astrocytes [15,16,24], as well as in brain homogenates from mouse models expressing the human ApoE4 isoform [43,44]. It has been hypothesized that ApoE4 levels may be lower due to rapid degradation [45], but the exact mechanisms of this degradation are not yet clear. However, it appears that ApoE can be degraded by autophagy and by an LC3-associated endocytosis-like process, and that ApoE4 is sequestered in enlarged endosomes [46]. On the other hand, although previous studies reported for reduced ApoE secretion by hiPSC-derived ApoE4 astrocytes [15,16,24], in our experimental model we observed a significantly increased ApoE secretion in the ACM of ApoE4 astrocytes (Figure 1B). The evident increase in secreted ApoE could be due to a higher number of ApoE molecules associated with each ApoE-containing lipoprotein in ApoE4 ACM, as observed by Gong and colleagues [47]; however, it is still not clear the exact stoichiometry of ApoE per ApoE-lipid particle, or if it is affected by the ApoE genotype [29]. 4. Discussion 4. Discussion Concerning astrocytes, they have been shown to mainly contain precursors of the Bloch pathway [54]. In order to maintain brain cholesterol homeostasis, excess cholesterol is stored in the endochylema after esterification, or it is oxidized and converted into oxysterols (Figure 3A). The main oxysterol involved in this process is 24-OHC, also called cerebrosterol, which derives from cholesterol oxidation by CYP46A1, a cytochrome P-450 enzyme mainly expressed by neurons but also by astrocytes [55]. The 24-OHC diffuses across the BBB into the systemic circulation (∼99%) driven by the concentration gradient, and it is finally metabolized in the liver [56,57]; less than 1% of 24-OHC flows into the CSF [58]. Moreover, 24-OHC, being an LXR ligand, regulates the expression and synthesis of the proteins involved in cholesterol synthesis and efflux, including ApoE and ABCA1/ABCG1 [59]. To a lesser extent, cholesterol is oxidized into 27-OHC by CYP27A1, although most of it comes from the systemic circulation [11,14]. In addition to these two oxysterols, others are produced via enzymatic reactions, such as 25-OHC and 7α-OHC deriving from cholesterol oxidation by CH25H and CYP7A1, respectively; however, these oxysterols may be also generated by cholesterol non-enzymatic oxidation mediated by reactive oxygen species (ROS). In fact, cholesterol autooxidation can also occur, induced by different compounds such as lipid peroxides, free radical species, and metal cations, giving rise to the formation of other oxysterols, such as 7-KC and 7β-OHC [60]. In the adult brain, mature neurons mostly depend on the rate of cholesterol supplied by astrocytes. Once synthesized by astrocytes, cholesterol is loaded into lipoproteins similar to high-density lipoproteins (HDL) containing ApoE, and then it is secreted into the extracellular matrix via ABCA1 and ABCG1; then, lipoproteins are transported to neurons where they are internalized through the LDLR and LRP1 placed on the nerve cell membrane. Following receptor-mediated endocytosis ApoE is recycled, and cholesterol is used for cell membrane turnover and repair, dendritic growth, myelin formation, synaptogenesis, and neurotransmitter release [61,62]. However, LDLR, LRP1 and ApoER2 receptors are also present in astrocytes [15,24,63] and have been also shown to be involved in the Aβ clearance mediated by astrocytes [50,64]. y y It has been shown that metabolite levels and gene expression associated with choles- terol de novo biosynthesis are reduced in AD brain regions vulnerable to AD pathology, probably due to impaired enzymatic cholesterol catabolism and efflux [25]. 4. Discussion 4. Discussion The observed increase in secreted ApoE may be also due to a reduced ApoE re-uptake through LDLR, LRP1, and ApoER2 [48] caused by the low levels of these receptors found in ApoE4 astrocytes (Figure 5); this is suggested by the increase in ApoE cerebrospinal fluid (CSF) levels observed in LDLR-/- mice [49], and by the increase in ApoE released in ACM by LDLR-/- primary astrocytes [50]. The maintenance of cholesterol homeostasis in the brain is essential for neuronal functioning and the abnormal brain cholesterol metabolism is associated with neurodegen- erative diseases, such as AD [25]. In order to have a broader view of the lipid-related gene expression in ApoE3 and ApoE4 cell lines, the expression levels of more than forty genes were analysed and lots of them resulted up- or down-regulated in ApoE4 astrocytes. In particular, among the identified differentially expressed genes, there are genes involved in the regulation of cholesterol synthesis (e.g., SREBF1, INSIG1, HMGCS1), oxidation (e.g., CYP27A1), and efflux (e.g., ABCA1). Interestingly, LXRα and PPARγ, key proteins involved in the regulation of lipid homeostasis [51,52], have been shown to be two of the most down-regulated genes (Figure 2). These results, together with those obtained by transcriptomic and proteomic analyses of hiPSC-derived astrocytes [15,16], further confirm that lipid metabolism is one of the main altered processes in ApoE4 astrocytes. Brain cholesterol homeostasis is highly regulated to ensure steady levels of the sterol, and it is almost entirely independent from that of the periphery because of the limited exchange between the central nervous system (CNS) and peripheral circulation due to the presence of the blood–brain barrier (BBB). Thus, cholesterol in the brain derives almost exclusively (>95%) from local de novo synthesis by astrocytes from Acetyl-CoA through Antioxidants 2022, 11, 2168 14 of 21 14 of 21 reactions catalyzed by over 20 enzymes. As shown in Figure 3A, in an early stage of cholesterol biosynthesis Acetyl-CoA is converted into the first sterol, lanosterol. The first rate-limiting enzyme involved in this stage is HMGCR, which catalyzes the reduction in HMG-CoA to mevalonate giving rise to a series of reactions that lead to the formation of lanosterol. Then, in the so-called “post-lanosterol stage”, sterol intermediates proceed through either the Bloch or the Kandutsch–Russell pathway, which both ultimately lead to cholesterol synthesis. The enzyme DHCR24 may act on any intermediate in the Bloch pathway and deflect it in the Kandutsch–Russell one [53]. 4. Discussion 4. Discussion Recently, it has been shown that cholesterol homeostasis is also impaired in ApoE4 astrocytes, although controversial data concerning intracellular and secreted cholesterol levels are present in lit- erature depending on the different experimental model employed [15,16,24]. Our analysis by GC-MS pointed out that total cholesterol levels are slightly but significantly reduced in ApoE4 astrocytes in comparison to ApoE3 astrocytes (Figure 3B), similarly to what was observed by de Leeuw and colleagues in hiPSC-derived ApoE4 astrocytes [24]. The modest reduction in the amount of intracellular cholesterol could be due to the reduction in the expression levels of the two key enzymes involved in cholesterol biosynthesis, i.e., HMGCR and DHCR24 (Figure 3C), also observed in AD brain samples [25]. Moreover, the analyses pointed out that the levels of the cholesterol precursors lanosterol and lath- osterol are much higher in ApoE4 astrocytes (Figure 3B), indicating a possible feedback regulation to compensate for reduced cholesterol. Conversely, desmosterol levels are lower in ApoE4 compared to ApoE3 astrocytes (Figure 3B). Based on this evidence, it could be assumed that in ApoE4 astrocytes cholesterol anabolism shifts from the Bloch pathway to the Kandutsch–Russell pathway, as suggested by desmosterol decrease and lathosterol Antioxidants 2022, 11, 2168 15 of 21 15 of 21 increase. Therefore, the presence of ApoE4 genotype might affect one or more enzymes involved in the post-lanosterol phases of cholesterol synthesis. As a consequence of the Bloch-pathway inhibition, lanosterol enhances; to avoid accumulation of the latter, both the switch to the Kandutsch–Russell pathway and a feed-back HMGCR downregulation could occur, resulting altogether in a moderate, although significant, cholesterol decrease. In this connection, it is recognized that HMGCR is negatively regulated by side-chain oxidized oxysterols such as 25-OHC and 27-OHC [10], which appear to augment in our cell model; this evidence points to these oxysterols as possible important co-factors in ApoE4 astrocyte cholesterol synthesis. In addition, it is worth noting that while the Bloch pathway generally appears as typical for astrocytes, microglia cells seem to preferentially resort to the Kandutsch–Russell pathway; thus, the shift to this enzymatic cascade might reflect a sort of astrocyte polarization to a cell-type more specialized in immune responses [53]. However, due to the many enzymes involved in cholesterol synthesis and to the complex regulation of this process, a more in-depth analysis is needed to understand whether and how ApoE3 and ApoE4 astrocytes synthetize cholesterol through different pathways. 4. Discussion 4. Discussion p p y y g p y Concerning cholesterol oxidation, in our experimental model ApoE4 astrocytes have been shown to produce higher levels of the oxysterols 25-OHC and 27-OHC, but lower levels of 24-OHC, 7α-OHC and 7β-OHC compared to ApoE3 astrocytes; 7-KC levels did not change significantly (Figure 4A). Interestingly, the expression trend of the enzymes involved in the oxysterol generation goes in parallel with the levels of the corresponding oxysterol. In fact, CYP46A1 and CYP7A1 reduction is followed by 24-OHC and 7α-OHC reduction, respectively; conversely, CYP27A1 and CH25H increase is followed by 27-OHC and 25-OHC increase (Figure 4B). Considerable evidence indicates that changes in oxysterol levels in the brain could be one of the factors contributing to AD progression. Indeed, it has been demonstrated that numerous oxysterols accumulate in the brain as AD progresses (e.g., 27-OHC, 25-OHC, 7α- and 7β-OHC, and 7-KC); conversely, the levels of 24-OHC drop dramatically in the late stages of the disease likely due to neuronal loss [19,65]. Oxysterols accumulating in the brain certainly play a crucial role in AD development by enhancing oxidative stress and inflammation, with consequent neurodegeneration [11]. Notably, although most oxysterols exert detrimental effects, 24-OHC has been shown to have mainly neuroprotective properties, such as preventing tau hyperphosphorylation and Aβ production [66,67]. This leads to the likely assumption that the physiological presence of this oxysterol in the brain is fundamental to guarantee brain health, suggesting the importance of preventing its loss in the brain during the course of AD [67]. Results showed that the changes in oxysterol levels in ApoE4 astrocytes somehow reflect some of those occurring in the AD brain (i.e., 24-OHC decrease, 27-OHC and 25-OHC increase) [19]; in fact, it should be considered that other ApoE4 cell types (i.e., neurons, microglia) could differently contribute to the brain oxysterol content, as well as other systemic and/or environmental factors, among which oxidative stress. Data from this study might highlight another feature of ApoE4 astrocytes that could further explain ApoE4 AD-promoting role by affecting the levels of cholesterol oxidized derivatives. In particular, we observed a reduction in both autoxidation (i.e., 7β-OHC) and enzymatic (i.e., 7α-OHC and 24-OHC) products which, at least in part, could be due to a less availability in ApoE4 astrocytes of the oxysterol precursor, namely cholesterol itself. 4. Discussion 4. Discussion Concerning the impact of the ApoE genotype on the three receptors, several studies showed that ApoE4 is able to reduce their recycling to the cell membrane, likely because of the aberrant endosomal acidification that traps receptors within the intracellular compartments and leads to their degradation [73–75]. Moreover, astrocytic LDLR and LRP1 have been shown to be involved in Aβ uptake and clearance, as confirmed by the significant change in Aβ accumulation observed in experimental models characterized by the knockdown or the overexpression of one of these two receptors [50,64,76]; thus, studies suggest that lower levels of ApoE receptors in the astrocytic membrane may lead to the reduced ability of ApoE4 astrocytes to clear Aβ [16,73]. Another key protein that we observed to be reduced in ApoE4 astrocytes is PPARγ (Figures 2 and 5). PPARγ is a ligand-modulated transcription factor which, through the formation of heterodimers with the retinoid X receptor (RXR), regulates the expression of genes involved in lipid and glucose metabolism [52]; in addition, it is known to exert a neu- roprotective and anti-inflammatory role in the CNS [77,78]. Interestingly, reduced PPARγ expression and protein levels have been observed to characterize the brain of ApoE4-TR mice [79]. Barrera and colleagues showed that PPARγ regulates the transcription of several genes involved in late-onset AD, including ApoE [80]. For instance, PPARγ has been shown to regulate LRP1 protein levels in adipocytes and hepatocytes [81,82], suggesting that the decrease in LRP1 observed in ApoE4 astrocytes may be also due to the dramatic decline in PPARγ levels that characterizes this cell line (Figure 5). In addition to LRP1, other genes are regulated by PPARγ such as ABCA1, LXRα, LPL, and GYK [52,83,84], and all were found to be down-regulated in ApoE4 astrocytes (Figures 2 and 5). Thiazo- lidinediones (TZDs) are PPARγ synthetic ligands with insulin-sensitizing properties, used for the treatment of type 2 diabetes. Since AD is also characterized by alterations of brain insulin signaling, studies on AD mouse models and AD patients have been carried out to investigate the potential of these drugs in AD treatment; however, contrasting results have been obtained so far [85,86]. In particular, a lower effectiveness of PPARγ-targeting therapies has been observed in ApoE4 carriers and may be due to the impairment of PPARγ signaling mediated by ApoE4 through still unknown mechanisms [87]. 4. Discussion 4. Discussion On the other hand, the oxysterols of enzymatic origin 25-OHC and 27-OHC are strikingly enhanced; these data indicate that factors, such as gene modulation, could affect the formation of these oxysterols more than cholesterol amount, as suggested by CH25H and CYP27A1 significant up-regulation. In this connection, it is interesting to note that while 7α-OHC, 7β-OHC, and 24-OHC are primarily involved in cholesterol homeostasis regulation, 25-OHC and 27-OHC are signalling molecules also involved in anti-viral, immunomodulant and inflammatory responses [20], further suggesting an ApoE4 astrocyte differentiation into a microglia-like phenotype, more involved in immune response than in cholesterol supply for neuronal function and development. Antioxidants 2022, 11, 2168 16 of 21 16 of 21 One of the main proteins responsible for ApoE lipidation in the CNS is the transporter ABCA1. The reduced ABCA1 protein levels observed in immortalized ApoE4 astrocytes (Figure 5) further confirm results obtained in mouse primary and hiPSC-derived ApoE4 astrocytes [68]. This marked reduction may be due to the down-regulation of the expression levels of the nuclear receptor LXRα (Figure 2), a key regulator of ABCA1 expression [51]. It has also been suggested that ApoE4 is able to alter ABCA1 protein levels by trapping it in the late-endosomes, thus reducing its recycling to the astrocytic membrane [68]. Furthermore, ABCA1 reduction is considered one of the reasons why ApoE4-containing lipoproteins are poorly lipidated [69,70]; indeed, the treatment of different mouse models expressing ApoE4 with the ABCA1 agonist CS-6253 or the LXR agonist T0901317 have been shown to increase ApoE4 lipidation, as well as to reduce ApoE4-driven Aβ accumulation and cognitive deficits [71,72]. g [ ] ApoE is able to bind various receptors such as LDLR, LRP1, and ApoER2, through which ApoE-containing lipoproteins are taken up by cells [9]. Data showed that ApoE4 astrocytes contain significantly lower levels of all three receptors (Figure 5), although conflicting data regarding some of them have been obtained in primary and hiPSC-derived ApoE4 astrocytes [15,24,73]. As previously discussed, the pronounced reduction in these receptors in ApoE4 astrocytes could be one of the reasons for the increased ApoE levels detected in ACM (Figure 1), as suggested by other studies [49,50]. 4. Discussion 4. Discussion Overall, these data suggest that PPARγ down-regulation could be one of the leading causes of the alterations in cholesterol metabolism that we observed in ApoE4 astrocytes. However, PPARγ response networks are very complex and differently regulated, depending on the specific cell type [52] and its role in the brain need to be further deepened. Antioxidants 2022, 11, 2168 17 of 21 17 of 21 5. Conclusions Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions Although we are aware of the limitations of the employed experimental model, this study highlighted new ApoE4-induced alterations that concern astrocytic choles- terol metabolism, providing support for further investigations on the specific molecular mechanisms involved and the potential consequences on AD pathology. In particular, we observed that ApoE4 astrocytes are characterized by altered expression of several genes involved in cholesterol homeostasis (e.g., synthesis, oxidation, efflux, and uptake). This wide range of differentially expressed genes leads to alterations of the levels of cholesterol precursors and total cholesterol, suggesting a potential shift toward the Kandutsch–Russell pathway, as well as of the levels of several oxysterols. Moreover, ApoE intracellular and extracellular content, as well as the levels of the ABCA1 transporter and the receptors LDLR, LRP1, and ApoER2 resulted to be impaired in ApoE4 astrocytes. One of the reasons for these alterations may be the significant down-regulation of LXRα and PPARγ, both key regulators of lipid homeostasis. g p Based on the obtained data, and given the many roles played by astrocytes in normal neuronal functioning and the considerable impact of astrocytic alterations in AD pathol- ogy, drug development aimed at restoring cholesterol homeostasis could be effective to counteract not only AD but also other types of dementia. Author Contributions: Conceptualization: E.S., P.G. and G.L.; Formal Analysis: E.S. and M.L.I.; Investigation: E.S., V.L., and G.T.; Resources: B.S.; Writing—Original Draft Preparation: E.S. and P.G.; Writing—Review and Editing: G.L. and B.S.; Visualization: E.S. and S.G.; Supervision: P.G.; Funding Acquisition: P.G., G.L. and V.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the University of Turin (grant ID: LEOG_RILO_21_01; GAMP_RILO_21_01) and the University of Milano-Bicocca (grant ID: 2019-ATE-0481). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in article Data Availability Statement: The data presented in this study are available in article. Acknowledgments: The authors would like to thank David Holtzman for kindly providing the ApoE3 and ApoE4 astrocytic cell lines. Acknowledgments: The authors would like to thank David Holtzman for kindly providing the ApoE3 and ApoE4 astrocytic cell lines. Acknowledgments: The authors would like to thank David Holtzman for kindly providing the ApoE3 and ApoE4 astrocytic cell lines. Conflicts of Interest: The authors declare no conflict of interest. References Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [CrossRef] 24. de Leeuw, S.M.; Kirschner, A.W.T.; Lindner, K.; Rust, R.; Budny, V.; Wolski, W.E.; Gavin, A.C.; Nitsch, R.M.; Tackenberg, C. APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes. Stem Cell Rep. 2022, 17, 110–126. [CrossRef] [PubMed] y p 25. Varma, V.R.; Busra Luleci, H.; Oommen, A.M.; Varma, S.; Blackshear, C.T.; Griswold, M.E.; An, Y.; Roberts, J.A.; O’Brien, R.; Pletnikova, O.; et al. Abnormal brain cholesterol homeostasis in Alzheimer’s disease-a targeted metabolomic and transcriptomic study. NPJ Aging Mech. Dis. 2021, 7, 11. [CrossRef] [PubMed] y g g 26. Xu, Q.; Bernardo, A.; Walker, D.; Kanegawa, T.; Mahley, R.W.; Huang, Y. Profile and regulation of apolipoprotein E (ApoE) expression in the CNS in mice with targeting of green fluorescent protein gene to the ApoE locus. J. Neurosci. 2006, 26, 4985–4994. [CrossRef] 27. Lane-Donovan, C.; Wong, W.M.; Durakoglugil, M.S.; Wasser, C.R.; Jiang, S.; Xian, X.; Herz, J. Genetic Restoration of Plasma ApoE Improves Cognition and Partially Restores Synaptic Defects in ApoE-Deficient Mice. J. Neurosci. 2016, 36, 10141–10150. [CrossRef] 28. Yin, C.; Guo, Z.D.; He, Z.Z.; Wang, Z.Y.; Sun, X.C. Apolipoprotein E Affects In Vitro Axonal Growth and Regeneration via the MAPK Signaling Pathway. Cell Transplant. 2019, 28, 691–703. [CrossRef] 27. Lane-Donovan, C.; Wong, W.M.; Durakoglugil, M.S.; Wasser, C.R.; Jiang, S.; Xian, X.; Herz, J. Genetic Restoration of Plasma ApoE Improves Cognition and Partially Restores Synaptic Defects in ApoE-Deficient Mice. J. Neurosci. 2016, 36, 10141–10150. [CrossRef] 28 Yi C G Z D H Z Z W Z Y S X C A li t i E Aff t I Vit A l G th d R ti i th 28. Yin, C.; Guo, Z.D.; He, Z.Z.; Wang, Z.Y.; Sun, X.C. Apolipoprotein E Affects In Vitro Axonal Growth a MAPK Signaling Pathway. Cell Transplant. 2019, 28, 691–703. [CrossRef] and, M.R.; Soranno, A.; Holtzman, D.M. Apolipoprotein E: Structural Insights and Links to Alzheimer Dise euron 2021, 109, 205–221. [CrossRef] 29. Chen, Y.; Strickland, M.R.; Soranno, A.; Holtzman, D.M. Apolipoprotein E: Structural Insights and Lin Pathogenesis. Neuron 2021, 109, 205–221. [CrossRef] 30. Frieden, C.; Wang, H.; Ho, C.M.W. A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions. Proc. Natl. Acad. Sci. USA 2017, 114, 6292–6297. [CrossRef] 31. References 1. Querfurth, H.W.; LaFerla, F.M. Alzheimer’s disease. N. Engl. J. Med. 2010, 362, 329–344. [CrossRef] ; LaFerla, F.M. Alzheimer’s disease. N. Engl. J. Med. 2010, 362, 329–344. [CrossRef] [PubMed] 1. Querfurth, H.W.; LaFerla, F.M. Alzheimer’s disease. N. Engl. J. Med. 2010, 362, 329–344. [CrossRef] [PubMed] 2. Corder, E.H.; Saunders, A.M.; Strittmatter, W.J.; Schmechel, D.E.; Gaskell, P.C.; Small, G.W.; Roses, A.D.; Haines, J.L.; Pericak- Vance, M.A. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993, 261, 921–923. [CrossRef] [PubMed] 2. Corder, E.H.; Saunders, A.M.; Strittmatter, W.J.; Schmechel, D.E.; Gaskell, P.C.; Small, G.W.; Roses, A.D.; Haines, J.L.; Pericak- Vance, M.A. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993, 261, 921–923. [CrossRef] [PubMed] 3. Reiman, E.M.; Arboleda-Velasquez, J.F.; Quiroz, Y.T.; Huentelman, M.J.; Beach, T.G.; Caselli, R.J.; Chen, Y.; Su, Y.; Myers, A.J.; Hardy, J.; et al. Exceptionally low likelihood of Alzheimer’s dementia in APOE2 homozygotes from a 5000-person neuropathological study. Nat. Commun. 2020, 11, 667. [CrossRef] [PubMed] 4. Di Paolo, G.; Kim, T.W. Linking lipids to Alzheimer’s disease: Cholesterol and beyond. Nat. Rev. Neurosci. 2011, 12, 284–296. [CrossRef] 5. Liu, C.C.; Liu, C.C.; Kanekiyo, T.; Xu, H.; Bu, G. Apolipoprotein E and Alzheimer disease: Risk, mechanisms and therapy. Nat. Rev. Neurol. 2013, 9, 106–118. [CrossRef] 6. Koriath, C.; Lashley, T.; Taylor, W.; Druyeh, R.; Dimitriadis, A.; Denning, N.; Williams, J.; Warren, J.D.; Fox, N.C.; Schott, J.M.; et al. ApoE4 lowers age at onset in patients with frontotemporal dementia and tauopathy independent of amyloid-beta copathology. Alzheimers Dement. 2019, 11, 277–280. [CrossRef] 7. Dickson, D.W.; Heckman, M.G.; Murray, M.E.; Soto, A.I.; Walton, R.L.; Diehl, N.N.; van Gerpen, J.A.; Uitti, R.J.; Wszolek, Z.K.; Ertekin-Taner, N.; et al. APOE epsilon4 is associated with severity of Lewy body pathology independent of Alzheimer pathology. Neurology 2018, 91, e1182–e1195. [CrossRef] gy 8. Mahley, R.W. Central Nervous System Lipoproteins: ApoE and Regulation of Cholesterol Metabolism. Arterioscler. Thromb. Vasc. Biol. 2016, 36, 1305–1315. [CrossRef] 18 of 21 18 of 21 Antioxidants 2022, 11, 2168 9. Holtzman, D.M.; Herz, J.; Bu, G. Apolipoprotein E and apolipoprotein E receptors: Normal biology and roles in Alzheimer disease. Cold Spring Harb. Perspect. Med. 2012, 2, a006312. [CrossRef] p g p 10. Staurenghi, E.; Giannelli, S.; Testa, G.; Sottero, B.; Leonarduzzi, G.; Gamba, P. Cholesterol Dysmetabolism in Alzheimer’s Disease: A Starring Role for Astrocytes? References [CrossRef] [PubMed] , F.; Feldman, H.M.; Wen, H.L.; Penney, J.; Cam, H.P.; Gjoneska, E.; Raja, W.K.; Cheng, J.; et al. APOE4 Causes 16. Lin, Y.T.; Seo, J.; Gao, F.; Feldman, H.M.; Wen, H.L.; Penney, J.; Cam, H.P.; Gjoneska, E.; Raja, W.K.; Cheng, J.; et al. APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer’s Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron 2018, 98, 1141–1154.e7. [CrossRef] Widespread Molecular and Cellular Alterations Associated with Alzheimer’s Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron 2018, 98, 1141–1154.e7. [CrossRef] 17. Farmer, B.C.; Kluemper, J.; Johnson, L.A. Apolipoprotein E4 Alters Astrocyte Fatty Acid Metabolism and Lipid Droplet Formation. Cells 2019, 8, 182. [CrossRef] 18. Morikawa, M.; Fryer, J.D.; Sullivan, P.M.; Christopher, E.A.; Wahrle, S.E.; DeMattos, R.B.; O’Dell, M.A.; Fagan, A.M.; Lashuel, H.A.; Walz, T.; et al. Production and characterization of astrocyte-derived human apolipoprotein E isoforms from immortalized astrocytes and their interactions with amyloid-beta. Neurobiol. Dis. 2005, 19, 66–76. [CrossRef] y y 19. Testa, G.; Staurenghi, E.; Zerbinati, C.; Gargiulo, S.; Iuliano, L.; Giaccone, G.; Fanto, F.; Poli, G.; Leonarduzzi, G.; Gamba, P. Changes in brain oxysterols at different stages of Alzheimer’s disease: Their involvement in neuroinflammation. Redox Biol. 2016, 10, 24–33. [CrossRef] , [ ] 20. Civra, A.; Leoni, V.; Caccia, C.; Sottemano, S.; Tonetto, P.; Coscia, A.; Peila, C.; Moro, G.E.; Gaglioti, P.; Bertino, E.; et al. Antiviral oxysterols are present in human milk at diverse stages of lactation. J. Steroid Biochem. Mol. Biol. 2019, 193, 105424. [CrossRef] 21. Leoni, V.; Nury, T.; Vejux, A.; Zarrouk, A.; Caccia, C.; Debbabi, M.; Fromont, A.; Sghaier, R.; Moreau, T.; Lizard, G. Mitochondrial dysfunctions in 7-ketocholesterol-treated 158N oligodendrocytes without or with alpha-tocopherol: Impacts on the cellular profil of tricarboxylic cycle-associated organic acids, long chain saturated and unsaturated fatty acids, oxysterols, cholesterol and cholesterol precursors. J. Steroid Biochem. Mol. Biol. 2017, 169, 96–110. [CrossRef] [PubMed] 22. Risso, D.; Leoni, V.; Canzoneri, F.; Arveda, M.; Zivoli, R.; Peraino, A.; Poli, G.; Menta, R. Presence chocolates and their correlation with milk powder freshness. PLoS ONE 2022, 17, e0264288. [CrossR sso, D.; Leoni, V.; Canzoneri, F.; Arveda, M.; Zivoli, R.; Peraino, A.; Poli, G.; Menta, R. Presence of choleste l t d th i l ti ith ilk d f h PLoS ONE 2022 17 0264288 [C R f] 23. Livak, K.J.; Schmittgen, T.D. References Antioxidants 2021, 10, 1890. [CrossRef] y ; Gargiulo, S.; Staurenghi, E.; Poli, G.; Leonarduzzi, G. Oxidized cholesterol as the driving force behind the heimer’s disease Front Aging Neurosci 2015 7 119 [CrossRef] [PubMed] g y P.; Testa, G.; Gargiulo, S.; Staurenghi, E.; Poli, G.; Leonarduzzi, G. Oxidized cholesterol as the driving force 11. Gamba, P.; Testa, G.; Gargiulo, S.; Staurenghi, E.; Poli, G.; Leonarduzzi, G. Oxidized cholesterol as the driving force behind the development of Alzheimer’s disease. Front. Aging Neurosci. 2015, 7, 119. [CrossRef] [PubMed] ba, P.; Testa, G.; Gargiulo, S.; Staurenghi, E.; Poli, G.; Leonarduzzi, G. Oxidized cholesterol as the driving fo opment of Alzheimer’s disease. Front. Aging Neurosci. 2015, 7, 119. [CrossRef] [PubMed] 12. Loera-Valencia, R.; Goikolea, J.; Parrado-Fernandez, C.; Merino-Serrais, P.; Maioli, S. Alterations in cholesterol metabolism as a risk factor for developing Alzheimer’s disease: Potential novel targets for treatment. J. Steroid Biochem. Mol. Biol. 2019, 190, 104–114. [CrossRef] [PubMed] [ ] [ ] 13. Zarrouk, A.; Debbabi, M.; Bezine, M.; Karym, E.M.; Badreddine, A.; Rouaud, O.; Moreau, T.; Cherkaoui-Malki, M.; El Ayeb, M.; Nasser B ; et al Lipid Biomarkers in Alzheimer’s Disease Curr Alzheimer Res 2018 15 303 312 [CrossRef] [PubMed] uk, A.; Debbabi, M.; Bezine, M.; Karym, E.M.; Badreddine, A.; Rouaud, O.; Moreau, T.; Cherkaoui-Malki, M B t l Li id Bi k i Al h i ’ Di C Al h i R 2018 15 303 312 [C R f] [P b Debbabi, M.; Bezine, M.; Karym, E.M.; Badreddine, A.; Rouaud, O.; Moreau, T.; Cherkaoui-Malki, M.; El Ay 13. Zarrouk, A.; Debbabi, M.; Bezine, M.; Karym, E.M.; Badreddine, A.; Rouaud, O.; Moreau, T.; Cherkaoui-Malki, M.; El Ayeb, M.; Nasser, B.; et al. Lipid Biomarkers in Alzheimer’s Disease. Curr. Alzheimer Res. 2018, 15, 303–312. [CrossRef] [PubMed] uk, A.; Debbabi, M.; Bezine, M.; Karym, E.M.; Badreddine, A.; Rouaud, O.; Moreau, T.; Cherkaoui-Malki, M r, B.; et al. Lipid Biomarkers in Alzheimer’s Disease. Curr. Alzheimer Res. 2018, 15, 303–312. [CrossRef] [Pub .; et al. Lipid Biomarkers in Alzheimer’s Disease. Curr. Alzheimer Res. 2018, 15, 303–312. [CrossRef] [PubMe 14. Bjorkhem, I.; Cedazo-Minguez, A.; Leoni, V.; Meaney, S. Oxysterols and neurodegenerative diseases. Mol. Aspects Med. 2009, 30, 171–179. [CrossRef] [PubMed] 15. Tcw, J.; Qian, L.; Pipalia, N.H.; Chao, M.J.; Liang, S.A.; Shi, Y.; Jain, B.R.; Bertelsen, S.E.; Kapoor, M.; Marcora, E.; et al. Cholesterol and matrisome pathways dysregulated in astrocytes and microglia. Cell 2022, 185, 2213–2233.e2225. References ApoE4 induces Abeta42, tau, and neuronal patho young targeted replacement apoE4 mice. Mol. Neurodegener. 2013, 8, 16. [CrossRef] ung targeted replacement apoE4 mice. Mol. Neurodegener. 2013, 8, 16. [CrossRef] y g g p p g 39. Wang, C.; Wilson, W.A.; Moore, S.D.; Mace, B.E.; Maeda, N.; Schmechel, D.E.; Sullivan, P.M. Human apoE4-targeted replacement mice display synaptic deficits in the absence of neuropathology. Neurobiol. Dis. 2005, 18, 390–398. [CrossRef] 39. Wang, C.; Wilson, W.A.; Moore, S.D.; Mace, B.E.; Maeda, N.; Schmechel, D.E.; Sullivan, P.M. Human apoE4-targeted replacement mice display synaptic deficits in the absence of neuropathology. Neurobiol. Dis. 2005, 18, 390–398. [CrossRef] 40. Koutsodendris, N.; Nelson, M.R.; Rao, A.; Huang, Y. Apolipoprotein E and Alzheimer’s Disease: Findings, Hypotheses, and Potential Mechanisms Annu Rev Pathol 2022 17 73–99 [CrossRef] g p g p mice display synaptic deficits in the absence of neuropathology. Neurobiol. Dis. 2005, 18, 390–398. [CrossRef] 40. Koutsodendris, N.; Nelson, M.R.; Rao, A.; Huang, Y. Apolipoprotein E and Alzheimer’s Disease: Findings, Hypotheses, and Potential Mechanisms. Annu. Rev. Pathol. 2022, 17, 73–99. [CrossRef] p y y p p gy 40. Koutsodendris, N.; Nelson, M.R.; Rao, A.; Huang, Y. Apolipoprotein E and Alzheimer’s Disease: Findings, Hypotheses, and Potential Mechanisms. Annu. Rev. Pathol. 2022, 17, 73–99. [CrossRef] 40. Koutsodendris, N.; Nelson, M.R.; Rao, A.; Huang, Y. Apolipoprotein E and Alzheimer s Disease: Fin Potential Mechanisms. Annu. Rev. Pathol. 2022, 17, 73–99. [CrossRef] , W.; Lee, H.; Cho, S.; Seo, J. ApoE4-Induced Cholesterol Dysregulation and Its Brain Cell Type-Specific Imp Jeong, W.; Lee, H.; Cho, S.; Seo, J. ApoE4-Induced Cholesterol Dysregulation and Its Brain Cell Type-Speci Pathogenesis of Alzheimer’s Disease. Mol. Cells 2019, 42, 739–746. [CrossRef] [PubMed] 41. Jeong, W.; Lee, H.; Cho, S.; Seo, J. ApoE4-Induced Cholesterol Dysregulation and Its Brain Cell Type-Specific Implications in the Pathogenesis of Alzheimer’s Disease. Mol. Cells 2019, 42, 739–746. [CrossRef] [PubMed] 41. Jeong, W.; Lee, H.; Cho, S.; Seo, J. ApoE4 Induced Cholesterol Dysregulation and Its Brain Cell Type Specific Implications in the Pathogenesis of Alzheimer’s Disease. Mol. Cells 2019, 42, 739–746. [CrossRef] [PubMed] genesis of Alzheimer’s Disease. Mol. Cells 2019, 42, 739–746. [CrossRef] [PubMed] g 42. Zhao, J.; Davis, M.D.; Martens, Y.A.; Shinohara, M.; Graff-Radford, N.R.; Younkin, S.G.; Wszolek, Z.K.; Kanekiyo, T.; Bu, G. APOE epsilon4/epsilon4 diminishes neurotrophic function of human iPSC-derived astrocytes. Hum. Mol. Genet. 2017, 26, 2690–2700. [CrossRef] [PubMed] [ ] [ ] 43. References Sullivan, P.M.; Han, B.; Liu, F.; Mace, B.E.; Ervin, J.F.; Wu, S.; Koger, D.; Paul, S.; Bales, K.R. Reduced levels of human apoE4 protein in an animal model of cognitive impairment. Neurobiol. Aging 2011, 32, 791–801. [CrossRef] 44. Bales, K.R.; Liu, F.; Wu, S.; Lin, S.; Koger, D.; DeLong, C.; Hansen, J.C.; Sullivan, P.M.; Paul, S.M. Human APOE isoform-dependent effects on brain beta-amyloid levels in PDAPP transgenic mice. J. Neurosci. 2009, 29, 6771–6779. [CrossRef] 45. Riddell, D.R.; Zhou, H.; Atchison, K.; Warwick, H.K.; Atkinson, P.J.; Jefferson, J.; Xu, L.; Aschmies, S.; Kirksey, Y.; Hu, Y.; et al. Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels. J. Neurosci. 2008, 28, 11445–11453. [CrossRef] 46. Fote, G.M.; Geller, N.R.; Efstathiou, N.E.; Hendricks, N.; Vavvas, D.G.; Reidling, J.C.; Thompson, L.M.; Steffan, J.S. Isoform- dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins. J. Cell Sci. 2022, Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels. J. Neurosci. 2008, 28, 11445–11453. [CrossRef] 46. Fote, G.M.; Geller, N.R.; Efstathiou, N.E.; Hendricks, N.; Vavvas, D.G.; Reidling, J.C.; Thompson, L.M.; Steffan, J.S. Isoform- dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins. J. Cell Sci. 2022, 135, jcs258687. [CrossRef] 47. Gong, J.S.; Kobayashi, M.; Hayashi, H.; Zou, K.; Sawamura, N.; Fujita, S.C.; Yanagisawa, K.; Michikawa, M. Apolipoprotein E (ApoE) isoform-dependent lipid release from astrocytes prepared from human ApoE3 and ApoE4 knock-in mice. J. Biol. Chem. 2002, 277, 29919–29926. [CrossRef] [PubMed] 48. Farkas, M.H.; Weisgraber, K.H.; Shepherd, V.L.; Linton, M.F.; Fazio, S.; Swift, L.L. The recycling of apolipoprotein E and its amino-terminal 22 kDa fragment: Evidence for multiple redundant pathways. J. Lipid Res. 2004, 45, 1546–1554. [CrossRef] 49 Fryer J D ; Demattos R B ; McCormick L M ; O’Dell M A ; Spinner M L ; Bales K R ; Paul S M ; Sullivan PM ; Parsadanian M ; 48. Farkas, M.H.; Weisgraber, K.H.; Shepherd, V.L.; Linton, M.F.; Fazio, S.; Swift, L.L. The recycling of amino-terminal 22 kDa fragment: Evidence for multiple redundant pathways. J. Lipid Res. 2004, 45, 15 48. Farkas, M.H.; Weisgraber, K.H.; Shepherd, V.L.; Linton, M.F.; Fazio, S.; Swift, L.L. The recycling of apolipoprotein E and its amino-terminal 22 kDa fragment: Evidence for multiple redundant pathways. J. Lipid Res. 2004, 45, 1546–1554. [CrossRef] 49. References Henry, N.; Krammer, E.M.; Stengel, F.; Adams, Q.; Van Liefferinge, F.; Hubin, E.; Chaves, R.; Efremov, R.; Aebersold, R.; Vandenbussche, G.; et al. Lipidated apolipoprotein E4 structure and its receptor binding mechanism determined by a combined cross-linking coupled to mass spectrometry and molecular dynamics approach. PLoS Comput. Biol. 2018, 14, e1006165. [CrossRef] [PubMed] 19 of 21 19 of 21 Antioxidants 2022, 11, 2168 32. Bussy, A.; Snider, B.J.; Coble, D.; Xiong, C.; Fagan, A.M.; Cruchaga, C.; Benzinger, T.L.S.; Gordon, B.A.; Hassenstab, J.; Bateman, R.J.; et al. Effect of apolipoprotein E4 on clinical, neuroimaging, and biomarker measures in noncarrier participants in the Dominantly Inherited Alzheimer Network. Neurobiol. Aging 2019, 75, 42–50. [CrossRef] y g g 33. Sperling, R.A.; Donohue, M.C.; Raman, R.; Sun, C.K.; Yaari, R.; Holdridge, K.; Siemers, E.; Johnson, K.A.; Aisen, P.S.; Team, A.S. Association of Factors With Elevated Amyloid Burden in Clinically Normal Older Individuals. JAMA Neurol. 2020, 77, 735–745. [CrossRef] [PubMed] 34. Mishra, S.; Blazey, T.M.; Holtzman, D.M.; Cruchaga, C.; Su, Y.; Morris, J.C.; Benzinger, T.L.S.; Gordon, B.A. Longitudinal brain imaging in preclinical Alzheimer disease: Impact of APOE epsilon4 genotype. Brain 2018, 141, 1828–1839. [CrossRef] 35. Elias-Sonnenschein, L.S.; Viechtbauer, W.; Ramakers, I.H.; Verhey, F.R.; Visser, P.J. Predictive value of APOE-epsilon4 allele for progression from MCI to AD-type dementia: A meta-analysis. J. Neurol. Neurosurg. Psychiatry 2011, 82, 1149–1156. [CrossRef] [PubMed] 36. Rodriguez, G.A.; Burns, M.P.; Weeber, E.J.; Rebeck, G.W. Young APOE4 targeted replacement mice exhibit poor spatial learning and memory, with reduced dendritic spine density in the medial entorhinal cortex. Learn. Mem. 2013, 20, 256–266. [CrossRef] 37. Salomon-Zimri, S.; Boehm-Cagan, A.; Liraz, O.; Michaelson, D.M. Hippocampus-related cognitive impairments in young apoE4 targeted replacement mice. Neurodegener. Dis. 2014, 13, 86–92. [CrossRef] 36. Rodriguez, G.A.; Burns, M.P.; Weeber, E.J.; Rebeck, G.W. Young APOE4 targeted replacement mice exhibit poor spatial learning and memory, with reduced dendritic spine density in the medial entorhinal cortex. Learn. Mem. 2013, 20, 256–266. [CrossRef] 37 S l Zi i S B h C A Li O Mi h l D M Hi l t d iti i i t i E4 y p y 37. Salomon-Zimri, S.; Boehm-Cagan, A.; Liraz, O.; Michaelson, D.M. Hippocampus-related cognitive impairments in young apoE4 targeted replacement mice. Neurodegener. Dis. 2014, 13, 86–92. [CrossRef] p g O.; Boehm-Cagan, A.; Michaelson, D.M. ApoE4 induces Abeta42, tau, and neuronal pathology in the hip 38. Liraz, O.; Boehm-Cagan, A.; Michaelson, D.M. References Fryer, J.D.; Demattos, R.B.; McCormick, L.M.; O’Dell, M.A.; Spinner, M.L.; Bales, K.R.; Paul, S.M.; Sullivan, P.M.; Parsadanian, M.; Bu G ; et al The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein 49. Fryer, J.D.; Demattos, R.B.; McCormick, L.M.; O’Dell, M.A.; Spinner, M.L.; Bales, K.R.; Paul, S.M.; Sullivan, P.M.; Parsadanian, M.; Bu, G.; et al. The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice. J. Biol. Chem. 2005, 280, 25754–25759. [CrossRef] y y p q p gy 50. Basak, J.M.; Verghese, P.B.; Yoon, H.; Kim, J.; Holtzman, D.M. Low-density lipoprotein receptor represents an apolipoprotein E-independent pathway of Abeta uptake and degradation by astrocytes. J. Biol. Chem. 2012, 287, 13959–13971. [CrossRef] 51. Courtney, R.; Landreth, G.E. LXR Regulation of Brain Cholesterol: From Development to Disease. Trends Endocrinol. Metab. 2016, 27, 404–414. [CrossRef] [PubMed] 52. Ahmadian, M.; Suh, J.M.; Hah, N.; Liddle, C.; Atkins, A.R.; Downes, M.; Evans, R.M. PPARgamma signaling and metabolism: The good, the bad and the future. Nat. Med. 2013, 19, 557–566. [CrossRef] [PubMed] 53. Qian, L.; Chai, A.B.; Gelissen, I.C.; Brown, A.J. Balancing cholesterol in the brain: From synthesis to dispos Ther. 2022, 2, 1–27. [CrossRef] Gelissen, I.C.; Brown, A.J. Balancing cholesterol in the brain: From synthesis to disposal. Explor. Neuroprotective CrossRef] 54. Nieweg, K.; Schaller, H.; Pfrieger, F.W. Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats. J. Neurochem. 2009, 109, 125–134. [CrossRef] [PubMed] 55. Milagre, I.; Nunes, M.J.; Gama, M.J.; Silva, R.F.; Pascussi, J.M.; Lechner, M.C.; Rodrigues, E. Transcriptional regulation of the human CYP46A1 brain-specific expression by Sp transcription factors. J. Neurochem. 2008, 106, 835–849. [CrossRef] 56. Bjorkhem, I.; Leoni, V.; Svenningsson, P. On the fluxes of side-chain oxidized oxysterols across blood-brain and blood-CSF barriers and origin of these steroids in CSF (Review). J. Steroid Biochem. Mol. Biol. 2019, 188, 86–89. [CrossRef] [PubMed] g 57. Dosch, A.R.; Imagawa, D.K.; Jutric, Z. Bile Metabolism and Lithogenesis: An Update. Surg. Clin. N. Am. 2019, 99, 215–229. [CrossRef] [PubMed] 20 of 21 20 of 21 Antioxidants 2022, 11, 2168 58. Lutjohann, D.; Breuer, O.; Ahlborg, G.; Nennesmo, I.; Siden, A.; Diczfalusy, U.; Bjorkhem, I. Cholesterol homeostasis in human brain: Evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc. Natl. Acad. Sci. USA 1996, 93, 9799–9804. References Rawat, V.; Wang, S.; Sima, J.; Bar, R.; Liraz, O.; Gundimeda, U.; Parekh, T.; Chan, J.; Johansson, J.O.; Tang, C.; et al. ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes. J. Neurosci. 2019, 39, 9611–9622. [CrossRef] [PubMed] 69. Hu, J.; Liu, C.C.; Chen, X.F.; Zhang, Y.W.; Xu, H.; Bu, G. Opposing effects of viral mediated brain expression of apolipoprotein E2 (apoE2) and apoE4 on apoE lipidation and Abeta metabolism in apoE4-targeted replacement mice. Mol. Neurodegener. 2015, 10, 6. [CrossRef] [PubMed] 70. Heinsinger, N.M.; Gachechiladze, M.A.; Rebeck, G.W. Apolipoprotein E Genotype Affects Size of ApoE Complexes in Cere- brospinal Fluid. J. Neuropathol. Exp. Neurol. 2016, 75, 918–924. [CrossRef] 71. Boehm-Cagan, A.; Michaelson, D.M. Reversal of apoE4-driven brain pathology and behavioral deficits by bexarotene. J. Neurosci. 2014, 34, 7293–7301. [CrossRef] [PubMed] 72. Carter, A.Y.; Letronne, F.; Fitz, N.F.; Mounier, A.; Wolfe, C.M.; Nam, K.N.; Reeves, V.L.; Kamboh, H.; Lefterov, I.; Koldamova, R. Liver X receptor agonist treatment significantly affects phenotype and transcriptome of APOE3 and APOE4 Abca1 haplo-deficient mice. PLoS ONE 2017, 12, e0172161. [CrossRef] [PubMed] 73. Prasad, H.; Rao, R. Amyloid clearance defect in ApoE4 astrocytes is reversed by epigenetic correction of endosomal pH. Proc. Natl. Acad. Sci. USA 2018, 115, E6640–E6649. [CrossRef] [PubMed] 74. Kim, M.; Bezprozvanny, I. Differences in Recycling of Apolipoprotein E3 and E4-LDL Recepto Hypothesis. Int. J. Mol. Sci. 2021, 22, 5030. [CrossRef] [PubMed] 75. Xian, X.; Pohlkamp, T.; Durakoglugil, M.S.; Wong, C.H.; Beck, J.K.; Lane-Donovan, C.; Plattner, F.; Herz, J. Reversal of ApoE4- induced recycling block as a novel prevention approach for Alzheimer’s disease. eLife 2018, 7, e40048. [CrossRef] [PubMed] y g p pp f 76. Kim, J.; Castellano, J.M.; Jiang, H.; Basak, J.M.; Parsadanian, M.; Pham, V.; Mason, S.M.; Paul, S.M.; Holtzman, D.M. Overexpres- sion of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular A beta clearance. Neuron 2009, 64, 632–644. [CrossRef] 77. Storer, P.D.; Xu, J.; Chavis, J.; Drew, P.D. Peroxisome proliferator-activated receptor-gamma agonists inhibit the activation of microglia and astrocytes: Implications for multiple sclerosis. J. Neuroimmunol. 2005, 161, 113–122. [CrossRef] 77. Storer, P.D.; Xu, J.; Chavis, J.; Drew, P.D. Peroxisome proliferator activated receptor gamma agonists inhibit the activation of microglia and astrocytes: Implications for multiple sclerosis. J. Neuroimmunol. 2005, 161, 113–122. [CrossRef] 78. Gillespie, W.; Tyagi, N.; Tyagi, S.C. Role of PPARgamma, a nuclear hormone receptor in neuroprotection. Indian J. Biochem. Biophys. 2011, 48, 73–81. References [CrossRef] [ ] 59. Abildayeva, K.; Jansen, P.J.; Hirsch-Reinshagen, V.; Bloks, V.W.; Bakker, A.H.; Ramaekers, F.C.; de Vente, J.; Groen, A.K.; Wellington, C.L.; Kuipers, F.; et al. 24(S)-hydroxycholesterol participates in a liver X receptor-controlled pathway in astrocytes that regulates apolipoprotein E-mediated cholesterol efflux. J. Biol. Chem. 2006, 281, 12799–12808. [CrossRef] g p p p 60. Brown, A.J.; Jessup, W. Oxysterols: Sources, cellular storage and metabolism, and new insights into their roles in cholesterol homeostasis. Mol. Aspects Med. 2009, 30, 111–122. [CrossRef] 61. Petrov, A.M.; Kasimov, M.R.; Zefirov, A.L. Brain Cholesterol Metabolism and Its Defects: Linkage to Neurodegenerative Diseases and Synaptic Dysfunction. Acta Nat. 2016, 8, 58–73. [CrossRef] im, J. Apolipoprotein E metabolism and functions in brain and its role in Alzheimer’s disease. Curr. Opin 67. [CrossRef] 62. Liao, F.; Yoon, H.; Kim, J. Apolipoprotein E metabolism and functions in brain and its role in Alzheim Lipidol. 2017, 28, 60–67. [CrossRef] 63. Fan, Q.W.; Iosbe, I.; Asou, H.; Yanagisawa, K.; Michikawa, M. Expression and regulation of apolipoprotein E receptors in the cells of the central nervous system in culture: A review. J. Am. Aging Assoc. 2001, 24, 1–10. [CrossRef] y g g 64. Liu, C.C.; Hu, J.; Zhao, N.; Wang, J.; Wang, N.; Cirrito, J.R.; Kanekiyo, T.; Holtzman, D.M.; Bu, G. Astrocytic LRP1 Mediates Brain Abeta Clearance and Impacts Amyloid Deposition. J. Neurosci. 2017, 37, 4023–4031. [CrossRef] [PubMed] 65. Hascalovici, J.R.; Song, W.; Liberman, A.; Vaya, J.; Khatib, S.; Holcroft, C.; Laferla, F.; Schipper, H.M. Neural HO-1/sterol interactions in vivo: Implications for Alzheimer’s disease. Neuroscience 2014, 280, 40–49. [CrossRef] 66. Testa, G.; Staurenghi, E.; Giannelli, S.; Gargiulo, S.; Guglielmotto, M.; Tabaton, M.; Tamagno, E.; Gamba, P.; Leonarduzzi, G. A silver lining for 24-hydroxycholesterol in Alzheimer’s disease: The involvement of the neuroprotective enzyme sirtuin 1. Redox Biol. 2018, 17, 423–431. [CrossRef] [ ] 67. Gamba, P.; Giannelli, S.; Staurenghi, E.; Testa, G.; Sottero, B.; Biasi, F.; Poli, G.; Leonarduzzi, G. The Controversial Role of 24-S-Hydroxycholesterol in Alzheimer’s Disease. Antioxidants 2021, 10, 740. [CrossRef] P.; Giannelli, S.; Staurenghi, E.; Testa, G.; Sottero, B.; Biasi, F.; Poli, G.; Leonarduzzi, G. The Controvers roxycholesterol in Alzheimer’s Disease Antioxidants 2021 10 740 [CrossRef] .; G a e , S.; Stau e g , .; esta, G.; Sotte o, .; as , .; o , G.; eo a du , G. e Co t o roxycholesterol in Alzheimer’s Disease. Antioxidants 2021, 10, 740. [CrossRef] 68. 83. Bujold, K.; Rhainds, D.; Jossart, C.; Febbraio, M.; Marleau, S.; Ong, H. CD36-mediated cholesterol efflux is associated with PPARgamma activation via a MAPK-dependent COX-2 pathway in macrophages. Cardiovasc. Res. 2009, 83, 457–464. [CrossRef] [PubMed] [ ] 84. Moosecker, S.; Pissioti, A.; Leidmaa, E.; Harb, M.R.; Dioli, C.; Gassen, N.C.; Yu, S.; Gazea, M.; Catania, C.; Anderzhanova, E.; et al. Brain Expression, Physiological Regulation and Role in Motivation and Associative Learning of Peroxisome Proliferator-activated Receptor gamma. Neuroscience 2021, 479, 91–106. [CrossRef] [PubMed] p g 85. Heneka, M.T.; Sastre, M.; Dumitrescu-Ozimek, L.; Hanke, A.; Dewachter, I.; Kuiperi, C.; O’Banion, K.; Klockgether, T.; Van Leuven, F.; Landreth, G.E. Acute treatment with the PPARgamma agonist pioglitazone and ibuprofen reduces glial inflammation and Abeta1-42 levels in APPV717I transgenic mice. Brain 2005, 128, 1442–1453. [CrossRef] g 86. Gold, M.; Alderton, C.; Zvartau-Hind, M.; Egginton, S.; Saunders, A.M.; Irizarry, M.; Craft, S.; Landreth, G.; Linnamagi, U.; Sawchak, S. Rosiglitazone monotherapy in mild-to-moderate Alzheimer’s disease: Results from a randomized, double-blind, placebo-controlled phase III study. Dement. Geriatr. Cogn. Disord. 2010, 30, 131–146. [CrossRef] References microglia and astrocytes: Implications for multiple sclerosis. J. Neuroimmunol. 2005, 161, 113 122. [CrossRef] 78. Gillespie, W.; Tyagi, N.; Tyagi, S.C. Role of PPARgamma, a nuclear hormone receptor in neuroprotection. Indian J. Biochem. Biophys. 2011, 48, 73–81. 79. Keeney, J.T.; Ibrahimi, S.; Zhao, L. Human ApoE Isoforms Differentially Modulate Glucose and Amyloid Metabolic Pathways in Female Brain: Evidence of the Mechanism of Neuroprotection by ApoE2 and Implications for Alzheimer’s Disease Prevention and Early Intervention. J. Alzheimers Dis. 2015, 48, 411–424. [CrossRef] y J 80. Barrera, J.; Subramanian, S.; Chiba-Falek, O. Probing the role of PPARgamma in the regulation of late-onset Alzheimer’s disease-associated genes. PLoS ONE 2018, 13, e0196943. [CrossRef] 81. Gauthier, A.; Vassiliou, G.; Benoist, F.; McPherson, R. Adipocyte low density lipoprotein receptor-related protein gene expression and function is regulated by peroxisome proliferator-activated receptor gamma. J. Biol. Chem. 2003, 278, 11945–11953. [CrossRef] [PubMed] 82. Rondon-Ortiz, A.N.; Lino Cardenas, C.L.; Martinez-Malaga, J.; Gonzales-Urday, A.L.; Gugnani, K.S.; Bohlke, M.; Maher, T.J.; Pino-Figueroa, A.J. High Concentrations of Rosiglitazone Reduce mRNA and Protein Levels of LRP1 in HepG2 Cells. Front. Pharmacol. 2017, 8, 772. [CrossRef] [PubMed] 21 of 21 21 of 21 Antioxidants 2022, 11, 2168 84. Moosecker, S.; Pissioti, A.; Leidmaa, E.; Harb, M.R.; Dioli, C.; Gassen, N.C.; Yu, S.; Gazea, M.; Catania, C.; Anderzhanova, E.; et al. Brain Expression, Physiological Regulation and Role in Motivation and Associative Learning of Peroxisome Proliferator-activated Receptor gamma. Neuroscience 2021, 479, 91–106. [CrossRef] [PubMed] p g 85. Heneka, M.T.; Sastre, M.; Dumitrescu-Ozimek, L.; Hanke, A.; Dewachter, I.; Kuiperi, C.; O’Banion, K.; Klockgether, T.; Van Leuven, F.; Landreth, G.E. Acute treatment with the PPARgamma agonist pioglitazone and ibuprofen reduces glial inflammation and Abeta1-42 levels in APPV717I transgenic mice. Brain 2005, 128, 1442–1453. [CrossRef] 86. Gold, M.; Alderton, C.; Zvartau-Hind, M.; Egginton, S.; Saunders, A.M.; Irizarry, M.; Craft, S.; Landreth, G.; Linnamagi, U.; Sawchak, S. Rosiglitazone monotherapy in mild-to-moderate Alzheimer’s disease: Results from a randomized, double-blind, placebo-controlled phase III study. Dement. Geriatr. Cogn. Disord. 2010, 30, 131–146. [CrossRef] p p y g 87. Iketani, R.; Ohno, K.; Kawasaki, Y.; Matsumoto, K.; Yamada, H.; Kishino, S. Apolipoprotein E Gene Polymorphisms Affect the Efficacy of Thiazolidinediones for Alzheimer’s Disease: A Systematic Review and Meta-Analysis. Biol. Pharm. Bull. 2018, 41, 1017–1023. [CrossRef]
https://openalex.org/W2101590408
http://juser.fz-juelich.de/record/7703/files/amt-3-339-2010.pdf
English
null
Tomographic retrieval approach for mesoscale gravity wave observations by the PREMIER Infrared Limb-Sounder
Atmospheric measurement techniques
2,010
cc-by
15,526
Tomographic retrieval approach for mesoscale gravity wave observations by the PREMIER Infrared Limb-Sounder J. Ungermann, L. Hoffmann, P. Preusse, M. Kaufmann, and M. Riese Forschungszentrum J¨ulich, Institut f¨ur Chemie und Dynamik der Geosph¨are (ICG-1), J¨ulich, Germany Received: 24 August 2009 – Published in Atmos. Meas. Tech. Discuss.: 30 October 2009 Revised: 21 February 2010 – Accepted: 26 February 2010 – Published: 10 March 2010 Abstract. PREMIER is one of three candidates for ESA’s 7th Earth Explorer mission that are currently undergoing fea- sibility studies. The main mission objective of PREMIER is to quantify processes controlling atmospheric composition in the mid/upper troposphere and lower stratosphere, a region of particular importance for climate change. To achieve this objective, PREMIER will employ the first satellite Fourier transform infrared limb-imager with a 2-D detector array combined with a millimetre-wave limb-sounder. The in- frared limb-imager can be operated in a high spatial reso- lution mode (“dynamics mode”) for observations of small- scale structures in atmospheric temperatures and trace gas fields with unprecedented 3-D sampling (0.5 km in the ver- tical direction, 50 km along track, 25 km across track). In this paper, a fast tomographic retrieval scheme is presented, which is designed to fully exploit the high-resolution radi- ance observations of the dynamics mode. Based on a de- tailed analysis of the “observational filter”, we show that the dynamics mode provides unique information on global distri- butions of gravity waves (GW). The achievable vertical res- olution for GW observations has values between the verti- cal sampling (0.5 km) of the dynamics mode and the vertical field of view (about 0.75 km). The horizontal across track resolution corresponds to the horizontal across track sam- pling of 25 km. Since the achievable along track horizontal resolution is about 70 km, the dynamics mode will provide GW limb-observations with a horizontal resolution compa- rable to nadir sounders. Compared to previous observations, PREMIER will therefore considerably extend the range of detectable GWs in terms of horizontal and vertical wave- length. Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Atmospheric Measurement Techniques 2 The PREMIER Infrared Limb Sounder The PREMIER IRLS essentially combines a Fourier trans- form infrared spectrometer with two two-dimensional (2-D) detector arrays measuring in the spectral regions from 770 to 980 cm−1 (band A) and from 1070 to 1650 cm−1 (band B), respectively. Each 2-D detector array will consist of about 100 × 100 pixels, providing about 10 000 simultaneous limb- views within the altitude range from ≈3 to 55 km and with a swath width of 320 km simultaneously. To increase the signal-to-noise ratio, individual array pixels will be co-added to obtain sampling patterns of specific scientific measure- ment modes. Global observations of GWs are a major objective of the PREMIER mission (ESA, 2008). GWs represent an impor- tant coupling mechanism for the middle atmosphere (Fritts and Alexander, 2003). They contribute about 50% to the driving of the quasi-biennial oscillation (Dunkerton, 1997), are the major forcing mechanism of the summer branch of the Brewer-Dobson circulation (Alexander and Rosenlof, 2003), and contribute 30 to 50% to the predicted increase of the Brewer-Dobson circulation due to climate change (McLan- dress and Shepherd, 2009). GWs are also the main driver of mesospheric winds and cause the cold summer mesopause (McLandress, 1998). Currently, uncertainties in the global representation of GWs and their effects on the circulation represent a major limitation for the predictive capabilities of chemistry climate models. Limb sounding exploits the radiation thermally emitted in the atmosphere along the line-of-sight (LOS) of the instru- ment, which is directed towards the limb of the Earth’s atmo- sphere. The point of the LOS closest to the surface is called the tangent point. Under optically-thin conditions, the tan- gent point of the LOS generally determines the point in the atmosphere that contributes most to the measured radiation. The radiance of a single measurement typically originates mainly from an air volume with about 500 km extent along the LOS (half width of horizontal weighting function). The vertical extent is mainly determined by the special viewing geometry of the limb observation and the vertical field-of- view (FOV), which has about 750 m full-width at half-max at the tangent point. See Sect. 4.2 for a more detailed dis- cussion of the two-dimensional radiative transfer weighting functions. The great potential of GW observations from space has been demonstrated in several studies (e.g. Eckermann and Preusse, 1999; Preusse et al., 2002). 1 Introduction Numerous previous satellite missions have used the infrared limb-emission sounding technique for atmospheric remote sensing. Global observation of atmospheric infrared limb- emissions represents a reliable technique to obtain vertically resolved profile data of temperature, a variety of trace gases, aerosols, and clouds simultaneously, at daytime and at night- time. First global infrared limb-emission observations of an extensive number of atmospheric trace species were made by LIMS (Limb Infrared Monitor of the Stratosphere; Gille and Russel III, 1984) and SAMS (Stratospheric and Mesospheric Sounder; Drummond et al., 1980) aboard the Nimbus 7 satel- lite. Trace gas fields obtained from these sensors and follow- on instruments such as the CRISTA instrument (Riese et al., 1999) greatly contributed to our understanding of the three- dimensional composition, structure and large-scale dynam- ics of the middle atmosphere. Early 2002 ESA launched Envisat, a polar-orbiting Earth observation satellite, which included the Michelson Interferometer for Passive Atmo- spheric Sounding (MIPAS) instrument (Fischer et al., 2008) for infrared limb-emission observations with high spectral resolution. MIPAS significantly expanded the number of detectable species and the corresponding height range com- pared to previous space missions. All previous instruments have used telescopes with scan- ning mirrors or one-dimensional detector arrays to obtain profile information of atmospheric trace species. While these previous instruments were rather restricted in terms of spatial resolution (and in particular horizontal sampling), the PRE- MIER mission (Process Exploration through Measurements of Infrared and millimetre-wave Emitted Radiation) (ESA, 2008) employs an InfraRed Limb-Sounder (hereafter IRLS) that builds on recent developments in infrared detector array technology providing the basis for an instrument capable of achieving the high three-dimensional (3-D) spatial resolution Correspondence to: J. Ungermann (j.ungermann@fz-juelich.de) Correspondence to: J. Ungermann (j.ungermann@fz-juelich.de) Published by Copernicus Publications on behalf of the European Geosciences Union. 340 J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations necessary to sense the upper troposphere/lower stratosphere (UTLS) region (Riese et al., 2005; Friedl-Vallon et al., 2006). temperature measurements and good altitude coverage of- fered by PREMIER will allow for simultaneous determina- tion of GW temperature amplitudes and associated horizontal and vertical wavelength (wave-vector). Preusse et al. (2009) showed that global 3-D observations by limb-imagers such as PREMIER provide the best perspective to obtain global information of GW momentum flux. ( ) g ( ) During the PREMIER mission the IRLS can be operated in two different modes. 1 Introduction The one discussed in this paper, the “dynamics mode”, is designed for unprecedented 3-D spa- tial sampling and is thereby optimised to resolve small and mesoscale atmospheric temperature and trace gas structures (e.g. Adams et al., 2009). The sampling is 0.5 km in the vertical direction, 25 km across track (in a 320 km swath), and 50 km in the along track direction. The across track sampling corresponds to the achievable across track reso- lution. To fully exploit the benefits of the dense sampling in the vertical and along track direction in terms of resolu- tion, we present a new tomographic retrieval scheme. Tomo- graphic retrievals for limb sounding measurements were first explored by a variety of authors for different purposes and in- struments (e.g. Solomon et al., 1984; Gurevich, 1995). Prac- tical implementations for the large-scale retrieval of atmo- spheric constituents from satellite measurements were first produced by Carlotti et al. (2001) for MIPAS and Livesey and Read (2000) for MLS (Microwave Limb Sounder). By us- ing a very fast and efficient forward model (Hoffmann et al., 2008; Hoffmann and Alexander, 2009) we are able to explore the approach at smallest spatial scales that were not accessed by previous studies (e.g. Carlotti et al., 2006; Steck et al., 2005). In particular, our analysis demonstrates the capability of the PREMIER IRLS to provide unique global information on mesoscale gravity waves (GWs). For the interpretation of the results, it is crucial to determine accurately the observational filter (Alexander, 1998) of the limb-imaging technique employed by the PRE- MIER IRLS. The observational filter describes the reproduc- tion of GW amplitudes by the retrieval scheme as function of vertical and horizontal wavelength. A precise character- isation of the observational filter is also essential for com- parisons with other measurement techniques (Preusse et al., 2000; Hertzog et al., 2008) as well as for comparisons with GW resolving models or results obtained from GW parame- terisation schemes (Ern et al., 2004, 2005). In Sect. 2, we briefly introduce the dynamics mode of the PREMIER IRLS. We present our simulation setup in Sect. 3 followed by the forward model in Sect. 4 and finally the to- mographic retrieval approach in Sect. 5. The results regard- ing achievable resolution and GW observational filter are presented in Sect. 6. J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations Each blue orb rep- resents a full profile and thereby the horizontal component of the measurement grid of ten consecutive images. In our simulations, we generally use only measurements of the central track, of which ten consecutive vertical measurement profiles are shown coloured in red. As the tangent points of a single profile are spaced at vertical intervals of 500 m, the orbs representing it are merged into a vertical line. For the outermost measurement tracks, the tangent-points lie generally not within the idealized 2-D measurement track, but are displaced from it by up to 2.2 km in the across-track direction. wavenumber [cm-1] (b) 0 0.001 0.002 0.003 0.004 0.005 1100 1200 1300 1400 1500 1600 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band B) / mid latitudes / zt = 30 km total aerosol CO2 CH4 N2O H2O HNO3 O3 Fig. 1. Simulated radiance spectra for the PREMIER IRLS band A and B. Calculations are based on mid-latitude atmospheric condi- tions (Remedios et al., 2007) and 30 km tangent altitude. The black curve indicates total radiance. The other curves indicate radiance for single emitter atmospheres (see legend). The noise level of the observations will be about 10−5 W/(m2 sr cm−1). The black arrow indicates the CO2 Q-branch analysed in the retrievals. wavenumber [cm 1] (b) 0 0.001 0.002 0.003 0.004 0.005 1100 1200 1300 1400 1500 1600 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band B) / mid latitudes / zt = 30 km total aerosol CO2 CH4 N2O H2O HNO3 O3 Fig. 1. Simulated radiance spectra for the PREMIER IRLS band A and B. Calculations are based on mid-latitude atmospheric condi- tions (Remedios et al., 2007) and 30 km tangent altitude. The black curve indicates total radiance. The other curves indicate radiance for single emitter atmospheres (see legend). The noise level of the observations will be about 10−5 W/(m2 sr cm−1). J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations The black arrow indicates the CO2 Q-branch analysed in the retrievals (b) [ ] (b) 0 0.001 0.002 0.003 0.004 0.005 1100 1200 1300 1400 1500 1600 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band B) / mid latitudes / zt = 30 km total aerosol CO2 CH4 N2O H2O HNO3 O3 PREMIER IRLS (band B) / mid latitudes / zt = 30 km PREMIER IRLS (band B) / mid latitudes / zt = 30 km The vertical coverage of the IRLS dynamics mode obser- vations is 48 km with a lower boundary that increases from 3 km at the poles to 7 km at the equator to account for the latitudinal variation of the tropopause height. Vertical sam- pling is 500 m. Limb images will be measured rearward to the flight direction of the satellite. Dense sampling along track is achieved by high measurement speed, i.e. about 7.5 s per image in the dynamics mode, corresponding to ≈50 km along track distance. The 2-D detector array will allow for 13 parallel measurement tracks. The across track sampling is 25 km and the swath width is 320 km. The three-dimensional measurement grid of the PREMIER IRLS dynamics mode is illustrated in Fig. 2. The green spheres represent the tan- gent points of all measurements of a single image. Each blue orb represents a single measurement profile of preceding and following images, providing a visual clue for the horizontal measurement density. Please note that tangent points of a sin- gle image are not placed exactly vertically above one another as higher tangent points are located closer to the satellite; in fact, consecutive images are spaced so closely that the top- most tangent points of one image are placed vertically above the lowermost tangent points of the previous image. Fig. 1. Simulated radiance spectra for the PREMIER IRLS band A and B. Calculations are based on mid-latitude atmospheric condi- tions (Remedios et al., 2007) and 30 km tangent altitude. The black curve indicates total radiance. The other curves indicate radiance for single emitter atmospheres (see legend). The noise level of the observations will be about 10−5 W/(m2 sr cm−1). The black arrow indicates the CO2 Q-branch analysed in the retrievals. The main advantage of the proposed instrument concept is its great flexibility, i.e. the trade-off between spatial and spectral resolution can be adapted to the scientific needs. J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations The across track samp (a) 0 0.001 0.002 0.003 0.004 0.005 0.006 800 850 900 950 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band A) / mid latitudes / zt = 30 km total aerosol CO2 HNO3 O3 (b) 0 0.001 0.002 0.003 0.004 0.005 1100 1200 1300 1400 1500 1600 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band B) / mid latitudes / zt = 30 km total aerosol CO2 CH4 N2O H2O HNO3 O3 Fig. 1. Simulated radiance spectra for the PREMIER IRLS band A and B. Calculations are based on mid-latitude atmospheric condi- tions (Remedios et al., 2007) and 30 km tangent altitude. The black curve indicates total radiance. The other curves indicate radiance for single emitter atmospheres (see legend). The noise level of the observations will be about 10−5 W/(m2 sr cm−1). The black arrow indicates the CO2 Q-branch analysed in the retrievals. Fig sure ind me rese me we ten red inte line gen disp T vat 3 k lati plin the trac per alo 13 Fig. 2. Illustration of the PREMIER IRLS dynamics mode mea- surement grid. Symbols indicate the locations of tangent points of individual measurements. The green coloured spheres represent the measurements of a single image of PREMIER. Each blue orb rep- resents a full profile and thereby the horizontal component of the measurement grid of ten consecutive images. In our simulations, we generally use only measurements of the central track, of which ten consecutive vertical measurement profiles are shown coloured in red. As the tangent points of a single profile are spaced at vertical intervals of 500 m, the orbs representing it are merged into a vertical line. For the outermost measurement tracks, the tangent-points lie generally not within the idealized 2-D measurement track, but are displaced from it by up to 2.2 km in the across-track direction. (a) 0 0.001 0.002 0.003 0.004 0.005 0.006 800 850 900 950 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band A) / mid latitudes / zt = 30 km total aerosol CO2 HNO3 O3 (b) (a) PREMIER IRLS (band A) / mid latitudes / zt = 30 km Fig. 2. Illustration of the PREMIER IRLS dynamics mode mea- surement grid. Symbols indicate the locations of tangent points of individual measurements. The green coloured spheres represent the measurements of a single image of PREMIER. 2 The PREMIER Infrared Limb Sounder However, the spatial resolution of previous global observations was insufficient for the determination of direction-resolved momentum flux (Ern et al., 2004) and so far only absolute values of momen- tum flux have been deduced from CRISTA (Ern et al., 2004, 2006) and HIRDLS (Alexander et al., 2008; Wright et al., 2010). In addition, there is a case study of momentum flux from an exceptional long-vertical wavelength mountain wave event over South Georgia Island using AIRS data (Alexan- der et al., 2009). The combination of high-resolution 3-D Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 341 (a) 0 0.001 0.002 0.003 0.004 0.005 0.006 800 850 900 950 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band A) / mid latitudes / zt = 30 km total aerosol CO2 HNO3 O3 (b) 0 0.001 0.002 0.003 0.004 0.005 1100 1200 1300 1400 1500 1600 radiance [W/(m2 sr cm-1)] wavenumber [cm-1] PREMIER IRLS (band B) / mid latitudes / zt = 30 km total aerosol CO2 CH4 N2O H2O HNO3 O3 Fig. 1. Simulated radiance spectra for the PREMIER IRLS band A and B. Calculations are based on mid-latitude atmospheric condi- tions (Remedios et al., 2007) and 30 km tangent altitude. The black curve indicates total radiance. The other curves indicate radiance for single emitter atmospheres (see legend). The noise level of the observations will be about 10−5 W/(m2 sr cm−1). The black arrow indicates the CO2 Q-branch analysed in the retrievals. Fig. 2. Illustration of the PREMIER IRLS dynamics mode surement grid. Symbols indicate the locations of tangent po individual measurements. The green coloured spheres repres measurements of a single image of PREMIER. Each blue or resents a full profile and thereby the horizontal component measurement grid of ten consecutive images. In our simul we generally use only measurements of the central track, of ten consecutive vertical measurement profiles are shown colou red. As the tangent points of a single profile are spaced at v intervals of 500 m, the orbs representing it are merged into a v line. For the outermost measurement tracks, the tangent-poi generally not within the idealized 2-D measurement track, b displaced from it by up to 2.2 km in the across-track direction The vertical coverage of the IRLS dynamics mode o vations is 48 km with a lower boundary that increases 3 km at the poles to 7 km at the equator to account f latitudinal variation of the tropopause height. Vertical pling is 500 m. Limb images will be measured rearw the flight direction of the satellite. Dense sampling track is achieved by high measurement speed, i.e. abou per image in the dynamics mode, corresponding to ≈ along track distance. The 2-D detector array will allo 13 parallel measurement tracks. J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations While currently only two modes are specified, the hardware poses few restrictions should a certain scientific question re- quire a different trade-off between spatial and spectral sam- pling. In this paper we will concentrate on the IRLS dy- namics mode. It is designed to measure a subset of atmo- spheric variables with very high spatial sampling but mod- erate spectral sampling. Spectral sampling requirements are relaxed to 1.25 cm−1 from its maximum envisioned sampling of 0.2 cm−1, which is employed in the other major operat- ing mode. As an example, simulated spectra for the IRLS dynamics mode for mid-latitude atmospheric conditions and 30 km tangent altitude are shown in Fig. 1. Even at this re- duced spectral resolution, infrared emission features of im- portant atmospheric constituents (e.g. CO2, H2O, O3, HNO3, CH4, or N2O) are clearly visible. In this study we are interested in determining the achiev- able resolution of the dynamics mode with a focus on resolv- ing GWs. As the across track resolution directly corresponds to the across track sampling, it suffices to examine a single track and determine its resolution in vertical and along track direction. Further, we examine how well monochromatic Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ 342 J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 800 850 900 950 1000 1050 1100 horizontal distance (km) 10 11 12 13 14 altitude (km) pencilbeams Fig. 3. Line-of-sight (LOS) of the central measurement track in the PREMIER IRLS dynamics mode. LOS belonging to the same image are assigned the same colour. Note that the plot shows only a minor subset of the full simulated atmospheric slice of 6000 km times 80 km. 800 850 900 950 1000 1050 1100 horizontal distance (km) 10 11 12 13 14 altitude (km) pencilbeams Fig. 3. Line-of-sight (LOS) of the central measurement track in the PREMIER IRLS dynamics mode. LOS belonging to the same image are assigned the same colour. Note that the plot shows only a minor subset of the full simulated atmospheric slice of 6000 km times 80 km. 800 850 900 950 1000 1050 1100 horizontal distance (km) 10 11 12 13 14 altitude (km) pencilbeams measurements overlap one another in the along track direc- tion. The same air volume is viewed from multiple angles, which suggests a tomographic retrieval problem. 4 Forward modelling The set of simulated measurements is designed according to the PREMIER specification as described in Sect. 2. For our analysis of the GW observational filter, we only use limb profiles of the central track of the 2-D array (depicted as red orbs in Fig. 2). In total 101 consecutive profiles along track with 91 measured altitudes each are used per simulation. The tangent point altitudes of one such profile range from 10 to 55 km spaced at intervals of ≈500 m. This is a simplification of the vertical range of the PREMIER IRLS, which covers 48 km but varies in height between the equator and the poles. Figure 3 demonstrates how the line-of-sight of these dense 3 Simulation setup We examine how well tomographic temperature retrievals using simulated PREMIER IRLS dynamics mode measure- ments are able to reproduce GW parameters like amplitude, horizontal and vertical wavelength, as well as phase shifts. To this end, we carry out linear and non-linear retrieval end- to-end tests. We first simulate the radiance observations the PREMIER IRLS would make for a given monochromatic GW field. Second, we use the simulated observations to per- form a tomographic temperature retrieval. Finally, retrieved temperatures are compared with the original field. We do not add noise or other instrument effects to the simulations so as not to obscure the principal capabilities of the approach; however, we do provide error estimates for retrieval noise. Please note that this setup is highly simplified compared to what an inversion scheme for actual PREMIER IRLS mea- surements would look like. In practice, additional values like pressure, contaminant trace gasses, etc. would need to be re- trieved in addition to temperature. We use typical cloudless mid-latitude atmospheric profiles from Remedios et al. (2007) as background for our input at- mosphere and as the “a priori” retrieval state. In practice, measurements contaminated by clouds will likely be filtered out, affecting the local retrieval result with a reduced resolu- tion and higher a priori influence. This input atmosphere con- tains temperature, pressure, aerosol, CO2, Ozone and several other trace gasses relevant for the 12.6 µm channel. Onto the temperature of this background and a priori atmosphere, waves with varying horizontal and vertical wavelength with a constant amplitude of 5 K are modulated to form the input atmospheres for our simulations; we refer to the latter at- mospheres as “true” atmospheric state. Ideally, the retrieval would perfectly reproduce the true state. J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations To accurately represent a wide spectrum of GWs with dif- ferent horizontal and vertical wavelength, we define a cross- section of the atmosphere 6000 km long and 82 km high. This accommodates the full LOS of 91 consecutive verti- cal measurement profiles of the central track constituting the measurement grid. For the initial calculation of simulated satellite measurements, this cross-section was finely gridded with a horizontal sampling of only 5 km and a vertical sam- pling of 50 m. For the retrieval of the baseline setup, we use a coarser sampling: the vertical sampling of the atmo- spheric retrieval grid corresponds to the measurement grid, i.e. 500 m between 10 km and 55 km height and 2 km outside this range. The horizontal sampling is set to 12.5 km (four times finer than the measurement grid) to avoid representa- tion errors (see Sect. 6.2). Fig. 3. Line-of-sight (LOS) of the central measurement track in the PREMIER IRLS dynamics mode. LOS belonging to the same image are assigned the same colour. Note that the plot shows only a minor subset of the full simulated atmospheric slice of 6000 km times 80 km. For tracks other than the central one, the tangent points of consecutive vertical measurement profiles do not lie within a plane. We therefore examined the effect of the angle of 2.6◦between the LOS of the outermost tracks and the along track direction using both a 3-D atmosphere and a 2-D plane- parallel approximation and found the error of the approxima- tion to be in the order of the instrument noise. In effect, the results presented below for the central track are assumed to be representative for all tracks of the PREMIER IRLS. GW waves can be reproduced by PREMIER measurements and tomographic retrieval techniques in accordance with one of the major scientific mission objectives. 4.1 Fast forward model for the PREMIER IRLS A fast radiative transfer model is essential to solve large in- verse problems in atmospheric remote sensing. We adapted the C++ based Juelich Rapid Spectral Simulation Code Ver- sion 2 (JURASSIC2) for the PREMIER IRLS tomographic temperature retrievals presented in this paper. An older ver- sion of JURASSIC (Hoffmann, 2006) was previously used as forward model for retrievals for several satellite- and air-borne remote sensing experiments (e.g. Hoffmann et al., Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ 343 J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations Fig. 4. Temperature weighting functions for the PREMIER IRLS at 790.75 cm−1 for mid-latitude atmospheric conditions. Weight- ing functions are superimposed by taking the maximum sensitivity at each atmospheric grid point. The plot shows weighting functions for 10, 15, . . . , 55 km tangent point altitude. Tangent points are sep- arated by 500 km horizontal distance. Note that the measurement grid of the instrument is ten times finer in the vertical and horizon- tal domain than presented in this figure. The instrument is located on the left. 2008, 2009). It was applied to dedicated GW studies for AIRS aboard NASA’s Aqua satellite (Eckermann et al., 2009; Hoffmann and Alexander, 2009). JURASSIC2 is an ad- vancement of JURASSIC including a sophisticated instru- ment model, various powerful inversion algorithms and sev- eral specific code optimisations for this study, which are out- lined in Appendix A. JURASSIC2 computes the radiative transfer based on the emissivity growth approximation (EGA) (Weinreb and Neuendorffer, 1973; Gordley and Russel, 1981; Marshall et al., 1994). Compared with conventional line-by-line cal- culations this approach is about a factor 1000 faster, since radiative transfer is calculated based on precalculated spec- trally averaged values of emissivity stored in lookup tables. JURASSIC2 computes the radiative transfer based on the emissivity growth approximation (EGA) (Weinreb and Neuendorffer, 1973; Gordley and Russel, 1981; Marshall et al., 1994). Compared with conventional line-by-line cal- culations this approach is about a factor 1000 faster, since radiative transfer is calculated based on precalculated spec- trally averaged values of emissivity stored in lookup tables. The emissivity look-up-tables for JURASSIC2 are prepared by means of exact line-by-line calculations utilising the Ref- erence Forward Model (RFM) (Dudhia et al., 2002). 4.2 Channel selection and weighting functions For the temperature retrieval studies presented in this pa- per we analyse radiance emissions of the CO2 Q-branch at 12.6 µm, which was successfully used by Riese et al. (1997) to retrieve stratospheric and mesospheric temperature dis- tributions from satellite observations. Retrieving tempera- ture from radiance measurements at individual spectral grid points of the PREMIER IRLS is technically feasible due to the rather low noise equivalent spectral radiance of the pro- posed instrument. For this project we apply a 2-D scheme with the grid de- scribed in Sect. 3. To sample sufficiently the atmospheric in- homogeneities along the ray paths the ray-tracing step length was set to a value of 0.4 km for generating the simulated mea- surements and 4 km for the actual retrieval. For pressure and aerosol extinction we apply a logarithmic vertical interpola- tion and a linear horizontal interpolation. Interpolation for temperature and trace gas volume mixing ratios is bi-linear. The temperature weighting functions presented here are computed by means of numerical perturbation. Figure 4 shows several weighting functions for the selected radiance channel at 12.6 µm (790.75 cm−1). To analyse the temper- ature weighting functions and provide a fast analytical rep- resentation for other studies, we developed a semi-empirical model. Preusse et al. (2002) provides an analytical model under the assumption of an optically transparent atmosphere. Our simulations indicate that in the 12.6 µm CO2-branch transmittance gets as low as 0.6 and cannot be neglected. We therefore modified the model of Preusse et al. (2002) by adding a term compensating for transmittance. Using the same nomenclature as in Preusse et al. (2002), the sensitiv- ity of the measured signal on temperature at given altitude z (measured from the ground) and the distance along the ray path s (measured from the tangent point) is obtained from a Each measurement is affected by the vertical field-of-view of the instrument. A single radiative transfer calculation of JURASSIC2 however simulates an infinitesimal thin beam of radiation, also called a “pencil beam”. To account for the ef- fect of the FOV, we perform pencil beam calculations with a finer vertical sampling than that of the actual measurements. A single measurement is then simulated by convolving the results of the finely spaced pencil beam calculations with a sensitivity function describing the FOV of the instrument. 4.1 Fast forward model for the PREMIER IRLS In most stratospheric applications the EGA typically has an accuracy of 1 to 2% or better and no further means were taken here to improve forward model accuracy for the simulation study. Processing real measurements at a later stage may require improvements, e.g. by means of regression techniques (e.g. Francis et al., 2006). The emissivity look-up-tables for JURASSIC2 are prepared by means of exact line-by-line calculations utilising the Ref- erence Forward Model (RFM) (Dudhia et al., 2002). In most stratospheric applications the EGA typically has an accuracy of 1 to 2% or better and no further means were taken here to improve forward model accuracy for the simulation study. Processing real measurements at a later stage may require improvements, e.g. by means of regression techniques (e.g. Francis et al., 2006). Fig. 4. Temperature weighting functions for the PREMIER IRLS at 790.75 cm−1 for mid-latitude atmospheric conditions. Weight- ing functions are superimposed by taking the maximum sensitivity at each atmospheric grid point. The plot shows weighting functions for 10, 15, . . . , 55 km tangent point altitude. Tangent points are sep- arated by 500 km horizontal distance. Note that the measurement grid of the instrument is ten times finer in the vertical and horizon- tal domain than presented in this figure. The instrument is located on the left. JURASSIC2 provides a flexible handling of different types of observation geometries, e.g. nadir, sub-limb, limb, or zenith viewing for different types of atmospheric input data, e.g. single vertical profiles (1-D), sets of profiles along a satellite track (2-D), and regular or irregular 3-D model data. This is achieved by providing different interpolation func- tions for atmospheric data as well as a flexible 3-D ray- tracing routine (Hase and H¨opfner, 1999). Atmos. Meas. Tech., 3, 339–354, 2010 Gaussian, Gaussian, matrix. The Jacobian matrix consists of the weighting func- tions of the measurements. With xa ∈Rn being the a priori state of the atmosphere, Sa ∈Rn×n a covariance matrix for xa, y ∈Rm the vector of measurements and Sϵ ∈Rm×m the covariance matrix of the measurement error, effectively the cost function J(x) with K(s,z)=K0exp −(s−µ)2 2ϕ2s −(z−zt−s2/2RE)2 2ϕ2z ! . (1) (1) Here K0 denotes the maximum sensitivity. Note that the ab- solute values of K0 presented below depend on the atmo- spheric grid sampling. The sensitivity will change when the grid sampling varies because the individual perturbations re- fer to smaller or larger air volumes. The standard deviations ϕz and ϕs measure the vertical and horizontal extent of the weighting function, respectively. The horizontal shift µ was introduced to deal with atmospheres which are not optically thin, as occurs in our setup. As a kind of first-order effect, the point of maximum sensitivity will shift towards the in- strument. In the exponential function, zt denotes the tangent altitude. The term −s2/2RE, with Earth radius RE, deter- mines the apparent curvature of the ray paths with respect to the ground-base altitude coordinate system (Preusse et al., 2002). J(x)=  (x−xa)T Sa−1(x−xa)+(F(x)−y)T Sϵ−1(F(x)−y)  (2) needs to be minimised to obtain the MAP solution. The min- imisation is performed using the iterative Gauss-Newton al- gorithm. The resulting iteration formula can be written in two forms, one that requires the inverse of the covariance matrices and the solution to a linear equation system with n unknowns in each iteration (n-form), or one that requires solely the covariance matrices and the solution to a linear equation system with m unknowns in each iteration (m-form) (Rodgers, 2000). The choice of the more efficient form for our problem is critical since the linear algebra determines the memory consumption and thereby the maximal problem size. In our simulations n is generally four times as large as m, so we chose the m-form to reduce memory consumption and processing time. We fitted the semi-empirical model to the temperature weighting functions shown in Fig. 4 to obtain statistics of the variables K0, µ, ϕs, and ϕz. Gaussian, From 10 to 55 km altitude the maximum sensitivity K0 decreases from 4×10−6 to 2×10−7 (W/(m2 sr cm−1))/K, the horizontal shift −µ towards the in- strument decreases from 140 to 40 km, the horizontal full- width at half-max along the line-of-sight 2 √ 2ln2ϕs de- creases from 575 to 475 km and the vertical full-width at half-max 2 √ 2ln2ϕz is nearly constant at 0.95 km. The semi- empirical model provides a reasonable description of the true weighting functions as the accuracy of the fits is about 2 to 3% of K0. As an initial guess or starting point x0 ∈Rn we se- lected the true atmospheric state to reduce simulation time. As this choice may sidestep potential convergence issues, it was confirmed for selected representative non-linear re- trievals that they also converge to the same solution from other starting points, e.g. a polar atmosphere. Using a different starting point approximately doubles the num- ber of required iteration steps. The covariance matrix Sϵ is assumed to be diagonal and takes into account noise (10−5 W/(m2 sr cm−1)) as well as the quasi-statistical part of the forward model errors (which is estimated to be 0.3% of radiance (Hoffmann, 2006), that is, it ranges from 0.3×10−4 W/(m2 sr cm−1) for the lowermost tangent heights and 0.8 × 10−6 W/(m2 sr cm−1) for the uppermost tangent heights). The a priori covariance matrix Sa is set up accord- ing to a first-order autoregressive model 4.2 Channel selection and weighting functions We found a sampling twice as dense as the original mea- surement spacing to be sufficiently accurate, i.e. the tangent points of the pencil beams are vertically spaced at intervals of ≈250 m. Including pencil beams above and below the upper- most and lowermost measurement, we calculate 193 pencil beams for a single vertical measurement profile of the PRE- MIER IRLS consisting of 91 measurements. Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ 344 J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 345 (a) (b) (c) (d) Fig. 5. Example of a retrieval end-to-end test used for generating the observational filter. The instrument flies from right to left and measures radiance coming from the right. A wave perturbation with 10 km vertical wavelength and 320 km horizontal wavelength tilted towards the instrument is super-imposed onto the a priori atmosphere depicted in (a). The difference between the disturbed atmosphere and the a priori is shown in (b). The difference between the retrieval result and the a priori – that is the retrieved wave perturbation – is depicted in (c) and the difference between the result of the retrieval and the true state is depicted in (d). The tangent points of the satellite measurements are shown as white circles. Please note that the horizontal retrieval grid is four times finer than the measurement grid. For better representation of the investigated structure only kilometres 2000 to 3000 of the simulated 6000 kilometres are shown. b) (a) ( (b) (a) (b) (c) (d) (a) (b) (c) (d) (d) (d) Fig. 5. Example of a retrieval end-to-end test used for generating the observational filter. The instrument flies from right to left and measures radiance coming from the right. A wave perturbation with 10 km vertical wavelength and 320 km horizontal wavelength tilted towards the instrument is super-imposed onto the a priori atmosphere depicted in (a). The difference between the disturbed atmosphere and the a priori is shown in (b). The difference between the retrieval result and the a priori – that is the retrieved wave perturbation – is depicted in (c) and the difference between the result of the retrieval and the true state is depicted in (d). The tangent points of the satellite measurements are shown as white circles. Please note that the horizontal retrieval grid is four times finer than the measurement grid. For better representation of the investigated structure only kilometres 2000 to 3000 of the simulated 6000 kilometres are shown. to 91 grid points with a 0.5 km spacing and 5 grid points above with a 2 km spacing per profile. provided by one additional limb-scan, but perform a simul- taneous update using all available measurement information in a single step. 5.1 Inversion technique Deriving atmospheric temperature values from (tomo- graphic) infrared spectral measurements presents a non- linear, ill-posed inversion problem. To obtain a unique and physically reasonable solution the problem needs to be regu- larised. (Sa)i,j=σiσje− |vi−vj | cv e− |hi−hj | ch , (3) (3) (Sa with vi being the height of an atmospheric grid point and hi its horizontal coordinate given as along track displacement in kilometres. The correlation lengths are used as tuning param- eters for the regularisation. The vertical correlation length cv is chosen to be 0.5 km and the horizontal correlation length ch is chosen to be 200 km (see Sect. 6.3 regarding the choice of value for these parameters). The standard deviations σi are taken from the reference atmosphere of Remedios et al. (2007) and vary between 10 K and 14 K depending on height. To perform the inversion, the JURASSIC2 retrieval pro- cessor implements a number of standard methods, of which the maximum a posteriori estimation (MAP) was selected (Rodgers, 1976). This method interprets measurements and atmospheric states statistically and determines an optimal weighted mean between an a priori atmospheric state and the measurements. Let n ∈N be the number of atmospheric state vector ele- ments and m ∈N the number of measurements. Simulations of the JURASSIC2 forward model can then be represented mathematically as the application of a (non-linear) function F : Rn→Rm. F’(x) is the Jacobian matrix of the forward model F at atmospheric state x ∈Rn, also known as Kernel Only temperature values at grid points between 10 km and 65 km are retrieved, the others are fixed to the a priori atmo- sphere. Thereby an additional 10 km are retrieved compared to the uppermost tangent point height of 55 km. As the verti- cal grid becomes more sparse above 55 km, this corresponds Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ characterises the retrieval. One row of the averaging kernel matrix of the baseline setup with no modulated wave structure is depicted in Fig. 6. As each element in the row describes the influence of one point of the true atmosphere on the result, it can be reordered two-dimensionally according to their horizontal and vertical coordinates. The figure is representative for rows belonging to measurements with tangent points located between 15 km and 50 km height. Aside from a strong centred peak, one can see small “ripples” in the vicinity of the peak resembling the shape of the weighting functions (compare with Fig. 4). The asymmetry is caused by the asymmetry of the weighting functions. The vertical resolution in the central area is slightly bet- ter than the assumed vertical FOV, which has a full-width at half-max of 750 m; however, without the effects of the a pri- ori vertical correlation a vertical resolution of nearly 500 m, corresponding to the vertical sampling of measurements, is achievable (see Sect. 6.3). This shows that the achievable resolution is not necessarily limited by the FOV. The hori- zontal resolution of about 70 km comes close to the measure- ment grid sampling (50 km) and is a significant improvement compared to the 475 to 575 km full-width at half-max along the line-of-sight of the individual weighting functions. Com- bined with the across track sampling and resolution of 25 km, PREMIER IRLS would achieve a horizontal resolution com- parable to nadir sounders. Using the averaging kernels it is possible to deduce mea- surement contribution and resolution of the retrieval. Typical values are plotted in Figs. 7 and 8. The contribution of mea- surement information to the retrieval results is estimated by summing the elements of each row of the averaging kernel matrix. A value of 1 indicates a high contribution of mea- surement information while a value of 0 indicated a high contribution of a priori information. The tangent points of measurements lie within the region of 10 to 55 km height and 500 to 5500 km horizontal distance, which closely cor- responds to the region where the measurement contribution in Fig. 7 is between 0.95 and 1.05. Outside this region re- trieval results are dominated by a priori information. J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations Shown is the measurement contribution for a baseline re- trieval with no modulated wave structure (i.e. the atmosphere of Fig. 5a). Tangent points of measurements lie between ≈10 km and 55 km vertically and ≈500 km and 5500 km horizontally. 600 400 200 0 200 400 600 along-track distance (km) 36 38 40 42 44 46 48 50 altitude (km) averaging kernel at 40.0 km 0.025 0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 averaging kernel (K/K) Fig. 6. Plot of the averaging kernel of a single atmospheric state vector element located in the middle of the atmosphere at 40 km height. Each coloured box represents the influence of the atmo- spheric grid point of the true atmosphere on a single sample of the t i l lt It h i t i l f i k l f l t Fig. 7. Shown is the measurement contribution for a baseline re- trieval with no modulated wave structure (i.e. the atmosphere of Fig. 5a). Tangent points of measurements lie between ≈10 km and 55 km vertically and ≈500 km and 5500 km horizontally. 600 400 200 0 200 400 600 along-track distance (km) 36 38 40 42 44 46 48 50 altitude (km) averaging kernel at 40.0 km 0.025 0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 averaging kernel (K/K) along-track distance (km) Fig. 7. Shown is the measurement contribution for a baseline re- trieval with no modulated wave structure (i.e. the atmosphere of Fig. 5a). Tangent points of measurements lie between ≈10 km and 55 km vertically and ≈500 km and 5500 km horizontally. Fig. 6. Plot of the averaging kernel of a single atmospheric state vector element located in the middle of the atmosphere at 40 km height. Each coloured box represents the influence of the atmo- spheric grid point of the true atmosphere on a single sample of the retrieval result. Its shape is typical for averaging kernels of elements between 15 km and 50 km height. are mainly interested in results with high measurement con- tent and to simplify data processing, only data points with a measurement contribution between 0.95 and 1.05 are evalu- ated further. 5.2 Characteristics and error analysis The averaging kernels can also be used to estimate vertical and horizontal resolution by mapping each row of the aver- aging kernel matrix associated with an atmospheric sample to the two-dimensional structure it represents, as depicted in Fig. 6. In this form, the full-width at half-max of the vertical and horizontal direction can be calculated (e.g. Steck et al., 2005; von Clarmann et al., 2009). Figure 8 shows exemplar- ily the estimated horizontal and vertical resolution. As can be seen in Fig. 6, the averaging kernels generally contain nu- merous very small elements that describe the influence of at- mospheric sample points that are not directly above or beside the atmospheric sample point in question; as these are disre- garded when calculating the resolution, this method provides an approximate measure of resolution. 5.2.1 Measurement contribution and resolution The averaging kernel matrix A ∈Rn×n of the final estimate ˆx A=SaF′( ˆx)T  F′( ˆx)SaF′( ˆx)T +Sϵ −1 F′( ˆx). (5) (5) characterises the retrieval. J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations Starting from the initial guess x0, each iteration of the employed Gauss-Newton algorithm generates a new atmo- spheric state xi+1 ∈Rn: Figure 5 shows exemplarily such an end-to-end test. The unperturbed atmosphere common to all tests is depicted in Fig. 5a. An temperature perturbation of the input atmosphere is depicted in Fig. 5b. The tangent points of measurements are shown as white circles. The outcome of the retrieval is shown in Fig. 5c. One can clearly see that the wave struc- ture is qualitatively well reproduced between 10 and 55 km height. In the area between 55 km and 65 km, the wave struc- ture degrades into the unperturbed a priori atmosphere due to a lack of measurement information. The amplitude of the retrieved wave sometimes slightly surpasses ±5 K, which is shown as white areas in the wave troughs and crests. Fig- ure 5d depicts the difference between the input atmosphere and the result; the error is overall less than 0.5 K between 20 and 50 km height. xi+1=xa+SaF′(xi)T  F′(xi)SaF′(xi)T +Sϵ −1 · y−F(xi)+F′(xi)(xi−xa)  , (4) (4) converging onto the optimal estimate ˆx ∈Rn. converging onto the optimal estimate ˆx ∈Rn. The approach outlined above is traditionally applied to re- trieve single vertical profiles of atmospheric data, but it is just as well-suited for 2-D tomographic retrieval problems, the only difference being the interpretation of the state vec- tor xi. Please note that we do not apply the approach of Steck et al. (2005), where the full 2-D state vector is updated sequentially by using in each step the radiance information Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 346 600 400 200 0 200 400 600 along-track distance (km) 36 38 40 42 44 46 48 50 altitude (km) averaging kernel at 40.0 km 0.025 0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 averaging kernel (K/K) Fig. 6. Plot of the averaging kernel of a single atmospheric state vector element located in the middle of the atmosphere at 40 km height. Each coloured box represents the influence of the atmo- spheric grid point of the true atmosphere on a single sample of the retrieval result. Its shape is typical for averaging kernels of elements between 15 km and 50 km height. Fig. 7. 6.1 Deduction from end-to-end simulations To determine the observational filter, end-to-end simulations must be performed for all waves of interest. To deduce the amplitude of the wave in the retrieved atmosphere, all atmo- spheric grid points between 15 and 50 km and along track 1500 to 4500 km are fitted against the modulated wave with amplitude as the only free parameter using a least-square fit. Please note that this does not compensate for eventual shifts in phase as is generally present in 1-D retrievals (Preusse et al., 2009); any phase shift present would reduce the sensi- tivity (even to zero for a phase shift of 180◦). However, tests including the phase shift in the fit indicated that no phase shift was present for tomographic retrievals. According to Shannon’s sampling theorem, it is impossible to perfectly re- produce waves with wavelength δ using a sampling distance longer than δ/2; for this reason we tested only waves from a vertical wavelength of 1 km upwards and from a horizon- tal wavelength of 100 km upwards. Detecting GW signatures from actually measured profiles will require a more sophis- ticated process, as it is generally neither known if a GW is present in the data, nor what wave length it may have, nor will it generally be monochromatic. (b) Fig. 8. Shown are the vertical (a) and horizontal (b) resolution for a baseline retrieval with no modulated wave structure (i.e. the at- mosphere of Fig. 5a). Tangent points of measurements lie between ≈10 km and 55 km vertically and ≈500 km and 5500 km horizon- tally. Fig. 8. Shown are the vertical (a) and horizontal (b) resolution for a baseline retrieval with no modulated wave structure (i.e. the at- mosphere of Fig. 5a). Tangent points of measurements lie between ≈10 km and 55 km vertically and ≈500 km and 5500 km horizon- tally. 6 Gravity wave observational filter The observational filter defines how a wave perturbation of given horizontal and vertical wavelength is reproduced by a retrieval. The retrieval may modify the wave amplitude, wavelength, and phase in a complicated way. As the ampli- tude is the most important feature of the wave with respect to its energy, the assessment presented here focuses on how well the amplitude of a given wave is reproduced. (a) (a) (b) characterises the retrieval. As we The simulations indicate that spatially over-sampled mea- surements (with respect to the weighting functions) can be separated and provide high spatial resolution in the vertical and horizontal domain. Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 347 (a) (b) Fig. 8. Shown are the vertical (a) and horizontal (b) resolution for a baseline retrieval with no modulated wave structure (i.e. the at- mosphere of Fig. 5a). Tangent points of measurements lie between ≈10 km and 55 km vertically and ≈500 km and 5500 km horizon- tally. (a) The retrieval noise is the most important error for gravity wave analysis as the effect of other, systematic errors can be more readily handled by detrending (e.g. Ern et al., 2004). It will be analysed and compared with GW amplitudes in Sect. 6.3. 5.2.2 Retrieval noise 0 GW tilted towards instrument (a) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted towards instrument 0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20 retrieved / true wave amplitude coarse retrieval grid 0 150 200 250 300 horizontal wave length (km) (a) g 0GW tilted away from instrument GW tilted away from instrument (b) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted away from instrument 0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20 retrieved / true wave amplitude Fig. 10. This shows the observational filter derived with a sampling grid of 50 km horizontal distance instead of the usually employed 12.5 km. Please note the different colour scale compared to Fig. 9. wave peaks and sampling grid. Changing the orientation of the waves from being tilted towards the instrument to being tilted away from it has a significant influence as is shown in Fig. 9b. The sensitivity to waves with a horizontal wave- length below 150 km is lost, but this is compensated by an in- creased sensitivity to waves with a larger vertical wavelength and an overall smaller transition region, in which waves be- come distorted. As the atmosphere is not completely trans- parent, the sensitivity of the ray to temperature changes is not only a function of height, but also of the horizontally trav- elled distance, which can be clearly seen in Fig. 4. As the sensitivity to waves largely depends on the relation between ray path and wave trough and crest (Preusse et al., 2002) this is to be expected and needs to be taken into account when GW parameters are deduced from retrieved tempera- tures. The slope of the tangent point locations does slightly influence the observational filter, but is not the cause for the observed asymmetry. (b) Fig. 9. Observational filter for the PREMIER IRLS. Depicted is the ratio of retrieved wave amplitude to true wave amplitude for the baseline retrieval setup with varying modulated waves. Anal- ysed wavelength combinations are indicated with white circles. (a) shows the filter for the baseline setup with waves tilted towards the instrument. (b) shows the filter for a negative horizontal wave num- ber (i.e. waves tilted away from the instrument). 5.2.2 Retrieval noise of a vertical wavelength of 2 km can be retrieved well, but the sensitivity drops quickly for shorter vertical wavelengths, as the measurement grid is not fine enough to properly re- solve them. Around two kilometre vertical wavelength and long horizontal wavelength, the retrieved amplitude becomes slightly larger than 1; this is caused by an insufficient vertical sampling of the atmospheric retrieval grid, similar to the hor- izontal sampling issue discussed below in Sect. 6.2 but less pronounced. For waves with large vertical wavelength, the sensitivity becomes smaller depending strongly on the hor- izontal wavelength. But for waves with 300 km horizontal wavelength and longer, a sensitivity of at least 0.5 is given for all examined vertical wavelength longer than 2 km. Sen- sitivity rises to 1 for all waves with horizontal wavelength longer than ≈500 km (not shown). 5.2.2 Retrieval noise The effect of noise on the retrieval can be estimated based on the effect of perturbations of the measurements on the retrieval result. The method of optimal estimation does not only provide an optimal estimate ˆx but also a corresponding covariance matrix of the estimate. However, as our a priori covariance matrix for the atmospheric state is not derived from a cli- matology, but chosen ad-hoc, the covariance matrix of the retrieval result ˆx should not be interpreted statistically. In- stead, we use the retrieval noise for diagnosis, i.e. the effect of measurement noise on the retrieval result. The gain ma- trix G ∈Rn×m, which can be interpreted as linear pseudo- inverse of F and maps measurements into the atmospheric state space, is defined as Figure 9a shows the filter for the baseline setup described in Sect. 3; it corresponds qualitatively with the shape of an- alytically derived sensitivity of Preusse et al. (2002), albeit most notably the region of good sensitivity is moved well towards shorter horizontal wavelengths. Due to the much denser atmospheric sampling and the tomographic approach, much smaller waves become visible. The finest horizontal wavelength resolvable is about 100 km for a vertical wave- length around 5 km and the wave being tilted towards the in- strument. This is a significant improvement compared to the roughly 250 km horizontal wavelength of previous sensitiv- ity studies involving the CRISTA instrument (Preusse et al., 2002). Please note that the resolvable horizontal wavelength is much smaller than the horizontal full-width at half-max of the weighting function, which is roughly 500 km. Waves G=SaF′( ˆx)T  F′( ˆx)SaF′( ˆx)T +Sϵ −1 . (6) (6) It can be used to estimate the retrieval noise covariance ma- trix: It can be used to estimate the retrieval noise covariance ma- trix: Snoise=GSϵGT . (7) Snoise=GSϵGT . (7) Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ www.atmos-meas-tech.net/3/339/2010/ J. 5.2.2 Retrieval noise Ungermann et al.: Tomographic retrieval approach for gravity wave observations 348 (a) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted towards instrument 0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20 retrieved / true wave amplitude (b) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted away from instrument 0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20 retrieved / true wave amplitude Fig. 9. Observational filter for the PREMIER IRLS. Depicted is the ratio of retrieved wave amplitude to true wave amplitude for the baseline retrieval setup with varying modulated waves. Anal- ysed wavelength combinations are indicated with white circles. (a) shows the filter for the baseline setup with waves tilted towards the instrument. (b) shows the filter for a negative horizontal wave num- ber (i.e. waves tilted away from the instrument). 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) coarse retrieval grid 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 retrieved / true wave amplitude Fig. 10. This shows the observational filter derived with a sampling grid of 50 km horizontal distance instead of the usually employed 12.5 km. Please note the different colour scale compared to Fig. 9. wave peaks and sampling grid. Changing the orientation of the waves from being tilted towards the instrument to being tilted away from it has a significant influence as is shown in Fig. 9b. The sensitivity to waves with a horizontal wave- length below 150 km is lost, but this is compensated by an in- creased sensitivity to waves with a larger vertical wavelength and an overall smaller transition region, in which waves be- come distorted. As the atmosphere is not completely trans- parent, the sensitivity of the ray to temperature changes is not only a function of height, but also of the horizontally trav- elled distance, which can be clearly seen in Fig. 4. 5.2.2 Retrieval noise As the sensitivity to waves largely depends on the relation between ray path and wave trough and crest (Preusse et al., 2002) this is to be expected and needs to be taken into account when GW parameters are deduced from retrieved tempera- tures The slope of the tangent point locations does slightly (a) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted towards instrument 0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20 retrieved / true wave amplitude (b) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted away from instrument 0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20 retrieved / true wave amplitude Fig. 9. Observational filter for the PREMIER IRLS. Depicted is the ratio of retrieved wave amplitude to true wave amplitude for the baseline retrieval setup with varying modulated waves. Anal- ysed wavelength combinations are indicated with white circles. (a) shows the filter for the baseline setup with waves tilted towards the instrument. (b) shows the filter for a negative horizontal wave num- ber (i.e. waves tilted away from the instrument). 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) coarse retrieval grid 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 retrieved / true wave amplitude Fig. 10. This shows the observational filter derived with a sampling grid of 50 km horizontal distance instead of the usually employed 12.5 km. Please note the different colour scale compared to Fig. 9. 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) coarse retrieval grid 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 retrieved / true wave amplitude Fig. 10. This shows the observational filter derived with a sampling grid of 50 km horizontal distance instead of the usually employed 12.5 km. Please note the different colour scale compared to Fig. 9. 6.3 Tradeoff between retrieval noise and resolution (b) The vertical and horizontal correlation length of the a priori covariance plays the most important role in the MAP regular- isation applied here. Different values were examined before the values of 0.5 km and 200 km were chosen to provide a satisfactory trade-off between resolution and retrieval noise. In the Bayesian scheme applied in this study the strength of the regularisation, i.e. smoothing via correlation lengths in the a priori covariance matrix model, does not depend on the actual measurements. As the noise error indicates, the effect of noise on the retrieval result is sufficiently small that the forward model can be assumed to be nearly linear so that the Jacobian matrix will vary only negligible. This means that the averaging kernel stays nearly identical if noise is added to the measurements and in turn the results of this section are also representative for actual measurements affected by noise even though they were derived using unperturbed mea- surements. Fig. 11. The effect on vertical correlation length on vertical resolu- tion and noise error. The average resolution and retrieval noise for the four major height ranges are depicted. One can clearly see the tradeoff between vertical resolution and retrieval noise. and therefore not depicted. The main effect of the horizon- tal correlation is to stabilise the underdetermined retrievals. Reducing the horizontal correlation length from 200 km to 100 km improves the horizontal resolution from 75 km to ≈70 km, but the non-linear retrievals do not converge for some wave structures with very small vertical and horizontal wavelength. Reducing the horizontal correlation further, the horizontal resolution derived from the averaging kernel ma- trix goes as low as 50 km for a horizontal correlation length of 12.5 km, i.e. is close to the horizontal measurement sam- pling grid at the cost of ever increasing convergence issues. To avoid any convergence problems, a correlation length of 200 km was chosen. The GW observational filter behaves as expected when varying the horizontal correlation length, as waves become increasingly dampened for larger correla- tion lengths. However, this barely affects the region of the GW observational filter close to one but influences mostly the transition region. Figure 11 shows the effect of the vertical correlation length on vertical resolution and retrieval noise. 6.2 Sensitivity to retrieval grid sampling The horizontal sampling of the retrieval grid has a significant influence on the retrieval results as shown in Fig. 10. Before settling on a horizontal sampling distance of 12.5 km for the retrieval grid, several other sampling grids were tested ranging from 6.75 km to 400 km. It was found that the larger the sampling distance became, the more pro- nounced the amplification of wave amplitudes for short GW lengths became. This amplification can be seen in Fig. 10, which depicts the filter for a “natural” retrieval grid being identical with the measurement grid. Apparently, at least for GW with short wavelengths, the natural sampling is not nec- essarily the best choice. Varying the phase of the modulated wave had no signifi- cant influence on the sensitivity filter derived by the 2-D re- trieval and is therefore not depicted. Small differences are present for waves with vertical wavelength below 2 km where the resolvability strongly depends on the relation between The amplification of retrieved waves is most likely caused by the bi-linear interpolation of the temperature between the grid points. The fine structure of wave perturbations with Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 349 (a) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 vertical correlation (km) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 noise error (K) 10-20 km 20-30 km 30-40 km 40-50 km (b) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 vertical correlation (km) 0.50 0.55 0.60 0.65 0.70 0.75 vertical resolution (km) 10-20 km 20-30 km 30-40 km 40-50 km Fig. 11. The effect on vertical correlation length on vertical resolu- tion and noise error. The average resolution and retrieval noise for the four major height ranges are depicted. One can clearly see the tradeoff between vertical resolution and retrieval noise. short wavelength cannot be sufficiently represented by linear interpolation, which the retrieval compensates for by rais- ing the wave amplitudes. Therefore, either a different atmo- spheric representation more suited to represent wave struc- tures is needed or the grid sampling needs to be refined to suf- ficiently capture the variations of the atmospheric state. Here we employ the second approach. If the retrieval grid sam- pling drops below the measurement grid sampling, the prob- lem becomes underdetermined and needs to be regularised appropriately. 6.2 Sensitivity to retrieval grid sampling Simulations have shown that resolution, mea- surement content and retrieval noise are not noticeably im- pacted by the refinement of the atmospheric grid. (a) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 vertical correlation (km) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 noise error (K) 10-20 km 20-30 km 30-40 km 40-50 km (a) For the examined waves, the artificial amplitude magnifi- cation is greatly diminished with a horizontal grid distance of 25 km and negligible at 12.5 km. The sampling distance of 12.5 km was chosen to obtain exactness of the results. ( ) (b) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 vertical correlation (km) 0.50 0.55 0.60 0.65 0.70 0.75 vertical resolution (km) 10-20 km 20-30 km 30-40 km 40-50 km As mentioned in Sect. 6.1, a similar effect occurs for waves with small vertical wavelength, which increases the retrieved amplitude by a factor of up to 1.2 for waves with about 2 km vertical wavelength. As the effect is much less pronounced and therefore easier to correct, we decided not to increase the vertical retrieval grid sampling for this study. 6.3 Tradeoff between retrieval noise and resolution Practically, a slightly larger vertical wavelength than 1 km is required, meaning that we expect to consistently resolve waves from about 2 km vertical wavelength upward, which is confirmed by the GW observational filter. The maximal amplitude of actual GWs of that vertical wavelength can be determined according to the temperature amplitude saturation limit (e.g. Preusse et al., 2008). This limit ˆTmax due to static instability is proportional to the vertical wavelength: ˆTmax= λz 2π T g N2. (8) ˆTmax= λz 2π T g N2. (8) (a) 0GW tilted away from instrum GW tilted away from instrument (b) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted away from instrument 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 retrieved / true wave amplitude Using N=0.02 s−1 and a background temperature T of 250 K, this gives a saturation amplitude of greater than 3.2 K for waves with vertical wavelength greater than or equal to 2 km at about 40 km height. Comparing this value to the 0.5 K of retrieval noise for that height range shows that such GWs are easily detectable also in the presence of noise. As the temperature amplitude saturation limit increases with ver- tical wavelength, any wave with vertical wavelength longer than 2 km is therefore also detectable with the given noise level. 6.3 Tradeoff between retrieval noise and resolution To decrease the re- trieval noise to ≈0.5 K for most of the vertical range, while still retaining a good vertical resolution, a correlation length of 0.5 km was finally chosen. The choice of vertical cor- relation parameter also influences the GW observational fil- ter. As expected, a large correlation length slightly reduces the region where sensitivity is very close to one. We further found that increasing the vertical correlation length beyond 2 km leads to unwanted oscillations in the averaging kernels, which confirms our choice of a much smaller value. GWs can be detected if their amplitude exceeds retrieval noise. Therefore, it is important that the retrieval noise of the retrieval is smaller than the amplitude of GW that shall be observed. According to the sampling theorem, with a The retrieval noise is not influenced significantly by the horizontal correlation factors we examined (12.5 to 400 km) Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 350 (a) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted towards instrument 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 retrieved / true wave amplitude (b) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted away from instrument 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 retrieved / true wave amplitude Fig. 12. The gravity wave filter as deduced from the averaging ker- nel. Each rectangle represents the mapping of a wave onto itself. (a) shows the diagonal elements for waves tilted towards the instru- ment (see also Fig. 9a), (b) shows the diagonal elements for waves tilted away from the instrument (see also Fig. 9b). 0 GW tilted towards instrument (a) 100 150 200 250 300 350 horizontal wave length (km) 10 20 30 40 50 60 vertical wave length (km) GW tilted towards instrument 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 retrieved / true wave amplitude vertical sampling grid of 500 m one cannot resolve waves with a vertical wavelength shorter than 1 km. 6.4 Deducing the gravity wave observational filter from the averaging kernel matrix (b) A more efficient way to derive the GW observational filter is to analyse the averaging kernel matrix of a retrieval for a climatological atmosphere of the region of interest. Fig. 12. The gravity wave filter as deduced from the averaging ker- nel. Each rectangle represents the mapping of a wave onto itself. (a) shows the diagonal elements for waves tilted towards the instru- ment (see also Fig. 9a), (b) shows the diagonal elements for waves tilted away from the instrument (see also Fig. 9b). Assuming the non-linearities of the forward model to be negligible and thereby the Jacobian matrix to be constant across the space spanned by the a priori atmosphere and all examined wave perturbations, the averaging kernel matrix A will be identical for all wave structures and the unperturbed atmosphere. Neglecting measurement errors, it follows that simulations. The latter are still required, as the linear approx- imation cannot be used to examine, e.g. the convergence of the non-linear retrieval for different atmospheric states. ˆx−xa=(A(xa+xδ)+(I−A)xa)−xa=Axδ, (9) 7 Summary and conclusions We performed various non-linear end-to-end simulations us- ing simulated measurements of the PREMIER IRLS with different input atmospheres consisting of a cloudless cli- matological background atmosphere with modulated wave perturbations. In this way, we determined the gravity wave (GW) observational filter that shows the amplitude with which a given wave perturbation is reproduced by our tomo- graphic retrieval setup. It was shown that the new, innovative PREMIER IRLS using the dense measurement grid of the dynamics mode combined with tomographic retrievals can resolve much finer GW structures than previously possible. GWs with a horizontal wavelength down to 100 km may be resolved depending on the vertical wavelength. (b) Fig. 13. This shows the retrieval result for (a) a series of conven- tional 1-D retrievals on the left and (b) a 2-D retrieval in the middle for a wave with 30 km vertical and 320 km horizontal wavelength. Please note the different color mapping. The climatological back- ground profile was subtracted from the result. A contour plot for the difference between the true modulated wave structure and the a priori has been included as black lines to visualize the phase shift of the 1-D retrieval. We examined different retrieval grids and found that using a coarse retrieval grid introduces an amplification of GWs with short wavelength. It is therefore essential to use a re- trieval grid that is sufficiently fine for the purpose of repre- senting examined and existing atmospheric structure. Other- wise, retrieval results may be tainted by artifacts. thereby much smaller than the expected 5 K. In addition, the amplitude has also a strong artificial height-dependence. The vertical wavelength is also slightly reduced. On the other hand, the 2-D retrieval shows a more pronounced wave struc- ture and fits well to the true wave (again shown as black con- tour plot) in the region between 25 km and 55 km; the 2-D re- trieval deviates less than 2 K from the true atmospheric state whereas the 1-D retrieval is subject to deviations of more than 6 K (the latter is partly caused by the 180◦phase shift). As depicted in Fig. 9, this retrieval has an amplitude damp- ening factor of 0.9. By tweaking the correlation length of the a priori covari- ance matrix, it is possible to reduce the retrieval noise at the cost of a reduced resolution. 6.5 Comparison of 2-D and 1-D retrievals (9) with xδ being the modulated wave structure. This shows that the averaging kernel matrix directly maps the true wave per- turbations xδ onto the retrieved wave structure. A 2-D retrieval reproduces wave structures with better sensi- tivity and less distortions than 1-D retrievals (which assume a homogeneously stratified atmosphere). Figure 12 shows the GW observational filter derived in this manner. Just as for the non-linear end-to-end simulations, only the part of the ˆx−xa vector is used to derive the sensi- tivity that has a good measurement contribution and lies well within the region covered by tangent points of measurements. The perturbations of up to 5 K are small enough so that the assumption about linearity of the forward model sufficiently holds and the filter derived in this manner replicates closely the structure of the filter derived from non-linear end-to-end tests. For example, Fig. 13 shows side-by-side the retrieval re- sults for a wave of 320 km horizontal wavelength and 30 km vertical wave length. Figure 13a was generated by a se- ries of 1-D retrievals, each using a single vertical measure- ment profile of the simulated measurements as input. The resulting temperature profiles were assembled to a 2-D struc- ture by adding each retrieved temperature profile at the mean horizontal location of the tangent points of the involved mea- surements. Comparing the retrieved wave structure with the true wave structure (which is depicted as the black contour plot in the figure), it becomes apparent that the phase of the wave structure of the 1-D retrieval is subject to a phase shift of nearly 180◦; the amplitude does not surpass 2.5 K and is This method is therefore an efficient way to quickly evalu- ate the effect of changes in the setup before proceeding to the much more computationally intensive non-linear end-to-end Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 351 (a) (b) Fi 13 Thi h h i l l f ( ) i f (a) Fig. 14. This depicts the gravity wave observational filter derived by a series of a 1-D retrievals. Fig. 14. This depicts the gravity wave observational filter derived by a series of a 1-D retrievals. (a) (b) Fig. 14. This depicts the gravity wave observational filter derived by a series of a 1-D retrievals. 7 Summary and conclusions We also found that conver- gence issues arise when the horizontal correlation length is reduced to or below 100 km. We examined various horizon- tal and vertical correlation lengths and settled for a horizontal correlation length of 200 km and a vertical correlation length of 0.5 km. With these figures, the expected retrieval noise is well below the temperature amplitude saturation limit of resolvable GWs. The GW observational filter deduced using 1-D retrievals is shown in Fig. 14. In contrast to Fig. 9, also the phase of the wave was fitted (not depicted), otherwise the sensitivity would be even worse. It is apparent that fewer wave patterns can be reliably retrieved. Especially waves with large ver- tical wavelength are affected. This comparison shows that tomographic retrievals are superior for the purpose of GW detection. A linear approximation of the observation filter derived from the averaging kernel matrix is a helpful tool when ex- amining different setups. One may generate the averaging kernel matrix for a climatological atmosphere and use this to calculate inexpensively an approximation of the observa- tion filter for a wide range of GWs. The observational filter Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 352 1.0 60 2.0 120 3.0 180 4.0 240 5.0 300 6.0 360 1 2 3 4 5 6 7 8 wall clock time (minutes) speedup factor number of processors consumed time speedup Fig. A1. Consumed wall clock time and achieved speedup factor for the baseline retrieval if an OpenMP parallelisation is applied. 1.0 60 2.0 120 3.0 180 4.0 240 5.0 300 6.0 360 1 2 3 4 5 6 7 8 wall clock time (minutes) speedup factor number of processors consumed time speedup derived in this manner resembles closely the observational filter derived by more accurate end-to-end simulations in- cluding the distinction between GWs tilted towards or away from the satellite. The observational filter derived by conventional 1-D re- trievals has a severely reduced sensitivity compared to the observational filter derived by our tomographic retrieval technique. Comparing the results for selected wave pertur- bations, we found the result of the tomographic retrieval to be much closer to the true state than the result of the 1-D re- trieval. Further, the tomographic retrieval is not subject to the massive shift in phase that affects 1-D retrievals. 7 Summary and conclusions p The PREMIER IRLS should provide 3-D temperature measurements with sufficient altitude coverage and resolu- tion in the stratosphere to allow for simultaneous determi- nation of GW temperature amplitudes and the associated horizontal and vertical wavelength. Therefore, the dynam- ics mode of the PREMIER satellite instrument will be well suited for examining the dynamic structure of the upper tro- posphere and stratosphere. The presented tomographic re- trieval scheme resolves the dense 3-D measurement of the PREMIER IRLS dynamics mode with a comparable 3-D res- olution and is thereby well suited for the retrieval of tempera- ture data for the analysis of GWs. The across track resolution of PREMIER IRLS corresponds to the across track sampling of 25 km. Further, it is possible to retrieve temperature data with a vertical resolution of 750 m; depending on the retrieval setup even a vertical resolution of 500 m is achievable, sur- passing the full-height-at-half-maximum of the vertical field- of-view (750 m). In addition, one can nearly fully exploit the dense measurement grid of 50 km horizontal distance with an achievable horizontal along track resolution of ≈70 km, which is unprecedented for limb sounders and approaches the horizontal resolution of nadir sounders. Fig. A1. Consumed wall clock time and achieved speedup factor for the baseline retrieval if an OpenMP parallelisation is applied. rather sparse Jacobian matrices compared to 1-D retrievals. For the specific PREMIER setup only between one and three percent of the entries of the Jacobian matrix are non-zero. By generating a data structure that tracks which points of the atmosphere influence which pencil beams, it is possible to simply skip the calculation of pencil beams unaffected by the current perturbation. In effect, zeros within the Jacobian matrix cost next to no processing time. Further, generally, only part of the pencil beams influencing a single changed measurement need to be recalculated, resulting in an overall speedup of ≈200 for the type of problems considered here. The second change involved reworking the linear algebra of the retrieval processor. For conventional 1-D retrievals, the processing time spent in the matrix multiplications and the solving of linear equation systems is negligible compared to the calculation of the Jacobian matrices. For increased problem sizes, it becomes much more important, beginning to noticeably increase the processing time and to dominate the memory consumption. 7 Summary and conclusions Using more efficient algorithms to solve the linear equation systems and the Automatically Tuned Linear Algebra Software (ATLAS) BLAS implemen- tation by Whaley et al. (2001), could halve the previous memory consumption and significantly reduce the process- ing time. References Adams, S., Spang, R., Preusse, P., and Heinemann, G.: The benefit of limb cloud imaging for infrared limb sounding of tropospheric trace gases, Atmos. Meas. Tech., 2, 287–298, 2009, http://www.atmos-meas-tech.net/2/287/2009/. Ern, M., Preusse, P., and Warner, C. D.: Some experimental con- straints for spectral parameters used in the Warner and McIntyre gravity wave parameterization scheme, Atmos. Chem. Phys., 6, 4361–4381, 2006, Alexander, J.: Interpretations of observed climatological patterns in stratospheric gravity wave variance, Geophys. Res. Lett., 103, 8627–8640, doi:10.1029/97JD03325, 1998. http://www.atmos-chem-phys.net/6/4361/2006/. ESA: Candidate Earth Explorer Core Missions – Report for Assess- ment: PREMIER – PRocess Exploitation through Measurements of Infrared and millimetre-wave Emitted Radiation, SP-1313/5, ESA Publications Division, ESTEC, Keplerlaan 1, 2200 AG No- ordwijk, The Netherlands, 2008. Alexander, M. J., Gille, J., Cavanaugh, C., Coffey, M., Craig, C., Eden, T., Francis, G., Halvorson, C., Hannigan, J., Khos- ravi, R., Kinnison, D., Lee, H., Massie, S., Nardi, B., Barnett, J., Hepplewhite, C., Lambert, A., Dean, V.: Global estimates of gravity wave momentum flux from high resolution dynam- ics limb sounder (HIRDLS) observations, J. Geophys. Res., 113, D15S18, doi:10.1029/2007JD008807, 2008. Fischer, H., Birk, M., Blom, C., Carli, B., Carlotti, M., von Clar- mann, T., Delbouille, L., Dudhia, A., Ehhalt, D., Endemann, M., Flaud, J. M., Gessner, R., Kleinert, A., Koopman, R., Langen, J., Lpez-Puertas, M., Mosner, P., Nett, H., Oelhaf, H., Perron, G., Remedios, J., Ridolfi, M., Stiller, G., and Zander, R.: MIPAS: an instrument for atmospheric and climate research, Atmos. Chem. Phys., 8, 2151–2188, 2008, http://www.atmos-chem-phys.net/8/2151/2008/. Alexander, M. J., Eckermann, S. D., Broutman, D., and Ma, J.:, Mo- mentum flux estimates for South Georgia Island mountain waves in the stratosphere observed via satellite, Geophys. Res. Lett., 36, L12816, doi:10.1029/2009GL038587, 2009. Alexander, J. and Rosenlof, K. H.: Gravity-wave forcing in the stratosphere: Observational constraints from the Upper At- mosphere Research Satellite and implications for parameter- ization in global models, Geophys. Res. Lett., 108, 4597, doi:10.1029/2003JD003373, 2003. http://www.atmos-chem-phys.net/8/2151/2008/. Francis, G. L., Edwards, D. P., Lambert, A., Halvorson, C. M., Lee-Taylor, J. M., and Gille, J. C.: Forward modeling and ra- diative transfer for the NASA EOS-Aura High Resolution Dy- namics Limb Sounder (HIRDLS) instrument, J. Geophys. Res., 111, D13301, doi:10.1029/2005JD006270, 2006. Carlotti, M., Dinelli, B. M., Raspollini, P., and Ridolfi, M.: Geo-fit approach to the analysis of limb-scanning satellite measurements, Appl. Optics, 40, 1872–1885, doi:10.1364/AO.40.001872, 2001. J. Ungermann et al.: Tomographic retrieval approach for gravity wave observations 353 Acknowledgements. We sincerely thank A. Dudhia, Univ. Oxford, for providing us with the reference forward model (RFM). Acknowledgements. We sincerely thank A. Dudhia, Univ. Oxford, for providing us with the reference forward model (RFM). Ern, M., Preusse, P., Alexander, M. J., and Warner, C. D.: Absolute values of gravity wave momentum flux de- rived from satellite data, J. Geophys. Res., 109, D20103, doi:10.1029/2004JD004752, 2004. Edited by: S. Buehler Ern, M., Preusse, P., and Warner, C. D.: A comparison between CRISTA satellite data and Warner and McIntyre gravity wave pa- rameterization scheme: horizontal and vertical wavelength filter- ing of gravity wave momentum flux, Adv. Space Res., 35, 2017– 2023, doi:10.1016/j.asr.2005.04.109, 2005. Code optimisation and performance Code optimisation and performance Conventional 1-D retrievals often depend on the assumption of horizontal homogeneity to represent the atmosphere by a single profile. For tomographic retrievals this assumption is discarded, requiring a much larger number of variables to properly represent the atmosphere. Combined with an in- creased number of measurements, the retrieval process be- comes technically more demanding. To enable many such re- trieval simulations, the JURASSIC2 forward model and the retrieval processor were optimised both for modern imager instruments and tomographic retrievals. For our simulations, a cluster of 16 computer nodes is used, each fitted with 64 Gb RAM and two AMD Quad- Core Opteron 2380 CPUs. To retrieve the baseline setup (19 493 pencil beams combined to 9191 measurements for retrieving 46 080 atmospheric state vector elements) with a single thread, roughly seven hours are needed. A simple OpenMP parallelisation of the embarrassingly parallel for- ward model together with the multi-threaded ATLAS back- end allows a faster execution on a multi-core host. Figure A1 shows the speedup realised with the OpenMP parallelisation. From the curve can be deduced that about 93% of the re- trieval code are parallelised. The first change improved the calculation speed of the Ja- cobian matrices involved in the retrieval, which JURASSIC2 calculates using a finite differences approach. For tomo- graphic retrievals, a single ray path crosses only a minor frac- tion of points of the full atmospheric grid, which results in www.atmos-meas-tech.net/3/339/2010/ Atmos. Meas. Tech., 3, 339–354, 2010 References Ungermann et al.: Tomographic retrieval approach for gravity wave observations Hoffmann, L.: Schnelle Spurengasretrieval f¨ur das Satellitenex- periment Envisat MIPAS, Forschungszentrum J¨ulich, Germany, Tech. Rep. JUEL-4207, ISSN 0944-2952, 2006. Riese, M., Spang, R., Preusse, P., Ern, M., Jarisch, M., Offermann, D., and Grossmann, K. U.: Cryogenic Infrared Spectrometers and Telescopes for the Atmosphere (CRISTA) data processing and atmospheric temperature and trace gas retrieval, J. Geophys. Res., 104, 16349–16367, doi:10.1016/S0273-1177(97)00172-5, 1999. Hoffmann, L. and Alexander, M. J.: Retrieval of stratospheric tem- peratures from Atmospheric Infrared Sounder radiance measure- ments for gravity wave studies, J. Geophys. Res., 114, D07105, doi:10.1029/2008JD011241, 2009. Riese, M., Preusse, P., Spang, R., Ern, M., Jarisch, M., Gross- mann, U., and Offermann, D.: Measurements of trace gases by the cryogenic infrared spectrometers and telescopes for the at- mosphere CRISTA experiment, Adv. Space Res., 19, 563–566, doi:10.1016/S0273-1177(97)00172-5, 1997. Hoffmann, L., Kaufmann, M., Spang, R., Mller, R., Remedios, J. J., Moore, D. P., Volk, C. M., von Clarmann, T., and Riese, M.: Envisat MIPAS measurements of CFC-11: retrieval, validation, and climatology, Atmos. Chem. Phys., 8, 3671–3688, 2008, http://www.atmos-chem-phys.net/8/3671/2008/. Riese, M., Friedl-Vallon, F., Spang, R., Preusse, P., Schiller, C., Hoffmann, L., Konopka, P., Oelhaf, H., von Clarmann, T., and H¨opfner, M.: GLObal limb Radiance Imager for the Atmosphere (GLORIA): Scientific objectives, Adv. Space Res., 36, 989–995, doi:10.1016/j.asr.2005.04.115, 2005. Hoffmann, L., Weigel, K., Spang, R., Schroeder, S., Arndt, K., Lehmann, C., Kaufmann, M., Ern, M., Preusse, P., Stroh, F., and Riese, M.: CRISTA-NF measurements of water vapor during the SCOUT-O3 Tropical Aircraft Campaign, Adv. Space Res., 43, 74–81, doi:10.1016/j.asr.2008.03.018, 2009. Rodgers, C. D.: Retrieval of Atmospheric Temperature and Composition From Remote Measurements of Ther- mal Radiation, Rev. Geophys. Space Ge., 14, 609–624, doi:10.1029/RG014i004p00609, 1976. Livesey, N. J. and Read, W. G.: Direct Retrieval of Line-of- Sight Atmospheric Structure from Limb Sounding Observations, Geophys. Res. Lett., 27, 891–894, doi:10.1029/1999GL010964, 2000. Rodgers, C. D.: Inverse Methods for Atmospheric Sounding: The- ory and Practice, vol. 2 of Series on Atmospheric, Oceanic and Planetary Physics, World Scientific, 2000. Marshall, B. T., Gordley, L. L., and Chu, D. A.: BANDPAK: Algorithms for Modeling Broadband Transmission and Radi- ance, J. Quant. Spectrosc. Ra., 52, 581–599, doi:10.1016/0022- 4073(94)90026-4, 1994. Solomon, S. C., Hays, P. B., and Abreu, V. J.: Tomographic in- version of satellite photometry, Appl. Opt., 23(19), 3409–3414, doi:10.1364/AO.23.003409, 1984. References Friedl-Vallon, F., Riese, M., Maucher, G., Lengel, A., Hase, F., Preusse, P., and Spang, R.: Instrument concept and preliminary performance analysis of GLORIA, Adv. Space Res., 37, 2287– 2291, doi:10.1016/j.asr.2005.07.075, 2006. Carlotti, M., Brizzi, G., Papandrea, E., Prevedelli, M., Ridolfi, M., Dinelli, B. M., and Magnani, L.: GMTR: Two-dimensional geo- fit multitarget retrieval model for Michelson Interferometer for Passive Atmospheric Sounding/Environmental Satellite observa- tions, Appl. Optics, 45, 716–727, doi:10.1364/AO.45.000716, 2006. Fritts, D. C. and Alexander, M. J.: Gravity wave dynamics and effects in the middle atmosphere, Rev. Geophys., 41, 1003, doi:10.1029/2001RG000106, 2003. Gille, J. C. and Russel III, J. M.: The Limb Infrared Mon- itor of the Stratosphere: Experiment Description, Perfor- mance, and Results, J. Geophys. Res., 89, 5125–5140, doi:10.1029/JD089iD04p05125, 1984. Drummond, J. R., Houghton, J. T., Peskett, G. D., Rodgers, C. D., Wale, M. J., Whitney, J., and Williamson, E. J.: The stratospheric and mesospheric sounder on Nimbus 7, Philos. T. R. Soc. Lond., 296, 219–241, doi:10.1098/rsta.1980.0166, 1980. Gordley, L. L. and Russel, J. M.: Rapid inversion of limb radiance data using an emissivity growth approximation, Appl. Optics, 20, 807–813, doi:10.1364/AO.20.000807, 1981. Dudhia, A., Morris, P. E., and Wells, R. J.: Fast monochromatic radiative transfer calculations for limb sounding, J. Quant. Spec- trosc. Ra., 74, 745–756, doi:10.1016/S0022-4073(01)00285-0, 2002. Gurevich, G.: Application of satellite tomography methods for the improvement of atmospheric sounding inversion algorithms. In: Geoscience and Remote Sensing Symposium, 1995, IGARSS 95, Quantitative Remote Sensing for Science and Applications, 2, 1195–1197, doi:210.1109/IGARSS.1995.521182, 1995. Dunkerton, T.: The role of gravity waves in the quasi- biennial oscillation, J. Geophys. Res., 102, 26053–26076, doi:10.1029/96JD02999, 1997. Hase, F. and H¨opfner, M.: Atmospheric ray path modeling for radiative transfer algorithms, Appl. Optics, 38, 3129–3133, doi:10.1364/AO.38.003129, 1999. Eckermann, S. D. and Preusse, P.: Global measurements of strato- spheric mountain waves from space, Science, 286, 1534–1537, doi:10.1126/science.286.5444.1534, 1999. Hertzog, A., Boccara, G., Vincent, R. A., Vial, F., and Coquerez, P.: Estimation of gravity-wave momentum flux and phase speeds from long-duration stratospheric balloon flights, 2: Results from the Vorcore campaign in Antarctica, J. Atmos. Sci., 65, 3056– 3070, doi:10.1175/2008JAS2710.1, 2008. Eckermann, S. D., Hoffmann, L., H¨opfner, M., Wu, D. L., and Alexander, M. J.: Antarctic NAT PSC belt of June 2003: Ob- servational validation of the mountain wave seeding hypothesis, Geophys. Res. Lett., 36, L02807, doi:10.1029/2008GL036629, 2009. Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/ 354 J. References McLandress, C.: On the importance of gravity waves in the middle atmosphere and their parameterization in general cir- culation models, J. Atmos. Sol.-Terr. Phy., 60, 1357–1383, doi:10.1016/S1364-6826(98)00061-3, 1998. Spang, R., Remedios, J. J., and Barkley, M. P.: Colour indices for the detection and differentiation of cloud type in infra- red limb emission spectra, Adv. Space Res., 33, 1041–1047, doi:10.1016/S0273-1177(03)00585-4, 2004. McLandress, C. and Shepherd, T. G.: Simulated anthro- pogenic changes in the Brewer-Dobson circulation, including its extension to high latitudes, J. Climate, 22, 1516–1540, doi:10.1175/2008JCLI2679.1, 2009. Steck, T., H¨opfner, M., von Clarmann, T., and Grabowski, U.: Tomographic retrieval of atmospheric parameters from infrared limb emission observations, Appl. Optics, 44, 3291–3301, doi:10.1364/AO.44.003291, 2005. Preusse, P., Eckermann, S. D., and Offermann, D.: Comparison of global distributions of zonal-mean gravity wave variance in- ferred from different satellite instruments, Geophys. Res. Lett., 27, 3877–3880, doi:10.1029/2000GL011916, 2000. von Clarmann, T., De Clercq, C., Ridolfi, M., Hpfner, M., and Lam- bert, J.-C.: The horizontal resolution of MIPAS, Atmos. Meas. Tech., 2, 47–54, 2009, http://www.atmos-meas-tech.net/2/47/2009/. Preusse, P., D¨ornbrack, A., Eckermann, S. D., Riese, M., Schaeler, B., Bacmeister, J. T., Broutman, D., and Grossmann, K. U.: Space-based measurements of stratospheric mountain waves by CRISTA, 1, Sensitivity, analysis method, and a case study, J. Geophys. Res., 107(D23), 8178, doi:10.1029/2001JD000699, 2002. Weinreb, M. P. and Neuendorffer, A. C.: Method to Apply Homogeneous-path Transmittance Models to Inhomogenous At- mospheres, J. Atmos. Sci., 30, 662–666, doi:10.1175/1520- 0469(1973)030<0662:MTAHPT>2.0.CO;2, 1973. Whaley, R. C., Petitet, A., and Dongarra, J. J.: Automated Empir- ical Optimization of Software and the ATLAS Project, Parallel Comput., 27, 3–35, doi:10.1145/1512762.1512773, 2001. Preusse, P., Eckermann, S. D., and Ern, M.: Transparency of the at- mosphere to short horizontal wavelength gravity waves, J. Geo- phys. Res., 113, D24194, doi:10.1029/2007JD009682, 2008. Wright, C. J., Osprey, S. M., Barnett, J. J., Gray, L. J., and Gille, J. C.: High Resolution Dynamics Limb Sounder measurements of gravity wave activity in the 2006 Arctic stratosphere, J. Geophys. Res., 115, D02105, doi:10.1029/2009JD011858, 2010 Preusse, P., Schroeder, S., Hoffmann, L., Ern, M., Friedl-Vallon, F., Ungermann, J., Oelhaf, H., Fischer, H., and Riese, M.: New per- spectives on gravity wave remote sensing by spaceborne infrared limb imaging, Atmos. Meas. Tech., 2, 299–311, 2009, http://www.atmos-meas-tech.net/2/299/2009/. Remedios, J. J., Leigh, R. J., Waterfall, A. M., Moore, D. References P., Sembhi, H., Parkes, I., Greenhough, J., Chipperfield, M.P., and Hauglustaine, D.: MIPAS reference atmospheres and compar- isons to V4.61/V4.62 MIPAS level 2 geophysical data sets, At- mos. Chem. Phys. Discuss., 7, 9973–10017, 2007, http://www.atmos-chem-phys-discuss.net/7/9973/2007/. http://www.atmos-chem-phys-discuss.net/7/9973/2007/ Atmos. Meas. Tech., 3, 339–354, 2010 www.atmos-meas-tech.net/3/339/2010/
https://openalex.org/W4384154771
https://mrj.ima-press.net/mrj/article/download/1437/1367
Russian
null
Revisiting the question of the safety of glucocorticoids use of in the treatment of rheumatoid arthritis
Sovremennaâ revmatologiâ
2,023
cc-by
6,504
Revisiting the question of the safety of glucocorticoids use of in the treatment of rheumatoid arthritis Aronova E.S., Belov B.S., Gridneva G.I. V.A. Nasonova Research Institute of Rheumatology, Moscow 34A, Kashirskoe Shosse, Moscow 115522, Russia Glucocorticoids (GCs) are one of the most commonly used drugs for the treatment of rheumatoid arthritis (RA), the effectiveness of which is beyond doubt. The review considers current literature data on the safety of GCs use, as well as the most common adverse events associated with such therapy. Most authors point to an increased risk of complications with an increase in the daily dose and/or duration of GCs treatment. At the same time, a safe dose of GCs has not been determined. Probably, the optimal tactic is the selection of an individual dose of GCs in each individual case, taking into account the activity of RA and the spectrum of comorbid conditions. In this case, the minimum effective doses and short courses of GCs should be used, regular monitoring of clinical and laboratory parameters should be carried out in order to detect adverse events early. Keywords: glucocorticoids; rheumatoid arthritis; methylprednisolone; pregnancy; complications; adverse events; osteoporosis; mortality. Contact: Evgenia Sergeevna Aronova; eugpozd@mail.ru For reference: Aronova ES, Belov BS, Gridneva GI. Revisiting the question of the safety of glucocorticoids use of in the treatment of rheumatoid arthritis. Sovremennaya Revmatologiya=Modern Rheumatology Journal. 2023;17(3):89–95. DOI: 10.14412/1996-7012-2023-3-89-95 Глюкокортикоиды (ГК) применяются в ревматологии с 1949 г., когда американский врач Филипп Хенч сообщил о выраженном клиническом эффекте «субстанции Е» у больной ревматоидным артритом (РА) [1]. Несмотря на появление инновационных методов лечения, таких как генно-инже- нерные биологические препараты (ГИБП), ГК по-прежнему используются в современных стратегиях лечения РА с целью быстрого подавления активности заболевания и создания терапевтического «моста» с медленно нарастающим эффектом базисных противовоспалительных препаратов (БПВП). При выборе тактики лечения предпочтение отдается схемам с минимальными эффективными дозами ГК и дальнейшим их снижением до полной отмены, если имеется клиническая возможность [2]. В организме синтетические ГК связываются с внутриклеточными стероидными рецепторами и регулируют экспрессию широкого спектра генов на транскрипционном и посттранскрипционном уровнях. Сложный механизм дей- ствия ГК, включающий влияние на различные метаболические пути организма, обусловливает широкий спектр нежелатель- ных явлений (НЯ). О Б З О Р Ы / R E V I E W S О Б З О Р Ы / R E V I E W S Современная ревматология. 2023;17(3):89–95 Аронова Е.С., Белов Б.С., Гриднева Г.И. ФГБНУ «Научно-исследовательский институт ревматологии им. В.А. Насоновой», Москва Россия, 115522, Москва, Каширское шоссе, 34А Глюкокортикоиды (ГК) – одно из наиболее часто применяемых средств для лечения ревматоидного артрита (РА), эффективность которого не подлежит сомнению. В обзоре рассмотрены современные данные литературы о безопасности применения ГК, а также наиболее частые неблагоприятные явления, связанные с такой терапией. Большинство авторов указывают на повышение риска развития осложнений при увеличении суточной дозы и/или продолжительности лечения ГК. В то же время безопасная доза ГК не определена. Вероятно, оптимальной тактикой является подбор индивидуальной дозы ГК в каждом отдельном случае с учетом активности РА и спектра коморбидных состояний. При этом следует использовать минимальные эффективные дозы и короткие курсы ГК, проводить регулярный мониторинг клинических и лабораторных показателей с целью раннего выявления не- благоприятных явлений. Ключевые слова: глюкокортикоиды; ревматоидный артрит; метилпреднизолон; беременность; осложнения; нежелательные явления; остеопороз; смертность. Контакты: Евгения Сергеевна Аронова; eugpozd@mail.ru р р ; gp z Для ссылки: Аронова ЕС, Белов БС, Гриднева ГИ. К вопросу о безопасности применения глюкокортикоидов в терапии ревматоидного артрита. Современная ревматология. 2023;17(3):89–95. DOI: 10.14412/1996-7012-2023-3-89-95 Revisiting the question of the safety of glucocorticoids use of in the treatment of rheumatoid arthritis Aronova E.S., Belov B.S., Gridneva G.I. V.A. Nasonova Research Institute of Rheumatology, Moscow 34A, Kashirskoe Shosse, Moscow 115522, Russia Аронова Е.С., Белов Б.С., Гриднева Г.И. ФГБНУ «Научно-исследовательский институт ревматологии им. В.А. Насоновой», Москва Россия, 115522, Москва, Каширское шоссе, 34А О Б З О Р Ы / R E V I E W S Таблица 1. Риск сердечно-сосудистых НЯ у больных РА при лечении ГК (данные наблюдательных исследований) Table 1. Risk of cardiovascular adverse events in RA patients treated with GCs (data from observational studies) Источник Схема применения ГК СОР (95% ДИ) J.C. Wilson и соавт., 2019 [3] M. Pujades-Rodriguez и соавт., 2020 [4] J.A. Avina-Zubieta и соавт., 2013 [5] A.M. van Sijl и соавт., 2014 [6] G. Ozen и соавт., 2021 [9] A.J. Ocon и соавт., 2021 [10] J.C. Wilson и соавт., 2019 [3] Применение в целом Применение в прошлом Текущее применение Кумулятивная доза: <700 мг ≥700<3500 мг ≥3500<7000 мг ≥7000 мг Применение в целом Текущее применение Текущее применение в дозе: 1–4,9 мг/сут 5,0–14,9 мг/сут 15,0–24,9 мг/сут ≥25 мг/сут Кумулятивная доза: 1–959,9 мг 960–3054,9 мг 3055–7299,9 мг ≥7300 мг Текущее применение Текущее применение в средней дозе 5 мг/сут Кумулятивная доза, равная 1 г Применение когда-либо Недавнее (<1 года назад) применение Текущее применение Длительность применения: ≤5 лет >5 лет Кумулятивная доза: ≤10 г >10 г Применение когда-либо Применение в дозе: <7,5 мг/сут <3 мес <7,5 мг/сут 3 мес ≥7,5 мг/сут <3 мес ≥7,5 мг/сут >3 мес Применение в дозе: 1 – <5 мг/сут ≥5–9 мг/сут ≥10 мг/сут Кумулятивная доза за предыдущие 6 мес: 1–380 мг 381–750 мг 751–1100 мг >1100 мг Применение в целом Применение в прошлом Текущее применение К 1,28 (1,07; 1,52) 1,09 (0,98; 1,21) 1,31 (1,05; 1,64) 1,35 (1,06; 1,72) 1,03 (0,80; 1,32) 1,56 (1,14; 2,14) 1,60 (1,13; 2,28) 1,63 (1,52; 1,73) 2,11 (1,98; 2,25) 1,84 (1,62; 2,10) 2,00 (1,85; 2,15) 2,79 (2,21; 3,51) 4,98 (4,11; 6,03) 1,47 (1,34; 1,61) 1.52 (1,36; 1,68) 1,72 (1,55; 1,19) 1,80 (1,65; 1,97) 1,68 (1,14; 2,47) 1,14 (1,05; 1,24) 1,06 (1,02; 1,10) 0,89 (0,26; 3,09) 1,11 (0,27; 4,53) 1,34 (0,31; 5,88) 0,71 (0,15; 3,27) 1,48 (0,21; 10,45) 0,42 (0,05; 3,30) 1,80 (0,37; 8,74) 1,15 (1,11; 1,19) 0,90 (0,40; 2,01) 0,90 (0,40; 2,01) 1,18 (0,63; 2,20) 1,47 (1,26; 1,71) 1,94 (0,55; 1,59) 1,56 (1,18; 2,05 ) 1,91 (1,31; 2,79 ) 0,86 (0,53; 1,40) 1,20 (0,81; 1,79) 1,43 (1,04; 1,98 ) 2,05 (1,42; 2,94 ) 0,93 (0,85; 1,01) 0,96 (0,91; 1,02) 1,02 (0,90; 1,16) Сердечно-сосудистые НЯ в целом АГ РА, которая также может быть незави- симым фактором риска. Авторы не со- общали об увеличении частоты сер- дечно-сосудистых событий для любой указанной продолжительности приме- нения или кумулятивной дозы ГК. О Б З О Р Ы / R E V I E W S При изолированной оценке частоты воз- никновения артериальной гипертензии (АГ) в большинстве работ отмечается негативное влияние ГК, в частности повышенный риск увеличения арте- риального давления у пациентов с АГ либо возникновение последней de novo [3–12]. T.F. Mebrahtu и соавт. [11] вы- явили связь риска развития АГ с лю- быми кумулятивными дозами ГК (СОР 1,11–1,39; 95% ДИ 1,03–1,48), кроме тех схем лечения, в которых суточная доза не превышала 7,5 мг. По данным R.E. Costello и соавт. [12], повышенный риск АГ сохранялся только в отноше- нии суточных доз ≥7,5 мг и кумуля- тивных доз 5000–9900 мг. Напротив, авторы других исследований не на- блюдали увеличения риска развития АГ на фоне применения ГК как в це- лом, так и различных кумулятивных доз [3], а также при ежедневном приеме ГК по сравнению с альтернирующей схемой [7]. АГ J.C. Wilson и соавт., 2019 [3] Применение в целом Применение в прошлом Текущее применение Кумулятивная доза: <700 мг ≥700<3500 мг ≥3500<7000 мг ≥7000 мг 0,93 (0,85; 1,01) 0,96 (0,91; 1,02) 1,02 (0,90; 1,16) 0,93 (0,82; 1,06) 0,93 (0,82; 1,06) 0,92 (0,76; 1,11) 0,91 (0,74; 1,12) Применение в целом Применение в прошлом Текущее применение Кумулятивная доза: <700 мг ≥700<3500 мг ≥3500<7000 мг ≥7000 мг 0,93 (0,85; 1,01) 0,96 (0,91; 1,02) 1,02 (0,90; 1,16) 0,93 (0,82; 1,06) 0,93 (0,82; 1,06) 0,92 (0,76; 1,11) 0,91 (0,74; 1,12) Сердечно-сосудистые НЯ По данным литературы, у пациентов, получающих ГК, нередко встречаются НЯ со стороны сердечно-сосудистой системы (табл. 1). Так, некоторые авторы сообщают о повы- шенном риске сердечно-сосудистых событий у пациентов, использующих ГК в любых дозах (скорректированное отно- шение рисков, СОР 1,02–4,98; 95% доверительный интервал, ДИ 1,00–6,03) [3–5]. A.M. van Sijl и соавт. [6] указывают на необходимость оценивать вероятность НЯ с учетом активности Современная ревматология. 2023;17(3):89–95 89 Остеопороз и остеопоротические переломы Глюкокортикоидный остеопороз (ОП) – самая частая форма вторичного ОП, связанного с приемом лекарст- венных препаратов [13, 14]. Пациенты любого возраста и пола, длительно (>3 мес) принимающие ГК, относятся к группе высокого риска развития ОП и переломов [7–9, 15, 16]. По данным некоторых исследователей, повышен- ный риск наблюдался только при на- значении высоких суточных или ку- мулятивных доз ГК, независимо от тактики лечения (ежедневный прием или альтернирующая схема) [7, 8]. Од- нако S. Abtahi и соавт. [15] отметили повышенный риск развития остеопо- ротических НЯ при использовании ГК в низких дозах (2,5 мг/сут). В другом исследовании сообщалось о риске при длительном применении ГК (>3 мес) вне связи с суточной дозой [9]. О Б З О Р Ы / R E V I E W S О Б З О Р Ы / R E V I E W S Источник Схема применения ГК СОР (95% ДИ) Примечание. Здесь и в табл. 2, 3: жирным шрифтом выделены статистически значимые ре- зультаты (p<0,05). T.F. Mebrahtu и соавт., 2020 [11] R.E. Costello и соавт., 2021 [12] Варьирующаяся по времени кумулятивная доза: <0–959,9 мг 960–3054,9 мг ≥3055 мг Варьирующаяся по времени суточная доза: 0–4,9 мг 5,0–7,4 мг ≥7,5 мг Недавнее применение Недавняя доза: 0–4,9 мг/сут 5–7,4 мг/сут 7,5–14,9 мг/сут ≥15 мг/сут Кумулятивная доза: 0–2,49 г 2,5–4,99 г 5–9,99 г ≥10 г 1,11 (1,03; 1,21 ) 1,16 (1,05; 1,29) 1,39 (1,30; 1,48) 1,04 (0,89; 1,22) 1,06 (0,93; 1,22) 1,07 (0,96; 1,19) 1,17 (1,10; 1,2 ) 1,10 (0,98; 1,24) 1,07 (0,93; 1,23) 1,18 (1,08; 1,29) 1,36 (1,18; 1,56) 1,00 (0,92; 1,08) 0,99 (0,90; 1,08) 1,12 (1,02; 1,22) 1,07 (0,97; 1,17) >10 мг/сут – 17,7%, у пациентов из базы Optimum – соответственно 4,0; 5,2; 8,1 и 10,6% (p<0,001 по сравнению с отсут- ствием приема ГК во всех случаях). M. Suda и соавт. [7] сравнивали риск развития инфекций при разных схемах назначения ГК. Авторы пришли к выводу, что инфекционные ослож- нения реже встречались у пациентов, получавших ГК по альтернирующей схеме, по сравнению с ежедневным приемом (отношение шансов 0,27; 95% ДИ 0,12–0,63). Однако риск возник- новения серьезных инфекций значимо не различался. Сахарный диабет и гипергликемия Индуцированный ГК сахарный диабет (СД) определяется как аномаль- ное повышение уровня глюкозы в кро- ви, связанное с применением ГК, у па- циента с предшествующим анамнезом СД или без него [28]. Отрицательное влияние ГК на гомеостаз глюкозы слож- но и вызвано множеством факторов, включая повышенную инсулинорезистентность с нарастанием непереносимости глюкозы, снижение массы β-клеток из-за их дисфункции и нарушение подавления глюкогенеза в печени за счет уве- личения в ней инсулинорезистентности. ески значимые ре- ( , ; , ) 99 (0,90; 1,08) 12 (1,02; 1,22) 07 (0,97; 1,17) Примечание. Здесь и в табл. 2, 3: жирным шрифтом выделены статистически значимые ре- зультаты (p<0,05). Примечание. Здесь и в табл. 2, 3: жирным шрифтом выделены статистически значимые ре- зультаты (p<0,05). фекциям и риску реактивации латентно протекающих ви- русных и бактериальных заболеваний, таких как ветряная оспа, опоясывающий лишай, микозы, пиелонефрит, остео- миелит, туберкулез [17], что подтверждается данными как наблюдательных, так и рандомизированных контролируемых исследований (РКИ) [18–25]. Изучение риска развития СД как одного из частых ослож- нений терапии ГК остается актуальным. Большинство авторов указывают, что риск СД возрастает при постоянном приеме ГК, особенно при использовании высоких суточных и ку- мулятивных доз [3, 29, 30], что подтверждается данными РКИ [19, 21, 22, 25, 31, 32]. Как видно из табл. 2, при ежедневном приеме ГК значимо возрастает риск серьезных инфекций, требующих стацио- нарного лечения. Более того, риск инфекционных НЯ уве- личивается с повышением кумулятивной дозы ГК [8, 26, 27]. C. Roubille и соавт. [8] отметили, что значимое повышение частоты серьезных инфекций было связано как с самим фак- том применения ГК в целом (р=0,009), так и с нарастанием их кумулятивной дозы (р=0,024). M.D. George и соавт. [27] проанализировали две крупные национальные базы данных Medicare и Optum. У пациентов из базы Medicare частота серьезных инфекций, требующих госпитализации, в течение года для лиц, не получавших ГК, составила 8,6%, у получавших ГК в дозе ≤5 мг/сут – 11%, 5–10 мг/сут – 14,4%, При изучении схем применения ГК значимых различий по риску развития СД при ежедневном приеме и альтерни- рующей тактике не выявлено [7]. Инфекционные НЯ Иммуносупрессивное действие ГК (подавление активности нейтрофилов и моноцитов, клеточных иммунологи- ческих реакций, лимфопения) приводит к повышенной восприимчивости к ин- Современная ревматология. 2023;17(3):89–95 90 Современная ревматология. 2023;17(3):89–95 О Б З О Р Ы / R E V I E W S В эпидемиологических исследова- ниях, проведенных в Великобритании, выявлена ассоциация низкой массы тела ребенка при рождении со значительным воз- растанием риска развития патологии сердечно-сосудистой системы во взрослом возрасте (до 20% после 45 лет), СД 2-го ти- па и дислипидемии [34]. C.N. Hales и D.J.P. Barker [35] вы- двинули предположение о том, что неблагоприятное воз- действие ГК во время беременности приводит к задержке внутриутробного развития и запуску механизмов адаптации плода. При этом у плода возникают необратимые изменения, вызывающие развитие патологических состояний во взрослом возрасте. внутриглазного давления (ВГД) [14, 22, 33]. В то же время это НЯ чаще встречается при местном, чем при системном применении ГК. Частота индуцированного ГК повышения уровня ВГД на фоне системной терапии неизвестна, поскольку у большинства этих пациентов не проводится измерение ВГД. Индуцированная ГК глаукома у них выявляется либо при обычном офтальмологическом обследовании, либо при прогрессировании поражения глаз до появления нарушений зрения. внутриглазного давления (ВГД) [14, 22, 33]. В то же время это НЯ чаще встречается при местном, чем при системном применении ГК. Частота индуцированного ГК повышения уровня ВГД на фоне системной терапии неизвестна, поскольку у большинства этих пациентов не проводится измерение ВГД. Индуцированная ГК глаукома у них выявляется либо при обычном офтальмологическом обследовании, либо при прогрессировании поражения глаз до появления нарушений зрения. По данным J.C. Wilson и соавт. [3], пациенты с РА, полу- чавшие ГК, имели больший риск развития глаукомы по сравнению с контрольной группой. При этом увеличение средней суточной дозы ГК ассоциировалось с нарастанием риска, однако эта связь носила нелинейный характер и, ве- роятно, зависела от схемы назначения и продолжительности терапии ГК. По мнению авторов, необходимо дальнейшее изучение гормональной офтальмопатии для обоснованной стратификации рисков. В перинатальном периоде применение ГК также может быть небезопасным. Так, по данным K. Palmsten и соавт. [36], ежедневный прием таблетированных ГК может спровоцировать преждевременные роды. Повышенный риск этого осложнения статистически значимо чаще возникал при пероральном при- менении ГК в дозе ≥10 мг/сут после 139-го дня беременности (относительный риск 2,47; 95% ДИ 1,32–4,63). О Б З О Р Ы / R E V I E W S Таблица 3. Риск летального исхода у больных РА при лечении ГК (данные наблюдательных исследований) Table 3. Risk of death in RA patients treated with GCs (data from observational studies) Источник Схема применения ГК СОР (95% ДИ) J.C. Wilson и соавт., 2019 [3] Применение в целом 1,33 (1,19; 1,48) Применение в прошлом 1,03 (0,97; 1,10) Текущее применение 1,80 (1,59; 2,04) Кумулятивная доза: <700 мг 0,90 (0,76; 1,07) ≥700<3500 мг 1,27 (1,10; 1,48) ≥3500<7000 мг 1,42 (1,18; 1,71) ≥7000 мг 2,33 (1,95; 2,77) M. Movahedi и соавт., 2016 [29] Применение в целом 1,97 (1,81; 2,15 ) Текущее применение 1,77 (1,62; 1,93) Текущее применение в дозе: 0–4,9 мг/сут 1,02 (0,87; 1,20) 5,0–7,4 мг/сут 1,44 (1,26; 1,64) 7,5–14,9 мг/сут 2,24 (1,98; 2,54) 15,0–24,9 мг/сут 4,50 (3,61; 5,62) ≥25 мг/сут 11,0 (8,87; 13,6) Кумулятивная доза: >9–959,9 мг 1,60 (1,42; 1,81) 960–3054,9 мг 1,83 (1,62; 2,07) 3055–7299,9 мг 2,11 (1,87; 2,39) ≥7300 мг 3,11 (2,74; 3,52) I. del Rincon и соавт., 2014 [37] Применение в дозе: <5 мг/сут 1,19 (0,74; 1,90) 5–7 мг/сут 1,21 (0,88; 1,66) 8–15 мг/сут 1,78 (1,22; 2,60) ≥15 мг/сут 2,83 (1,41; 5,66) Кумулятивная доза: <9 мг 0,59 (0,36; 0,95) 9–39,9 мг 1,12 (0,76; 1,64) ≥40 мг 1,74 (1,25; 2,44) Применение в соотношении суточная доза/время: <1,98 мг/год 0,48 (0,29; 0,80) 1,98–5,08 мг/год 0,99 (0,67; 1,45) ≥5,08 мг/год 2,11 (1,51; 2,94) Таблица 3. Риск летального исхода у больных РА при лечении ГК (данные наблюдательных исследований) Table 3. Risk of death in RA patients treated with GCs (data from observational studies) последствий является основным пунктом при решении во- проса о назначении ГК беременным. В настоящее время об- суждается влияние этих препаратов на поведенческие и ме- таболические нарушения, а также на раннее развитие ги- пертонической болезни. В эпидемиологических исследова- ниях, проведенных в Великобритании, выявлена ассоциация низкой массы тела ребенка при рождении со значительным воз- растанием риска развития патологии сердечно-сосудистой системы во взрослом возрасте (до 20% после 45 лет), СД 2-го ти- па и дислипидемии [34]. C.N. Hales и D.J.P. Barker [35] вы- двинули предположение о том, что неблагоприятное воз- действие ГК во время беременности приводит к задержке внутриутробного развития и запуску механизмов адаптации плода. При этом у плода возникают необратимые изменения, вызывающие развитие патологических состояний во взрослом возрасте. последствий является основным пунктом при решении во- проса о назначении ГК беременным. В настоящее время об- суждается влияние этих препаратов на поведенческие и ме- таболические нарушения, а также на раннее развитие ги- пертонической болезни. Глаукома По данным как наблюдательных исследований, так и РКИ, на фоне лечения ГК отмечается увеличение уровня ых инфекций у больных РА при лечении ГК (данные наблюдательных исследований) ections in RA patients treated with GCs (data from observational studies) Таблица 2. Риск серьезных инфекций у больных РА при лечении ГК (данные наблюдательных исследований) Table 2. Risk of serious infections in RA patients treated with GCs (data from observational studies) Table 2. Risk of serious infections in RA patients treated with GCs (data from observational studies) Источник Схема применения ГК СОР (95% ДИ) J.C. Wilson и соавт., Применение в целом 1,41 (1,25; 1,59 ) 2019 [3] Применение в прошлом 1,02 (0,95; 1,09) Текущее применение 1,77 (1,54; 2,04) Кумулятивная доза: <700 мг 1,31 (1,11; 1,54) ≥700<3500 мг 1,52 (1,30; 1,77) ≥3500<7000 мг 1,43 (1,16; 1,76) ≥7000 мг 1,56 (1,24; 1,97) W.G. Dixon и соавт., Применение в целом 1,72 (1,53; 1,94) 2012 [26] Текущее применение 1,84 (1,64; 2,06 ) Любое применение за последние 30 дней 2,08 (1,86; 2,33) Любое применение за последние 90 дней 2,26 (2,02; 2,54) Средняя суточная доза за последние 30 дней (при нарастании на каждые 5 мг преднизолона) 1,07 (1,06; 1,08) Средняя суточная доза за последние 90 дней (при нарастании на каждые 5 мг преднизолона) 1,09 (1,08; 1,11) Максимальная доза за последние 30 дней (при нарастании на каждые 5 мг преднизолона) 1,03 (1,02; 1,03) Максимальная доза за последние 90 дней (при нарастании на каждые 5 мг преднизолона) 1,02 (1,01; 1,02) Современная ревматология. 2023;17(3):89–95 91 Современная ревматология. 2023;17(3):89–95 matic drugs: 2022 update. Ann Rheum Dis. 2023 Jan;82(1):3-18. doi: 10.1136/ard-2022- 223356 Другие НЯ Некоторые НЯ встречались реже и зарегистрированы только в РКИ: выпадение волос, общее недомогание, онко- логическая, гематологическая патология, нарушение функции мочевыделительной и пищеварительной систем, неинфек- ционные поражения кожи, депрессивные расстройства, ка- таракта, головная боль и др. [14, 16, 18–24, 31, 38]. В целом риск остеопоротических переломов, развития серьезных инфекционных НЯ и СД у пациентов с РА, полу- чающих ГК, был выше, чем в контрольной группе, и прямо коррелировал с увеличением суточных доз и сроков лечения. В то же время достоверные данные о возможной «безопасной» дозе ГК отсутствуют [42]. В целом риск остеопоротических переломов, развития серьезных инфекционных НЯ и СД у пациентов с РА, полу- чающих ГК, был выше, чем в контрольной группе, и прямо коррелировал с увеличением суточных доз и сроков лечения. В то же время достоверные данные о возможной «безопасной» дозе ГК отсутствуют [42]. Результаты рандомизированных исследований, посвященных изучению общей безопасности длительного применения ГК Что касается применения ГК у беременных, то продол- жение лечения оправданно в тех ситуациях, когда прекращение приема препаратов нецелесообразно для матери по клини- ческим показаниям и возможный риск не превышает по- тенциальную пользу. Во всех случаях следует отдавать пред- почтение преднизолону или метилпреднизолону и исполь- зовать минимальные дозы ГК. В РКИ SEMIRA пациенты со стабильно низкой актив- ностью заболевания, получавшие тоцилизумаб, были рас- пределены на группы: снижение дозы и полная отмена пред- низолона через 16 нед или продолжение приема преднизолона в дозе 5 мг/сут. После 24 нед наблюдения в группе больных, у которых была снижена доза ГК вплоть до отмены, по сравнению с пациентами, продолжавшими принимать пред- низолон, отмечено большее число НЯ как в целом (80 и 64 случая соответственно), так и серьезных НЯ (7 и 4 случая соответственно) [39]. Только в одном из четырех приведенных исследований не отмечалось повышенного риска смертности от разных причин [8], вместе с тем в большинстве работ указано, что риск наступления неблагоприятного исхода возрастал при увеличении суточных и/или кумулятивных доз ГК [3, 29, 37]. Однако следует отметить, что во всех исследованиях не учитывались активность РА и системность заболевания. Безопасность применения преднизолона в дозе 5 мг/сут в сочетании с БПВП у пациентов с активным РА в возрасте ≥65 лет изучалась в течение 2 лет в двойном слепом плаце- бо-контролируемом исследовании GLORIA. Было показано, что НЯ, требовавшие прекращения лечения либо назначения противодействующих мероприятий, значимо чаще наблю- дались в группе преднизолона по сравнению с группой плацебо (60 и 49% соответственно; p=0,02). У пациентов, принимавших преднизолон, зарегистрировано большее число серьезных (26 и 16 соответственно) и прочих (124 и 91 соот- ветственно) инфекционных НЯ. Частота возникновения сер- дечно-сосудистых НЯ также была выше в группе преднизолона по сравнению с группой плацебо [40]. Необходимо еще раз подчеркнуть, что большинство ав- торов признают повышение риска развития НЯ при уве- личении суточной дозы и/или продолжительности лечения. В то же время имеющиеся данные не позволяют дать опре- деление безопасной дозы ГК. Вероятно, оптимальной тактикой является подбор индивидуальной дозы ГК в каждом отдельном случае с учетом активности РА и спектра коморбидных со- стояний. При этом следует назначать минимальные эффек- тивные дозы ГК, использовать короткие курсы и проводить регулярный мониторинг клинических и лабораторных по- казателей с целью раннего выявления НЯ. Заключение при котором прием ГК жизненно необходим и не может быть прекращен, предпочтение должно отдаваться натураль- ным ГК (преднизолон, метилпреднизолон). Большинство исследователей склоняются к мнению об относительной безопасности ГК. Данные о сердечно-сосу- дистых НЯ, возникающих на фоне терапии ГК, неоднозначны и требуют дополнительного изучения для уточнения дозоза- висимого эффекта. Некоторые авторы отмечают повышенный риск НЯ только при использовании ГК в более высоких ку- мулятивных или суточных дозах – от 5 до 7,5 мг/сут в экви- валенте преднизолона [9, 10, 12] с поправкой на активность заболевания [6, 10]. Оценка последнего фактора представляет особый интерес, поскольку оказывает влияние на интер- претацию результатов в тех работах, в которых исследуемая группа представлена пациентами с высокой активностью РА и невозможностью полной отмены ГК вследствие развития обострения. Кроме того, для получения достоверных ре- зультатов необходимы комплаентность пациента и правильная оценка дозы ГК. Так, R.M. Joseph и соавт. [41] полагают, что ошибки, возникающие в наблюдательных исследованиях из-за несоответствия рекомендаций клинической практике, приводят к недостоверным статистическим результатам при изучении эффектов ГК и могут еще больше осложнить ин- терпретацию полученных данных. Смертность В большинстве исследований показано, что у пациентов с РА, получающих ГК, риск смертности от разных причин выше, чем в контрольной группе [3, 8, 14, 15, 22, 29, 37]. В частности, некоторые авторы отмечают, что риск наступ- ления этого неблагоприятного события возрастает при ис- пользовании ГК в дозах ≥5 мг/сут [29, 37] или при увеличении кумулятивных доз [3, 29, 37] (табл.3). C. Roubille и соавт. [8] изучали отдаленный риск развития осложнений, в том числе летального исхода, у больных РА, длительно получавших низкие дозы ГК. По данным этого исследования, риск воз- растал с течением времени, однако авторы указывают на не- обходимость проведения более объективного анализа с по- правкой на активность и вариант течения РА, а также ко- морбидные заболевания. Неблагоприятные исходы беременности Обсуждая использование ГК во время беременности, необходимо принимать во внимание, что их применение в данном периоде влечет за собой не только ближайшие (рож- дение детей с низкой массой тела и тератогенный эффект), но и отдаленные (влияние на заболеваемость во взрослом возрасте) последствия для матери и плода. Оценка отдаленных Следовательно, беременность остается одним из главных ограничений для назначения ГК или продолжения такой терапии. В случае наличия у матери хронического заболевания, Современная ревматология. 2023;17(3):89–95 92 3. Wilson JC, Sarsour K, Gale S, et al. Inci- dence and risk of Glucocorticoid-associated matic drugs: 2022 update. Ann Rheum Dis. 2023 Jan;82(1):3-18. doi: 10.1136/ard-2022- 223356 3. Wilson JC, Sarsour K, Gale S, et al. Inci- dence and risk of Glucocorticoid-associated Современная ревматология. 2023;17(3):89–95 О Б З О Р Ы / R E V I E W S Balasubramanian A, Wade SW, Adler RA, et al. Glucocorticoid exposure and fracture risk in patients with new-onset rheumatoid arthritis. Osteoporos Int. 2016 Nov;27(11): 3239-49. doi: 10.1007/s00198-016-3646-z. Epub 2016 Jun 8. g doi: 10.1093/rheumatology/kes353. Epub 2012 Nov 28. 25. Stouten V, Westhovens R, Pazmino S, et al. Five-year treat-to-target outcomes after met- hotrexate induction therapy with or without other csDMARDs and temporary glucocorti- coids for rheumatoid arthritis in the CareRA trial. Ann Rheum Dis. 2021 Aug;80(8):965-73. doi: 10.1136/annrheumdis-2020-219825. 6. Van Sijl AM, Boers M, Voskuyl AE, et al. Confounding by indication probably distorts the relationship between steroid use and car- diovascular disease in rheumatoid arthritis: 6. Van Sijl AM, Boers M, Voskuyl AE, et al. Confounding by indication probably distorts the relationship between steroid use and car- diovascular disease in rheumatoid arthritis: results from a prospective cohort study. PLoS One. 2014 Jan 30;9(1):e87965. doi: 10.1371/ journal.pone.0087965. eCollection 2014. 6. Van Sijl AM, Boers M, Voskuyl AE, et al. Confounding by indication probably distorts the relationship between steroid use and car- diovascular disease in rheumatoid arthritis: results from a prospective cohort study. PLoS One. 2014 Jan 30;9(1):e87965. doi: 10.1371/ journal.pone.0087965. eCollection 2014. 7. Suda M, Ohde S, Tsuda T, et al. Safety and efficacy of alternate-day corticosteroid trea- tment as adjunctive therapy for rheumatoid arthritis: a comparative study. Clin Rheumatol. 2018 Aug;37(8):2027-34. doi: 10.1007/ s10067-018-4073-7. Epub 2018 Mar 26. 17. Борисова ЕО. Побочные эффекты си- стемной глюкокортикостероидной тера- пии. Практическая пульмонология 2004; (3):14-9. results from a prospective cohort study. PLoS One. 2014 Jan 30;9(1):e87965. doi: 10.1371/ journal.pone.0087965. eCollection 2014. Epub 2021 Apr 2. [Borisova EO. Side effects of systemic gluco- corticosteroid therapy. Prakticheskaya pul'mo- nologiya. 2004;(3):14-9 (In Russ.)]. 26. Dixon WG, Abrahamowicz M, Beauc- hamp ME, et al. Immediate and delayed im- pact of oral glucocorticoid therapy on risk of serious infection in older patients with rheu- matoid arthritis: a nested case-control analy- sis. Ann Rheum Dis. 2012 Jul;71(7):1128-33. doi: 10.1136/annrheumdis-2011-200702. 7. Suda M, Ohde S, Tsuda T, et al. Safety and efficacy of alternate-day corticosteroid trea- tment as adjunctive therapy for rheumatoid arthritis: a comparative study. Clin Rheumatol. 2018 Aug;37(8):2027-34. doi: 10.1007/ s10067-018-4073-7. Epub 2018 Mar 26. 18. Nam JL, Villeneuve E, Hensor EMA, et al. Remission induction comparing inflixi- mab and high-dose intravenous steroid, follo- wed by treat-to-target: a double-blind, rando- mised, controlled trial in new-onset, trea- tment-naive, rheumatoid arthritis (the idea study). Ann Rheum Dis. 2014 Jan;73(1):75-85. doi: 10.1136/annrheumdis-2013-203440. О Б З О Р Ы / R E V I E W S adverse effects in patients with rheumatoid arthritis. Arthritis Care Res (Hoboken). 2019 Apr;71(4):498-511. doi: 10.1002/acr.23611. 4. Pujades-Rodriguez M, Morgan AW, Cub- bon RM, et al. Dose-dependent oral gluco- corticoid cardiovascular risks in people with immune-mediated inflammatory diseases: a population-based cohort study. PLoS Med. 2020 Dec 3;17(12):e1003432. doi: 10.1371/ journal.pmed.1003432. eCollection 2020 Dec. 5. Avina-Zubieta JA, Abrahamowicz M, De Vera MA, et al. Immediate and past cumu- lative effects of oral glucocorticoids on the risk of acute myocardial infarction in rheumatoid arthritis: a population-based study. Rheumato- logy (Oxford). 2013 Jan;52(1):68-75. doi: 10.1093/rheumatology/kes353. Epub 2012 Nov 28 adverse effects in patients with rheumatoid arthritis. Arthritis Care Res (Hoboken). 2019 Apr;71(4):498-511. doi: 10.1002/acr.23611. 4. Pujades-Rodriguez M, Morgan AW, Cub- bon RM, et al. Dose-dependent oral gluco- corticoid cardiovascular risks in people with immune-mediated inflammatory diseases: a population-based cohort study. PLoS Med. 2020 Dec 3;17(12):e1003432. doi: 10.1371/ journal.pmed.1003432. eCollection 2020 Dec 5. Avina-Zubieta JA, Abrahamowicz M, De Vera MA, et al. Immediate and past cumu- lative effects of oral glucocorticoids on the risk of acute myocardial infarction in rheumatoid arthritis: a population-based study. Rheumato- logy (Oxford). 2013 Jan;52(1):68-75. doi: 10.1093/rheumatology/kes353. Epub 2012 Nov 28 14. Grossman JM, Gordon R, Ranganath VK, et al. American College of Rheumatology 2010 Recommendations for the Prevention and Treatment of Glucocorticoid-Induced Osteoporosis. Arthritis Care Res (Hoboken). 2010 Nov;62(11):1515-26. doi: 10.1002/acr. 20295. Epub 2010 Jul 26. 23. Verschueren P, De Cock D, Corluy L, et al. Methotrexate in combination with other DMARDs is not superior to methotrexate alo- ne for remission induction with moderate-to- high-dose glucocorticoid bridging in early rheumatoid arthritis after 16 weeks of treat- ment: the CareRA trial. Ann Rheum Dis. 2015 Jan;74(1):27-34. doi: 10.1136/annrheumdis- 2014-205489. Epub 2014 Oct 30. 2010 Recommendations for the Prevention and Treatment of Glucocorticoid-Induced 2010 Nov;62(11):1515-26. doi: 10.1002/acr. 20295. Epub 2010 Jul 26. 15. Abtahi S, Driessen JHM, Burden AM, et al. Concomitant use of oral glucocorticoids and proton pump inhibitors and risk of osteo- porotic fractures among patients with rheu- matoid arthritis: a population-based cohort study. Ann Rheum Dis. 2021 Apr;80(4):423-31. doi: 10.1136/annrheumdis-2020-218758. Epub 2020 Dec 11. 24. Verschueren P, De Cock D, Corluy L, et al. Patients lacking classical poor prognostic mar- kers might also benefit from a step-down glu- cocorticoid bridging scheme in early rheuma- toid arthritis: week 16 results from the rando- mized multicenter CareRA trial. Arthritis Res Ther. 2015 Apr 9;17(1):97. doi: 10.1186/ s13075-015-0611-8. 16. О Б З О Р Ы / R E V I E W S Epub 2013 Aug 2. 8. Roubille C, Coffy A, Rincheval N, et al. Ten-year analysis of the risk of severe outco- mes related to low-dose glucocorticoids in ear- ly rheumatoid arthritis. Rheumatology (Oxford). 2021 Aug 2;60(8):3738-46. doi: 10.1093/ rheumatology/keaa850. Epub 2012 Jan 12. 27. George MD, Baker JF, Winthrop K, et al. Risk for serious infection with low-dose glu- cocorticoids in patients with rheumatoid arth- ritis: a Cohort study. Ann Intern Med. 2020 Dec 1;173(11):870-8. doi: 10.7326/M20- 1594. Epub 2020 Sep 22. 19. De Jong PH, Hazes JM, Barendregt PJ, et al. Induction therapy with a combination of DMARDs is better than methotrexate mono- therapy: first results of the tREACH trial. Ann Rheum Dis. 2013 Jan;72(1):72-8. doi: 10.1136/ annrheumdis-2011-201162. Epub 2012 Jun 7. 20. Verschueren P, De Cock D, Corluy L, et al. Effectiveness of methotrexate with step- down glucocorticoid remission induction (cobra slim) versus other intensive treatment strategies for early rheumatoid arthritis in a treat-to-target approach: 1-year results of Ca- reRA, a randomised pragmatic open-label su- periority trial. Ann Rheum Dis. 2017 Mar; 76(3):511-20. doi: 10.1136/annrheumdis- 2016-209212. Epub 2016 Jul 18. 9. Ozen G, Pedro S, Michaud K. The risk of cardiovascular events associated with disease- modifying antirheumatic drugs in rheumatoid arthritis. J Rheumatol. 2021 May;48(5): 648-55. doi: 10.3899/jrheum.200265. 28. Сабиров ИС, Муркамилов ИТ, Фо- мин ВВ и др. Стероидно- индуцирован- ный диабет: современный взгляд на про- блему и возможности терапии. The Scien- tific Heritage.2021;(70): 35-41. 28. Сабиров ИС, Муркамилов ИТ, Фо- мин ВВ и др. Стероидно- индуцирован- ный диабет: современный взгляд на про- блему и возможности терапии. The Scien- tific Heritage.2021;(70): 35-41. [Sabirov IS, Murkamilov IN, Fomin VV, et al. Steroid-induced diabetes: a modern perspecti- ve on the problem and treatment options. The Scientific Heritage.2021;70: 35-41 (In Russ.)]. 29. Movahedi M, Beauchamp M-E, Abraha- mowicz M, et al. Risk of incident diabetes mellitus associated with the dosage and dura- tion of oral glucocorticoid therapy in patients with rheumatoid arthritis. Arthritis Rheumatol. 2016 May;68(5):1089-98. doi: 10.1002/ art.39537. Epub 2020 Aug 15. 10. Ocon AJ, Reed G, Pappas DA, et al. Short-term dose and duration-dependent glu- cocorticoid risk for cardiovascular events in glucocorticoid-naive patients with rheumatoid arthritis. Ann Rheum Dis. 2021 Dec;80(12): 1522-9. doi: 10.1136/annrheumdis-2021- 220577. Epub 2021 Jul 2. [Sabirov IS, Murkamilov IN, Fomin VV, et al. Steroid-induced diabetes: a modern perspecti- ve on the problem and treatment options. The Scientific Heritage.2021;70: 35-41 (In Russ.)]. 6736(05)74374-9. 2. Smolen JS, Landewй RBM, Bergstra SA, et al. EULAR recommendations for the ma- nagement of rheumatoid arthritis with synthe- tic and biological disease-modifying antirheu- 1. Raju TN. The Nobel chronicles. 1950: Ed- ward Calvin Kendall (1886–1972); Philip Showalter Hench (1896–1965); and Tadeus Reichstein (1897–1996). Lancet. 1999 Apr 17;353(9161):1370. doi: 10.1016/s0140- Современная ревматология. 2023;17(3):89–95 93 О Б З О Р Ы / R E V I E W S [Sabirov IS, Murkamilov IN, Fomin VV, et al. Steroid-induced diabetes: a modern perspecti- ve on the problem and treatment options. The Scientific Heritage.2021;70: 35-41 (In Russ.)]. 29. Movahedi M, Beauchamp M-E, Abraha- mowicz M, et al. Risk of incident diabetes mellitus associated with the dosage and dura- tion of oral glucocorticoid therapy in patients with rheumatoid arthritis. Arthritis Rheumatol. 2016 May;68(5):1089-98. doi: 10.1002/ art.39537. 29. Movahedi M, Beauchamp M-E, Abraha- mowicz M, et al. Risk of incident diabetes 11. Mebrahtu TF, Morgan AW, West RM, et al. Oral glucocorticoids and incidence of hypertension in people with chronic inflam- matory diseases: a population-based cohort study. CMAJ. 2020 Mar 23;192(12):E295- E301. doi: 10.1503/cmaj.191012. 2016 209212. Epub 2016 Jul 18. 21. Den Uyl D, ter Wee M, Boers M, et al. A non-inferiority trial of an attenuated combi- nation strategy (’COBRA-light’) compared to the original cobra strategy: clinical results af- ter 26 weeks. Ann Rheum Dis. 2014 Jun;73(6): 1071-8. doi: 10.1136/annrheumdis-2012- 202818. Epub 2013 Apr 19. 22. Konijn NPC, van Tuyl LHD, Boers M, et al. Similar efficacy and safety of initial COBRA-light and COBRA therapy in rheu- matoid arthritis: 4-year results from the COBRA-light trial. Rheumatology (Oxford). 2017 Sep 1;56(9):1586-1596. doi: 10.1093/ rheumatology/ kex223. 21. Den Uyl D, ter Wee M, Boers M, et al. A non-inferiority trial of an attenuated combi- nation strategy (’COBRA-light’) compared to the original cobra strategy: clinical results af- ter 26 weeks. Ann Rheum Dis. 2014 Jun;73(6): 1071-8. doi: 10.1136/annrheumdis-2012- 202818. Epub 2013 Apr 19. 30. Wu J, Mackie SL, Pujades-Rodriguez M. Glucocorticoid dose-dependent risk of type 2 diabetes in six immune-mediated inflamma- tory diseases: a population-based cohort ana- lysis. BMJ Open Diabetes Res Care. 2020 Jul;8 (1):e001220. doi: 10.1136/bmjdrc-2020- 001220. 12. Costello RE, Yimer BB, Roads P, et al. Glucocorticoid use is associated with an in- creased risk of hypertension. Rheumatology (Oxford). 2021 Jan 5;60(1):132-9. 22. Konijn NPC, van Tuyl LHD, Boers M, et al. Similar efficacy and safety of initial doi: 10.1093/rheumatology/keaa209. doi: 10.1093/rheumatology/keaa209. 13. Joint IOF-ECTS GIO Guidelines Wor- king Group. A framework for the development of guidelines for the management of glucocor- ticoid-induced osteoporosis. Osteoporos Int. 2012 Sep;23(9):2257-76. doi: 10.1007/s00198- 012-1958-1. Epub 2012 Mar 21. 31. Ding CZ, Yao Y, Feng XB, et al. Clinical analysis of Chinese patients with rheumatoid arthritis treated with leflunomide and metho- trexate combined with different dosages of glu- cocorticoid. Curr Ther Res Clin Exp. О Б З О Р Ы / R E V I E W S Вестник репродуктивного здоровья. 2008; (1-2):7-17. [M l' i h k GA S i h TV F d VV 39. Burmester GR, Buttgereit F, Bernasconi C, et al. Continuing versus tapering glucocorti- coids after achievement of low disease activity or remission in rheumatoid arthritis (SEMIRA): a double-blind, multicentre, randomised cont- rolled trial. Lancet. 2020 Jul 25;396(10246): 267-76. doi: 10.1016/S0140-6736(20)30636-X. 40. Boers M, Hartman L, Opris-Belinski D, et al. Low dose, add-on prednisolone in pati- кокортикоидов во время беременности. Вестник репродуктивного здоровья. 2008; (1-2):7-17. [Mel'nichenko GA, Semicheva TV, Fadeev VV, Chebotnikova TV. The use of glucocorticoids during pregnancy. Vestnik reproduktivnogo zdo- rov'ya. 2008;(1-2):7-17. (In Russ.)]. [Mel'nichenko GA, Semicheva TV, Fadeev VV, Chebotnikova TV. The use of glucocorticoids during pregnancy. Vestnik reproduktivnogo zdo- rov'ya. 2008;(1-2):7-17. (In Russ.)]. 35. Hales CN, Barker DJP. Type 2 (non-insu- lin-dependent) diabetes mellitus: the thrifty О Б З О Р Ы / R E V I E W S 73(4-5):123-33. doi: 10.1016/j.curtheres.2012. 06.001. ents with rheumatoid arthritis aged 65+: the pragmatic randomised, double-blind pla- cebo-controlled GLORIA trial. Ann Rheum Dis. 2022 Jul;81(7):925-36. doi: 10.1136/ annrheumdis-2021-221957. Epub 2022 May 31. 41. Joseph RM, van Staa TP, Lunt M, et al. Exposure measurement error when assessing current glucocorticoid use using UK primary care electronic prescription data. Pharmaco- epidemiol Drug Saf. 2019 Feb;28(2):179-86. doi: 10.1002/pds.4649. Epub 2018 Sep 28. 42. Bergstra SA, Sepriano A, Kerschbaumer A, et al. Efficacy, duration of use and safety of glucocorticoids: a systematic literature review informing the 2022 update of the EULAR re- commendations for the management of rheu- matoid arthritis. Ann Rheum Dis. 2023 Jan; 82(1):81-94. doi: 10.1136/ard-2022-223358. phenotype hypothesis. Diabetologia. 1992 Jul; 35(7):595-601. doi: 10.1007/BF00400248. 36. Palmsten K, Bandoli G, Vazquez-Benitez G, et al. Oral corticosteroid use during pregnancy and risk of preterm birth. Rheumatology (Oxford). 2020 Jun 1;59(6):1262-71. doi: 10.1093/ rheumatology/kez405. phenotype hypothesis. Diabetologia. 1992 Jul; 35(7):595-601. doi: 10.1007/BF00400248. 36. Palmsten K, Bandoli G, Vazquez-Benitez G, et al. Oral corticosteroid use during pregnancy and risk of preterm birth. Rheumatology (Oxford). 32. Sadra V, Khabbazi A, Kolahi S, et al. Ran- domized double-blind study of the effect of dexamethasone and methylprednisolone pulse in the control of rheumatoid arthritis flare-up: a preliminary study. Int J Rheum Dis. 2014 May;17(4):389-93. doi: 10.1111/1756-185X. 12278. Epub 2014 Jan 9. 37. Del Rincon I, Battafarano DF, Restrepo JF, et al. Glucocorticoid dose thresholds associa- ted with all-cause and cardiovascular mortali- ty in rheumatoid arthritis. Arthritis Rheumatol. 2014 Feb;66(2):264-72. doi: 10.1002/art.38210. 38. Buttgereit F, Mehta D, Kirwan J, et al. Low-dose prednisone chronotherapy for rheumatoid arthritis: a randomised clinical trial (CAPRA-2). Ann Rheum Dis. 2013 Feb; 72(2):204-10. doi: 10.1136/annrheumdis- 2011-201067. Epub 2012 May 5. 33. Bakker MF, Jacobs JWG, Welsing PMJ, et al. Low-dose prednisone inclusion in a methotrexate-based, tight control strategy for early rheumatoid arthritis: a randomized trial. Ann Intern Med. 2012 Mar 6;156(5):329-39. doi: 10.7326/0003-4819-156-5-201203060- 00004. 34. Мельниченко ГА, Семичева ТВ, Фаде- ев ВВ, Чеботникова ТВ. Применение глю- кокортикоидов во время беременности. Вестник репродуктивного здоровья. 2008; (1-2):7-17. [Mel'nichenko GA, Semicheva TV, Fadeev VV, Chebotnikova TV. The use of glucocorticoids during pregnancy. Vestnik reproduktivnogo zdo- rov'ya. 2008;(1-2):7-17. (In Russ.)]. 35. Hales CN, Barker DJP. Type 2 (non-insu- 34. Мельниченко ГА, Семичева ТВ, Фаде- ев ВВ, Чеботникова ТВ. Применение глю- кокортикоидов во время беременности. 34. Мельниченко ГА, Семичева ТВ, Фаде- ев ВВ, Чеботникова ТВ. Применение глю- кокортикоидов во время беременности. О Б З О Р Ы / R E V I E W S 2012 Sep; COBRA-light and COBRA therapy in rheu- matoid arthritis: 4-year results from the Современная ревматология. 2023;17(3):89–95 94 Аронова Е.С. https://orcid.org/0000-0002-1833-5357 Белов Б.С. https://orcid.org/0000-0001-7091-2054 Гриднева Г.И. https://orcid.org/0000-0002-0928-3911 ents with rheumatoid arthritis aged 65+: the pragmatic randomised, double-blind pla- cebo-controlled GLORIA trial. Ann Rheum Dis. 2022 Jul;81(7):925-36. doi: 10.1136/ annrheumdis-2021-221957. Epub 2022 May 31. 41. Joseph RM, van Staa TP, Lunt M, et al. Exposure measurement error when assessing current glucocorticoid use using UK primary care electronic prescription data. Pharmaco- epidemiol Drug Saf. 2019 Feb;28(2):179-86. doi: 10.1002/pds.4649. Epub 2018 Sep 28. 42. Bergstra SA, Sepriano A, Kerschbaumer A, et al. Efficacy, duration of use and safety of glucocorticoids: a systematic literature review informing the 2022 update of the EULAR re- commendations for the management of rheu- matoid arthritis. Ann Rheum Dis. 2023 Jan; 82(1):81-94. doi: 10.1136/ard-2022-223358. Современная ревматология. 2023;17(3):89–95 Поступила/отрецензирована/принята к печати Received/Reviewed/Accepted 12.01.2023/15.03.2023/20.03.2023 Заявление о конфликте интересов/Conflict of Interest Statement ление о конфликте интересов/Conflict of Interest Statement ья подготовлена в рамках государственного задания по теме №1021051503137-7. / Статья подготовлена в рамках государственного задания по теме №1021051503137-7. у Исследование не имело спонсорской поддержки. Конфликт интересов отсутствует. Авторы несут полную ответственность за предоставление окончательной версии рукописи в печать. Все авторы принимали участие в разработке концепции статьи и написании рукописи. Окончательная версия рукописи была одобрена всеми авторами. The investigation has been conducted within scientific topic №1021051503137-7. The investigation has not been sponsored. There are no conflicts of interest. The authors are solely responsible for submitting the final version of the manuscript for publication. All the authors have participated in developing the concept of the article and in writing the manuscript. The final version of the manuscript has been approved by all the authors. Аронова Е.С. https://orcid.org/0000-0002-1833-5357 Белов Б.С. https://orcid.org/0000-0001-7091-2054 Гриднева Г.И. https://orcid.org/0000-0002-0928-3911 Современная ревматология. 2023;17(3):89–95 95
https://openalex.org/W2031810954
https://zenodo.org/records/2014794/files/article.pdf
English
null
SOME REMARKS UPON " INTERNAL DERANGEMENT " OF THE KNEE-JOINT:
Lancet
1,902
public-domain
3,721
731 We know now that the trouble within the joint is by no means always of the nature mentioned above. larynx very great. I will now mention some things which I think ought to be considered when we witness a laryngeal crisis of a tabetic. The limbs are, it is said, convulsed in some severe attacks. But perhaps in some of these attacks there occur only writh- ings of the limbs as an indirect consequence of arrest of respiration ; these writhings, being movements proper, ought not to be confounded with convulsion proper, as I believe is done in accounts of some slight cerebral paroxysms commonly called epileptic. If the limbs were the subject of such an intense discharge as produces convulsion there could not be movements even so little elaborate as those of writhing : there would be tonic or clonic spasm of them and not movements properly so-called. In any case of fits, epileptic or of any other sort, we ought to note the condition of the chest and whether or not the fit begins tkoracically- begins by fixation of the chest walls or by mere cessation of respiration-with subsequent involvement of the limbs. Variety of lesion.-The fact is that a considerable variety of lesions in the knee-joint may all cause the symptoms common to cases known as examples of internal derangement. Moreover, some cases present these symptoms and improve- after the joint is opened, although no lesion at all is discerned. This seems just analogous to what happens in certain cases. of pain and discomfort in the abdomen in which laparotomy leads to a cure, and presumably the effect upon the two. large serous surfaces, the peritoneum and the lining of the- knee-joint, when they are exposed is similar and at present. equally obscure. In the case of the abdomen such instances. are most commonly seen in connexion with tuberculous peritonitis. Possibly similar cases in the knee-joint may represent very early instances of tuberculous affection of the synovial membrane. Or, for want of more definite know- ledge, perhaps we may explain some of these cases of symptoms without apparent lesion but with excess of fluid in the joint, in the same way that we veil our ignorance in the matter of hydrocele and say that the proper balance between secretion and absorption is upset in the joints in question. 731 731 731 731 The same general principle applies, I think, to all cases oi insanity except total dementia and to cases of partial aphasia The same general principle applies, I think, to all cases oi insanity except total dementia and to cases of partial aphasia attacks of laryngismus stridulus in rickety infants are, I have tried to show (Brain, April, 1886), lowest level fits caused by very great stimulation of the Respiratory centre by supervenous blood. n. aphasia. I have not forgotten that laryngeal crises occur in some tabetic patients who have no discoverable paralysis of the abductor muscles of the vocal cords (Semon) ; to these lJ, the foregoing hypothesis does not, it may well be said, apply. But there may in them be the "irritative" state of the sensory, medulla, elements suggested. And once more I urge, having regard to the effects of experimental stimulation of the superior laryngeal nerve, that the nisus of the Respiratory centre when it is stimulated from the "irritative’ sensory elements representing the laryngeal mucous mem- brane, is for expiration, for narrowing of the glottis. I suggest that in the cases where the abductor muscles are not discoverably paralysed the" irritative state " of the medulla sensory elements representing part of the mucous membrane of the larynx is very great. p SOME REMARKS UPON " INTERNAL DE- RANGEMENT " OF THE KNEE-JOINT: BASED UPON 59 CASES IN WHICH OPERATION WAS PERFORMED. BY HERBERT W. ALLINGHAM, F.R.C.S. ENG., SURGEON-IN-ORDINARY TO H.R.H. THE PRINCE OF WALES; SURGEON TO THE HOUSEHOLD OF HIS MAJESTY THE KING; ASSISTANT SURGEON TO ST. GEORGE’S HOSPITAL. p SOME REMARKS UPON " INTERNAL DE- RANGEMENT " OF THE KNEE-JOINT: BASED UPON 59 CASES IN WHICH OPERATION WAS PERFORMED. BY HERBERT W. ALLINGHAM, F.R.C.S. ENG., SURGEON-IN-ORDINARY TO H.R.H. THE PRINCE OF WALES; SURGEON TO THE HOUSEHOLD OF HIS MAJESTY THE KING; ASSISTANT SURGEON TO ST. GEORGE’S HOSPITAL. " INTERNAL derangement of the knee-joint" is a term that was used originally to describe cases having a well-defined series of symptoms, in which the lesion was held to be always damage or displacement of one of the semilunar cartilages. Since the publication of my book on this subject in 1889 I have met with a variety of cases in point which it may now be of use to put forward. are owing to suspension of cerebral influence ; in infants the Rolandic cells for the small muscles of the hand will, I submit, not be well developed, whilst those for the larger muscles moving the hand and fingers will be more developed, hence carpo-pedal contractions. In older people the cells for the small as well as the large muscles will be- developed, hence tetany when the function of these cells is annulled. Moreover in infants the pyramidal tract is ill-developed. 731 If then we continue to use the term " internal derangement," we must realise that in so describ- ing a case we do not infer that the lesion is necessarily one of a semilunar cartilage. Having now had the opportunity of operating upon a considerable number of such cases, it seemed to me that to put them on record with a brief state- ment of the actual lesion present in each case might serve a. useful purpose by illustrating the various conditions that may produce similar symptoms and demonstrating the consequent difficulty of accurate diagnosis in these cases before the joint is opened. It must not therefore be assumed that I am advocating immediate and indiscriminate opening of the knee-joint in all these cases. The very opposite is, in fact, my aim, one of the main objects of this paper being to lay stress upon what I consider the proper treatment of all these cases if they are seen when the trouble has first arisen. respiration with subsequent I here state the hypothesis regarding patients rendered hemiplegic in infancy by a cerebral lesion and who later in life become subject to fits, that in some of the cases the fits are not cerebral but bulbar (lowest level fits). Certainly very careful attention should be given to the paroxysms in this clinical group of "epilepsies" to determine whether the onset is thoracic, more generally by convulsion of the bilaterally acting muscles (trunk) or by convulsion of the limbs. (So, too, for observations of fits in cases of general paralysis.) paralysis.) I think it possible-this is merely a speculative inference from reports of friends of patients-that there may occur in some stage of a laryngeal crisis terminal tonic spasms like those of tetany. Such terminal tonic spasms are certainly, in no kind of malady, parts of convulsion proper.]5 We should note whether or not the patient perspires very much during the crisis (supervenosity would account for sweating). If he be deeply asphyxiated, we should note whether his knee-jerks are present or absent (no doubt, however, in nearly every case they will be absent at all times, as part of the tabetic symptomatology). Is there passage of urine or fasces in or just after severe seizures ? 14 It is possible that in these cases there may be (to borrow a term from ophthalmology) a slight degree of weakness, which we may call " insufficiency," of the abductors of the vocal cords. 15 I have, however, been said to regard carpo-pedal contractions as rudimentary epileptiform convulsions. I admit that I have (Brain, vol. ix., p. 17, April, 1886) written so carelessly that it was naturally inferrible that I thought so. I there wrote " cargo-pedal contractions as well as the severer universal convulsions." 16 I have suggested that both carpo-pedal contractions and tetany 14 It is possible that in these cases there may be (to borrow a term from ophthalmology) a slight degree of weakness, which we may call " insufficiency," of the abductors of the vocal cords. insufficiency, t e abducto s o t e vocal cords. 15 I have, however, been said to regard carpo-pedal contractions as rudimentary epileptiform convulsions. I admit that I have (Brain, vol. ix., p. 17, April, 1886) written so carelessly that it was naturally inferrible that I thought so. I there wrote " cargo-pedal contractions as well as the severer universal convulsions." u e sa co u s o s. 16 I have suggested that both carpo-pedal contractions and tetany 731 The passage of fseces might be a result of great venosity and thus not a part of the fit proper, or at least a very indirect result of the nervous discharges in the fit. discharges In some cases of whooping-cough there occur convulsions which are, I suppose, caused by highly supervenous blood (consequent on severe cough-paroxysms) over-stimulating the respiratory centre. Whether, when in these cases there is considerable supervenosity but no convulsion, there are ever carpo-pedal contractions, I do not know. I believe that these persisting terminal tonic spasms are owing, indirectly, to suspension of the influence of certain cells of the Rolandic cortex, and that when they occur in rickety children in association with, in the intervals of, attacks of laryngismus stridulus, and in some other cases, the cerebral influence is suspended owing to supervenosity annulling the function of small cells of the hand area of the Rolandic cortex. The spasm itself is owing directly to permitted over-action of cells of anterior horns for the hands and, as I think, also by non-antagonised cerebellar influence upon those horns, cerebral influence on them being suspended.]6 The they Early treatment.-Suppose a young man to have received a blow upon, or to have twisted, one of his knees. The joint. became fixed for a time, very painful, and rapidly swelled. Perhaps something was felt to move or to lock in the joint. Possibly even something was felt protruding from the joint and on pushing this in the joint could be moved again. The- treatment to be pursued is briefly this : splinting for a period varying from one to three weeks, followed by massage, passive movements, and properly selected exercises for the joint. The rationale of such treatment is that first of all rest is given so that the damage may heal, the split cartilage- join, the displaced one grow firmly fixed again, the torn alar ligament mend, &c., and then the slackness of ligaments and muscles consequent upon the distension and the disuse of the joint is remedied by massage and movement. Thus the supporting structures of the joint being thoroughly restored al L 2 732 ’ A TABLE OF 59 CASES IN WHICH THE KNEE-JOINT WAS OPENED FOR SYMPTOMS OF INTERNAL DERANGEMENT. 733 A TABLE OF 59 CASES IN WHICH THE KNEE-JOINT WAS OPENED FOR SYMPTOMS OF INTERNAL DERANGEMENT&mdash; MM/MJ. 731 Such a line of treatment is that which has been followed for years with the greatest success by Dr. Wharton P. Hood. In a certain percentage of cases it will fail and the trouble will recur. These must be operated upon. Similarly chronic cases that have already had numerous attacks when first seen require operation. Those cases, also, in which a quite loose body can be felt to move to widely different positions in the joint and those in which a loosened cartilage protrudes from the joint will not improve without operation. In all other cases such a course of treatment as has been sketched shoul be thoroughly tried before the question of operation is considered. g o i c i d c p y body In the cases of damage to a semilunar cartilage there are generally these features : (1) distinct history of injury originating the trouble ; (2) a well-defined site of pain on the inner or outer side of the knee according to the cartilage- damaged ; (3) no foreign body palpable ; and (4) no creak- ing in the joint. These same features also characterise- generally cases in which the lesion affects an alar ligament, or consists in localised hypertrophy of synovial membrane. &eacute; ( g &eacute; ( e r e n yp p y y In the cases where a loose body is present the symptoms, again, may be exactly similar to those caused by semilunar damage (Case 59). Sometimes, however, the body will be able to be moved into widely separated portions of the joint (Case 45) and felt in its different situations. Similarly a very loose semilunar cartilage may sometimes be diagnosed with certainty through its being protruded at the side of the joint. Such a cartilage, however, may be most confusingly imitated by an excrescence due to rheumatoid arthritis. The operation.-A few particulars concerning the operation itself may be mentioned. A vertical incision of about three inches, one inch to the inner side of the patella and going down to an inch below the head of the tibia is the incision which I choose for opening the joint, unless, of course, there is special indication, as the presence of a foreign body, for incising at some other situation. 731 An advantage over the transverse incision is that by the vertical cut, the extensio of the vasti on to the tibia, an important supporting struc- ture is not divided ; moreover, the vertical incision is easily prolonged upwards when this is necessary for examination of the large synovial pouch about the patella. Care must be taken to clip and to tie every bleeding point, and the joint is to be literally squeezed dry before it is closed. The joint should not be washed out with antiseptics and no drainage should be employed. One exception to the rule of not washing out is presented by such cases as Case 35. Here a loose body, the presence of which had been determined with certainty before the joint was opened, could not afterwards be found in spite of the freest digital searching. On flushing the joint with boiled water the body literally floated out. Such a manoeuvre may occasionally be neces- sary and has led to no harm. In another case (Case 45) even this proceeding failed to discover a loose body abont the- presence of which there was absolutely no doubt. It became fixed presumably in the posterior part of the joint behind the crucial ligaments and gave no further trouble. The course of this case to a perfect cure seems to prove conclusively that rest, exercise, and massage may lead to the remedying of a condition within the knee-joint which might well have seemed incurable except by operation. It is, however, possible that the body became fixed owing to influences which would not have come into play had the joint not been opened. 5 : by -Viewed generally these cases show that operation for internal derangement of the knee-joint is in most cases highly satisfactory. There are generally quick recovery and no recurrence of the trouble. In one case after several years of freedom another operation was required and a large number of loose bodies were removed from the joint. I am, however, very anxious not to minimise the gravity of the operation, which I hold to be considerable, and would lay stress upcn Cases 22 and 51, which illustrate failure such as is probably inseparable from a certain proportion of all serious operations. BY C. A. HINGSTON, M.D., B.Sc. LOND., CONSULTING PHYSICIAN TO THE PLYMOUTH PUBLIC DISPENSARY ; AND W. H. B. STODDART, M.D., M.R.C.P. LOND., ASSISTANT MEDICAL OFFICER TO BETHLEM AND BRIDEWELL. ROYAL HOSPITALS. opened. It is hardly necessary to insist upon the strictest asepsis, which must, of course, be observed, but I cannot agree with Mr A. E. Barker that the finger should not be used within the joint. Without such use I cannot believe it possible in some cases properly to estimate or to remedy the fault that may be present. It is necessary in some cases to examine with the finger every part of the joint that can be reached. Even then a small loose body may escape detection as in Case 45 WE have thought it desirable to place the following case on record, partly on account of the rarity of the condition and partly on account of the fact that our case presented some unusual features. The patient, aged 71 years, was a contractor of Plymouth. He had lived a life of considerable mental and physical activity for about 30 years and during this time his health was above the average. At the age of 60 years he relin- quished the greater part of his work ; he became easily excited and fatigued and his business capacity obviously lessened. At the age of 63 years he began to suffer severe neuralgic pains in various parts of his body and limbs, for which there was no apparent cause. Finally, however, these pains became limited to the left leg and were sufficiently severe to confine him to bed for many weeks. At this time the urine was found to be of high specific gravity and to contain a considerable quantity of sugar. The glycosuria never quite disappeared, but it became so inconsiderab as to be scarcely appreciable by the ordinary tests. My experience favours removing loose semilunar carti- lages rather than fixing them, and the same applies to torn or hypertrophied alar ligaments or local hypertrophy of synovial membrane. synovial In closing the wound the sutures are to be passed so as to include the cut synovial membrane. synovial After-treatment.-The splints are to be taken off within a week. Passive movements are begun as soon as the skin wound is healed, and these are combined with massage. If in spite of exercises and massage there appears some stiff- ness of the joint it must be freely moved under an anses- thetic and then daily massaged and freely moved to prevent stiffness from reappearing. reappearing. 731 Even short of the unhappy event of permanent stiffness, considerable trouble may be caused in convalescence by profuse secretion of synovia (Case 37) and by a tendency to stiffness of the joint (Case 23). Such cases cause great anxiety and require the most early careful and persistent treatment if a perfect joint is to be finally secured. persistent perfect joint finally Grosvenor-street, W. pe s ste t Grosvenor-street, W. 731 734 ’ 734 ’ A TABLE OF 59 CASES IN WHICH THE KNEE-JOINT WAS OPENED FOR SYMPTOMS OF INTERNAL DERANGEMENT-( (continued). 735 A TABLE OF 59 CASES IN WHICH THE KNEE-JOINT WAS OPENED FOR SYMPTOMS OF INTERNAL DERANGEMENT&mdash; (" 6’OMMtM). A TABLE OF 59 CASES IN WHICH THE KNEE-JOINT WAS OPENED FOR SYMPTOMS OF INTERNAL DERANGEMENT- (Continued). CASE 51.-There was pain in the joint from the day after the operation. The temperature was never above 100&deg; F., but the condition of the joint was unsatisfactory. There were a pulpy condition of the synovial membrane and cellulitis superficially. The stitches were removed and the joint was washed out. There were always pain and discharge of synovial-fluid. The knee remained swollen and painful and was opened to let out th& discharge oh two occasions. There was not much discharge of synovia, but there was constant pain. Finally the joint quieted down but it remained absolutely ankylosed. 737 to their natural strength, when the joint is again fully used in the ordinary way there will be no tendency to a re- currence of the accident. Such a line of treatment is that which has been followed for years with the greatest success by Dr. Wharton P. Hood. In a certain percentage of cases it will fail and the trouble will recur. These must be operated upon. Similarly chronic cases that have already had numerous attacks when first seen require operation. Those cases, also, in which a quite loose body can be felt to move to widely different positions in the joint and those in which a loosened cartilage protrudes from the joint will not improve without operation. In all other cases such a course of treatment as has been sketched shoul be thoroughly tried before the question of operation is considered. p d n d e a points which help us in differentiating between thesel con- ditions, although it will happen not seldom that symptoms- most strongly suggestive of one lesion will prove to be due to another. Thus Case 59 was apparently a typical example of semilunar damage; yet the lesion proved to; be a perfectly loose body and the semilunars were natural. to their natural strength, when the joint is again fully used in the ordinary way there will be no tendency to a re- currence of the accident. BY C. A. HINGSTON, M.D., B.Sc. LOND., CONSULTING PHYSICIAN TO THE PLYMOUTH PUBLIC DISPENSARY ; AND W. H. B. STODDART, M.D., M.R.C.P. LOND., ASSISTANT MEDICAL OFFICER TO BETHLEM AND BRIDEWELL. ROYAL HOSPITALS. Points concerning diagnosis.-The 59 cases here tabulated, all of which presented either the characteristic symptoms with locking of the joint, or else weakness with recurrence of pain and swelling, which is often the only evidence of damaged semilunars, illustrate the following conditions : (1) semilunar cartilage, internal or external, split, loosened or displaced, torn from either or both extremities or from coronary ligaments ; (2) loose bodies-the body may be quite free or may be attached by a pedicle ; (3) a torn or hypertrophied alar ligament or hypertrophied fringe of synovial membrane ; (4) rheumatoid arthritis-roughened cartilage, thickened synovial membrane, and osteophytic outgrowth ; and (5) no obvious derangement. There are some scarcely appreciable by ordinary About 12 months before his death albuminuria set in and continued to the end. During the 12 months prior to his death he steadily lost energy and strength ; but no local symptoms presented themselves till seven weeks before his death. He then relaxed his diabetic diet and his final illness appeared to date from this time. He developed a gradually increasing general weakness without malaise. He became exhausted during the process of dressing, the distance which he was able to walk grew daily shorter, and slight bilateral ptosis developed so that he was unable to read for any length of
https://openalex.org/W2799562580
https://se.copernicus.org/articles/9/985/2018/se-9-985-2018.pdf
English
null
Syn-kinematic hydration reactions, dissolution-precipitation creep and grain boundary sliding in experimentally deformed plagioclase-pyroxene mixtures
null
2,018
cc-by
21,464
Correspondence: Sina Marti (sina.marti@ed.ac.uk) Correspondence: Sina Marti (sina.marti@ed.ac.uk) Received: 29 April 2018 – Discussion started: 7 May 2018 Revised: 11 July 2018 – Accepted: 14 July 2018 – Published: 9 August 2018 Received: 29 April 2018 – Discussion started: 7 May 2018 Revised: 11 July 2018 – Accepted: 14 July 2018 – Published: 9 August 2018 Abstract. It is widely observed that mafic rocks are able to accommodate high strains by viscous flow. Yet, a number of questions concerning the exact nature of the involved deformation mechanisms continue to be debated. In this contribution, rock deformation experiments on four different water-added plagioclase–pyroxene mixtures are presented: (i) plagioclase(An60–70)–clinopyroxene–orthopyroxene, (ii) plagioclase(An60)–diopside, (iii) plagioclase(An60)– enstatite, and (iv) plagioclase(An01)–enstatite. Samples were deformed in general shear at strain rates of 3 × 10−5 to 3 × 10−6 s−1, 800 ◦C, and confining pressure of 1.0 or 1.5 GPa. Results indicate that dissolution–precipitation creep (DPC) and grain boundary sliding (GBS) are the dominant deformation mechanisms and operate simultaneously. Co- inciding with sample deformation, syn-kinematic mineral reactions yield abundant nucleation of new grains; the resulting intense grain size reduction is considered crucial for the activity of DPC and GBS. In high strain zones domi- nated by plagioclase, a weak, nonrandom, and geometrically consistent crystallographic preferred orientation (CPO) is observed. Usually, a CPO is considered a consequence of dislocation creep, but the experiments presented here demonstrate that a CPO can develop during DPC and GBS. This study provides new evidence for the importance of DPC and GBS in mid-crustal shear zones within mafic rocks, which has important implications for understanding and modeling mid-crustal rheology and flow. Solid Earth, 9, 985–1009, 2018 https://doi.org/10.5194/se-9-985-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Solid Earth, 9, 985–1009, 2018 https://doi.org/10.5194/se-9-985-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Viscous deformation of crustal rocks is usually dominated either by intracrystalline deformation (dislocation creep) or by a form of diffusion creep together with grain boundary sliding. Two cases of diffusion creep might thereby be dif- ferentiated, one in which diffusive mass transfer is caus- ing a change in grain shape and grain boundary sliding is a local accommodating mechanism (Lifshitz sliding Lang- don, 2006) and another in which grain boundary sliding is the dominate strain-accommodating mechanism with diffu- sive mass transfer ensuring strain compatibility at the grain scale (Rachinger sliding, Langdon, 2006). The relative im- portance of these processes has been discussed by Paterson (1990, 1995), and for the sake of simplicity, we implicitly include the operation of grain boundary sliding when we speak of diffusion creep. Apart from being rate and temper- ature sensitive, the rheology of viscously deforming rocks is also observed to be material dependent (for a compre- hensive list of flow-law parameters for different rock types see, e.g., Kohlstedt et al., 1995, Shaocheng and Bin, 2002, Bürgmann and Dresen, 2008, Burov, 2011, and references therein). Flow laws for viscous creep exist for different types of rocks, with the majority of these flow laws being deter- mined for monomineralic materials. In monomineralic aggregates at mid-crustal to lower crustal conditions, grain growth in monomineralic aggre- gates is assumed to be extensive and the resulting large grain size is expected to render diffusion creep less effi- Syn-kinematic hydration reactions, grain size reduction, and dissolution–precipitation creep in experimentally deformed plagioclase–pyroxene mixtures Sina Marti1, Holger Stünitz2,3, Renée Heilbronner1, Oliver Plümper4, and Rüdiger Kilian1 1Department of Environmental Sciences, University of Basel, Basel, Switzerland 2Department of Geosciences, University of Tromsø, Tromsø, Norway 3Institut des Sciences de la Terre d’Orléans (ISTO), Université d’Orléans, Orléans, France 4Department of Earth Sciences, Utrecht University, Utrecht, the Netherlands Sina Marti1, Holger Stünitz2,3, Renée Heilbronner1, Oliver Plümper4, and Rüdiger Kilian1 1Department of Environmental Sciences, University of Basel, Basel, Switzerland 2Department of Geosciences, University of Tromsø, Tromsø, Norway 3Institut des Sciences de la Terre d’Orléans (ISTO), Université d’Orléans, Orléans, France 4Department of Earth Sciences, Utrecht University, Utrecht, the Netherlands S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures The strain rate of the two-phase aggregates is thereby suggested to be a combina- tion of the strain rates of the individual phases; e.g., Dimanov et al. (2003). No mineral reactions were observed in these experiments. In somewhat lower-temperature experiments and mostly under hydrous conditions, Rutter et al. (1985), Getsinger and Hirth (2014), and Stünitz and Tullis (2001) performed deformation experiments with syn-kinematic hy- dration reactions. Phase mixing was found to be (partly) due to the nucleation of new phases. The authors suggest that the dominant deformation mechanism is grain-size-sensitive creep by a mix of diffusion creep and grain boundary sliding. Rutter et al. (1985) state explicitly that they interpret diffu- sion creep in the sense of dissolution–precipitation creep. In the presence of fluids, mafic rocks are particularly susceptible to reactions during changing temperatures and pressures, representing a suitable material to study the in- terplay between reaction and deformation. That high strain zones such as ultramylonites usually consist of a phase mixture indicates their ability to deform at higher strain rates (or lower stresses) than monomineralic aggregates and emphasizes their importance for localizing deformation. In this study, we present results from deformation experiments on water-added plagioclase–pyroxene mixtures. At the im- posed pressure–temperature conditions of ∼1.0–1.5 GPa and 800 ◦C, deformation takes place within the lower tem- perature range of the viscous regime. The metastability of the starting material in the H2O-present system causes syn- kinematic mineral reactions, thus facilitating the interplay between reaction and deformation in the experiments. In polymineralic mixtures, several processes are known to influence the deformability and the dominating deforma- tion mechanism of the bulk aggregate. The occurrence of mineral reactions and nucleation causes grain size reduction (e.g., Brodie and Rutter, 1987; Fitz Gerald and Stünitz, 1993; Newman et al., 1999; Handy and Stünitz, 2002; de Ronde et al., 2005) and can lead to (further) phase mixing, whereas grain pinning due to secondary phases is likely to impede grain growth (e.g., Olgaard and Evans, 1986; Berger and Herwegh, 2004; Linckens et al., 2011). Furthermore it has been suggested that under certain conditions during diffu- sion creep, the convergence and divergence rate between two different mineral phases can be faster than between grains of the same mineral phase (e.g., Hickman and Evans, 1991; Wheeler, 1992; Sundberg and Cooper, 2008). The result is more efficient strain accumulation in polymineralic ar- eas compared to monomineralic ones. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 986 In the absence of fluids, metastable mineral assemblages can be preserved over long time periods (e.g., Jamtveit et al., 2016). When fluids infiltrate, mineral reactions take place. Under deviatoric stress conditions, deformation is frequently localized along these zones of fluid infiltration and metamor- phic reactions (e.g., Austrheim, 1987). A positive feedback between deformation and metamorphic reactions has been recognized for some time but the exact mechanisms of the interaction are still not sufficiently understood. The positive feedback with metamorphic reactions may not be the same for all deformation mechanisms and thus the syn-kinematic occurrence of mineral reactions is a factor that can influ- ence the dominance of a certain deformation mechanism. It has been shown by the experiments of, e.g., Linckens et al. (2014) and Cross and Skemer (2017) that in the absence of fluids and mineral reactions, phase mixing is seen to be in- efficient and necessitates shear strains of > 17 in the case of Cross and Skemer (2017). This is in contrast to fluid-assisted deformation in polymineralic rocks, which are described to have a strong tendency for phase mixing (Kruse and Stünitz, 1999; Kilian et al., 2011; Precigout and Stünitz, 2016). Thus, strain may preferentially localize into wet and/or reactive re- gions of the lithosphere, promoted by (as mentioned previ- ously) phase mixing and grain size reduction. cient than dislocation creep (e.g., Brodie and Rutter, 1987; Paterson, 1990). Insights into deformation mechanisms, slip systems, and flow-law parameters have been obtained from experimental studies, e.g., for plagioclase: Tullis and Yund (1985), Shaocheng and Mainprice (1987), Tullis and Yund (1991), Dimanov et al. (1999), Rybacki and Dresen (2000), Stünitz and Tullis (2001), Stünitz et al. (2003), Ji et al. (2004), and Barreiro et al. (2007); for pyroxene: Lallemant (1978), Kolle and Blacic (1982), Raterron and Jaoul (1991), Mauler et al. (2000), Bystricky and Mackwell (2001), Hier- Majumder et al. (2005), Chen et al. (2006), and Zhang et al. (2006). For polyphase mixtures of gabbroic composition, data from high-temperature deformation experiments are pub- lished by Dimanov et al. (2003, 2007) and Dimanov and Dresen (2005). Depending on the grain size, the differential stress, and the volume fraction of pyroxene (as the stronger phase in their pyroxene–plagioclase mixtures), the dominant deformation mechanism identified by these authors is either diffusion creep or dislocation creep. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures All these factors en- hance diffusion creep rates and may thus lead to a switch of the dominant deformation mechanism from dislocation creep in monomineralic layers to diffusion creep in polymineralic ones (Etheridge and Wilkie, 1979; Mehl and Hirth, 2008; Linckens et al., 2011; Kilian et al., 2011). i. MD: crushed Maryland diabase (Kronenberg and Shel- ton, 1980; Marti et al., 2017) using a grain size fraction ≤125 µm. The Maryland diabase starting material has a Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 2.1.2 Experimental conditions and sample assembly Experiments are performed using the Griggs-type deforma- tion apparatus at the University of Tromsø, Norway. Ex- periments are run at confining pressures (Pc) of ∼1.0 and 1.5 GPa, temperatures (T ) of 800 ◦C, and (axial) displace- ment rates of ∼2 × 10−8 to 2 × 10−9 ms−1, resulting in bulk strain rates of ∼3 × 10−5 to 3 × 10−6 s−1. General shear type of flow is achieved by placing the rock powder (0.11 g) between cylindrical alumina forcing blocks (diame- ter of 6.33 mm) precut at 45◦with respect to the load axis (Appendix Fig. A1). Descriptions of the experimental setup, data recording, and data treatment can be found in the Ap- pendix Sect. A1–A3, and experimental conditions are listed in Table 2. ii. An60+En: synthetic mixture of Sonora labradorite (∼An60) and Damaping enstatite powder; grain size fraction of ∼2–125 and 40–180 µm. iii. An60+Di: synthetic mixture of Sonora labradorite and Damaping diopside powder; grain size fraction of ∼2– 125 µm. iv. An60+Di: synthetic mixture of Sonora labradorite and Cranberry Lake diopside powder; grain size fraction of ∼2–125 and 40–125 µm. v. Ab+En: synthetic mixture of Alpe Rischuna albite (∼Ab98) and Damaping enstatite powder; grain size fraction of ≤125 and 40–180 µm. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures An60+En: synthetic mixture of Sonora labradorite (∼An60) and Damaping enstatite powder; grain size fraction of ∼2–125 and 40–180 µm. iii. An60+Di: synthetic mixture of Sonora labradorite and Damaping diopside powder; grain size fraction of ∼2– 125 µm. iv. An60+Di: synthetic mixture of Sonora labradorite and Cranberry Lake diopside powder; grain size fraction of ∼2–125 and 40–125 µm. v. Ab+En: synthetic mixture of Alpe Rischuna albite (∼Ab98) and Damaping enstatite powder; grain size fraction of ≤125 and 40–180 µm. A detailed description of the sample preparation can be found in the Appendix. Synthetic plagioclase–pyroxene pow- ders are mixed with phase proportions of ∼57 % vol pla- gioclase to 43 % vol pyroxene. Either 0.2 µL (0.18 wt %) or 0.12 µL (0.11 wt %) H2O is added to the sample. 2.1.2 Experimental conditions and sample assembly Experiments are performed using the Griggs-type deforma tion apparatus at the University of Tromsø, Norway. Ex periments are run at confining pressures (Pc) of ∼1.0 and 1.5 GPa, temperatures (T ) of 800 ◦C, and (axial) displace ment rates of ∼2 × 10−8 to 2 × 10−9 ms−1, resulting in bulk strain rates of ∼3 × 10−5 to 3 × 10−6 s−1. Genera shear type of flow is achieved by placing the rock powde (0.11 g) between cylindrical alumina forcing blocks (diame ter of 6.33 mm) precut at 45◦with respect to the load axi (Appendix Fig. A1). Descriptions of the experimental setup data recording, and data treatment can be found in the Ap pendix Sect. A1–A3, and experimental conditions are listed in Table 2. 2.2 Strain determination The thickness of the shear zone (measured normal to the shear plane) at the hit point is th0 = 0.75 ± 0.03 mm. Dur ing the experiment, ∼86 ± 3 % of the axial displacement i accommodated as shear displacement within the shear zone and ∼14±3 % is accommodated as plane strain thinning o 2.1.1 Starting materials Experiments are performed on five different starting materi- als (the composition of starting material and chemical com- position of minerals are given in Table 1; mineral abbrevia- tions after Whitney and Evans, 2010). i. MD: crushed Maryland diabase (Kronenberg and Shel- ton, 1980; Marti et al., 2017) using a grain size fraction ≤125 µm. The Maryland diabase starting material has a www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 987 Table 1. Mineral composition. Representative mineral measurements as normalized oxide wt % and as calculated stoichiometric mineral composition for the different starting materials. All Fe is taken as Fe2+ due to the reducing environment in the sample assembly. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 987 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 987 Table 1. Mineral composition. Representative mineral measurements as normalized oxide wt % and as calculated stoichiometric minera composition for the different starting materials. All Fe is taken as Fe2+ due to the reducing environment in the sample assembly. Rischuna Sonora Maryland diabase Cranberry Damaping Damaping Maryland Maryland albite labradorite plagioclase Lake diopside diopside enstatite diabase Cpx diabase Opx wt % Core Rim wt % SiO2 67.87 53.66 51.86 55.67 SiO2 57.18 54.39 55.98 51.58 52.61 Al2O3 20.14 30.37 29.92 27.72 Al2O3 0.85 6.37 4.01 1.77 0.75 CaO 0.21 11.09 13.39 10.57 CaO 22.54 17.93 0.80 14.71 1.44 Na2O 11.71 3.90 3.63 5.11 Na2O 0.39 1.71 0.27 0.28 0.00 K2O 0.10 0.39 0.26 0.37 K2O 0.00 0.00 0.00 0.00 0.00 MgO 0.00 0.00 0.00 0.00 MgO 17.93 16.13 33.28 14.03 19.36 TiO2 0.00 0.00 0.00 0.00 TiO2 0.00 0.34 0.00 0.76 0.28 FeO 0.00 0.00 0.00 0.00 FeO 1.11 2.37 5.13 16.40 25.55 MnO 0.00 0.00 0.00 0.00 MnO 0.00 0.00 0.00 0.48 0.00 Cr2O3 0.00 0.00 0.00 0.00 Cr2O3 0.00 0.76 0.53 0.00 0.00 Total 100.00 100.00 100.00 99.99 Total 100.00 100.00 100.00 100.01 99.99 Atoms per 8 oxygen Atoms per 6 oxygen Si 2.97 2.42 2.36 2.51 Si 2.04 1.94 1.92 1.95 1.99 Al 1.04 1.61 1.61 1.47 Al 0.04 0.27 0.16 0.08 0.03 Ca 0.01 0.54 0.65 0.51 Ca 0.86 0.68 0.03 0.60 0.06 Na 0.99 0.34 0.32 0.45 Na 0.03 0.12 0.02 0.02 0.00 K 0.01 0.02 0.02 0.02 K 0.00 0.00 0.00 0.00 0.00 Mg 0.00 0.00 0.00 0.00 Mg 0.95 0.86 1.70 0.79 1.09 Ti 0.00 0.00 0.00 0.00 Ti 0.00 0.01 0.00 0.02 0.01 Fe2+ 0.00 0.02 0.04 0.02 Fe2+ 0.03 0.07 0.15 0.52 0.81 Mn 0.00 0.00 0.00 0.00 Mn 0.00 0.00 0.00 0.02 0.00 Cr 0.00 0.00 0.00 0.00 Cr 0.00 0.02 0.01 0.00 0.00 Total 5.01 4.96 5.00 4.97 Total 3.95 3.97 4.00 4.00 3.99 An 0.98 0.60 0.66 0.52 En 0.52 0.53 0.91 0.42 0.56 Ab 98.47 0.38 0.32 0.46 Fe 0.02 0.04 0.08 0.27 0.41 Or 0.55 0.02 0.02 0.02 Wo 0.47 0.42 0.02 0.31 0.03 modal composition of plagioclase ∼57 % vol, clinopy- roxene ∼32 % vol, orthopyroxene ∼8 % vol, and ac- cessories ∼3 % vol (Qz, Kfs, Ilm, Mag, Bt, Ap). ii. 2.1.1 Starting materials Rischuna Sonora Maryland diabase Cranberry Damaping Damaping Maryland Maryland albite labradorite plagioclase Lake diopside diopside enstatite diabase Cpx diabase Opx wt % Core Rim wt % SiO2 67.87 53.66 51.86 55.67 SiO2 57.18 54.39 55.98 51.58 52.61 Al2O3 20.14 30.37 29.92 27.72 Al2O3 0.85 6.37 4.01 1.77 0.75 CaO 0.21 11.09 13.39 10.57 CaO 22.54 17.93 0.80 14.71 1.44 Na2O 11.71 3.90 3.63 5.11 Na2O 0.39 1.71 0.27 0.28 0.00 K2O 0.10 0.39 0.26 0.37 K2O 0.00 0.00 0.00 0.00 0.00 MgO 0.00 0.00 0.00 0.00 MgO 17.93 16.13 33.28 14.03 19.36 TiO2 0.00 0.00 0.00 0.00 TiO2 0.00 0.34 0.00 0.76 0.28 FeO 0.00 0.00 0.00 0.00 FeO 1.11 2.37 5.13 16.40 25.55 MnO 0.00 0.00 0.00 0.00 MnO 0.00 0.00 0.00 0.48 0.00 Cr2O3 0.00 0.00 0.00 0.00 Cr2O3 0.00 0.76 0.53 0.00 0.00 Total 100.00 100.00 100.00 99.99 Total 100.00 100.00 100.00 100.01 99.99 Atoms per 8 oxygen Atoms per 6 oxygen Si 2.97 2.42 2.36 2.51 Si 2.04 1.94 1.92 1.95 1.99 Al 1.04 1.61 1.61 1.47 Al 0.04 0.27 0.16 0.08 0.03 Ca 0.01 0.54 0.65 0.51 Ca 0.86 0.68 0.03 0.60 0.06 Na 0.99 0.34 0.32 0.45 Na 0.03 0.12 0.02 0.02 0.00 K 0.01 0.02 0.02 0.02 K 0.00 0.00 0.00 0.00 0.00 Mg 0.00 0.00 0.00 0.00 Mg 0.95 0.86 1.70 0.79 1.09 Ti 0.00 0.00 0.00 0.00 Ti 0.00 0.01 0.00 0.02 0.01 Fe2+ 0.00 0.02 0.04 0.02 Fe2+ 0.03 0.07 0.15 0.52 0.81 Mn 0.00 0.00 0.00 0.00 Mn 0.00 0.00 0.00 0.02 0.00 Cr 0.00 0.00 0.00 0.00 Cr 0.00 0.02 0.01 0.00 0.00 Total 5.01 4.96 5.00 4.97 Total 3.95 3.97 4.00 4.00 3.99 An 0.98 0.60 0.66 0.52 En 0.52 0.53 0.91 0.42 0.56 Ab 98.47 0.38 0.32 0.46 Fe 0.02 0.04 0.08 0.27 0.41 Or 0.55 0.02 0.02 0.02 Wo 0.47 0.42 0.02 0.31 0.03 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 988 Table 2. List of experiments. Table 2. List of experiments. Exp. Material Pc Peak τ τ at end Mean µL H2O γa th0 thF ds γ k Wk Stretching 9 (◦) R no. (GPa) (MPa) (MPa) ˙γa (s−1) added (mm) (mm) (mm) ISA (◦) 414 MD 0.97 407 192 2.1e−5 0.20 5.12 0.75 0.50 2.56 3.32 1.50 0.972 38.1 9.3 14.3 449 MD 1.50 479 337 2.3e−5 0.20 4.51 0.75 0.61 2.75 3.64 1.23 0.994 41.8 11.3 15.6 468a MD 1.07 348 1.2e−5 0.20 0.70 0.75 0.69 0.48 0.64 1.09 0.968 37.7 28.8 1.9 470a MD 1.50 446 1.3e−5 0.20 0.86 0.75 0.69 0.65 0.86 1.09 0.982 39.5 27.9 2.3 484b MD 1.02 371 316 1.9e−5 0.20 0.75 233 9.5e−6 1.97 0.56 1.33 489 MD 1.05 428 286 1.9e−5 0.20 3.04 0.75 0.63 1.91 2.54 1.19 0.991 41.1 15.3 8.5 490b MD 1.00 350 2.4e−5 0.20 0.75 1.2e−5 130 2.5e−6 5.37 0.53 2.84 3.72 1.42 0.983 39.7 9.3 16.8 491b MD 1.52 388 1.7e−5 0.20 0.75 8.0e−6 82 2.3e−6 4.97 0.54 2.68 3.52 1.39 0.983 39.7 10.0 15.2 492 MD 1.01 468 197 2.6e−5 0.20 8.95 0.75 0.44 3.94 5.01 1.70 0.978 39.0 5.8 30.8 502b MD 1.52 391 1.8e−5 0.20 0.75 8.1e−6 33 1.7e−6 3.90 0.58 2.26 2.98 1.29 0.985 40.1 12.3 11.3 503 An60+En 1.03 530 367 2.6e−5 0.12 6.55 0.75 0.55 3.60 4.73 1.36 0.992 41.3 8.1 25.4 505 An60+Di 1.01 460 253 2.5e−5 0.12 6.17 0.75 0.55 3.39 4.45 1.36 0.990 41.0 8.5 22.8 (CrLk) 507 MD 1.04 479 191 2.4e−5 0.12 5.82 0.75 0.49 2.85 3.69 1.53 0.974 38.5 8.4 17.2 518 Ab+En 1.02 511 263 2.4e−5 0.12 5.83 0.75 0.54 3.15 4.12 1.39 0.988 40.5 8.8 20.0 519 En60+Di 1.02 517 289 2.2e−5 0.12 5.60 0.75 0.54 3.02 3.96 1.39 0.987 40.3 9.1 18.6 (D) Pc: confining pressure, τ: shear stress, ˙γa: apparent shear strain rate, th0: shear zone thickness at experiment start, thF: shear zone thickness at experiment end, ds: shear displacement, γ : simple shear component of total strain, k: pure shear component of total strain, Wk: vorticity number, 9: finite stretching direction, R: ratio strain ellipse major vs. minor axis. Angles for stretching ISA and 9 are given with from east (0◦) increasing counterclockwise. MD: Maryland diabase, slab: Sonora labradorite, En: Damaping enstatite, Di(CrLk): Cranberry Lake diopside, Di(D): Damaping diopside, Ab: Alpe Rischuna albite. a Experiment terminated at peak stress. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures b Displacement rate stepping test. Pc: confining pressure, τ: shear stress, ˙γa: apparent shear strain rate, th0: shear zone thickness at experiment start, thF: shear zone thickness at experiment end, ds: shear displacement, γ : simple shear component of total strain, k: pure shear component of total strain, Wk: vorticity number, 9: finite stretching direction, R: ratio strain ellipse major vs. minor axis. Angles for stretching ISA and 9 are given with from east (0◦) increasing counterclockwise. MD: Maryland diabase, slab: Sonora labradorite, En: Damaping enstatite, Di(CrLk): Cranberry Lake diopside, Di(D): Damaping diopside, Ab: Alpe Rischuna albite. a Experiment terminated at peak stress. b Displacement rate stepping test. geneous sample deformation and do not take into account strain localization. the shear zone. As in previous experiments, the shear zone thickness decreases linearly with the applied axial displace- ment (see Marti et al., 2017). The shear strain is presented as apparent shear strain, γa, and calculated as the sum of the incremental shear displace- ments divided by the instantaneous shear zone thickness; strain rates are given as apparent shear strain rates, ˙γa (see Marti et al., 2017). γa is not a direct measure of the sim- ple shear component in the general shear progressive defor- mation and should not be used to derive the strain ellipsoid or other strain-related parameters. Instead, the procedure de- scribed by Fossen and Tikoff (1993) and Tikoff (1995) is adopted to calculate the true simple shear component (γ ) from the general shear deformation and used to calculate pa- rameters such as the instantaneous stretching axes (ISAs), the orientation of the finite stretching direction, the kinematic vorticity number, and the strain ratio given by the ratio of the long to short axis of the strain ellipsoid (Table 2). All strains and strain-related parameters are calculated for bulk homo- 2.2 Strain determination A detailed description of the sample preparation can be found in the Appendix. Synthetic plagioclase–pyroxene pow- ders are mixed with phase proportions of ∼57 % vol pla- gioclase to 43 % vol pyroxene. Either 0.2 µL (0.18 wt %) or 0.12 µL (0.11 wt %) H2O is added to the sample. The thickness of the shear zone (measured normal to the shear plane) at the hit point is th0 = 0.75 ± 0.03 mm. Dur- ing the experiment, ∼86 ± 3 % of the axial displacement is accommodated as shear displacement within the shear zone, and ∼14±3 % is accommodated as plane strain thinning of www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures www.solid-earth.net/9/985/2018/ 3 Results (b) (c) 1100 1200 1300 1400 1500 1600 −450 −400 −350 −300 −250 −200 x coordinate [px] y coordinate [px] θ° Center of gravity θ = 68° thc = 23 px Px Amp (b) (a) (c) 1100 1200 1300 1400 1500 1600 −450 −400 −350 −300 −250 −200 x coordinate [px] y coordinate [px] θ° Center of gravity θ = 68° thc = 23 px Figure 1. Analysis of amphibole corona thickness. (a) Digital phase map of segmented pyroxene (Px) clasts and associated amphibole (Amp) coronas. Where adjacent coronas are in contact, they are sep- arated manually (close-up, black arrows). (b) Long “tails” of Amp growing in low stress sites around clasts are eventually “cut” (black arrow) if they extend too far away from the clast. (c) Corona thick- ness, thc(θ), is determined from the polar coordinates of the aggre- gate and clast outline as a function of the angle θ (0◦< θ < 360◦), with θ running counterclockwise from the horizontal. Px Amp (a) S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 989 3.1 Mechanical data This approach yields good results in which coronas follow the clast shape, but tends to underestimate corona thickness at which the corona becomes very elongated as, e.g., in “tails” around the clasts. Note that where tails grew extensively long, they were eventually cut so that the analysis does not include the whole tail length (Fig. 1b). Strain rate stepping tests on Maryland diabase sample ma- terial at Pc ≈1.0 and 1.5 GPa have been performed (Fig. 2c) to test the sensitivity of shear stress on strain rate. Stress ex- ponents, n, of n = 1.9 and n = 1.4 are obtained for experi- ments at Pc ≈1.0 and 1.5 GPa, respectively (Fig. 3). 3.1 Mechanical data formed by visual inspection and correlation of the origi- nal BSE image with the corresponding segmentation. Where necessary, the segmentation was corrected and cleaned with the pencil tool in Photoshop®. Clean phase maps contain segmented pyroxene and amphibole phases, and each pyrox- ene grain is in contact only with its own amphibole corona. The x–y coordinates of the clast (pyroxene) and the aggre- gate (pyroxene + corona) outlines are measured and ex- ported using Fiji and a modified version of the Jazy XY export macro (by Rüdiger Kilian, available at https://github. com/kilir/Jazy_macros; last access: September 2016). Using a MATLAB script (available from the author upon request), the x–y coordinates of clast and aggregate outlines are con- verted to polar coordinates (r–θ), and the corona thickness, thc(θ), is determined at each point along the pyroxene clast as the shortest distance between the clast to the aggregate outline (Fig. 1c) as a function of θ. The angle runs coun- terclockwise from the horizontal. This approach yields good results in which coronas follow the clast shape, but tends to underestimate corona thickness at which the corona becomes very elongated as, e.g., in “tails” around the clasts. Note that where tails grew extensively long, they were eventually cut so that the analysis does not include the whole tail length (Fig. 1b). formed by visual inspection and correlation of the origi- nal BSE image with the corresponding segmentation. Where necessary, the segmentation was corrected and cleaned with the pencil tool in Photoshop®. Clean phase maps contain segmented pyroxene and amphibole phases, and each pyrox- ene grain is in contact only with its own amphibole corona. The x–y coordinates of the clast (pyroxene) and the aggre- gate (pyroxene + corona) outlines are measured and ex- ported using Fiji and a modified version of the Jazy XY export macro (by Rüdiger Kilian, available at https://github. com/kilir/Jazy_macros; last access: September 2016). Using a MATLAB script (available from the author upon request), the x–y coordinates of clast and aggregate outlines are con- verted to polar coordinates (r–θ), and the corona thickness, thc(θ), is determined at each point along the pyroxene clast as the shortest distance between the clast to the aggregate outline (Fig. 1c) as a function of θ. The angle runs coun- terclockwise from the horizontal. 3.2 Microstructures In all experiments strain is partitioned into a network of shear bands (Figs. 4, 5). Their thickness is variable but the main shear band strands usually have a thickness of the order of 40–150 µm (e.g., Fig. 5d, j) and are characterized by strong grain size reduction (Fig. 5b, e, k). The following hydration reactions are observed within shear bands and in low strain lenses: For the interpretation of the amphibole corona evolution it is important to have knowledge of the course and the differ- ent stages of an experiment (the details of which are listed in the Appendix). Due to the experimental procedure, the de- formation stage is always preceded by an initial hydrostatic part (“lead run-in”; Appendix Fig. A1c) in which the sample is held at approximately hydrostatic conditions for 24–30 h. During the initial lead run-in mineral reactions commence prior to sample deformation. Px + Pl + H2O −→Amp + Qz, (R1) Pl1 + H2O −→Pl2 + Zo + Qz + Ky, (R2) (R1) (R2) (R1) (R2) (R2) where Pl2 has a lower anorthite component than Pl1. In Maryland diabase samples, both Reactions (R1) and (R2) occur pervasively, with Reaction (R1) being the more 3.1 Mechanical data For all experiments, the mechanical data plotted as shear stress, τ, vs. apparent shear strain, γa, show a curve with an initial steep increase in shear stress, reaching a peak value usually after ∼γa of 0.8–1.0 (Fig. 2). Peak stress is fol- lowed by a slow decrease, often approaching a quasi-steady- state shear stress value from γa ≈4 onwards. The samples with 0.12 µL H2O added show higher peak stresses and a more rapid shear stress decrease thereafter compared to sam- ples with 0.2 µL H2O added. For the Maryland diabase sam- ples (Fig. 2a) at Pc ≈1.0 GPa, the sample with 0.12 µL H2O reaches a higher peak stress, but after an additional ∼0.5γa, it drops to a value similar to the samples with 0.2 µL H2O. Sample strengths of Maryland diabase at 1.0 and 1.5 GPa reach the same peak stress, but the 1.0 GPa experiments weaken more rapidly within the attained strain range. (a) 1300 1400 15 x coordinate [px] Figure 1. Analysis of amphibole corona thickness. (a) Digital phase map of segmented pyroxene (Px) clasts and associated amphibole (Amp) coronas. Where adjacent coronas are in contact, they are sep- arated manually (close-up, black arrows). (b) Long “tails” of Amp growing in low stress sites around clasts are eventually “cut” (black arrow) if they extend too far away from the clast. (c) Corona thick- ness, thc(θ), is determined from the polar coordinates of the aggre- gate and clast outline as a function of the angle θ (0◦< θ < 360◦), with θ running counterclockwise from the horizontal. The synthetic plagioclase–pyroxene mixtures (Fig. 2b) show similar peak stress values (460–530 MPa) and all but the An60+En mixture attain similar flow stresses. The synthetic mixtures generally support ∼60–110 MPa higher shear stresses than the Maryland diabase samples (compare Fig. 2a and b). At peak stress, the synthetic mixtures (sam- ples 503, 518, and 519) reach differential stress values near the Goetze criterion. According to Kohlstedt et al. (1995), the Goetze criterion, 1σ ≤Pc, is an empirically defined stress range at which rocks are expected to deform plasti- cally. However, due to the significant weakening subsequent to peak stress, many samples that start above the Goetze cri- terion then fall substantially below it. The Maryland diabase samples all stay below the Goetze criterion for all stages of deformation. 2.3 Image analysis After the experiments, samples are immersed in epoxy, cut parallel (in some cases also normal to the shear direction), and prepared to polished thin sections. A polarized light mi- croscope, scanning electron microscope (SEM), and trans- mission electron microscope (TEM) are used for sample analysis. Grain size and surface fabric are determined as de- scribed in Appendix Sect. A5. A special method is developed to study the amphibole coronas that grow on pyroxene porpyhroclasts. Corona thick- ness is measured as a function of orientation around the clasts (Fig. 1). To this end, phase maps of pyroxene and amphibole are created (as described in Appendix Sect. A5). Where am- phibole coronas of neighboring pyroxene clasts are in con- tact, individual pyroxene–amphibole pairs have to be sep- arated manually. Manual separation and cleaning was per- Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures For experiments at confining pressures, Pc = 1.0 GPa, n = 1.9; for Pc = 1.5 GPa, n = 1.4. Data for Pc = 1.0 GPa from Marti et al. (2017). 10 2 10 3 τ (MPa) n=1 n=3 γ stepping Single γ Pc = 1.5 GPa Pc = 1.0 GPa γ stepping Single γ n = 1.4 n = 1.9 γa 10 -6 10 -5 10 -4 Figure 4. Shear zone overview. Micrograph of sample 492, plane polarized light. Strain localizes into a network of shear bands, anas- tomosing around low strain lenses identifiable by the large porphy- roclasts. Sketch in the upper left shows the orientation of the micro- graph with respect to the loading direction (LD) of the sample setup (Appendix Fig. A1). Figure 3. Determination of stress exponents. Shear stress, τ (MPa), versus apparent shear strain rate, ˙γa. Two stress exponents, n, are obtained using constant strain rate data and strain rate stepping ex- periments. For experiments at confining pressures, Pc = 1.0 GPa, n = 1.9; for Pc = 1.5 GPa, n = 1.4. Data for Pc = 1.0 GPa from Marti et al. (2017). and ∼19 % for 1.5 GPa Pc experiments (sample 470, dura- tion 46.5 h: lead run-in and subsequent deformation to peak stress). In 1.0 GPa Pc experiments, the volume of hydrous re- action products reaches about 15–25 % for experiments with durations of ∼60–70 h (lead run-in and subsequent deforma- tion to γa ≈4 to 6). In 1.5 GPa Pc experiments, the volume of hydrous reaction products reaches up to 31 %. Shear bands in Maryland diabase experiments are broad and subparallel to the shear zone boundaries (Fig. 5c), with an angle φ = 3◦between the preferred orientation of shear bands and shear zone boundaries (see Fig. 5 for reference frame). Shear bands are mainly formed by grains with < 1 µm diameter and frequently show a compositional layering between plagioclase-dominated and amphibole-dominated layers (Figs. 5b, 6a, b). Plagioclase layers are either monomineralic or show mixing with zoisite. In amphibole- prominent one. Amphibole grows as reaction coronas on py- roxene clasts and as aggregates, often mixed with quartz, in- side shear bands (Figs. 5b, 6a, c). Both Reactions (R1) and (R2) initiated during the hydrostatic “lead run-in” (Appendix Fig. A1c). At the hit point, samples are fully compacted with only a few submicron-sized pores remaining. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 484 γa: ~ 1 10-5 s-1 γa: ~ 2 10-5 s-1 Shear stress (MPa) Shear stress (MPa) Apparent gamma Apparent gamma 0 1 2 3 4 0 1 2 3 4 0 200 400 0 200 400 0 200 400 0 200 400 Pc 1.0 GPa Pc 1.5 GPa γa: ~ 2 10-5 s-1 γa: ~ 1 10-5 s-1 γa: ~ 3 10-6 s-1 γa: ~ 2 10-5 s-1 γa: ~ 8 10-6 s-1 γa: ~ 2 10-6 s-1 MD MD Pc 1.0GPa, 0.20µl H2O Pc 1.0GPa, 0.12µl H2O MD 0 1 2 3 4 5 6 Apparent gamma 0 Shear stress (MPa) 200 400 600 414 492 507 468 489 Pc 1.5GPa, 0.20µl H2O 0 1 2 3 4 Apparent gamma 502 491 449 MD 0 Shear stress (MPa) 200 400 600 0 1 2 3 4 5 507 518 Ab+En Apparent gamma 0 1 2 3 4 5 Pc 1.0GPa, 0.12µl H2O 507 505 519 An60+Di Apparent gamma 0 1 2 3 4 5 503 Pc 1.0GPa, 0.12µl H2O 507 An60+En Apparent gamma T = 800°C Pc 1.0GPa, 0.12µl H2O (a) (b) (b) (c) Apparent gamma 2 3 Figure 2. Mechanical data. Shear stress, τ (MPa), versus apparent shear strain, γa. Stippled line: experiment 507 (MD) for reference. (a) Maryland diabase (MD) experiments for different confining pressures, Pc (GPa), and water contents. (b) Experiments using different Pl–Px mixtures and constant Pc and water content. (c) Displacement rate stepping tests on MD sample material for experiments performed at different confining pressures. LD Figure 4. Shear zone overview. Micrograph of sample 492, plane polarized light. Strain localizes into a network of shear bands, anas- tomosing around low strain lenses identifiable by the large porphy- roclasts. Sketch in the upper left shows the orientation of the micro- graph with respect to the loading direction (LD) of the sample setup (Appendix Fig. A1). 10 2 10 3 τ (MPa) n=1 n=3 γ stepping Single γ Pc = 1.5 GPa Pc = 1.0 GPa γ stepping Single γ n = 1.4 n = 1.9 γa 10 -6 10 -5 10 -4 Figure 3. Determination of stress exponents. Shear stress, τ (MPa), versus apparent shear strain rate, ˙γa. Two stress exponents, n, are obtained using constant strain rate data and strain rate stepping ex- periments. www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 990 Pc 1.0GPa, 0.20µl H2O Pc 1.0GPa, 0.12µl H2O MD 0 1 2 3 4 5 6 Apparent gamma 0 Shear stress (MPa) 200 400 600 414 492 507 468 489 Pc 1.5GPa, 0.20µl H2O 0 1 2 3 4 Apparent gamma 502 491 449 MD 0 Shear stress (MPa) 200 400 600 0 1 2 3 4 5 507 518 Ab+En Apparent gamma 0 1 2 3 4 5 Pc 1.0GPa, 0.12µl H2O 507 505 519 An60+Di Apparent gamma 0 1 2 3 4 5 503 Pc 1.0GPa, 0.12µl H2O 507 An60+En Apparent gamma T = 800°C Pc 1.0GPa, 0.12µl H2O (a) (b) (c) Exp.nr. 491 γa: ~ 2 10-5 s-1 γa: ~ 8 10-6 s-1 γa: ~ 2 10-6 s-1 Exp.nr. 502 Exp.nr. 490 Exp.nr. 484 γa: ~ 1 10-5 s-1 γa: ~ 2 10-5 s-1 Shear stress (MPa) Shear stress (MPa) Apparent gamma Apparent gamma 0 1 2 3 4 0 1 2 3 4 0 200 400 0 200 400 0 200 400 0 200 400 Pc 1.0 GPa Pc 1.5 GPa γa: ~ 2 10-5 s-1 γa: ~ 1 10-5 s-1 γa: ~ 3 10-6 s-1 γa: ~ 2 10-5 s-1 γa: ~ 8 10-6 s-1 γa: ~ 2 10-6 s-1 MD MD Figure 2. Mechanical data. Shear stress, τ (MPa), versus apparent shear strain, γa. Stippled line: experiment 507 (MD) for reference. (a) Maryland diabase (MD) experiments for different confining pressures, Pc (GPa), and water contents. (b) Experiments using different Pl–Px mixtures and constant Pc and water content. (c) Displacement rate stepping tests on MD sample material for experiments performed at different confining pressures. (c) Exp.nr. 491 γa: ~ 2 10-5 s-1 γa: ~ 8 10-6 s-1 γa: ~ 2 10-6 s-1 Exp.nr. 502 Exp.nr. 490 Exp.nr. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures At peak stress, the volume percentage of hydrous reaction products for experiments at 1.0 GPa Pc is ∼7 % (sample 468, duration 38 h: lead run-in and subsequent deformation to peak stress) www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 991 10µm Zo+Pl Px Zo+Pl 50µm BSE image Pl Px 100µm Px Pl BSE image 5µm Pl porph Zo Pl fine- grained Px 100µm Px Pl BSE image Px Pl Qz Amp+Qz Px Amp 5µm 50µm Pl Px BSE image 25µm EBSD band contrast image + phase map overlay Pl Px Overview Shear band close-up SB ODF (b) (a) Maryland diabase (c) (e) (d) An60 + En An60 + Di (f) (g) (h) (i) (j) (k) (l) 9° 6° 6° 3° Ab + En φ LD. gure 5. Microstructures of experiments at Pc ≈1.0 GPa. (a–c) Maryland diabase sample material. (b) Shear bands are fine-grained an en polymineralic, with the main constituents Pl, Amp, and Qz. (d–f) An60+En sample material. Due to the low iron content, pyroxen pears darker than the plagioclase in BSE–SEM images. In panels (d) and (j), shear bands are traced with white dotted lines. (e) Fin ained Pl + Zo in a shear band next to a Pl porphyroclast. (g–i) An60+Di sample material. (j–l) Ab+En sample material. (h) EBSD ban ntrast image with transparent phase map overlay. Plagioclase appears blueish, pyroxene yellowish. Rose diagrams represent the orientatio the shear bands, and black dots indicate the preferred trend of shear band segments. Red arrows indicate the direction of loading. Th gle, φ, between the shear zone boundary (or forcing block) and the preferred shear band trend is indicated. Shear band close-up SB ODF (c) 3° φ LD. Overview (c) (a) (f) 9° (f) (i) 6° (f) (i) (h) (g) (l) (k) Figure 5. Microstructures of experiments at Pc ≈1.0 GPa. (a–c) Maryland diabase sample material. (b) Shear bands are fine-grained and often polymineralic, with the main constituents Pl, Amp, and Qz. (d–f) An60+En sample material. Due to the low iron content, pyroxene appears darker than the plagioclase in BSE–SEM images. In panels (d) and (j), shear bands are traced with white dotted lines. (e) Fine- grained Pl + Zo in a shear band next to a Pl porphyroclast. (g–i) An60+Di sample material. (j–l) Ab+En sample material. (h) EBSD band contrast image with transparent phase map overlay. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures Plagioclase appears blueish, pyroxene yellowish. Rose diagrams represent the orientation of the shear bands, and black dots indicate the preferred trend of shear band segments. Red arrows indicate the direction of loading. The angle, φ, between the shear zone boundary (or forcing block) and the preferred shear band trend is indicated. www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 992 BSE contrast 20 µm Px Pl Amp Amp+Zo+Qz+Pl Amp Pc = 1.5 GPa Pl+Qz Px Amp+Qz Amp+Qz+Pl 5 µm BSE contrast Pc = 1.0 GPa Pc = 1.0 GPa 20 µm Px Pl BSE contrast (b) (a) (c) Figure 6. Distribution of phases in Maryland diabase. (a) Area of extensive phase mixing in a shear band: mixing of Pl + Qz, Pl + Zo, and Amp + Qz. Mixing between Amp and Pl is less frequent. Px clasts show Amp coronas and asymmetric Amp tails. (b) Area where shear bands (outlined with dashed orange lines) are predominantly composed of polycrystalline Pl. (c) Extensive phase mixing between Amp + Zo + Qz (+ Pl) within shear bands. Px clasts show Amp coronas and asymmetric Amp tails. Pl+Qz Px Amp+Qz Amp+Qz+Pl 5 µm BSE contrast Pc = 1.0 GPa ( (a) BSE contrast 20 µm Px Pl Amp Amp+Zo+Qz+Pl Amp Pc = 1.5 GPa (c) (c) (a) (b) Figure 6. Distribution of phases in Maryland diabase. (a) Area of extensive phase mixing in a shear band: mixing of Pl + Qz, Pl + Zo, and Amp + Qz. Mixing between Amp and Pl is less frequent. Px clasts show Amp coronas and asymmetric Amp tails. (b) Area where shear bands (outlined with dashed orange lines) are predominantly composed of polycrystalline Pl. (c) Extensive phase mixing between Amp + Zo + Qz (+ Pl) within shear bands. Px clasts show Amp coronas and asymmetric Amp tails. dominated layers, amphibole frequently occurs together with quartz. Mixing between amphibole and plagioclase is subor- dinate. cleating mainly along porphyroclast rims and along straight internal trails, which are thought to represent former frac- tures (Fig. 5k). The newly nucleated grains generally show a lower anorthite component than the plagioclase in the starting material (Fig. 7). In experiments on Maryland di- abase, pyroxene grain size reduction is largely caused by the pyroxene-consuming reaction to Amp (Reaction R1, Figs. 5b, 6). In the synthetic mixtures, however, much of the grain size reduction of pyroxene is caused by fracturing. In synthetic mixtures of An60+En and An60+Di (Fig. 5d– i), Reaction (R2) is the dominant hydration reaction. www.solid-earth.net/9/985/2018/ The vol- ume of hydrous reaction products reaches 1–9 % for exper- iments with durations of ∼66–69 h (lead run-in and subse- quent deformation). In the An60+En mixture, shear bands are somewhat narrower and more anastomosing. At an an- gle of φ = 9◦, they are also more inclined to the shear zone boundaries compared to the other samples (Fig. 5f). Shear bands in both An60+En and An60+Di mixtures are mainly formed by fine-grained (< 1 µm) plagioclase and zoisite. The main difference between the microstructures devel- oped at 1.0 and 1.5 GPa Pc (Maryland diabase experiments) is the increased amount of reaction products at higher Pc (Fig. 6). Zoisite and amphibole form more abundantly at 1.5 GPa and amphibole corona surround pyroxene porphy- roclasts in early stages of the experiments. Shear bands at Pc ≈1.0 GPa are mainly composed of a fine-grained mix- ture of Pl + Amp + Qz + Zo (in order of abundance) com- pared to Amp + Pl + Zo + Qz (again in order of abundance) at 1.5 GPa. Additionally, shear bands are somewhat narrower and more inclined to the shear zone boundaries at higher Pc (compare Marti et al., 2017). The zoisite reaction predicts the formation of a new pla- gioclase with a lower anorthite component. The fine grain size within shear bands does not allow for quantitative EDS measurements, but backscattered electron (BSE) images re- veal lower Z contrast (lower anorthite contents) for plagio- clase within shear bands compared to plagioclase porphyro- clasts (Fig. 5e). Semiquantitative EDS measurements yield a decrease in anorthite component from ∼An(60) (starting composition) to ∼An(52–55) for grains within shear bands (Fig. 7). www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 993 p p p p y p g py An40 Kfs30 An70 Sonora Lab., deformed Fine-grained fraction within shear bands Porphyroclasts MD starting material An40 Kfs40 An80 MD deformed An40 Kfs40 An80 Core Rim Porphyroclast Fine-grained fraction within shear bands (a) (b) (c) Figure 7. Plagioclase chemical compositions. (a) Sonora labradorite in An60+Di experiment runs. (b) Maryland diabase starting material. (c) Maryland diabase after the experiment. Porphyroclasts vs. small new grain fraction found in shear bands. (b) Figure 7. Plagioclase chemical compositions. (a) Sonora labradorite in An60+Di experiment runs. (b) Maryland diabase starting material. (c) Maryland diabase after the experiment. Porphyroclasts vs. small new grain fraction found in shear bands. 0.20 0.30 0.15 0.25 Na (p.f.u.) Al_tot (p.f.u.) 1.0 1.5 2.0 2.5 3.0 3.5 Al_tot (p.f.u.) (b) (a) (c) (d) Mg (p.f.u.) 1.0 2.0 Ca (p.f.u.) 1.0 1.5 2.0 2.5 0.3 0.4 0.5 0.6 Na (p.f.u.) 6.5 7.0 7.5 Si (p.f.u.) 1.0 2.0 Ca (p.f.u.) 1.0 2.0 Aliv (p.f.u.) 0.0 1.0 2.0 Ca (p.f.u.) 1.5 (e) (f) 1.0 2.0 Alvi (p.f.u.) 0.0 1.0 2.0 Ca (p.f.u.) 1.5 3.0 Px-Px Px-Pl Pl-Pl Figure 8. Amphibole chemistry. Amphibole grains of Maryland diabase experiments performed at Pc ≈1.0 GPa. Measurements are grouped according to their neighborhood: Px-Px: pyroxene-dominated neighborhood. Px-Pl: amphibole grown between pyroxene and plagioclase grains; Pl-Pl: plagioclase-dominated neighborhood. (a) Ca vs. Si per formula unit (pfu). (b) Ca vs. Mg. (c) Estimated Aliv vs. Ca. (d) Esti- mated Alvi vs. Ca. (e) Na vs. Al (total). (f) Na per Al (total) ratio vs. Al (total). 0.20 0.30 0.15 0.25 Na (p.f.u.) Al_tot (p.f.u.) 1.0 1.5 2.0 2.5 3.0 3.5 Al_tot (p.f.u.) 0.3 0.4 0.5 0.6 Na (p.f.u.) (e) (f) (c) 1.0 2.0 Aliv (p.f.u.) 0.0 1.0 2.0 Ca (p.f.u.) 1.5 Px Pl l (d) 1.0 2.0 Alvi (p.f.u.) 0.0 1.0 2.0 Ca (p.f.u.) 1.5 3.0 (b) (a) Mg (p.f.u.) 1.0 2.0 Ca (p.f.u.) 1.0 1.5 2.0 2.5 6.5 7.0 7.5 Si (p.f.u.) 1.0 2.0 Ca (p.f.u.) Px-P Px-P Pl-P (f) (d) Figure 8. Amphibole chemistry. Amphibole grains of Maryland diabase experiments performed at Pc ≈1.0 GPa. Measurements are grouped according to their neighborhood: Px-Px: pyroxene-dominated neighborhood. Px-Pl: amphibole grown between pyroxene and plagioclase grains; Pl-Pl: plagioclase-dominated neighborhood. (a) Ca vs. Si per formula unit (pfu). (b) Ca vs. Mg. (c) Estimated Aliv vs. Ca. (d) Esti- mated Alvi vs. Ca. (e) Na vs. Al (total). S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures (f) Na per Al (total) ratio vs. Al (total). Two groups of amphibole compositions are present, which can be distinguished by their Na per Al content ratio (Fig. 8e, f). The plagioclase of the Maryland diabase starting material has an anorthite component of ∼An(65–70), with thin rims of ∼An(52–56) (Fig. 7). The core’s Na to Al ratio is thus ∼0.18–0.21. Plagioclase is the sole provider for Na and Al in amphibole as the pyroxene in the starting material shows only trace amounts of these elements. Most amphibole mea- surements show an Na : Al ratio of 0.16–0.21 (Fig. 8f), con- sistent with reaction (R1) and the consumption of a plagio- clase with a composition of ∼An(65–70). The second type of amphibole, with Na : Al ratios > 0.25, is comparable to the Na : Al ratio of the starting plagioclase rim composition of ∼An(52–56) (resulting in Na : Al ratios of ∼0.28–0.32) and thus again would be compatible with the plagioclase- consuming, amphibole-forming Reaction (R1). 3.3 Amphibole chemistry In the Ab+En sample, shear bands are broad and subpar- allel to the shear zone boundaries, with φ = 6◦(Fig. 5l). Shear bands are predominantly composed of fine-grained plagioclase (Fig. 5k) with sizes < 2 µm. No difference in composition between plagioclase porphyroclasts and fine- grained plagioclase within shear bands was detected. In high- resolution BSE images, a fine-grained phase with a Z con- trast similar to enstatite is observed. Due to the small grain size EDS measurements are extremely challenging but point to a new type of pyroxene with a somewhat higher Si and Na component. For Maryland diabase experiments at 1.0 GPa, two groups of amphibole are recognized, differing in their Al and Mg per formula unit (pfu), and in their Na to Al ratio (Fig. 8, Tables 3 and S1 in the Supplement). The amphiboles are classified as ranging between tschermakite and Mg hornblende. When la- beling the amphibole measurements according to their 2-D neighborhood as observed in the thin section, the Al and Mg contents shows a consistent pattern in which high Al–low Mg amphiboles grow in plagioclase-dominated areas (Fig. 8b– d). The Si and Ca contents thereby show no systematic dif- ference between the different grain neighborhoods. Table S1 lists amphibole and plagioclase (starting material and newly nucleated) compositions. In all experiments (Maryland diabase and synthetic mix- tures), plagioclase shows extensive grain size refinement. Porphyroclasts are replaced by fine-grained plagioclase, nu- www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 3.4.3 Albite crystallographic preferred orientation Three EBSD maps are collected along one shear band in the Ab+En sample 518. Orientation data of plagioclase from three distinct sites (Fig. 12) are used to test for the presence of a crystallographic preferred orientation (CPO). Grains are calculated with a threshold angle of 10◦and merged across boundaries consistent with the albite twin law. Porphyro- clasts and their adjacent fragments as well as grains with < 2 µm equivalent diameter and < 3 points are excluded (Fig. 12b). g The pole figures (Fig. 12c), which combine the data of all three sites, show a weak but distinct CPO for which es- pecially [010] and (010) show systematic arrangements of a maximum ∼35◦off the periphery and with a high angle to the trace of the shear band. [100] axes and poles to (110) form local maxima along the trace of the shear band plane. Inverse pole figures (IPF) (Fig. 12d) are plotted for each site individually. Reference directions for the IPF are 0◦(parallel to the global shear direction) and 90◦(normal to the global shear plane). Choosing, e.g., the bulk finite stretching direc- tion (+10◦) or the shear band trace (−10◦) and their normals did not result in significantly different results. Maxima of all distributions have moderate strengths and occupy nearly identical positions for all sites and at each reference direc- tion (showing the close similarity of the CPO developed in all three individual sites). For all distributions, the positions of maxima do not coincide with any common poles to low index planes or directions. Distributions with respect to the shear plane normal are strongest with pole maxima at (−121) (90◦reference frame). Amphibole classification after Hawthorne et al. (2012). Tschermak.: tschermakite, Mg hornbl.: magnesium hornblende. All Fe is taken as Fe2+ due to the reducing conditions in the sample assembly. Amphibole classification after Hawthorne et al. (2012). Tschermak.: tschermakite, Mg hornbl.: magnesium hornblende. All Fe is taken as Fe2+ due to the reducing conditions in the sample assembly. small pores are seen as pore trails along grain boundaries (Fig. 9c, d) oriented at a small angle to the expected σ1. The shear band formed in the Maryland diabase sam- ple shows the typical compositional layering between plagioclase-dominated layers and amphibole (+ Qz) aggre- gates (Fig. 10a). Bright-field TEM images reveal largely defect-free grains (Fig. 10b, d) and grain sizes are similar for amphibole and plagioclase. 3.5 Amphibole coronas Amphibole grows abundantly in experiments on Maryland diabase, especially at the higher Pc (e.g., Fig. 6c) for which pyroxene clasts are already surrounded by amphibole coro- nas in early stages of the experiment. It has been noted that whereas coronas seem to grow symmetrically during the hy- drostatic stage of an experiment, they evolve to become nar- rower in high stress sites around their host porphyroclast and grow larger in the clast strain shadow during sample deformation (Fig. 13a). To quantify this corona thickness evolution, pyroxene clast–amphibole rim pairs from exper- iments performed at Pc ≈1.5 GPa were analyzed, studying the average amphibole corona thickness as a function of ori- entation around pyroxene porphyroclasts (Fig. 13). Coronas are measured at three different stages: at hydrostatic condi- S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 994 Table 3. EDS measurements of Amp chemical compositions from samples deformed at Pc ≈1.0 GPa. Amphibole wt %, norma- 414 sm 414 sm 490 sm 490 sm lized to 98 % tschermak. Mg hornbl. Mg hornbl. tschermak. SiO2 45.18 45.72 47.76 47.31 Al2O3 17.13 14.13 13.06 17.73 CaO 9.24 8.92 9.45 10.02 Na2O 1.63 1.74 2.12 1.84 K2O 1.26 0.86 0.89 0.90 MgO 7.56 9.95 11.24 7.39 TiO2 0.00 1.78 0.00 0.00 FeO 15.99 14.90 13.48 12.81 MnO 0.00 0.00 0.00 0.00 Cr2O3 0.00 0.00 0.00 0.00 Total: 97.99 98.00 97.99 98.00 Formula per 23 oxygen Si 6.59 6.76 6.89 6.77 Ti 0.00 0.00 0.00 0.00 Al 2.95 2.46 2.22 2.99 Fe3+ 0.00 0.00 0.00 0.00 Cr 0.00 0.00 0.00 0.00 Mg 1.65 2.19 2.42 1.58 Ca 1.45 1.41 1.46 1.54 Mn 0.00 0.00 0.00 0.00 Fe2+ 1.95 1.84 1.63 1.53 Na 0.46 0.50 0.59 0.51 K 0.23 0.16 0.16 0.16 Total 15.28 15.34 15.38 15.08 Amphibole classification after Hawthorne et al. (2012). Tschermak.: tschermakite, Mg hornbl.: magnesium hornblende. All Fe is taken as Fe2+ due to the reducing conditions in the sample assembly. Table 3. EDS measurements of Amp chemical compositions from samples deformed at Pc ≈1.0 GPa. Table 3. EDS measurements of Amp chemical compositions from samples deformed at Pc ≈1.0 GPa. cerning the measured GSDs inherent from the grain segmen- tation. Nonetheless, grains segmented from TEM and SEM images correlate well for the highest frequency bins. Mea- sured on TEM and SEM images, the GSDs for sample 518 have modes at 0.51 and 0.36 µm, respectively. In sample 414, the GSDs have modes at 0.23 and 0.30 µm (Fig. 11). 3.4.3 Albite crystallographic preferred orientation Grain and phase boundaries are tight and porosity is scarce (Fig. 10c, d). Plagioclase grains are weakly anisotropic in shape (not perfectly equant) with a shape preferred orientation subparallel to the shear zone boundaries (Fig. 10b, d; compare Marti et al., 2017). 3.4.1 Nanostructure of plagioclase within shear bands TEM images are presented from shear bands formed within the Ab+En sample 518 (Fig. 9) and the Maryland diabase sample 414 (Fig. 10). For both samples, micrographs are ob- tained from foils cut normal to the shear zone boundaries and parallel to the shear direction. Figure 9a shows the interface between an albite porphyroclast and the fine-grained albite matrix of an adjacent shear band. The albite clast has a high defect density, whereby intragranular domains develop mis- orientations to each other, as seen in the bright-field image or from the rotation of diffraction spots (Fig. 9b). However, no recovery to form sub-grain walls is observed. Furthermore, the interface between the clast and the shear band is sharp and no bulges are observed (Fig. 9a). Within the shear band, www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 995 σ 1 µm BF Porphyroclast Fine-grained shear band 1 µm HAADF Fine-grained shear band 1 µm BF 200 nm 100 nm HAADF (c) (d) (a) (b) (e) 49.5° 1 Figure 9. Nanostructures of shear bands in Ab+En sample. For sample 518, shear zone boundaries are horizontal and shear sense is dextral. (a) Bright-field (BF) image of a plagioclase porphyroclasts adjacent to a fine-grained shear band. White arrows mark the porphyroclast– shear band interface. Black arrow points to a high defect density band within the clast. (b) BF image of the internal structure of porphyroclast showing high defect density. Twin lamellae run from the upper left to lower right. (c) HAADF image of a fine-grained plagioclase in a shear band. Black rectangle marks close-up view in panel (d). (d) HAADF image of a pore trail following several aligned grain boundaries. The local orientation of σ1 is derived from the orientation of the ISA (Table 2). (e) HAADF image. White arrows point to porosity or opening sites developed along two triple junctions and a grain boundary. (b) (a) BF σ 1 µm BF 1 µm HAADF Fine-grained shear band 200 nm (c) (d) (a) 49.5° 1 (c) (e) Figure 9. Nanostructures of shear bands in Ab+En sample. For sample 518, shear zone boundaries are horizontal and shear sense is dextral. (a) Bright-field (BF) image of a plagioclase porphyroclasts adjacent to a fine-grained shear band. White arrows mark the porphyroclast– shear band interface. Black arrow points to a high defect density band within the clast. (b) BF image of the internal structure of porphyroclast showing high defect density. Twin lamellae run from the upper left to lower right. (c) HAADF image of a fine-grained plagioclase in a shear band. Black rectangle marks close-up view in panel (d). (d) HAADF image of a pore trail following several aligned grain boundaries. The local orientation of σ1 is derived from the orientation of the ISA (Table 2). (e) HAADF image. White arrows point to porosity or opening sites developed along two triple junctions and a grain boundary. At hydrostatic conditions (e.g., during the lead run-in), corona growth is symmetrical around the clasts, with an av- erage thickness of 2.4 to 3.1 µm (Fig. 13b). In the deformed case, the average corona thickness shows an overall mon- oclinic shape. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures Assuming that the microstructure after lead run-in (hydrostatic part) is approximately the same as that of the hydrostatic case sample, the corona thickness in the de- formed sample is reduced by ∼0.5–2 µm in directions close to the loading direction. Thickness is reduced in most direc- tions except in the range of 346–53◦and 186–232◦, where it is increased. On average, the corona thickness is reduced on clast surfaces facing the loading direction and increased at high angles to the loading direction. At peak stress the av- tions (1σ ≈0), at peak stress (1σ > 0; γa ≈1), and after considerable deformation (1σ > 0; γa ≈4) corresponding to three evolutionary stages of a typical high strain experiment (Appendix Fig. A1c). Accordingly, three cases are distin- guished. The hydrostatic case represents the microstructural state at the hit point after the lead run-in. The peak stress case records the microstructural state at the time the sam- ple has reached its maximum strength (including lead run-in and initial sample loading) and the deformed case represents the microstructure evolved after the sample underwent high shear strain (including lead run-in, sample yielding, and de- formation; for an explanation on the nomenclature used see Appendix Fig. A1c). 3.4.2 Plagioclase grain size distribution within shear bands 2-D grain size distributions (GSDs) are determined for pla- gioclase inside shear bands of the Ab+En experiment 518 and the MD experiment 414 (Fig. 11). The distributions in the two samples are similar with somewhat higher frequen- cies in bins > 1 µm for albite compared to the labradorites of the Maryland diabase sample. Due to the small grain sizes and extremely narrow grain boundaries, grains are difficult to identify on SEM images and there is some uncertainty con- www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures www.solid-earth.net/9/985/2018/ www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 996 Ky Amp 200 nm BF 500 nm BF 1 µm HAADF (b) (d) 500 nm HAADF Pl Amp (d) (c) (d) (a) (b) Amp Pl Qz Figure 10. Nanostructure of shear bands in Maryland diabase sample. For sample 414, shear zone boundaries are horizontal and shear sense is dextral. (a) HAADF image, overview. Amp aggregates are traced with black lines for better visibility. Rectangles indicate areas shown in panels (b) and (d). (b) BF-TEM image of small (usually ≤600 nm) plagioclase grains with low internal defect densities. Grain boundaries are tight and porosity is scarce. (c) HAADF image, overview. Black rectangle indicates area shown in panel (d). (d) BF-TEM image of a few Ky and Amp grains growing between Pl grains. The size of all phases is a few hundred nanometers, and grains have a low internal defect density. Ky Amp 200 nm BF 500 nm BF 1 µm b) 500 nm (d) (b) Qz base sample. For sample 414, shear zone boundaries are horizontal and shear sense are traced with black lines for better visibility. Rectangles indicate areas shown in ≤600 nm) plagioclase grains with low internal defect densities. Grain boundaries iew. Black rectangle indicates area shown in panel (d). (d) BF-TEM image of a few e of all phases is a few hundred nanometers, and grains have a low internal defect (b) (d) (a) Amp Pl Qz 1 µm HAADF (b) (d) 500 nm HAADF Pl Amp (d) (c) (a) Amp Pl Qz (b) (b) BF (a) Pl (d) (d) (c) BF BF 500 nm Figure 10. Nanostructure of shear bands in Maryland diabase sample. For sample 414, shear zone boundaries are horizontal and shear sense is dextral. (a) HAADF image, overview. Amp aggregates are traced with black lines for better visibility. Rectangles indicate areas shown in panels (b) and (d). (b) BF-TEM image of small (usually ≤600 nm) plagioclase grains with low internal defect densities. Grain boundaries are tight and porosity is scarce. (c) HAADF image, overview. Black rectangle indicates area shown in panel (d). (d) BF-TEM image of a few Ky and Amp grains growing between Pl grains. www.solid-earth.net/9/985/2018/ The size of all phases is a few hundred nanometers, and grains have a low internal defect density. between the stretching ISA and the finite stretching direc- tion. The short diameter corresponds to the shortening sector around the pyroxene clast. The direction of the maximum corona thickness is at a higher angle with respect to the shear plane than the finite stretching direction (Fig. 13b). erage corona thickness in the direction of loading is the same as in the hydrostatic case (∼2.7 µm) but already increased in almost all other directions. Furthermore, despite the 23 h longer duration of the hydrostatic run compared to the peak stress run (Fig. 13d), coronas did not grow to larger thick- nesses in the former. Instantaneous stretching and shortening axes (ISAs), finite stretching directions, and vorticity numbers (Wk) are calcu- lated for the peak stress and the deformed case (Fig. 13c). The orthorhombic shape of the peak stress corona curve is well described by the ISA, e.g., such that the long side is normal to the shortening ISA. After deformation, the long diameter of the monoclinic-shaped corona curve is oriented 4.2 Derivation of the stress exponent The determined n values are low, with n = 1.4 and 1.9 (Fig. 3), and are thus within the range of expected values for diffusion creep (including grain boundary sliding) with theoretical values between 1 (e.g., Ashby and Verrall, 1973; Coble, 1963; Karato, 2008; Kohlstedt and Hansen, 2015; Pa- terson, 2013) and 2 (e.g., Gratier et al., 2009, 2013; Paterson, 2013). The stress exponents determined in this study have to be taken with some caution as deformation of the samples is inhomogeneous. While the shear bands are able to accom- modate higher strain rates, the lesser deformed domains in between seem to still control the overall bulk stress (Marti et al., 2017). Nevertheless, the low stress exponents strongly suggest an absence of frictional deformation and make dislo- cation creep unlikely. duction is only observed in the pyroxene grains of the syn- thetic mixtures (e.g., Fig. 12b). In these samples, pyroxene only participates in mineral reactions to a minor degree, and the plagioclase hydration Reaction (R2) is dominant (except for the Ab+En sample). In contrast, pyroxene grains in ex- periments on Maryland diabase show grain size reduction by dissolution during the pyroxene-consuming Reaction (R1) to amphibole. Fracturing only minorly contributes to the grain size re- duction of plagioclase. Instead, grain size reduction is pri- marily caused by mineral reactions and abundant nucleation of new grains. New plagioclase grains have a different com- position from that of the original clasts (e.g., Fig. 7). The low defect densities, the narrow grain size range, and the lozenge- shaped grains of the very fine-grained pure plagioclase ag- gregates within shear bands (Figs. 9–11) are in accordance with formation by nucleation and limited growth. Of the two initial mineral phases, plagioclase and pyroxene, plagioclase is particularly susceptible to grain size reduction via the re- action and nucleation of new grains. For all new phases, like zoisite and amphibole, it is clear that reaction and nucleation are the mechanisms leading to a small grain size and to phase mixing. 4.3 Dissolution–precipitation creep and grain boundary sliding From the mechanical data (including stress exponents), the determined grain sizes, and from the nucleation of new grains, it is concluded that in all samples the dominant de- formation mechanism cannot be frictional or crystal plastic (dislocation creep). Instead, the dominant deformation mech- anism is inferred to be DPC, accompanied and/or accom- modated by mineral reactions. Pyroxene is less involved in accommodating strain but plays an important part by being involved in mineral reactions and thereby aiding grain size reduction by nucleation of new grains. In the special case of the Ab+En experiment, it is difficult to observe an obvious change in the chemical composition of the plagioclase and to connect it to grain size reduction in shear bands. Qualitative EDX measurements reveal pos- sible new pyroxene grains with higher Si and Na contents compared to the starting pyroxene. Due to the very small grain size, however, chemical measurements are challenging. No measurable change in plagioclase composition is detected but in order for the new pyroxene to grow with higher Si and Na contents compared to the starting material, a plagioclase with a higher anorthite component is expected to grow. Mineral reactions change the initial phase assemblage of Px + Pl to mostly Pl + Px + Zo in the synthetic mixtures and Pl + Px + Amp + Qz + Zo in Maryland diabase samples. Disregarding the differences in the amount and type of min- eral reactions, strain is always localized into a network of shear bands characterized by intense grain size reduction and phase mixing (to a lesser extent in the Ab+En sample; Fig. 5). The small size of grains in shear bands (Fig. 11) clearly favors a grain-size-sensitive deformation mechanism such as DPC and related grain boundary sliding. This interpretation Mineral reactions change the initial phase assemblage of Px + Pl to mostly Pl + Px + Zo in the synthetic mixtures and Pl + Px + Amp + Qz + Zo in Maryland diabase samples. Disregarding the differences in the amount and type of min- eral reactions, strain is always localized into a network of shear bands characterized by intense grain size reduction and phase mixing (to a lesser extent in the Ab+En sample; Fig. 5). The small size of grains in shear bands (Fig. 11) clearly favors a grain-size-sensitive deformation mechanism such as DPC and related grain boundary sliding. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures n: number of grains; solid black (TEM) and dashed grey lines (SEM) are kernel density estimate fits to the GSDs and modes determined from the fit are given in the graph. g p g (a) Ab+En experiment 518 and (b) the Maryland diabase experi- ment 414. 2-D grain size distributions (GSDs) in both samples are determined on BSE–SEM images and TEM images separately. n: number of grains; solid black (TEM) and dashed grey lines (SEM) are kernel density estimate fits to the GSDs and modes determined from the fit are given in the graph. 4.1 Physics and chemistry of grain size reduction There is a drastic grain size decrease (down to diameters < 2 µm) accompanied by the shear band formation (Figs. 5, 9–11). Fracturing as an important process of grain size re- Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 997 0 0.4 0.8 1.2 1.6 Dequ (µm) 0 5 10 15 20 25 30 Frequency [%] 0 0.2 0.4 0.6 0.8 1.0 1.2 Dequ (µm) SEM (n:89) TEM (n:46) mode : 0.51 µm mode : 0.36 µm SEM (n:417) TEM (n:190) mode : 0.23 µm mode : 0.30 µm Ab+En (exp.nr. 518) MD (exp.nr. 414) (a) (b) Figure 11. Grain size distributions of plagioclase in shear bands. (a) Ab+En experiment 518 and (b) the Maryland diabase experi- ment 414. 2-D grain size distributions (GSDs) in both samples are determined on BSE–SEM images and TEM images separately. n: number of grains; solid black (TEM) and dashed grey lines (SEM) are kernel density estimate fits to the GSDs and modes determined from the fit are given in the graph. tation or bulging recrystallization) of plagioclase is not ob- served (see, e.g., Fig. 9). In addition, had the recrystallization of plagioclase in monomineralic domains taken place by dy- namic recrystallization, the resulting grain sizes would imply very high stresses. Using the normalized grain size–stress re- lationship by Derby (1991), the observed plagioclase grain size mode of ∼0.4 µm (Fig. 11) would require differential stresses of 2 to 2.5 GPa. The observed differential stresses are ∼500 MPa in the last stages of this experiment (Fig. 2); they are clearly far too low to produce such a small grain size in equilibrium. As in other samples, the grain size reduction of plagioclase in the Ab+En sample is considered to take place by the dissolution of original porphyroclasts and nucleation (i.e. “neo-crystallization”) of new grains. 0 0.4 0.8 1.2 1.6 Dequ (µm) 0 5 10 15 20 25 30 Frequency [%] 0 0.2 0.4 0.6 0.8 1.0 1.2 Dequ (µm) SEM (n:89) TEM (n:46) mode : 0.51 µm mode : 0.36 µm SEM (n:417) TEM (n:190) mode : 0.23 µm mode : 0.30 µm Ab+En (exp.nr. 518) MD (exp.nr. 414) (a) (b) (a) Figure 11. Grain size distributions of plagioclase in shear bands. . Grain size distributions of plagioclase in shear band Figure 11. Grain size distributions of plagioclase in shear bands. (a) Ab+En experiment 518 and (b) the Maryland diabase experi- ment 414. 2-D grain size distributions (GSDs) in both samples are determined on BSE–SEM images and TEM images separately. 4.3 Dissolution–precipitation creep and grain boundary sliding This interpretation Microstructural evidence for grain size reduction by frac- turing or the dynamic recrystallization (e.g., sub-grain ro- www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 998 998 S. a e a .: sso u o p ec p a o c eep e pe e a y de o ed p ag oc ase py o e e u σ IPF reference direction 0° IPF reference direction 90° Site 3 Px Pl Site 2 Site 1 50 µm (a) -49.5° sz boun- dary II 1 0° 90° -90° Specimen coordinates [201] [100] [111] [112] [001] [110] [110] [010] [112] [111] (100) (110) (111) (010) (001) (010) (110) (100) 25 µm stpsz: 250 nm Site 3 25 µm stpsz: 220 nm Site 1 25 µm stpsz: 200 nm Site 2 Crystal coordinates 0 1 2 3 4 (b) Site 1 Site 2 Site 3 Max: 3.4 Max: 4.0 Max: 3.2 Max: 2.7 Max: 2.0 Max: 2.3 Upper Lower Upper [100] Max: 2.6 [010] Max: 2.7 [001] Max: 1.6 (010) pfJ:3.9 Max: 2.8 (110) pfJ:3.9 Max: 2.3 pfJ:3.8 (110) Max: 2.3 (c) (d) pfJ:3.9 Max: 2.3 pfJ:3.9 Max: 2.2 pfJ:3.7 Max: 1.8 0 1 2 3 Figure 12. EBSD analysis of Ab+En sample. (a) Reflected light image. EBSD map locations are indicated; Pl: plagioclase, Px: pyroxe (b) Band contrast images and EBSD map (for site 1, inverse pole figure coloring, x direction); stpsz: step size. (c) Pole figures (equal a projections) plotted from combined data of all sites. Orientation data of grains < 2 µm in diameter (and min. > 3 pixels) are plotted. Sm black lines at pole figure rims indicate approximate trace of shear band (plunging with 15◦to the right). (d) Inverse pole figures (IP for directions and poles to planes, respectively, for reference directions 90 and 0◦(normal to the global shear plane and parallel to sh direction, respectively). For the specimen coordinate reference sketch, sz: shear zone. Number of grains analyzed: site 1: 2492, site 2: 16 site 3: 3513. 4.4 Evidence for dissolution–precipitation creep of amphibole 2002; Marsh et al., 2009; Stokes et al., 2012), but has only rarely been reproduced in experimental studies (Rutter et al., 1985; Getsinger and Hirth, 2014). g ) The direction of the shortening ISA (equal to the direc- tion of the instantaneous maximum principal stress, σ1) lies within the range of directions of minimum average corona thickness. A correlation between the shortening ISA and the minimum in average corona thickness is consistent with the interpretation of DPC in which material is preferentially dis- solved along high stress sites (and reprecipitated along low stress sites). During DPC, the grain shape change is deter- mined by the stress field, whereby the instantaneous (i.e. for very low strains) shape change is expected to result in an orthorhombic grain shape with the short and long axis at 90◦and parallel to σ1 and σ3, respectively. With progressive deformation during DPC, grain boundary sliding will take place alongside, causing grain rotation and a deviation from this orthorhombic-shaped fabric. At the peak stress where shear strain is still small, the rim thickness shows an over- all ∼orthorhombic shape (see corona curve in Fig. 13c) well aligned with the ISAs. As the ISAs indicate the minimum and maximum principal stress directions, the fact that the corona curve follows the ISAs correlates well with the inter- pretation that DPC determines the amphibole corona thick- ness evolution. The monoclinic shape of the corona curve in the deformed case can be explained by a superposition of the coaxial geometry of deformation by diffusion creep and rigid Microstructural evidence for DPC is usually scarce but the truncation of grains (or other markers such as chemical zona- tion) and their overgrowths are sometimes correlated with deformation by DPC (e.g., Schwarz and Stöckhert, 1996; Wintsch and Yi, 2002; Svahnberg, 2010). The evolution of amphibole corona thicknesses has been analyzed to investi- gate if it could be explained in terms of amphibole deforma- tion by DPC. y During the experiments, amphibole grows as a reaction product and pyroxene porphyroclasts are replaced at their rims by amphibole growth coronas. During all stages of an experiment, amphibole is observed to grow stably. Clasts with coronas are predominantly found in low strain lenses for which shear strains are lower than in shear bands. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures (a) Phase map of microstructure developed when no 1σ was applied (“hydrostatic”) and in the case of sample deformation (1σ > 0, “deformed”). (b) Average Amp corona thickness presented as a rose diagram; n: number of analyzed coronas. Analysis for three different samples are presented:“hydrostatic”, “peak stress” (γa ∼0.6), and “deformed” (γa ∼4). (c) Duration of experiments in hours (h) and minutes (′). (d) Kinematic analysis of panel (b); blue lines indicate calculated instan- taneous stretching directions. In red is the calculated finite stretching direction; LD: loading direction. Wk: kinematic vorticity number. The error range in the ISA direction, finite stretching direction, and Wk is caused by the uncertainty of the starting thickness of the shear zone. Figure 13. Analysis of amphibole coronas. Thickness of amphibole (Amp) corona on pyroxene (Px) porphyroclasts as a function of orien- tation from Maryland diabase experiments deformed at Pc ≈1.5 GPa. (a) Phase map of microstructure developed when no 1σ was applied (“hydrostatic”) and in the case of sample deformation (1σ > 0, “deformed”). (b) Average Amp corona thickness presented as a rose diagram; n: number of analyzed coronas. Analysis for three different samples are presented:“hydrostatic”, “peak stress” (γa ∼0.6), and “deformed” (γa ∼4). (c) Duration of experiments in hours (h) and minutes (′). (d) Kinematic analysis of panel (b); blue lines indicate calculated instan- taneous stretching directions. In red is the calculated finite stretching direction; LD: loading direction. Wk: kinematic vorticity number. The error range in the ISA direction, finite stretching direction, and Wk is caused by the uncertainty of the starting thickness of the shear zone. 4.3 Dissolution–precipitation creep and grain boundary sliding (a) IPF reference direction 0° IPF reference direction 90° s 25 µm stpsz: 220 nm 0 1 2 3 4 Site 1 Site 2 Site 3 Max: 3.4 Max: 4.0 Max: 3.2 Max: 2.7 Max: 2.0 Max: 2.3 (d) Crystal coordinates [201] [100] [111] [112] [001] [110] [110] [010] [112] [111] (100) (110) (111) (010) (001) (010) (110) (100) Crystal coordinates (110) pfJ:3.9 Max: .3 2 pfJ:3.8 Max: .3 (c) (d) (c) Figure 12. EBSD analysis of Ab+En sample. (a) Reflected light image. EBSD map locations are indicated; Pl: plagioclase, Px: pyroxene. (b) Band contrast images and EBSD map (for site 1, inverse pole figure coloring, x direction); stpsz: step size. (c) Pole figures (equal area projections) plotted from combined data of all sites. Orientation data of grains < 2 µm in diameter (and min. > 3 pixels) are plotted. Small black lines at pole figure rims indicate approximate trace of shear band (plunging with 15◦to the right). (d) Inverse pole figures (IPFs) for directions and poles to planes, respectively, for reference directions 90 and 0◦(normal to the global shear plane and parallel to shear direction, respectively). For the specimen coordinate reference sketch, sz: shear zone. Number of grains analyzed: site 1: 2492, site 2: 1647, site 3: 3513. terpretation; the pore trail in Fig. 9d is interpreted to have formed by precipitation of plagioclase and entrapment of residual fluid along grain boundaries with a trace subparal- lel to the estimated σ1 direction (expected opening direction of dilatancy normal to σ1). is also supported by the strain-free interior and grain mor- phology of the small grains in shear bands (Figs. 9 and 10). The activity of solution–mass transport processes is clearly indicated by the vast extent of mineral reactions, which ne- cessitate the movement of chemical components over several tens of micrometers at least. The morphology of pores pre- sented in Fig. 9 is further supporting evidence for DPC in- www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 999 Hydrostatic Deformed γ = 4.1 10µm 90 270 180 0° 5 4 Average corona thickness [µm] LD 2 1 Hydrostatic n=185 Peak stress n=127 Deformed n=129 ISA Peak stress 28° ± 2° Wk=0.996 - 0.950 90 270 180 0° LD 39 ± 3.5 Deformed ISA 42° ± 1° Finite stretching direction 11.3° ± 0.02° Wk=0.996 - 0.990 270 90 180 0° LD Px Amp Pl Zo Grt At hydrostatic conditions Δσ applied Total Hydrostatic Deformed Peak stress 69h 46’ - 69h 46’ 32h 06’ 14h 25’ 46h 31’ 28h 01’ 39h 42’ 67h 43’ (a) (b) (d) (c) Figure 13. Analysis of amphibole coronas. Thickness of amphibole (Amp) corona on pyroxene (Px) porphyroclasts as a function of orien- tation from Maryland diabase experiments deformed at Pc ≈1.5 GPa. (a) Phase map of microstructure developed when no 1σ was applied (“hydrostatic”) and in the case of sample deformation (1σ > 0, “deformed”). (b) Average Amp corona thickness presented as a rose diagram; n: number of analyzed coronas. Analysis for three different samples are presented:“hydrostatic”, “peak stress” (γa ∼0.6), and “deformed” (γa ∼4). (c) Duration of experiments in hours (h) and minutes (′). (d) Kinematic analysis of panel (b); blue lines indicate calculated instan- taneous stretching directions. In red is the calculated finite stretching direction; LD: loading direction. Wk: kinematic vorticity number. The error range in the ISA direction, finite stretching direction, and Wk is caused by the uncertainty of the starting thickness of the shear zone. Hydrostatic Deformed γ = 4.1 10µm 90 270 180 0° 5 4 Average corona thickness [µm] LD 2 1 Hydrostatic n=185 Peak stress n=127 Deformed n=129 Px Amp Pl Zo Grt (a) (b) ISA Peak stress 28° Wk=0.99 90 270 180 0 LD 39 ± 3.5 (c) .1 ISA Peak stress 28° ± 2° Wk=0.996 - 0.950 90 270 180 0° LD 39 ± 3.5 Deformed ISA 42° ± 1° Finite stretching direction 11.3° ± 0.02° Wk=0.996 - 0.990 270 90 180 0° LD (c) 2° - 0.950 Deformed ISA 42° ± 1° Finite stretching direction 11.3° ± 0.02° Wk=0.996 - 0.990 270 90 180 0° LD Deformed (c) (b) Figure 13. Analysis of amphibole coronas. Thickness of amphibole (Amp) corona on pyroxene (Px) porphyroclasts as a function of orien- tation from Maryland diabase experiments deformed at Pc ≈1.5 GPa. 4.5 Albite crystallographic preferred orientation Dislocation creep and dynamic recrystallization are not con- sidered to occur in our experiments. The large Burgers vec- tors in plagioclase are unfavorable for intracrystalline de- formation, especially at the comparatively low experimen- tal temperatures of 800 ◦C. Dislocation glide and climb have been suggested to be active in plagioclase under both natu- ral and experimental conditions (e.g., Tullis and Yund, 1985; Shaocheng and Mainprice, 1987; Yund and Tullis, 1991; Rybacki and Dresen, 2000; Shigmeatsu and Tanaka, 2000; Kruse et al., 2001; Lapworth et al., 2002; Stünitz et al., 2003; Ji et al., 2004; Barreiro et al., 2007; Mehl and Hirth, 2008) but usually are not considered to accommodate large amounts of strain. Recrystallization takes place by different mechanisms including neo-crystallization (e.g., Fitz Gerald and Stünitz, 1993; Rosenberg and Stünitz, 2003; Brander et al., 2012; Fukuda and Okudaira, 2013; Mukai et al., 2014) or by growth of fragments formed by fracturing (e.g., Stünitz et al., 2003; Viegas et al., 2016). In fine-grained aggregates, diffusion creep (in the broadest sense), often dissolution– precipitation creep (DPC), is the main strain-accommodating process described for polycrystalline plagioclase aggregates (e.g., Yund and Tullis, 1991; Fitz Gerald and Stünitz, 1993; Jiang et al., 2000; Lapworth et al., 2002; Rosenberg and Stünitz, 2003; Brander et al., 2012; Fukuda and Okudaira, 2013; Mukai et al., 2014; Viegas et al., 2016). The exact CPO-forming mechanism could not be deter- mined. It may be speculated that the CPO formation could be due to directed and anisotropic growth during DPC and grain boundary sliding of grains with a crystallographically con- trolled shape. Studies on plagioclase crystal growth in mag- matic environments usually determine [100] and/or [001] as fast growth directions and crystals forming with pronounced (010) planes (e.g., Smith, 1974). In hydrothermal growth ex- periments on albite, Franke and Ghobarkar (1982) report an increasing growth velocity of (110) at higher experimental temperatures (up to ∼700 ◦C). The alignment of (110) poles within the trace of the shear band (pole figures, Fig. 12c) and that of (010) poles at a high angle is conspicuous and may in- dicate that the CPO is caused by the combined mechanisms of grain rotation during deformation and anisotropic growth. The CPO measured in a shear band of the Ab+En sample 518 is generally weak, but the independent sites 1–3 show very similar CPO patterns (Fig. 12c, d). 4.5 Albite crystallographic preferred orientation This similarity in- dicates that these CPOs, although weak, are not random and that there must be a mechanism leading to this weak but sys- tematic CPO. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1000 body rotation induced by the rotational component of simple shear. crystallographically controlled grain shape) during diffusion- accommodated grain boundary sliding. For plagioclase, the most commonly reported slip plane in dislocation glide is (010) (e.g., Kruse et al., 2001). However, although (010) aligns at a high angle to the shear plane (as seen from the pole figures, Fig. 12c), the position of the max- ima in the IPF is significantly off (∼35◦) the pole to (010). Maxima in the IPF are close to [3–14] (for reference, direc- tion is shear direction) and (−121) (for reference, direction is normal to shear plane). They do not correlate with the [100] or [001] slip directions and the (010) slip plane commonly reported for feldspars in the literature. It is therefore argued that the CPO is not due to dislocation creep but rather caused by some other process(es) during grain-size-sensitive creep in the predominantly monomineralic albite layers. 4.4 Evidence for dissolution–precipitation creep of amphibole No microstructure can be found to indicate that the reduction in average corona thickness at the compressional sites of the clasts is due to “shearing-off” of amphibole from compres- sional to extensional sites by some sort of granular flow. On the contrary, the amphibole coronas, which grow symmetri- cally in thickness during the hydrostatic “lead run-in”, be- come partly dissolved in high stress sites while simultane- ously growing thicker in low stress sites during deformation (Fig. 13b). These results indicate grain-scale DPC of amphi- bole. It is frequently observed for amphiboles in naturally deformed rocks (e.g., Berger and Stünitz, 1996; Imon et al., Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ 5 Summary and conclusions i. Viscous deformation in experiments with mafic com- positions at temperatures of 800 ◦C and confining pres- sures of 1.0 and 1.5 GPa at strain rates of ∼10−5 s−1 is dominantly achieved by dissolution–precipitation creep (DPC) and grain boundary sliding accompanied by syn- deformational mineral reactions. No evidence for fric- tional deformation or significant contributions of dislo- cation glide or creep to the accommodation of strain can be found for any of the mineral phases. Strain is frequently localized into shear bands, which con- sist of fine-grained mixtures of neo-crystallized plagio- clase and the syn-kinematic reaction products amphi- bole, quartz, and zoisite, none of which are present in the starting material. i. Viscous deformation in experiments with mafic com- positions at temperatures of 800 ◦C and confining pres- sures of 1.0 and 1.5 GPa at strain rates of ∼10−5 s−1 is dominantly achieved by dissolution–precipitation creep (DPC) and grain boundary sliding accompanied by syn- deformational mineral reactions. No evidence for fric- tional deformation or significant contributions of dislo- cation glide or creep to the accommodation of strain can be found for any of the mineral phases. Strain is frequently localized into shear bands, which con- sist of fine-grained mixtures of neo-crystallized plagio- clase and the syn-kinematic reaction products amphi- bole, quartz, and zoisite, none of which are present in the starting material. With the dominant deformation mechanisms, DPC and grain boundary sliding, the samples presented here deform viscously. At the experimentally induced conditions of T = 800 ◦C and strain rates of ∼3×10−5 s−1, deformation takes place at the lower end of the viscous field, close to the brittle– viscous transition of the studied material (e.g., Marti et al., 2017). However, it is probable that the observed deformation mechanisms are also active at higher temperatures in these types of rocks. The principal constituent phases of mafic rocks (plagioclase + pyroxene + amphibole) all have high strengths in terms of intracrystalline plasticity, even at high temperatures. Where water is absent and DPC suppressed, the buildup of high stresses can be expected in such rocks, at least transiently (e.g., Okudaira et al., 2015). If local stresses are high enough to induce cracking, dilatancy and fluid in- filtration may be facilitated. 5 Summary and conclusions Once reactions start to operate (metastability of mafic rocks is very common because the mineral compositions are very variable and critically depen- dent on P , T , and fluid composition), a switch to DPC is likely to occur. The resulting deformation takes place with low stress exponents. ii. Intense grain size reduction is produced by high nucle- ation rates, probably caused by a large overstepping of reaction boundaries. Both deformation and nucleation are localized in shear bands, implying a positive feed- back between the two mechanisms. iii. Amphibole is seen to accommodate displacement via dissolution–precipitation creep, as interpreted from the evolution and distribution of amphibole coronas on py- roxene porphyroclasts. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1001 Wayte et al., 1989; Krabbendam et al., 2000; Austrheim, 2013; Jamtveit et al., 2016). Only where fluid infiltrates are mineral reactions enabled and equilibrium can be attained. gone to completion, the driving potential for the dissolution of phases is reduced, and it may be expected that the de- formation rate is reduced, too. Interestingly, the measured amphibole compositions show variations, which are most probably influenced by the local mineral composition of the other neighboring mineral phases (Fig. 8). Shear offset and neighbor switching during deformation by diffusion creep and grain boundary sliding will continuously change the lo- cal neighborhood of grains. As the neighborhood of a given grain changes, new surfaces will be brought into contact and become involved in reactions, potentially providing a rela- tively constant chemical driving force for reaction and nucle- ation if local disequilibrium prevails. Thus, it may take con- siderable time until the deforming assemblage has reached complete equilibrium, and some local chemical driving po- tentials may persist, even when the nominal bulk equilibrium assemblage has formed. q TEM analyses revealed high defect densities in the albite porphyroclasts (Fig. 9a, b). It has been proposed that a high intragranular defect density results in an increased rate of grain dissolution, speeding up the reaction and/or deforma- tion rate (e.g., Wintsch, 1985; Schott et al., 1989; Stünitz, 1998). However, the observation of, e.g., abundant nucle- ation along former fractures (Fig. 5k) cannot simply be ac- credited to enhanced reaction rates due to locally increased strain energy (in the sense of high defect densities locally in- troduced by fracturing according to, e.g., Fitz Gerald et al., 1991; Fitz Gerald and Stünitz, 1993; Stünitz et al., 2003; Trepmann et al., 2007). Fracturing is always accompanied by dilatancy. Fluid infiltrates the fractures, leading to higher solution–mass transport rates (e.g., Fitz Gerald and Stünitz, 1993; Precigout and Stünitz, 2016). Thus higher reaction rates can be expected. The significance of strain energy as a possible rate-enhancing contributor to reaction and nucle- ation is thus difficult to separate from the effects of enhanced fluid flow. 4.6 Importance of dissolution–precipitation creep in natural rocks Grain size reduction is energetically unfavorable as it in- creases the total grain surface area. DPC does not necessar- ily require the formation of new grains; instead, precipita- tion could take place as overgrowth rims on existing grains. The intense grain size reduction observed in shear bands within our experiments is most probably caused by high nu- cleation rates. High nucleation rates are typically attained by a large overstepping of a reaction boundary (e.g., Ru- bie, 1998; Putnis, 1992), introducing a high driving potential. The start of our experiments represents such an instance of large overstepping of reaction boundaries. The starting ma- terials + H2O are not in equilibrium at the experimental Pc– T conditions. As the pressurization and heating procedure required to attain the experimental Pc–T conditions takes place within 5–8 h, the sample material is brought rapidly to a metastable state. Although this rapid change in P –T condi- tions is unique to experiments, there is widespread evidence from observations of natural rocks that, similarly, metastable mineral assemblages can be sustained even at high-grade conditions and to large overstepping of reaction boundaries when rocks are dry (e.g., Rubie, 1986; Austrheim, 1987; There are a number of mechanisms that can lead to a CPO within an aggregate. Examples are dislocation glide (e.g., Schmid and Boas, 1950) or directed growth (possibly to- gether with rigid body rotation; e.g., Shelley, 1994; Berger and Stünitz, 1996; Rosenberg and Stünitz, 2003; Getsinger and Hirth, 2014; Viegas et al., 2016). Furthermore, a CPO can form via host-controlled nucleation (e.g., Jiang et al., 2000) or caused by interface-controlled diffusion creep (e.g., Bons and den Brok, 2000; Sundberg and Cooper, 2008). Sim- ilar CPOs as the ones shown here have been found in exper- imentally deformed anorthite and basalt (e.g., Ji et al., 2004; Barreiro et al., 2007) and in naturally deformed basaltic and peridotitic rocks (e.g., Mehl and Hirth, 2008; Viegas et al., 2016; Xie et al., 2003; Drury et al., 2011). Ji et al. (2004) and Mehl and Hirth (2008) interpret the CPOs to be due to dislocation creep in monomineralic plagioclase lay- ers. Viegas et al. (2016) observe no evidence for disloca- tion creep and suggest that the CPOs may be the result of directed growth and rigid body rotation of grains (with a www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 4.7 Continued operation of deformation mechanisms at higher strain iv. A weak but systematic crystallographic preferred orien- tation (CPO) of albite – unrelated to any known slips system operating in dislocation creep – is formed in the shear bands. The CPO is thereby interpreted to have de- veloped as a result of deformation via DPC and grain boundary sliding. In the case of the experimental setup described here, in which the starting mineral assemblages are not in equilibrium at the imposed P –T fluid conditions, the chemical driving poten- tial for attaining a lower-energy assemblage partially con- trols the reaction rate (although for a stressed system, there is no unique driving force, i.e., Gibbs free energy, defined; e.g., Kamb, 1961; Wheeler, 2014, 2018). In principle, when the stable assemblage is reached, i.e., when the reaction has v. The deformation in the samples takes place under condi- tions of pronounced weakening in all cases. The weak- ening is induced by strain localization into shear bands www.solid-earth.net/9/985/2018/ www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures show strong grain size reduction. Grain size re- on, in turn, is due to nucleation of new phases, onstrating the direct relationship between mineral ion, grain size refinement, and the operation of and grain boundary sliding (as grain-size-sensitive Code and data availability. The MATLAB code for analyzing am- phibole reaction corona thicknesses is available from the author upon request (sina.marti@ed.ac.uk). Mechanical data and chemical analyses are available from the author upon request. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures how strong grain size reduction Grain size re Code and data availability The MATLAB code for analyzing am 1002 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures that show strong grain size reduction. Grain size re- duction, in turn, is due to nucleation of new phases, demonstrating the direct relationship between mineral reaction, grain size refinement, and the operation of DPC and grain boundary sliding (as grain-size-sensitive Code and data availability. The MATLAB code for analyzing am- phibole reaction corona thicknesses is available from the author upon request (sina.marti@ed.ac.uk). Mechanical data and chemical analyses are available from the author upon request. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures show strong grain size reduction. Grain size re- on, in turn, is due to nucleation of new phases, onstrating the direct relationship between mineral ion, grain size refinement, and the operation of d i b d lidi ( i i i i Code and data availability. The MATLAB code for analyzing am- phibole reaction corona thicknesses is available from the author upon request (sina.marti@ed.ac.uk). Mechanical data and chemical analyses are available from the author upon request. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1002 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures how strong grain size reduction Grain size re Code and data availability The MATLAB code for analyzing am that show strong grain size reduction. Grain size re- duction, in turn, is due to nucleation of new phases, demonstrating the direct relationship between mineral reaction, grain size refinement, and the operation of DPC and grain boundary sliding (as grain-size-sensitive mechanisms), resulting in viscous deformation with low stress exponents. Code and data availability. Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ A2 Experimental procedure Maryland diabase rock powder is fabricated by crushing Maryland diabase pieces with a handpress and subsequently with an alumina hand mortar. The resulting powder is dry- sieved to extract a grain size fraction ≤125 µm. The plagio- clase in Maryland diabase shows a relatively homogeneous composition (∼An65–70) except for a thin rim with lower anorthite component (∼An50–55). The core to rim area ra- tio is 83 : 17 (±3). Some of the clinopyroxene grains show a Mg-enriched core and clinopyroxene grains generally show orthopyroxene exsolution lamellae. Confining pressure, axial load, and displacement are con- stantly recorded. The force on the load piston is measured using an external load cell, whereas the displacement of the load piston is measured with either a direct-current displace- ment transducer (resolution ∼1 µm) (Rig 1) or a noiseless digital linear-transformation measurement system (resolu- tion 0.1 µm) (Rig 2). Pc is measured via the oil pressure in the hydraulic pumping system and T is monitored and regu- lated to within ±1 ◦C via a Eurotherm controller. To bring the sample to the desired Pc–T conditions during pressurization, the independently movable Pc and load pis- tons are alternately advanced (thereby raising the pressure), accompanied by stepwise increases in temperature until the desired Pc–T conditions are reached (Fig. 2). The pressur- ization procedure usually takes 5–8 h. During the actual de- formation experiment, only the load piston is advanced. The experimental setup necessitates that each deformation exper- iment starts with a so-called “lead run-in”, whereby the load piston is advanced through a thin (∼1.5 mm) top lead layer. During this stage, the sample is expected to experience ap- proximate isotropic pressure. During the initial lead run-in, the sample is under approximate hydrostatic conditions for several hours (usually between 24–30 h). Once the hit point is reached, sample loading initiates. At the end of an exper- iment, the sample is quenched to 200 ◦C within 2 min while simultaneously removing the differential load. Subsequently, Pc, load, and T are slowly and simultaneously reduced to room conditions within about 5 h. The diopside and enstatite material is a mineral powder provided by Jacques Precigout (University d’Orléans) and Holger Stünitz (University of Tromsø, University d’Orléans) with grain sizes of 40–125 µm for Cranberry Lake diop- side and 40–180 µm for Damaping enstatite. A2 Experimental procedure Damaping en- statite and diopside are derived from a peridotite xenolith and the Cranberry Lake diopside from a calc–silicate rock. The albite material is extracted from an albite–quartz vein formed along a joint from the Alpe Rischuna area, Switzer- land. Sonora labradorite consists of labradorite megacrysts formed in basaltic deposits from the Pinacate volcanic field, Sonora, Mexico. From Sonora labradorite, Alpe Rischuna al- bite, and Damaping diopside a powder (grain size fraction ≤125 µm) is produced in the same manner as described for the Maryland diabase powder. As the Sonora labradorite ma- terial shows some accessory calcite, the powder is cleaned with HClaq (10 %). Subsequently, the powder is placed in a funnel with a grade 602 h qualitative filter paper with a pore size of 2 µm and rinsed thoroughly with distilled wa- ter. The powder retained by the filter is then dried in an oven at ∼110 ◦C. After this treatment, no calcite is detected in the material. Appendix A: Methods used, as the mullite tubing is more durable in the corrosive environment of the heated salt. www.solid-earth.net/9/985/2018/ The MATLAB code for analyzing am- phibole reaction corona thicknesses is available from the author upon request (sina.marti@ed.ac.uk). Mechanical data and chemical analyses are available from the author upon request. Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ 1003 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures as zero and the deviation of σ1 by 3–6◦from 45◦is neglected as well. at the nano-imaging lab of the Swiss Nanoscience Insti- tute (SNI) at Basel University. Chemical analyses are per- formed using standardless energy-dispersive X-ray spec- trometry (EDS) at 15 keV of acceleration voltage with a ZAF matrix correction for spectra quantification. The stress exponent, n, is calculated for a relationship τ = ˙γ 1/n a (A3) τ = ˙γ 1/n a (A3) Scanning transmission electron microscopy (STEM) anal- yses are performed at Utrecht University using a FEI Ta- los 200FX equipped with a high-sensitivity Super-EDX sys- tem. TEM images are recorded in bright-field (BF) mode, whereas STEM images are in high-angular annular dark-field (HAADF) mode. BF-TEM images are highly sensitive to crystallographic orientation, whereas contrasts in HAADF- STEM images are sensitive to the average atomic number (Z contrast) of the material. Focused ion beam (FIB) foils for TEM investigations are prepared using an FEI Helios NanoLab G3. The FIB foils are cut parallel to the shear direc- tion and normal to the shear plane. Both the FEI Talos 200FX and the NanoLab G3 are located at the electron microscopy square at Utrecht University. between shear stress, τ, and strain rate, ˙γa (Eq. A3), using data of displacement rate stepping and single-displacement- rate experiments. Over the years and between the different laboratories, dif- ferent data treatment routines have been developed and are in use. The variations in calculated stresses using the different methods can influence the determined stress exponents (n) from the data, causing variations of the order of 16–27 % in determined n values (see Marti et al., 2017) A4 Strain and flow descriptors The ISA and the finite stretching direction can be derived be- cause the initial thickness of the shear zone is known (with an error of ±0.03 mm) and the end thickness can be measured after the experiment from the thin section. This uncertainty associated with the initial thickness leads to some uncertainty concerning the amount of thinning and therefore also to a range of values for the ISA, the finite stretching direction, and Wk. A5.2 EBSD analysis For electron backscatter diffraction (EBSD) measurements, thin sections were polished with colloidal silica suspension and subsequently coated with a thin layer of carbon. Sam- ples were analyzed in the Zeiss Merlin SEM at the Univer- sity of Tromsø with a Nordlys nano-camera in high vac- uum at 15 keV of acceleration voltage and probe currents of ∼15 nA. Data are acquired with the Oxford AZtec soft- ware and processed with Channel 5 and the MATLAB tool- box MTEX (available at https://mtex-toolbox.github.io, last access: January 2018; Bachmann et al., 2010). Orientation S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1004 (a) (b) 0 0.5 1.0 1.5 2.0 2.5 Time (s) × 105 0 10 20 30 40 50 60 Load (kN) Lead run-in Sample deformation Hit point Pressurization Quenching + depressurization IF Peak stress Pc piston Load piston Lead Cu disk Salt (NaCl) Furnace Pyrophyllite Tungsten carbide plug 25.4 mm Sample material 45° Al2O3 Pt jacket (c) 45° Pc F th0 d ds thF Figure A1. General shear experiments. (a) Sample assembly in cross section. (b) Schematic sample cross section at the start of experiment (left) and after sample deformation (right). F: load induced by load piston, Pc: confining pressure, d: axial displacement, ds: shear displace- ment, th0: shear zone thickness at start, thF: shear zone thickness at end of experiment. (c) Phases of an experiment; red line: load F(t). During “pressurization”, the sample is brought to the desired Pc–T conditions. Black dot denotes the start of experiment. IF: initial load in- crease caused by machine friction. Phase 1: “lead run-in”; the sample is not loaded. Phase 2: sample supports load and deforms. “Hit point” denotes the onset of sample loading. During “quenching + depressurization”, the load is released and the temperature brought to ambient conditions. (b) 45° Pc F th0 d ds thF (a) Pc piston Load piston Lead Cu disk Salt (NaCl) Furnace Pyrophyllite Tungsten carbide plug 25.4 mm Sample material 45° Al2O3 Pt jacket P 0 0.5 1.0 1.5 2.0 2.5 Time (s) × 105 0 10 20 30 40 50 60 Load (kN) Lead run-in Sample deformation Hit point Pressurization Quenching + depressurization IF Peak stress (c) (b) 0 (c) Figure A1. General shear experiments. (a) Sample assembly in cross section. (b) Schematic sample cross section at the start of experiment (left) and after sample deformation (right). F: load induced by load piston, Pc: confining pressure, d: axial displacement, ds: shear displace- ment, th0: shear zone thickness at start, thF: shear zone thickness at end of experiment. (c) Phases of an experiment; red line: load F(t). During “pressurization”, the sample is brought to the desired Pc–T conditions. Black dot denotes the start of experiment. IF: initial load in- crease caused by machine friction. Phase 1: “lead run-in”; the sample is not loaded. Phase 2: sample supports load and deforms. “Hit point” denotes the onset of sample loading. During “quenching + depressurization”, the load is released and the temperature brought to ambient conditions. A3 Evaluation of mechanical data Axial displacement is corrected for apparatus stiffness. σ3 is assumed to be equal to the confining pressure, Pc (Eq. A1). With increasing advancement of the load piston, the pressure inside the vessel increases and Pc is corrected for this (see e.g., Richter et al., 2016). The differential stress, 1σ, acting on the sample is derived from the difference between the ax- ial load (F) with reference to the load at the hit point (F0) (Eq. A2) and the cross-sectional area of the forcing block (31.47 mm2). Synthetic plagioclase–pyroxene powders are mixed with a phase distribution of ∼57 % vol plagioclase to 43 % vol py- roxene. To produce the synthetic mixtures, the powders are placed in a 5 mL glass beaker with acetone and mixed us- ing an ultrasonic stirrer (procedure of de Ronde et al., 2004). When most of the acetone is evaporated, the slurry is dried in an oven at 110 ◦C. This procedure prevents grain size and density sorting of the minerals. The sample is then placed in a platinum jacket (0.15 mm wall thickness) with a 0.025 mm nickel foil insert. The Pt jacket is weld-sealed with a Lampert welding apparatus while the sample is encased in a cooled brass piece (T ∼ 4 ◦C) to minimize sample heating and resulting potential wa- ter loss. σ3 = Pc (A1) 1σ = (F −F0)/31.47mm2 (A2) σ3 = Pc (A2) 1σ in the sample is corrected for the decreasing overlap of the forcing blocks (see procedure described in Marti et al., 2017). The shear and normal stresses, τ and σn, supported by the sample inclined at 45◦are obtained by Mohr circle construction from 1σ. The effective pore fluid pressures in our experiments are assumed to be negligible, i.e. are taken Solid sodium chloride (NaCl) is used as a confining medium (Appendix Fig. A1a). K-type thermocouples (with metal tubing) positioned next to the sample are used for most experiments. Only for long-duration experiments of more than 6 days are S-type thermocouples (with mullite tubing) www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1005 Grain maps were produced by manually tracing (super- vised segmentation) grains from SEM or TEM images. Grains were analyzed for their 2-D area using Fiji, and for each grain the diameter of the area equivalent circle, dequ, is calculated. data are analyzed and plotted using MTEX. The de la Vallée Poussin kernel with a half-width of 10◦was used for pole fig- ure contouring, whereas a kernel half-width of 12◦was used for contouring of inverse pole figures (IPFs). All pole figures and IPFs are calculated with Laue symmetry. Pole figure J index (PFJ; e.g., Bunge, 1982; Mainprice and Silver, 1993) values are given as a measure of texture strength. The index has a value of 1 for a random distribution and is infinite for a single orientation. g ) g g Grains were analyzed for their 2-D area using Fiji, and for each grain the diameter of the area equivalent circle, dequ, is calculated. dequ = 2 · p (A/π) (A4) (A4) A is the area of the digitized shape. Grain size distributions are presented as histograms of the 2-D number-weighted dis- tribution of equivalent diameters. A5.1 Data acquisition SEM analyses are performed either with the Zeiss Merlin SEM at Tromsø University or with a Philips XL30 ESEM Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ www.solid-earth.net/9/985/2018/ References Dimanov, A. and Dresen, G.: Rheology of synthetic anorthite-diopside aggregates: Implications for duc- tile shear zones, J. Geophys. Res., 110, B07203, https://doi.org/10.1029/2004JB003431, 2005. Ashby, M. F. and Verrall, R. A.: Diffusion-accommodated Flow and Superplasticity, Acta Metall. Mater., 21, 149–163, 1973. Austrheim, H.: Eclogitization of lower crustal granulites by fluid migration through shear zones, Earth Planet. Sc. Lett., 81, 221– 232, 1987. Dimanov, A., Dresen, G., Xiao, X., and Wirth, R.: Grain boundary diffusion creep of synthetic anorthite aggregates: The effect of water, J. Geophys. Res., 104, 10483–10497, 1999. Austrheim, H.: Fluid and deformation induced metamorphic pro- cesses around Moho beneath continent collision zones: Exam- ples from the exposed root zone of the Caledonian mountain belt, W-Norway, Tectonophysics, 609, 620–635, 2013. Dimanov, A., Lavie, M. P., Dresen, G., Ingrin, J., and Jaoul, O.: Creep of polycrystalline anorthite and diopside, J. Geophys. Res., 108, B001815, https://doi.org/10.1029/2002JB001815, 2003. W-Norway, Tectonophysics, 609, 620–635, 2013. Bachmann, F., Hielscher, R., and Schaeben, H.: Texture Analysis with MTEX – Free and Open Source Software Toolbox, Sol. St. Phen., 160, 63–68, 2010. Dimanov, A. E., Rybacki, E., Wirth, R., and Dresen, G.: Creep and strain-dependent microstructures of synthetic anorthite-diopside aggre-gates, J. Struct. Geol., 29, 1049–1069, 2007. Barreiro, J. G., Lonardelli, I., Wenk, H., Dresen, G., Rybacki, E., Ren, Y., and Tome, C.: Preferred orientation of anorthite de- formed experimentally in Newtonian creep, Earth Planet. Sc. Lett., 264, 188–207, 2007. Drury, M., Avé Lallemant, H., Pennock, G., and Palasse, L.: Crys- tal preferred orientation in peridotite ultramylonites deformed by grain size sensitive creep, Étang de Lers, Pyrenees, France, J. Struct. Geol., 33, 1776–1789, 2011. Berger, A. and Herwegh, M.: Grain coarsening in contact metamor- phic carbonates: effects of second-phase particles, fluid flow and thermal pertubations, J. Metamorph. Geol., 22, 459–474, 2004. Etheridge, M. A. and Wilkie, J. C.: Grainsize reduction, grain boundary sliding and the flow strength of mylonites, Tectono- physics, 58, 159–178, 1979. Berger, A. and Stünitz, H.: Deformation mechanisms and reaction of hornblende: examples from the Bergell tonalite (Central Alps), Tectonophysics, 257, 149–174, 1996. Fitz Gerald, J. D. and Stünitz, H.: Deformation of Granitoids at low Metamorphic Grade. 1. Reactions and Grain-size Reduction, Tectonophysics, 221, 269–297, 1993. Bons, P. D. and den Brok, B.: Crystallographic preferred orientation development by dissolution-precipitation creep, J. Struct. Geol., 22, 1713–1722, 2000. Fitz Gerald, J. D., Boland, J. N., McLaren, A. Competing interests. The authors declare that they have no conflict of interest. Competing interests. The authors declare that they have no conflict of interest. Burov, E. B.: Rheology and strength of the lithosphere, Mar. Petrol. Geol., 28, 1402–1443, 2011. Bystricky, M. and Mackwell, S.: Creep of dry clinopyroxene aggre- gates, J. Geophys. Res., 106, 13443–13454, 2001. Acknowledgements. We thank the team of the center for nano- imaging (SNI) at the University of Basel and Tom Eilertsen at Tromsø University for help and assistance with the electron microscopy. Terry Tullis is thanked for providing the Maryland diabase material, and the Cranberry Lake diopside was kindly provided by Jacques Précigout. Willy Tschudin is thanked for excellent thin section preparation. We gratefully acknowledge the funding provided by the Swiss National Foundation grant NF 200020_144448 and financial support from the Freiwillige Akademische Gesellschaft, Basel, during the last stages of finishing this paper. John Wheeler and Andrew Cross are thanked for thorough reviews and their suggestions and comments to improve the paper. Chen, S., Hiraga, T., and Kohlstedt, D. L.: Water weakening of clinopyroxene in the dislocation creep regime, J. Geophys. Res., 111, B08203, https://doi.org/10.1029/2005JB003885, 2006. Coble, R. L.: A Model for Boundary Diffusion Controlled Creep in Polycrystalline Materials, J. Appl. Phys., 34, 1679–1682, 1963. Cross, A. J. and Skemer, P.: Ultramylonite generation via phase mixing in high-strain experiments, J. Geophys. Res.-Sol. Ea., 122, 1744–1759, 2017. Derby, B.: The dependence of grain size on stress during dynamic recrystallisation, Acta Metall. Mater., 39, 955–962, 1991. de Ronde, A. A., Heilbronner, R., Stünitz, H., and Tullis, J.: Spatial correlation of deformation and mineral reaction in experimen- tally deformed plagioclase-olivine aggregates, Tectonophysics, 389, 93–109, 2004. Edited by: Michael Heap Reviewed by: John Wheeler and Andrew Cross Reviewed by: John Wheeler and Andrew Cross de Ronde, A. A., Stünitz, H., Tullis, J., and Heilbronner, R.: Reaction-induced weakening of plagioclase-olivine composites, Tectonophysics, 409, 85–106, 2005. A5.3 Image analysis Shear bands are traced and digitized on BSE–SEM im- ages. The x–y coordinates of the outlines of the digitized structures are smoothed to remove pixel artifacts and ex- ported using Fiji. The smoothed x–y coordinates are used as input for the SURFOR program (Panozzo Heilbronner, 1984; Heilbronner and Barrett, 2014), which determines the orien- tation distribution function (ODF) of the boundary segments. The ODFs are presented as rose diagrams. Phase segmentation is performed on BSE–SEM images, whereby individual minerals are identified by their Z con- trast. Using the software Fiji (link at: http://fiji.sc/Fiji; last ac- cess: August 2017) and the plugin Statistical Region Merger, automatic pre-segmentation was achieved. The automatic segmentation was then manually inspected and corrected where necessary. Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1006 The Supplement related to this article is available online at https://doi.org/10.5194/se-9-985-2018-supplement. The Supplement related to this article is available online at https://doi.org/10.5194/se-9-985-2018-supplement. Brodie, K. H. and Rutter, E. H.: The role of transiently fine-grained reaction prodcuts in syntectonic metamorphism: natural and ex- perimental examples, Can. J. Earth Sci., 24, 556–564, 1987. Bunge, H. J.: Texture Analysis in Materials Sciences, Buttleworth, London, UK, 1982. Bürgmann, R. and Dresen, G.: Rheology of the Lower Crust and Upper Mantle: Evidence from Rock Mechanics, Geodesy, and Field Observations, Annu. Rev. Earth Pl. Sc., 36, 531–567, 2008. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1007 Kohlstedt, D. L. and Hansen, L. N.: Constitutive equations, rheolog- ical behavior, and viscosity of rocks, in: Treatise on Geophysics, edited by: Schubert, G., Elsevier, Oxford, UK, 441–472, 2015. plication to transpression-transtension tectonics, J. Struct. Geol., 15, 413–422, 1993. Franke, W. and Ghobarkar, H.: The Morphology of Hydrothermally grown Plagioclase, Cryst. Res. Technol., 17, 459–464, 1982. Kohlstedt, D. L., Evans, B., and Mackwell, S. J.: Strength of the Litosphere: Constraints imposed by laboratory experimetns, J. Geophys. Res.-Sol. Ea., 100, 517–587, https://doi.org/10.1029/95JB01460, 1995. Fukuda, J.-I. and Okudaira, T.: Grain-size-sensitive Creep of Pla- gioclase accompanied by Solution-Precipitation and Mass Trans- fer under Mid-crustal Conditions, J. Struct. Geol., 51, 61–73, 2013. Kolle, J. J. and Blacic, J. D.: Deformation of Single-Crystal Clinopyroxenes: Mechanical Twinning in Diopside and Heden- bergite, J. Geophys. Res., 87, 4019–4034, 1982. Getsinger, A. J. and Hirth, G.: Amphibole fabric formation during diffusion creep and the rheology of shear zones, Geology, 42, 535–538, 2014. Krabbendam, M., Wain, A., and Andersen, T. B.: Pre-Caledonian granulite and gabbro enclaves in the Western Gneiss Region, Norway: indications of incomplete transition at high pressure, Geol. Mag., 137, 235–255, 2000. Gratier, J.-P., Guiget, R., Renard, F., Jenatton, L., and Bernard, D.: A pressure solution creep law for quartz from in- dentation experiments, J. Geophys. Res., 114, B03403, https://doi.org/10.1029/2008JB005652, 2009. Kronenberg, A. K. and Shelton, G. L.: Deformation microstructures in experimentally deformed Maryland Diabase, J. Struct. Geol., 2, 341–353, 1980. Gratier, J.-P., Dysthe, D. K., and Francois, R.: The role of pressure solution creep in the ductility of the Earth’s upper crust, Adv. Geophys., 54, 47–179, 2013. Kruse, R. and Stünitz, H.: Deformation mechanisms and phase dis- tribution in mafic high-temperature mylonites from the Jotun Nappe, southern Norway, Tectonophysics, 303, 223–249, 1999. Handy, M. R. and Stünitz, H.: Strain localization by fracturing and reaction weakening – a mechanism for initiating exhumation of subcontinental mantle beneath rifted margins, Geol. Soc. Lon., Spec. Pub., 200, 387–407, 2002. Kruse, R., Stünitz, H., and Kunze, K.: Dynamic recrystallization processes in plagioclase porphyroclasts, J. Struct. Geol., 23, 1781–1802, 2001. Hawthorne, F., Oberti, R., Harlow, G., Maresch, W., Martin, R., Schumacher, J., and Welch, M.: Nomenclature of the amphibole supergroup, Am. Mineral., 97, 2031–2048, 2012. Lallemant, H. A.: Experimental Deformation of Diopside and Web- sterite, Tectonophysics, 48, 1–27, 1978. Langdon, T. G.: Grain boundary sliding revisited: Developments in sliding over four decades, J. Mater. Sci., 41, 597–609, 2006. Heilbronner, R. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures and Barrett, S.: Image Analysis in Earth Sciences - Microstructures and Textures of Earth Materials, Springer- Verlag, Berlin, Germany, 2014. Lapworth, T., Wheeler, J., and Prior, D. J.: The Deformation of Plagioclase investigated using Electron Backscatter Diffraction Crystallographic Preferred Orientation Data, J. Struct. Geol., 24, 387–399, 2002. Hickman, S. H. and Evans, B.: Experimental pressure solution in halite: the effect of grain/interphase boundary structure, J. Geol. Soc. London, 148, 549–560, 1991. Hier-Majumder, S., Mei, S., and Kohlstedt, D. L.: Water weaken- ing of clinopyroxenite in diffusin creep, J. Geophys. Res., 110, B07406, https://doi.org/10.1029/2004JB003414, 2005. Linckens, J., Herwegh, M., Müntener, O., and Mercolli, I.: Evolu- tion of a polymineralic mantle shear zone and the role of second phases in the localization of deformation, J. Geophys. Res., 116, B06210, https://doi.org/10.1029/2010JB008119, 2011. Imon, R., Okudaira, T., and Fujimoto, A.: Dissolution and precipi- tation processes in deformed amphibolites: an example from the ductile shear zone of the Ryoke metamorphic belt, SW Japan, J. Metamorph. Geol., 20, 297–308, 2002. Linckens, J., Bruijn, R. H., and Skemer, P.: Dynamic recrystalliza- tion and phase mixing in experimentally deformed peridotite, Earth Planet. Sc. Lett., 388, 134–142, 2014. Mainprice, D. and Silver, P. G.: Interpretation of SKS-waves using samples from the subcontinental lithosphere, Phys. Earth Planet. In., 78, 257–280, 1993. Jamtveit, B., Austrheim, H., and Putnis, A.: Disequilibrium meta- morphism of stressed lithosphere, Earth-Sci. Rev., 154, 1–13, 2016. Marsh, J., Johnson, S., Yates, M., and West, J.: Coupling of defor- mation and reactions during mid-crustal shear zone development: an in situ frictional-viscous transition, J. Metamorph. Geol., 27, 531–553, 2009. Ji, S., Jiang, Z., Rybacki, E., Wirth, R., Prior, D. J., and Xia, B.: Strain softening and microstructural evolution of anorthite aggre- gates and quartz-anorthite layered composites deformed in tor- sion, Earth Planet. Sc. Lett., 222, 377–390, 2004. Marti, S., Stünitz, H., Heilbronner, R., Plümper, O., and Drury, M.: Experimental investigation of the brittle-viscous transition in mafic rocks – Interplay between fracturing, reaction, and viscous deformation, J. Struct. Geol., 105, 62–79, 2017. Jiang, Z., Prior, D. J., and Wheeler, J.: Albite crystallographic pre- ferred orientation and grain misorientation distribution in a low- grade mylonite: implications for granular flow, J. Struct. Geol., 22, 1663–1674, 2000. Mauler, A., Bystricky, M., Kunze, K., and Mackwell, S.: Mi- crostructures and lattice preferred orientations in experimentally deformed clinopyroxene aggregates, J. Struct. Geol., 22, 1633– 1648, 2000. Kamb, W. References C., Ord, A., and Hobbs, B.: Microstructures in water-weakened single crystals of quartz, J. Geophys. Res.-Sol. Ea., 96, 2139–2155, 1991. Brander, L., Svahnberg, H., and Piazolo, S.: Brittle-plastic deforma- tion in initially dry rocks at fluid-present conditions: transient be- haviour of feldspar at mid-crustal levels, Contrib. Mineral. Petr., 163, 403–425, 2012. Fossen, H. and Tikoff, B.: The deformation matrix for simultaneous simple shearing, pure shearing and volume change, and its ap- Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1008 tions for Deformation Mechanism and Rock Strength, J. Petrol., 55, 1457–1477, 2014. tions for Deformation Mechanism and Rock Strength, J. Petrol., 55, 1457–1477, 2014. Shaocheng, J. and Mainprice, D.: Experimental deformation of sin- tered albite above and below the ordered-disorder transition, Geodin. Acta, 1, 113–124, 1987. Newman, J., Lamb, W. M., Drury, M. R., and Vissers, R. L. M.: De- formation processes in a peridotite shear zone: reaction-softening by an H2O-deficient, continuous net transfer reaction, Tectono- physics, 303, 193–222, 1999. Shelley, E.: Spider texture and amphibole preferred orientations, J. Struct. Geol., 16, 709–717, 1994. Shigmeatsu, N. and Tanaka, H.: Dislocation creep of fine-grained recrystallized plagioclase under low-temperature conditions, J. Struct. Geol., 22, 65–79, 2000. Okudaira, T., Jerabek, P., H., S., and Fusseis, F.: High-temperature fracturing and subsequent grain-size-sensitive creep in lower crustal gabbros: Evidence for coseismic loading followed by creep during decaying stress in the lower crust?, J. Geophys. Res.-Sol. Ea., 120, 3119–3141, 2015. Smith, J. V.: Feldspar Minerals, vol. 2, Springer, Berlin, Germany, 1974. Stokes, M., Wintsch, R., and Southworth, C.: Deformation of am- phibolites via dissolution-precipitation creep in the middle and lower crust, J. Metamorph. Geol., 30, 723–737, 2012. Olgaard, D. L. and Evans, B.: Effect of Second-Phase Particles on Grain Growth in Calcite, J. Am. Ceram. Soc., 69, 272–277, 1986. Stünitz, H.: Syndeformational recrystallization – dynamic or com- positionally induced?, Contrib. Mineral. Petr., 131, 219–236, 1998. Panozzo Heilbronner, R.: Two-dimensional strain from the orienta- tion of lines in a plane, J. Struct. Geol., 6, 215–221, 1984. Paterson, M. S.: Superplasticity in Geological Materials, Mat. Res. Soc. Symp. Proc., 196, 1341–1358, 1990. Stünitz, H. and Tullis, J.: Weakening and strain localization pro- duced by syn-deformational reaction of plagioclase, Int. J. Earth Sci., 90, 136–148, 2001. Paterson, M. S.: A theory for granular flow accommodated by mate- rial transfer via an intergranular fluid, Tectonophysics, 245, 135– 151, 1995. Stünitz, H., Fitz Gerald, J. D., and Tullis, J.: Dislocation genera- tion, slip systems, and dynamic recrystallization in experimen- tally deformed plagioclase single crystals, Tectonophysics, 372, 215–233, 2003. Paterson, M. S.: Materials Science for Structural Geology, Springer, Dordrecht, the Netherlands, 2013. Precigout, J. and Stünitz, H.: Evidence of phase nucleation during olivine diffusion creep: A new perspective for mantle strain lo- calisation, Earth Planet. Sc. Lett., 455, 94–105, 2016. Sundberg, M. and Cooper, R. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures F.: Crystallographic preferred orientation produced by diffusional creep of harzbur- gite: Effects of chemical interactions among phases during plastic flow, J. Geophys. Res., 113, B12208, https://doi.org/10.1029/2008JB005618, 2008. Putnis, A.: Introduction to mineral sciences, Cambridge University Press, New York, USA, 1992. Raterron, P. and Jaoul, O.: High-temperature deformation of diop- side single crystal, 1. Mechanical data, J. Geophys. Res., 96, 14277–14286, 1991. Svahnberg, H.: Deformation behaviour and chemical signatures of anorthosites: Examples from southern West Greenland and south-central Sweden, PhD thesis, Stockholm University, Stock- holm, Sweden, 2010. Richter, B., Stünitz, H., and Heilbronner, R.: Stresses and pres- sures at the quartz-to-coesite phase transformation in shear- deformation experiments, J. Geophys. Res.-Sol. Ea., 121, JB013084, https://doi.org/10.1002/2016JB013084, 2016. Tikoff, B.: The limitations of three-dimensional kinematic vorticity analysis, J. Struct. Geol., 17, 1771–1784, 1995. Trepmann, C. A., Stöckert, B., Dorner, D., Moghadam, R. H., Küster, M., and Röller, K.: Simulating coseismic deformation of quartz in the middle crust and fabric evolution during postseis- mic stress relaxation – An experimental study, Tectonopyhsics, 442, 83–104, 2007. Rosenberg, C. L. and Stünitz, H.: Deformation and recrystallization of plagioclase along a temperature gradient: an example from the Bergell tonalite, J. Struct. Geol., 25, 389–408, 2003. Rubie, D. C.: The catalysis of mineral reaction by water and re- strictions of the presence of aqueous fluid during metamorphism, Mineral. Mag., 50, 399–415, 1986. Tullis, J. and Yund, A.: Dynamic recrystallization of feldspar: A mechanism for ductile shear zone formation, Geology, 13, 238– 241, 1985. Rubie, D. C.: Disequilibrium during metamorphism: the role of nu- cleation kinetics, Geol. Soc. SP, 138, 199–214, 1998. Tullis, J. and Yund, A.: Diffusion creep in feldspar aggregates: ex- perimental evidence, J. Struct. Geol., 13, 987–1000, 1991. Rutter, E. H., Peach, C. J., White, S. H., and Johnston, D.: Experi- mental “syntectonic” hydration of basalt, J. Struct. Geol., 7, 251– 266, 1985. Viegas, G., Menegon, L., and Archanjo, C.: Brittle grain-size reduc- tion of feldspar, phase mixing and strain localization in granitoids at mid-crustal conditions (Pernambuco shear zone, NE Brazil), Solid Earth, 7, 375–396, https://doi.org/10.5194/se-7-375-2016, 2016. Rybacki, E. and Dresen, G.: Dislocation and diffusion creep of synthetic anorthithe aggregates, J. Geophys. Res.-Sol. Ea., 105, 26017–26036, 2000. Schmid, E. and Boas, W.: Plasticity of Crystals with Special Refer- ence to Metals, F.A. Hughes, London, UK, 1950. Wayte, G. J., Worden, R. H., Rubie, D. C., and Droop, G. T. S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures B.: The thermodynamic theory of nonhydrostatically stressed solid, J. Geophys. Res., 66, 259–271, 1961. Karato, S.-I.: Deformation of Earth Materials – an introduction to the rheology of solid earth, Cambridge University Press, Cam- bridge, UK, 2008. Mehl, L. and Hirth, G.: Plagioclase preferred orientation in layered mylonites: Evaluation of flow laws for the lower crust, J. Geophys. Res.-Sol. Ea., 113, B05202, https://doi.org/10.1029/2007JB005075, 2008. Kilian, R., Heilbronner, R., and Stünitz, H.: Quartz grain size re- duction in a granitoid rock and the transition from dislocation to diffusion creep, J. Struct. Geol., 33, 1265–1284, 2011. Mukai, H., Austrheim, H., Putnis, C. V., and Putnis, A.: Textural Evolution of Plagioclase Feldspar across a Shear Zone: Implica- www.solid-earth.net/9/985/2018/ Solid Earth, 9, 985–1009, 2018 S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures R.: A TEM study of disequilibrium plagioclase breakdown at high pressure: the role of infiltrating fluid, Contrib. Mineral. Petr., 101, 426–437, 1989. Schott, J., Brantley, S., Crerar, D., Guy, C., Borcsik, M., and Willaime, C.: Dissolution kinetics of strained calcite, Geochem. Cosmochim. Ac., 53, 373–382, 1989. Schwarz, S. and Stöckhert, B.: Pressure solution in siliciclastic HP- LT metamorphic rocks – constraints on the state of stress in deep levels of accretionary complexes, Tectonophysics, 255, 203–209, 1996. Wheeler, J.: Importance of Pressure Solution and Coble Creep in the Deformation of Polymineralic Rocks, J. Geophys. Res., 97, 4579–4586, 1992. Wheeler, J.: Dramatic effects of stress on metamorphic reactions, Geology, 42, 647–650, 2014. Shaocheng, J. and Bin, X.: Rheology of polyphase earth materials, Polytechnic International Press, Montreal, Canada, 2002. Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/ www.solid-earth.net/9/985/2018/ Wheeler, J.: The effects of stress on reactions in the Earth: some- times rather mean, usually normal, always important, J. Meta- morph. Geol., 36, 439–461, 2018. www.solid-earth.net/9/985/2018/ S. Marti et al.: Dissolution precipitation creep in experimentally deformed plagioclase–pyroxene mixtures 1009 Xie, Y., Wenk, H.-R., and Matthies, S.: Plagioclase preferred ori- entation by TOF neutron diffraction and SEM-EBSD, Tectono- physics, 370, 269–286, 2003. p Whitney, D. and Evans, B. W.: Abbrevations for names of rock- forming minerals, Am. Mineral., 95, 185–187, 2010. Yund, R. A. and Tullis, J.: Compositional change of minerals as- sociated with dynamic recrystallization, Contrib. Mineral. Petr., 108, 346–355, 1991. Wintsch, R. P.: The Possible Effects of Deformation on Chemical Processes in Metamorphic Fault Zones, in: Advances in Physical Geochemistry, 4, Springer-Verlag, New York, USA, 1985. Zhang, J., Green, H. W., and Bozhilov, K. N.: Rheology of om- phacite at high temperature and pressure and significance of its lattice preferred orientations, Earth Planet. Sc. Lett., 246, 432– 443, 2006. Wintsch, R. P. and Yi, K.: Dissolution and replacement creep: a sig- nificant deformation mechanism in mid-crustal rocks, J. Struct. Geol., 24, 1179–1193, 2002. Solid Earth, 9, 985–1009, 2018 www.solid-earth.net/9/985/2018/
https://openalex.org/W3011255683
http://www.scielo.br/pdf/bjps/v56/2175-9790-bjps-56-e18271.pdf
English
null
Nonsteroidal anti-inflammatory drugs as potential ecto-nucleotide phosphodiesterase inhibitors
Brazilian Journal of Pharmaceutical Sciences
2,020
cc-by
4,205
Article Brazilian Journal of Pharmaceutical Sciences http://dx.doi.org/10.1590/s2175-97902019000318271 ABBREVIATIONS 2001). Including all analgesics and antipyretics, these are the most widely used medications globally, i.e. 30% of all medicines used (Litalien, Jacqz-Aigrain, 2001). NSAIDs are used in management of fever, acute or chronic pain and inflammation in a wide spectrum of diseases. Their effectiveness has been proven in various clinical conditions, including osteoarthritis, rheumatoid arthritis, gout, dysmenorrhea, ankylosing spondylitis, headache and dental pain (Zochling et al., 2006; Kean, Buchanan, 2005). NSAIDs- Non-steroidal Antiinflammatory Drugs cAMP- Cyclic Adenosine monophosphate cGMP-Cyclic guanosine monophosphate COX-Cyclooxygenase PDE- Phosphodiesterase NSAIDs- Non-steroidal Antiinflammatory Drugs cAMP- Cyclic Adenosine monophosphate cGMP-Cyclic guanosine monophosphate COX-Cyclooxygenase PDE- Phosphodiesterase NSAIDs- Non-steroidal Antiinflammatory Drugs cAMP- Cyclic Adenosine monophosphate cGMP-Cyclic guanosine monophosphate COX-Cyclooxygenase PDE- Phosphodiesterase NSAIDs- Non-steroidal Antiinflammatory Drugs cAMP- Cyclic Adenosine monophosphate cGMP-Cyclic guanosine monophosphate COX-Cyclooxygenase PDE- Phosphodiesterase PDE- Phosphodiesterase Shumaila Tasneem 1,3*, Muhammad Saleem2,4, Sheikh Arshad Saeed1 (Late) 1Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan, 2H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan, 3Department of Pharmacology, Faculty of Pharmaceutical Sciences, Dow College of Pharmacy, Dow University of Health Sciences. Gulzar-e-Hijri, Karachi, Pakistan, 4Department of Chemistry, University of Education, Lahore, Dera Ghazi Khan Campus, Dera Ghazi Khan, Punjab, Pakistan Phosphodiesterases (PDE) are group of enzymes which catalyze the hydrolysis of cAMP and cGMP. Since these cyclic phosphate moieties worked as intracellular second messengers in numerous physiological processes, their inhibition can affect normal physiology of living system. NSAIDs are among the frequently prescribed medications, because of their efficacy as analgesic, antipyretic and anti-inflammatory agents. They are known to block cyclooxygenase pathway. In limited data NSAIDs has been shown anti-tumor potential, and phosphodiesterase inhibition has assumed to be one of the mechanism. To date no further evaluation being done. Further, NSAIDs are classified as cyclooxygenase inhibitors and phosphodiesterase inhibition can imprint its side effects. This study first time investigates the effects of NSAIDs on phosphodiesterase 1 inhibition. The activity against snake venom phosphodiesterase 1 was assayed on a microtitre plate reader spectrophotometer. Selective COX-2 inhibitor, celecoxib, exhibited a potent PDE1 inhibitory activity, at therapeutic doses, with an IC50 value of 29.4 µM. The findings of our study are indicative of new pharmacological actions of cyclooxygenase inhibitors. This article presents the PDE inhibitory properties as a new effects of already existing drugs. These additional effects could be potentially helpful for researchers to assess other physiological and pathological states. Keywords: Non-steroidal antiinflammatory drugs. Cyclooxygenase. Phosphodiesterase. Inflammation cAMP. *Correspondence: S. A. Tasneem. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Dow College of Pharmacy, Dow University of Health Sciences, Gulzar-e-Hijri, Ojha Campus, Suparco Road, KDA Scheme-33, Karachi, Pakistan. Tel.: +92 21 38771111, +92 21 99232660 (2427), +92 21 99261472-9 021-99261495-6 (2427). E-mail: pharmacist_online@hotmail.com *Correspondence: S. A. Tasneem. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Dow College of Pharmacy, Dow University of Health Sciences, Gulzar-e-Hijri, Ojha Campus, Suparco Road, KDA Scheme-33, Karachi, Pakistan. Tel.: +92 21 38771111, +92 21 99232660 (2427), +92 21 99261472-9 021-99261495-6 (2427). E-mail: pharmacist_online@hotmail.com Nonsteroidal anti-inflammatory drugs as potential ecto-nucleotide phosphodiesterase inhibitors Shumaila Tasneem 1,3*, Muhammad Saleem2,4, Sheikh Arshad Saeed1 (Late) Phosphodiesterase Ecto-nucleotide pyrophosphatases or Cyclic nucleotide pyrophosphatases or cyclic nucleotide phosphodiesterases are isoenzymes that catalyze the hydrolysis of the 3′, 5′-cyclic phosphate moiety of cAMP and/or cGMP to their 5′-nucleotide monophosphatase (Figure 1). Ligand binding to G-coupled protein receptor leads to activation of adenylate cyclase which is involved in conversion of ATP to cAMP. This action is terminated by PDE which hydrolyze the cAMP thus blocking the numerous physiological events (Figure 2). cAMP and cGMP are substrates for PDE1, and served as intracellular second messengers in the regulation of various physiological stimuli. This includes cardiac contractility, cardiac output, smooth muscle relaxation, neurodegeneration, vascular resistance, visceral motility, reduced immune and inflammatory activity of cells, neuroplasticity, reproduction and vision (Li, Yee, Beavo, et al., 1999; Dousa, 1999). FIGURE 2 - G-Protein coupled receptor binding to ligand activates adenylate cyclase which form cAMP by ATP. Reaction is catalyzed by phosphodiesterase. Inhibition of phosphodiesterase leads to various physiological functions. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; 5’AMP, 5’ Adenosine monophosphate. nucleotide phosphodiesterase, was first reported by Kakiuchi and Yamazaki in 1970 in rat brain (Kakiuchi, Yamazaki, 1970). In early 1970’s, the existence of protein activator “calmodulin” (CaM) was established which stimulates the PDE activity several folds. Three genes (PDE1A, PDE1B and PDE1C), with various splice variants, constitute PDE1 family. For the stimulation of PDE1 activity, the presence of both Ca2+ and calmodulin is required. Activity of PDE1 is triggered by binding of Ca2+ to free CaM to convert protein from its inactive state to activated conformation, which then associates with PDE1 to form active PDE. Activity of PDE1 is regulated by several different mechanisms, e.g. stimulate PDE1 phosphorylation, block phosphorylation and reverse phosphorylation. nucleotide phosphodiesterase, was first reported by Kakiuchi and Yamazaki in 1970 in rat brain (Kakiuchi, Yamazaki, 1970). In early 1970’s, the existence of protein activator “calmodulin” (CaM) was established which stimulates the PDE activity several folds. Three genes (PDE1A, PDE1B and PDE1C), with various splice variants, constitute PDE1 family. For the stimulation of PDE1 activity, the presence of both Ca2+ and calmodulin is required. Activity of PDE1 is triggered by binding of Ca2+ to free CaM to convert protein from its inactive state to activated conformation, which then associates with PDE1 to form active PDE. Activity of PDE1 is regulated by several different mechanisms, e.g. stimulate PDE1 phosphorylation, block phosphorylation and reverse phosphorylation. INTRODUCTION In brief, the NSAIDs include two groups, nonselective NSAIDs that block both cyclooxygenases-1 and -2, and COXIBs are selective COX-2 inhibitors. Previous studies with NSAIDs suggested that cGMP specific phosphodiesterase inhibition is an important COX-independent mechanism to suppress β-catenin signaling (Thompson et al., 2000). FIGURE 1 - PDE catalyzes the cleavage in the 3′- to 5′-direction to yield nucleoside 5′-phosphates. FIGURE 1 - PDE catalyzes the cleavage in the 3′- to 5′-direction to yield nucleoside 5′-phosphates. FIGURE 2 - G-Protein coupled receptor binding to ligand activates adenylate cyclase which form cAMP by ATP. Reaction is catalyzed by phosphodiesterase. Inhibition of phosphodiesterase leads to various physiological functions. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; 5’AMP, 5’ Adenosine monophosphate. Phosphodiesterase Eleven members of phosphodiesterase (PDE) superfamily are identified uptil now. PDE4, PDE7 and PDE8 are specific for catalyzing the cleavage of cAMP, PDE5, PDE6 and PDE9 are specific for cGMP only. While PDE1, PDE2, PDE3, PDE10 and PDE11 can handle both the nucleotides as substrates (Dousa, 1999). Phosphodiesterase 1 INTRODUCTION The basic mode of antiinflammatory actions of NSAIDs has been attributed to inhibition of the biosynthesis of prostaglandins, through the inhibition of key enzyme namely ‘cyclooxygenase (COX)’ (Vane, Botting, 1998; Vane, 1971). The inhibition of COX by NSAIDs yields clinical benefits and low toxicity (Fries et al., 2004). Cyclooxygenases (COX) are involved in the formation of prostaglandins (PG), which further role in many physiological conditions, such as the NSAIDs are among the most frequently prescribed medications because of their demonstrated efficacy as analgesic, antipyretic and antiinflammatory agents (Laine, Page 1 / 6 Page 1 / 6 Page 1 / 6 Braz. J. Pharm. Sci. 2020;56:e18271 S. Tasneem, M. Saleem, S. A. Saeed FIGURE 1 - PDE catalyzes the cleavage in the 3′- to 5′-direction to yield nucleoside 5′ phosphates dilation and constriction of blood vessels, contraction and relaxation of smooth muscles, control of blood pressure, and regulation of inflammation (Tasneem et al., 2019). It was previously believed that the effects of NSAIDs are due to the blockade of a single cyclooxygenase enzyme, later, the two distinct isoenzymes, namely COX-1 and COX-2 were discovered. Extensive studies now indicate that the clinical efficacy of NSAIDs are primarily due to the inhibition of COX-2, whereas the undesirable effects, e.g. gastric and intestinal mucosal damage and renal toxicity are due to the inhibition of COX-1 (Vane and Botting, 1998). Thus the specific COX-2 inhibitors lack the toxic effects of conventional NSAIDs. In brief, the NSAIDs include two groups, nonselective NSAIDs that block both cyclooxygenases-1 and -2, and COXIBs are selective COX-2 inhibitors. Previous studies with NSAIDs suggested that cGMP specific phosphodiesterase inhibition is an important COX-independent mechanism to suppress β-catenin signaling (Thompson et al., 2000). dilation and constriction of blood vessels, contraction and relaxation of smooth muscles, control of blood pressure, and regulation of inflammation (Tasneem et al., 2019). It was previously believed that the effects of NSAIDs are due to the blockade of a single cyclooxygenase enzyme, later, the two distinct isoenzymes, namely COX-1 and COX-2 were discovered. Extensive studies now indicate that the clinical efficacy of NSAIDs are primarily due to the inhibition of COX-2, whereas the undesirable effects, e.g. gastric and intestinal mucosal damage and renal toxicity are due to the inhibition of COX-1 (Vane and Botting, 1998). Thus the specific COX-2 inhibitors lack the toxic effects of conventional NSAIDs. Material Inhibition of PDE1 may induce apoptosis in human leukemic cells (Zhang, Wang, Sharma, 1993). It is highly expressed in proliferating smooth muscle cells and is believed to be a major regulator of smooth muscle proliferation (Rotella, 2002). It also promotes arterial smooth muscle cell proliferation, hence could be beneficial in pathophysiology of atherosclerosis. According to some studies, it may also be involved in neuronal signaling (Sharma, Kalra, 1994). Clinically available PDE1 inhibitor, vinpocetine, is known to improve memory. Recently phosphodiesterases have attracted a major scientific attention as potential targets for PDE inhibitor- based antiparasitic drugs.l Bis(p-nitrophenyl) phosphate sodium salt and phosphodiesterase 1 enzyme were purchased from Sigma Chemical Co. (USA). EDTA was purchased from Merck (Germany). Aspirin, diclofenac, etodolac, flurbiprofen, meloxicam, naproxen, nimesulide, and piroxicam were purchased from Sigma chemical Co. (USA). Celecoxib was purchased from Bosch Pharmaceuticals (Pakistan). Ibuprofen was purchased from MP Biomedicals, Inc. (France). Indomethacin was purchased from TCI (Japan). NS-398 was purchased from Wako (Japan). The instrument used was microtitre plate reader spectrophotometer, Molecular Devices (USA). All the reagents used for buffer preparation were of analytical grade. It is well known fact that numerous inflammatory mediators are produced during pro- and anti- inflammatory pathways, and among them TNFα and IL1β are well known to alter the cAMP levels and they are thought to do so primarily by way of an upregulation of cAMP- specific PDE expression and activity (Ghosh et al., 2012). There are several lines of evidences suggest the role of COX independent targets for NSAIDs during cancer, apoptosis and neurodegenration (Grösch et al., 2006; Elder et al., 1997; Weggen et al., 2001). One of the COX- independent mechanisms that has been proposed for the anticancer activity of NSAIDs involves inhibition of cGMP PDEs, resulting in increased intracellular cGMP levels and activation of cGMP signaling (Tinsley et al., 2010). There are several studies that demonstrate NSAIDs attenuating their pleiotropic effects by altering the cyclic monophosphates. Tinsley and his co-workers have shown that the NSAID inhibits PDE5 activity in colon tumor cell lysates (Tinsley et al., 2010). Another report suggest that NSAIDs inhibit the PDE and tumor cell growth (Piazza et al., 2005; Thompson et al., 2000). There are studies that show the role of COX inhibitors as PDE inhibitors but, to date, no data has been reported on extra-pharmacological effects of NSAIDs, as specific PDE1 inhibitors. Material During this study, we aim to find out the effects of COX inhibitors on phosphodiesterase1 enzyme. Phosphodiesterase 1 Nucleotide pyrophosphatases/phosphodiesterases (NPP1) or oligonucleate 5′- nucleotidohydrolase or PDE1, also called as Ca2+/calmodulin – dependent cyclic Existence of PDE1 has been established in most mammalian tissues. Mammalian brain contains the highest Page 2 / 6 Page 2 / 6 Braz. J. Pharm. Sci. 2020;56:e18271 Nonsteroidal anti-inflammatory drugs as potential ecto-nucleotide phosphodiesterase inhibitors CaM-PDE activities. Additionally, PDE1 activity has been detected in other tissues, including heart, smooth muscles, lungs, and pancreas as well as in several other eukaryotes. This enzyme has also been presented in numerous immune cell types, such as lymphocytes and monocytes. The presence of PDE1 in many tissues and organisms highlights its importance and its pivotal role in signal transduction. PDE1 plays a significant role in a numerous physiological and pathological functions. It serves to regulate vascular smooth muscle contraction and participates in some interleukin regulation related allergic diseases (Sharma, Wang, 1986). CaM-PDE activities. Additionally, PDE1 activity has been detected in other tissues, including heart, smooth muscles, lungs, and pancreas as well as in several other eukaryotes. This enzyme has also been presented in numerous immune cell types, such as lymphocytes and monocytes. The presence of PDE1 in many tissues and organisms highlights its importance and its pivotal role in signal transduction. PDE1 plays a significant role in a numerous physiological and pathological functions. It serves to regulate vascular smooth muscle contraction and participates in some interleukin regulation related allergic diseases (Sharma, Wang, 1986). The aim of the work has been to find out the pleiotropic effects of the drugs that could possibly be interfering with their known or intended therapeutic effects. We believe that these findings will contribute both in basic research, as well as in clinical development. Further clinical study for the PDE enzyme inhibition activity of celecoxib can provide beneficial effects in thrombosis and neurodegenerative conditions. Method Nucleotide pyrophosphatases / phosphodiesterases 1 inhibition assay RESULTS AND DISCUSSION Addition of celecoxib to PDE mixture at pharmacological concentrations (1 – 400 µM) inhibited the phosphodiesterase enzyme. The results show that celecoxib inhibits PDE with an IC50= 29.43 ± 0.23 µM. 50 To examine whether the celecoxib mediated inhibition of snake venom PDE is a general property of COX inhibitors or is specific to celecoxib, we evaluated other inhibitors of COX-1 and COX-2 such as aspirin, diclofenac, etodolac, flurbiprofen, ibuprofen, indomethacin, meloxicam, naproxen, nimesulide, NS-398 and piroxicam. None of these compounds in a wide range of concentration showed any inhibitory effects on PDE. Instead, nimesulide, a selective COX-2 inhibitor, exhibited significant inhibitory activity against PDE1. The results show that nimesulide inhibits the PDE enzyme with an IC50 = 440.4 ± 5.92 µM (Table I). TABLE I - Effects of COX inhibitors on phosphodiesterase 1 inhibition TABLE I - Effects of COX inhibitors on phosphodiesterase 1 inhibition Drugs IC50 (µM) ± SEM % Inhibition 1. Celecoxib 29.43 ± 0.23 84.8 2. Nimesulide 440.40 ± 5.92 97.2 EDTA Std. 277.69 ± 2.72 69.0 g y p Celecoxib and nimesulide are specific COX‑2 inhibitors, thus are more specific in reducing the inflammation. Celecoxib is a selective COX-2 inhibitor which was tested previously for its effects on phosphodiesterases types 4 and 5 (Soh et al., 2008; Klein et al., 2007). In particular, our results demonstrate the inhibitory effects of both COX-2 inhibitors on PDE1. Neuronal transmission through glutamate leads to an increase of intracellular Ca++, and cAMP/cGMP levels, which further triggers the signaling pathways that will eventually lead to the activation of the transcription factors serum response factor and CREB (cAMP responsive element binding protein) (Vitolo et al, 2002). Since PDE I inhibition potentiates the glutamatergic transmission and improves the neuronal plasticity and memory functions and our study shows the celecoxib as strong inhibitor of PDE, lead to the conclusion that celecoxib may also be involved in potentiating glutamatergic transmissions. Similarly, Nimesulide is NSAID belong to the aryl group (sulfonanilide class) and it seems to exert its effects through several mechanisms. Our data suggest strong inhibitory activity of nimesulide on PDE1 in dose-response dependent manner. A better understanding and more studies on clinical patients might help to identify the role Progression of inflammation involve certain pro- and anti- inflammatory signaling pathways which results in the synthesis and activation of numerous inflammatory mediators (Serhan, Chiang, Van Dyke, 2008). Nucleotide pyrophosphatases / phosphodiesterases 1 inhibition assay In this assay, the activity against the snake venom phosphodiesterase 1 (EC 3.1.4.1) was assayed by using the reported method (Razzell, Khorana, 1959) with the following modifications. Tris-HCl buffer 33 mM (pH 8.8), 30 mM Mg-acetate as a cofactor was added with 0.000742 U of enzyme phosphodiesterase 1 as a final concentrations using 96-well flat bottom plate and 0.33 mM bis(p‑nitrophenyl) phosphate as a substrate. EDTA was used as positive controls having IC50 = 277.69 ± 2.52 µM. After 30 min of incubation, the enzyme activity was monitored at 37 °C on a microtitre plate reader spectrophotometer (Molecular Devices, USA) by following the release of p-nitrophenol from p-nitrophenyl phosphate at 410 nm. All the reactions were performed in triplicate and the initial rates were measured as the rates of changes in the OD/min (optical density/min) and used in subsequent calculations. The % inhibition was calculated by using the formula: Braz. J. Pharm. Sci. 2020;56:e18271 Page 3 / 6 Page 3 / 6 S. Tasneem, M. Saleem, S. A. Saeed % Inhibition = 100-(OD of test/OD of control × 100). to regulate the vascular smooth muscles contraction and proliferation and can be effective in atherosclerosis (Chan, Yan, 2011). Presence of PDE1 in mammalian brain makes it a target for the discovery of drugs for treatment or management of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases (Medina, 2011). PDE1 inhibition increases the levels of cAMP/cGMP in brain which leads to increase expression of neuronal plasticity-related genes and neuroprotective molecules. These make PDE1 inhibitory agents important drug candidates for the treatment of neurological conditions. On the other hand, inflammation also has a close relation with the onset and progression of various neurodegenerative diseases. For instance, activation of astrocytes, microglia, lymphocytes, and macrophages results in an increase release of cytokines, chemokines, neurotransmitters and ROS (Tansey, McCoy, Frank-Cannon, 2007). Uptil now the suggested possible mechanisms of action of NSAIDs in Alzheimer’s disease are to alter the levels of Aβ1-42 (Weggen et al., 2001), activation of PPARγ (Jaradat et al., 2001), inhibition of Ras and Ras-like GTP-binding proteins, and inhibiting multidrug resistance protein-4 (MRP-4). These evidences supported the role of inflammation in the pathogenesis of Alzheimer’s disease. The classical inhibitor of PDE1, vinpocetine, has also been used in the treatment of cerebrovascular disorders and age-related memory impairments. IC50 Values were calculated by using the EZ-fit enzyme kinetics software from USA. RESULTS AND DISCUSSION Among those mediators, TNFα and IL1β are well known to alter the cAMP levels and they are thought to do so primarily by way of an upregulation of cAMP-specific PDE expression and activity (Ghosh et al., 2012). To date, no data has been reported about NSAIDs extra-pharmacological effects including PDE1 inhibition. During our studies we observed that NSAIDs are inhibitors of PDE1 isoenzyme. PDE are isoenzymes which catalyze the hydrolysis of 3’, 5’-cyclic phosphate moiety from cAMP and cGMP. PDE1 activity has been detected in many tissues which highlights its role in signal transduction in numerous physiological and pathological conditions (Dousa, 1999). Controlling or modulating the activity of PDE1 could be beneficial in many ways. For example, PDE1 serves Page 4 / 6 Braz. J. Pharm. Sci. 2020;56:e18271 Nonsteroidal anti-inflammatory drugs as potential ecto-nucleotide phosphodiesterase inhibitors Jaradat MS, Wongsud B, Phornchirasilp S, Rangwala SM, Shams G, Sutton M, Romstedt KJ, Noonan DJ, Feller DR. Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H2 synthases by ibuprofen, naproxen, and indomethacin. Biochem Pharmacol. 2001;62(12):1587-1595. of celecoxib and nimesulide in neurodegenerative diseases and atherosclerosis and to discover new indications of old drugs in the treatment of such diseases. f Jaradat MS, Wongsud B, Phornchirasilp S, Rangwala SM, Shams G, Sutton M, Romstedt KJ, Noonan DJ, Feller DR. Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H2 synthases by ibuprofen, naproxen, and indomethacin. Biochem Pharmacol. 2001;62(12):1587-1595. Our study clearly demonstrates the inhibitory effects of COX-2 inhibitors over phosphodiesterase enzyme. A better understanding and more studies on clinical patients might help to identify the role of celecoxib and nimesulide in Alzheimer’s disease and atherosclerosis and to discover new indications of old drugs in the treatment of these diseases. Kakiuchi S, Yamazaki R. Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain: studies on cyclic 3′, 5′-nucleotide phosphodiesterase (III). Biochem Biophys Res Comm. 1970;41(5):1104-1110. ACKNOWLEDGEMENTS Kean WF, Buchanan WW. The use of NSAIDs in rheumatic disorders: a global perspective. Inflammopharmacology. 2005;13(4):343-370. We show our special gratitude for International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan, for all technical support and funding. Klein T, Eltze M, Grebe T, Hatzelmann A, Kömhoff M. Celecoxib dilates guinea-pig coronaries and rat aortic rings and amplifies NO/cGMP signaling by PDE5 inhibition. Cardiovasc Res. 2007;75(2):390-397. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature. 1971;231(25):232-235. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature. 1971;231(25):232-235. Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol. 2008;8(5):349-361. Vane JR, Botting RM. Mechanism of action of nonsteroidal anti- inflammatory drugs. Am J Med. 1998;104(3):2S-8S. Sharma RK, Kalra J. Characterization of calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes. Biochem J. 1994;299(Pt 1):97-100. Vitolo OV, Sant’Angelo A, Costanzo V, Battaglia F, Arancio O, Shelanski M. Amyloid β-peptide inhibition of the PKA/ CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling. Proc Nat Acad Sci. 2002;99(20):13217-21. Sharma RK, Wang J. Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit. J Biol Chem. 1986;261(30):14160-66. Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietrzik CU, et al. A subset of NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase activity. Nature. 2001;414(6860):212-216. Soh JW, Kazi JU, Li H, Thompson WJ, Weinstein IB. Celecoxib‐ induced growth inhibition in SW480 colon cancer cells is associated with activation of protein kinase G. Mol Carcinogen. 2008;47(7):519-525. Zhang GY, Wang JH, Sharma RK. Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme. Mol Cell Biochem. 1993;122(2):159-169. Tansey MG, McCoy MK, Frank-Cannon TC. Neuroinflammatory mechanisms in Parkinson’s disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp Neurol. 2007;208(1):1-25. Tasneem S, Liu B, Li B, Choudhary MI, Wang W. Molecular pharmacology of inflammation: Medicinal plants as anti- inflammatory agents. Pharmacological research. 2019;139:126- 40. Zochling J, van der Heijde D, Dougados M, Braun J. Current evidence for the management of ankylosing spondylitis: a systematic literature review for the ASAS/EULAR management recommendations in ankylosing spondylitis. Ann Rheum Dis. 2006;65(4):423-432. Thompson WJ, Piazza GA, Li H, Liu L, Fetter J, Zhu B. Exisulind induction of apoptosis involves guanosine 3’,5’-cyclic monophosphate phosphodiesterase inhibition, protein kinase G activation, and attenuated beta-catenin. Cancer Res. 2000;60(13):3338-42. Received for publication on 10th April 2018 Accepted for publication on 23th September 2018 Received for publication on 10th April 2018 Accepted for publication on 23th September 2018 Tinsley HN, Gary BD, Thaiparambil J, Li N, Lu W, Li Y, Maxuitenko YY, Keeton AB, Piazza GA. Colon tumor cell growth-inhibitory activity of sulindac sulfide and other nonsteroidal anti-inflammatory drugs is associated with phosphodiesterase 5 inhibition. Cancer Prev Res. Braz. J. Pharm. Sci. 2020;56:e18271 REFERENCES Chan S, Yan C. PDE1 isozymes, key regulators of pathological vascular remodeling. Curr Opin Pharmacol. 2011;11(6):720- 724. Chan S, Yan C. PDE1 isozymes, key regulators of pathological vascular remodeling. Curr Opin Pharmacol. 2011;11(6):720- 724. Laine L. Approaches to nonsteroidal anti-inflammatory drug use in the high-risk patient. Gastroenterology. 2001;120(3):594-606. Li L, Yee C, Beavo JA. CD3-and CD28-dependent induction of PDE7 required for T cell activation. Science. 1999;283(5403):848-851. Dousa TP. Cyclic-3′, 5′-nucleotide phosphodiesterase isozymes in cell biology and pathophysiology of the kidney. Kidney Int. 1999;55(1):29-62. Litalien C, Jacqz-Aigrain E. Risks and benefits of nonsteroidal anti-inflammatory drugs in children. Paediatric Drugs. 2001;3(11):817-58. Elder DJ, Halton DE, Hague A, Paraskeva C. Induction of apoptotic cell death in human colorectal carcinoma cell lines by a cyclooxygenase-2 (COX-2)-selective nonsteroidal anti-inflammatory drug: independence from COX-2 protein expression. Clin Cancer Res. 1997;3(10):1679-83. Medina AE. Therapeutic utility of phosphodiesterase type I inhibitors in neurological conditions. Front Neurosci. 2011;5:21. Fries JF, Murtagh KN, Bennett M, Zatarain E, Lingala B, Bruce B. The rise and decline of nonsteroidal antiinflammatory drug– associated gastropathy in rheumatoid arthritis. Arthr Rheum. 2004;50(8):2433-2440. Piazza GA, Keeton A, Able C, Ross L, Payne P, Maxuitenko YY, et al. Correlation between colon tumor cell growth inhibition and phosphodiesterase inhibition among specific chemical classes of NSAIDs and COX-2 inhibitors. Cancer Res. 2005;65(9):175. Ghosh M, Garcia-Castillo D, Aguirre V, Golshani R, Atkins CM, Bramlett HM, Dietrich WD, Pearse DD. Proinflammatory cytokine regulation of cyclic AMP-phosphodiesterase 4 signaling in microglia in vitro and following CNS injury. Glia. 2012;60(12):1839-59. Ghosh M, Garcia-Castillo D, Aguirre V, Golshani R, Atkins CM, Bramlett HM, Dietrich WD, Pearse DD. Proinflammatory cytokine regulation of cyclic AMP-phosphodiesterase 4 signaling in microglia in vitro and following CNS injury. Glia. 2012;60(12):1839-59. Razzell W, Khorana H. Studies on polynucleotides III. Enzymic degradation. Substrate specificity and properties of snake venom phosphodiesterase. J Biol Chem. 1959;234(8):2105-13. Rotella DP. Phosphodiesterase 5 inhibitors: current status and potential applications. Nat Rev Drug Discov. 2002;1(9):674- 682. Grösch S, Maier TJ, Schiffmann S, Geisslinger G. Cyclooxygenase-2 (COX-2)–independent anticarcinogenic effects of selective COX-2 inhibitors. J Nat Cancer Inst. 2006;98(11):736-747. Braz. J. Pharm. Sci. 2020;56:e18271 Page 5 / 6 Page 5 / 6 This is an open-access article distributed under the terms of the Creative Commons Attribution License. S. Tasneem, M. Saleem, S. A. Saeed Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature. 1971;231(25):232-235. 2010;8:1940- 6207. Page 6 / 6 Braz. J. Pharm. Sci. 2020;56:e18271
https://openalex.org/W2800316743
https://mural.maynoothuniversity.ie/14778/1/15-1-3.pdf
English
null
Study of Higgs effective couplings at electron-proton colliders
Physical review. D/Physical review. D.
2,018
cc-by
9,221
(Received 24 June 2017; revised manuscript received 7 March 2018; published 30 May 2018) We perform a search for beyond-the-Standard-Model (BSM) dimension-six operators relevant to the Higgs boson at the Large Hadron Electron Collider (LHeC) and the Future Circular Hadron Electron Collider (FCC-he). With a large amount of data (few ab−1) and collisions at the TeV scale, both LHeC and FCC-he provide excellent opportunities to search for the BSM effects. The study is done through the process e−p →hjνe, where the Higgs boson decays into a pair of b¯b, and we consider the main sources of background processes, including a realistic simulation of detector effects. For the FCC-he case, in some signal scenarios, to obtain an efficient event reconstruction and to have a good background rejection, jet substructure techniques are employed to reconstruct the boosted Higgs boson in the final state. In order to assess the sensitivity to the dimension-six operators, a shape analysis on the differential cross sections is performed. Stringent bounds are found on the Wilson coefficients of dimension-six operators with the integrated luminosities of 1 ab−1 and 10 ab−1, which in some cases show improvements with respect to the high-luminosity LHC results. DOI: 10.1103/PhysRevD.97.095041 PHYSICAL REVIEW D 97, 095041 (2018) PHYSICAL REVIEW D 97, 095041 (2018) Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. DOI: 10.1103/PhysRevD.97.095041 Published by the American Physical Society Study of Higgs effective couplings at electron-proton colliders Hoda Hesari,1,* Hamzeh Khanpour,2,1,† and Mojtaba Mohammadi Najafabadi1,‡ 1School of Particles and Accelerators, Institute for Research in Fundamental Sciences (IPM P.O. Box 19395-5531, Tehran, Iran 2Department of Physics, University of Science and Technology of Mazandaran, P.O. Box 48518-78195, Behshahr, Iran *h.hesari@ipm.ir †Hamzeh.Khanpour@mail.ipm.ir ‡mojtaba@ipm.ir Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. I. INTRODUCTION The inclusive Higgs boson production cross section at the high-energy FCC-he is *h.hesari@ipm.ir †Hamzeh.Khanpour@mail.ipm.ir ‡mojtaba@ipm.ir 095041-1 2470-0010=2018=97(9)=095041(12) Published by the American Physical Society HESARI, KHANPOUR, and NAJAFABADI PHYS. REV. D 97, 095041 (2018) this section. Finally, Sec. VII presents the summary and conclusions. expected to be about 5 times larger than at the future proposed high-energy and high-luminosity electron- positron collider TLEP/FCC-ee [41]. In comparison with the LHC or FCC-hh, the LHeC or FCC-he have the advantage of providing a clean environment with small background contributions from QCD strong interactions. Furthermore, no effects from pileup or multiple interactions exist in these machines, and they are able to provide precise measurements of the proton structure, and electroweak and strong interactions. I. INTRODUCTION The operators are composed of all possible combinations of SM fields respecting the SUð3Þc × SUð2ÞL × Uð1ÞY gauge symmetries and Lorentz invariance. In the EFT approach, potential deviations from the SM could be described using the following Lagrangian: So far, the Standard Model (SM) of particle physics has been found to be a successful theory describing nature up to the electroweak scale. However, there are reasons to believe that the SM is not the ultimate theory of particle physics at the TeV scale. The Higgs boson discovery by the LHC experiments [1,2] has been a milestone in understanding the mechanism of electroweak symmetry breaking (EWSB). After that, one of the main goals would be to precisely measure the Higgs boson properties that would provide the possibility of searching for new physics effects beyond the SM. LSM EFT ¼ LSM þ X i ci Λ2 Oi þ H:c:; ð1Þ ð1Þ where Oi is the ith dimension-six operator, Λ is the scale at which new physics is expected to appear, and c0 i’s are arbitrary Wilson coefficients. These dimension-six oper- ators have been listed and studied in Refs. [3–6]. There have been many studies that probe these operators, and so far much work has been devoted to constraining these operators, which can be found in Refs. [7–33]. Given the absence of any signature of new physics in the present data, one can parametrize the effects of beyond-the- SM in an effective field theory (EFT) expansion. This approach is a powerful tool that parametrizes possible new physics effects via a systematic expansion in a series of higher-dimensional operators composed of SM fields [3,4]. The aim of this study is to explore Wilson coefficients of dimension-six operators, as described in Refs. [34–36], contributing to the Higgs production in association with a jet and a neutrino at the LHeC and FCC-he [37–40]. The LHeC is a proposed deep inelastic electron-nucleon scat- tering (DIS) machine, which has been designed to collide electrons with an energy from 60 GeV to possibly 140 GeV with protons with an energy of 7 TeV. The future circular collider (FCC) has the option of colliding electrons with protons with an electron energy Ee ¼ 60 GeV and a proton energy of Ep ¼ 50 TeV. II. THEORETICAL FRAMEWORK The SM effective Lagrangian can be obtained by including higher-dimensional operators that take into account the new physics effects beyond the SM which may appear at the energy scale much larger than the SM energy scale. Under the assumption of baryon and lepton number conservation and keeping only dimension-six operators, the most general SUð3ÞC × SUð2ÞL × Uð1ÞY gauge-invariant Lagrangian can be constructed from the SM fields. We concentrate on the dimension-six inter- actions of the Higgs boson, fermions, and the electroweak gauge bosons in the strongly interacting light Higgs (SILH) basis conventions, which can be written as [34,35,42] This paper is structured as follows: In Sec. II, we present the theoretical framework of our analysis by recalling the relevant aspects of the effective SM in which dimension-six operators are considered. We review the higher-dimensional operators and highlight the operators contributing to the Higgs boson production processes at the LHeC and FCC-he. Section III describes the details of our analysis, including the simulation tools and analysis strategy for both LHeC and FCC-he. We explain the event selection criteria and stat- istical method by which we obtain the constraints on the Wilson coefficients. The analysis strategy for the LHeC collider and its sensitivity to the dimension-six operators is presented in Sec. IV. Section V is dedicated to presenting the sensitivity of the FCC-he collider to the related Wilson coefficients in the hjνe process. Our results and the con- straints on the Wilson coefficients are given in Sec. VI. Comparisons with the LHC bounds are also made in Leff ¼ LSM þ X i ¯ciOi ≡LSM þ ΔLF1 þ ΔLF2 þ ΔLSILH; ð2Þ where ¯ci coefficients are dimensionless Wilson coefficients, and Oi are dimension-six operators made up of SM fields. The first term in the effective Lagrangian of Eq. (2) is the SM Lagrangian, LSM. The second term ΔLF1 in Eq. (2) addresses the interactions between two Higgs fields and a pair of quarks or leptons. STUDY OF HIGGS EFFECTIVE COUPLINGS AT … STUDY OF HIGGS EFFECTIVE COUPLINGS AT … Finally, the last term of this Lagrangian corresponds to the Higgs field, which is part of a SILH. The ΔLSILH term can be expressed as ΔLSILH ¼ ¯cH 2v2 ∂μðH†HÞ∂μðH†HÞ þ ¯cT 2v2 ðH†Dμ ↔ HÞðH†D ↔ μHÞ −¯c6λ v2 ðH†HÞ3 þ ¯cu v2 yuH†H¯qLHcuR þ ¯cd v2 ydH†H¯qLHdR þ ¯cl v2 ylH†H ¯LLHlR  þ H:c:  þ i¯cWg 2m2 W ðH†σiDμ ↔ HÞðDνWμνÞi þ i¯cBg0 2m2 W ðH†Dμ ↔ HÞð∂νBμνÞ þ i¯cHWg m2 W ðDμHÞ†σiðDνHÞWiμν þ i¯cHBg0 m2 W ðDμHÞ†ðDνHÞBμν þ ¯cγg02 m2 W H†HBμνBμν þ ¯cgg2 S m2 W H†HGaμνGaμν þ i˜cHWg m2 W ðDμHÞ†σiðDνHÞ ˜Wiμν þ i˜cHBg0 m2 W ðDμHÞ†ðDνHÞ ˜Bμν þ ˜cγg02 m2 W H†HBμν ˜Bμν þ ˜cgg2 S m2 W H†HGaμν ˜Gaμν þ ˜c3Wg3 m2 W ϵijkWi ν μ Wj ρ ν ˜Wk μ ρ þ ˜c3Gg3 S m2 W fabcGa ν μ Gb ρ ν ˜Gc μ ρ ; ΔLSILH ¼ ¯cH 2v2 ∂μðH†HÞ∂μðH†HÞ þ ¯cT 2v2 ðH†Dμ ↔ HÞðH†D ↔ μHÞ −¯c6λ v2 ðH†HÞ3 þ ¯cu v2 yuH†H¯qLHcuR þ ¯cd v2 ydH†H¯qLHdR þ ¯cl v2 ylH†H ¯LLHlR  þ H:c:  þ i¯cWg 2m2 W ðH†σiDμ ↔ HÞðDνWμνÞi þ i¯cBg0 2m2 W ðH†Dμ ↔ HÞð∂νBμνÞ þ i¯cHWg m2 W ðDμHÞ†σiðDνHÞWiμν þ i¯cHBg0 m2 W ðDμHÞ†ðDνHÞBμν þ ¯cγg02 m2 W H†HBμνBμν þ ¯cgg2 S m2 W H†HGaμνGaμν ΔLSILH ¼ ¯cH 2v2 ∂μðH†HÞ∂μðH†HÞ þ ¯cT 2v2 ðH†Dμ ↔ HÞðH†D ↔ μHÞ −¯c6λ v2 ðH†HÞ3 þ ¯cu v2 yuH†H¯qLHcuR þ ¯cd v2 ydH†H¯qLHdR þ ¯cl v2 ylH†H ¯LLHlR  þ H:c:  þ i¯cWg 2m2 W ðH†σiDμ ↔ HÞðDνWμνÞi þ i¯cBg0 2m2 W ðH†Dμ ↔ HÞð∂νBμνÞ þ i¯cHWg m2 W ðDμHÞ†σiðDνHÞWiμν þ i¯cHBg0 m2 W ðDμHÞ†ðDνHÞBμν þ ¯cγg02 m2 W H†HBμνBμν þ ¯cgg2 S m2 W H†HGaμνGaμν þ i˜cHWg m2 W ðDμHÞ†σiðDνHÞ ˜Wiμν þ i˜cHBg0 m2 W ðDμHÞ†ðDνHÞ ˜Bμν þ ˜cγg02 m2 W H†HBμν ˜Bμν þ ˜cgg2 S m2 W H†HGaμν ˜Gaμν þ ˜c3Wg3 m2 W ϵijkWi ν μ Wj ρ ν ˜Wk μ ρ þ ˜c3Gg3 S m2 W fabcGa ν μ Gb ρ ν ˜Gc μ ρ ; ð5Þ where Φ is a weak doublet containing the Higgs boson field, and Gμν, Bμν, Wμν are the strong and electroweak field strength tensors. Here, Φ†D ↔μΦ ¼ Φ†ðDμΦÞ −ðDμΦÞ†Φ is the Hermitian covariant derivative. In Eq. II. THEORETICAL FRAMEWORK This term has the following form: ΔLF1 ¼ i¯cHQ v2 ð¯qLγμqLÞðH†D ↔ μHÞ þ i¯c0 HQ v2 ð¯qLγμσiqLÞðH†σiD ↔ μHÞ þ i¯cHu v2 ð¯uRγμuRÞðH†D ↔ μHÞ þ i¯cHd v2 ð¯dRγμdRÞðH†D ↔ μHÞ þ i¯cHud v2 ð¯uRγμdRÞðHc†D ↔ μHÞ þ H:c:  þ i¯cHL v2 ð ¯LLγμLLÞðH†D ↔ μHÞ þ i¯c0 HL v2 ð ¯LLγμσiLLÞðH†σiD ↔ μHÞ þ i¯cHl v2 ð¯lRγμlRÞðH†D ↔ μHÞ: ð3Þ þ i¯c0 HL v2 ð ¯LLγμσiLLÞðH†σiD ↔ μHÞ þ i¯cHl v2 ð¯lRγμlRÞðH†D ↔ μHÞ: ð3Þ ð3Þ The third term ΔLF2 of the effective Lagrangian in Eq. (2) contains the interactions of a pair of quarks or leptons, a Higgs field, and a gauge boson. This term reads The third term ΔLF2 of the effective Lagrangian in Eq. (2) contains the interactions of a pair of quarks or leptons, a Higgs field, and a gauge boson. This term reads ΔLF2 ¼ ¯cuBg0 m2 W yu ¯qLHcσμνuRBμν þ ¯cuWg m2 W yu ¯qLσiHcσμνuRWiμν þ ¯cuGgS m2 W yu ¯qLHcσμνλauRGaμν þ ¯cdBg0 m2 W yd ¯qLHσμνdRBμν þ ¯cdWg m2 W yd ¯qLσiHσμνdRWiμν þ ¯cdGgS m2 W yd ¯qLHσμνλadRGaμν þ ¯clBg0 m2 W yl ¯LLHσμνlRBμν þ ¯clWg m2 W yl ¯LLσiHσμνlRWiμν þ H:c: ð4Þ Wg m2 W yl ¯LLσiHσμνlRWiμν þ H:c: ð4Þ ð4Þ 095041-2 095041-2 PHYS. REV. D 97, 095041 (2018) STUDY OF HIGGS EFFECTIVE COUPLINGS AT … (5), λ is the Higgs boson quartic coupling and v is the vacuum expectation value defined as v ¼ 1=ð ffiffiffi 2 p GFÞ1=2 ¼ 246 GeV. electron-proton collisions [43–51]. In Ref. [47], it has been shown that the production cross section of the charged current process is larger than the neutral current process by a factor of a around 5 for the incoming electron energy of 140 GeV and the proton energy of 7 TeV. In this work, our focus is on the charged current production process due to its larger production cross section. Also, this process has a clean signature as it is comprised of significant missing transverse energy and an energetic jet which tends to be forward. In this analysis, we concentrate on the Higgs boson decay into a pair of bottom quarks because of its large branching fraction. In the electron-proton colliders, the Higgs bosons are produced through two main channels: either via charged current e−q →Hq0νe or neutral current e−q →eHq. The leading order diagram for the production of a Higgs boson in the electron-proton collisions for the charged current process is depicted in Fig. 1. There have already been several studies on different aspects of the Higgs boson production via charged and neutral currents in the The present work is dedicated to considering the effects of Leff presented in Eq. (2) on the Higgs boson production through the charged current process e−q →Hq0νe. The contributions originating from other possible effective operators are neglected for simplicity. The representative Feynman diagrams for e−q →Hq0νe are displayed in Fig. 2. The vertices that receive contributions from the Leff are shown by filled circles. It is remarkable that the SM tree-level contribution does not have any dependence on the momenta of the involved particles; however, when considering Leff, momentum-dependent interactions enter the calculations. This leads to changes in the production cross sections as well as the shape of differential distribu- tions. In this paper, differences in the shapes of distribu- tions are used to constrain the involved Wilson coefficients in this process. FIG. 1. Leading order Feynman diagram Higgs boson produc- tion via e−p →hjνe processes. The e−p →hjνe process is sensitive to the following set of Leff parameters: FIG. 1. Leading order Feynman diagram Higgs boson produc- tion via e−p →hjνe processes. 095041-3 HESARI, KHANPOUR, and NAJAFABADI PHYS. REV. D 97, 095041 (2018) FIG. 2. STUDY OF HIGGS EFFECTIVE COUPLINGS AT … Representative Feynman diagrams at tree level for the e−p →hjνe process in electron-proton collisions in the presence of dimension-six operators. FIG. 2. Representative Feynman diagrams at tree level for the e−p →hjνe process in electron-proton collisions in the presence of dimension-six operators. ¯cHW; ˜cHW; ¯cW; ¯cH; ¯cd; ¯cu; ¯cl; ¯cHud; ¯c0HL; ¯c0HQ; ¯cuW; ¯cdW; ¯ceW: ð6Þ view is by the effective Lagrangian in the mass basis. In particular, it has been found to be an applicable approach in the study of electroweak precision tests (EWPT). The anomalous Higgs interactions in the mass basis have been presented in Ref. [35]. The relationship between the mass basis couplings and the dimension-six coefficients that are involved in this analysis are given in Table I. ð6Þ The cross section of the e−p →hjνe process is found to be almost insensitive to the parameters ¯cl, ¯ceW, ¯cd; ¯cu; ¯cuW; ¯cdW. This is because of the very small Yukawa couplings of light quarks and electrons. As a result, our analysis is restricted to the remaining seven parameters: There have already been many studies about con- straining the Wilson coefficients discussed above in differ- ent colliders using various channels; these can be found in Refs. [7–25,52–54]. Although the obtained limits on some of the coefficients in the previous studies are tight, we examine possible improvements for these limits in the future high-energy electron-proton colliders via a careful investigation of the Higgs production mechanism in the framework of effective field theory. In the next section, the details of simulation for probing the effective Lagrangian using the e−p →hjνe process in the future LHeC and FCC-he colliders will be discussed. ¯cH; ¯cHud; ¯cHW; ¯c0HL; ¯c0HQ; ¯cW; and ˜cHW: An interesting way to represent the effective Lagrangian from the experimental and phenomenological points of TABLE I. Anomalous Higgs boson couplings in the mass basis and their relation to the dimension-six coefficients. Mass basis Gauge basis gð1Þ hww 2g mW ¯cHW ˜ghww 2g mW ˜cHW gð2Þ hww g mW f¯cW þ ¯cHWg gðLÞ hwud ffiffi 2 p g v ¯c0 HQVCKM gðRÞ hwud ffiffi 2 p g v ¯cHud ghwνe ffiffi 2 p g v ¯c0 HL Mass basis IV. LHeC SENSITIVITY In this section, we present the analysis strategy and the results for the LHeC. The strategy for choosing the basic cuts is similar to the one proposed in [38]. Jets are reconstructed with a distance parameter for the jet reconstruction algorithm R ¼ 0.7. We require at least three jets with pjets T > 20 GeV, from which two are required to be b tagged. A minimum cut of 20 GeV is imposed on the missing transverse energy, and the total transverse energy of all final states needs to be greater than 100 GeV. The electromagnetic and hadronic calorimeter resolu- tions are considered by the energy smearing of 5% ffiffiffiffiffiffiffiffiffiffiffiffi EðGeVÞ p (plus 1% of constant term) and 60% ffiffiffiffiffiffiffiffiffiffiffiffi EðGeVÞ p , respectively [38]. The electromagnetic and hadronic calorimeter resolu- tions are considered by the energy smearing of 5% ffiffiffiffiffiffiffiffiffiffiffiffi EðGeVÞ p (plus 1% of constant term) and 60% ffiffiffiffiffiffiffiffiffiffiffiffi EðGeVÞ p , respectively [38]. The b-tagging efficiency is assumed to be 60%, while mistag probabilities of 10% and 1% for c-quark jets and light-quark jets are considered, respectively [38]. The b-tagging efficiency is assumed to be 60%, while mistag probabilities of 10% and 1% for c-quark jets and light-quark jets are considered, respectively [38]. The Higgs boson is reconstructed using the two b-tagged jets that give the closest mass to the nominal Higgs mass, i.e., 125 GeV. Among the light jets, the highest pT one is taken as the light flavor jet. Figure 3 shows the recon- structed Higgs boson mass (left) and the invariant mass distribution of the Higgs þ jet (MHiggs;j) (right) for signal with ¯cH ¼ 0.1 and the main backgrounds after the pre- selection cuts. The tracker of the LHeC detector is expected to cover a pseudorapidity range up to 3.0 [38]. Therefore, the b-tagging performance is valid up to jηb-jetj < 3. For the light jets, the calorimeter coverage is considered to be jηlight−jetj < 5. In the reconstructed distribution of the Higgs boson mass, the mass peak is lower than the right Higgs boson mass because of the energy carried by the neutrino from the b-quark decays. Based on the signal final state that consists of missing transverse energy, a pair of b¯b from the Higgs boson decay, and a forward jet, the backgrounds include processes with three jets and large missing energy in the final state. III. SIGNAL AND BACKGROUND PRODUCTION AND SIMULATION The chains we have used to perform the generation and simulation of the signal and background processes are described in this section. The full set of interactions generated by the dimension-six operators mentioned in the Higgs effective Lagrangian LSILH of Eq. (5), ΔLF1 in 095041-4 PHYS. REV. D 97, 095041 (2018) STUDY OF HIGGS EFFECTIVE COUPLINGS AT … Eq. (3), and ΔLF2 in Eq. (4) have been implemented in FeynRules [55,56], and the model is imported to a universal FeynRules output (UFO) module [35,57]. Then, the UFO model files are inserted into the MadGraph5-aMC@NLO [58,59] Monte Carlo (MC) event generator to calculate the cross sections and generate the signal events. The CTEQ6L1 PDF set [60] is used to describe the proton structure functions. The renormalization and factorization scales are dynamical in MadGraph5-aMC@NLO. considered; j0 refers to the light flavor jets except the b quark, and j denotes all light flavor quarks including the b quark. The background contributions from photoproduc- tion processes, which include the subprocesses gγ →b¯b and t¯t, are considered in the analysis. In the next sections, we present the analysis strategies for LHeC and FCC-he separately in more detail. As mentioned before, we consider the LHeC with electron energies of 60 GeV and 140 GeV colliding with the 7 TeV protons, while for the FCC-he case, the 60 GeV electrons collide with 50 TeV protons. The next-to-leading order QCD correction to the signal process e−p →hjνe is found to be small [61]. Therefore, in this work the k factor for the signal is assumed to be 1. The events of the signal process e−p →hjνe are generated with MadGraph5-aMC@NLO; then the Higgs boson decay into a b¯b pair via the MadSpin module [62,63]. The Pythia 6 [64,65] package is utilized to perform fragmentation, hadronization, and initial- and final-state parton showers. Jets are clustered using FastJet3.2.0 [66] with the kT algorithm [67]. IV. LHeC SENSITIVITY In particular, the following processes have been taken into account: bbj0νe, bbbνe, j0j0j0νe, tνe, Wjνe, and Zjνe. The hadronic decays of the top quark, W boson, and Z boson are The cross sections (in fb) after each cut for the signal, SM production of Higgs boson via the hjνe process, and the main background processes are presented in Table II for [GeV] Higgs M 20 40 60 80 100 120 140 160 180 200 Normalized distribution 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 =0.1) H c Signal ( e ν bbj’ e νt e ν Wj e ν Zj [GeV] Higgs,j M 100 200 300 400 500 600 Normalized distribution 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 =0.1) H c Signal ( e ν bbj’ e νt e ν Wj e ν Zj FIG. 3. The Higgs boson invariant mass distribution (left) and invariant mass distributions of Higgs þ jet (right) after the basic cuts. [GeV] Higgs,j M 100 200 300 400 500 600 Normalized distribution 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 =0.1) H c Signal ( e ν bbj’ e νt e ν Wj e ν Zj [GeV] Higgs M 20 40 60 80 100 120 140 160 180 200 Normalized distribution 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 =0.1) H c Signal ( e ν bbj’ e νt e ν Wj e ν Zj Normalized distribution [GeV] Higgs M FIG. 3. The Higgs boson invariant mass distribution (left) and invariant mass distributions of Higgs þ variant mass distribution (left) and invariant mass distributions of Higgs þ jet (right) after the basic cuts. FIG. 3. The Higgs boson invariant mass distribution (left) and invariant mass distributions of Higgs þ jet (right) after the basic cuts. 095041-5 PHYS. REV. D 97, 095041 (2018) HESARI, KHANPOUR, and NAJAFABADI TABLE II. Cross section (in fb) for signal and background events after applied kinematic cuts used for this analysis at the LHeC with Ee ¼ 60 GeV. The details of the basic cuts applied are presented in the text. TABLE II. Cross section (in fb) for signal and background events after applied kinematic cuts used for this analysis at the LHeC with Ee ¼ 60 GeV. The details of the basic cuts applied are presented in the text. IV. LHeC SENSITIVITY LHeC collider Signal Standard model Backgrounds Cuts ¯cH ¼ 0.1 hjνe bbj0νe tνe Wjνe Zjνe Cross sections (in fb) 84.8 94.3 639.5 1287 1885 379.6 Acceptance cuts 18.10 20.12 12.15 96.76 37.81 16.33 95 ≤MHiggs ≤135 ðGeVÞ 9.69 13.07 1.28 23.60 10.08 1.52 260 < MHiggs;j < 1000 ðGeVÞ 6.37 7.05 0.45 1.77 0.76 0.72 the LHeC operating with an electron energy of Ee ¼ 60 GeV. In addition to the basic kinematical cuts, a cut on the invariant mass of the Higgs boson 95 ≤MHiggs ≤135 GeV, as well as the invariant mass of the Higgs-jet system 260 < MHiggs;j < 1000 GeV, is also applied for this analysis. The impact of these cuts is presented in Table II. It clearly shows that the selection criteria are effective in enhancing signal and suppression of the background contributions. As one can see, cuts on the Higgs boson invariant mass and the Higgs-jet system affect all backgrounds and reduce their contributions signifi- cantly. The cross section of the photoproduction back- ground after all cuts is found to be 0.18 fb. the LHeC operating with an electron energy of Ee ¼ 60 GeV. In addition to the basic kinematical cuts, a cut on the invariant mass of the Higgs boson 95 ≤MHiggs ≤135 GeV, as well as the invariant mass of the Higgs-jet system 260 < MHiggs;j < 1000 GeV, is also applied for this analysis. The impact of these cuts is presented in Table II. It clearly shows that the selection criteria are effective in enhancing signal and suppression of the background contributions. As one can see, cuts on the Higgs boson invariant mass and the Higgs-jet system affect all backgrounds and reduce their contributions signifi- cantly. The cross section of the photoproduction back- ground after all cuts is found to be 0.18 fb. In Fig. 4, we show the expected normalized distribution of ΔEpz for the signal and the main sources of background processes after applying all cuts presented in Table II. As we can see, the shape of the ΔEpz signal with ¯cH ¼ 0.1 is quite different from the sum of all background processes. As a result, it is useful to obtain the exclusion limits on the Wilson coefficients defined in Eqs. (3) and (5). IV. LHeC SENSITIVITY q ( ) ( ) To set upper limits at the 95% CL, we use a χ2 criterion from the distribution of ΔEpz defined as χ2ðfcngÞ ¼ X N i¼bins ðfiðfcngÞ −sSM i Þ2 Δ2 i ; ð10Þ ð10Þ where fcng denotes the Wilson coefficient fcn ¼ ¯cH; ¯cHud; ¯cHW; ¯c0HL; ¯c0HQ; ¯cW; ˜cHWg considered in the present analy- sis, sSM i refers to the SM expectation in the ith bin of the ΔEpz distribution, and fiðfcngÞ is the number of signal events in the ith bin. In the χ2ðfcngÞ definition, Δi is the statistical uncertainty. We consider the most general for- mulation of fiðfcngÞ as second-degree polynomials according to the following: In this analysis, one might be concerned about the validity of the effective field theory. Several authors have discussed this issue in Refs. [68–70]. The Wilson coef- ficients of the dimension-six operators could be related to the new physics characteristic scale M via where fcng denotes the Wilson coefficient fcn ¼ ¯cH; ¯cHud; ¯cHW; ¯c0HL; ¯c0HQ; ¯cW; ˜cHWg considered in the present analy- sis, sSM i refers to the SM expectation in the ith bin of the ΔEpz distribution, and fiðfcngÞ is the number of signal events in the ith bin. In the χ2ðfcngÞ definition, Δi is the statistical uncertainty. We consider the most general for- mulation of fiðfcngÞ as second-degree polynomials according to the following: ¯c ∼g2v2 M2 ; ð7Þ ð7Þ where g is the coupling constant of the heavy degrees of freedom with the SM particles. Additional suppression factors appear in the case where an operator is generated at loop level. An upper bound can be put on the new mass scale M using the fact that the underlying theory is strongly coupled by setting g ¼ 4π. Assuming ¯c ¼ Oð1Þ, we find fiðfcngÞ ¼ sSM i þ X N n¼1 ðαn¯cn þ βn¯c2nÞ: ð11Þ ð11Þ (GeV) Z EP Δ 1000 1500 2000 2500 3000 3500 4000 4500 5000 Normalized distribution 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 =0.1) H c Signal( Backgrounds FIG. 4. Normalized distribution for ΔEpz for signal and all background processes after applying all cuts presented in Table II. IV. LHeC SENSITIVITY (GeV) Z EP Δ 1000 1500 2000 2500 3000 3500 4000 4500 5000 Normalized distribution 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 =0.1) H c Signal( Backgrounds M < 4πvffiffiffi¯c p ∼3.2 TeV: ð8Þ ð8Þ This upper bound is not violated in this analysis, as we have MHiggs;j < 1 TeV. A. Sensitivity estimate This subsection is dedicated to estimating the sensitivity of the e−p →hjνe process to the Wilson coefficients. The sensitivity is obtained using a χ2 analysis over all bins of ΔEpz distribution. The ΔEpz variable is defined as ΔEpz ¼ ðEb−jet1 −pz;b−jet1Þ þ ðEb−jet2 −pz;b−jet2Þ þ ðElight-jet1 −pz;light-jet1Þ: FIG. 4. Normalized distribution for ΔEpz for signal and all background processes after applying all cuts presented in Table II. ð9Þ 095041-6 PHYS. REV. D 97, 095041 (2018) STUDY OF HIGGS EFFECTIVE COUPLINGS AT … TABLE III. Predicted constraints at 95% CL on dimension-six Wilson coefficients for the LHeC with electrons energies of Ee ¼ 60 GeV and Ee ¼ 140 GeV, and for integrated luminosities of 300 fb−1 and 3000 fb−1. Wilson coefficients LHeC-300 (Ee ¼ 140 GeV) LHeC-3000 (Ee ¼ 140 GeV) LHeC-300 (Ee ¼ 60 GeV) LHeC-3000 (Ee ¼ 60 GeV) LHC-3000 [20] ¯cH½×100 ½−0.90; 0.95 ½−0.29; 0.29 ½−7.8; 8.8 ½−2.5; 2.6 ½−4.40; 3.50 ¯cHud½×100 ½−0.80; 0.80 ½−0.25; 0.25 ½−6.26; 8.33 ½−2.40; 2.86    ¯cHW½×100 ½−1.40; 1.70 ½−0.47; 0.50 ½−2.3; 2.8 ½−0.79; 0.83 ½−0.4; 0.4 ¯c0HL½×100 ½−1.30; 1.40 ½−0.40; 0.40 ½−2.6; 2.7 ½−0.85; 0.82    ¯c0HQ½×100 ½−1.50; 1.60 ½−0.50; 0.50 ½−2.20; 2.70 ½−0.79; 0.76    ¯cW½×100 ½−1.00; 1.00 ½−0.36; 0.37 ½−1.20; 1.40 ½−0.42; 0.44 ½−0.40; 0.40 ˜cHW½×100 ½−0.70; 0.70 ½−0.20; 0.20 ½−11.4; 9.2 ½−4.2; 3.6    TABLE III. Predicted constraints at 95% CL on dimension-six Wilson coefficients for the LHeC with electrons energies of Ee ¼ 60 GeV and Ee ¼ 140 GeV, and for integrated luminosities of 300 fb−1 and 3000 fb−1. opening angle θb¯b versus the parent mass Higgs boson and the momenta of the b and ¯b quarks. Using kinematic relations, the angular separation of a b¯b pair produced in a Higgs boson decay can also be written as opening angle θb¯b versus the parent mass Higgs boson and the momenta of the b and ¯b quarks. Using kinematic relations, the angular separation of a b¯b pair produced in a Higgs boson decay can also be written as Considering only one coefficient in the fit, one can obtain exclusion limits for the individual constraints on the Wilson coefficient fcn ¼ ¯cH; ¯cHud; ¯cHW; ¯c0HL; ¯c0HQ; ¯cW; ˜cHWg. The corresponding results for the integrated luminosities of 300 fb−1, 3000 fb−1, and 1 ab−1 are pre- sented in Tables III and V with electron energies of 60 GeV and 140 GeV. V. FCC-HE SENSITIVITY In this section, the sensitivity of the FCC-he to the related Wilson coefficients in the hjνe process is studied. As we mentioned before, FCC-he employs a 50 TeV proton beam of a proposed circular proton-proton collider. Similar to the LHeC case, FCC-he is sensitive to ¯cH; ¯cHud; ¯cHW; ¯c0HL; ¯c0HQ; ¯cW, and ˜cHW Wilson coeffi- cients. The same analysis strategy as presented for the LHeC is followed for the FCC-he. The Higgs boson decay into a b¯b pair is considered, and a χ2 fit is performed to estimate the sensitivities. The ratio of the cross section of e−p →hjνe at the FCC-he to the LHeC in terms of the Wilson coefficients is presented in Fig. 5. As we can see, when the couplings vary in the range −0.03 to 0.03, the cross section at the FCC-he increases by a factor of around 6 with respect to the LHeC. For the Higgs bosons with substantial momentum and pT, from Eq. (12) and (13) it is expected that the angular separation of the Higgs boson decay products decreases. Figure 7 shows the normalized distribution of ΔR between two b quarks from the Higgs boson decay for the FCC-he. We present the distributions for two signal scenarios ¯cH ¼ 0.1 and ¯cHW ¼ 0.1. The plot clearly confirms that for the ic 0.03 − 0.02 − 0.01 − 0 0.01 0.02 0.03 LHeC σ / FCC-he σ 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 H c H c H c HW c Hud c HL c' HQ c' W c HW c~ ic 0.03 − 0.02 − 0.01 − 0 0.01 0.02 0.03 LHeC σ / FCC-he σ 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 H c H c H c HW c Hud c HL c' HQ c' W c HW c~ FIG. 5. The ratio of the signal cross section at the FCC-he (Ee ¼ 60 GeV and Ep ¼ 50 TeV) to the LHeC (Ee ¼ 60 GeV and Ep ¼ 7 TeV) versus various Wilson coefficients. At this step, we mention one of the interesting character- istics of the signal events at the FCC-he, which requires using a particular strategy for reconstruction of the Higgs boson. A. Sensitivity estimate As an example, LHeC would be able to constrain ¯cH by more than 1 order of magnitude with respect to the LHC in the high-luminosity regime. ΔRb¯b ≃ 1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi xð1 −xÞ p mH pT ; ð13Þ ð13Þ where pT is the transverse momentum of the Higgs boson, and x and 1 −x are the momentum fractions of the b and ¯b quarks. Figure 6 shows the distributions of the Higgs boson momentum and transverse momentum for the signal scenario of ¯cHW ¼ 0.1 for the LHeC and FCC-he. As we can see, at the FCC-he, Higgs bosons reside at large values of momentum and pT. V. FCC-HE SENSITIVITY Thus, the two subjets must satisfy an arbitrary parameter that shows the degree of mass drop. Also, to avoid including high-pT light jets, two subjets need to be symmetrically split. Thus, the two subjets must satisfy signal scenario of ¯cHW, a considerable fraction of Higgs bosons are produced in the boosted regime, while this is not valid for the signal scenario of ¯cH. As a result, the high-pT Higgs bosons produce a collimated jet with substructure. Because of the small angular separation between two b jets from the Higgs decay and large boost, the common jet reconstruction with a cone size of ΔR ¼ 0.4–0.5 would not be usable for most of the signal events with a nonzero value of ¯cHW. An alternative method of the fat jet algorithm is applied [71] for these boosted events. minðp2 T;J1; p2 T;J2Þ m2 J ΔR2 J1;J2 < ycut ð14Þ ð14Þ where ycut is a parameter of the algorithm which determines the limit of asymmetry between two subjets, and p2 T;J1 and p2 T;J2 are the squares of the transverse momentum of each subjet. Finally, if the above criteria are not satisfied, the algorithm takes J ¼ J1 and returns to the first step for performing decomposition. The explained algorithm for boosted object reconstruction has been implemented in the FastJet package [66] by which our analysis is done. To reconstruct the signal events with two boosted b jets in the final state, first we reconstruct the fat jets by using the Cambridge/Aachen (CA) jet algorithm [72,73] assuming a jet cone size of R ¼ 1.2. Then, in order to identify the boosted Higgs boson, the methods described in the fat jet reconstruction algorithm [71] are explained in the following. In the beginning, a reconstructed fat jet J is split into two subjets J1, J2 with masses mJ1 > mJ2. Then, the method requires a significant mass drop of mJ1 < μMDmJ with μ ¼ 0.667. It should be mentioned that μMD is In this analysis, jets of both the signal and background are first reconstructed with the kT algorithm with a cone size of R ¼ 0.7. Then, if jets with pT > 250 GeV are found, the fat jet algorithm is applied; otherwise, the event is considered to be a normal event. V. FCC-HE SENSITIVITY The exclusion limits for the Wilson coefficients of the dimension-six operators at the FCC-he are obtained using a method similar to what was described in Sec. IVA. b b R Δ 0 0.5 1 1.5 2 2.5 3 3.5 Normalized distribution 2 − 10 1 − 10 = 0.1 H c =0.1 HW c FIG. 7. Normalized distribution of ΔR between two b quarks coming from the decay of Higgs bosons for two signal scenarios of ¯cH ¼ 0.1 and ¯cHW ¼ 0.1 at the FCC-he. b b R Δ 0 0.5 1 1.5 2 2.5 3 3.5 Normalized distribution 2 − 10 1 − 10 = 0.1 H c =0.1 HW c V. FCC-HE SENSITIVITY At the FCC-he, Higgs bosons for some scenarios of the signal are highly boosted, and from the topological point of view, they decay differently compared to the Higgs bosons which are not boosted. For instance, Higgs bosons produced in the signal scenario with a nonzero value of ¯cHW are highly boosted because of the momentum-dependent interaction. For the splitting of the Higgs boson into a pair of b¯b, one can write m2 H ≃2j⃗pbjj⃗p¯bjð1 −cos θb¯bÞ; ð12Þ ð12Þ FIG. 5. The ratio of the signal cross section at the FCC-he (Ee ¼ 60 GeV and Ep ¼ 50 TeV) to the LHeC (Ee ¼ 60 GeV and Ep ¼ 7 TeV) versus various Wilson coefficients. where ⃗pbð⃗p¯bÞ is the momentum of the bð¯bÞ quark and the bottom quark mass has been neglected. One can express the 095041-7 095041-7 PHYS. REV. D 97, 095041 (2018) HESARI, KHANPOUR, and NAJAFABADI (GeV) Higgs P 0 500 1000 1500 2000 2500 3000 3500 4000 Normalized distribution 4 − 10 3 − 10 2 − 10 1 − 10 = 0.1 HW c = 7 TeV p = 60 GeV, E e E = 50 TeV p = 60 GeV, E e E (GeV) T Higgs P 0 100 200 300 400 500 600 Normalized distribution 5 − 10 4 − 10 3 − 10 2 − 10 1 − 10 1 = 0.1 HW c = 50 TeV p = 60 GeV, E e E = 7 TeV p = 60 GeV, E e E FIG. 6. The distributions of the Higgs boson momentum (left) and the Higgs boson transverse momentum (right) at the LHeC and FCC-he for the signal scenario of ¯cHW ¼ 0.1. (GeV) T Higgs P 0 100 200 300 400 500 600 Normalized distribution 5 − 10 4 − 10 3 − 10 2 − 10 1 − 10 1 = 0.1 HW c = 50 TeV p = 60 GeV, E e E = 7 TeV p = 60 GeV, E e E FIG. 6. The distributions of the Higgs boson momentum (left) and the Higgs boson transverse momentum (right) at the LHeC and FCC-he for the signal scenario of ¯cHW ¼ 0.1. an arbitrary parameter that shows the degree of mass drop. Also, to avoid including high-pT light jets, two subjets need to be symmetrically split. VI. RESULTS In this section, the results are presented for the electron- proton collisions at the LHeC when the 60 GeV and 140 GeV electrons collide with the 7 TeV protons, and at the FCC-he when the 60 GeV electrons collide with the 50 TeV protons. The limits are presented for the integrated luminosities of 300 fb−1, 3000 fb−1, and 1 ab−1 in Tables III–V. In Tables III and IV, we present the constraints on the Wilson coefficients that have been obtained at the LHC at 14 TeV with an integrated luminosity of 3000 fb−1 [20]. As we can see from these tables, more sensitivity is FIG. 7. Normalized distribution of ΔR between two b quarks coming from the decay of Higgs bosons for two signal scenarios of ¯cH ¼ 0.1 and ¯cHW ¼ 0.1 at the FCC-he. 095041-8 PHYS. REV. D 97, 095041 (2018) STUDY OF HIGGS EFFECTIVE COUPLINGS AT … TABLE IV. Predicted constraints at 95% CL on dimension-six Wilson coefficients for the LHeC and FCC-he colliders and for integrated luminosity of 300 fb−1 and 3000 fb−1. Wilson coefficients LHeC-300 (Ee ¼ 60 GeV) LHeC-3000 (Ee ¼ 60 GeV) FCC-he-300 (Ee ¼ 60 GeV) FCC-he-3000 (Ee ¼ 60 GeV) LHC-3000 [20] ¯cH½×100 ½−7.8; 8.8 ½−2.5; 2.6 ½−8.70; 8.70 ½−2.75; 2.75 ½−4.40; 3.50 ¯cHud½×100 ½−6.26; 8.33 ½−2.40; 2.86 ½−4.00; 4.00 ½−1.26; 1.26    ¯cHW½×100 ½−2.3; 2.8 ½−0.79; 0.83 ½−1.00; 1.10 ½−0.32; 0.35 ½−0.4; 0.4 ¯c0HL½×100 ½−2.6; 2.7 ½−0.85; 0.82 ½−4.50; 4.90 ½−1.42; 1.54    ¯c0HQ½×100 ½−2.20; 2.70 ½−0.79; 0.76 ½−5.70; 6.00 ½−1.80; 1.90    ¯cW½×100 ½−1.20; 1.40 ½−0.42; 0.44 ½−1.20; 1.30 ½−0.38; 0.41 ½−0.40; 0.40 ˜cHW½×100 ½−11.4; 9.2 ½−4.2; 3.6 ½−4.70; 4.70 ½−1.49; 1.49    TABLE IV. Predicted constraints at 95% CL on dimension-six Wilson coefficients for the LHeC and FCC-he colliders and for integrated luminosity of 300 fb−1 and 3000 fb−1. achievable on the coefficient ¯cH at the electron-proton colliders with respect to the LHC. Comparison between LHeC and FCC-he sensitivities shows that more sensitivity to most of the Wilson coefficients can be obtained in the FCC-he. From these results, one can conclude that the LHeC and FCC-he are suitable platforms to complement the LHC results in the search for dimension-six effective couplings in the Higgs boson sector. VI. RESULTS 10 ab−1, the sensitivity to the Wilson coefficients is much better than the other options analyzed in this study, and in some cases, it is better than the ones expected to be achieved by the HL-LHC with an integrated luminosity of 3000 fb−1. VII. SUMMARY AND CONCLUSIONS The effects of physics beyond the SM may appear in the Higgs sector, which requires one to measure the Higgs boson couplings with the SM particles precisely. Any deviation of the Higgs boson interactions with respect to the predictions of the SM would be a hint to new physics. The LHeC with a rich physics program would be able to provide a lot of information on physics beyond the SM as well as precise measurements of the SM. In electron-proton collisions, there are two clean production mechanisms for the Higgs boson, either in neutral current interactions or in charged current inter- actions. In this paper, we present an analysis to constrain new physics in the Higgs boson sector by adopting an effective Lagrangian approach. The analysis is based on Higgs boson production in charged current interactions (via the WþW−H coupling), i.e., the e−p →hjνe process for the electrons with energies of Ee ¼ 60 GeV and Ee ¼ 140 GeV colliding with the 7 TeV protons. We also perform the same analysis for the FCC-he in which 60 GeV electrons collide with very high-energy protons, with energy of 50 TeV. For the FCC-he case, to efficiently reconstruct the Higgs boson and to achieve a reasonable background rejection, jet substructure tech- niques are used in order to capture the signal events that are boosted objects. In order to study the effect arising from different energies of colliding electrons, we present the results for the LHeC with the electron energies of Ee ¼ 60 GeV and Ee ¼ 140 GeV. From Table III, it can be seen that going to higher energies of the electron-proton collisions, from 60 GeV to 140 GeV, would lead to improvements for the Wilson coefficients. For example, the constraints obtained from Ee ¼ 60 GeV with the integrated luminosity of 3000 fb−1 on ¯cH are −0.025 < ¯cH < 0.026, which is tightened to −0.0029 < ¯cH < 0.0029 at a Ee ¼ 140 GeV machine. The results for the LHeC and FCC-he at very high integrated luminosities are presented in Table V. The bounds are given for maximum achievable integrated luminosities of 1 ab−1 and 10 ab−1 for the LHeC and FCC-he, respectively. Based on this analysis for the FCC- he with Ee ¼ 60 GeV for an integrated luminosity of TABLE V. ACKNOWLEDGMENTS Wudka, Patterns of deviation from the standard model, Nucl. Phys. B433, 41 (1995). [20] C. Englert, R. Kogler, H. Schulz, and M. Spannowsky, Higgs coupling measurements at the LHC, Eur. Phys. J. C 76, 393 (2016). [7] C. Hartmann, W. Shepherd, and M. Trott, The Z decay width in the SMEFT: yt and λ corrections at one loop, J. High Energy Phys. 03 (2017) 060. [21] J. Ellis, V. Sanz, and T. You, Complete Higgs sector constraints on dimension-6 operators, J. High Energy Phys. 07 (2014) 036. [8] S. Kuday, H. Saygin, I. Hos, and F. Cetin, Projections for neutral Di-Boson and Di-Higgs interactions at FCC-he collider, arXiv:1702.00185. [22] H. Khanpour and M. M. Najafabadi, Constraining Higgs boson effective couplings at electron-positron colliders, Phys. Rev. D 95, 055026 (2017). [9] W. Kilian, S. Sun, Q. S. Yan, X. Zhao, and Z. Zhao, New physics in multi-Higgs boson final states, J. High Energy Phys. 06 (2017) 145. [23] H. Khanpour, S. Khatibi, and M. M. Najafabadi, Probing Higgs boson couplings in H þ γ production at the LHC, Phys. Lett. B 773, 462 (2017). [10] J. Ellis, P. Roloff, V. Sanz, and T. You, Dimension-6 operator analysis of the CLIC sensitivity to new physics, J. High Energy Phys. 05 (2017) 096. [24] A. Buckley, C. Englert, J. Ferrando, D. J. Miller, L. Moore, M. Russell, and C. D. White, Constraining top quark effective theory in the LHC run II era, J. High Energy Phys. 04 (2016) 015. [11] S. Fichet, A. Tonero, and P. R. Teles, Sharpening the shape analysis for higher-dimensional operator searches, Phys. Rev. D 96, 036003 (2017). [25] A. Buckley, C. Englert, J. Ferrando, D. J. Miller, L. Moore, M. Russell, and C. D. White, Global fit of top quark effective theory to data, Phys. Rev. D 92, 091501 (2015). [12] C. Sigismondi, Measuring the position of the center of the Sun at the Clementine Gnomon of Santa Maria degli Angeli in Rome, J. Occult. Astron. 1N5, 20 (2012). [26] H. Denizli and A. Senol, Constraints on Higgs effective couplings in Hν¯ν production of CLIC at 380 GeV, Adv. High Energy Phys. 2018, 1627051 (2018). [13] A. Arbey, S. Fichet, F. Mahmoudi, and G. Moreau, The correlation matrix of Higgs rates at the LHC, J. High Energy Phys. 11 (2016) 097. [27] T. Barklow, K. Fujii, S. Jung, R. Karl, J. List, T. Ogawa, M. E. ACKNOWLEDGMENTS Table IV, show a great sensitivity, and in some cases, improvements are expected with respect to the potential constraints for the LHC [20,23]. We also show that the FCC-he collider with Ee ¼ 60 GeV and with an integrated luminosity of L ¼ 10 ab−1 or even with 3 ab−1 would be able to probe the Wilson coefficients of dimension-six operators of the Higgs boson (especially ¯cH, ¯cHW, and ¯cW couplings) beyond the HL-LHC. The authors thank the School of Particles and Accelerators, Institute for Research in Fundamental Sciences (IPM) for financial support of this project. H. K. is also grateful to the University of Science and Technology of Mazandaran for financial support provided for this research. M. M. N. acknowledges the Iran National Science Foundation (INSF) for support. [15] S. Banerjee, T. Mandal, B. Mellado, and B. Mukhopad- hyaya, Cornering dimension-6 HVV interactions at high luminosity LHC: The role of event ratios, J. High Energy Phys. 09 (2015) 057. [1] G. Aad et al. (ATLAS Collaboration), Observation of a new particle in the search for the Standard Model Higgs boson with the ATLAS detector at the LHC, Phys. Lett. B 716, 1 (2012). [16] N. Craig, J. Gu, Z. Liu, and K. Wang, Beyond Higgs couplings: Probing the Higgs with angular observables at future eþ e−colliders, J. High Energy Phys. 03 (2016) 050. [2] S. Chatrchyan et al. (CMS Collaboration), Observation of a new boson at a mass of 125 GeV with the CMS experiment at the LHC, Phys. Lett. B 716, 30 (2012). [3] W. Buchmuller and D. Wyler, Effective Lagrangian analysis of new interactions and flavor conservation, Nucl. Phys. B268, 621 (1986). [17] T. Corbett, O. J. P. Eboli, D. Goncalves, J. Gonzalez-Fraile, T. Plehn, and M. Rauch, The Higgs legacy of the LHC run I, J. High Energy Phys. 08 (2015) 156. [4] B. Grzadkowski, M. Iskrzynski, M. Misiak, and J. Rosiek, Dimension-six terms in the standard model Lagrangian, J. High Energy Phys. 10 (2010) 085. [18] J. Ellis, V. Sanz, and T. You, The effective standard model after LHC run I, J. High Energy Phys. 03 (2015) 157. [5] J. A. Aguilar-Saavedra, A minimal set of top anomalous couplings, Nucl. Phys. B812, 181 (2009). [19] L. Berthier and M. Trott, Consistent constraints on the standard model effective field theory, J. High Energy Phys. 02 (2016) 069. [6] C. Arzt, M. B. Einhorn, and J. PHYS. REV. D 97, 095041 (2018) PHYS. REV. D 97, 095041 (2018) HESARI, KHANPOUR, and NAJAFABADI VII. SUMMARY AND CONCLUSIONS Predicted constraints at 95% CL on Wilson coef- ficients for the LHeC and FCC-he with Ee ¼ 60 GeV. The limits are presented for the integrated luminosities of 1 ab−1 for the LHeC and 10 ab−1 for the FCC-he, respectively. Wilson coefficients LHeC (Ee ¼ 60 GeV) 1 ab−1 FCC-he (Ee ¼ 60 GeV) 10 ab−1 ¯cH½×100 ½−4.28; 4.60 ½−1.5; 1.5 ¯cHud½×100 ½−3.88; 4.96 ½−0.69; 0.69 ¯cHW½×100 ½−0.89; 0.96 ½−0.17; 0.19 ¯c0HL½×100 ½−1.43; 1.58 ½−0.78; 0.85 ¯c0HQ½×100 ½−1.29; 1.4 ½−0.98; 1.01 ¯cW½×100 ½−0.71; 0.76 ½−0.21; 0.22 ˜cHW½×100 ½−7.05; 6.03 ½−0.81; 0.81 To obtain the sensitivity to the involved Wilson coef- ficients of dimension-six operators, an analysis on the kinematic distribution of ΔEpz [defined in Eq. (9)] is performed. The Higgs boson production via charged current interaction, e−p →hjνe, is, in particular, sensitive to a variety of Wilson coefficients, namely, fci ¼ ¯cH; ¯cHud; ¯cHW; ¯c0HL; ¯c0HQ; ¯cW; ˜cHWg. The extracted bounds for both the LHeC and FCC-he, which are presented in 095041-9 ACKNOWLEDGMENTS High Energy Phys. 04 (2014) 110. [56] A. Alloul, N. D. Christensen, C. Degrande, C. Duhr, and B. Fuks, FeynRules 2.0–A complete toolbox for tree-level phenomenology, Comput. Phys. Commun. 185, 2250 (2014). [36] P. Artoisenet et al., A framework for Higgs characterisation, J. High Energy Phys. 11 (2013) 043. [36] P. Artoisenet et al., A framework for Higgs c J. High Energy Phys. 11 (2013) 043. J. High Energy Phys. 11 (2013) 043. [57] C. Degrande, C. Duhr, B. Fuks, D. Grellscheid, O. Mattelaer, and T. Reiter, UFO–The universal FeynRules output, Comput. Phys. Commun. 183, 1201 (2012). [37] O. Bruening and M. Klein, The Large Hadron Electron Collider, Mod. Phys. Lett. A 28, 1330011 (2013). [38] J. L. A. Fernandez et al. (LHeC Study Group), A Large Hadron Electron Collider at CERN: Report on the physics and design concepts for machine and detector, J. Phys. G 39, 075001 (2012). [58] J. Alwall, M. Herquet, F. Maltoni, O. Mattelaer, and T. Stelzer, MadGraph 5: Going beyond, J. High Energy Phys. 06 (2011) 128. [59] J. Alwall, R. Frederix, S. Frixione, V. Hirschi, F. Maltoni, O. Mattelaer, H.-S. Shao, T. Stelzer, P. Torrielli, and M. Zaro, The automated computation of tree-level and next-to- leading order differential cross sections, and their matching to parton shower simulations, J. High Energy Phys. 07 (2014) 079. [39] J. L. A. Fernandez et al. (LHeC Study Group), On the relation of the LHeC and the LHC, arXiv:1211.5102. [40] J. L. A. Fernandez et al., A large hadron electron collider at CERN, arXiv:1211.4831. [41] M. Kumar, X. Ruan, R. Islam, A. S. Cornell, M. Klein, U. Klein, and B. Mellado, Probing anomalous couplings using di-Higgs production in electron-proton collisions, Phys. Lett. B 764, 247 (2017). [60] J. Pumplin, D. R. Stump, J. Huston, H. L. Lai, P. M. Nadolsky, and W. K. Tung, New generation of parton distributions with uncertainties from global QCD analysis, J. High Energy Phys. 07 (2002) 012. [42] A. Pomarol and F. Riva, Towards the ultimate SM fit to close in on Higgs physics, J. High Energy Phys. 01 (2014) 151. [61] B. Jager, Next-to-leading order QCD corrections to Higgs production at a future lepton-proton collider, Phys. Rev. D 81, 054018 (2010). [43] J. R. Ellis, M. K. Gaillard, and D. V. Nanopoulos, A phenomenological profile of the Higgs boson, Nucl. Phys. B106, 292 (1976). [62] P. Artoisenet, R. Frederix, O. Mattelaer, and R. ACKNOWLEDGMENTS Peskin, and J. Tian, Improved formalism for precision Higgs coupling fits, Phys. Rev. D 97, 053003 (2018). [14] G. Amar, S. Banerjee, S. von Buddenbrock, A. S. Cornell, T. Mandal, B. Mellado, and B. Mukhopadhyaya, Exploration of the tensor structure of the Higgs boson coupling to weak bosons in eþ e−collisions, J. High Energy Phys. 02 (2015) 128. [28] S. Jana and S. Nandi, New Physics Scale from Higgs Observables with Effective Dimension-6 Operators, arXiv:1710.00619. 095041-10 STUDY OF HIGGS EFFECTIVE COUPLINGS AT … PHYS. REV. D 97, 095041 (2018) [49] H. Sun and X. Wang, Searches for the anomalous FCNC top-Higgs couplings at the LHeC, Eur. Phys. J. C 78, 281 (2018). [29] A. Dedes, W. Materkowska, M. Paraskevas, J. Rosiek, and K. Suxho, Feynman rules for the Standard Model Effective Field Theory in Rξ −gauges, J. High Energy Phys. 06 (2017) 143. [50] X. Wang, H. Sun, and X. Luo, Searches for the anomalous FCNC top-Higgs couplings with polarized electron beam at the LHeC, Adv. High Energy Phys. 2017, 4693213 (2017). [30] A. Dedes, M. Paraskevas, J. Rosiek, K. Suxho, and L. Trifyllis, The decay h →γ γ in the Standard-Model Effec- tive Field Theory, arXiv:1805.00302. [51] A. Senol, Anomalous Higgs couplings at the LHeC, Nucl. Phys. B873, 293 (2013). [31] S. Bar-Shalom and A. Soni, A universally enhanced light- quarks Yukawa couplings paradigm, arXiv:1804.02400. [52] G. Passarino and M. Trott, The standard model effective field theory and next to leading order, arXiv:1610.08356. [32] J. Gu, H. Li, Z. Liu, S. Su, and W. Su, Learning from Higgs Physics at Future Higgs Factories, J. High Energy Phys. 12 (2017) 153. [53] G. F. Giudice, C. Grojean, A. Pomarol, and R. Rattazzi, The strongly-interacting light Higgs, J. High Energy Phys. 06 (2007) 045. [33] C. W. Murphy, Statistical approach to Higgs boson cou- plings in the standard model effective field theory, Phys. Rev. D 97, 015007 (2018). [54] J. Ellis and T. You, Sensitivities of prospective future eþe− colliders to decoupled new physics, J. High Energy Phys. 03 (2016) 089. [34] R. Contino, M. Ghezzi, C. Grojean, M. Muhlleitner, and M. Spira, Effective Lagrangian for a light Higgs-like scalar, J. High Energy Phys. 07 (2013) 035. [55] N. D. Christensen and C. Duhr, FeynRules–Feynman rules made easy, Comput. Phys. Commun. 180, 1614 (2009). [35] A. Alloul, B. Fuks, and V. Sanz, Phenomenology of the Higgs effective Lagrangian via FEYNRULES, J. ACKNOWLEDGMENTS Rietkerk, Automatic spin-entangled decays of heavy resonances in Monte Carlo simulations, J. High Energy Phys. 03 (2013) 015. [44] J. M. LoSecco, Higgs Boson production in neutrino scatter- ing, Phys. Rev. D 14, 1352 (1976). [45] Z. Hioki, S. Midorikawa, and H. Nishiura, Higgs Boson production in high-energy lepton-nucleon scattering, Prog. Theor. Phys. 69, 1484 (1983). [63] S. Frixione, E. Laenen, P. Motylinski, and B. R. Webber, Angular correlations of lepton pairs from vector boson and top quark decays in Monte Carlo simulations, J. High Energy Phys. 04 (2007) 081. [46] J. Blumlein, G. J. van Oldenborgh, and R. Ruckl, QCD and QED corrections to Higgs boson production in charged current ep scattering, Nucl. Phys. B395, 35 (1993). [64] T. Sjostrand, L. Lonnblad, S. Mrenna, and P. Z. Skands, Pythia 6.3 physics and manual, arXiv:hep-ph/0308153. [47] T. Han and B. Mellado, Higgs Boson searches and the Hb¯b coupling at the LHeC, Phys. Rev. D 82, 016009 (2010). [65] T. Sjostrand, S. Mrenna, and P. Z. Skands, A brief intro- duction to PYTHIA 8.1, Comput. Phys. Commun. 178, 852 (2008). [48] S. S. Biswal, R. M. Godbole, B. Mellado, and S. Raychaudhuri, Azimuthal Angle Probe of Anomalous HWW Couplings at a High Energy ep Collider, Phys. Rev. Lett. 109, 261801 (2012). [66] M. Cacciari, G. P. Salam, and G. Soyez, FastJet user manual, Eur. Phys. J. C 72, 1896 (2012). [67] G. Soyez, The SISCone, and anti-kt jet algorithms, arXiv:0807.0021. 095041-11 PHYS. REV. D 97, 095041 (2018) HESARI, KHANPOUR, and NAJAFABADI HESARI, KHANPOUR, and NAJAFABADI [71] J. M. Butterworth, A. R. Davison, M. Rubin, and G. P. Salam, Jet Substructure as a New Higgs Search Channel at the LHC, Phys. Rev. Lett. 100, 242001 (2008). [68] R. Contino, A. Falkowski, F. Goertz, C. Grojean, and F. Riva, On the validity of the effective field theory approach to SM precision tests, J. High Energy Phys. 07 (2016) 144. [69] C. Englert and M. Spannowsky, Effective theories and measurements at colliders, Phys. Lett. B 740, 8 (2015). [72] Y. L. Dokshitzer, G. D. Leder, S. Moretti, and B. R. Webber, Better jet clustering algorithms, J. High Energy Phys. 08 (1997) 001. [70] M. Farina, G. Panico, D. Pappadopulo, J. T. Ruderman, R. Torre, and A. Wulzer, Energy helps accuracy: Electroweak precision tests at hadron colliders, Phys. Lett. B 772, 210 (2017). [73] M. Wobisch and T. Wengler, Hadronization corrections to jet cross-sections in deep inelastic scattering, arXiv:hep-ph/ 9907280. 095041-12
https://openalex.org/W2585640870
https://hal.archives-ouvertes.fr/hal-01467861/file/Workshop_ISWC2016.pdf
English
null
Autonomy through knowledge: how IoT-O supports the management of a connected apartment
HAL (Le Centre pour la Communication Scientifique Directe)
2,016
cc-by
5,350
Autonomy through knowledge: how IoT-O supports the management of a connected apartment Nicolas Seydoux1,2,3, Khalil Drira2,3, Nathalie Hernandez1, Thierry Monteil2,3 1 IRIT Maison de la Recherche, Univ. Toulouse Jean Jaur`es, 5 all´ees Antonio Machado, F-31000 Toulouse {nseydoux,hernande}@irit.fr 2 CNRS, LAAS, 7 avenue du Colonel Roche, F-31400 Toulouse, France {nseydoux,khalil,monteil}@laas.fr 3 Univ de Toulouse, INSA, LAAS, F-31400, Toulouse, France 1 IRIT Maison de la Recherche, Univ. Toulouse Jean Jaur`es, 5 all´ees Antonio Machado, F-31000 Toulouse {nseydoux,hernande}@irit.fr 2 CNRS, LAAS, 7 avenue du Colonel Roche, F-31400 Toulouse, France {nseydoux,khalil,monteil}@laas.fr 3 Univ de Toulouse, INSA, LAAS, F-31400, Toulouse, France Abstract. The IoT is a domain in exponential growth: both the num- ber of connected devices and the quantity of data they produce are in- creasing. The heterogeneity of technologies involved, and the diversity of domains impacted raise interoperability concerns. The semantic web principles and technologies help tackling these interoperability issues, and ontologies like SSN have been used in several IoT projects. However, many existing IoT ontologies fail to comply with the good practices of the semantic web. After detailing such good practices, this paper proposes IoT-O, a modular core-domain IoT ontology. IoT-O is then showcased in a home automation use case: it is used to semantically describe the devices of the system, and to guide the decisions of an autonomic agent. 1 Achieving semantic interoperability in the IoT Connected devices, forming a so-called Internet of Things (IoT), are becoming part of our everyday lives: it is estimated that up to 50 billion of them will be exchanging data by the next five to ten years [1]. Such an increase in the number of devices, combined to the heterogeneity of applications domains (agriculture, domotics, smart cities, e-health...) and technologies, raises interoperability issues in the IoT. These issues can be divided in two main classes: syntactic and seman- tic, brought respectively by the diversity of domains and data models [2]. Two systems are semantically interoperable when they attribute the same meaning to the data they exchange, and it is this type of interoperability that is ad- dressed in this paper. Initiatives such as [3] show the importance of semantic interoperability in the development of the IoT. Semantic interoperability requires the use of shared, unambiguous, machine- understandable vocabularies that allow to transform raw data issued by sensors to be transformed into self-sufficient, interoperable knowledge. Such vocabular- ies can be defined using semantic web principles and technologies, and if they are expressed with specific formalisms are called ontologies. Among semantic web principles are good practices that should be followed in order to create semantic models that can be reused in various use cases, that can be extended according to needs discovered a posteriori, that can be maintained over time, and that are compatible with existing ontologies at the time of design. Be- ing essential to semantic interoperability, many ontologies have been built within IoT projects, and are not always compliant with the aforementioned guidelines. This is why we propose IoT-O4, an IoT core-domain modular ontology engi- neered for reusability and extensibility. IoT-O is available on the LOV5, and based on the initial contribution of [4]. In the remainder of this paper, section 2 introduces a motivating use case that will serve to instantiate portions of IoT-O. Section 3 presents the design process of IoT-O, and gives an overview of the ontology. Section 4 details how IoT-O is instantiated in the use case, and finally section 5 concludes this paper and provides some insight about future works. 4 http://www.irit.fr/recherches/MELODI/ontologies/IoT-O 5 http://lov.okfn.org/dataset/lov/vocabs/ioto 6 h // l f / bl / / d // / / / / 6 http://www.laas.fr/public/en/adream 2 Motivating use case The automation of the home, or domotics, is a domain of the IoT with direct impact on citizens. At LAAS-CNRS, the ADREAM project6 aims at conducting research thanks to an instrumented, energy-positive building. This building is equipped with more than 4500 sensing devices, producing up to 500,000 mea- sures a day. Inside the building, there is a mock-up apartment equipped with commercial devices from diverse vendors. Deployed devices include sensors (tem- perature, luminosity, humidity, pressure), actuators (fan, space heater, diverse lamps), which communicate using different technologies (phidget, ethernet, zig- bee) with gateways connected to a central server (see fig. 1). Fig. 1. The connected appartment inside ADREAM p://www.irit.fr/recherches/MELODI/ontologies/IoT-O p://lov.okfn.org/dataset/lov/vocabs/ioto p://www.laas.fr/public/en/adream Fig. 1. The connected appartment inside ADREAM 4 http://www.irit.fr/recherches/MELODI/ontologies/IoT-O 5 http://lov.okfn.org/dataset/lov/vocabs/ioto 6 http://www.laas.fr/public/en/adream Our use case is defined as follows: the user should be able to define simple high-level policies to manage its environment (”the temperature in the living room should stay between 19oC and 25oC”), without having to select specific sensors or actuators to perform the task. He should also be able to extend the capabilities of the apartment by adding devices without restarting the system. To fulfill these requirements, both syntactic and semantic interoperability among devices are required. Syntactic interoperability is ensured using OM2M7, an open-source horizontal integration platform implementing the oneM2M stan- dard. On top of OM2M, another platform, SemIoTics, is in charge of ensuring semantic interoperability and of implementing the policies defined by the user. SemIoTics is guided by a knowledge base containing information about the de- vices, described with our ontology, IoT-O. This use case is applied to home automation and is described in a dedicated knowledge base extending IoT-O, ADREAM-Model8, but the genericity of IoT-O makes it relevant to any domain impacted by the IoT. 3.1 Identifying IoT core concepts Conceptual requirements The conceptual requirements aim at capturing knowledge that should be present in an IoT ontology. They are deduced from a bottom-up analysis of the IoT domain. Even if a typical IoT application is presented in section 2, IoT-O is not be limited to this use case. To be reusable in a wider scope, an IoT ontology should necessarily contain some identified concepts tightly associated to the IoT, independently of the applicative context. This approach makes the ontology horizontal and core-domain to the IoT, and suitable for applications in different domains of the IoT that can extend it accordingly. We distinguish namely: We distinguish namely: – ”Device” and ”software agent” constitute the two basic components of an IoT system, composed of both physical and virtual elements. The devices can be of two principle types, not mutually exclusive, that are listed below. – ”Sensor” are devices acquiring data, and ”observation” describe the ac- quisition context and the data collected by the system. – ”Actuator” are the devices that enable the system to act on the physical world, and ”action” represents what they can perform. – ”Service”: In many cases, the IoT and the programmable web are very close. Connected devices can be seen as service providers and consumers, and by specifying a notion of service, every aspect of an IoT system can be represented. – ”Energy”: In the paradigm of pervasive computing, many distributed Things perform computations. Most of these Things being physical devices, a com- plete modelling of the system will include a description of their energy con- sumption. Energy management is a crucial topic in IoT systems. – ”Lifecycle”: Be it data, devices or services, IoT components are all included in different scales of lifecycles. Devices are switched on and off, services are deployed or updated, pieces of data become outdated... The evolution through a set of discrete states representing a lifecycle is an important con- cept for IoT systems. Concept coverage by existing ontologies Table 1 sums up the assessment of existing IoT ontologies regarding the presence of key concepts. One star means that the concept is superficially represented (few specializations, data/object properties), two stars that the requirement is covered, and stars between paren- theses indicate that the requirement is met by an included ontology. IoT-O, the ontology we propose, is also included for comparison. 3 IoT-O, an IoT ontology designed in a requirement-driven process To ensure the respect of good practices, IoT-O design is compliant with the NeOn methodology, presented in [5]. Its first step is to define ontology requirements. We split these in two categories: conceptual, regarding the concepts that should be present in the ontology (detailed in section 3.1), and functional, regarding the ontology structure and design principles (detailed in section 3.2). These requirements are used to analyze existing IoT ontologies: Semantic Sensor Network (SSN)9, Smart Appliance REFerence (SAREF)10, iot-ontology 11, IoT-lite 12, Spitfire 13, IoT-S14, SA15 and the oneM2M base ontology16. Had one of these ontologies matched all the requirements, we would not have proposed a new ontology. Even more, ontologies compliant with parts of the requirements and covering concepts useful to IoT-O are reused to limit redefini- tion, as it is recommanded in NeOn. Details about such ontologies are given in section 3.3. Studied ontologies are the ones for which we have found information on the web. Further details are available on the Linked Open Vocabularies for the IoT (LOV4IoT)17, a recent initiative that lists IoT ontologies, even if they are 7 om2m.org 8 http://www.irit.fr/recherches/MELODI/ontologies/Adream-Model 9 http://purl.oclc.org/NET/ssnx/ssn 10 http://sites.google.com/site/smartappliancesproject/ontologies 11 http://ai-group.ds.unipi.gr/kotis/ontologies/IoT-ontology 12 http://iot.ee.surrey.ac.uk/fiware/ontologies/iot-lite 13 http://sensormeasurement.appspot.com/ont/sensor/spitfire.owl 14 http://personal.ee.surrey.ac.uk/Personal/P.Barnaghi/ontology/OWL-IoT-S.owl 15 http://sensormeasurement.appspot.com/ont/sensor/hachem onto.owl 16 http://www.onem2m.org/ontology/Base Ontology/ 17 http://www.sensormeasurement.appspot.com/?p=ontologies 17 http://www.sensormeasurement.appspot.com/?p=ontologies not referenced on the LOV because they fail to comply with its requirements recalled in [2]. Ontologies related to specific domains impacted by IoT (domotics, agriculture, smart cities...) are out of the scope of this study. 18 https://sites.google.com/site/smartappliancesproject/ontologies/ediana-ontology Table 1. Key concept coverage in IoT ontologies Table 1. Key concept coverage in IoT ontologies Table 1. Key concept coverage in IoT ontologies Table 1. Key concept coverage in IoT ontologies Actuator Action Service Sensor Observation Energy Lifecycle Device Software agent iot-ontology * * ** (**) (**) (*) (**) ** saref * * ** * ** ** ** OWL-IoT-S (**) (**) (**) (*) (**) SA * * (**) (**) (**) (**) (**) iot-lite * * (*) (*) spitfire (*) (*) ** (*) ssn ** ** * ** oneM2M ** * IoT-O ** ** (**) (**) (**) (**) (**) (**) * Actuator Action Service Sensor Observation Energy Lifecycle Device Software agent iot-ontology * * ** (**) (**) (*) (**) ** saref * * ** * ** ** ** OWL-IoT-S (**) (**) (**) (*) (**) SA * * (**) (**) (**) (**) (**) iot-lite * * (*) (*) spitfire (*) (*) ** (*) ssn ** ** * ** oneM2M ** * IoT-O ** ** (**) (**) (**) (**) (**) (**) * 3.1 Identifying IoT core concepts Note that we focus on con- nected device ontologies, and exclude, on purpose, the ontologies SSN is based on, since they are only focused on sensors and observation, which is only a subset of the identified key concepts. We can observe that some of the IoT ontologies cover most of the key concepts but none of them covers them all. Moreover, the different concepts are not represented with the same level of expressivity. In iot- ontology and SAREF, key concepts such as Actuator or Action are present but their representation is limited. For example, an actuator is defined as a device that modifies a property. This is less expressive than what can be expressed for a sensor with SSN which proposes a deep modeling of the sensors and the property they observe, but also of the relations between the sensors and their observa- tions, and of the observations themselves. In eDIANA18, an ontology referenced by SAREF, some specializations of actuator are given, but the mappings from these specializations to the saref:Actuator concept are not available directly. This analysis highlights the fact that an ontology for Actuators and Actions is needed (c.f. section 3.3). This analysis also highlights the failure of existing IoT ontologies in representing correctly all IoT key concepts. As these concepts are not limited to the IoT domain, reusing ontologies dedicated to them (such as SSN for sensor) could help gain in expressivity, as shown in section 3.2. 19 http://lov.okfn.org 20 3.2 Designing ontologies according to good practices Functional requirements The functional requirements aim at capturing good practices for ontology design and general semantic web guidelines. Reusability: One of the most important aspects of an ontology in such a broad domain as IoT is reusability: if an ontology is ad-hoc to a project, the work done in its definition will not benefit further projects. It is a critical issue that can be solved by different, non-mutually exclusive approaches: Reusability: One of the most important aspects of an ontology in such a broad domain as IoT is reusability: if an ontology is ad-hoc to a project, the work done in its definition will not benefit further projects. It is a critical issue that can be solved by different, non-mutually exclusive approaches: – Modularization: as stated in [6], designing ontologies in separated modules makes them easier to reuse and/or extend. IoT applications are related to many various domains, and it is difficult to capture all these application domains in the same ontology. Modular ontologies can be combined together according to specific needs, which is a more scalable approach. – Ontology Design Patterns: were introduced in [7]. Designing ontologies that respect Ontology Design Pattern (ODP) increases reusability and their potential for alignment, as shown in [8]. ODPs capture modelling efforts: using them is a way to capitalize on previous work, and to take advantage of the maturity of the semantic web compared to the IoT. 18 https://sites.google.com/site/smartappliancesproject/ontologies/ediana-ontology – Reuse of existing sources: avoids redefinition, and prevents from having to align a posteriori the redefined concepts to the existing sources for inter- operability. It is a key requirement for interoperability, which is a real issue in heterogeneous systems. – Alignment to upper ontologies: Upper-level ontologies define very ab- stract concepts in a horizontal manner. They articulate very diverse domain- specific ontologies, which is crucial for broad domains like IoT. – Compliance with the LOV requirements: The LOV19 is an online vo- cabulary register that increases visibility of vocabularies, and favours reuse by ensuring the respect of good practices listed in [2]. Level of formalism: To use the full advantages of the semantic description of devices and data, the description should enable reasoning and inference. 20 http://www.ontologydesignpatterns.org/ont/dul/DUL.owl 21 http://sweet.jpl.nasa.gov/ // gy g 21 http://sweet.jpl.nasa.gov/ 20 http://www.ontologydesignpatterns.org/ont/dul/DUL.owl 21 3.2 Designing ontologies according to good practices This choice is motivated by the possibilities it opens: – Applied to data, it is a way to bring context-awareness, as presented in [9] – Applied to data, it is a way to bring context-awareness, as presented in [9] – Applied to devices, it enables Thing discovery or self-configuration [10] – Applied to devices, it enables Thing discovery or self-configuration [10] – Applied to services it enables automatic composition as in [11] However, for concrete applications, the model should also by decidable, and in reasonable time, which de facto excludes an OWL-full model: OWL-DL is therefore the best choice. All surveyed ontologies are expressed in OWL-DL. Table 2. Reusability of IoT ontologies Structured by ODP Modular Reuses external ontologies Aligned with upper ontologies One the LOV Available online iot-ontology * ** N Y saref ** * Y Y OWL-IoT-S (*) * ** * N Y SA (*) * ** ** N N iot-lite N Y spitfire * ** Y N ssn ** ** * ** Y Y oneM2M N Y IoT-O (**) ** ** ** Y Y Assessment of existing IoT ontologies Table 2 shows that the semantic web best practices for reusability are not always followed: some ontologies are not available online, and the majority is not compliant with the requirements of the LOV. External ontologies are generally not reused, with the exception of SSN. OWL-S, a service ontology is reused in only one case. The other surveyed ontologies propose redefinitions of the service concept. For example, SAREF redefines the concepts present in multiple ontologies, and proposes alignments in an external, textual document. Design patterns have only been used in ontologies importing SSN. Upper ontologies used are DUL20 (especially used by SSN) and SWEET21 (for SA). The limited reuse of ontologies shows a lack of federating 19 http://lov.okfn.org 20 http://www.ontologydesignpatterns.org/ont/dul/DUL.owl Table 2. Reusability of IoT ontologies Structured by ODP Modular Reuses external ontologies Aligned with upper ontologies One the LOV Available online iot-ontology * ** N Y saref ** * Y Y OWL-IoT-S (*) * ** * N Y SA (*) * ** ** N N iot-lite N Y spitfire * ** Y N ssn ** ** * ** Y Y oneM2M N Y IoT-O (**) ** ** ** Y Y Table 2. 3.2 Designing ontologies according to good practices Both concept coverage and compliance with the functional requirements of existing ontologies fail to match the requirements we expect from an IoT ontol- ogy, which provides the motivation for our proposal of IoT-O. 3.3 Integrating existing ontologies in IoT-O Identification of existing ontologies is part of the NeOn process. Some concepts, which are part of the conceptual requirements, are defined by existing ontologies that are imported in IoT-O to avoid redefinition. SSN is a widely used W3C recommended ontology for sensors and observations. However, no ontology de- scribes the concept of actuator the way SSN describes the concept of sensor. This is why we propose Semantic Actuator Network (SAN)22, the Semantic Ac- tuator Network ontology. Actuators are devices that transform an input signal into a physical output, making them the exact opposite of sensors. SAN is built around Action-Actuator-Effect (AAE)23, a design pattern we propose, inspired from the Stimulus Sensor Observation (SSO) design pattern described in [12]. ( ) [ ] To define the notion of service, IoT-O imports Minimal Service Model (MSM), a lightweight service ontology which is generic enough to represent both REST and WSDL services (contrary to OWL-S24). The notion of energy consump- tion dedicated to the IoT is specified in PowerOnt, an ontology referenced by SAREF. The concepts of lifecycle are described using Lifecycle25, a lightweight vocabulary defining state machines. We extended Lifecycle in the IoT-lifecycle26 ontology with classes and properties specific to the IoT. Finally, to maximize extensibility and reusability, IoT-O imports DUL27, a top-level ontology, and aligns all its concepts and imported modules with it. 3.4 IoT-O, a modular core-domain IoT ontology IoT-O, the core-ontology we propose is composed of several modules. IoT-O’s architecture is summarized in figure 2. The names of the resources created from scratch are in red and highlighted, the names of the reengineered resources are underlined, and the arrows show dependencies. Solid arrows represent imports, and dashed arrows the reuse of concepts without import. 3.2 Designing ontologies according to good practices Reusability of IoT ontologies Structured by ODP Modular Reuses external ontologies Aligned with upper ontologies One the LOV Available online iot-ontology * ** N Y saref ** * Y Y OWL-IoT-S (*) * ** * N Y SA (*) * ** ** N N iot-lite N Y spitfire * ** Y N ssn ** ** * ** Y Y oneM2M N Y IoT-O (**) ** ** ** Y Y Table 2. Reusability of IoT ontologies Table 2. Reusability of IoT ontologies Assessment of existing IoT ontologies Table 2 shows that the semantic web best practices for reusability are not always followed: some ontologies are not available online, and the majority is not compliant with the requirements of the LOV. External ontologies are generally not reused, with the exception of SSN. OWL-S, a service ontology is reused in only one case. The other surveyed ontologies propose redefinitions of the service concept. For example, SAREF redefines the concepts present in multiple ontologies, and proposes alignments in an external, textual document. Design patterns have only been used in ontologies importing SSN. Upper ontologies used are DUL20 (especially used by SSN) and SWEET21 (for SA). The limited reuse of ontologies shows a lack of federating Assessment of existing IoT ontologies Table 2 shows that the semantic web best practices for reusability are not always followed: some ontologies are not available online, and the majority is not compliant with the requirements of the LOV. External ontologies are generally not reused, with the exception of SSN. OWL-S, a service ontology is reused in only one case. The other surveyed ontologies propose redefinitions of the service concept. For example, SAREF redefines the concepts present in multiple ontologies, and proposes alignments in an external, textual document. Design patterns have only been used in ontologies importing SSN. Upper ontologies used are DUL20 (especially used by SSN) and SWEET21 (for SA). The limited reuse of ontologies shows a lack of federating ontologies, apart from SSN. SSN being compliant with the semantic web good practices, it is possible to say that these guidelines favour reuse. Both concept coverage and compliance with the functional requirements of existing ontologies fail to match the requirements we expect from an IoT ontol- ogy, which provides the motivation for our proposal of IoT-O. 22 https://www.irit.fr/recherches/MELODI/ontologies/SAN 23 http://ontologydesignpatterns.org/wiki/Submissions:Actuation-Actuator-Effect 24 https://www.w3.org/Submission/OWL-S/, more dedicated to WSDL services 25 http://vocab.org/lifecycle/schema 26 https://www.irit.fr/recherches/MELODI/ontologies/IoT-Lifecycle 27 http://www.ontologydesignpatterns.org/ont/dul/DUL.owl 22 https://www.irit.fr/recherches/MELODI/ontologies/SAN 28 http://www.irit.fr/recherches/MELODI/ontologies/IoT-O.owl 4.1 Implementing the MAPE-K loop with SemIoTics Autonomic computing is a programming paradigm proposed in [13] focused on allowing a system to control an entity thanks to high-level policies and intro- spective knowledge. A classic control structure in autonomic computing is the MAPE-K loop (see fig. 3), separated in four steps : Monitoring, Analysis, Plan- ning and Execution, all exchanging Knowledge with the same knowledge base. Fig. 3. The MAPE-K loop, adapted to our use case Fig. 3. The MAPE-K loop, adapted to our use case SemIoTics implements the MAPE-K loop to control the connected devices in the apartment according to the policies fixed by the user. It is Java-based, and uses Apache Jena to manage the knowledge base and query it in SPARQL. The remainder of this section describes the usage of IoT-O at each step of the MAPE-K loop representing the introductory use case, from the temperature sensor measure to the actuator action. 4 Using IoT-O to manage a smart building This section gives an overview of the technical choices to implement our use case, and a detailed analysis of the scenario execution that instantiates IoT-O, allowing to see the interest of our requirements. The modules of IoT-O: – The Sensing module describes the input data. Its main classes come from SSN: ssn:Sensor and ssn:Observation. ssn:Device and its characteristics (ssn:- OperatingRange, ssn:Deployment...) provide a generic device description. 26 https://www.irit.fr/recherches/MELODI/ontologies/IoT-Lifecycle 27 27 http://www.ontologydesignpatterns.org/ont/dul/DUL.owl Fig. 2. Overview of IoT-O’s architecture Fig. 2. Overview of IoT-O’s architecture Fig. 2. Overview of IoT-O’s architecture – The Acting module describes how the system can interact with the physical world. Its main classes come from SAN: san:Actuator and san:Actuation. It also reuses SSN classes that are not specific to sensing, such as ssn:Device. g – The Lifecycle module models state machines to specify system life cycles and device usage. Its main classes are lifecycle:State and lifecycle:Transition. – The Service module represents web service interfaces. Its main classes come from MSM: msm:Service and msm:Operation. Services produce and consume msm:Messages, and RESTful services can be described with hRest. g , – Energy module: IoT-O’s energy module is defined by PowerOnt. It pro- vides the poweront:PowerConsumption class, and a set of properties to ex- press power consumption profiles for appliances. The core of IoT-O: IoT-O28 is both the name of the ontology and of the top module. It gives a conceptualization of the IoT domain, independent of the appli- cation, providing classes and relationships to link the underlying modules. Since many concepts are already defined in the modules, IoT-O’s core is limited: it de- fines 14 classes (out of 1126 including all modules), 18 object properties (out of 249) and 4 data properties (out of 78). IoT-O key class is iot-o:IoT Thing, which can be either an ssn:Device or an iot-o:SoftwareAgent. The power consumption of ssn:Devices is associated to lifecycle:State and poweront:PowerConsumption. iot-o:IoT Thing is a provider of msm:Service, and an msm:Operation can have an iot-o:ImpactOnProperty on an ssn:Property, linking abstract services to the physical world through devices. As a core domain ontology, IoT-O is meant to be extended regarding specific applicative needs and real-life devices and services. This design, inspired by SSN, makes IoT-O independent of the application. 4.2 Monitoring: Enrichment of sensor data into Observations Monitoring is the collection of signals regarding the controlled entity, here the apartment. The connected sensors collect observations, and produce raw data. This data is enriched to become a reusable piece of knowledge. Enrichment of sensor data is performed using the SSN ontology, which is in the Sensing module of IoT-O, as well as upper ontologies like QUDT (for the units), and possibly more sensor-specific domain ontologies, necessary due to the horizontal nature of IoT-O and SSN. An application-dedicated extension is for instance proposed in the Adream-Model module29. Each ssn:Sensor produces ssn:Observation, themselves composed of ssn:- SensorOutput whose value is described by ssn:ObservationValue. To maintain provenance knowledge, a ssn:SensorOutput can be linked to its raw representa- tion with the iot-o:hasRawRepresentation data property. The sensor’s character- istics (ssn:MeasurementProperty, the ssn:Property of the measured ssn:Feature- OfInterest) can lift the observation as well. f ) In the use case, the data initially observed by the sensor is standardized according to oneM2M, and then enriched. The enrichment process is ad-hoc, being performed by a dedicated enrichment script. For the example’s sake, the sensor observes a temperature of 26oC, converted into a ssn:ObservationValue. Once enriched, the observation is stored in the knowledge base to be used in the Analysis step. 4.3 Analysis: Abstraction of Observations in symptoms Analysis is the identification of observed signals as hiegher-level symptoms. In the use case, enriched observations are processed and matched against rules rep- resenting user preferences. These preferences are described using the concepts de- fined in yet another module: Autonomic30. The user creates autonomic:Property- Constraints (seamlessly through a graphical interface), transforming a ssn:- Property into a autonomic:ConstrainedProperty. Specifically, the ssn:Property temperature of the ssn:FeatureOfInterest living room air has two constraints, instances of autonomic:MaximumValue (25oC) and autonomic:MinimumValue (19oC). The last ssn:ObservationValue of the autonomic:ConstrainedProperty is out of the bounds defined by the autonomic:PropertyConstraint (26oC instead of 25), so the temperature is classified by the reasoner as an autonomic:Out- OfBoundsProperty thanks to custom rules expressed in the Jena rule engine language. 29 http://www.irit.fr/recherches/MELODI/ontologies/Adream-Model 30 http://www.irit.fr/recherches/MELODI/ontologies/Autonomic 31 http://delicias.dia.fi.upm.es/ontologies/ObjectWithStates.owl 32 http://vocab.org/lifecycle/schema 33 http://www.irit.fr/recherches/MELODI/ontologies/IoT-Lifecycle 4.5 Execution: Transmitting the Actions to the actuators Execution is the concrete implementation of the plan by the agent on the controlled system. An san:Action can be executed if it iot-o:isGroundedBy an msm:Operation. The agent converts the san:ActuationValue format that target devices can process, here REST commands. The translation of knowledge into a simpler data format (the opposite process of enrichment) can be driven by the semantic description of Operations, or dedicated annotations as in [14], where XML schemas are annotated for transformation from RDF to XML. This trans- lation enables the agent to interact with low-level, constrained devices that are not able to process complex knowledge representations. The example cycle is complete: the agent calls the adream-model:turnOn operation, and the fan cools the apartment. 4.4 Planning: Deducing Actions from symptoms Planing is the computation to an appropriate response to the monitored sys- tem’s symptoms. To determine its actions, the autonomic agent relies on its knowledge base, which contains a priori defined policies. For semIoTics, actions to be implemented by the agent are described using SAN, the action and ac- tuator ontology. The agent queries the knowledge base to look for san:Actuator instances that san:actsOn the autonomic:OutOfBoundsProperty, and which san:- receivesActuation an actuation that iot-o:hasImpact an autonomic:ImpactOn- Property that is coherent with the symptom. In the example, since it is too hot, the adream-model:fan can be used, but also potentially the adream-model:space- Heater, since its adream-model:turnOffoperation has a adream-model:Negative- Impact on the temperature. If multiple, sequential actions have to be performed, they are orchestrated using the Lifecycle module of IoT-O, which rrelies on the the Objects with States (ows)31 ontology design pattern to represent devices as state machines. ssn:Device (superclass of both ssn:SensingDevice and san:ActuatingDevice) are objects that ows:hasState exactly 1 ows:State, because objects should only be in one state at a time. The ows:State is equivalent to the lifecycle:State (from the Lifecycle32 vocabulary, extended by the IoT-Lifecycle33 ontology), and life- cycle:State are connected by lifecycle:Transition instances. This description of devices allows for the representation of transitions only available in certain states of the device. Only msm:Operation instances that iot-o:isGroundedBy a san:- Actuation that iot-lifecycle:triggersTransition a lifecycle:Transition that is a life- cycle:possibleTransition of the device current lifecycle:State can be called at a given time. For instance, the space heater adream-model:turnOffoperation will only be available if the space heater is on. In our example it is off, so the agent plans to turn on the fan and creates the corresponding san:ActuationValue. The selection of devices and their operations is driven by necessity (only the devices impacting the right property are selected), but it can also be driven by policies based on knowledge about the system, to minimize energy consumption, to optimize reaction time... 5 Conclusion and future works This paper introduces both functional and conceptual requirements to build an IoT ontology usable in a wide scope of projects and compliant with the seman- tic web good practices. These requirements drove the development of the core contribution of the paper: IoT-O, a modular core-domain IoT ontology. IoT-O’s modules are presented in details, to show their compliance with the requirements. IoT-O is then used by semIoTics, a system implementing the MAPE-K loop, an autonomic computing structure. SemIoTics is described in a home automation use case that instantiates knowledge described using IoT-O’s modules. In the IoT, data is produced raw by sensors, and needs to be enriched to become reusable knowledge. However, enriched data is heavier to exchange and process than raw data, making it unsuitable to be consumed by the more con- strained devices (typically sensors and actuators). To allow these IoT nodes to be semantically interoperable with more powerful ones (e.g. gateways, servers, laptops), lowering techniques, transforming knowledge into raw data, are re- quired. We are currently working on such an approach. Other perspectives of our work will be to define an intuitive way to help end users/administrators express constraints and policies to drive the system. References 1. Ganz, F., Puschmann, D., Barnaghi, P., Carrez, F.: A Practical Evaluation of Information Processing and Abstraction Techniques for the Internet of Things. IEEE Internet of Things Journal 2(4) (2015) 340–354 2. Gyrard, A., Serrano, M., Atemezing, G.A.: Semantic web methodologies, best practices and ontology engineering applied to Internet of Things. In: 2015 IEEE 2nd World Forum on Internet of Things (WF-IoT), IEEE (2015) 412–417 g ( ), ( ) 3. Murdock, P.: White paper: Semantic interoperability for the web of things (2016) 4. Ben-Alaya, M., Medjiah, S., Monteil, T., Drira, K.: Toward semantic interoper- ability in oneM2M architecture. IEEE Communications Magazine 53(12) (2015) 5. del Carmen Suarez de Figueroa Baonza, M.: NeOn methodology for building ontology networks : specification, sheduling and reuse. PhD thesis (2010) 6. Aquin, M.: Modularizing ontologies. Ontology engineering in a networked world Springer B (2012) 9–34 7. Gangemi, A.: Ontology Design Patterns for Semantic Web Content. History 3729(4) (2005) 262–276 8. Scharffe, F., Euzenat, J., Fensel, D.: Towards design patterns for ontology align- ment. In: Proceedings of the 2008 ACM symposium on Applied computing - SAC ’08, New York, New York, USA, ACM Press (mar 2008) 2321 ( ) 9. Henson, C., Sheth, A., Thirunarayan, K.: Semantic perception: Converting sensory observations to abstractions. IEEE Internet Computing 16(2) (2012) 26–34 10. Chatzigiannakis, I., Hasemann, H., Karnstedt, M., Kleine, O., Kr¨oller, A., Leggieri, M., Pfisterer, D., R¨omer, K., Truong, C.: True Self-Configuration for the loT. In: 3rd International Conference on the Internet of Things (IOT). (2012) 11. Han, S.N., Lee, G.M., Crespi, N.: Towards Automated Service Composition Using Policy Ontology in Building Automation System. In: 2012 IEEE Ninth Interna- tional Conference on Services Computing. (2012) 685–686 12. Janowicz, K., Compton, M.: The Stimulus-Sensor-Observation Ontology Design Pattern and its Integration into the Semantic Sensor Network Ontology. In: Pro- ceedings of the 9th International Semantic Web Conference, 3rd International Workshop on Semantic Sensor Networks. (2010) 7–11 13. Kephart, J., Chess, D.: The vision of autonomic computing. Computer 36(1) (jan 2003) 41–50 14. Kopeck´y, J., Vitvar, T., Bournez, C., Farrell, J.: SAWSDL: Semantic Annotations for WSDL and XML Schema. IEEE Internet Computing 11(6) (2007) 60–67
https://openalex.org/W1977190268
https://link.springer.com/content/pdf/10.1007/s10863-012-9479-3.pdf
English
null
A study of molecular changes relating to energy metabolism and cellular stress in people with Huntington’s disease: looking for biomarkers
Journal of bioenergetics and biomembranes
2,012
cc-by
10,861
J Bioenerg Biomembr (2013) 45:71–85 DOI 10.1007/s10863-012-9479-3 J Bioenerg Biomembr (2013) 45:71–85 DOI 10.1007/s10863-012-9479-3 A study of molecular changes relating to energy metabolism and cellular stress in people with Huntington’s disease: looking for biomarkers Jolanta Krzysztoń-Russjan & Daniel Zielonka & Joanna Jackiewicz & Sylwia Kuśmirek & Irena Bubko & Aneta Klimberg & Jerzy T. Marcinkowski & Elżbieta L. Anuszewska Received: 26 June 2012 /Accepted: 4 October 2012 /Published online: 16 October 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Huntington’s disease (HD) is a neurodegenerative disorder characterized by a progressive motor and cognitive decline and the development of psychiatric symptoms. The origin of molecular and biochemical disturbances in HD is a mutation in the HTT gene, which is autosomally dominantly inherited. The altered huntingtin protein is ubiquitously expressed in the CNS, as well as in peripheral tissues. In this study we measured the metabolism changes in gene transcrip- tion in blood of HD gene carriers (premanifest and manifest combined) versus 28 healthy controls. The comparison revealed statistically significant Global Pattern Recognition Fold Change (FC) for 6 mRNA transcripts, reflecting an in- crease of: MAOB (FC03.07; p00.0005) which encodes an outer mitochondrial membrane-bound enzyme called mono- amine oxidase type B; TGM2 (FC01.8; p00.02) encoding a transglutaminase 2 that mediates cellular stress; SLC2A4 (FC0 1.64; p00.02) solute carrier family 2 (facilitated glucose trans- porter) member 4; branched chain ketoacid dehydrogenase kinase (BCKDK) (FC01.34; p00.02); decrease of LDHA (FC0−1.16; p00.03) lactate dehydrogenase A; and brain- derived neurotrophic factor (BDNF) (FC0−2,11; p00.03). These distinguished changes coincided with HD progress. The analyses of gene transcription levels in sub-cohorts con- firmed these changes and also revealed 28 statistically signifi- cant FCs of gene transcripts involved in ATP production and BCAA metabolism. The analyses of gene transcription levels in sub-cohorts con- firmed these changes and also revealed 28 statistically signifi- cant FCs of gene transcripts involved in ATP production and BCAA metabolism. Keywords Huntington’s disease . Energy metabolism . Transcriptomic biomarkers Keywords Huntington’s disease . Energy metabolism . Transcriptomic biomarkers J. Krzysztoń-Russjan (*): J. Jackiewicz: S. Kuśmirek: I. Bubko: E. L. Anuszewska Department of Biochemistry and Biopharmaceuticals, National Medicines Institute, Chelmska 30/34 Str., 00-725 Warsaw, Poland e-mail: jrussjan@il.waw.pl D. Zielonka: A. Klimberg: J. T. Marcinkowski Chair of Social Medicine, Poznan University of Medical Science, Rokietnicka 5C Str., 60-806 Poznan, Poland D. Zielonka: A. Klimberg: J. T. Marcinkowski Chair of Social Medicine, Poznan University of Medical Science, Rokietnicka 5C Str., 60-806 Poznan, Poland J. Krzysztoń-Russjan (*): J. Jackiewicz: S. Kuśmirek: I. Bubko: E. L. Anuszewska Department of Biochemistry and Biopharmaceuticals, National Medicines Institute, Chelmska 30/34 Str., 00-725 Warsaw, Poland e-mail: jrussjan@il.waw.pl Subjects Altogether 57 people were included in this study. The HD group – HDG (n029) consisted of pre-manifest HD (n06) and manifest HD (n023) subjects and the control group (CG) comprised healthy subjects (n028). Subject’s key character- istics are presented in Table 1a and b. All HDG individuals were confirmed carriers of the HTT gene mutation (ranged from 38 to 81 CAG (mean 45, SD±15) in the larger allele) Subjects were also characterized by their body mass index (BMI), calf circumference (CC; cm), to reflect condition of muscle mass, and a Mini Nutritional Assessment (MNA) evaluated the risk of malnutrition. HD motor symptoms were rated using the Unified Huntington Disease Rating Scale (UHDRS), and motor and functional impairment were evalu- ated using the Total Functional Capacity (TFC) assessment. The activity of the peroxisome proliferator-activated recep- tor γ (PPARγ) coactivator 1α (PGC-1α) in human and mouse HD striatal and motor neurons is reduced due to the mHTT/ HTT related decrease of its transcription level. This reduction results in impairment of mitochondrial functions (Cui et al. 2006; St-Pierre et al. 2006; Weydt et al. 2006) as PGC-1α, a transcriptional co-factor, encoded by PPARGC1A gene, plays a key role regulating the expression of genes responsible for energy metabolism. It participates in energy homeostasis, adaptive thermogenesis, β-oxidation of fatty acids, and glu- cose metabolism by regulating the expression of mitochondrial OXPHOS genes and endogenous antioxidants (Puigserver et al. 2003). PGC-1α also regulates the activity of histone deace- tylase in muscles. Co-localization of HTT and PPARGC1A genes can influence transcription deregulation and is therefore considered to play an important role in HD pathogenesis (Kozlowski et al. 2010; Weydt et al. 2006). Mitochondrial malfunction in the basal ganglia of HD patients results in glucose metabolism reduction and lactate increase (Milakovic and Johnson 2005). Furthermore, biochemical studies revealed a reduced activity of several key components of oxidative phosphorylation, including complexes II, III, and IV of the electron transport chain in the mitochondria of striatal neurons in HD patients at an advanced stage of the disease (Beal et al. 1993; Browne and Beal 2004). Subjects were identified using the REGISTRY Database of European Huntington’s Disease Network. This study was approved by Bioethical Committee at Poznan University of Medical Science, Poland on September 03. 2009. Agreement No, 770/09. Subjects All human studies approved by this Agreement have been performed in accordance with Good Clinical Practice laid down in the Declaration of Helsinki. Introduction Huntington’s disease (HD) is an autosomal dominant, neuro- degenerative disorder with no effective treatment (The Huntington’s Disease Collaborative Research Group 1993; Perez-De La Cruz and Santamaria 2007). The course of HD is slow and characterized by a gradual progression of motor, emotional and cognitive dysfunction leading to speech cessa- tion, swallowing difficulties, walking problems and finally to the loss of independence and the whole organism devastation (Davies and Ramsden 2001; Ross and Tabrizi 2011). CAG triplet repeat expansion in the first exon of the HTT gene results in an expansion of the polyglutamine (polyQ) tract in the N- terminus of the huntingtin protein (HTT) (Atwal et al. 2007); expansion above 35 polyQ results in the “mutant huntingtin” (mHTT). The unchanged polyQ tract is an active point of HTT in binding to other protein partners, but an elongated polyQ tract leads to the formation of nuclear and cytoplasmic aggre- gates, confers a gain-of-function as well as a loss-of-function of huntingtin and causes pathological changes on the cellular level inside and outside the CNS (Bjorkqvist et al. 2008; Sathasivam et al. 1999). Both the number of CAG repeats in the larger allele and epigenetic factors contribute to HD onset age and disease progression. 72 J Bioenerg Biomembr (2013) 45:71–85 Polymerase Chain Reaction (qPCR), of HD (pre-manifest and manifest) subjects compared to healthy subjects. Polymerase Chain Reaction (qPCR), of HD (pre-manifest and manifest) subjects compared to healthy subjects. Metabolic abnormalities in HD, including an impairment of cellular energy production and mitochondrial dysfunc- tion, result in lower adenosine 5′-triphosphate (ATP) pro- duction (Chaturvedi et al. 2010; Cui et al. 2006; Liang and Ward 2006; Milakovic and Johnson 2005; Mochel et al. 2007, 2012; Weydt et al. 2006). Bioenergetics defects lead to weight loss and muscle wastage despite increased caloric intake in HD patients (Chaturvedi et al. 2010; Cui et al. 2006; Kosinski et al. 2007; Weydt et al. 2006). In addition, a systemic metabolic defect is closely related to the low level of branched chain amino acids (BCAA) in the serum or plasma of HD patients, which also results in early weight loss (Chaturvedi et al. 2010; Cui et al. 2006; Kosinski et al. 2007; Mochel et al. 2007, 2011; Weydt et al. 2006). Blood sample collection Blood samples were collected from fasting (since 6.00 p.m. of the previous day) subjects between 8 and 10 a.m. General blood tests required 15 ml and the Tempus collection tube (with RNA stabilizing buffer) (Applied Biosystems, Foster City, CA, USA) for total RNA isolation required 3 ml. To exclude disorders affecting metabolism, general blood tests of all participants were preformed for blood smear and morphology, CRP, ESR, triiodothyronine (FT3), tetraiodo- thyronine (FT4), TSH, cortisol, urea, creatinine, uric acid, cholesterol, protein fractions pattern and glucose levels. It is argued that the BCAA level decrease in the sera of HD subjects contributes to a BMI reduction and accelerates the disease progression (Mochel et al. 2007, 2011). BCAAs are involved in the mitochondrial intermediary metabolism as substrates in tricarboxilic acid (TCA), and their decreased level may indicate a systemic attempt to compensate for an early energy deficit in HD (Mochel et al. 2012). Therefore an identification of molecular changes associated with energy production and BCAA metabolism at the gene transcription level could identify biomarkers for the early detection of the disease as well as potential drug targets for effective therapies. Total RNA extraction Total RNA was isolated using Tempus™Spin RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manual instructions. Genomic DNA was eliminated by treating each sample with RNase-free DNase I (Fermentas Thermo Fisher Scientific Inc., Vilnius, Lithuania) according to the manual instructions. The concentration of isolated total RNA was calculated from absorbance at 260 nm with a BioPhotometer (Eppendorf, Hamburg, Germany), the purity In this study we focused on energy metabolism pathways in blood cells in order to evaluate energy metabolism changes at the gene transcription level, using quantitative J Bioenerg Biomembr (2013) 45:71–85 73 Table 1 The study group and sub-cohorts clinical and genetic characteristics a) Study group and sub-cohorts HDG n029 CG n028 b) HDG sub-cohorts HDG Gender-cohort CAG–cohort Female n016 n022 CAG number repeats Male n013 n06 Mean value (SD) 45 (±15.0) Age-cohort CAG1: 38–41 n09 Age (years) 46 40 CAG2: 42–44 n010 Mean value (SD)* (±15.0) (±15.0) CAG3: 46–81 n010 Age1: ≤35 n08 n012 TFC-cohort Age2: 36–55 n015 n011 TFC (score in points) Age3: ≥56 n06 n05 Mean value (SD) 9.7 (±3.1) BMI-cohort TFC1: 1–8 n07 BMI (kg/m2) 23.8 23.2 TFC2: 9–10 n09 Mean value (SD) (±4) (±4.4) TFC3: 11–13 n013 BMI1: up to 18.4 (underweight) n02 n01 UHDRS-cohort BMI2: 18.5–24.9 (standard) n017 n019 UHDRS (score in points) BMI3: 25.0–29.9 (overweight) n06 n07 Mean value (SD) 38.5 (±24.3) BMI4: 30.0–34.9 (obesity) n04 n01 UHDRS1: 1– 8 (presymptomatic) n06 CC-cohort UHDRS2: 22–31 n05 Calf circumference CC (cm) 36.0 36.7 UHDRS3: 36–43 n08 Mean value (SD) (±3.6) (±3.1) UHDRS4: 45–55 n06 CC1: 27–30 n04 n00 UHDRS5: 61–91 n05 CC2: 31–35 n06 n012 HD disease duration (dd)-cohort CC3: 36–38 n014 n010 HD duration period (years) CC4 HDG/CG: 39–41/39–45 n05 n06 Mean value (SD) 6.3 (±4.7) MNA-cohort HD dd1; 0 (presymptomatic) n06 MNA (score in points) 11.1 12.6 HD dd2; 1–5 n06 Mean value (SD) (±2.0) (±1.3) HD dd3; 6–10 n012 MNA1: 0–7 (malnourished) n04 n02 HD dd4; 11–15 n05 MNA2: 8–11 (risk of malnutrition) n010 n05 MNA3: 12–14 ( normal nutritional status) n015 n021 SD* standard deviation Table 1 The study group and sub-cohorts clinical and genetic characteristics was verified by optical density (OD) absorption ratio OD260 nm/ OD280 nm between 1.60 and 1.8, and OD260 nm/OD230 nm ranging from 1.8 to 2.00 and the integrity was evaluated by electrophoresis on Gel – RNA Flash Gel System (Lonza Rockland Inc., Rockland, ME, USA). First strand cDNA synthesis First strand cDNA synthesis Total RNA extraction Total RNA subunits of 18S and 28S were observed on the gel and absence of smears indicating minimal degradation of the RNA, RNA template was suspended in RNase and DNase free water and stored at −80 °C with RNase inhibitor (Fermentas Thermo Fisher Scientific Inc, Vilnius, Lithuania). priming method according to the manufacturer’s recommen- dations. The first strand cDNA synthesis was started with the incubation of the transcription mixture at 37 °C for 30 min, to begin the reverse transcriptase reaction. Finally, the Prime Script Reverse Transcriptase was inactivated by heating the reaction mixture for 5 s. at 85 °C. Each RNA sample was controlled for genomic DNA contamination by incubation without the addition of reverse transcriptase into the cDNA synthesis mixture. All cDNA samples were stored at −20 °C and diluted with RNase and DNase free water before being used as a template in the RT-qPCR (Reverse Transcriptase quantitative Real-Time Polymerase Chain Reaction) analysis. SD* standard deviation qPCR at 60 °C (for annealing and elongation). A dissocia- tion protocol with a gradient from 95 °C to 55 °C and to 95 °C was used for each primer pair to verify the specificity of the RT-qPCR reaction and the absence of primer-dimer control. In addition, each PCR reaction included a reverse transcription negative control to check for potential genomic DNA contam- ination. Reagent contamination was also detected by a reac- tion mix without template. reference to the CG (calibrator) using cycle threshold (Ct) values obtained for all the genes tested. The GPR FC value was calculated for each gene tested. FC with p≤0.05 was defined as statistically significant. For all the genes tested in the distinguished cohorts, several comparisons were per- formed in order to screen for statistical significance in FCs. The statistically significant FCs across sub-cohorts were ana- lyzed by the Kruskal Wallis (Chi2) test and compared using the Mann–Whitney U test - (Table 3a–i). The Spearman’s coefficient was calculated to verify the correlation for analyt- ical results with gene transcription level changes expressed by FC (in the sub-cohorts), (Tables 4 and 5). SPSS v. 19.0 software (SPSS Inc., USA) and Statistica v. 9. software, (StatSoft Inc, USA) were used for statistical calculations. qPCR qPCR One microgram RNA was reverse-transcribed using the SYBR PrimeScript RT-PCR kit II (Takara Bio Inc., Otsu, Shiga, Japan) for first-strand cDNA synthesis using 2.5 μM oligonucleotides primer and 5 μM random hexamer for the Quantitative PCR was performed using the MxPro 3005P apparatus (Stratagene, LaJolla, CA, USA) and the SYBR Quantitative PCR was performed using the MxPro 3005P apparatus (Stratagene, LaJolla, CA, USA) and the SYBR J Bioenerg Biomembr (2013) 45:71–85 74 Premix Ex TaqTM II (Takara Bio Inc., Otsu, Shiga, Japan). The PCR Mix solution was previously made using the 974.68 μl SYBR Premix, 1031.32 μl RNase and DNase free water, and the 106 μl diluted template contained 400 ng of cDNA. The single PCR mix reaction volume was 20 μl. Gene expression analysis was conducted with the StellARray™Gene Expression System (Lonza, Walkersville, MD, USA), a type of qPCR array, with previ- ously optimized primer concentrations applied into the 96 wells plates. Thermocycling conditions were set as follows: an initial PCR mix activation was for 2 min. at 50 °C, the next polymerase activation step was for 15 s. at 95 °C, then 40 cycles of 15 s. at 95 °C (template denaturation) and 1 min. at 60 °C (for annealing and elongation). A dissocia- tion protocol with a gradient from 95 °C to 55 °C and to 95 °C was used for each primer pair to verify the specificity of the RT-qPCR reaction and the absence of primer-dimer control. In addition, each PCR reaction included a reverse transcription negative control to check for potential genomic DNA contam- ination. Reagent contamination was also detected by a reac- tion mix without template. Premix Ex TaqTM II (Takara Bio Inc., Otsu, Shiga, Japan). The PCR Mix solution was previously made using the 974.68 μl SYBR Premix, 1031.32 μl RNase and DNase free water, and the 106 μl diluted template contained 400 ng of cDNA. The single PCR mix reaction volume was 20 μl. Gene expression analysis was conducted with the StellARray™Gene Expression System (Lonza, Walkersville, MD, USA), a type of qPCR array, with previ- ously optimized primer concentrations applied into the 96 wells plates. Thermocycling conditions were set as follows: an initial PCR mix activation was for 2 min. at 50 °C, the next polymerase activation step was for 15 s. at 95 °C, then 40 cycles of 15 s. at 95 °C (template denaturation) and 1 min. Analytical tests Comparison between analytical test results in both HDG and CG was performed. Apart from cholesterol, HDL and LDL levels in HDG, and HDL in CG, all other values of the analytical test results in both groups did not exceed the range of reference values. The cholesterol and LDL levels were slightly higher (~210.1 and 131.6 respectively) in HDG than in the control (198.7 and 119.4 respectively), but not significant in the t-Student test. Total proteins, β2 fraction levels, alfa-1 globulins level and FT3 level were significantly different between the CG and HDG, but were within the range of reference values. Positive significant correlations in the HGD among the HGB, HCT, ESR, CRP MNA, MHCH levels as well as lymphocytes amount and BMI were found. In the CG, a significant positive correlation between CC and WBC and also BMI and CRP was obtained (Table 5). The panel of 95 genes, including normalizers, was previ- ously selected for this study. The gene panel focused on several pathways involved in energy metabolism (ATP pro- duction) such as: glycolysis/Krebs cycle, electron transport chain, mitochondria biogenesis and construction, BCAA metabolism, transcription factors related to energy produc- tion, cell cycle, cell stress/immune response and other cel- lular pathways (Table 2). An initial analysis was performed using the MxPro 3005P system software for determining the Ct (cycle thresh- old) value after threshold assessment. The relative transcrip- tion level was calculated based on ΔΔCt type of analysis using the 2−ΔΔCt method in order to Fold Change (FC) value determination between compared groups. The data obtained after qPCR was analyzed by Global Pattern Recognition, statistical software (GPR; https://array.bhbio. com/BHB/GUI/AP/GPR.aspx). Normalization to 18S rRNA, was used as internal control, and 9 additional genes normal- izers simultaneously distinguished by GPR software analy- sis were performed for each gene tested (Akilesh et al. 2003). The GPR algorithm allows the detection of signifi- cant changes in gene expression patterns by comparing the expression of each gene to every other gene in the array. Significant changes are identified and ranked providing relative normalization in qPCR experiments. The relations between HD defining parameters, anthropometric tests and MNA score Correlations among clinical HD defining parameters, such as: TFC, disease duration and CAG repeats number in the larger allele and BMI, CC and MNA score were calculated based on the Spearman’s coefficient factor analysis (Table 4). The sta- tistically significant positive correlations were found between the age of subjects and the age of HD onset (R00.718; p< 0.01) and between the calf circumference and TFC score (R0 0.386; p00.039) and also between MNA and TFC score (R0 0.525; p00.003). Results of these correlations indicate that as HD progresses, the calf circumference and MNA score decreases. A negative correlation was only found between the age of HD subjects and the number of CAG repeats in the larger allele (R0−0.551; p00.002). Statistical methods and calculations Statistical methods and calculations A normal distribution of analytical results was verified and confirmed. Then the t-Student’s test for differences between HDG and CG was preformed. Statistical methods and calculations The GPR was used to deter- mine the gene transcript level changes for the HDG with 75 J Bioenerg Biomembr (2013) 45:71–85 Table 2 A list of genes tested in this study Gene ID Gene Symbol Function Glycolysis/Krebs cycle 1 50 ACO2a Aconitase 2, mitochondrial 2 226 ALDOA Aldolase A, fructose-bisphosphate 3 2597 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 4 3417 IDH1 Isocitrate dehydrogenase 1 (NADP+) 5 3939 LDHA Lactate dehydrogenase A 6 4967 OGDH Oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) 7 5160 PDHA1 Pyruvate dehydrogenase (lipoamide) alpha 1 8 5163 PDK1 Pyruvate dehydrogenase kinase, isozyme 1 9 5223 PGAM1 Phosphoglycerate mutase 1 (brain) Electron transport chain/ATP production/mitochondria biogenesis and construction 10 488 ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 11 1327 COX4I1 Cytochrome c oxidase subunit IV isoform 1 12 10328 COX4NB COX4 neighbor 13 4513 COX2 Cytochrome c oxidase subunit II 14 1374 CPT1A Carnitine palmitoyltransferase 1A 15 4129 MAOB Monoamine oxidase B 16 6390 SDH Succinate dehydrogenase complex, subunit B, iron sulfur (Ip) 17 7351 UCP2 Uncoupling protein 2 (mitochondrial, proton carrier) 18 7352 UCP3 Uncoupling protein 3 (mitochondrial, proton carrier) BCAA metabolism 19 27034 ACAD8 Acyl-Coenzyme A dehydrogenase family, member 8 20 4329 ALDH6A1 Aldehyde dehydrogenase 6 family, member A1 21 549 AUH AU RNA binding protein/enoyl-Coenzyme A hydratase 22 587 BCAT2 Branched chain aminotransferase 2, mitochondrial 23 593 BCKDHA Branched chain keto acid dehydrogenase E1, alpha polypeptide 24 594 BCKDHB Branched chain keto acid dehydrogenase E1, beta polypeptide 25 10295 BCKDK Branched chain ketoacid dehydrogenase kinase 26 1629 DBT Dihydrolipoamide branched chain transacylase E2 27 1738 DLD Dihydrolipoamide dehydrogenase, E3 28 11112 HIBADH 3-hydroxyisobutyrate dehydrogenase 29 56922 MCCC1 Methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) 30 64087 MCCC2 Methylcrotonoyl-Coenzyme A carboxylase 2 (beta) Transcription factors related to the energy production 31 10891 PPARGC1A Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha 32 23082 PPRC1 Peroxisome proliferator-activated receptor gamma, coactivator-related 1 33 5465 PPARA Peroxisome proliferator-activated receptor alpha 34 5467 PPARD Peroxisome proliferator-activated receptor delta 35 5468 PPARG Peroxisome proliferator-activated receptor gamma 36 6506 SLC1A2 Solute carrier family 1 (glial high affinity glutamate transporter), member 2 37 6517 SLC2A4 Solute carrier family 2 (facilitated glucose transporter), member 4 38 7019 TFAM Transcription factor A, mitochondrial Cell cycle/cell death/apoptosis/immune response 39 366 AQP9 Aquaporin 9 40 581 BAX BCL2-associated X protein 41 598 BCL2L1 BCL2-like 1 42 627 BDNF Brain-derived neurotrophic factor 43 834 CASP1 Caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase) 44 836 CASP3 Caspase 3 apoptosis related cysteine peptidase Table 2 A list of genes tested in this study Table 2 (continued) Gene ID Gene Symbol Function 45 839 CASP6 Caspase 6, apoptosis-related cysteine peptidase 46 841 CASP8 Caspase 8, apoptosis-related cysteine peptidase 47 1020 CDK5 Cyclin-dependent kinase 5 48 1211 CLTA Clathrin, light chain A 49 1213 CLTC Clathrin, heavy chain (Hc) 50 3309 HSPA5 Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) 51 3329 HSPD1 Heat shock 60 kDa protein 1 (chaperonin) 52 8870 IER3 Immediate early response 3 53 9927 MFN2 Mitofusin 2 54 27030 MLH3 MutL homolog 3 55 2475 MTOR Mechanistic target of rapamycin (serine/threonine kinase) 56 4792 NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 57 5921 RASA1 RAS p21 protein activator (GTPase activating protein) 1 58 8767 RIPK2 Receptor-interacting serine-threonine kinase 2 59 7124 TNF-a Tumor necrosis factor (TNF superfamily, member 2) 60 608 TNFRSF17 Tumor necrosis factor receptor superfamily, member 17 61 7157 TP53 Tumor protein p53 62 7316 UBC Ubiquitin C 63 3093 UBE2K Ubiquitin-conjugating enzyme E2K (UBC1 homolog, yeast) 64 7345 UCHL1 Ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) Cell stress/Immune response 65 875 CBS Cystathionine-beta-synthase 66 948 CD36 CD36 molecule (thrombospondin receptor) 67 1639 DCTN1 Dynactin 1 (p150, glued homolog, Drosophila) 68 1843 DUSP1 Dual specificity phosphatase 1 69 3117 HLA-DQA1 Major histocompatibility complex, class II, DQ alpha 1 70 3576 IL8 Interleukin 8 71 3553 IL1B Interleukin 1, beta 72 8564 KMO KMO kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) 73 6648 SOD2 Manganese superoxide dismutase Other transcription factors 74 1386 ATF2 Activating transcription factor 2 75 1387 CREBBP CREB binding protein 76 1958 EGR1 Early growth response 1 77 9611 NCOR1 Nuclear receptor co-repressor 1 78 283131 NEAT1 Nuclear paraspeckle assembly transcript 1 (non-protein coding) 79 4899 NRF1 Nuclear respiratory factor 1 80 56731 SLC2A4RG SLC2A4 regulator 81 6667 SP1 Sp1 transcription factor 82 6874 TAF4 TAF4 RNA polymerase II, TATA box binding protein 83 6908 TBP TATA box binding protein 84 7052 TGM2 Transglutaminase 2 (C polypeptide, protein-glutamine-gamma-glutamyltransferas 85 9322 TRIP10 Thyroid hormone receptor interactor 10 Calcium modulated proteins and HD related genes 86 801 CALM1 Calmodulin 1 (phosphorylase kinase, delta) 87 805 CALM2 Calmodulin 2 (phosphorylase kinase, delta) 88 808 CALM3 Calmodulin 3 (phosphorylase kinase, delta) 89 51806 CALML5 Calmodulin-like 5 90 9001 HAP1 Huntingtin-associated protein 1 76 J Bioenerg Biomembr (2013) 45:71–8 J Bioenerg Biomembr (2013) 45:71–85 76 77 J Bioenerg Biomembr (2013) 45:71–85 Table 2 (continued) Gene ID Gene Symbol Function 91 3092 HIP1 Huntingtin interacting protein 1 92 3064 HTT Huntingtin Preliminary normalizers and genomic DNA control 93 3251 HPRT1 94 60 ACTB 95 100008588 18S rRNA 96 gDNA control a Statistically significant gene transcripts changes obtained in this study were distinguished by the bold type a Statistically significant gene transcripts changes obtained in this study were distinguished by the bold type Gene expression general classification of obesity (Table 1a). Statistical methods and calculations A comparison of the qPCR results between the HDG and control yielded 5 transcripts FCs with p<0.05 based on GPR including an increase of IL8 transcript (from −2.48 for CC1 up to 1.41 for CC3), MAOB transcript (from 3.72 for CC1 up to 4.16 for CC3), and a decrease of COX2 transcript (from −1.05 for CC1 up to −1.27 for CC3), and TGM transcript (from 2.62 for CC1 up to 1.36 for CC4), however COX2 FC ranges were not statistically significant. The HDG and CG were stratified according to the following characteristics: gender, age, BMI, CC, MNA and gene tran- scription rates were compared. Significant changes in transcrip- tion were detected between the various sub-cohorts (Table 1a). Comparison of gender revealed significant FCs for 3 transcripts (Table 3a): CD36, MAOB and SLC2A4. In the HDG the FCs for CD36 and MAOB were significantly higher in women than in men (1.1 vs. −1.78; p<0.05) and (2.34 vs. 4.32; p<0.05) respectively. This was in contrast to SLC2A4, where the FC was higher in men (1.89 vs. 1.15; p<0.05). The age sub-cohort comparison (Table 3b) between the HDG and CG based on the GPR analysis showed significant FCs in 16 genes. The FCs range across age1- to age3-cohorts showed a decrease of CREBBP, SLC2A4RG and an increase of HSPA5, ATP2A2 in HDG. These changes were found in all three HDG age sub- cohorts; however the FC ranges across the age sub-cohorts were not statistically significant. The transcription changes of IL8, MTOR, BCKDHA, BCKDHB, MLH3 and UCP2 genes found across the age cohorts presented a FCs decrease together with age increase. The increased level of mRNA transcripts concerned RASA1, ACO2, UBE2K, HSPD1, PPARG and CASP6 was also found in the age2- and age3-cohorts regarding the reduced level only in the age1-cohort. g y g The MNA-cohorts reflected a study group differentiation performed on the basis of the nutrition status assessment according to the MNA scale (Table 1a) (Rubenstein et al. 2001). Due to the relatively small number of healthy subjects found in the MNA1-cohort, control MNA1 and 2-cohorts (with MNA score 8–11) were pooled and used as a reference for the MNA1- and MNA2-cohort GPR FC calculations (Table 3e). The MNA-cohorts comparison between the HDG and control showed only two significant FCs, namely in BDNF and MAOB. Statistical methods and calculations Due to the relatively small number of HD and control subjects collect- ed in the BMI1- and BMI4-cohorts, for the FCs calculation the BMI1- and BMI2-cohorts and also BMI3- and BMI4- cohorts were pooled. Analysis of transcription level changes revealed 4 transcripts with statistically significant GPR FCs including PPRC1, LDHA, SLC2A and MAOB (Table 3c). The simultaneous increase of PPRC1 and decrease of MAOB transcripts across the BMI-cohorts were statistically significant according to the Kruskal Wallis test and appeared together with the increase of BMI index value. In order to identify the transcription level changes between HDG and CG three types of comparisons were performed in this study, the first, between undifferentiated HDG and CG, and the second, between HDG- and CG-cohorts, were both differentiated according to gender, age, BMI, calf circum- ference (CC) and Mini Nutritional Assessment (MNA). In addition, HDG-cohorts differentiated according to CAG number repeats, TFC, UHDRS and HD duration were com- pared with undifferentiated CG. The first transcript levels measurement in undifferentiated HDG (n029) and CG (n0 28) revealed a statistically significant GPR FCs (p<0.05) for MAOB (3.07), TGM2 (1.8), SLC2A4 (1.64) BCKDK (1.34), LDHA (−1.16) and BDNF (−2.11) (Fig. 1). In order to identify the transcription level changes between HDG and CG three types of comparisons were performed in this study, the first, between undifferentiated HDG and CG, and the second, between HDG- and CG-cohorts, were both differentiated according to gender, age, BMI, calf circum- ference (CC) and Mini Nutritional Assessment (MNA). In addition, HDG-cohorts differentiated according to CAG number repeats, TFC, UHDRS and HD duration were com- pared with undifferentiated CG. The first transcript levels measurement in undifferentiated HDG (n029) and CG (n0 28) revealed a statistically significant GPR FCs (p<0.05) for MAOB (3.07), TGM2 (1.8), SLC2A4 (1.64) BCKDK (1.34), LDHA (−1.16) and BDNF (−2.11) (Fig. 1). The CC-cohort (Table 3d) was differentiated based on the size of calf circumference reflecting an increase of the approximate muscular mass amount into four CC-cohorts (Table 1a.). Statistical methods and calculations The range of FC values for BDNF showed a transcript level decrease from −1.56 for the MNA1 to −2.55 for the MNA3-cohort and an increase of the MAOB level from 3.86 in the MNA1 up to 8.15 for MNA2, but a decrease of this gene transcript level in the MNA3 to 1.61. Statistical methods and calculations Depending on the number of CAG repeats in larger allele of HTT gene, the HDG was divided into CAG1-3 cohorts (Table 1b). Significant FCs in 9 transcripts: BDNF, IL8, PPRC1, PPARGC1A, TNF-α, DCTN1, SLC2A4, TGM2 and MAOB were observed (Table 3f). Across the CAG-cohorts, statistically significant FC range changes were found only for the IL8, TNF-α and MAOB transcripts, however, IL8 FC values simultaneously in- creased together with the CAG number repeats in CAG1- and CAG2-cohorts. The TNF transcript levels presented almost the same level in the CAG1- and 2-cohorts (1.06 and 1.14 respectively), but significantly decreased up to −1.34 in the CAG3-cohort and the FC range was statistically significant. The increase of MAOB transcript level from 2.08 in the CAG1- into 4.19 in the CAG2- was simulta- neously observed with the number of CAG repeats. The highest decrease of the BDNF FCs value was observed in the CAG2- (−2.85) in contrast to the CAG1 cohort (−2.09), however the BDNF FC range across the CAG1-3 cohorts was not statistically significant. The TFC-cohorts reflected the stage of HD from functional ability perspective (Table 1b) and corresponded to HD pro- gression. HD subjects with the most advanced functional decline were included in the TFC1-cohort (score: 1–8), the TFC2-cohort contains intermediate stage (score: 9–10) and the TFC3-cohort presymptomatic stage (score: 11–13). A comparative TFC-cohorts analysis (Table 3g) showed a sta- tistically significant GPR FC concerning 4 transcripts (SLC2A4, CBS, TGM2 and MAOB). However Kruskal Wallis only confirmed significance for CBS and MAOB and showed the CBS FC decrease (from 1.55 in the TFC1- to −1.29 in the TFC3-cohort) and the MAOB FC decrease (from 6.47 in the TFC1- up to 1.92 in the TFC3-cohorts). The strongest increase of the CBS and MAOB FCs was found among HD subjects with the most advanced HD-stage (Table 3g). Movement disorders may contribute to metabolic HD patients’ status as well. Therefore UHDRS motor scale assess- ment was included in this analysis (Table 1b). The presymp- tomatic mutation carriers were located in the UHDRS1-cohort with the scores’ range (1–8) what is an equivalent of mild motor symptoms. Subjects with most severe motor symptoms were included to the next UHDRS2-5-cohorts. Highest scored patients were included in the UHDRS5-cohort with the UHDRS motor scores from 61 to 91. The FC results obtained for the UHDRS (1–5)-cohorts showed 5 transcripts with sig- nificant GPR FCs: IL8, HIP1, TGM2, SLC2A4 and MAOB. Statistical methods and calculations The third type of the qPCR result analysis was performed for HDG sub-cohorts differentiated by the CAG number repeats, TFC, UHDRS motor scale assessment, the HD duration period and compared to no varied CG in order to The BMI-cohort was differentiated into four sub-cohorts (BMI1-4) based on the BMI score ranges and reflected the J Bioenerg Biomembr (2013) 45:71–85 78 Table 3 (a–i) Gene expression changes in blood cells found in distinguished cohorts, including: a) gender-, b) age-, c) BMI-, d) CC-, e) MNA-, f) CAG number repeats-, g) TFC-, h) UHDRS-, i) HD disease duration (dd)-cohort a) GPR statistical analysis of qPCR results Statistical analysis results of Gender-cohort GPR FCs Cohorts: Female Male Kruskal Wallis Z-value Kruskal Wallis p-value Mann Whitney U test HDG n016 HDG n013 CG n022 CG n06 Gene p-value FCa p-value FCb CD36 0.51 1.10 0.02 −1.78 70.71 <0.05 a> b MAOB 0.00 2.34 0.07 4.32 21.09 <0.05 a< b SLC2A4 0.03 1.89 0.51 1.15 17.21 <0.05 a> b b) GPR statistical analysis of qPCR results Statistical analysis results of Age-cohort GPR FCs Cohorts: Age1 Age2 Age3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann Whitney U test ≤35 years 36–55 years ≥56 years Gene HDG n08 CG n012 HDG n015 CG n011 HDG n06 CG n05 p-value FCa p-value FCb p-value FCc CREBBP 0.29 −1.13 0.04 −1.24 0.50 −1.12 1.93 NS – SLC2A4RG 0.27 −1.22 0.00 −1.48 0.59 −1.02 6.21 NS – HSPA5 0.51 1.03 0.03 1.29 0.55 1.16 3.78 NS – ATP2A2 0.35 1.10 0.03 1.42 0.41 1.12 5.04 NS – IL8 0.62 1.01 0.26 −1.27 0.04 −2.00 36.67 <0.05 a>b, a>c, b>c MTOR 0.27 1.09 0.02 −1.35 0.54 −1.10 37.93 <0.05 a>b, a>c, b<c BCKDHA 0.48 1.00 0.03 −1.33 0.32 −1.26 36.94 <0.05 a>b, a>c, b<c BCKDHB 0.30 1.26 0.02 −1.65 0.49 −1.13 38.41 <0.05 a>b, a>c, b<c MLH3 0.56 1.12 0.01 −1.69 0.72 −1.04 38.22 <0.05 a>b, a>c, b<c UCP2 0.39 1.11 0.01 −1.37 0.26 −1.29 37.39 <0.05 a>b, a>c, b<c RASA1 0.44 −1.02 0.01 −1.45 0.63 1.03 37.85 <0.05 a>b, a<c, b<c ACO2 0.47 −1.00 0.05 1.29 0.35 1.16 37.29 <0.05 a<b, a<c UBE2K 0.48 −1.00 0.02 1.44 0.49 1.44 36.32 <0.05 a<b, a<c HSPD1 0.47 −1.06 0.00 1.51 0.35 1.22 37.13 <0.05 a<b, a<c PPARG 0.42 −1.15 0.05 1.66 0.37 1.38 36.95 <0.05 a<b, a<c CASP6 0.31 −1.28 0.01 1.59 0.45 1.18 38.31 <0.05 a<b, a<c c) GPR statistical analysis of qPCR results Statistical analysis results of BMI-cohort GPR FCs Cohorts: BMI1-2 BMI 2 BMI3 BMI3-4 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test Gene BMI score: 18.4–24.9 BMI score: 18,5–24,9 BMI score: 25,0–29,9 BMI score: 25,0–34,9 HDG n019 HDG n017 HDG n06 HDG n010 CG n020 CG n019 CG n07 CG n08* 78 J Bioenerg Biomembr (2013) 45:71 8 (a–i) Gene expression changes in blood cells found in distinguished cohorts, including: a) gender-, b) age-, c) BMI-, d) CC-, e) MNA-, f) CAG number repeats-, g) TFC-, h) UHDRS-, i) HD duration (dd)-cohort Table 3 (a–i) Gene expression changes in blood cells found in distinguished cohorts, including: a) gender-, b) age-, c) BMI-, d) CC-, e) disease duration (dd)-cohort a) GPR statistical analysis of qPCR results Statistical analysis results of Gender-cohort GPR FCs Cohorts: Female Male Kruskal Wallis Z-value Kruskal Wallis p-value Mann Whitney U test HDG n016 HDG n013 CG n022 CG n06 Gene p-value FCa p-value FCb CD36 0.51 1.10 0.02 −1.78 70.71 <0.05 a> b MAOB 0.00 2.34 0.07 4.32 21.09 <0.05 a< b SLC2A4 0.03 1.89 0.51 1.15 17.21 <0.05 a> b b) GPR statistical analysis of qPCR results Statistical analysis results of Age-cohort GPR FCs Cohorts: Age1 Age2 Age3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann Whitney U test ≤35 years 36–55 years ≥56 years Gene HDG n08 CG n012 HDG n015 CG n011 HDG n06 CG n05 p-value FCa p-value FCb p-value FCc CREBBP 0.29 −1.13 0.04 −1.24 0.50 −1.12 1.93 NS – SLC2A4RG 0.27 −1.22 0.00 −1.48 0.59 −1.02 6.21 NS – HSPA5 0.51 1.03 0.03 1.29 0.55 1.16 3.78 NS – ATP2A2 0.35 1.10 0.03 1.42 0.41 1.12 5.04 NS – IL8 0.62 1.01 0.26 −1.27 0.04 −2.00 36.67 <0.05 a>b, a>c, b>c MTOR 0.27 1.09 0.02 −1.35 0.54 −1.10 37.93 <0.05 a>b, a>c, b<c BCKDHA 0.48 1.00 0.03 −1.33 0.32 −1.26 36.94 <0.05 a>b, a>c, b<c BCKDHB 0.30 1.26 0.02 −1.65 0.49 −1.13 38.41 <0.05 a>b, a>c, b<c MLH3 0.56 1.12 0.01 −1.69 0.72 −1.04 38.22 <0.05 a>b, a>c, b<c UCP2 0.39 1.11 0.01 −1.37 0.26 −1.29 37.39 <0.05 a>b, a>c, b<c RASA1 0.44 −1.02 0.01 −1.45 0.63 1.03 37.85 <0.05 a>b, a<c, b<c ACO2 0.47 −1.00 0.05 1.29 0.35 1.16 37.29 <0.05 a<b, a<c UBE2K 0.48 −1.00 0.02 1.44 0.49 1.44 36.32 <0.05 a<b, a<c HSPD1 0.47 −1.06 0.00 1.51 0.35 1.22 37.13 <0.05 a<b, a<c PPARG 0.42 −1.15 0.05 1.66 0.37 1.38 36.95 <0.05 a<b, a<c CASP6 0.31 −1.28 0.01 1.59 0.45 1.18 38.31 <0.05 a<b, a<c c) GPR statistical analysis of qPCR results Statistical analysis resu Cohorts: BMI1-2 BMI 2 BMI3 BMI3-4 Kruskal Wallis Chi2 test Kr Gene BMI score: 18.4–24.9 BMI score: 18,5–24,9 BMI score: 25,0–29,9 BMI score: 25,0–34,9 HDG n019 HDG n017 HDG n06 HDG n010 CG n020 CG n019 CG n07 CG n08* J Bioenerg Biomembr (2013) 45:71–85 79 value FCb p-value FCc p-value FCd 06 −1.26 0.48 1.08 0.55 1.03 28.87 <0.05 a<b, a<c, a>d, b>d, c>d 03 −1.27 0.34 −1.09 0.36 −1.12 1.80 NS – 02 1.71 0.34 1.48 0.40 1.22 3.93 NS – 01 4.28 0.13 1.56 0.31 1.44 14.11 <0.05 a>c, a>d, b>c, b>d, c<d Statistical analysis results of CC-cohort GPR FCs C2 diameter (cm) CC3 diameter (cm) CC4 diameter (cm) Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test DG&CG HDG&CG HDG039–41 –11 36–38 CG039–45 DG n019 HDG n019 HDG n019 G n020 CG n020 CG n020 value FCb p-value FCc p-value FCd 43 −1.29 0.33 1.41 0.61 1.01 30.03 <0.05 a<b, a<c, a<d, b<c, b<d 23 −1.27 0.03 −1.23 0.43 −1.03 2.40 NS – 45 −1.01 0.04 −1.27 0.42 −1.06 2.65 NS – 01 1.83 0.52 1.62 0.25 1.36 25.88 <0.05 a<b, a<c, a<d, b<c, b<d, c>d 27 4.39 0.01 4.16 0.56 1.37 10.18 <0.05 a<c, a>d, b>d, c>d Statistical analysis results of MNA-cohort GPR FCs NA2 MNA3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test ore: score: DG: 10–11 12–14 G: 8–11 DG n010 HDG n015 G n07 CG n021 value FCb p-value FCc 78 −1.28 0.02 −2.55 12.33 <0.05 a>c, b>c 02 8.15 0.08 1.61 24.38 <0.05 a<b, a>c, b>c Statistical analysis results of CAG-cohort GPR FCs AG2 CAG3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann- Whitney U test –44* 46–81* DG n010 HDG n010 G n028 CG n028 value FCb P-value FCc 05 −2.85 0.15 −2.17 5.85 NS – 8 1.30 0.19 1.30 41.06 <0.05 a<b, a<c Bioenerg Biomembr (2013) 45:71 85 79 ( ) p-value FCa p-value FCb p-value FCc p-value FCd PPRC1 0.03 −1.30 0.06 −1.26 0.48 1.08 0.55 1.03 28.87 <0.05 a<b, a<c, a>d, b>d, c>d LDHA 0.03 −1.23 0.03 −1.27 0.34 −1.09 0.36 −1.12 1.80 NS – SLC2A4 0.01 1.74 0.02 1.71 0.34 1.48 0.40 1.22 3.93 NS – MAOB 0.00 4.43 0.01 4.28 0.13 1.56 0.31 1.44 14.11 <0.05 a>c, a>d, b>c, b>d, c<d d) GPR statistical analysis of qPCR results Statistical analysis results of CC-cohort GPR FCs Cohorts: CC1 diameter (cm) CC2 diameter (cm) CC3 diameter (cm) CC4 diameter (cm) Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test HDG03–9 HDG&CG HDG&CG HDG039–41 CG08–11 10–11 36–38 CG039–45 Gene HDG n019 HDG n019 HDG n019 HDG n019 CG n020 CG n020 CG n020 CG n020 p-value FCa p-value FCb p-value FCc p-value FCd IL8 0.03 −2.48 0.43 −1.29 0.33 1.41 0.61 1.01 30.03 <0.05 a<b, a<c, a<d, b<c, b<d LDHA 0.37 −1.09 0.23 −1.27 0.03 −1.23 0.43 −1.03 2.40 NS – COX2 0.45 −1.05 0.45 −1.01 0.04 −1.27 0.42 −1.06 2.65 NS – TGM2 0.08 2.61 0.01 1.83 0.52 1.62 0.25 1.36 25.88 <0.05 a<b, a<c, a<d, b<c, b<d, c>d MAOB 0.00 3.72 0.27 4.39 0.01 4.16 0.56 1.37 10.18 <0.05 a<c, a>d, b>d, c>d e) GPR statistical analysis of qPCR results Statistical analysis results of MNA-cohort GPR FCs Cohorts: MNA1 MNA2 MNA3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test score: score: score: HDG: 3–9 HDG: 10–11 12–14 CG: 8–11 CG: 8–11 Gene HDG n04 HDG n010 HDG n015 CG n07 CG n07 CG n021 p-value FCa p-value FCb p-value FCc BDNF 0.47 −1.56 0.78 −1.28 0.02 −2.55 12.33 <0.05 a>c, b>c MAOB 0.00 3.86 0.02 8.15 0.08 1.61 24.38 <0.05 a<b, a>c, b>c f) GPR statistical analysis of qPCR results Statistical analysis results of CAG-cohort GPR FCs Cohorts: CAG1 CAG2 CAG3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann- Whitney U test 38–41* 42–44* 46–81* Gene HDG n09 HDG n010 HDG n010 CG n028 CG n028 CG n028 P-value FCa P-value FCb P-value FCc BDNF 0.20 −2.09 0.05 −2.85 0.15 −2.17 5.85 NS – IL8 0.01 −1.90 0.38 1.30 0.19 1.30 41.06 <0.05 a<b, a<c J Bioenerg Biomembr (2013) 45:71–85 80 ( ) 1 0.42 −1.06 0.43 −1.00 0.03 −1.43 6.65 NS – GC1A 0.75 1.04 0.24 1.37 0.03 1.54 6.40 NS – 0.52 1.06 0.43 1.14 0.03 −1.34 39.80 <0.05 a>c, b>c 1 0.28 1.17 0.30 1.14 0.03 1.41 3.94 NS – A4 0.13 1.47 0.03 1.98 0.06 1.60 5.31 NS – 0.64 1.68 0.00 2.06 0.06 1.64 4.24 NS – 0.10 2.08 0.00 4.19 0.02 3.11 11.22 <0.05 a<b, a<c G number repeats tatistical analysis of qPCR results Statistical analysis results of TFC-cohort GPR FCs ts: TFC1 TFC2 TFC3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test 1–8* 9–10* 11–13* HDG n07 HDG n09 HDG n013 CG n028 CG n028 CG n028 P-value FCa P-value FCb P-value FCc A4 0.23 1.37 0.01 2.27 0.08 1.49 9.49 NS – 0.02 1.55 0.53 1.41 0.26 −1.29 37.69 <0.05 a>c, b>c 0.03 2.00 0.02 1.85 0.38 1.66 3.15 NS – 0.02 6.47 0.00 2.47 0.06 1.92 22.87 <0.05 a>b, a>c, b>c score value tatistical analysis of qPCR results Statistical analysis results of UHDRS-cohort GPR FCs ts: UHDRS1 UHDRS2 UHDRS3 UHDRS4 UHDRS5 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test 1–8* 22–31 36–43 45–55 61–91 HDG n06 HDG n05 HDG n08 HDG n06 HDG n05 CG n028 CG n028 CG n028 CG n028 CG n028 p-value FCa p-value FCb p-value FCc p-value FCd p-value FCe 0.03 −1.98 0.34 1.25 0.22 1.35 0.63 1.11 0.37 −1.24 22.85 <0.05 a<b, a<c, a<d, a<e, b>e, c>e, d>e 0.05 −1.66 0.53 −1.06 0.23 1.39 0.23 −1.28 0.42 1.56 22.37 <0.05 a<b, a<c, a<d, a<e, b<c, b>d, b<e, c>d, d<e 0.65 1.15 0.00 2.06 0.04 1.75 0.03 2.05 0.06 2.05 4.34 NS – A4 0.44 1.19 0.08 1.44 0.04 2.18 0.11 1.61 0.07 1.71 4.56 NS – 0.21 1.87 0.33 2.13 0.01 3.75 0.00 2.47 0.09 5.97 10.43 <0.05 a<b, a<c, a<d, a<e, b<c, b<d, b<e, c>d, d<e RS score value J oe e g o e b ( 0 3) 5:7 Table 3 (continued) PPRC1 0.42 −1.06 0.43 −1.00 0.03 −1.43 6.65 NS – PPARGC1A 0.75 1.04 0.24 1.37 0.03 1.54 6.40 NS – TNF 0.52 1.06 0.43 1.14 0.03 −1.34 39.80 <0.05 a>c, b>c DCTN1 0.28 1.17 0.30 1.14 0.03 1.41 3.94 NS – SLC2A4 0.13 1.47 0.03 1.98 0.06 1.60 5.31 NS – TGM2 0.64 1.68 0.00 2.06 0.06 1.64 4.24 NS – MAOB 0.10 2.08 0.00 4.19 0.02 3.11 11.22 <0.05 a<b, a<c * CAG number repeats g) GPR statistical analysis of qPCR results Statistical analysis results of TFC-cohort GPR FCs Cohorts: TFC1 TFC2 TFC3 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test 1–8* 9–10* 11–13* Gene HDG n07 HDG n09 HDG n013 CG n028 CG n028 CG n028 P-value FCa P-value FCb P-value FCc SLC2A4 0.23 1.37 0.01 2.27 0.08 1.49 9.49 NS – CBS 0.02 1.55 0.53 1.41 0.26 −1.29 37.69 <0.05 a>c, b>c TGM2 0.03 2.00 0.02 1.85 0.38 1.66 3.15 NS – MAOB 0.02 6.47 0.00 2.47 0.06 1.92 22.87 <0.05 a>b, a>c, b>c * TFC score value h) GPR statistical analysis of qPCR results Sta Cohorts: UHDRS1 UHDRS2 UHDRS3 UHDRS4 UHDRS5 Kr C 1–8* 22–31 36–43 45–55 61–91 Gene HDG n06 HDG n05 HDG n08 HDG n06 HDG n05 CG n028 CG n028 CG n028 CG n028 CG n028 p-value FCa p-value FCb p-value FCc p-value FCd p-value FCe IL8 0.03 −1.98 0.34 1.25 0.22 1.35 0.63 1.11 0.37 −1.24 22 HIP1 0.05 −1.66 0.53 −1.06 0.23 1.39 0.23 −1.28 0.42 1.56 22 TGM2 0.65 1.15 0.00 2.06 0.04 1.75 0.03 2.05 0.06 2.05 4.3 SLC2A4 0.44 1.19 0.08 1.44 0.04 2.18 0.11 1.61 0.07 1.71 4.5 MAOB 0.21 1.87 0.33 2.13 0.01 3.75 0.00 2.47 0.09 5.97 10 *UHDRS score value i) GPR statistical analysis of qPCR results Statistical analysis results of HD period-cohort GPR FCs Cohorts: HD dd1 HD dd2 HD dd3 HD dd4 Kruskal Wallis Chi2 test Kruskal Wallis p-value Mann–Whitney U test 0* 1–5* 6–10* 11–15* Gene HDG n06 HDG n06 HDG n012 HDG n05 CG n028 CG n028 CG n028 CG n028 p-value FCa p-value FCb p-value FCc p-value FCd IL8 0.03 −1.98 0.03 1.62 0.53 1.09 0.51 −1.11 29.74 <0.05 a<b, a<c, a<d, b>d, c>d BDNF 0.63 −1.66 0.12 −2.59 0.01 −3.60 0.69 −1.56 10.12 <0.05 a>b, a>c, b>c, b<d, c<d AQP9 0.17 −1.37 0.04 1.70 0.07 1.65 0.16 1.27 24.50 <0.05 a<b, a<c, a<d SOD2 0.22 −1.26 0.03 1.44 0.31 1.23 0.28 1.16 25.04 <0.05 a<b, a<c, a<d SLC2A4 0.44 1.19 0.01 2.05 0.02 1.90 0.19 1.35 6.47 NS – BCAT2 0.39 1.85 0.04 −1.42 0.20 −1.10 0.39 −1.16 27.91 <0.05 a>b, a>c, a>d, b<c b<d, c>d MAOB 0.21 1.87 0.05 2.64 0.01 2.14 0.02 7.89 19.65 <0.05 a<b, a<c, a<d, b>c b<d, c<d *HD dd - HD disease duration in years oenerg Biomembr (2013) 45:71–85 J Bioenerg Biomembr (2013) 45:71–85 81 identify a panel of gene transcript changes related to the HD progress. Statistical methods and calculations The Kruskal Wallis test confirmed significance in 3 FCs: IL8, HIP1 and MAOB (Table 3h). The HD disease duration – cohort (dd) was differentiated into 5 sub-cohorts, the HD dd1-cohort consisting of pre- symptomatic carriers, the HD dd2-cohort (up to 5 years J Bioenerg Biomembr (2013) 45:71–85 82 Table 4 Spearman’s coefficient correlation in HDG and parame- ters related to Huntington disease R- Spearman’s coefficient corre- lation value; There are presented only statistically significant correlations HD – sub-cohorts HD - subjects Age (years) BMI- (kg/m2) Calf circumference (cm) MNA -score HD onset age -years R* 0.718 0.227 −0.029 0.081 P <0.01 0.236 0.879 0.677 CAG-repeats number R −0.551 −0.318 −0.341 −0.249 P 0.002 0.093 0.071 0.192 TFC score R −0.022 0.263 0.386 0.525 P 0.909 0.168 0.039 0.003 Table 4 Spearman’s coefficient correlation in HDG and parame- ters related to Huntington disease After sub-cohort comparisons 34 gene transcripts were dis- tinguished (Table 3a–i), including genes involved in: from onset), HD dd3 (5–10 years from onset), HD dd4 (10– 15 years from onset), and HD dd5 (15–20 years from onset) respectively (Table 1b). The qPCR results comprised 7 tran- scripts with statistically significant FCs including IL8, BDNF, AQP9, SOD2, SLC2A4, BCAT2, and MAOB (Table 3i). The widest statistically significant FCs concerned BDNF (from −1.66 to −3.6) and BCAT2 (from 1.85 to −1.42) where a gradual decrease was observed and MAOB (from 1.87 to 7.89) where a gradual increase was observed following increase of HD duration. – energy metabolism: ACO2, ATP2A2, COX2, LDHA, MAOB, PPARG, PPARGC1A, PPRC1, UCP2; – BCAA metabolism: BCAT2, BCKDHA, BCKDHB, BCKDK; – other types of cellular processes: AQP9, CREBBP, BDNF, CASP6, CBS, CD36, DCTN1, HIP1, HSPA5, HSPD1, IL8, MLH3, MTOR, RASA1, SOD2, SLC2A4, SLC2A4RG, TGM2, TNF-α and UBE2K. To summarize, the first comparison results performed between undifferentiated HD- and Control Groups revealed 6 transcript genes, including the increase of MAOB, TGM2, SLC2A4, BCKDK and the decrease of LDHA and BDNF transcripts amount (Fig. 1) with statistically significant FC. The widest FCs revealed in this study (Table 3a–i) concerned 7 transcripts including: MAOB (min. 1.44; max. 8.15), IL8 (min. −2.48; max. 1.41), HIP1 (min. −1.66; max. 1.56), BDNF (min. −3.6; max.−1.28), SLC2A4 (min. R- Spearman’s coefficient correlation value; p- value. There are presented only statistically significant correlations Discussion The number of CAG repeats in the larger allele is responsi- ble for up to 73 % of the variability of HD onset age (Langbehn et al. 2004). It is therefore important to find other genetic and non-genetic factors such as gender, age, nutrition state, lifestyle, diet, activity or several etc. responsible for the remaining 27 % of variability. This study was designed to identify possible metabolic biomarkers of HD at the gene transcription level. The relatively small study cohort limits the ability to achieve significant results; however, among the 95 genes tested, the transcription rate of 34 of them was significantly changed in the HDG. Statistically significant GPR FC values obtained after the comparison between undif- ferentiated HDG and CG revealed an increased level of four transcripts; MAOB, TGM2, SLC2A4 and BCKDK, and de- creased levels of two transcripts; BDNF, LDHA, compared to healthy subjects. The MAOB protein location in the outer mitochondrial membrane can be related to a gradual increase of the MAOB gene transcript level. Several mitochondrial abnormalities have been revealed in HD cells, including a decreased mem- brane resting potential, impaired calcium ion homeostasis, and marked morphological abnormalities with derangement of the mitochondrial matrix and cristae (Panov et al. 2002; Squitieri et al. 2006) and these mitochondrial abnormalities caused by mHTT can be recapitulated in normal lymphocytes treated with a fusion protein composed of a peptide containing a pathogenic polyglutamine tract (Panov et al. 2002). There is also evidence from HD mouse models (YAC72) and from cultured HD striatal neurons expressing endogenous mutant huntingtin (STHdhQ111) that the mHTT protein associates directly with the outer mitochondrial membrane (Choo et al. 2004). The direct relationship between the increase of the MAOB transcription levels in blood cells and its gradual increase concurrent with HD progression found in this study suggest a possible link with HD pathology. The most significant transcription changes found in this study concerned the MAOB gene encoding monoamine ox- idase B. The MAOB protein is an outer mitochondrial membrane-bound enzyme that catalyzes the oxidative de- amination of arylalkylamine neurotransmitters such as do- pamine and serotonin and xenobiotic monoamines (Binda et al. 2003). Statistical methods and calculations 1.19; Table 5 Spearman’s coefficient correlation between analytical tests results determined for HDG and CG Analytical tests HDG - subjects CG - healthy subjects Age (years) BMI (kg/m2) Calf circumference (cm) MNA -score Age (years) BMI (kg/m2) Calf circumference (cm) MNA- score WBC R* −0.054 0.163 0.209 0.135 −0.213 0.213 0.378 0.055 p 0.781 0.398 0.276 0.485 0.276 0.277 0.047 0.781 RBC R 0.329 0.436 0.227 0.111 0.009 0.040 0.101 0.139 p 0.081 0.018 0.235 0.567 0.964 0.839 0.608 0.482 HGB R 0.322 0.506 0.361 0.251 0.008 0.065 0.166 0.166 p 0.088 0.005 0.055 0.189 0.967 0.741 0.400 0.399 HCT R 0.334 0.439 0.305 0.087 0.004 −0.016 0.149 0.006 p 0.077 0.017 0.108 0.654 0.985 0.937 0.459 0.977 MCHC R 0.043 0.353 0.274 0.494 0.086 0.189 0.315 0.356 p 0.825 0.060 0.151 0.006 0.665 0.336 0.103 0.063 Lymphocytes R 0.031 0.220 0.116 0.440 −0.234 0.103 0.294 −0.091 p 0.875 0.252 0.549 0.017 0.231 0.601 0.130 0.646 ESR R 0.504 0.422 0.207 0.286 0.190 0.175 −0.048 −0.213 p 0.005 0.023 0.282 0.133 0.332 0.373 0.808 0.277 CRP R 0.230 0.511 0.331 0.151 0.079 0.408 0.361 −0.044 p 0.229 0.005 0.080 0.433 0.690 0.031 0.059 0.823 R Spearman’s coefficient correlation value; p value There are presented only statistically significant correlations Table 5 Spearman’s coefficient correlation between analytical tests results determined for HDG and CG J Bioenerg Biomembr (2013) 45:71–85 83 max. 2.7), TGM2 (min. 1.36; max. 2.61), LDHA (min. −1.27 -2.11 -1.16 1.34 1.64 1.80 3.07 -3 -2 -1 0 1 2 3 4 GPR Fold Change MAOB TGM2 SLC2A4 BCKDK LDHA BDNF Gene GPR p-value 0.0005 0.02 0.02 0.02 0.03 0.03 Fig. 1 A gene transcription level comparison between HDG (n029) and CG (n028) based on q-PCR results obtained by Global Pattern Recognition Software -2.11 -1.16 1.34 1.64 1.80 3.07 -3 -2 -1 0 1 2 3 4 GPR Fold Change MAOB TGM2 SLC2A4 BCKDK LDHA BDNF Gene GPR p-value 0.0005 0.02 0.02 0.02 0.03 0.03 course of HD duration from FC01.87 in the HD dd1 up to FC07.89 in the HD dd4. Interesting alterations were also found for the MAOB gene transcript across the BMI-cohorts. The highest MAOB FC level was in the cohort of subject with the lowest BMI (BMI1-cohort, FC04.43) in contrast to the cohort with the highest BMI (BMI3-4 cohort, FC01.44). Statistical methods and calculations While this study is the first description of increased levels of the MAOB gene transcript outside the CNS in HD-subjects, an abnormally high level of the MAOB activity has previously been demonstrated in the putamen, basal ganglia and other brain areas of HD subjects (Richards et al. 2011). Increased MAOB levels have also been described in the plasma of Alzheimer’s disease patients, however MAOB gene transcrip- tion was also increased in the brain. Increases of brain MAOB levels has also been shown in Parkinson’s disease as well as in psychotics disorders, where it is accompanied by MAOB polymorphism (Bergen et al. 2009; Emilsson et al. 2002; Naoi and Maruyama 2009). MAOB inhibitors are often clin- ically used in Parkinson’s disease to improve motor function (Binda et al. 2003; Schapira 2010). In several neurodegener- ative disorders, increased MAOB levels lead to neuronal damage caused by the neurotransmitters disturbances and an increase of the oxidative stress locally in specific brain regions (Bergen et al. 2009). Fig. 1 A gene transcription level comparison between HDG (n029) and CG (n028) based on q-PCR results obtained by Global Pattern Recognition Software max. 2.7), TGM2 (min. 1.36; max. 2.61), LDHA (min. −1.27 max. −1.09). Discussion In this study, the MAOB FC levels were the highest in the sub-cohorts of subjects with the most ad- vanced HD level, such as the TFC1- (6.47), UHDRS motor5 (5.97) -cohorts, compared to the MAOB FC levels in the subcohorts of presymptomatic HD subjects such as TFC3- (1.92) and UHDRS motor1 (1.87) -cohorts. .It also showed a gradual increase of the MAOB gene transcript level in the The next significant changes found in this study concern the tissue transglutaminase 2 (TGM2, TG2). TGM2 is a calcium sensitive multifunctional enzyme with guanosine tri- phosphate signaling activity as well as transamidating activity (Munsie et al. 2011), and is present in the cytosol and nuclear fractions of the brain tissue (Cooper et al. 1999). It has previously been shown that the polyglutamine expansion in huntingtin results in cellular stresses including endoplasmatic reticulum disruption, increased calcium levels and activation of TGM2 resulting in aberrant cofilin–actin covalent cross- J Bioenerg Biomembr (2013) 45:71–85 84 links (Munsie et al. 2011). In our study, an increase of the TGM2 gene transcription was found in a basic comparison and in some sub-cohorts analysis, including the CC-, CAG-, TFC- and UHDRS-cohorts. An increased level of TGM2 was pre- viously found in the HD brain and lymphocytes (Munsie et al. 2011), but in here that was confirmed only on the transcription level. The TGM2 FCs value alterations found in the sub- cohorts indicate a correlation between transcription changes and HD progress, indicated by an increase of TGM2 transcript level together with UHDRS-score and the number of CAG repeats, and with a decrease of the TFC-score. Additionally, the highest of TGM2 FCs values (2.61) was found in the CC1- cohort with the smallest calf circumference diameter. Our data confirms that the quantifiable TGM2 gene transcription alter- ations in blood cells may be used as biomarkers for HD. and has remained on the decreased level together with the disease duration. To clarify the BCAA metabolism disturban- ces in HD further studies are needed on the cellular level with cells derived from HD and healthy subjects. The slight decrease of the LDHA gene transcript level found in this study (−1.16) might reflect an impairment of glycolysis by inhibition of lactate acid conversion to pyru- vate. The lactate level elevation due to the lactate dehydro- genase level decrease was previously described in the HD brain tissue and other tissues (Milakovic and Johnson 2005). Discussion A decrease of the LDHA gene transcript level was clearly expressed across the BMI- and CC- cohorts with the lowest LDHA transcript level (−1.23-fold) in the BMI1-cohort and the CC2-cohort. The decrease of these transcript levels seems to be deepening together with the lower BMI- and CC-scores, reflected in lower body and muscle mass. This study is the first to describe the increased level of the SLC2A4 transcription in HD subjects with reference to the control and also the transcript level increased across CAG1-2, TFC1-2, UHDRS1-3cohorts together with the HD progression (HD dd1-2). The SLC2A4 gene encodes an insulin-sensitive glucose transporter 4 (GLUT4) with a critical role in glucose homeostasis (Korgun et al. 2002). The clinical meaning of the SLC2A4 transcript alteration in the HD-progress is unknown so far and it needs further study, but it’s potential as a biomarker in the monitoring of the HD progress outside the CNS could be useful. The strongest decrease found in this study concerned the BDNF gene, encoding Brain-Derived Neurotrophic Factor protein and was obtained in a basic comparison between the HD and control as well as in the sub-cohorts. It was previ- ously shown that the normal huntingtin protein positively influences the BDNF level, which protects and stimulates neuronal cells growth (Borrell-Pages et al. 2006). The stron- gest decrease in BDNF transcript levels was obtained in the CAG2-cohort (with 42–44 CAG repeats number) and in the dd-2-cohort. The reduction of the BDNF level had previ- ously been described on a protein and transcript level in HD and also other neurodegenerative disorders, but a recent study confirms the phenomenon in HD only (Zuccato et al. 2011). Many contemporary studies indicate that mito- chondrial dysfunction and oxidative stress play a crucial role in the majority of neurogenerative diseases. Mitochondria are the major source of intracellular reactive oxygen species and are particularly vulnerable to oxidative stress. In addi- tion, the impairment of mitochondrial function disturbs the cellular energy homeostasis. PGC1α stimulates the mito- chondrial biogenesis and respiration, and this study has shown significant changes concerning two transcript levels, PPARGC1A and PPARC1, which are involved in energy metabolism. The FCs values showed a slight increase of PPARGC1A (1.54) and decrease of PPARC1 (−1.43) simul- taneously with an increase of the number of CAG repeats. Discussion Additionally a decrease of the PPRC1 transcript level simul- taneously occurred with the BMI decrease in the HDG when compared to the CG. These results are in line with the early energy deficits described in HD subjects, with the exception of PPARGC1A transcript levels which have been previously reported to decrease in HD (Strand et al. 2005; Weydt et al. 2006; Yoon et al. 2003). It is difficult to explain this phe- nomenon, but the HD-subjects who participated in this study had previously used a variety of diet supplements that might have influenced these gene transcripts that regulate energy metabolism on the transcription level. On the other The comparison between HDG and CG also showed the increase of the BCKDK (branched chain ketoacid dehydro- genase kinase) transcript level (1.34), while the sub-cohorts comparisons revealed the alteration of three other transcripts (BCKDHA, BCKDHB, and BCAT2) encoding enzymes involved in BCAA catabolism. BCKDK controls the catab- olism of BCAA by branched-chain β-ketoacid dehydroge- nase complex (BCKDC) regulation (Harris et al. 2004). An increase of the BCKDHB transcript level was found only in the age1-cohort (1.26), but across the age2-3-cohorts a decrease both of the BCKDHA and BCKDHB transcripts was found. The decrease of BCAA level and muscle mass, described previously in HD (Mochel et al. 2007), might be related also to the increased level of BCKDHA and BCKDHB (α-ketoacid dehydrogenase E1, alfa- and beta polypeptide). However the gene transcript changes involved in the BCAA metabolism haven’t been found as statistically significant in the CC- and BMI-cohorts. The regulation of BCKDC is related to BCKDK and BCKD phosphatase (BCKDP) activity and linked to BCKDC level. Skeletal muscles are considered an initial site for BCAA catabolism because of a high activity of BCAA aminotransferase (BCAT2), a mitochondrial en- zyme, responsible for the first step of BCAA catabolism (Brosnan and Brosnan 2006). In the HD dd1-cohort an in- creased level (1.85-fold) of the BCAT2 gene transcript encod- ing aminotransferase was found, however in the HD dd2- cohort this transcript level has been decreased to −1.42-fold 85 J Bioenerg Biomembr (2013) 45:71–85 Cooper AJ, Sheu KF, Burke JR, Strittmatter WJ, Gentile V, Peluso G, Blass JP (1999) J Neurochem 72:889–899 hand, it has been proposed that aberrant transcriptional regulation of nuclear-encoded mitochondrial genes may be involved in HD pathogenesis. Discussion Cui L, Jeong H, Borovecki F, Parkhurst CN, Tanese N, Krainc D (2006) Cell 127:59–69 The HD related metabolic changes suggested by this study need to be confirmed in a larger cohort before they can be considered HD specific biomarkers. The gene tran- scription level expressed by FCs and presented in this pre- liminary report indicates that it may be possible to select a HD biomarker from nuclear blood cells. Also, multiple analyses of gene transcript levels (sub-cohorts comparisons) showed more statistically significant FCs than the general comparison between the HDG and GC. This implies the multiple impact of particular genes on energy production and BCAA metabolism. The results of the study are prom- ising both for the identification of biomarkers for HD and also for determining potential pharmacological targets for the improvement of these altered metabolic pathways. Davies S, Ramsden DB (2001) Mol Pathol 54:409–413 Emilsson L, Saetre P, Balciuniene J, Castensson A, Cairns N, Jazin EE (2002) Neurosci Lett 326:56–60 Harris RA, Joshi M, Jeoung NH (2004) Biochem Biophys Res Commun 313:391–396 Korgun ET, Demir R, Sedlmayr P, Desoye G, Arikan GM, Puerstner P, Hahn T (2002) Blood Cells Mol Dis 28:152–159 Kosinski CM, Schlangen C, Gellerich FN, Gizatullina Z, Deschauer M, Schiefer J, Lindenberg KS (2007) Mov Disord 22:1637–1640. doi:10.1002/mds.21550 Kozlowski P, Sobczak K, Krzyzosiak WJ (2010) Genome Med 2:29 Langbehn DR, Brinkman RR, Falush D, Paulsen JS, Hayden MR (2004) Clin Genet 65:267–277. doi:10.1111/j.1399-0004.2004.00241.x Liang H, Ward WF (2006) Adv Physiol Educ 30:45–151 Milakovic T, Johnson GV (2005) J Biol Chem 280:30773–30782 Mochel F, Charles P, Seguin F, Barritault J, Coussieu C, Perin L, Durr A (2007) PLoS One 2:e647. doi:10.1371/journal.pone. 0000647 Acknowledgments We are grateful to all the subjects who participated in the present study. This study was supported by European Huntington’s Disease Network (Research project No, 0133). Mochel F, Benaic S, Rabier D, Durr A (2011) Arch Neurol 68:265–267 Mochel F, Durant B, Meng X, O’Callaghan J, Yu H, Brouillet E, Durr A (2012) J Biol Chem 287:1361–1370 Authors are also grateful to the Central Coordination EHDN for support in manuscript revision and thanks to Dr Anita Chadha-Patel for assistance with English. Munsie L, Caron N, Atwal RS, Marsden I, Wild EJ, Bamburg JR, Truant R (2011) Hum Mol Genet 20:937–1951 Naoi M, Maruyama W (2009) Expert Rev Neurother 9:1233–1250. Discussion doi:10.1586/ern.09.68 Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribu- tion, and reproduction in any medium, provided the original author(s) and the source are credited. Panov AV, Gutekunst CA, Leavitt BR, Hayden MR, Burke JR, Strittmatter WJ, Greenamyre JT (2002) Nat Neurosci 5:731–736. doi:10.1038/nn884 Perez-De La Cruz V, Santamaria A (2007) Physiol Res 56:513–526 Perez-De La Cruz V, Santamaria A (2007) Physiol Res 56:513–526 Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, Spiegelman BM (2003) Nature 423:550–555. doi:10.1038/ nature01667 References Richards G, Messe J, Waldvogel HJ, Gibbons HM, Dragunow M, Faull RL, Saura J (2011) Brain Res 1370:204–214 Akilesh S, Shaffer DJ, Roopenian D (2003) Genome Res 13:1719– 1727. doi:10.1101/gr.533003 RL, Saura J (2011) Brain Res 1370:204–214 Ross CA, Tabrizi SJ (2011) Lancet Neurol 10:83–98 , ( ) Ross CA, Tabrizi SJ (2011) Lancet Neurol 10:83–98 Atwal RS, Xia J, Pinchev D, Taylor J, Epand RM, Truant R (2007) Hum Mol Genet 16:2600–2615. doi:10.1093/hmg/ddm217 Rubenstein LZ, Harker JO, Salva A, Guigoz Y, Vellas B (2001) J Gerontol A Biol Sci Med Sci 56:M366–M372 Sathasivam K, Hobbs C, Turmaine M, Mangiarini L, Mahal A, Bertaux F, Bates GP (1999) Hum Mol Genet 8:813–822 Beal MF, Brouillet E, Jenkins BG, Ferrante RJ, Kowall NW, Miller JM, Hyman BT (1993) J Neurosci 13:4181–4192 Schapira AH (2010) Expert Opin Pharmacother 11:2261–2268. doi:10.1517/14656566.2010.511612 Bergen SE, Fanous AH, Walsh D, O’Neill FA, Kendler KS (2009) Schizophr Res 109:94–97 Squitieri F, Cannella M, Sgarbi G, Maglione V, Falleni A, Lenzi P, Fornai F (2006) Mech Ageing Dev 127:217–220 Binda C, Li M, Hubalek F, Restelli N, Edmondson DE, Mattevi A (2003) Proc Natl Acad Sci U S A 100:9750–9755. doi:10.1073/ pnas.1633804100 St-Pierre J, Drori S, Uldry M, Silvaggi JM, Rhee J, Jager S, Spiegelman BM (2006) Cell 127:397–408 Bjorkqvist M, Wild EJ, Thiele J, Silvestroni A, Andre R, Lahiri N, Tabrizi SJ (2008) J Exp Med 205:1869–1877 Strand AD, Aragaki AK, Shaw D, Bird T, Holton J, Turner C, Olson JM (2005) Hum Mol Genet 14:1863–1876 Borrell-Pages M, Canals JM, Cordelieres FP, Parker JA, Pineda JR, Grange G, Humbert S (2006) J Clin Invest 116:1410–1424. doi:10.1172/JCI27607 ages M, Canals JM, Cordelieres FP, Parker JA, Pineda JR The Huntington’s Disease Collaborative Research Group (1993) Cell 72:971–983 Brosnan JT, Brosnan ME (2006) J Nutr 136:207S–211S Weydt P, Pineda VV, Torrence AE, Libby RT, Satterfield TF, Lazarowski ER, La Spada AR (2006) Cell Metab 4:349–362 Browne SE, Beal MF (2004) Neurochem Res 29:531–546 Chaturvedi RK, Calingasan NY, Yang L, Hennessey T, Johri A, Beal MF (2010) Hum Mol Genet 19:3190–3205 Yoon JC, Xu G, Deeney JT, Yang SN, Rhee J, Puigserver P, Spiegelman BM (2003) Dev Cell 5:73–83 Choo YS, Johnson GV, MacDonald M, Detloff PJ, Lesort M (2004) Hum Mol Genet 13:1407–1420. doi:10.1093/hmg/ddh162 Zuccato C, Marullo M, Vitali B, Tarditi A, Mariotti C, Valenza M, Cattaneo E (2011) PLoS One. doi:10.1371/journal.pone.0022966
https://openalex.org/W4312792884
https://www.redalyc.org/journal/381/38172849028/38172849028.pdf
Portuguese
null
Maternidade e cuidado na pandemia entre brasileiras de classe média e média alta
Revista Estudos Feministas
2,022
cc-by
9,660
Sistema de Informação Científica Redalyc Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto rtigo alyc.org Sistema de Informação Científica Redalyc Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto Como citar este artigo Número completo Mais informações do artigo Site da revista em redalyc.org Maternidade e cuidado na pandemia entre brasileiras de classe média e média alta Valeska Zanello1 0000-0002-2531-5581 Carla Antloga1 0000-0003-4105-6708 Eileen Pfeiffer-Flores2 0000-0002-7440-8872 Iara Flor Richwin3 0000-0002-9230-9018 1Universidade de Brasília, Departamento de Psicologia Clínica, Brasília, DF, Brasil. 70910-900 – pcl@unb.br 2Universidade de Brasília, Instituto de Psicologia, Departamento de Processos Psicológicos Básicos, Brasília, DF, Brasil. 70910-900 – ppb@unb.br 3Universidade de Brasília, Programa de Pós-Graduação em Psicologia Clínica e Cultura, Brasília, DF, Brasil. 70910-900 – secpsicc@gmail.com Resumo: A pandemia de Covid-19 incidiu diretamente sobre os arranjos e exercício do cuidado interpelado às mulheres. Investigou-se como mulheres mães exerceram esses cuidados, as mudanças ocorridas nesse exercício e os efeitos ressentidos, por meio de um survey online (N = 5.643). Responderam principalmente mulheres brancas de classe média a alta com escolarização avançada. Mesmo para esse segmento privilegiado, constatou-se que a pandemia exacerbou as desigualdades de gênero no cuidado doméstico e familiar. As mulheres encontravam-se sobrecarregadas e cansadas; sozinhas na encruzilhada entre trabalho profissional e trabalho de cuidados múltiplos; cuidando muito e sendo pouco cuidadas; muito disponíveis para outros e pouco disponíveis para si; e relataram culpa e sentimento de inadequação no cuidado e relação com os filhos. Palavras chave: maternidade; cuidado; gênero; pandemia de Covid 19 p p p ç ç Mesmo para esse segmento privilegiado, constatou-se que a pandemia exacerbou as desigualdades de gênero no cuidado doméstico e familiar. As mulheres encontravam-se sobrecarregadas e cansadas; sozinhas na encruzilhada entre trabalho profissional e trabalho de cuidados múltiplos; cuidando muito e sendo pouco cuidadas; muito disponíveis para outros e pouco disponíveis para si; e relataram culpa e sentimento de inadequação no cuidado e relação com os filhos. e se e o e eq ç o o c o e e ç o co os os Palavras-chave: maternidade; cuidado; gênero; pandemia de Covid-19. Motherhood and care in the pandemic among upper-middle class women in Brazil Abstract: The Covid-19 pandemic has directly affected the configuration of unpaid care work that women are routinely called upon to exercise. We investigated changes in how women mothers exercised this care and the effects of these changes on them, using an online questionnaire (N = 5.643). Respondents were mainly middle and higher class, white, with high educational levels. Even for this privileged segment, the pandemic was found to exacerbate gender inequalities in domestic and family care. Revista Estudos Feministas ISSN: 0104-026X ISSN: 1806-9584 Centro de Filosofia e Ciências Humanas e Centro de Comunicação e Expressão da Universidade Federal de Santa Catarina Zanello, Valeska; Antloga, Carla; Pfeiffer-Flores, Eileen; Richwin, Iara Flor Maternidade e cuidado na pandemia entre brasileiras de classe média e média alta Revista Estudos Feministas, vol. 30, núm. 2, e86991, 2022 Centro de Filosofia e Ciências Humanas e Centro de Comunicação e Expressão da Universidade Federal de Santa Catarina DOI: https://doi.org/10.1590/1806-9584-2022v30n286991 Disponível em: https://www.redalyc.org/articulo.oa?id=38172849028 Revista Estudos Feministas ISSN: 0104-026X ISSN: 1806-9584 Centro de Filosofia e Ciências Humanas e Centro de Comunicação e Expressão da Universidade Federal de Santa Catarina Zanello, Valeska; Antloga, Carla; Pfeiffer-Flores, Eileen; Richwin, Iara Flor Maternidade e cuidado na pandemia entre brasileiras de classe média e média alta Revista Estudos Feministas, vol. 30, núm. 2, e86991, 2022 Centro de Filosofia e Ciências Humanas e Centro de Comunicação e Expressão da Universidade Federal de Santa Catarina DOI: https://doi.org/10.1590/1806-9584-2022v30n286991 Disponível em: https://www.redalyc.org/articulo.oa?id=38172849028 Revista Estudos Feministas ISSN: 0104-026X ISSN: 1806-9584 Centro de Filosofia e Ciências Humanas e Centro de Comunicação e Expressão da Universidade Federal de Santa Catarina Disponível em: https://www.redalyc.org/articulo.oa?id=38172849028 Como citar este artigo Número completo Mais informações do artigo Site da revista em redalyc.org Seção Temática Fazendo Gênero em tempos de pandemia Maternidade e cuidado na pandemia entre brasileiras de classe média e média alta Valeska Zanello1 0000-0002-2531-5581 Carla Antloga1 0000-0003-4105-6708 Eileen Pfeiffer-Flores2 0000-0002-7440-8872 Iara Flor Richwin3 0000-0002-9230-9018 1Universidade de Brasília, Departamento de Psicologia Clínica, Brasília, DF, Brasil. 70910-900 – pcl@unb.br 2Universidade de Brasília, Instituto de Psicologia, Departamento de Processos Psicológicos Básicos, Brasília, DF, Brasil. 70910-900 – ppb@unb.br 3Universidade de Brasília, Programa de Pós-Graduação em Psicologia Clínica e Cultura, Brasília, DF, Brasil. 70910-900 – secpsicc@gmail.com Seção Temática Fazendo Gênero em tempos de pandemia 1 As propostas, dentro da psicanálise, de mudança do termo ‘mãe’ para ‘função materna’ resolvem o problema apenas em parte, sobretudo no que tange à questão da diversidade de possibilidades de parentesco. No entanto, como apontado por Zanello (2016), é muito importante problematizarmos o uso de certas palavras para exprimir certas ideias: ainda que a função materna possa ser exercida pelo pai ou qualquer figura masculina, cria-se uma proximidade semântica entre a função cuidadora e a mãe biológica, de modo que é comum nos círculos psis, ao ouvir alguém afirmar que o pai exerce a função materna, parecer haver algum problema ou distúrbio naquele arranjo parental. Por que não usar termos tais como “função cuidadora”? É preciso descolonizar a maternidade nas teorias psicológicas, bem como o vocabulário utilizado (ZANELLO, 2018). VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN A maternidade, tal qual a conhecemos, centrada sobretudo na figura da procriadora, nem sempre existiu. Ela é fruto de uma construção histórica, que remonta ao início do século XIX e é inscrita nos avanços e fortalecimento do capitalismo. Esse sistema econômico trouxe grandes novidades para o mundo ocidental, entre elas, a separação do mundo público e privado, a intensificação da hierarquização entre homens e mulheres e a afirmação do binarismo, ou seja, a ideia de que as diferenças entre ambos se justificariam no nível material, do corpo. Como Thomas Laqueur (2001) demonstrou, foi nesse momento que diferenças físicas foram tomadas para justificar desigualdades sociais. Nessa perspectiva, o mundo público foi relacionado aos homens, lugar por excelência para o exercício das atividades reconhecidas como trabalho, dignas de reconhecimento e de remuneração. Por outro lado, o mundo privado, da esfera doméstica, foi delegado às mulheres, sobretudo em função de sua capacidade de procriação, ou seja, pelo fato de elas serem portadoras de um útero. Foi nesse momento histórico que se deu a construção de um borramento ideológico entre a capacidade de cuidar e a capacidade de procriar (Valeska ZANELLO, 2018). Cuidar é uma habilidade humana, que pode ser exercida pela maioria das pessoas, independentemente do sexo, idade, fenótipo, condição social, etc. No entanto, ela foi naturalizada como algo que seria “instintivo” (Elizabeth BADINTER, 1985) nas pessoas portadoras de útero, lidas como mulheres. É É importante ressaltar que, até fins do século XVII, era comum que mulheres, de diferentes classes sociais, dessem seus filhos para serem amamentados por amas de leite (BADINTER, 1985). A mortalidade infantil era altíssima e, obviamente, isso afetava o crescimento e o tamanho da população. O capitalismo demandava um excedente populacional, para que fosse exequível seu projeto de mais valia e acumulação de capital. Visando garantir esse excedente, Igreja e Estado deram as mãos para convencerem as próprias mulheres que pariram a amamentarem suas crias. Como isso representaria perda de liberdade pessoal, gasto de tempo e renúncia aos próprios interesses, a estratégia utilizada foi menos de repressão e imposição, e mais de construção de um discurso que exaltava as habilidades maternas e o ideal da própria maternidade, ou seja, que criava um lugar desejável e digno de admiração para as mulheres. O que se deu, seguindo as ideias de Michel Foucault (1996), foi a passagem de um poder repressivo para outro, constitutivo. Maternidade e cuidado na pandemia entre brasileiras de classe média e média alta The women were overworked and tired; alone at the crossroads between professional work and multiple care work; caring a lot and being little cared for; constantly available to others and very little to themselves; and reported feelings of guilt and inadequacy related to the care of and relationship with their children. p Keywords: Maternity; Care, Gender; Covid-19 pandemic. Maternidad y cuidado en la pandemia entre brasileñas de clase media y media alta Resumen: La pandemia de Covid-19 ha afectado directamente a la configuración del trabajo de cuidados no remunerado que las mujeres son habitualmente llamadas a realizar. Investigamos los cambios en cómo las madres ejercían este cuidado y los efectos de estos cambios en ellas, utilizando un cuestionario en línea (N = 5.643). Las encuestadas fueron principalmente mujeres blancas, de clase media y media alta, con altos niveles educativos. Incluso para este segmento privilegiado, se encontró que la pandemia exacerbó las desigualdades de género en el cuidado doméstico y familiar. Las mujeres reportaron cansancio y soledad en la encrucijada entre el trabajo profesional y el trabajo de cuidados múltiples; se percibían cuidando mucho y recibiendo poco cuidado; se veían como siempre disponibles para los demás y muy poco disponibles para ellas mismas; y reportaron sentimientos de culpa e insuficiencia relacionados con el cuidado y la relación con sus hijos. Palabras clave: maternidad; cuidado; género; pandemia de Covid-19. Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 1 Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA Para sustentar isso, as hierarquias e opressões se reproduzem entre as próprias mulheres, de modo que, sobretudo às mulheres brancas, é possível terceirizar esses cuidados (a elas imputados pelo simples fato de serem mulheres) por meio da contratação de babás, empregadas domésticas e empresas de limpeza. Essa reorganização interna, dentro do grupo de mulheres, não deve nos fazer esquecer, no entanto, que quem mais usufrui de todos esses privilégios são os homens em geral, sobretudo os brancos, pois deles não é demandado nem que se preocupem com essa questão. Ou seja, mesmo que haja terceirização dos cuidados, ainda resta à mulher o planejamento, a carga mental da gestão entre tempo, tarefas e serviços e o trabalho emocional envolvido na gestão da família e do cotidiano, na administração dos afetos, antecipação de necessidades e provisão de suportes emocionais (Rebecca ERICKSON, 2005). No início de 2020, tivemos a eclosão de uma grande crise sanitária e social, de abrangência mundial: a pandemia de Covid-19. Essa pandemia foi e tem sido marcada por muitas dúvidas e incertezas, principalmente, em um primeiro momento, acerca dos modos de contágio e de cura. De todos os métodos usados em 2020 para combater o contágio, dois se destacaram: o uso de máscaras e o isolamento social. Sobretudo esse último provocou amplo debate no Brasil e foi combatido, por razões políticas, por certo setor negacionista. No entanto, vários estados o adotaram, levando milhares de pessoas a exercer, quando possível, suas atividades laborais a partir de casa. Paralelamente, crianças e jovens passaram a ter aulas presenciais suspensas e seguiram, em maior ou menor medida, o processo educativo por meio do ensino a distância. A Covid-19 escancarou aqui as profundas fissuras sociais já existentes em nossa sociedade. De um lado, no que diz respeito a quais parcelas da população conseguiram manter seus empregos e exercer sua profissão na modalidade de home office, assim como no acesso a condições mínimas de higiene, tais como água encanada e rede de esgoto. De outro lado, isso também se evidenciou na possibilidade de continuidade dos estudos a distância, sobretudo no acesso à internet, luz elétrica e condições mínimas de sobrevivência (como alimentação). ç ( ç ) Com os filhos de volta à casa em período integral, novos desafios, que teoricamente deveriam ter sido colocados aos diferentes membros das famílias, recaíram sobretudo nas mulheres dessas famílias: quem cuidaria dos filhos? E de que formas? MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA o que se aprende é a priorizar sempre os próprios interesses, anseios e desejos. Neste sentido, homens brasileiros têm aprendido muito pouco a cuidar e têm cuidado muito mal. o que se aprende é a priorizar sempre os próprios interesses, anseios e desejos. Neste sentido, homens brasileiros têm aprendido muito pouco a cuidar e têm cuidado muito mal. Além dos homens, o sistema capitalista expropria o dispositivo materno das mulheres. Como afirma Silvia Federici (2019), trata-se de um amor que é construído, mas esconde um trabalho não remunerado. Mas não apenas isso, todas as profissões relacionadas ao cuidado passaram, em nosso país, por um processo de feminização, ou seja, de entrada massiva de mulheres, com a decorrente precarização das condições de trabalho e dos salários (Cláudia VIANNA, 2013). Isto é, subentende-se que é trabalho, mas também algo “vocacional”, cuja suposta realização já seria em parte pagamento do próprio serviço. Podemos ver esse mecanismo histórico em profissões tais como: professora, psicóloga, médica da atenção básica, enfermeira, técnica de enfermagem, nutricionista etc. Mas, também, em profissões historicamente ligadas ao processo de escravização no país e relacionadas sobretudo a mulheres negras: babá, empregada doméstica, faxineira etc. É importante destacar que o cuidado deve ser pensado como uma forma de economia paralela, invisibilizada (Joan TRONTO, 2009), mas fundamental para a manutenção da vida e da cultura humana. E são as mulheres que carregam esse ofício nas costas. Porém, nem todas as mulheres o fazem da mesma forma. Em um país, além de sexista, racista como o nosso, faz- se fundamental pensar na distribuição racializada do cuidado, na qual, de um lado, temos as pessoas que mais oferecem cuidado e menos o recebem: as mulheres negras. Do outro lado, quem mais recebe cuidados e menos os exercita ou oferece: homens brancos (ZANELLO, 2018).2 ( ) Mulheres brancas também são demandadas, enquanto mulheres, a cuidarem de seus filhos, maridos e a se responsabilizarem pela gestão da manutenção da casa, e pelo bem- estar dos outros familiares. No entanto, pela existência da profunda desigualdade social, fruto de uma sociedade que foi escravocrata e na qual há uma racialização da pobreza3 (Sueli CARNEIRO, 2011), mulheres brancas usufruem de certos privilégios, conferidos por um acesso a maiores níveis de escolaridade, cargos e profissões mais altas e com melhor remuneração. 3 O Brasil foi escravocrata, no entanto, a mentalidade continua escravocrata. Essa parte de nossa história ainda não foi passada a limpo, como a nosso ver deveria ter sido. 2 Grande parte da introdução desse artigo se baseia no livro Saúde mental, gênero e dispositivos: Cultura e processos de subjetivação, de Valeska Zanello, publicado no ano de 2018. VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN Nesse momento, começou a ser construída uma subjetividade materna criada por um desejo produzido, interpelado e incentivado. Primeiramente, mulheres foram demandadas a amamentarem seus filhos. Em um segundo momento, a educá-los. Por fim, a partir do início do século XX, foi construída a ‘maternidade científica’, através dos discursos da pediatria e dos campos da psicanálise e das psicologias, que defendiam a ideia de que a mãe seria a grande responsável pela personalidade ou estrutura emocional dos filhos.1 Como apontado por Badinter (1985), foi produzida aqui, historicamente, a culpa materna. Em sociedades sexistas, como a brasileira, a maternidade e as qualidades ditas maternais são bastante associadas à feminilidade e a uma performance desejável por parte das mulheres. Mas para além das performances, importa pensar nas pedagogias afetivas presentes em nossa cultura, que ensinam não apenas os comportamentos, mas quais emocionalidades são desejáveis. Faz-se necessária, portanto, uma análise gendrada, crítica e feminista das emoções e de suas configurações (ZANELLO, 2018). Nesse sentido, é importante compreender que a construção das emocionalidades das mulheres passa por um heterocentramento que é demandado desde cedo às meninas: o que se aprende é a priorizar sempre, e em primeiro lugar, os desejos, anseios e necessidades dos outros, em detrimento dos próprios. Esse heterocentramento é o mecanismo fulcral de constituição do “dispositivo materno” (ZANELLO, 2018) e se realiza não apenas na relação entre mãe e filho, mas em todas as relações sociais das quais as mulheres participam. O que se espera é que elas estejam disponíveis e sejam, de certa forma, sempre solícitas. Se isso ocorre com as mulheres em geral, temos uma interseccionalidade importante que intensifica ainda mais essa demanda: a racial. Das mulheres negras se espera mais que uma disponibilidade, espera-se uma servidão voluntária. Quem mais se beneficia do dispositivo materno das mulheres são os homens. Enquanto elas cuidam deles, por eles e para eles (não só da casa, dos filhos, mas colocando energia pessoal nos projetos deles), os homens podem cuidar e investir sua energia em si mesmos e em seus próprios projetos. Em termos de pedagogia afetiva e processos psicodinâmicos de constituição subjetiva dos homens, trata-se do egocentramento. Ou seja, no tornar-se homem, Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 2 Grande parte da introdução desse artigo se baseia no livro Saúde mental, gênero e dispositivos: Cultura e processos de subjetivação, de Valeska Zanello, publicado no ano de 2018. 3 O Brasil foi escravocrata, no entanto, a mentalidade continua escravocrata. Essa parte de nossa história ainda não foi passada a limpo, como a nosso ver deveria ter sido. 2 Grande parte da introdução desse artigo se baseia no livro Saúde mental, gênero e dispositivos: Cultura e processos de subjetivação, de Valeska Zanello, publicado no ano de 2018. 3 O Brasil foi escravocrata, no entanto, a mentalidade continua escravocrata. Essa parte de nossa história ainda não Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA A literatura científica internacional vem apontando de modo consistente como a pandemia ampliou e acentuou as desigualdades de gênero na divisão das tarefas domésticas e na economia do cuidado: comparativamente aos homens, as mulheres continuaram a cuidar mais dos filhos e da casa (mesmo quando mantiveram seus trabalhos remunerados); assumiram majoritariamente as tarefas e responsabilidades adicionais colocadas pelo contexto pandêmico, como o acompanhamento escolar dos filhos e os cuidados de higiene; tiveram de reduzir o tempo dedicado ao trabalho remunerado e à carreira; e sofreram maior redução em seu tempo de lazer (Gema ZAMARRO; María PRADOS, 2021; Margaret KERR; Hannah RASMUSSEN; Kerrie FANNING; Sarah BRAATEN, 2021; Melanie ARNTZ; Sarra YAHMED; Francesco BERLINGIERI, 2020; Alison ANDREW et al., 2020; Caitlyn COLLINS; Liana LANDIVAR; Leah RUPPANNER; William SCARBOROUGH, 2021). Esses estudos também revelam que as mulheres, sobretudo aquelas que são mães de filhos em idade escolar, sofreram um significativo aumento do sofrimento psíquico (ZAMARRO; PRADOS, 2021, ansiedade, sintomas de esgotamento e questionamentos acerca da própria capacidade de maternar (KERR; RASMUSSEN; FANNING; BRAATEN, 2021). Como indica o estudo de Ben Etheridge e Lisa Spantig (2020), as mulheres sofreram um declínio em seu bem-estar psíquico significativamente maior do que os homens, o que, entre outros fatores, relaciona-se diretamente com o acúmulo de trabalho, tarefas domésticas, cuidados e responsabilidades familiares. Os estudos científicos nacionais, embora mais incipientes do que a literatura internacional, também apontam que o cruzamento entre a esfera laboral e a esfera doméstica e familiar constitui um dos principais pontos de vulnerabilização das mulheres, engendrado ou maximizado pela pandemia de Covid-19 (Anita OLIVEIRA, 2020; MELO, 2020; SOF, 2021; Juliana SANTOS et al., 2021; Lorena de SOUZA; Luiza MACHADO, 2021). Essas pesquisas revelam que, se a divisão sexual do trabalho e do cuidado já era muito desigual no Brasil, isso se agravou exponencialmente no contexto de quarentena (OLIVEIRA, 2020; SOUZA; MACHADO, 2021). Somada ao acúmulo e sobreposição do trabalho remunerado e do cuidado doméstico e familiar, a perda das redes de apoio – escolas e creches, avós e outros familiares (sobretudo mulheres), terceirização do cuidado – de que dispunham anteriormente implicou em sobrecarga de trabalho e em severo esgotamento físico e psicológico para as mulheres, com impactos prejudiciais sobre sua saúde física e mental (Fernanda INSFRAN; Ana MUNIZ, 2020; DINIZ, 2020; SANTOS et al., 2021; Rita de Cássia FREITAS; Carla ALMEIDA; Ana LOLE, 2020; SOUZA; MACHADO, 2021). MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA Quem os ajudaria nas aulas online e na necessidade de estudar em casa o que outrora aprenderiam na escola? Também aqui essas dificuldades foram postas de maneira desigual às mulheres: aquelas que poderiam exercer o home office, apesar de terem maior possibilidade de estarem perto dos filhos, viram seu trabalho exponencialmente aumentado, com o acréscimo de novas responsabilidades como mães e, também, com as tarefas domésticas, muitas das quais anteriormente eram terceirizadas. Às mulheres em condição de maior vulnerabilidade social, sem a possibilidade de Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 3 VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN manter o emprego em modelo de home office, apresentou-se o dilema: ficar em casa e passar necessidade, ou ir para a rua (com chance de se contaminar, bem como à família), mas ter de deixar os filhos em casa. Dentre as inúmeras consequências da pandemia para a vida das mulheres, destacam-se a alta incidência de desemprego, a precarização das condições de sobrevivência e a insegurança alimentar de muitas famílias, levando a um empobrecimento ainda maior de mulheres pobres. Como evidenciado por pesquisas de abrangência nacional (IPEA, 2021) e regional (OBSERVADF, 2021), as mulheres, em comparação com os homens, foram as que mais perderam empregos e que tiveram mais prejuízos em sua participação no mercado de trabalho durante a pandemia de Covid-19. Como destacado por Hildete de Melo (2020, p. 3) “escancarou a pobreza feminina, trouxe para o debate nacional o fardo das tarefas domésticas e a difícil conciliação das mulheres em ir para o mercado de trabalho e o cuidado com a família.” Somadas a essa exacerbação da feminização da miséria, também se destacam como importantes consequências gendradas da pandemia de Covid-19: a sobrecarga de trabalhos e tarefas domésticas e de cuidado; o esgotamento físico e psíquico; a vulnerabilização psicológica e os prejuízos à saúde mental das mulheres. MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA Nesse sentido, uma pesquisa realizada no primeiro semestre de 2020 com homens e mulheres de várias regiões do Brasil apontou que as mulheres foram mais afetadas emocionalmente do que os homens, apresentando maior sofrimento psíquico e sintomas de depressão, ansiedade e estresse (Antonio SERAFIM et al., 2021). Destaca-se, portanto, que a pandemia colocou em xeque, de forma diferenciada, a depender sobretudo da raça e da classe social, os arranjos do exercício do cuidado interpelado às mulheres e suas possibilidades, provocando consequências em seu bem-estar geral e em sua saúde mental. Diante desse panorama, a presente pesquisa teve como objetivo fazer um levantamento, no primeiro semestre da pandemia, sobre como as mulheres mães estavam exercendo esses cuidados, o que mudou em suas vidas e quais os efeitos que ressentiam em função dessas mudanças. MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA o preenchessem: ser mãe; estar fazendo distanciamento social em casa há pelo menos 3 (três) semanas; e ter pelo menos um(a) de seus(uas) filhos(as), que fosse dependente (física, emocional e/ou economicamente), morando com ela. Antes que a participante entrasse no questionário, era solicitada a leitura do TCLE e a concordância em participar do estudo. Vinte e duas questões relacionavam-se a características sociodemográficas como idade, raça, orientação sexual, escolaridade, se morava sozinha ou com cônjuge, etc. Cinquenta e uma questões eram relativas ao desempenho de tarefas domésticas e de cuidado com os filhos durante a pandemia, divisão ou não das tarefas com outras pessoas, tempo para si, cansaço e emoções envolvidas nessas atividades. Foi usada a escala Likert em quarenta e seis questões, apresentadas gramaticalmente sempre como asserções (sem frases com negação), porém representando, em todos os casos, os dois lados de cada dimensão (afirmativa ou negativa), das quais a respondente poderia assinalar uma das cinco opções seguintes: a) não se aplica; b) discordo completamente; c) discordo; d) não concordo, nem discordo; e) concordo; e) concordo completamente. Todas as questões da escala Likert foram apresentadas em ordem randômica. Adicionalmente, foram apresentadas quatro questões abertas opcionais. Essas questões esmiuçavam a pergunta que as antecedia e deveriam ser respondidas caso a mulher houvesse concordado (parcial ou totalmente) com a asserção. Os temas questionados foram: novas habilidades que a respondente estaria aprendendo na pandemia; quem cuidava dela quando ela se sentia cansada; sentimentos que a faziam se sentir culpada em relação aos filhos; atividades que executava quando se sentia ansiosa; quem era sua rede de apoio. Ao final, havia um espaço aberto para que, caso a mulher quisesse compartilhar alguma experiência e/ou sentimento (como mãe em momento de isolamento social) que ainda não havia sido abordado, pudesse fazê-lo. Para o presente estudo, foi realizada uma análise estatística descritiva dos dados e informações coletados (em trabalho futuro, também será realizada análise estatística inferencial). Para o tratamento e análise dos dados, no que se refere à questão racial, o grupo de mulheres foi abordado como um todo, mas também foi analisada e comparada a incidência das respostas especificamente no grupo de mulheres brancas e no grupo de mulheres negras, que constituíram os principais grupos raciais da amostra. MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA Ressaltamos, ainda, que, na análise e discussão dos resultados apresentadas a seguir, quando abordada a concordância das mulheres com determinada questão ou asserção, trata- se da soma das respostas “concordo parcialmente” e “concordo completamente”. Método No período compreendido entre 5 de março de 2020 e 7 de julho de 2020, foi disponibilizado um Survey online (Google Forms), com um cabeçalho, um Termo de Consentimento Livre e Esclarecido (TCLE) e 76 questões. A pesquisa foi publicizada por meio das redes sociais das autoras e dos grupos de WhatsApp. O cabeçalho do questionário apresentava explicações sobre a pesquisa e seus objetivos. Foi solicitado que apenas mulheres com as seguintes características Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 4 Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN vivendo como casada/união estável ou namorando/em relação não matrimonial. No período de preenchimento do questionário, 81,3% das mulheres estavam habitando com o cônjuge. g 82,5% concordaram com a asserção de que sua casa era um lugar confortável para se passar a quarentena. Entende-se essa afirmativa de acordo com a classe social que o preencheu, revelando uma experiência específica de privilégios (em relação à população brasileira) no período de isolamento social. A maioria também concordou (61,5%) que sua casa tinha espaço suficiente para as pessoas que nela habitavam. Outro dado intimamente relacionado à classe social contemplada no presente estudo diz respeito às novas possibilidades enxergadas por essas mulheres em função do isolamento social e da necessidade de fi carem em casa: 57% delas concordaram parcial ou completamente com a afirmação de que a quarentena estava se apresentando como uma possibilidade positiva de aprender novas habilidades. Na questão aberta sobre quais seriam essas habilidades, destacaram-se, sobretudo, os trabalhos domésticos. Dentre eles, o que mais se destacou foi cozinhar, mas também fazer reparos em casa, costurar etc. A nova ‘oportunidade’ de aprender a cozinhar revela justamente que essa não era uma atividade cotidiana para boa parte dessas mulheres, as quais provavelmente pagavam para outras pessoas (geralmente mulheres pobres) exercerem esses ofícios. Além dos trabalhos domésticos, também foram apontadas as seguintes habilidades aprendidas: exercer paciência; organizar e planejar o tempo; uso de tecnologias; hobbies (tocar um instrumento, desenhar, pintar, cuidar de plantas, falar uma nova língua). Sobre as crenças acerca da maternidade, foi encontrado que, segundo 38% das participantes, a maternidade ainda é vista como a maior realização na vida de uma mulher. Essa percentagem foi um pouco maior entre as mulheres brancas (39,5%) do que entre as negras (35,2%). Ao mesmo tempo, 76,1% delas discordaram, parcial ou totalmente, com a afirmação de que uma mulher precisa ter filhos para estar “completa”. Ou seja, já há uma desconstrução do papel da maternidade como algo essencial para a realização na vida de uma mulher, coadunando com pesquisa realizada pelo IPEA (2014), que apontou uma correlação inversa entre escolaridade e tendência a sustentar essa crença. No entanto, há boa parcela de mulheres nesse grupo populacional que ainda a mantém. No que se refere ao nascimento do filho, concepções tradicionais sobre a maternidade se mostraram ainda mais presentes: 55,4% das respondentes afirmaram acreditar que, a partir desse momento, essa deve ser a grande prioridade em sua vida. VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN E 39,6% delas concordaram (total ou parcialmente) que a chegada do filho faz o instinto materno aflorar. Isto é, mesmo discordando da necessidade de se ter um filho para estar completa, ainda se mantém uma crença na ‘naturalidade’ da maternidade, como um instinto. Essas respostas demonstram que, apesar de termos tido avanços na desconstrução da naturalização tanto da relação entre maternidade e mulheres, quanto da maternidade em si mesma como um instinto, não é pequena a parcela de mulheres mães que ainda mantém crenças tradicionalistas sobre a maternidade. Estudos demonstram que, quanto mais tradicionais e romantizadas são essas crenças, maiores são as chances de intensidade do sofrimento no exercício da maternidade, bem como da autoculpabilização por não conseguir cumprir seus ideais (Katia AZEVEDO; Alessandra ARRAIS, 2006; Shari TURER, 2007; Orna DONATH, 2017). Sobre a divisão de tarefas domésticas, 55,2% indicaram que lidar com as demandas da família, dos cuidados com a casa e de sustento estava quase impossível naquele momento, sendo que 88,9% concordaram que estavam realizando mais trabalho doméstico do que antes. Isso acabou por levá-las a se sentirem mais sobrecarregadas pelas tarefas que tinham que desempenhar (82,3%). Provavelmente, essa mudança trazida pela pandemia deve-se à perda do privilégio da possibilidade de terceirização de certos serviços de cuidado. Porém, se essa redistribuição do exercício do cuidado colocou em xeque o classismo e o racismo, tão presentes em nossa sociedade, por outro lado, não desconstruiu o sexismo, pois foi às mulheres que essas tarefas foram atribuídas. Ou seja, homens pouco ou nada foram questionados acerca de seus privilégios, seja de classe raça ou gênero. Pelo contrário, em muitos setores, acessíveis sobretudo a homens de classe social privilegiada, a pandemia se configurou como oportunidade de impulsionar a vida laboral e produtiva. Por exemplo, como mostra um levantamento feito pelo grupo Parent in Science (2020), enquanto mulheres negras (com ou sem filhos) e mulheres brancas (com filhos) constituíram o grupo cuja produtividade acadêmica foi mais prejudicada pela pandemia, os homens não tiveram sua produtividade afetada ou, como editores de diversos periódicos científicos vêm apontando, apresentaram inclusive um aumento no número de submissões de artigos (PARENT IN SCIENCE, 2020; Maria Beatriz CARUSO; Manuela RAMALHO; Juliana PHILIPP; Cibele BRAGAGNOLO, 2020). Resultados e Discussões Ao todo, 5.643 mulheres brasileiras responderam ao questionário. Descrevemos, inicialmente, as principais informações sociodemográficas das respondentes. Em seguida, apresentamos a análise estatística descritiva realizada a partir dos seguintes núcleos, por meio dos quais foram organizadas as respostas dos questionários: a) possibilidades enxergadas em função do isolamento social e da necessidade de ficar em casa; b) crenças acerca da maternidade; c) divisão de tarefas domésticas; d) cuidados e relação com os filhos; e) relação consigo mesmas e sentimentos experimentados no momento da pandemia; f) encruzilhada da relação trabalho x cuidado de casa/filhos. 99,7% das respondentes identificaram-se com o gênero feminino e 94,5% como heterossexual ou, principalmente, heterossexual. Com relação ao segmento socioeconômico, as respondentes eram majoritariamente de classe média e classe média alta, com nível escolar alto (68,8% com pós-graduação e 23,2% com nível superior). Esse recorte social não foi intencional na pesquisa, mas ele se explica justamente por serem essas mulheres pertencentes à classe social privilegiada as que dispõem de maior possibilidade de exercício do home office (como veremos em dado adiante) e que têm maior acesso à internet (local onde a pesquisa foi divulgada e o questionário disponibilizado). Com relação à comparação entre as incidências das respostas no grupo de mulheres brancas e no grupo de mulheres negras, não foi encontrada diferença significativa em nenhum dos aspectos e dimensões analisados. Grande parte das respondentes era moradora de cidades/áreas urbanas (94,1%) das seguintes regiões: Centro-Oeste (42,9%, sendo a grande maioria habitante do Distrito Federal); Sudeste (31,4%), Nordeste (11,9%), Sul (10,5%), Norte (1,6%). Uma pequena parcela foi constituída por brasileiras morando em outros países (1,7%). Em relação à faixa etária, a maioria (56,1%) foi de mulheres entre 35 – 44 anos; seguida por mulheres entre 25 – 34 anos (22%). A maioria tinha um filho (49,8%) ou dois (40,2%) e grande parte desses filhos eram não adotivos (97,4%). No que tange à religião, 36,4% identificaram-se como católicas; 22,3% como espiritualistas, mas sem religião; 12,7% espíritas; 6,7% evangélicas; 5,2% agnósticas; 4,6% ateístas; 3,6% protestantes. g g g Antes da pandemia, 82,1% dessas mulheres trabalhavam fora de casa e 69,7% delas passaram a usar o teletrabalho. 89% reportaram não haver perdido (pelo menos até julho de 2020) o emprego ou a atividade remunerada. A maior parte dessas mulheres (85,1%) estava Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 5 MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA Apesar das ambivalências experimentadas na relação com o(s) filho(s) e do cansaço pela hiperconcentração (ainda maior na pandemia) de responsabilidades sobre si mesmas em relação a eles, 82% apontaram que, se pudessem voltar no tempo, não deixariam de ter filhos. É importante ressaltar que, embora sejam plausíveis e inerentes à própria maternidade, as ambivalências, conflitos e sentimentos de insatisfação, raiva, desgosto, sofrimento e arrependimento; nessa esfera, são socialmente julgados e estigmatizados como aberrações, como sentimentos “antinaturais” ou como distúrbios psiquiátricos (ZANELLO, 2018; Jane USSHER, 2011; DONATH, 2017). Especificamente sobre o arrependimento, domina uma proibição social de que essa postura emocional seja associada à maternidade. De um lado, o arrependimento é encarado como inexistente e inconcebível; de outro, quando não denegado, é visto com espanto, como algo ilegítimo e condenável. Portanto, já que é considerado um sentimento aberrante, ilícito e inconfessável, o arrependimento relacionado à maternidade é sistematicamente silenciado (DONATH, 2017). Há poucos espaços onde o mal-estar da maternidade possa ser nomeado, sem julgamentos sociais, mesmo nas psicologias (ZANELLO, 2016). Sobre a relação consigo mesmas e sentimentos experimentados naquele momento da pandemia, 79,7% concordaram parcial ou totalmente que estavam mais cansadas do que o normal. Grande parte do cansaço ligava-se a estar disponível para os outros e 78,3% concordaram que queriam ter mais tempo sozinhas. Ou seja, apesar de estarem em casa, a maioria (79,2%) quase não tinha tempo para si mesma e nem mesmo para fazer as coisas de que gostavam (77,1%). O sentimento de se sentir sozinha foi partilhado por 43,2% das respondentes e 35,3% das mulheres apontaram que, quando estavam tristes, exaustas ou ansiosas, não conseguiam compartilhar seus sentimentos com os demais membros da casa, tentando não os demonstrar (54,4%). Em geral, as atividades apontadas pelas mulheres para ‘descontar’ a ansiedade foram sobretudo as seguintes: comer (a que mais se destacou), beber e fumar. Quando cansadas, 45,3% das mulheres apontaram não haver ninguém que cuidasse delas. Porém, em caso de adoecimento, 65,7% concordaram que poderiam contar com alguém, caso precisassem se isolar da família (por exemplo, em caso de se contaminarem pela Covid). Ou seja, em situações cotidianas, somente cerca de 50% das mulheres sentiam que podiam contar com suportes e cuidados, mas na hipótese de situações extremas ou emergenciais, essa proporção mostrou-se um pouco maior. MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA parte delas, 68,7% concordaram que, caso não houvesse essa cobrança, as tarefas domésticas muitas vezes não seriam feitas. Ou seja, mesmo que outras pessoas da casa as ajudassem com as tarefas, ainda seria necessário verificar se elas foram devidamente feitas (55,6%). Assim, planejar, organizar e garantir que as tarefas domésticas diárias fossem realizadas era entendido como algo que dependia principalmente delas (76,5%). parte delas, 68,7% concordaram que, caso não houvesse essa cobrança, as tarefas domésticas muitas vezes não seriam feitas. Ou seja, mesmo que outras pessoas da casa as ajudassem com as tarefas, ainda seria necessário verificar se elas foram devidamente feitas (55,6%). Assim, planejar, organizar e garantir que as tarefas domésticas diárias fossem realizadas era entendido como algo que dependia principalmente delas (76,5%). Isto é, ainda que contassem com alguma participação dos homens ou de outro familiar na execução das atividades de cuidado diário com a casa e com a família, cabia a elas a famosa “carga mental” implicada na gestão doméstica e no trabalho mental invisibilizado e intangível de planejar, prever, delegar, cobrar e supervisionar (Rafaela CYRINO, 2011; Kimberly FISCHER; John ROBSON, 2010; Margaret MARUANI, 2003). g ) Sobre os cuidados e relação com os fi lhos, 60,6% das mulheres concordaram parcial ou completamente que era delas a maior parte da responsabilidade de auxiliar o filho nas tarefas escolares nesse período. Essa intensa demanda trouxe, muitas vezes, o sentimento de culpa quando elas não se sentiam disponíveis para tal (65,2%). Além disso, também trouxe culpa, pelos sentimentos experimentados em relação aos próprios filhos (52,9%) e que não gostariam de sentir. Dentre esses sentimentos, sobressaiu-se a raiva, mas também foram relatados o cansaço, a impaciência, a frustração, a vontade de querer fazer coisas para si mesma e de ter um tempo sozinha e, inclusive, a dúvida sobre se deveria ter tido filho e o arrependimento por tê-lo concebido. Frente a muitos desses sentimentos, sobretudo à raiva, 43,4% concordaram que, em certos momentos, mesmo que não passassem ao ato, tinham vontade de bater nos seus filhos. A expectativa de que as crianças voltassem logo para as aulas, de forma segura, se apresentou em 64,2% delas e 43,4% concordaram que estavam preocupadas com a interrupção da educação das crianças devido às medidas de isolamento social (32,2% disseram não estar). Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN Nesse sentido, apesar de a maioria das respondentes habitar com o cônjuge ou parceiro, 59,1% delas concordaram que as tarefas domésticas eram distribuídas de forma injusta entre os membros da casa e, mesmo quando divididas, cabia a elas a responsabilidade de cobrá-las (65,7%). Além de cobrar a execução das tarefas, mesmo quando havia divisão e delegação de Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 6 Considerações fi nais Embora não tenha sido intencional, o fato de que a participação nesta pesquisa dependesse do acesso à internet, somado ao critério de que a mulher estivesse fazendo distanciamento social em casa, engendrou um importante recorte social: a maioria das respondentes era pertencente aos segmentos socioeconômicos mais favorecidos, de classe média e classe média alta, com elevado nível de escolaridade. Além disso, as respondentes foram majoritariamente brancas, constituindo quase 70% da totalidade de mulheres, evidenciando como, em nossa sociedade, a configuração das classes socioeconômicas é informada e organizada pelo racismo. Portanto, é necessário salientar que nossos achados e discussões referem-se ao exercício da maternidade e do cuidado na pandemia de Covid-19, única e especificamente entre mulheres desse segmento socioeconômico que, comparativamente a grande parte da população brasileira, tem vivenciado essa crise sanitária e social a partir de um lugar de múltiplos privilégios. De modo diretamente relacionado a essa experiência de privilégio, a análise estatística descritiva evidenciou que quase 60% das mulheres identificaram, na situação de quarentena e isolamento social, uma oportunidade positiva para aprender novas habilidades, relacionadas principalmente aos afazeres domésticos, como cozinhar. Esse dado mostra que, anteriormente à pandemia, grande parte das mulheres terceirizavam esses serviços e atividades, que, em geral, continuam recaindo sobre mulheres, principalmente negras e pobres. Com relação às crenças acerca da maternidade, os dados indicaram que está em marcha um processo de desconstrução da concepção de maternidade como condição sine qua non para o sentido de completude das mulheres. No entanto, observa-se que esse processo de desconstrução convive intimamente com a permanência de concepções idealizadas e tradicionalistas de maternidade, como: a ideia de que ela consiste na maior realização das mulheres; a compreensão de que o filho se torna a grande prioridade em suas vidas; ou a naturalização da maternidade, compreendida como instintiva. Os dados relativos à divisão das tarefas domésticas, ao revelarem que a maioria das mulheres estava sobrecarregada e com dificuldades para lidar com o aumento dessas demandas imposto pela pandemia, permitiu-nos observar dois aspectos principais sobre essa questão. De um lado, os resultados apontam para a perda do privilégio da terceirização do cuidado doméstico e familiar, produzindo alguma fissura, ainda que discreta, no racismo e no classismo estruturais em nossa sociedade. VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN Ainda sobre essa autoexigência de cuidar e estar disponível para os outros, bem como de cuidar do bem-estar alheio (característico do dispositivo materno), 46,1% reportaram se sentirem responsáveis por tentar manter os outros membros da casa felizes e entretidos. Trata-se aqui do “trabalho emocional” que recai sobre as mulheres e que, segundo Rebecca Erickson (2005), refere-se às inúmeras atividades (visíveis e invisíveis) envolvidas na promoção do bem-estar emocional dos outros; em fazê-los se sentirem cuidados, reconfortados, amados ou encorajados; na facilitação e gestão das relações interpessoais; em suma, na provisão de suporte afetivo e emocional. Além das consequências físicas das atividades domésticas e do cuidar requeridos na pandemia, e do funcionamento no dispositivo materno (autoculpabilização e autorresponsabilização pelo bem-estar do outros, a despeito de si mesmas), 66,1% das mulheres apontaram queda da libido nesse período. E, também, 80,2% das mulheres reportaram que sentiam falta de ter contato físico com outras pessoas. Por fim, na encruzilhada (e tentativa de balanço) da relação trabalho x cuidado de casa/fi lhos, apenas 38,5% apontaram que, quando estavam trabalhando em casa, podiam contar com outros membros da família para cuidar do(s) seu(s) filho(s). Ou seja, a maioria viveu, nesse momento, a invasão, em sua vida profissional, dos papéis historicamente construídos e atribuídos às mulheres, como responsáveis e cuidadoras dos filhos. Nesse momento inicial da pandemia, ainda não era possível perguntar e avaliar as consequências desse fenômeno para suas carreiras. No entanto, se as mulheres, de fato, tiverem reduzido o tempo de dedicação ao trabalho remunerado em virtude das demandas domésticas e de cuidado, como indicado por estudos internacionais (ZAMARRO; PRADOS, 2021; COLLINS; LANDIVAR; RUPPANNER; SCARBOROUGH, 2021; ANDREW et al., 2020), cabe supor que essa situação pode ter afetado negativamente suas vidas profissionais. Além disso, apesar de a maioria ter relatado morar em lugares confortáveis para passarem o isolamento social, 42,7% disseram não ter um lugar confortável e tranquilo para trabalhar. Por fim, 68,2% das mulheres reportaram que mesmo se sentindo sobrecarregadas, achavam difícil dizer não para as demandas do trabalho. MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA A existência de uma rede de apoio foi questionada por 46,6% das mulheres e, quando apontaram quem constituiria essa rede (caso sentissem que existia), destacaram sobretudo familiares e marido, mas ainda apareceu, mesmo que de forma discreta, a figura da diarista e da babá, revelando que, mesmo na situação de risco sanitário, foi cogitada a possibilidade de terceirização do cuidado e do trabalho doméstico. A maioria das mulheres (77,2%) se disse mais propensa a ajudar os outros do que a pedir ajuda. E 51,3% apontaram não considerar suas necessidades e desejos antes de concordar em fazer o que os outros lhes pedem. Esses dados evidenciam nitidamente como, pela via do dispositivo materno, as mulheres se subjetivam em um heterocentramento, a partir do qual são ensinadas a priorizar os outros em detrimento dos próprios anseios, desejos e necessidades, engendrando uma distribuição muito desigual do cuidado e do ser cuidada (ZANELLO, 2018). Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 7 Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA No que se refere aos cuidados e à relação com os filhos durante o isolamento social, os dados mostraram que as novas demandas colocadas pela pandemia, particularmente o acompanhamento escolar das crianças, também recaiu de forma majoritária sobre as mulheres. Frente a essas novas demandas e à sobrecarga física e psíquica decorrente do acúmulo de múltiplas tarefas e funções, muitas mulheres indicaram sentimentos de culpa, raiva, cansaço, impaciência, frustração e ambivalências relacionadas à maternidade. Não obstante, a maioria das mulheres indicou que, mesmo se pudessem refazer essa escolha, não deixariam de ter filhos. Isso aponta para o não arrependimento, o que, em alguma medida, precisa ser analisado em diálogo com a denegação, condenação e silenciamento do arrependimento relacionado à maternidade em nossa sociedade. Os dados sobre os sentimentos das mulheres na pandemia indicaram que, em sua maioria, elas estavam sobrecarregadas e estafadas, o que se relaciona diretamente com a exacerbação da situação de acúmulo e sobreposição do trabalho remunerado, doméstico, trabalho de cuidado e trabalho emocional. Além disso, também foi significativa a proporção de mulheres que indicaram sentir-se sozinhas; sem um ponto de acolhimento e escuta para falarem de suas tristezas, cansaço e ansiedade; sem uma rede de apoio consistente; e pouco cuidadas. Vale ressaltar que, evidenciando o heterocentramento que marca a subjetividade das mulheres, a maioria delas apontou estar mais propensa a ajudar os outros do que a pedir ajuda, e mais disponível e atenta às necessidades e anseios dos outros em detrimento dos próprios. Por último, os dados revelaram que as mulheres, em sua maioria, não podiam contar com outras pessoas e familiares para lidar com os desafios e sobrecargas do entrecruzamento entre o trabalho remunerado e o trabalho de cuidado da casa, dos filhos e da família. Além disso, tampouco contavam com espaço adequado e reservado para o desempenho do trabalho profissional. Nesse sentido, destaca-se que elas viveram uma sobreposição dos diferentes tipos de demandas e de trabalho – profissional e remunerado, de cuidado da casa e dos filhos –, tendo sua dimensão profissional invadida pelo papel de cuidadora. No momento atual, passados cerca de um ano e oito meses de pandemia, seria importante a realização de pesquisas que buscassem compreender os impactos e consequências dessa situação sobre a profissão e a carreira das mulheres, em distintos segmentos socioeconômicos. MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA Em suma, corroborando a literatura internacional e nacional existentes, os dados do presente levantamento revelaram que a situação de pandemia e isolamento social exacerbou as desigualdades de gênero que atravessam a economia do cuidado doméstico e familiar. Mesmo para o segmento socioeconomicamente favorecido contemplado por este estudo, que dispõe de uma série de privilégios, identificou-se que as mulheres, majoritariamente, encontravam-se sobrecarregadas e cansadas; sozinhas na encruzilhada entre trabalho profissional e trabalho de cuidados múltiplos; cuidando muito e sendo muito pouco cuidadas; muito disponíveis para os outros e pouco disponíveis para si mesmas; e, a despeito de tudo isso, culpadas na relação e cuidado com os filhos. Diante desse quadro, reiteramos a importância de outros estudos que examinem, depois de decorrido um período mais prolongado nessa situação, suas consequências não apenas sobre a dimensão profissional das mulheres, mas também sobre sua saúde mental. Sobre as limitações do presente estudo, ressaltamos, principalmente, aquelas que decorrem do próprio recorte socioeconômico que foi delineado pela trajetória metodológica adotada. Salientamos, assim, que os resultados desta pesquisa não abarcam e não nos permitem pensar sobre as outras múltiplas formas de maternidade existentes, atravessadas por diferentes interseccionalidades, hierarquias, opressões e distintas formas de interpelação e exercício do cuidado. Nessa perspectiva, faz-se fundamental a realização de outras pesquisas que contemplem essa diversidade de maternidades e os modos como elas foram impactadas pela pandemia, nos diferentes entrelaçamentos das estruturas de raça, classe e gênero. Essa compreensão faz-se ainda mais urgente para as mulheres que vêm experienciando a crise social e sanitária nas posições mais vulnerabilizadas e expostas, como as mulheres negras e pobres; as que perderam o emprego; empregadas domésticas; mulheres que tiveram de conciliar o trabalho fora e as demandas de casa; e, ainda, as mulheres que se encontram na linha de frente do combate à Covid-19, como as profissionais de saúde e de limpeza. Considerações fi nais De outro lado, eles mostram como o sexismo e os privilégios dos homens mantiveram-se, no mínimo, intocados, uma vez que o adicional de atividades e trabalhos (visíveis e invisíveis, físicos e mentais, de gestão e execução) relacionados ao cuidado da casa e da família, continuou recaindo predominantemente sobre as mulheres. 8 ARNTZ, Melanie; YAHMED, Sarra; BERLINGIERI, Francesco. “Working from Home and COVID-19: The Chances and Risks for Gender Gaps”. Intereconomics, v. 55, n. 6, p. 381-386, 2020. Referências ANDREW, Alison et al. How are mothers and fathers balancing work and family under lockdown? The Institute for Fiscal Studies, 2020. ANDREW, Alison et al. How are mothers and fathers balancing work and family under lockdown? The Institute for Fiscal Studies, 2020. ARNTZ, Melanie; YAHMED, Sarra; BERLINGIERI, Francesco. “Working from Home and COVID-19: The Chances and Risks for Gender Gaps”. Intereconomics, v. 55, n. 6, p. 381-386, 2020. ARNTZ, Melanie; YAHMED, Sarra; BERLINGIERI, Francesco. “Working from Home and COVID-19: The Chances and Risks for Gender Gaps”. Intereconomics, v. 55, n. 6, p. 381-386, 2020. AZEVEDO, Kátia; ARRAIS, Alessandra. “O Mito da Mãe Exclusiva e seu Impacto na Depressão Pós- Parto”. Psicologia: Reflexão e Crítica, v. 19, n. 2, p. 269-276, 2006. Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 9 VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN BADINTER, Elizabeth. Um amor conquistado: o mito do amor materno. Rio de Janeiro: Nova Fronteira, 1985. CARNEIRO, Sueli. Racismo, sexismo e desigualdade no Brasil. São Paulo: Selo Negro, 2011. CARUSO, Maria Beatriz; RAMALHO, Manuela; PHILIPP, Juliana; BRAGAGNOLO, Cibele. “Maternity, science and pandemic: an urgent call for action!” Hoehnea, v. 47, n. 1, e812020, 2020. COLLINS, Caitlyn; LANDIVAR, Liana; RUPPANNER, Leah; SCARBOROUGH, William. “COVID-19 and the gender gap in work hours”. Gender Work and Organization, v. 28, n. S1, p. 101-112, 2021. CYRINO, Rafaela. “A gestão do trabalho doméstico entre as mulheres executivas. Um exemplo de combinação de dados de uma pesquisa de Usos do Tempo com metodologia qualitativa”. Revista de ciências sociais – Política & Trabalho, [S. l.] v. 34, 2011. Disponível em https://periodicos. ufpb.br/ojs/index.php/politicaetrabalho/article/view/12187. Acesso em 21/11/2021. DINIZ, Débora. “Mundo pós-pandemia terá valores feministas no vocabulário comum, diz antropóloga Débora Diniz”. Folha de S. Paulo, 06 abr. 2020. Disponível em https://www1. folha.uol.com.br/equilibrioesaude/2020/04/mundo-pos-pandemia-tera-valores-feministas-no- vocabulario-comum-diz-antropologa-debora-diniz.shtml. Acesso em 21/11/2021. DONATH, Orna. Mães arrependidas: uma outra visão da maternidade. Rio de Janeiro: Civilização Brasileira, 2017. ERICKSON, Rebecca. “Why Emotion Work Matters: Sex, Gender, and the Division of Household Labor”. Journal of Marriage and Family, v. 67, p. 337-351, May, 2005. ETHERIDGE, Bem; SPANTIG, Lisa. “The Gender Gap in Mental Well-Being During the Covid-19 Outbreak: Evidence from the UK”. Institute for Social & Economic Research, n. 2020-08, June, 2020. FEDERICI, Silvia. O Ponto Zero da Reprodução: Trabalho Doméstico, Reprodução e Luta Feminista. São Paulo: Editora Elefante, 2019. FISCHER, Kimberly; ROBSON, John. Referências Daily Routines in 22 Countries: Diary Evidence of Average Daily Time in Thirty Activities. Oxford, UK: Center for Time Use Research, University of Oxford, 2010. FOUCAULT, Michel. Microfísica do poder. Rio de Janeiro: Graal, 1996. FREITAS, Rita de Cássia; ALMEIDA, Carla; LOLE, Ana. “As mulheres e a pandemia da COVID-19 na encruzilhada do cuidado”. In: LOLE, Ana; STAMPA, Inez; GOMES, Rodrigo (Orgs.). Para além da quarentena: reflexões sobre crise e pandemia. Rio de Janeiro: Mórula Editorial, 2020. INSFRAN, Fernanda; MUNIZ, Ana. “Maternagem e Covid-19: desigualdade de gênero sendo reafirmada na pandemia”. Diversitates International Journal, v. 12, n. 2, p. 26-47, julho/dezembro 2020. IPEA - INSTITUTO DE PESQUISA ECONÔMICA APLICADA. SIPS – Sistema de Indicadores de Percepção Social. Tolerância social à violência contra as mulheres. Brasília: IPEA, 2014. IPEA - INSTITUTO DE PESQUISA ECONÔMICA APLICADA. Desigualdades no mercado de trabalho e pandemia da covid-19. Brasília: IPEA, 2021. KERR, Margaret; RASMUSSEN, Hannah; FANNING, Kerrie; BRAATEN, Sarah. “Parenting During COVID-19: A Study of Parents’ Experiences Across Gender and Income Levels”. Family Relations, v. 70, n. 5, p. 1327-1342, December, 2021. LAQUEUR, Thomas. Inventando o sexo: corpo e gênero dos gregos a Freud. Rio de Janeiro: Relume - Dumará, 2001. MARUANI, Margaret. “Les Sciences Sociales du Travail à L´épreuve des Différences de Sexe”. In: Jacqueline LAUFER; Catherine MARRY; Margaret MARUANI (Orgs.). Le travail du genre: les sciences sociales du travail à l’épreuve des différences de sexe. Paris: La Découverte/MAGE, 2003. p.163- 180. MARUANI, Margaret. “Les Sciences Sociales du Travail à L´épreuve des Différences de Sexe”. In: Jacqueline LAUFER; Catherine MARRY; Margaret MARUANI (Orgs.). Le travail du genre: les sciences sociales du travail à l’épreuve des différences de sexe. Paris: La Découverte/MAGE, 2003. p.163- 180. MELO, Hildete. A vida das mulheres em tempos de pandemia. Friedrich Ebert Stiftung, 13 de abril de 2020. Disponível em https://brasil.fes.de/detalhe/a-vida-das-mulheres-em-tempos-de- pandemia/. Acesso em 21/11/2021. MELO, Hildete. A vida das mulheres em tempos de pandemia. Friedrich Ebert Stiftung, 13 de abril de 2020. Disponível em https://brasil.fes.de/detalhe/a-vida-das-mulheres-em-tempos-de- pandemia/. Acesso em 21/11/2021. Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 10 MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA OBSERVADF. DF: mulheres foram as que mais perderam emprego durante pandemia. 11 de novembro de 2021. Disponível em https://observadf.org.br/2021/11/11/unb-esta-entre-as-dez- instituicoes-publicas-com-mais-graduacoes-cinco-estrelas-em-ranking-nacional/. Acesso em 21/11/2021. OLIVEIRA, Anita. “A espacialidade aberta e relacional do lar: a arte de conciliar maternidade, trabalho doméstico e remoto na pandemia de covid-19”. Rev. Tamoios, v. 16, n. 1, Especial COVID19, p. 154-166, maio 2020. OLIVEIRA, Anita. “A espacialidade aberta e relacional do lar: a arte de conciliar maternidade, trabalho doméstico e remoto na pandemia de covid-19”. Rev. Tamoios, v. 16, n. 1, Especial COVID19, p. 154-166, maio 2020. PARENT IN SCIENCE. Produtividade acadêmica durante a pandemia: efeitos de gênero, raça e parentalidade. Parent in Science, 2020. Disponível em https://327b604e-5cf4-492b- 910b-e35e2bc67511.filesusr.com/ugd/0b341b_81cd8390d0f94bfd8fcd17ee6f29bc0e. pdf?index=true. Acesso em 21/11/2021. SANTOS, Juliana et al. “A vivência da maternidade em meio à pandemia”. Global Academic Nursing Journal, n. 2 (Spe.1), e95, 2021. DOI: 10.5935/2675-5602.20200095. Disponível em https:// globalacademicnursing.com/index.php/globacadnurs/article/view/175. Acesso em 21/11/2021. SOF - SEMPRE VIVA ORGANIZAÇÃO FEMINISTA. SEM PARAR: o trabalho e a vida das mulheres na pandemia. SOF, 2021. Disponível em https://mulheresnapandemia.sof.org.br/. Acesso em 21/11/2021. SERAFIM, Antonio et al. “Exploratory study on the psychological impact of COVID-19 on the general Brazilian population”. PLOS ONE, v. 16, n. 2, e0245868, 2021. DOI: 10.1371/journal. pone.0245868. Disponível em https://journals.plos.org/plosone/article?id=10.1371/journal. pone.0245868#sec012. Acesso em 21/11/2021. SOUZA, Lorena de; MACHADO, Luiza. “Casa, maternidade e trabalho no distanciamento social: a “pandemia” da sobrecarga de trabalho para as mulheres”. Revista da ANPEGE, v. 17, n. 32, p. 281-308, 2021. TRONTO, Joan. “Care démocratique et démocraties du care”. In: MOLINIER, Pascal; LAUGIER, Sandra; PAPERMAN, Patrícia. Qu’est-ce que le care?. Paris: Payot & Rivage, 2009. p. 35-55. TURER, Shari. “The Myths of Motherhood”. In: O’REILLY, Andrea (Org.). Maternal Theory: Essential Readings. Toronto: Demeter Press, 2007, Edição do Kindle, cap. 21. USSHER, Jane. The madness of women. Nova York: Routledge, 2011. VIANNA, Cláudia Pereira. “A feminização do magistério na educação básica e os desafios para a prática e a identidade coletiva docente”. In: YANNOULAS, Silvia. (Org). Trabalhadoras. Análise da feminização das profissões e ocupações. Brasília: Abaré, 2013, p. 159-180. ZAMARRO, Gema; PRADOS María. “Gender differences in couples’ division of childcare, work and mental health during COVID-19”. Review of Economics of Household, v. 19, p. 11-40, Janeiro 2021. ZANELLO, Valeska. “Dispositivo materno e processos de subjetivação: desafios para a Psicologia”. In: ZANELLO, Valeska; PORTO, Madge (Orgs.). Aborto e (Não) Desejo de Maternidade(s): questões para a Psicologia. Brasília: CFP, 2016. p. 103-122. ZANELLO, Valeska. MATERNIDADE E CUIDADO NA PANDEMIA ENTRE BRASILEIRAS DE CLASSE MÉDIA E MÉDIA ALTA Saúde mental, gênero e dispositivos: cultura e processos de subjetivação. Curitiba: Appris, 2018. ZANELLO, Valeska. Saúde mental, gênero e dispositivos: cultura e processos de subjetivação. Curitiba: Appris, 2018. Valeska Zanello (valeskazanello@unb.br; valeskazanello@uol.com.br) é psicóloga e filósofa, doutora em Psicologia pela Universidade de Brasília. Orientadora de mestrado e doutorado no Programa de Pós-Graduação em Psicologia Clínica e Cultura da UnB. Coordena o grupo “Saúde Mental e Gênero”, no CNPq. Membro do NEPEM - Núcleo de Estudos e Pesquisas da Mulher/UnB e do GT “Psicologia e Estudos de Gênero” da Associação Nacional de Pesquisa e Pós-Graduação em Psicologia - ANPEPP. http://lattes.cnpq.br/0163069128352529 Carla Antloga (antloga@unb.br; antlogacarla@gmail.com) é psicóloga, pós-doutora em Psicologia do Social e do Trabalho pela Universidade de São Paulo, com estágio técnico no Conservatoire d’Arts et Métiers, de Paris. Orientadora de mestrado e doutorado no Programa de Pós-Graduação em Psicologia Clínica e Cultura/UnB. Coordenadora do Grupo de Estudos em Psicodinâmica do Trabalho Feminino – Psitrafem, no CNPq. Mãe de dois filhos. http://lattes.cnpq. br/1693120835730857 Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 11 , p , ( ) DOI: 10.1590/1806-9584-2022v30n286991 VALESKA ZANELLO, CARLA ANTLOGA, EILEEN PFEIFFER-FLORES E IARA FLOR RICHWIN Eileen Pfeiffer-Flores (eileen@unb.br; eileenpfeiffer@gmail.com) é doutora em Psicologia pela Universidade de Brasília, com Pós-Doutorado em Filosofia no King’s College, em Londres. Orientadora de mestrado e doutorado no Programa de Pós-Graduação em Ciências do Comportamento/UnB. Fundadora e coordenadora do Projeto de Extensão Livros Abertos/UnB. http:// lattes.cnpq.br/4007163045058892 Iara Flor Richwin (iara.flor@unb.br; iararaflor@gmail.com) é doutora em Psicologia Clínica e Cultura pela Universidade de Brasília e pela Université Paris Diderot, psicóloga do sistema socioeducativo do Distrito Federal (SEJUS/GDF), pesquisadora colaboradora do Programa de Pós-Graduação em Psicologia Clínica e Cultura da Universidade de Brasília, onde participa do grupo “Saúde Mental e Gênero”. http://lattes.cnpq.br/7131076646582970 COMO CITAR ESTE ARTIGO DE ACORDO COM AS NORMAS DA REVISTA ZANELLO, Valeska; ANTLOGA, Carla; PFEIFFER-FLORES, Eileen; RICHWIN, Iara Flor. “Maternidade e cuidado na pandemia entre brasileiras de classe média e média alta”. Revista Estudos Feministas, Florianópolis, v. 30, n. 2, e86991, 2022. Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991 CONTRIBUIÇÃO DE AUTORIA Valeska Zanello: Concepção, coleta de dados, análise de dados, discussão dos resultados, elaboração do manuscrito e redação. Carla Antloga: Concepção, coleta de dados, análise de dados e discussão dos resultados. Eileen Pfeiffer-Flores: Concepção, coleta de dados, discussão dos resultados e redação. Iara Flor Richwin: Análise de dados, discussão dos resultados, elaboração do manuscrito e redação. FINANCIAMENTO Não se aplica. CONSENTIMENTO DE USO DE IMAGEM Não se aplica. APROVAÇÃO DE COMITÊ DE ÉTICA EM PESQUISA Não se aplica. CONFLITO DE INTERESSES Não se aplica. LICENÇA DE USO Este artigo está licenciado sob a Licença Creative Commons CC-BY 4.0 International. Com essa licença você pode compartilhar, adaptar, criar para qualquer fim, desde que atribua a autoria da obra. HISTÓRICO Recebida em 12/04/2022 Reapresentado em 09/05/2022 Aceita em 27/05/2022 Eileen Pfeiffer-Flores: Concepção, coleta de dados, discussão dos resultados e redação. 12 Revista Estudos Feministas, Florianópolis, 30(2): e86991 DOI: 10.1590/1806-9584-2022v30n286991
https://openalex.org/W4293795657
https://journals.plos.org/plosmedicine/article/file?id=10.1371/journal.pmed.1003974&type=printable
English
null
Presentations of children to emergency departments across Europe and the COVID-19 pandemic: A multinational observational study
PLoS medicine
2,022
cc-by
16,205
OPEN ACCESS Citation: Nijman RG, Honeyford K, Farrugia R, Rose K, Bognar Z, Buonsenso D, et al. (2022) Presentations of children to emergency departments across Europe and the COVID-19 pandemic: A multinational observational study. PLoS Med 19(8): e1003974. https://doi.org/ 10.1371/journal.pmed.1003974 1 Department of Pediatric Emergency Medicine, Division of Medicine, St. Mary’s hospital—Imperial College NHS Healthcare Trust, London, United Kingdom, 2 Faculty of Medicine, Department of Infectious Diseases, Section of Pediatric Infectious Diseases, Imperial College London, London, United Kingdom, 3 Centre for Pediatrics and Child Health, Imperial College London, London, United Kingdom, 4 Faculty of Medicine, School of Public Health, Imperial College London, London, United Kingdom, 5 Department of Child and Adolescent Health, Mater Dei Hospital, Msida, Malta, 6 Division of Emergency Medicine–Pediatrics, University College London NHS Foundation Trust, London, United Kingdom, 7 Department of Pediatric Emergency Medicine, Heim Pal National Pediatric Institute, Budapest, Hungary, 8 Department of Woman and Child Health and Public Health, Fondazione Policlinico Universitario A. PLOS MEDICINE PLOS MEDICINE Presentations of children to emergency departments across Europe and the COVID- 19 pandemic: A multinational observational study Ruud G. NijmanID1,2,3*, Kate HoneyfordID4, Ruth FarrugiaID5, Katy RoseID1,6, Zsolt BognarID7, Danilo BuonsensoID8,9, Liviana Da DaltID10, Tisham DeID2, Ian K. MaconochieID1,3, Niccolo ParriID11, Damian RolandID12,13, Tobias AlfvenID14, Camille AupiaisID15, Michael BarrettID16,17,18, Romain Basmaci19, Dorine BorensztajnID20,21, Susana CastanhinhaID22, Corinne VasilicoID23, Sheena DurninID24, Paddy Fitzpatrick25, Laszlo FodorID26, Borja GomezID27,28, Susanne Greber-PlatzerID29,30, Romain Guedj31, Stuart HartshornID32,33, Florian HeyID34, Lina JankauskaiteID35, Daniela KohlfuerstID36, Mojca KolnikID37, Mark D. LyttleID38,39, Patrı´cia Mac¸ão40, Maria Inês Mascarenhas41, Shrouk MessahelID42, Esra Akyu¨z O¨ zkan43, Zanda PučukaID44, Sofia Reis45, Alexis RybakID46,47,48, Malin Ryd RinderID49, Ozlem Teksam50, Caner TuranID51, Valty´r Stefa´nsson ThorsID52, Roberto VelascoID53, Silvia Bressan10, Henriette A. MollID20, Rianne OostenbrinkID20, Luigi TitomanlioID46,47,48, in association with the REPEM network (Research in European Pediatric Emergency Medicine) as part of the EPISODES study group¶ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Gemelli IRCCS, Rome, Italy, 9 Università Cattolica del Sacro Cuore, Rome, Italy, 10 Division of Pediatric Emergency Medicine, Department of Women’s and Children’s Health, University Hospital of Padova, Padova, Italy, 11 Emergency Department & Trauma Center, Ospedale Pediatrico Meyer Firenze, Florence, Italy, 12 SAPPHIRE Group, Health Sciences, Leicester University, Leicester, United Kingdom, 13 Pediatric Emergency Medicine Leicester Academic (PEMLA) Group, Leicester Hospitals, Leicester, United Kingdom, 14 Pediatric emergency department, Sachs’ Children and Youth Hospital, Stockholm, Sweden, 15 Pediatric Emergency Department, Jean Verdier Hospital, Bondy, France, 16 Pediatric Emergency Department, Children’s Health Ireland at Crumlin, Dublin, Ireland, 17 Women’s and Children’s Health, School of Medicine, University College Dublin, Dublin, Ireland, 18 National Children’s Research Centre, Crumlin, Dublin, Ireland, 19 Pediatric Emergency Department, Louis Mourier Hospital, Colombes, France, 20 Department of General Pediatrics, Erasmus MC–Sophia, Rotterdam, the Netherlands, 21 Emergency Department, Medisch Centrum Alkmaar, Noordwest Ziekenhuisgroep, Alkmaar, the Netherlands, 22 Hospital Dona Estefania, Centro Hospitalar de Lisboa Central, Lisbon, Portugal, 23 Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria, 24 Pediatric Emergency Department, Children’s Health Ireland at Tallaght, Dublin, Ireland, 25 Pediatric Emergency Department, Children’s Health Ireland at Temple Street, Dublin, Ireland, 26 Pediatric Emergency Department, Szent Gyorgy University Teaching Hospital of Fejer County, Szekesfehervar, Hungary, 27 Pediatric emergency department, Cruces University Hospital, Barakaldo, Spain, 28 Biocruces Bizkaia Health Research Institute, Cruces University Hospital, Barakaldo, Spain, 29 Pediatric Emergency Outpatient Clinic, Clinical Division of Pediatric Pulmonology, Allergology and Endocrinology, Department of Pediatrics and Adolescent Medicine, Medical University Vienna, Vienna, Austria, 30 Clinical Division of Pediatric Pulmonology, Allergology and Endocrinology, Department of Pediatrics and Adolescent Medicine, Comprehensive Centre for Pediatrics, Medical University of Vienna, Vienna, Austria, 31 Pediatric Emergency Department, Armand Trousseau Hospital, Paris, France, 32 Pediatric emergency department, Birmingham women’s and children’s NHS Foundation Trust, Birmingham, United Kingdom, 33 Birmingham Clinical Trials Unit, Institute of Applied Health Research, Background During the initial phase of the Coronavirus Disease 2019 (COVID-19) pandemic, reduced numbers of acutely ill or injured children presented to emergency departments (EDs). Con- cerns were raised about the potential for delayed and more severe presentations and an increase in diagnoses such as diabetic ketoacidosis and mental health issues. This multina- tional observational study aimed to study the number of children presenting to EDs across Europe during the early COVID-19 pandemic and factors influencing this and to investigate changes in severity of illness and diagnoses. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Abbreviations: CI, confidence interval; COVID-19, Coronavirus Disease 2019; ECDC, European Centre for Disease Prevention and Control; ED, emergency department; EPISODES, Epidemiology, severity and outcomes of children presenting to emergency departments across Europe during the SARS-CoV- 2 pandemic; IRR, incidence rate ratio; LRTI, lower respiratory tract infection; MIS-C, Multi Inflammatory Syndrome in Children; PERUKI, Pediatric Emergency Research in the United Kingdom and Ireland; PICU, pediatric intensive care unit; REPEM, Research in European Pediatric Medicine; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. ¶ Membership of the EPISODES study group is provided in S1 Appendix * r.nijman@imperial.ac.uk ¶ Membership of the EPISODES study group is provided in S1 Appendix * r.nijman@imperial.ac.uk PLOS MEDICINE PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic University of Birmingham, Birmingham, United Kingdom, 34 Pediatric emergency department and pediatric intensive care unit, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians-University Munich, Munich, Germany, 35 Hospital of Lithuanian University of Health Sciences Kauno Klinikos, Lithuania, 36 Department of General Pediatrics, Medical University of Graz, Graz, Austria, 37 University Medical Centre Ljubljana, Univerzitetni Klinični Center, Department of Infectious Diseases, Ljubljana, Slovenia, 38 Emergency Department, Bristol Royal Hospital for Children, Bristol, United Kingdom, 39 Faculty of Health and Applied Sciences, University of the West of England, Bristol, United Kingdom, 40 Pediatric Emergency Service, Hospital Pedia´trico, Centro Hospitalar e Universita´rio de Coimbra, Coimbra, Portugal, 41 Departamento da Crianc¸a e do Jovem- Urgencia Pediatrica, Hospital Prof. Doutor Fernando da Fonseca, Amadora, Portugal, 42 Pediatric emergency department, Alder Hey Children’s NHS Foundation Trust, Liverpool, United Kingdom, 43 Pediatric Emergency Department, Ondokuz Mayıs University, Samsun, Turkey, 44 Pediatric emergency department, Children’s Clinical University Hospital, Riga Stradins University, Riga, Latvia, 45 Pediatric Department, Centro Hospitalar Tondela-Viseu, Viseu, Portugal, 46 Pediatric Emergency Department, Hopital Universitaire Robert-Debre, Paris, France, 47 ACTIV, Association Clinique et The´rapeutique Infantile du Val-de-Marne, Cre´teil, France, 48 INSERM, ECEVE, Universite´ de Paris, Paris, France, 49 Pediatric emergency department, Astrid Lindgrens Children’s hospital, Karolinska University, Solna, Sweden, 50 Division of Pediatric Emergency Medicine, Department of Pediatrics, Hacettepe University School of Medicine, Ankara, Turkey, 51 Department of Pediatrics, Division of Emergency Medicine, Mersin City Training and Research Hospital, Toroslar, Mersin, Turkey, 52 Children´s Hospital, Barnaspitali Hringsins, Reykjavik, Iceland, 53 Pediatric emergency unit, Hospital Universitario Rı´o Hortega, Valladolid, Spain Funding: RGN was supported by National Institute of Health Research, award number ACL-2018-021- 007. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Academic Editor: Zulfiqar A. Bhutta, The Hospital for Sick Children, CANADA Academic Editor: Zulfiqar A. Bhutta, The Hospital for Sick Children, CANADA Received: March 18, 2022 Accepted: July 28, 2022 Published: August 26, 2022 Academic Editor: Zulfiqar A. Bhutta, The Hospital for Sick Children, CANADA Received: March 18, 2022 Accepted: July 28, 2022 Published: August 26, 2022 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pmed.1003974 Copyright: © 2022 Nijman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All study data are available from via https://doi.org/10.14469/hpc/ 10685. 1 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Conclusions Reductions in ED attendances were seen across Europe during the first COVID-19 lock- down period. More severely ill children continued to attend hospital more frequently com- pared to those with minor injuries and illnesses, although absolute numbers fell. Methods and findings Routine health data were extracted retrospectively from electronic patient records of chil- dren aged 18 years and under, presenting to 38 EDs in 16 European countries for the period January 2018 to May 2020, using predefined and standardized data domains. Observed and predicted numbers of ED attendances were calculated for the period February 2020 to May 2020. Poisson models and incidence rate ratios (IRRs), using predicted counts for each site as offset to adjust for case-mix differences, were used to compare age groups, diagnoses, and outcomes. Reductions in pediatric ED attendances, hospital admissions, and high triage urgencies were seen in all participating sites. ED attendances were relatively higher in countries with lower SARS-CoV-2 prevalence (IRR 2.26, 95% CI 1.90 to 2.70, p < 0.001) and in children aged <12 months (12 to <24 months IRR 0.86, 95% CI 0.84 to 0.89; 2 to <5 years IRR 0.80, 95% CI 0.78 to 0.82; 5 to <12 years IRR 0.68, 95% CI 0.67 to 0.70; 12 to 18 years IRR 0.72, 95% CI 0.70 to 0.74; versus age <12 months as reference group, p < 0.001). The lowering 2 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic of pediatric intensive care admissions was not as great as that of general admissions (IRR 1.30, 95% CI 1.16 to 1.45, p < 0.001). Lower triage urgencies were reduced more than higher triage urgencies (urgent triage IRR 1.10, 95% CI 1.08 to 1.12; emergent and very urgent triage IRR 1.53, 95% CI 1.49 to 1.57; versus nonurgent triage category, p < 0.001). Reductions were highest and sustained throughout the study period for children with com- municable infectious diseases. The main limitation was the retrospective nature of the study, using routine clinical data from a wide range of European hospitals and health systems. Why was this study done? • Reduced numbers of children visiting urgent and emergency care services were reported following the introduction of infection prevention measures during the first phase of the Coronavirus Disease 2019 (COVID-19) pandemic. • Concerns were raised about potential delays in, and higher acuity of, emergency depart- ment (ED) presentations. • Concerns were raised about potential delays in, and higher acuity of, emergency depart- ment (ED) presentations. What did the researchers do and find? • This study compared routine clinical data from children aged 18 years and under pre- senting to EDs of 38 study sites in 16 European countries between January 2018 until May 2020. • This study compared routine clinical data from children aged 18 years and under pre- senting to EDs of 38 study sites in 16 European countries between January 2018 until May 2020. • Reductions in ED attendances were seen for all age groups, with smaller reductions for younger children in some sites. • More severely ill children continued to attend hospital more frequently compared to those with minor injuries and illnesses, although absolute numbers fell. Trial registration ISRCTN91495258 https://www.isrctn.com/ISRCTN91495258. Author summary PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Introduction Healthcare systems across Europe continue to be greatly affected by the Coronavirus Disease 2019 (COVID-19) pandemic. Early in the COVID-19 pandemic, urgent and emergency facili- ties prepared for a potential influx of acutely unwell children and young people [1]. However, evidence emerged that children were less likely to develop symptoms of Severe Acute Respira- tory Syndrome Coronavirus 2 (SARS-CoV-2) infection, when compared with adults [2–6]. Moreover, reduced numbers of unwell or injured children visiting urgent and emergency care Healthcare systems across Europe continue to be greatly affected by the Coronavirus Disease 2019 (COVID-19) pandemic. Early in the COVID-19 pandemic, urgent and emergency facili- ties prepared for a potential influx of acutely unwell children and young people [1]. However, evidence emerged that children were less likely to develop symptoms of Severe Acute Respira- tory Syndrome Coronavirus 2 (SARS-CoV-2) infection, when compared with adults [2–6]. Moreover, reduced numbers of unwell or injured children visiting urgent and emergency care services were reported, and these seemed to be greatest for children with infectious communi- cable diseases [7–11]. Typically, these studies did not compare patterns between countries or in relation to different public health strategies. y y p Moreover, reduced numbers of unwell or injured children visiting urgent and emergency care services were reported, and these seemed to be greatest for children with infectious communi- cable diseases [7–11]. Typically, these studies did not compare patterns between countries or in relation to different public health strategies. At the same time, concerns were raised about potential delays in, and higher acuity of, pre- sentations to appropriate healthcare services, as a result of difficulties accessing these services, changes in healthcare provision preferencing virtual consultations, fear of exposure to SARS- CoV-2 in healthcare facilities, and blanket “Stay at Home” statements [12–14]. In the United Kingdom, this resulted in a statement from the Royal Society of Pediatrics and Child Health to reassure parents and caregivers, urging them to seek appropriate urgent and emergency medi- cal attention when worried about the acute illness or injury of their child [15]. Additionally, mostly anecdotal evidence reported increased numbers of specific childhood diagnoses, such as diabetic ketoacidosis [16] and intussusception [17]. These hypothesized a possible link with acute or prior SARS-CoV-2 infection, yet evidence from large-scale cohorts is lacking. Con- cerns were also raised for the mental health of children resulting from school closures and stay at home orders [18,19]. What do these findings mean? • The findings suggest that the introduction of infection prevention measures can decrease the burden of acute childhood illnesses and injuries. • The findings suggest that the introduction of infection prevention measures can decrease the burden of acute childhood illnesses and injuries. 3 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic • There was no clear association of infection prevention measures with an increase in more severe, possibly delayed, presentations. • There was no clear association of infection prevention measures with an increase in more severe, possibly delayed, presentations. • For this first phase of the COVID-19 pandemic, the relative increase in cases of diabetic ketoacidosis or mental health issues might have contributed to a biased perception about increased occurrence. Introduction In this study, we aimed to compare the number of children presenting to emergency departments (EDs) across Europe during the first phase of the COVID-19 pandemic with the 2 previous years; investigating any change in severity of illness and describing the associations with specific diagnoses potentially related to SARS-CoV-2. Study design, setting, and participants This retrospective, observational study included 38 sites from 16 European countries as part of the “Epidemiology, severity and outcomes of children presenting to emergency departments across Europe during the SARS-CoV-2 pandemic” (EPISODES) study (trial registration num- ber: ISRCTN91495258) (S1 and S2 Files). Sites were selected from the Research in European Pediatric Medicine (REPEM) and the Pediatric Emergency Research in the United Kingdom and Ireland (PERUKI) networks following the earlier work of Bressan and colleagues [1]. Rou- tine clinical data from all children presenting to the ED were extracted from electronic health records for the period January 1, 2018 to May 17, 2020. The upper age limit varied between 4 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic sites at between 16 and 18 years old. This study is reported as per the REporting of studies Conducted using Observational Routinely collected health Data (RECORD) statement (S1 Checklist) and the study protocol is available in the Supporting information (S1 File). Aggregated, standardized data were uploaded using the REDCap online platform. For the period January 1, 2018 and February 1, 2020, data were collected on a monthly basis. For the period February 2, 2020 to May 17, 2020, on a weekly basis. This amounted to a total of 40 time windows (S1 Table). The clinical report form included 10 different data domains: (1) moment of presentation; (2) patient characteristics; (3) mode of arrival and referral pathway; (4) triage urgency; (5) type of presenting problem and vital signs; (6) diagnostics performed in the ED; (7) treatment in the ED; (8) diagnosis; (9) hospital admission; and (10) duration of ED and hospital stay (S3 File); data availability varied between sites (S1 Fig). Triage urgency levels, used to determine the urgency of care in the ED, were categorized in 3 predefined categories, defined as emergent-very urgent (or RED-ORANGE, or level 1 to 2), urgent (or YELLOW, or level 3), and standard-nonurgent (or GREEN-BLUE, or level 4 to 5) to allow uniform coding between sites. For diagnosis coding, ICD-10 codes were issued for guidance (S2 Table), but an internally and temporally consistent coding approach was encour- aged for each of the individual sites, acknowledging different coding systems and strategies in the ED. This was checked by plotting the diagnoses coding in time as percentage of total num- ber of attendances for each site. Study design, setting, and participants To achieve reliable and accurate transformation of local (non- ICD-10) coding systems into the predefined diagnosis categories, training sessions were held and support offered to study sites by the lead investigators. Diagnoses were selected to reflect the broad spectrum of presenting problems to EDs, and their perceived change in incidences during the initial phase of the COVID-19 pandemic, following a consensus methodology among the study steering group. Final selection of diagnoses for analyses, after completion of data quality control process, included (1) common communicable diseases (e.g., tonsillitis, oti- tis media, lower respiratory tract infection (LRTI), gastrointestinal infection); (2) common minor injuries (e.g., minor head injury, radius fracture); (3) mental health issue; (4) diabetic ketoacidosis; and (5) surgical presentations (e.g., appendicitis, volvulus-intussusception-mal- rotation, testicular torsion). Severity was defined based on level 1 to 2 urgency classification at triage, any hospital admissions, pediatric intensive care unit (PICU) admission, or death in ED. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Data analyses The completeness, quality, and internal consistency of data were checked by plotting the abso- lute numbers, as well as percentage of total attendances, for each variable of interest in time for the whole study period 2018 to 2020. In order to quantify changes in attendances, we com- pared observed attendances with predicted numbers of attendances. Predicted numbers of attendances were estimated using monthly data for the 25 months prior to February 3, 2020. As the data had both a trend and seasonal component, we used Holt–Winters exponential smoothing to make short-term monthly forecasts for February, March, April, and May 2020. We adjusted these to weekly estimates of predicted numbers. We plotted predicted ED atten- dances against the introduction of national infection prevention measures [20]. We also calcu- lated 28-day mean numbers for selected diagnoses, PICU and hospital admission, and death in ED for each month from January through April for the years 2018 to 2020. We used a Poisson model, adjusted for time since February 3, 2020, to determine if there were differences between age groups, diagnoses, and disposition for patients. For each model, the outcome was the count of attendances per week from the week beginning February 3, 2020 to the week beginning May 4, 2020, with an offset of the predicted number of attendances in 5 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic each week. An incidence rate ratio (IRR) >1 indicates higher numbers compared with the ref- erence group, whereas an IRR <1 reflects a higher reduction in numbers. For age groups, the analysis was adjusted for site; for diagnoses and disposition, numbers were too small to make forecasts at site level and we therefore aggregated these across the whole sample. For diagnoses, we completed 2 models, one with 8 separate diagnoses and one where these were divided into 3 groups: surgical presentation (i.e., appendicitis), communicable diseases (i.e., tonsillitis, otitis media, LRTI, and gastroenteritis) and “other” (i.e., mental health issue, radius fracture, and minor head trauma). For 3 diagnosis groups, the number of attendances was too low to make sensible forecasts, namely diabetic ketoacidosis, testicular torsion, and the combined group of intussusception, volvulus, and malrotation. Ethics Following initial approval by the UK Health Research Authority, all participating sites obtained approval from their national/local institutional review boards (S3 Table). The need for individual patient informed consent was waived. Data sharing agreements were in place. Data analyses In addition, we determined if there were associa- tions between the change in hospital attendances and the prevalence of SARS-CoV-2 in the country, as per the European Centre for Disease Prevention and Control (ECDC), and the number of COVID-19 measures that were introduced in each hospital in response to the pan- demic as previously detailed by Rose and colleagues [21]. Rose and colleagues performed a sur- vey study describing changes in local and regional healthcare pathways, including the diverting of patient groups to or away from the ED, and service provision. The survey covered a total of 37 possible points of change in provision of care for sites without a short stay unit (20 pertaining to service provision and 17 to patient pathways) and 38 possible points of change for those that did (21 service provision and 17 patient pathways). High-prevalence countries were defined as a cumulative 14-day rate of >80 new cases per 100,000 of the population. For countries with multiple sites, we used an ANOVA to determine if there was evidence that within country differences were greater than between country differ- ences, for total attendances in March and April, adjusted for predicted numbers to account for differences in site sizes. One site (MAL001) was unable to provide information on diagnosis so it was excluded from this section of analysis; 3 sites (SLO001, POR005, and TUR001) did not provide triage data. Two sites were excluded (NL002 and HUN002) from the forecasting anal- yses and Poisson models as they had missing data in the period before the pandemic (2018). One site (IRE003) was excluded from the Poisson models because it closed to pediatric atten- dances in response to the pandemic. One site (TUR003) accounted for 18% of all attendances, and we carried out sensitivity analysis to confirm the changes to our findings when including this site. Analyses were performed using R v4.0.0. Description of sites, infection prevention measures, and SARS-CoV-2 prevalence Sites included in the study varied in terms of size and service provision (S4 Table and S2 Fig). The annual number of ED attendances ranged from 4,961 (NL001, 2019) to 295,787 (TUR003, 2019) (S3 Fig). All but 3 sites were tertiary academic hospitals with specialized pediatric EDs; the remaining 3 sites were general teaching hospitals, two of which had dedicated pediatric sections and staff, and one of which had a mixed ED. Sites in Austria, Slovenia, and the Neth- erlands mainly saw medical presentations, whereas the other sites saw both medical and surgi- cal/trauma presentations. Timing and degree of infection prevention measures were similar 6 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Fig 1. Timelines of first phase of COVID-19 pandemic in participating countries. Timelines of the introduction of national infection prevention measures (“Response measures”), as well as dates for the first and first 100 cases of SARS-CoV-2 for each of the countries participating in the EPISODES study. The black circle depicts the date of the highest 14-day cumulative rate of new SARS-CoV-2 cases per 100,000, with the size reflecting the actual case rate. COVID-19, Coronavirus Disease 2019; EPISODES, Epidemiology, severity and outcomes of children presenting to emergency departments across Europe during the SARS-CoV-2 pandemic; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi org/10 1371/journal pmed 1003974 g001 Fig 1. Timelines of first phase of COVID-19 pandemic in participating countries. Timelines of the introduction of national infection prevention measures (“Response measures”), as well as dates for the first and first 100 cases of SARS-CoV-2 for each of the countries participating in the EPISODES study. The black circle depicts the date of the highest 14-day cumulative rate of new SARS-CoV-2 cases per 100,000, with the size reflecting the actual case rate. COVID-19, Coronavirus Disease 2019; EPISODES, Epidemiology, severity and outcomes of children presenting to emergency departments across Europe during the SARS-CoV-2 pandemic; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. across European countries (Fig 1 and S5 Table). Notably, Iceland and Sweden did not close day care, nurseries, or primary education; Germany and the UK kept higher education open; Sweden did not close any public spaces; Hungary and Sweden did not advocate use of face masks; Malta, Iceland, and Sweden did not introduce stay-at-home recommendations; and Germany, Hungary, and Iceland did not formally close workspaces. Description of sites, infection prevention measures, and SARS-CoV-2 prevalence Highest national preva- lence of SARS-CoV-2 varied between countries (Fig 1 and S6 Table). PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Changes in total attendances All 38 sites had significant reductions in attendances in spring 2020 (Table 1 and Figs 2 and S4). The largest reduction was seen in AUS001 with observed numbers at 5% (95% CI 5% to 6%) of predicted in the week starting March 30, 2020; the smallest peak reduction in ED atten- dances was at 56% (95% CI 52% to 60%) of predicted in SWE001 during the same week. IRE003 closed for pediatric visits from March 30, 2020 onwards, with most patients diverted to IRE001. Poisson models, adjusted for time since intervention and predicted numbers of attendances, showed that there were significant differences between sites. Observed atten- dances, with respect to predicted, were relatively higher in sites in France, Sweden, Ireland, Iceland, Latvia, and the Netherlands, where observed attendance rates were greater than 50% of predicted. However, there was considerable overlap between all sites when 95% confidence intervals were considered. Results of the Poisson models suggest that attendances in Spring 2020 were higher in EDs in countries with lower SARS-CoV-2 prevalence (IRR 2.26, 95% CI PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 7 / 27 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Table 1. Summary data on the lowest observed number of ED attendances during COVID-19 for each participating center. Changes in total attendances Site Week of first public health measure Week of highest reduction Observed number of ED attendances Total % of predicted numbers (95% CI) Overall reduction$ Date of highest 14-day cumulative rate of new cases per 100,000 Highest cumulative 14-day rate of new cases per 100,000 Total changes to health system Total changes as % of possible changes Austria 09-03-2020 75% 02-04-2020 102.33 AUS001 13-04-2020 16 5% (5% - 6%) 85% 5 13.5% AUS003 30-03-2020 119 17% (15% - 18%) 72% 1 2.7% AUS004 30-03-2020 114 21% (19% - 23%) 71% 0 0.0% France 24-02-2020 18% 11-04-2020 86.12 FR001 30-03-2020 470 41% (35% - 48%) 28% 2 5.3% FR002 30-03-2020 154 52% (40% - 77%) -6% 1 2.6% FR003 30-03-2020 285 42% (36% - 49%) 28% 0 0.0% FR004 13-04-2020 115 34% (27% - 48%) 23% 0 0.0% Germany 02-03-2020 09-04-2020 86.36 GER001 30-03-2020 165 36% (33% - 39%) 53% 0 0.0% Hungary 09-03-2020 23-04-2020 13.34 HUN001 23-03-2020 279 35% (33% - 37%) 52% 4 10.5% Iceland 16-03-2020 03-04-2020 277.04 ICE001 06-04-2020 132 49% (44% - 55%) 40% 3 7.9% Ireland 09-03-2020 56% 23-04-2020 213.02 IRE001 23-03-2020 404 50% (46% - 55%) 27% 3 7.9% IRE002 30-03-2020 333 32% (28% - 36%) 55% 4 10.5% IRE003 30-03-2020 0 0%+ 86% 13 34.2% Italy 02-03-2020 70% 02-04-2020 124.03 IT001 23-03-2020 105 20% (20% - 21%) 67% 1 2.6% IT002 23-03-2020 36 12% (11% - 13%) 73% 8 21.1% Latvia 16-03-2020 06-04-2020 20.52 LAT001 30-03-2020 491 38% (37% - 39%) 53% 0 0.0% Lithuania 09-03-2020 04-04-2020 25.12 LIT001 30-03-2020 164 26% (24% - 27%) 62% 9 23.7% Malta 09-03-2020 12-04-2020 46.20 MAL001 30-03-2020 68 13% (12% - 15%) 80% 1 2.7% The Netherlands 09-03-2020 19-04-2020 86.57 NL001 16-03-2020 38 36% (33% - 38%) 44% 1 2.7% Portugal 09-03-2020 71% 11-04-2020 109.02 POR001 13-04-2020 305 25% (23% - 28%) 70% 1 2.6% POR003 30-03-2020 365 22% (20% - 24%) 70% 2 5.3% POR004 30-03-2020 132 13% (12% - 15%) 74% 6 15.8% POR005 20-04-2020 117 21% (18% - 23%) 69% 1 2.6% Slovenia 02-03-2020 05-04-2020 28.55 SLO001 30-03-2020 12 8% (7% - 8%) 74% 3 7.9% Spain 09-03-2020 70% 05-04-2020 217.56 SP001 23-03-2020 256 26% (24% - 28%) 62% 1 2.6% SP002 30-03-2020 51 11% (10% - 11%) 77% 0 0.0% Sweden 09-03-2020 36% 01-05-2020 83.60 SWE001 30-03-2020 661 56% (52% - 60%)^ 31% 0 0.0% SWE002 06-04-2020 253 48% (44% - 52%) 40% 0 0.0% Turkey 09-03-2020 68% 22-04-2020 74.97 (Continued) ary data on the lowest observed number of ED attendances during COVID-19 for each participating center. Changes in total attendances ED attendances across all age groups significantly reduced (S5 and S6 Figs). Attendances in children aged above 12 months were reduced more than children below 12 months (12 to <24 months IRR 0.86, 95% CI 0.84 to 0.89; 2 to <5 years IRR 0.80, 95% CI 0.78 to 0.82; 5 to <12 years IRR 0.68, 95% CI 0.67 to 0.70; 12 to 18 years IRR 0.72, 95% CI 0.70 to 0.74; versus age <12 months as reference group, all P < 0.001) (Table 2). There was insufficient evidence to conclude that this pattern continued with increasing age for children aged 12 months and older. Patterns between sites within the same country appeared similar (S7 Fig) with strong evidence that between country differences were greater than within coun- try differences (F value: 6.453; p: 0.002). Changes in total attendances PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 8 / 27 paediatric emergency medicine in Europe and the COVID-19 pandemic PLOS MEDICINE Table 1. (Continued) Site Week of first public health measure Week of highest reduction Observed number of ED attendances Total % of predicted numbers (95% CI) Overall reduction$ Date of highest 14-day cumulative rate of new cases per 100,000 Highest cumulative 14-day rate of new cases per 100,000 Total changes to health system Total changes as % of possible changes TUR001 27-04-2020 98 33% (29% - 38%) 59% 3 7.9% TUR002 06-04-2020 233 15% (14% - 17%) 72% 6 15.8% TUR003 13-04-2020 882 14% (13% - 15%) 75% 5 13.5% United Kingdom 16-03-2020 63% 01-05-2020 99.25 UK001 30-03-2020 387 30% (28% - 32%) 63% 3 7.9% UK002 30-03-2020 72 18% (17% - 21%) 71% 3 7.9% UK004 23-03-2020 487 36% (34% - 39%) 55% 2 5.3% UK005 13-04-2020 76 14% (12% - 17%) 71% 1 2.6% UK006 30-03-2020 401 33% (31% - 35%) 54% 2 5.3% The starting date of the week where the observed numbers of ED attendances had the largest difference from predicted numbers expressed in % with 95% confidence intervals. With  AUS001 having the largest reduction from predicted numbers and ^ SWE001 the least change from predicted numbers. In addition, highest national SARS-CoV-2 infection rates are given for each of the study sites, as reported by the ECDC [20],with a threshold of 80 cases per 100,000 to indicate low (green) and high (orange) prevalence. Total changes to health system as per Rose and colleagues [21].  AUS001 having the largest reduction from predicted numbers and ^ SWE001 the least change from predicted numbers. In addition, highest national SARS-CoV-2 infection rates are given for each of the study sites, as reported by the ECDC [20],with a threshold of 80 cases per 100,000 to indicate low (green) and high (orange) prevalence. Total changes to health system as per Rose and colleagues [21]. $Overall reduction calculated from introduction of first national public health measure until end of study period. If multiple sites per country, overall reduction for the country was estimated using the average of the overall reductions of each individual site. +IRE003 was closed for pediatric attendances from March 30, 2020 onwards. COVID-19, Coronavirus Disease 2019; ECDC, European Centre for Disease Prevention and Control; ED, emergency department; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. Changes in total attendances https://doi.org/10.1371/journal.pmed.1003974.t001 1.90 to 2.70, P < 0.001) (Table 2). We found a relationship between the number of introduced organizational COVID-19 measures and ED attendances and more organizational COVID-19 measures were associated with lower numbers of ED attendances when adjusted for predicted ED attendances (IRR 0.13, 95% CI 0.11 to 0.16, when sites with 4 or more measures were com- pared to sites with no measures, P < 0.001). Similarly, larger reductions in ED attendances were seen in mixed adult and pediatric academic hospitals (versus standalone children’s hospi- tal, IRR 3.49, 95% CI 2.89 to 4.24, P < 0.001; general nonuniversity hospital, IRR 2.73, 95% CI 2.28 to 3.30, P < 0.001) and urban hospitals (versus mixed urban and rural hospitals, IRR 5.33, 95% CI 4.44 to 6.46, P < 0.001). ED attendances across all age groups significantly reduced (S5 and S6 Figs). Attendances in children aged above 12 months were reduced more than children below 12 months (12 to <24 months IRR 0.86, 95% CI 0.84 to 0.89; 2 to <5 years IRR 0.80, 95% CI 0.78 to 0.82; 5 to <12 years IRR 0.68, 95% CI 0.67 to 0.70; 12 to 18 years IRR 0.72, 95% CI 0.70 to 0.74; versus age <12 months as reference group, all P < 0.001) (Table 2). There was insufficient evidence to conclude that this pattern continued with increasing age for children aged 12 months and older. Patterns between sites within the same country appeared similar (S7 Fig) with strong evidence that between country differences were greater than within coun- try differences (F value: 6.453; p: 0.002). 1.90 to 2.70, P < 0.001) (Table 2). We found a relationship between the number of introduced organizational COVID-19 measures and ED attendances and more organizational COVID-19 measures were associated with lower numbers of ED attendances when adjusted for predicted ED attendances (IRR 0.13, 95% CI 0.11 to 0.16, when sites with 4 or more measures were com- pared to sites with no measures, P < 0.001). Similarly, larger reductions in ED attendances were seen in mixed adult and pediatric academic hospitals (versus standalone children’s hospi- tal, IRR 3.49, 95% CI 2.89 to 4.24, P < 0.001; general nonuniversity hospital, IRR 2.73, 95% CI 2.28 to 3.30, P < 0.001) and urban hospitals (versus mixed urban and rural hospitals, IRR 5.33, 95% CI 4.44 to 6.46, P < 0.001). Triage urgency Overall, there was a higher reduction (observed compared to predicted) in children with lower triage urgency when compared to children with higher triage classification (urgent triage, IRR 9 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Fig 2. Observed versus predicted ED attendances (%). The observed versus predicted number of children presenting to EDs in countries across Europe in the weeks following February 2, 2020 until May 11, 2020, for all sites combined. The color and the size of the dots reflect the actual number of ED attendances for each site and for each time window. The line connects the mean of the observed vs. predicted point estimates for each of the individual sites for each time window. Fig 2. Observed versus predicted ED attendances (%). The observed versus predicted number of children presenting to EDs in countries across Europe in the weeks following February 2, 2020 until May 11, 2020, for all sites combined. The color and the size of the dots reflect the actual number of ED attendances for each site and for each time window. The line connects the mean of the observed vs. predicted point estimates for each of the individual sites for each time window https://doi.org/10.1371/journal.pmed.1003974.g002 https://doi.org/10.1371/journal.pmed.1003974.g002 1.10, 95% CI 1.08 to 1.12, P < 0.001; emergent and very urgent triage IRR 1.53, 95% CI 1.49 to 1.57, P < 0.001; versus nonurgent triage category), even though clear reductions were seen for all triage categories (S8 Fig). Hospital and PICU admissions Hospital and PICU admissions were fewer than predicted (Figs 3, 4 and S9). We did not observe an increase in the number of deaths in ED (IRR 1.75, 95% CI 0.88 to 3.07, p = 0.08). The change in PICU admissions (IRR 1.30, 95% CI 1.16 to 1.45, versus general admissions) was not as great as the change in general admissions. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Diagnoses The 28-day mean numbers for common communicable diseases decreased in absolute and rel- ative frequencies (Table 3 and Fig 5A), in particular for tonsillitis, otitis media, gastrointestinal infections, and LRTIs. Decreases were also seen in common childhood injuries such as minor head injuries and radius fractures (Fig 5B). No increase in absolute numbers were seen for sev- eral uncommon diagnoses suggested to be linked with SARS-CoV-2 infection, such as diabetic ketoacidosis (Fig 5C), intussusception, and testicular torsion (Fig 5B), even when stratified for high-SARS-CoV-2 prevalence countries (S10 Fig). Mental health attendances declined during the first phase of the COVID-19 pandemic in absolute terms, but this corresponded with an increase in relative frequency (Fig 5C). Fig 6, reflecting the observed versus predicted numbers for the 8 selected diagnoses, shows that the change of children and young people with PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 10 / 27 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Table 2. Poisson regression models for ED attendances. https://doi.org/10.1371/journal.pmed.1003974.t002 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Diagnoses IRR (95% Confidence Interval) p-value Number of COVID-19 measures in hospital$ No measures–reference group 1 measure 0.426 (0.391 - 0.463) <0.001 2 measures 0.760 (0.717 - 0.806) <0.001 3 measures 0.454 (0.413 - 0.499) <0.001 4+ measures 0.130 (0.108 - 0.155) <0.001 SARS-CoV-2 prevalence^ Low prevalence 2.256 (1.904 - 2.700) <0.001 Type of hospital Mixed tertiary hospital–reference group Standalone tertiary children’s hospital 3.490 (2.894 - 4.241) <0.001 General nonuniversity teaching hospital 2.733 (2.280 - 3.303) <0.001 Urban–reference group Urban and rural mixed 5.333 (4.439 - 6.460) <0.001 Age group 0-<12 months–reference group 12-<24 months 0.862 (0.838 - 0.886) <0.001 2-<5 years 0.799 (0.780 - 0.819) <0.001 5-<12 years 0.682 (0.666 - 0.698) <0.001 12–18 years 0.719 (0.698 - 0.739) <0.001 Triage urgency classification Nonurgent and standard triage categories–reference group Urgent 1.096 (1.076 - 1.116) <0.001 Emergent and very urgent 1.530 (1.488 - 1.573) <0.001 Diagnosis I Appendicitis–reference group Gastro-intestinal infections 0.279 (0.253 - 0.308) <0.001 Minor head injury 0.783 (0.709 - 0.866) <0.001 LRTI 0.357 (0.323 - 0.396) <0.001 Mental health issues 0.688 (0.609 - 0.777) <0.001 Otitis media 0.231 (0.206 - 0.260) <0.001 Radius fracture 0.732 (0.654 - 0.819) <0.001 Tonsillitis 0.189 (0.172 - 0.208) <0.001 Diagnosis II Surgical presentation–appendicitis–reference group Communicable diseases 0.238 (0.253 - 0.308) <0.001 Other 0.754 (0.709 - 0.866) <0.001 Outcome Admission–reference group Death 1.749 (0.876 - 3.069) 0.077 PICU Admission 1.295 (1.157 - 1.445) <0.001 To derive IRRs, the predicted counts for each individual site were used as an offset in the Poisson model to account f i diff b t it Table 2. Poisson regression models for ED attendances. 11 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Fig 3. Observed versus predicted hospital admissions for patients attending the ED (%). The observed versus predicted number of children admitted to hospital from the ED in countries across Europe in the weeks following February 2, 2020 until May 11, 2020, for all sites combined. The color and the size of the dots reflect the actual number of ED attendances for each site and for each time window. The line connects the mean of the observed vs. predicted point estimates for each of the individual sites for each time window. h //d i /10 1371/j l d 1003974 003 Fig 3. Observed versus predicted hospital admissions for patients attending the ED (%). Sensitivity analyses The sensitivity analyses for the Poisson modelling without TUR003 resulted in IRRs slightly nearer to one, meaning all associations were slightly weaker (S7 Table). The change of the coef- ficient for tonsillitis was notable, increasing the IRR from 0.19 (95% CI 0.17 to 0.21) to 0.37 (95% CI 0.34 to 0.41). There was also a change between PICU admissions (IRR 1.13, 95% CI 1.00 to 1.28, p = 0.045) and admissions in general. Though the comparison remained statisti- cally significant, the association was weaker. Diagnoses The observed versus predicted number of children admitted to hospital from the ED in countries across Europe in the weeks following February 2, 2020 until May 11, 2020, for all sites combined. The color and the size of the dots reflect the actual number of ED attendances for each site and for each time window. The line connects the mean of the observed vs. predicted point estimates for each of the individual sites for each time window. https://doi.org/10.1371/journal.pmed.1003974.g003 appendicitis was less than for the other diagnoses groups. Mental health issues, radius frac- tures, and minor head injuries were all affected, but there was evidence that attendances increased from the end of March. In contrast, attendances for LRTI, otitis media, gastrointesti- nal infections, and tonsillitis remained low. Poisson models showed no significant difference between mental health, minor head trauma, and radius fracture. There was evidence of signifi- cant difference between infections and trauma and mental health, with bigger reductions in infections. When communicable diseases were combined, there was a clear difference between surgical presentation (appendicitis), communicable diseases, and “other” (mental health, radius fracture, and head trauma) (Table 2). Discussion Reductions in the numbers of children attending EDs were consistently seen across Europe during the first phase of the COVID-19 pandemic. There was variation between countries, but 12 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Fig 4. Hospital admissions, intensive care admissions, and deaths in the ED for the period January–April over a 3-year period. Percentages of total ED attendances (left) and absolute numbers (right) of children admitted to hospital (top), PICUs (middle), or died in the ED (bottom); comparing the 28-day mean numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; PICU, pediatric intensive care unit. https://doi.org/10.1371/journal.pmed.1003974.g004 Fig 4. Hospital admissions, intensive care admissions, and deaths in the ED for the period January–April over a 3-year period. Percentages of total ED attendances (left) and absolute numbers (right) of children admitted to hospital (top), PICUs (middle), or died in the ED (bottom); comparing the 28-day mean numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; PICU, pediatric intensive care unit. https://doi org/10 1371/journal pmed 1003974 g004 https://doi.org/10.1371/journal.pmed.1003974.g004 within countries patterns were similar. The levels to which ED attendances decreased appeared to be related to the introduction and extend of infection prevention measures, changes made to local health systems, type of hospital, and national SARS-CoV-2 prevalence. Attendances were relatively higher in some sites with fewer or less strict national infection prevention mea- sures (e.g., Sweden, Iceland), but this was not true for others (e.g., France). ED attendances were seen for all age groups, with smaller reductions in children aged below 1 year. The reduc- tion in numbers was largest and sustained for communicable diseases, whereas other groups of diagnoses trended towards normal levels of ED attendances by the end of the study period after initial reduced ED attendance rates. Our findings of reduced pediatric ED attendances are consistent with other studies from around the world [7–11,22–24]. The observed reduction in ED attendances will likely be mul- tifactorial, including changed parental health-seeking behavior, modified and newly intro- duced healthcare pathways, and fewer circulating and reduced transmission of infectious pathogens. For example, children with asthma often frequent EDs, but they had fewer exacer- bations needing ED visits during the first phase of the COVID-19 pandemic. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Discussion Proposed reasons include reduced air pollution, reduced social mixing with exposures to viral trigger, and improved compliance with medication at home [25,26]. All this appeared to have affected pre- sentations of children and young people with a low triage urgency and with minor injuries and illnesses most. Earlier studies suggested that infection prevention measures may have resulted in delayed presentations to hospitals [12,13,27–29]. In our study, children with more severe conditions, as measured by triage urgency, need for hospital admission, and PICU and death, continued 13 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 paediatric emergency medicine in Europe and the COVID-19 pandemic PLOS MEDICINE Table 3. Totals of selected clinical diagnoses in the ED for the period January–April over a 3-year period. Discussion Absolute numbers (and % of total children seen in the ED) of children with diagnosis of (a) tonsillitis; (b) otitis media; (c) LRTIs; (d) gastrointestinal infections; (e) appendicitis; (f) testicular torsion; (g) intussusception, volvulus, and malrotation; (h) mental health issues; (i) diabetic ketoacidosis; (j) radius fracture; and (k) minor head injury; comparing the 28-day mean numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; LRTI, lower respiratory tract infection. to attend hospital more frequently compared to those with minor injuries and illnesses, although overall absolute numbers fell. This was in line with other studies reporting similar reductions in children with high triage urgency or need for hospital admission [7,30–38]. Defining the harm of delayed presentations, as well as establishing what contributed to a possible delay, can be difficult [39]. In an attempt to distinguish the delay in seeking care from harm sustained, Roland and colleagues concluded that only a minority (6 out of 51 (11.8%)) of children with a potential delay in presentation were admitted to 1 of 7 hospitals [40]. Contra- dictory conclusions have been reported for the delay in presentations and for potential harm sustained for diagnoses of appendicitis [41,42] and testicular torsion [43–46], portraying a pic- ture that organizing regional healthcare delivery is important to ensure continued access to pediatric urgent and emergency care during a pandemic. In addition, our data showed that, despite overall falling ED attendances, presentations requiring surgical interventions remained stable, reiterating that access to surgical teams and the ability to perform emergent surgical procedures are crucial. Evidence is mounting that SARS-CoV-2 is directly involved in the pathogenesis of new onset diabetes [47,48]. Unsworth and colleagues first reported an increase of new onset type 1 diabetes in children and a possible link with SARS-CoV-2 in the UK [16]. Additional cohort studies found divergent associations between SARS-CoV-2, new onset diabetes, and decom- pensation of preexisting diabetes [49–52]. Our data did not identify increased incidence of https://doi.org/10.1371/journal.pmed.1003974.t003 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 14 / 27 to attend hospital more frequently compared to those with minor injuries and illnesses, although overall absolute numbers fell. This was in line with other studies reporting similar reductions in children with high triage urgency or need for hospital admission [7,30–38]. Defining the harm of delayed presentations, as well as establishing what contributed to a possible delay, can be difficult [39]. Discussion 2018 2019 2020 January February March April January February March April January February March April Tonsillitis 17,627 (12.9%) 16,781 (12.9%) 15,249 (12.4%) 14,722 (12.0%) 24,939 (16.8%) 16,231 (12.1%) 15,898 (12.3%) 18,751 (14.6%) 26,118 (18.4%) 17,668 (14.0%) 10,639 (13.5%) 2,382 (6.3%) Otitis media 4,917 (3.6%) 4,814 (3.7%) 4,063 (3.3%) 3,639 (3.0%) 4,843 (3.3%) 4,370 (3.2%) 3,900 (3.0%) 3,719 (2.9%) 4,115 (2.9%) 4,452 (3.5%) 2,156 (2.7%) 407 (1.1%) LRTIs 9,129 (6.7%) 8,981 (6.9%) 7,197 (5.8%) 5,407 (4.4%) 10,684 (7.2%) 10,294 (7.7%) 6,519 (5.1%) 5,415 (4.2%) 9,374 (6.6%) 9,028 (7.2%) 5,667 (7.2%) 1,059 (2.8%) Gastrointestinal infections 7,809 (5.7%) 8,488 (6.5%) 8,946 (7.3%) 9,103 (7.4%) 10,313 (6.9%) 9,949 (7.4%) 11,345 (8.8%) 11,066 (8.6%) 9,158 (6.4%) 8,726 (6.9%) 4,719 (6.0%) 1,905 (5.1%) Appendicitis 399 (0.3%) 412 (0.3%) 434 (0.4%) 424 (0.3%) 455 (0.3%) 437 (0.3%) 505 (0.4%) 443 (0.3%) 431 (0.3%) 435 (0.3%) 332 (0.4%) 307 (0.8%) Testicular torsion 119 (0.1%) 118 (0.1%) 124 (0.1%) 117 (0.1%) 143 (0.1%) 111 (0.1%) 147 (0.1%) 136 (0.1%) 125 (0.1%) 125 (0.1%) 99 (0.1%) 103 (0.3%) Intussusception, volvulus, and malrotation 33 (0.0%) 37 (0.0%) 56 (0.0%) 58 (0.0%) 40 (0.0%) 58 (0.0%) 63 (0.0%) 60 (0.0%) 55 (0.0%) 44 (0.0%) 53 (0.1%) 30 (0.1%) Mental health conditions 705 (0.5%) 652 (0.5%) 718 (0.6%) 736 (0.6%) 811 (0.5%) 821 (0.6%) 853 (0.7%) 757 (0.6%) 791 (0.6%) 802 (0.6%) 603 (0.8%) 388 (1.0%) Diabetic ketoacidosis 61 (0.0%) 60 (0.0%) 56 (0.0%) 64 (0.1%) 68 (0.0%) 54 (0.0%) 61 (0.0%) 62 (0.0%) 62 (0.0%) 64 (0.1%) 55 (0.1%) 50 (0.1%) Radius fracture 783 (0.6%) 891 (0.7%) 853 (0.7%) 1,311 (1.1%) 886 (0.6%) 1,036 (0.8%) 1,153 (0.9%) 1,362 (1.1%) 809 (0.6%) 935 (0.7%) 730 (0.9%) 592 (1.6%) Minor head injury 2,709 (2.0%) 2,976 (2.3%) 2,985 (2.4%) 3,392 (2.8%) 2,682 (1.8%) 2,730 (2.0%) 3,043 (2.4%) 3,169 (2.5%) 2,372 (1.7%) 2,415 (1.9%) 1,728 (2.2%) 1,472 (3.9%) Absolute numbers (and % of total children seen in the ED) of children with diagnosis of (a) tonsillitis; (b) otitis media; (c) LRTIs; (d) gastrointestinal infections; (e) appendicitis; (f) testicular torsion; (g) intussusception, volvulus, and malrotation; (h) mental health issues; (i) diabetic ketoacidosis; (j) radius fracture; and (k) minor head injury; comparing the 28-day mean numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; LRTI, lower respiratory tract infection. tals of selected clinical diagnoses in the ED for the period January–April over a 3-year period. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 https://doi.org/10.1371/journal.pmed.1003974.t003 Absolute numbers (and % of total children seen in the ED) of children with diagnosis of (a) tonsillitis; (b) otitis media; (c) LRTIs; (d) gastrointestinal infections; (e) appendicitis; (f) testicular torsion; (g) intussusception, volvulus, and malrotation; (h) mental health issues; (i) diabetic ketoacidosis; (j) radius fracture; and (k) minor head injury; comparing the 28-day mean numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; LRTI, lower respiratory tract infection. Discussion In an attempt to distinguish the delay in seeking care from harm sustained, Roland and colleagues concluded that only a minority (6 out of 51 (11.8%)) of children with a potential delay in presentation were admitted to 1 of 7 hospitals [40]. Contra- dictory conclusions have been reported for the delay in presentations and for potential harm sustained for diagnoses of appendicitis [41,42] and testicular torsion [43–46], portraying a pic- ture that organizing regional healthcare delivery is important to ensure continued access to pediatric urgent and emergency care during a pandemic. In addition, our data showed that, despite overall falling ED attendances, presentations requiring surgical interventions remained stable, reiterating that access to surgical teams and the ability to perform emergent surgical procedures are crucial. Evidence is mounting that SARS-CoV-2 is directly involved in the pathogenesis of new onset diabetes [47,48]. Unsworth and colleagues first reported an increase of new onset type 1 diabetes in children and a possible link with SARS-CoV-2 in the UK [16]. Additional cohort studies found divergent associations between SARS-CoV-2, new onset diabetes, and decom- pensation of preexisting diabetes [49–52]. Our data did not identify increased incidence of 14 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic urnal.pmed.1003974 August 26, 2022 1 rnal.pmed.1003974 August 26, 2022 15 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 15 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 15 / 27 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Fig 5. Selected clinical diagnoses in the ED for the period January–April over a 3-year period. Percentages of total ED attendances (left) and absolute numbers (right) of children with diagnosis of (A) common communicable diseases (tonsillitis, otitis media, LRTI, GI infections), (B) minor injuries and surgical presentations (radius fracture, minor head injury, appendicitis, intussusception, volvulus and malrotation (combined group), testicular torsion,), and (C) mental health issues and diabetic ketoacidosis; comparing the 28-day mean numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; GI, gastrointestinal; LRTI, lower respiratory tract infection. https://doi.org/10.1371/journal.pmed.1003974.g005 https://doi.org/10.1371/journal.pmed.1003974.g005 https://doi.org/10.1371/journal.pmed.1003974.g005 diabetic ketoacidosis during the first phase of the COVID-19 pandemic. It might well be that clusters of new onset diabetes can be found in high-prevalence areas and that we failed to cap- ture this in our study. Discussion Likewise, if SARS-CoV-2 acts as a precipitator, there might be a delay in the manifestation of new onset diabetes, and with reduced prevalence of typical viral triggers, this increase might only become apparent later in the pandemic [53]. We were not able to dif- ferentiate between new onset diabetes and decompensation of preexisting diabetes. We found a reduction of children with mental health conditions presenting to the EDs dur- ing the first phase of the COVID-19 pandemic in Europe, similar to findings from studies else- where [9,54–57]. This is unlikely to reflect the considerable mental health issues encountered in the wider pediatric and adolescent populations [58] and of the experiences later in the pan- demic, with, among others, reported increases in eating disorders in children and young peo- ple [59]. Joyce and colleagues observed an overall decrease in mental health issues in ED, albeit an increase in self-harm and deliberate ingestions [60]. Despite the reduction in absolute num- bers, there was an increase in the proportion of attendances attributable to mental health potentially contributing to the heightened awareness for mental health issues in the first COVID-19 wave. Fig 6. Observed versus predicted number of selected diagnoses (%). The observed versus predicted numbers of 8 selected diagnoses for all sites combined, for the period following February 2, 2020 until May 4, 2020. The error bars indicate the 80% prediction intervals. https://doi.org/10.1371/journal.pmed.1003974.g006 Fig 6. Observed versus predicted number of selected diagnoses (%). The observed versus predicted numbers of 8 selected diagnoses for all sites combined, for the period following February 2, 2020 until May 4, 2020. The error bars indicate the 80% prediction intervals. Fig 6. Observed versus predicted number of selected diagnoses (%). The observed versus predicted numbers of 8 selected diagnoses for all sites combined, for the period following February 2, 2020 until May 4, 2020. The error bars indicate the 80% prediction intervals. https://doi.org/10.1371/journal.pmed.1003974.g006 https://doi.org/10.1371/journal.pmed.1003974.g006 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 16 / 27 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Prior to the current COVID-19 pandemic, limited data were available describing the associ- ation of infection prevention measures on urgent and emergency pediatric care in high- income countries. One study found a decrease in respiratory infections of 42% and decreased ED attendances of 28% following school closures for an influenza outbreak in Israel [61]. Discussion Simi- lar patterns were seen during the SARS outbreak in 2003 [62–64] and the MERS outbreak in 2015 [65]. These studies also reported a larger reduction in ED utilization for children than for adults. In contrast, the 2009 H1N1 influenza pandemic generally led to increased ED utiliza- tion, with higher levels of acuity [66–68]. One previous study had reported reduced pediatric ED attendance rates for flu-like illness and respiratory tract infections following school clo- sures [69]. Another study reported increased pediatric ED attendance numbers following media reports on health threats of the H1N1 virus [70]. Altogether, previous evidence of infec- tious disease outbreaks suggests a similar impact on pediatric urgent and emergency care fol- lowing the introduction of public health and infection prevention measures. However, this is to a lesser extent than what was observed with the COVID-19 pandemic and one that is depen- dent on childhood susceptibility for the infectious pathogen. Strengths and limitations Our study presents multinational data enabling the comparison between infection prevention measures, national SARS-CoV-2 prevalence, and the association with acute illness and injuries in children between European countries. Most participating sites were tertiary institutions, with dedicated pediatric emergency medicine teams, with potential implications for the gener- alizability of our findings. At present, no standardized data extraction system for pediatric urgent and emergency care exists between European countries; and the EPISODES study is, to our knowledge, the first to navigate the difficulties of dealing with different data systems, data availability, and varying coding practices. Hence, also limited by the time restrictions caused by the COVID-19 pandemic, some sites were not able to provide data for all domains, and 2 sites (NL002 and HUN002) were only able to provide data for part of the study duration. Limitations of electronic health records to describe patients’ diagnoses are well known [71]. Some of the participating study sites had unique non-ICD-10-based coding systems, and we urged all study teams to be consistent in transforming local data to fit the study clinical report form and we implemented a rigorous data quality process to ensure validity of coding in time. Although most diagnoses linked to SARS-CoV-2 in children were included in the predefined clinical report form, other diagnoses might be of interest in future studies. Of note, coding for children with Multi Inflammatory Syndrome in Children (MIS-C) proved unreliable, with no unique diagnostic codes available for this new disease in automated coding systems. As the data were collected in aggregated form, thereby negating some of the difficulties with data protection regulations, we were not able to stratify for severity of specific diagnosis or age groups. This also introduced risks of overstratification during analyses. We observed large differences between sites for the number of annual ED attendances and the number of patients with high triage urgency and hospital admissions, reflecting both case-mix and diver- sity of patient management. We analyzed data mostly on a site-by-site basis, by using predicted versus observed ratios, and thus dealing with heterogeneity between sites. In addition, although very few study sites restructured local healthcare pathways diverting urgent and emergency care to alternative healthcare facilities, this does not fully rule out changes to access to healthcare or parental health-seeking behavior. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Conclusions Reductions in overall ED attendances were seen across our study sites during the first phase of the COVID-19 pandemic, with health systems across Europe affected similarly. In most sites, there was no suggestion of disproportionate numbers of more severely unwell children. In the first phase of the COVID-19 pandemic, the relative increase in cases of diabetic ketoacidosis or mental health issues might have contributed to a biased perception about increased occurrence, yet this is not supported by an increase in absolute numbers of cases in our data. Our study informs how pediatric emergency medicine can prepare for future pandemics, taking into account that differ- ent infectious diseases outbreaks can affect children differently, and illustrates the potential of elec- tronic health records to monitor trends in urgent and emergency care for children. Strengths and limitations Mongru and colleagues showed that, for one of the sites included in this study, the distribution of patients across primary and secondary care was similar before and during the first year of the COVID-19 pandemic, suggesting the observed reductions in patient numbers, especially for those with minor injuries and illnesses, As the data were collected in aggregated form, thereby negating some of the difficulties with data protection regulations, we were not able to stratify for severity of specific diagnosis or age groups. This also introduced risks of overstratification during analyses. We observed large differences between sites for the number of annual ED attendances and the number of patients with high triage urgency and hospital admissions, reflecting both case-mix and diver- sity of patient management. We analyzed data mostly on a site-by-site basis, by using predicted versus observed ratios, and thus dealing with heterogeneity between sites. In addition, although very few study sites restructured local healthcare pathways diverting urgent and emergency care to alternative healthcare facilities, this does not fully rule out changes to access to healthcare or parental health-seeking behavior. Mongru and colleagues showed that, for one of the sites included in this study, the distribution of patients across primary and secondary care was similar before and during the first year of the COVID-19 pandemic, suggesting the observed reductions in patient numbers, especially for those with minor injuries and illnesses, PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 17 / 27 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic were a true reflection of fewer children in the community in need of urgent and emergency care [72]. We used the cumulative 14-day rate of new cases per 100,000 of the population to identify high-prevalence countries, but indications for SARS-CoV-2 testing and reporting mechanisms differed between countries, and this could have led to under- or overestimation of national prevalence rates. Moreover, national prevalence numbers might wrongly reflect any regional variation, but, for example, in the UK, identical patterns in ED attendances were seen across the 5 sites, despite large variations in SARS-CoV-2 prevalence during the first phase of the COVID-19 pandemic [73]. Due to the rapid escalation and near-universal introduction of infection prevention measures in European countries during the study period, we were not able determine the role of each of the individual measures on reducing ED attendances. Supporting information Supporting information S1 Appendix. Membership of the EPISODES study group. (PDF) S1 File. EPISODES study protocol. (PDF) S2 File. EPISODES study registration. (PDF) S3 File. Clinical report form. (PDF) S1 Checklist. RECORD checklist. The RECORD statement—checklist of items, extended from the STROBE statement, which should be reported in observational studies using rou- tinely collected health data. (PDF) S1 Table. List of time windows for data entry. (PDF) S2 Table. ICD-10 guidance for coding of diagnosis. (PDF) S3 Table. List of approvals. (PDF) S4 Table. Overview of participating study sites. (PDF) S1 Appendix. Membership of the EPISODES study group. (PDF) S1 Appendix. Membership of the EPISODES study group. (PDF) S1 File. EPISODES study protocol. (PDF) S2 File. EPISODES study registration. (PDF) S2 File. EPISODES study registration. (PDF) S3 File. Clinical report form. (PDF) S1 Fig. Spiderplot for availability of data. (PDF) S1 Fig. Spiderplot for availability of data. (PDF) S2 Fig. Map of European with all participating study sites. All participating study sites are highlighted with their study site ID; represented countries in red. (PDF) S2 Fig. Map of European with all participating study sites. All participating study sites are highlighted with their study site ID; represented countries in red. (PDF) S3 Fig. Annual ED attendance numbers in 2018 and 2019. The total number of pediatric ED attendances for each of the study sites for 2018 and 2019 (pre-COVID-19). Overall, the numbers of ED attendances in 2018 and 2019 were similar for each of the study sites, with considerable diversity between the study sites and TUR003 seeing more children in their ED than the other study sites. (PDF) S4 Fig. Annual ED attendance numbers 2018 to 2020 for all sites separately. The total num- ber of pediatric ED attendances for each of the study sites for the entire study duration (Janu- ary 2018–May 2020). The y-axis is depicted in log scale. (PDF) S5 Fig. Observed versus predicted ED attendances (%) for different age categories. The observed versus predicted number of children presenting to EDs in countries across Europe in the weeks following February 2, 2020 until May 11, 2020, for all sites combined, for children (a) aged 0–1 years; (b) 1–2 years; (c) 2–5 years; (d) 5–12 years; and (e) 12–18 years. The color and the size of the dots reflect the actual number of ED attendances for each site and for each time window. The line connects the mean of the observed vs. predicted point estimates for each of the individual sites for each time window. (PDF) S3 File. Clinical report form. (PDF) Overall, the numbers of ED attendances in 2018 and 2019 were similar for each of the study sites, with considerable diversity between the study sites and TUR003 seeing more children in their ED than the other study sites. (PDF) S4 Fig. Annual ED attendance numbers 2018 to 2020 for all sites separately. The total num- ber of pediatric ED attendances for each of the study sites for the entire study duration (Janu- ary 2018–May 2020). The y-axis is depicted in log scale. (PDF) S5 Fig. Observed versus predicted ED attendances (%) for different age categories. The observed versus predicted number of children presenting to EDs in countries across Europe in the weeks following February 2, 2020 until May 11, 2020, for all sites combined, for children (a) aged 0–1 years; (b) 1–2 years; (c) 2–5 years; (d) 5–12 years; and (e) 12–18 years. The color and the size of the dots reflect the actual number of ED attendances for each site and for each time window. The line connects the mean of the observed vs. predicted point estimates for each of the individual sites for each time window. (PDF) S6 Fig. Observed versus predicted ED attendances (%) for different age categories, for indi- vidual sites. (PDF) S7 Fig. Observed versus predicted ED attendances (%) for each country. The observed ver- sus predicted number of children presenting to EDs in countries across Europe for which data from only 1 study site were available in the weeks following February 2, 2020 until May 11, 2020. A timeline is plotted (dashed line) to show the dates of the introduction of national social distancing measures.[20] One site from the Netherlands and 1 site from Hungary were excluded from these analyses as these sites could not provide data for the entire study dura- tion S3 File. Clinical report form. (PDF) S1 Checklist. RECORD checklist. The RECORD statement—checklist of items, extended from the STROBE statement, which should be reported in observational studies using rou- tinely collected health data. (PDF) S1 Table. List of time windows for data entry. (PDF) S2 Table. ICD-10 guidance for coding of diagnosis. (PDF) S4 Table. Overview of participating study sites. (PDF) PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 18 / 27 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic S5 Table. List of national social distancing measures. (PDF) S6 Table. List of national SARS-CoV-2 rates. The dates and numbers of SARS-CoV02 infec- tions in each of the study sites’ countries participating in the EPISODES study. (PDF) S7 Table. Sensitivity analyses: Poisson regression models for ED attendances without TUR003. (PDF) S1 Fig. Spiderplot for availability of data. (PDF) S2 Fig. Map of European with all participating study sites. All participating study sites are highlighted with their study site ID; represented countries in red. (PDF) S3 Fig. Annual ED attendance numbers in 2018 and 2019. The total number of pediatric ED attendances for each of the study sites for 2018 and 2019 (pre-COVID-19). Overall, the numbers of ED attendances in 2018 and 2019 were similar for each of the study sites, with considerable diversity between the study sites and TUR003 seeing more children in their ED than the other study sites. (PDF) S4 Fig. Annual ED attendance numbers 2018 to 2020 for all sites separately. The total num- ber of pediatric ED attendances for each of the study sites for the entire study duration (Janu- ary 2018–May 2020). The y-axis is depicted in log scale. S5 Table. List of national social distancing measures. (PDF) S6 Table. List of national SARS-CoV-2 rates. The dates and numbers of SARS-CoV02 infec- tions in each of the study sites’ countries participating in the EPISODES study. (PDF) S7 Table. Sensitivity analyses: Poisson regression models for ED attendances without TUR003. (PDF) S1 Fig. Spiderplot for availability of data. (PDF) S2 Fig. Map of European with all participating study sites. All participating study sites are highlighted with their study site ID; represented countries in red. (PDF) S3 Fig. Annual ED attendance numbers in 2018 and 2019. The total number of pediatric ED attendances for each of the study sites for 2018 and 2019 (pre-COVID-19). S6 Fig. Observed versus predicted ED attendances (%) for different age categories, for indi- vidual sites. (PDF) Percentages of total ED attendances (left) and absolute numbers (right) of children admitted to hospital (top) and PICUs (bottom); comparing the 28-day standardized numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; PICU, pediatric intensive care unit. (PDF) S10 Fig. Selected clinical diagnoses in the ED for the period January–April over a 3-year period, for high-prevalence countries. Percentages of total ED attendances (left) and absolute numbers (right) of children with diagnosis of (a) tonsillitis; (b) otitis media; (c) LRTIs; (d) GI infections; (e) appendicitis; (f) testicular torsion; (g) intussusception; (h) mental health issues; (i) diabetic ketoacidosis; (j) radius fracture; and (k) minor head injury; comparing the 28-day standardized numbers for the months of January–April for 2018 vs. 2019 vs. 2020, shown for countries with of a cumulative 14-day rate of new SARS-CoV-2 cases per 100,000 of 80 or more. ED, emergency department; GI, gastrointestinal; LRTI, lower respiratory tract infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. (PDF) S6 Fig. Observed versus predicted ED attendances (%) for different age categories, for indi- vidual sites. (PDF) S7 Fig. Observed versus predicted ED attendances (%) for each country. The observed ver- sus predicted number of children presenting to EDs in countries across Europe for which data from only 1 study site were available in the weeks following February 2, 2020 until May 11, 2020. A timeline is plotted (dashed line) to show the dates of the introduction of national social distancing measures.[20] One site from the Netherlands and 1 site from Hungary were excluded from these analyses as these sites could not provide data for the entire study dura- tion. (PDF) S7 Fig. Observed versus predicted ED attendances (%) for each country. The observed ver- sus predicted number of children presenting to EDs in countries across Europe for which data from only 1 study site were available in the weeks following February 2, 2020 until May 11, 2020. A timeline is plotted (dashed line) to show the dates of the introduction of national social distancing measures.[20] One site from the Netherlands and 1 site from Hungary were excluded from these analyses as these sites could not provide data for the entire study dura- tion. (PDF) S8 Fig. Observed versus predicted triage categories (%). The observed versus predicted num- ber of children presenting to EDs in countries across Europe in the weeks following February 2, 2020 until May 11, 2020, for all sites combined, for children (a) nonurgent and standard 19 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic triage classification; (b) urgent triage classification; (c) emergency and very urgent triage classi- fication. The color and the size of the dots reflect the actual number of ED attendances for each site and for each time window. The line connects the mean of the observed vs. predicted point estimates for each of the individual sites for each time window. UK001 did not use a tri- age system with the emergency and very urgent triage category. (PDF) S9 Fig. Percentage of children admitted to hospital for individual sites. Percentages of total ED attendances (left) and absolute numbers (right) of children admitted to hospital (top) and PICUs (bottom); comparing the 28-day standardized numbers for the months of January–April for 2018 vs. 2019 vs. 2020. ED, emergency department; PICU, pediatric intensive care unit. (PDF) S9 Fig. Percentage of children admitted to hospital for individual sites. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Author Contributions Conceptualization: Ruud G. Nijman, Kate Honeyford, Ruth Farrugia, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Tisham De, Ian K. Maconochie, Niccolo Parri, Damian Roland, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Conceptualization: Ruud G. Nijman, Kate Honeyford, Ruth Farrugia, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Tisham De, Ian K. Maconochie, Niccolo Parri, Damian Roland, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Data curation: Ruud G. Nijman, Kate Honeyford, Ruth Farrugia, Katy Rose, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Tisham De, Ian K. Maconochie, Niccolo Parri, Damian Roland, Tobias Alfven, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Formal analysis: Ruud G. Nijman, Kate Honeyford, Tisham De, Damian Roland, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Funding acquisition: Ruud G. Nijman, Ian K. Maconochie, Damian Roland, Henriette A. Acknowledgments We would like to thank and acknowledge the support and their contributions to the EPI- SODES study of the following persons: Celia Nekrouf (Pediatric Emergency Department, Hopital Universitaire Robert-Debre, Paris, France); Marcello Covino (Emergency Depart- ment, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy); Benita Lund (Medical Secretary; Pediatric emergency department, Sachs’ Children and Youth Hospital, Stockholm, Sweden); Izabella Lottiger (Pediatric Emergency department, Astrid Lindgren Children Hospital, Stockholm, Sweden); Sharna Crosdale (Contracts office; Imperial College London, UK); Sarah Sheedy (Research nurse; Emergency Department, Bristol Royal Hospital for Children, Bristol, UK); James Allbones (Information & Performance Analyst; Birmingham Women’s and Children’s NHS Foundation Trust, UK), William Jones (University Hospitals of Leicester NHS Trust, UK); Frazer Snowdon and Matthew Ryan (Pediatric emergency depart- ment, Alder Hey Children’s NHS Foundation Trust, Liverpool, UK), Carlos Saiz-Hernando (IT analyst; Department of Medical Documentation, Cruces University Hospital, Bilbao, Spain); Ellen Barry (research nurse; National Children’s Research Centre, Dublin, Ireland) and Fiona Leonard (data analyst, Children’s Health Ireland, Ireland); Ernst Eigenbauer (data ana- lyst) and Katharina Lieb (medical student; Department of Pediatrics and Adolescent Medicine, Vienna, Austria); Sanne Vrijland (medical student; Erasmus MC–Sophia Children’s hospital, Rotterdam, The Netherlands); Catarina Cordeiro (Pediatric Emergency Service, Hospital Pedia´trico, Centro Hospitalar e Universita´rio de Coimbra, Portugal); Mark Camenzuli (Senior systems administrator; Mater Dei Hospital, Malta); Sandra Distefano (Clinical Performance Unit, Mater Dei Hospital, Malta); Karin Kittl-Mitteregger (HIS Management and Clinical Pro- cesses, Paracelsus Medical University, Salzburg, Austria); Marta Arpone (Research Assistant, University of Padova, Italy) 20 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 Author Contributions Moll, Rianne Oostenbrink, Luigi Titomanlio. Investigation: Ruud G. Nijman, Kate Honeyford, Ruth Farrugia, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Tisham De, Ian K. Maconochie, Niccolo Parri, Damian Roland, Tobias Alfven, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Methodology: Ruud G. Nijman, Kate Honeyford, Ruth Farrugia, Katy Rose, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Tisham De, Ian K. Maconochie, Niccolo Parri, Damian Roland, Tobias Alfven, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 21 / 27 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Project administration: Ruth Farrugia, Katy Rose, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Tisham De, Ian K. Maconochie, Niccolo Parri, Damian Roland, Tobias Alfven, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Resources: Ruud G. Nijman, Kate Honeyford, Liviana Da Dalt, Tisham De, Ian K. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 References 1. Bressan S, Buonsenso D, Farrugia R, Parri N, Oostenbrink R, Titomanlio L, et al. Preparedness and Response to Pediatric COVID-19 in European Emergency Departments: A Survey of the REPEM and PERUKI Networks. Ann Emerg Med. 2020 Jun 21; 76(6):788–800. Available from: https://doi.org/10. 1016/j.annemergmed.2020.05.018 PMID: 32419713 2. Go¨tzinger F, Santiago-Garcı´a B, Noguera-Julia´n A, Lanaspa M, Lancella L, Calò Carducci FI, et al. COVID-19 in children and adolescents in Europe: a multinational, multicentre cohort study. Lancet Child Adolesc Heal. 2020 Jun 29; 4(9):653–61. Available from: https://doi.org/10.1016/S2352-4642(20) 30177-2 PMID: 32593339 3. Swann O V., Holden KA, Turtle L, Pollock L, Fairfield CJ, Drake TM, et al. Clinical characteristics of chil- dren and young people admitted to hospital with covid-19 in United Kingdom: Prospective multicentre observational cohort study. Baillie JK, Semple MG, Openshaw PJM, Alex B, Bach B, Barclay WS, et al., editors. BMJ. 2020; 370. Available from: https://www.bmj.com/content/370/bmj.m3249 https://doi.org/ 10.1136/bmj.m3249 PMID: 32960186 4. Lu X, Zhang L, Du H, Zhang J, Li YY, Qu J, et al. SARS-CoV-2 Infection in Children N Engl J Med. 2020 Mar 18; 382(17):1663–5. Available from: https://doi.org/10.1056/NEJMc2005073 5. Parri N, Lenge M, Buonsenso D. Children with Covid-19 in Pediatric Emergency Departments in Italy. N Engl J Med. 2020 May 1; 383(2):187–90. Available from: https://doi.org/10.1056/NEJMc2007617 PMID: 32356945 6. DeBiasi RL, Song X, Delaney M, Bell M, Smith K, Pershad J, et al. Severe Coronavirus Disease-2019 in Children and Young Adults in the Washington, DC, Metropolitan Region. J Pediatr. 2020 Aug 1; 223:199–203.e1. Available from: https://doi.org/10.1016/j.jpeds.2020.05.007 PMID: 32405091 7. Isba R, Edge R, Jenner R, Broughton E, Francis N, Butler J. Where have all the children gone? Decreases in paediatric emergency department attendances at the start of the COVID-19 pandemic of 2020. Arch Dis Child. 2020 May 5; 105(7):704. Available from: http://adc.bmj.com/content/early/2020/ 05/05/archdischild-2020-319385.abstract https://doi.org/10.1136/archdischild-2020-319385 PMID: 32376695 8. Angoulvant F, Ouldali N, Yang DD, Filser M, Gajdos V, Rybak A, et al. Coronavirus Disease 2019 Pan- demic: Impact Caused by School Closure and National Lockdown on Pediatric Visits and Admissions for Viral and Nonviral Infections—A Time Series Analysis. Clin Infect Dis. 2021; 72(2):319–22. Available from: https://doi.org/10.1093/cid/ciaa710 PMID: 33501967 9. Finkelstein Y, Maguire B, Zemek R, Osmanlliu E, Kam AJ, Dixon A, et al. Effect of the COVID-19 Pan- demic on Patient Volumes, Acuity, and Outcomes in Pediatric Emergency Departments: A Nationwide Study. Pediatr Emerg Care. 2021 Jun; 37(8):427–34. https://doi.org/10.1097/PEC.0000000000002484 PMID: 34074990 10. Author Contributions Maconochie, Damian Roland, Tobias Alfven, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Supervision: Ruud G. Nijman, Kate Honeyford, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Niccolo Parri, Damian Roland, Tobias Alfven, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. Validation: Kate Honeyford, Ruth Farrugia, Katy Rose, Zsolt Bognar, Danilo Buonsenso, Ian K. Maconochie, Esra Akyu¨z O¨ zkan, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors. Visualization: Ruud G. Nijman, Ruth Farrugia, Katy Rose, Liviana Da Dalt, Stuart Hartshorn, Roberto Velasco, Rianne Oostenbrink, Luigi Titomanlio. Writing – original draft: Ruud G. Nijman, Kate Honeyford, Ruth Farrugia, Katy Rose, Ian K. Maconochie, Rianne Oostenbrink, Luigi Titomanlio. Writing – review & editing: Ruud G. Nijman, Kate Honeyford, Ruth Farrugia, Katy Rose, Zsolt Bognar, Danilo Buonsenso, Liviana Da Dalt, Tisham De, Ian K. Maconochie, Niccolo Parri, Damian Roland, Tobias Alfven, Camille Aupiais, Michael Barrett, Romain Basmaci, Dorine Borensztajn, Susana Castanhinha, Corinne Vasilico, Sheena Durnin, Paddy Fitzpatrick, Laszlo Fodor, Borja Gomez, Susanne Greber-Platzer, Romain Guedj, Stuart Hartshorn, Florian Hey, Lina Jankauskaite, Daniela Kohlfuerst, Mojca Kolnik, Mark D. Lyttle, Patrı´cia Mac¸ão, Maria Inês Mascarenhas, Shrouk Messahel, Esra Akyu¨z O¨ zkan, Zanda Pučuka, Sofia Reis, Alexis Rybak, Malin Ryd Rinder, Ozlem Teksam, Caner Turan, Valty´r Stefa´nsson Thors, Roberto Velasco, Silvia Bressan, Henriette A. Moll, Rianne Oostenbrink, Luigi Titomanlio. 22 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic References European Centre for Disease Prevention and Control. COVID-19 [Internet] 2020. [cited 2022 Jun 10] Available from: https://www.ecdc.europa.eu/en/covid-19 21. Rose K, Bressan S, Honeyford K, Bognar Z, Buonsenso D, Da Dalt L, et al. Responses of paediatric emergency departments to the first wave of the COVID-19 pandemic in Europe: a cross-sectional sur- vey study. Rybak A, Simões AS, Chiaretti A, Haraldsson A, Gomez B, Aupiais C, et al., editors. BMJ Paediatr Open. 2021; 5(1). Available from: https://bmjpaedsopen.bmj.com/content/5/1/e001269 22. Keyes D, Hardin B, Sweeney B, Shedden K. Change in urban and non-urban pattern of ED use during the COVID-19 pandemic in 28 Michigan hospitals: An observational study. BMJ Open. 2021; 11(2). Available from: https://bmjopen.bmj.com/content/11/2/e043024 https://doi.org/10.1136/bmjopen-2020- 043024 PMID: 33550257 23. Pines JM, Zocchi MS, Black BS, Carlson JN, Celedon P, Moghtaderi A, et al. Characterizing pediatric emergency department visits during the COVID-19 pandemic. Am J Emerg Med. 2021; 41:201–4. Avail- able from: https://www.sciencedirect.com/science/article/pii/S0735675720310615 https://doi.org/10. 1016/j.ajem.2020.11.037 PMID: 33257144 24. Westgard BC, Morgan MW, Vazquez-Benitez G, Erickson LO, Zwank MD. An Analysis of Changes in Emergency Department Visits After a State Declaration During the Time of COVID-19. Ann Emerg Med. 2020; 76 (5):595–601. https://doi.org/10.1016/j.annemergmed.2020.06.019 PMID: 33008651 25. Gupta A, Bush A, Nagakumar P. Asthma in children during the COVID-19 pandemic: lessons from lock- down and future directions for management. Lancet Respir Med. 2020 Jun; 8(11):1070–1. https://doi. org/10.1016/S2213-2600(20)30278-2 PMID: 32593314 26. Taquechel K, Diwadkar AR, Sayed S, Dudley JW, Grundmeier RW, Kenyon CC, et al. Pediatric Asthma Health Care Utilization, Viral Testing, and Air Pollution Changes During the COVID-19 Pandemic. J Allergy Clin Immunol Pract. 2020; 8(10):3378–3387.e11. https://doi.org/10.1016/j.jaip.2020.07.057 PMID: 32827728 27. Valitutti F, Zenzeri L, Mauro A, Pacifico R, Borrelli M, Muzzica S, et al. Effect of Population Lockdown on Pediatric Emergency Room Demands in the Era of COVID-19. Front Pediatr. 2020; 8:521. https:// doi.org/10.3389/fped.2020.00521 PMID: 33072657 28. Raucci U, Musolino AM, Di Lallo D, Piga S, Barbieri MA, Pisani M, et al. Impact of the COVID-19 pan- demic on the Emergency Department of a tertiary children’s hospital. Ital J Pediatr. 2021 Jan; 47(1):21. https://doi.org/10.1186/s13052-021-00976-y PMID: 33514391 29. Isba R, Edge R, Auerbach M, Cicero MX, Jenner R, Setzer E, et al. COVID-19: Transatlantic declines in pediatric emergency admissions. Pediatr Emerg Care. 2020 Nov; 36(11):551–3. https://doi.org/10. 1097/PEC.0000000000002260 PMID: 32925702 30. Vierucci F, Bacci C, Mucaria C, Dini F, Federico G, Maielli M, et al. References Kruizinga MD, Peeters D, van Veen M, van Houten M, Wieringa J, Noordzij JG, et al. The impact of lock- down on pediatric ED visits and hospital admissions during the COVID19 pandemic: a multicenter anal- ysis and review of the literature. Eur J Pediatr. 2021 Jul; 180 (7):2271–2279. https://doi.org/10.1007/ s00431-021-04015-0 PMID: 33723971 11. DeLaroche AM, Rodean J, Aronson PL, Fleegler EW, Florin TA, Goyal M, et al. Pediatric emergency department visits at US children’s hospitals during the COVID-19 pandemic. Pediatrics. 2021; 147(4). Available from: https://pediatrics.aappublications.org/content/147/4/e2020039628 https://doi.org/10. 1542/peds.2020-039628 PMID: 33361360 12. Lynn RM, Avis JL, Lenton S, Amin-Chowdhury Z, Ladhani SN. Delayed access to care and late presen- tations in children during the COVID-19 pandemic: A snapshot survey of 4075 paediatricians in the UK and Ireland. Arch Dis Child. 2021; 106(2):archdischild-2020-319848. Available from: https://adc.bmj. com/content/early/2020/06/24/archdischild-2020-319848 https://doi.org/10.1136/archdischild-2020- 319848 PMID: 32586927 13. Lazzerini M, Barbi E, Apicella A, Marchetti F, Cardinale F, Trobia G. Delayed access or provision of care in Italy resulting from fear of COVID-19. Lancet Child Adolesc Heal. 2020 May 1; 4(5):e10–1. Avail- able from: https://doi.org/10.1016/S2352-4642(20)30108-5 PMID: 32278365 14. Honeyford K, Coughlan C, Nijman RG, Expert P, Burcea G, Maconochie I, et al. Changes in emergency department activity and the first covid-19 lockdown: A cross-sectional study. West J Emerg Med. 2021 May; 22(3):603–7. https://doi.org/10.5811/westjem.2021.2.49614 PMID: 34125034 15. Royal College for Paediatrics and Child Health. Advice for parents and young people during coronavi- rus—posters [Internet]. 2020. [cited 2022 Jun 10]. Available from: https://www.rcpch.ac.uk/resources/ advice-parents-young-people-during-coronavirus-posters 16. Unsworth R, Wallace S, Oliver NS, Yeung S, Kshirsagar A, Naidu H, et al. New-onset type 1 diabetes in children during COVID-19: Multicenter regional findings in the U.K. Diabetes Care. 2020 Nov; 43(11): e170–1. https://doi.org/10.2337/dc20-1551 PMID: 32816997 23 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic 17. Giovanni JE, Hrapcak S, Melgar M, Godfred-Cato S. Global Reports of Intussusception in Infants with SARS-CoV-2 Infection. Pediatr Infect Dis J. 2020 Jan; 40(1):35–6. 18. Orben A, Tomova L, Blakemore SJ. The effects of social deprivation on adolescent development and mental health. Lancet Child Adolesc Heal. 2020 Aug; 4(8):634–40. https://doi.org/10.1016/S2352-4642 (20)30186-3 PMID: 32540024 19. Newlove-Delgado T, McManus S, Sadler K, Thandi S, Vizard T, Cartwright C, et al. Child mental health in England before and during the COVID-19 lockdown. Lancet Psychiatry. 2021 May 1; 8(5):353–4. Available from: https://doi.org/10.1016/S2215-0366(20)30570-8 PMID: 33444548 20. References Children’s emergency presentations during the COVID-19 pandemic. J Clean Prod. 2020 Jun 30; Available from: https://doi.org/10.1016/ S2352-4642(20)30206-6 PMID: 32598871 41. Delgado-Miguel C, Munõz-Serrano AJ, Miguel-Ferrero M, De Ceano-Vivas M, Calvo C, Martı´nez L. Complicated Acute Appendicitis during COVID-19 Pandemic: The Hidden Epidemic in Children. Eur J Pediatr Surg. 2022 Jun 22; 32(03):268–73. Available from: http://www.thieme-connect.de/DOI/DOI?10. 1055/s-0041-1723992 PMID: 33618382 42. Gaitero Trista´n J, Souto Romero H, Escalada Pellitero S, Espiñera CR, Andina Martı´n D, Espinosa Go´ngora R, et al. Acute Appendicitis in Children During the COVID-19 Pandemic: Neither Delayed Diagnosis Nor Worse Outcomes. Pediatr Emerg Care. 2021 Mar; 37(3):185–90. https://doi.org/10. 1097/PEC.0000000000002364 PMID: 33651763 43. Nelson CP, Kurtz MP, Logvinenko T, Venna A, McNamara ER. Timing and outcomes of testicular tor- sion during the COVID-19 crisis. J Pediatr Urol. 2020 Dec; 16(6):841.e1-841.e5. https://doi.org/10. 1016/j.jpurol.2020.10.021 PMID: 33223456 44. Littman AR, Janssen KM, Tong L, Wu H, Wang MD, Blum E, et al. Did COVID-19 Affect Time to Presen- tation in the Setting of Pediatric Testicular Torsion? Pediatr Emerg Care. 2021 Feb; 37(2):123–5. https://doi.org/10.1097/PEC.0000000000002333 PMID: 33512891 45. Holzman SA, Ahn JJ, Baker Z, Chuang KW, Copp HL, Davidson J, et al. A multicenter study of acute testicular torsion in the time of COVID-19. J Pediatr Urol. 2021 Aug; 17(4):478.e1-478.e6. https://doi. org/10.1016/j.jpurol.2021.03.013 PMID: 33832873 46. Pogorelić Z, Milanović K, Versˇić AB, Pasini M, Divković D, Pavlović O, et al. Is there an increased inci- dence of orchiectomy in pediatric patients with acute testicular torsion during COVID-19 pandemic?–A retrospective multicenter study. J Pediatr Urol. 2021 Aug; 17(4):479.e1–479.e6. Available from: https:// linkinghub.elsevier.com/retrieve/pii/S1477513121002254 https://doi.org/10.1016/j.jpurol.2021.04.017 PMID: 33994321 47. Mu¨ller JA, Groß R, Conzelmann C, Kru¨ger J, Merle U, Steinhart J, et al. SARS-CoV-2 infects and repli- cates in cells of the human endocrine and exocrine pancreas. Nat Metab. 2021 Feb; 3(2):149–65. https://doi.org/10.1038/s42255-021-00347-1 PMID: 33536639 48. Fignani D, Licata G, Brusco N, Nigi L, Grieco GE, Marselli L, et al. SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic β-Cells and in the Human Pan- creas Microvasculature. Front Endocrinol (Lausanne). 2020; 11:596898. https://doi.org/10.3389/fendo. 2020.596898 PMID: 33281748 49. Salmi H, Heinonen S, Ha¨stbacka J, La¨a¨peri M, Rautiainen P, Miettinen PJ, et al. New-onset type 1 dia- betes in Finnish children during the COVID-19 pandemic. Arch Dis Child. 2022 Feb; 107(2):180–5. Available from: https://doi.org/10.1136/archdischild-2020-321220 PMID: 34045208 50. Tittel SR, Rosenbauer J, Kamrath C, Ziegler J, Reschke F, Hammersen J, et al. References How COVID-19 Pandemic Changed Children and Adolescents Use of the Emergency Department: the Experience of a Secondary Care Pediatric Unit in Central Italy. SN Compr Clin Med. 2020 Sep; 2(11):1959–69. https://doi.org/10.1007/ s42399-020-00532-5 PMID: 32984767 31. Morello F, Bima P, Ferreri E, Chiarlo M, Balzaretti P, Tirabassi G, et al. After the first wave and beyond lockdown: long-lasting changes in emergency department visit number, characteristics, diagnoses, and hospital admissions. Intern Emerg Med. 2021 Mar; 16(6):1683–90. https://doi.org/10.1007/s11739- 021-02667-2 PMID: 33683538 32. Shanmugavadivel D, Liu JF, Gilhooley C, Elsaadany L, Wood D. Changing patterns of emergency pae- diatric presentations during the first wave of COVID-19: Learning for the second wave from a UK tertiary emergency department. BMJ Paediatr Open. 2021; 5(1):e000967. https://doi.org/10.1136/bmjpo-2020- 000967 PMID: 34192192 33. Sano K, Nakamura M, Ninomiya H, Kobayashi Y, Miyawaki A. Large decrease in paediatric hospitalisa- tions during the COVID-19 outbreak in Japan. BMJ Paediatr Open. 2021; 5(1):e001013. https://doi.org/ 10.1136/bmjpo-2020-001013 PMID: 34192195 34. Raman R, Madhusudan M. Impact of the COVID-19 Pandemic on Admissions to the Pediatric Emer- gency Department in a Tertiary Care Hospital. Indian J Pediatr. 2021 Apr; 88(4):392. https://doi.org/10. 1007/s12098-020-03562-y PMID: 33146879 24 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic 35. McIntosh A, Bachmann M, Siedner MJ, Gareta D, Seeley J, Herbst K. Effect of COVID-19 lockdown on hospital admissions and mortality in rural KwaZulu-Natal, South Africa: Interrupted time series analysis. BMJ Open. 2021 Mar; 11(3):e047961. https://doi.org/10.1136/bmjopen-2020-047961 PMID: 33737445 36. McDonnell T, Nicholson E, Conlon C, Barrett M, Cummins F, Hensey C, et al. Assessing the impact of COVID-19 public health stages on paediatric emergency attendance. Int J Environ Res Public Health. 2020 Sep; 17(18):1–25. https://doi.org/10.3390/ijerph17186719 PMID: 32942698 37. Dann L, Fitzsimons J, Gorman KM, Hourihane J, Okafor I. Disappearing act: COVID-19 and paediatric emergency department attendances. Arch Dis Child. 2020; 105(8):810–1. Available from: https://adc. bmj.com/content/105/8/810 https://doi.org/10.1136/archdischild-2020-319654 PMID: 32518141 38. Chong SL, Soo JSL, Allen JC, Ganapathy S, Lee KP, Tyebally A, et al. Impact of COVID-19 on pediatric emergencies and hospitalizations in Singapore. BMC Pediatr. 2020 Dec; 20(1):562. https://doi.org/10. 1186/s12887-020-02469-z PMID: 33353540 39. Roland D, Nijman RG, Ponmani C, Munro APS. Arriving late, delayed, or not at all—presentations to paediatric emergency departments during covid-19 pandemic [Internet]. [cited 2022 Jun 10]. BMJ. 2020. Available from: https://blogs.bmj.com/bmj/2020/08/15/arriving-late-delayed-or-not-at-all- presentations-to-paediatric-emergency-departments-during-covid-19-pandemic/ 40. Roland D, Harwood R, Bishop N, Hargreaves D, Patel S, Sinha I. References Did the COVID-19 lock- down affect the incidence of pediatric type 1 diabetes in Germany? Diabetes Care. 2020 Nov; 43(11): e172–3. https://doi.org/10.2337/dc20-1633 PMID: 32826282 51. Rabbone I, Schiaffini R, Cherubini V, Maffeis C, Scaramuzza A. Has covid-19 delayed the diagnosis and worsened the presentation of type 1 diabetes in children? Diabetes Care. 2020 Nov; 43(11):2870– 2. https://doi.org/10.2337/dc20-1321 PMID: 32778554 52. Jacob R, Weiser G, Krupik D, Takagi D, Peled S, Pines N, et al. Diabetic Ketoacidosis at Emergency Department Presentation During the First Months of the SARS-CoV-2 Pandemic in Israel: A Multicenter 25 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic Cross-Sectional Study. Diabetes Ther. 2021 May; 12(5):1569–74. https://doi.org/10.1007/s13300-021- 01049-3 PMID: 33730335 Cross-Sectional Study. Diabetes Ther. 2021 May; 12(5):1569–74. https://doi.org/10.1007/s13300-021- 01049-3 PMID: 33730335 53. Gottesman BL, Yu J, Tanaka C, Longhurst CA, Kim JJ. Incidence of New-Onset Type 1 Diabetes Among US Children During the COVID-19 Global Pandemic. JAMA Pediatr. 2022. Available from: https://doi.org/10.1001/jamapediatrics.2021.5801 PMID: 35072727 54. Leeb RT, Bitsko RH, Radhakrishnan L, Martinez P, Njai R, Holland KM. Mental Health–Related Emer- gency Department Visits Among Children Aged <18 Years During the COVID-19 Pandemic—United States, January 1–October 17, 2020. MMWR Morb Mortal Wkly Rep. 2020; 69(45):1675–1680. https:// doi.org/10.15585/mmwr.mm6945a3 PMID: 33180751 55. Ougrin D, Wong BH, Vaezinejad M, Plener PL, Mehdi T, Romaniuk L, et al. Pandemic-related emer- gency psychiatric presentations for self-harm of children and adolescents in 10 countries (PREP-kids): a retrospective international cohort study. Eur Child Adolesc Psychiatry. 2021 Mar:1–13. https://doi.org/ 10.1007/s00787-021-01741-6 PMID: 33677628 56. Carr MJ, Steeg S, Webb RT, Kapur N, Chew-Graham CA, Abel KM, et al. Effects of the COVID-19 pan- demic on primary care-recorded mental illness and self-harm episodes in the UK: a population-based cohort study. Lancet Public Health. 2021 Feb; 6(2):e124–35. https://doi.org/10.1016/S2468-2667(20) 30288-7 PMID: 33444560 57. Leff RA, Setzer E, Cicero MX, Auerbach M. Changes in pediatric emergency department visits for men- tal health during the COVID-19 pandemic: A cross-sectional study. Clin Child Psychol Psychiatry. 2021 Jan; 26(1):33–8. https://doi.org/10.1177/1359104520972453 PMID: 33183097 58. Ford T, John A, Gunnell D. Mental health of children and young people during pandemic. BMJ. 2021 Mar 10; 372:n614. Available from: https://www.bmj.com/content/372/bmj.n614 https://doi.org/10.1136/ bmj.n614 PMID: 33692087 59. Lin JA, Hartman-Munick SM, Kells MR, Milliren CE, Slater WA, Woods ER, et al. The Impact of the COVID-19 Pandemic on the Number of Adolescents/Young Adults Seeking Eating Disorder-Related Care. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 70. McDonnell WM, Nelson DS, Schunk JE. Should we fear “flu fear” itself? Effects of H1N1 influenza fear on ED use. Am J Emerg Med. 2012 Feb; 30(2):275–282. https://doi.org/10.1016/j.ajem.2010.11.027 PMID: 21208765 References J Adolesc Health. 2021 Jul; 69(4):660–3. https://doi.org/10.1016/j.jadohealth.2021.05.019 PMID: 34266715 60. Joyce LR, Richardson SK, McCombie A, Hamilton GJ, Ardagh MW. Mental health presentations to Christchurch Hospital Emergency Department during COVID-19 lockdown. Emerg Med Australas. 2021 Apr; 33(2):324–30. https://doi.org/10.1111/1742-6723.13667 PMID: 33078509 61. Heymann A, Chodick G, Reichman B, Kokia E, Laufer J. Influence of school closure on the incidence of viral respiratory diseases among children and on health care utilization. Pediatr Infect Dis J. 2004 Jul; 23(7):675–7. https://doi.org/10.1097/01.inf.0000128778.54105.06 PMID: 15247610 62. Boutis K, Stephens D, Lam K, Ungar WJ, Schuh S. The impact of SARS on a tertiary care pediatric emergency department. CMAJ. 2004 Nov; 171(11):1353–8. https://doi.org/10.1503/cmaj.1031257 PMID: 15557588 63. Heiber M, Lou WYW. Effect of the SARS outbreak on visits to a community hospital emergency depart- ment. Can J Emerg Med. 2006 Sep; 8(5):323–8. https://doi.org/10.1017/s148180350001397x PMID: 17338843 64. Huang HH, Yen DHT, Kao WF, Wang LM, Huang CI, Lee CH. Declining emergency department visits and costs during the severe acute respiratory syndrome (SARS) outbreak. J Formos Med Assoc. 2006 Jan; 105(1):31–7. https://doi.org/10.1016/S0929-6646(09)60106-6 PMID: 16440068 65. Paek SH, Kim DK, Lee JH, Kwak YH. The impact of middle east respiratory syndrome outbreak on trends in emergency department utilization patterns. J Korean Med Sci. 2017 Oct; 32(10):1576–80. https://doi.org/10.3346/jkms.2017.32.10.1576 PMID: 28875599 66. Progression and impact of the first winter wave of the 2009 pandemic H1N1 influenza in New South Wales, Australia. Euro Surveill. 2009 Oct; 14(42). https://doi.org/10.2807/ese.14.42.19365-en PMID: 19883546 67. Graham J, Shirm S, Storm E, Lyle K, Linam WM, Romero J. Challenges and solutions: pandemic 2009 H1N1 influenza A in a pediatric emergency department. Am J Disaster Med. 2011; 6(4):211–218. https://doi.org/10.5055/ajdm.2011.0060 PMID: 22010598 68. Blumental S, Huisman E, Cornet MC, Ferreiro C, De Schutter I, Reynders M, et al. Pandemic A/H1N1v influenza 2009 in hospitalized children: A multicenter Belgian survey. BMC Infect Dis. 2011 Nov; 11:313. https://doi.org/10.1186/1471-2334-11-313 PMID: 22060843 69. Copeland DL, Basurto-Davila R, Chung W, Kurian A, Fishbein DB, Szymanowski P, et al. Effectiveness of a school district closure for pandemic influenza A (H1N1) on acute respiratory illnesses in the commu- nity: A natural experiment. Clin Infect Dis. 2013 Feb; 56(4):509–16. https://doi.org/10.1093/cid/cis890 PMID: 23087391 26 / 27 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1003974 August 26, 2022 PLOS MEDICINE paediatric emergency medicine in Europe and the COVID-19 pandemic 70. McDonnell WM, Nelson DS, Schunk JE. Should we fear “flu fear” itself? Effects of H1N1 influenza fear on ED use. Am J Emerg Med. References 2012 Feb; 30(2):275–282. https://doi.org/10.1016/j.ajem.2010.11.027 PMID: 21208765 71. Armstrong BG. Effect of influenza vaccination on excess deaths occurring during periods of high circula- tion of influenza: Cohort study in elderly people. BMJ. 2004; 329(7467)):660. Available from: https:// www.bmj.com/content/341/bmj.c4226 https://doi.org/10.1136/bmj.38198.594109.AE PMID: 15313884 72. Mongru R, Rose DF, Costelloe C, Cunnington A, Nijman RG. Retrospective analysis of North West Lon- don healthcare utilisation by children during the COVID-19 pandemic. BMJ Paediatr Open. 2022 Jan 13; 6(1):e001363. Available from: https://bmjpaedsopen.bmj.com/content/6/1/e001363 73. GOV.UK Coronavirus (COVID-19) in the UK [Internet]. [cited 2022 Jun 10]. Available from: https:// coronavirus.data.gov.uk/details/download 27 / 27
https://openalex.org/W2783307812
https://www.cambridge.org/core/services/aop-cambridge-core/content/view/E006B1039AB19E70712B94840C387EF5/S1062798717000485a.pdf/div-class-title-introduction-to-the-special-issue-on-university-governance-and-creativity-div.pdf
English
null
Introduction to the Special Issue on ‘University Governance and Creativity’
European review
2,018
cc-by
2,657
European Review, Vol. 26, No. S1, S1–S5 © 2018 Academia Europæa. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. European Review, Vol. 26, No. S1, S1–S5 © 2018 Academia Europæa. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S1062798717000485 doi:10.1017/S1062798717000485 G E O R G K R Ü C K E N * , L A R S E N G W A L L * * a n d E R I K D E C O R T E † G E O R G K R Ü C K E N * , L A R S E N G W A L L * * a n d E R I K D E C O R T E † *University of Kassel, Germany. Email: kruecken@incher.uni-kassel.de **Uppsala University, Sweden. Email: Lars.Engwall@fek.uu.se †University of Leuven, Belgium. Email: erik.decorte@kuleuven.be Higher education institutions in Europe have undergone remarkable transformations over the last two to three decades. Of particular importance here are the far-reaching changes in university governance structures. Changes related to higher education governance have also attracted the attention of researchers, as these changes have been broadly discussed and widely studied, quite often in an international compara- tive and interdisciplinary fashion.1 New Public Management reforms have challenged the traditional mode of university governance in Europe that was based on the interplay between strong state regulation and academic self-governance. In its place, a stronger sense of institutional accountability has now emerged. Various reasons explain the increased focus on institutional accountability. First, state regulations are changing as they are now increasingly exercised through target agreements with universities, in which the allocation of state resources follows concrete performance indicators. Detailed prescriptions of university behaviour by direct rules and regulations, on the other hand, play a weaker role in state governance. Second, intermediary organizations such as accreditation agencies, evaluation bodies, and university governance boards as well as the media, through ranking lists, increas- ingly shape the external governance of universities. These organizations do not sub- stitute state governance however, but constitute an additional governance layer. Third, and along with these changes, the universities themselves are internally transforming into organizational actors, by becoming integrated, goal oriented and competitive entities wherein management and leadership play an ever-important role.2 Con- ceptualizing the university as an organizational actor is at odds with the traditional conception of European universities, both in academic writing and in universities’ day- to-day practices. Up until the past few decades, universities were caught between the state and academic self-governance and, therefore, traditionally there was not much legitimate space for organizational management and leadership within universities. https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press S2 Georg Krücken et al. Both external and internal governance changes were spurred by a series of devel- opments in national and European policymaking, which have strengthened the institutional and individual accountability in higher education while weakening professional self-control. G E O R G K R Ü C K E N * , L A R S E N G W A L L * * a n d E R I K D E C O R T E † Not surprisingly, these changes have generated a plethora of unintended consequences, and the criticism of New Public Management reforms has grown stronger over time, questioning the alleged de-bureaucratization and related positive spill-over effects on overall performance, university autonomy, and individual motivation. Numerous critical studies exist on accreditation and evalua- tion processes that came with the Bologna process, on university leadership and management, on new regulatory frameworks and the changing role of state therein, and on related changes within universities that come with such regulatory frame- works.3 These studies have generated a lot of insights, in particular by comparing different countries and universities. However, and this is striking, the existing studies barely address the effects of governance reforms on creativity, even though creativity is indispensable for conducting high-quality academic research, and high-quality academic teaching requires creativity as well. Studying creativity in the context of governance seems to be a veritable ‘obstacle épistémologique’, to make use of a concept introduced by the French philosopher of science, Gaston Bachelard.4 Not always entirely consciously, we make assumptions concerning a subject that do not allow us to fully explore that subject. Studying creativity in the context of governance is such an ‘obstacle épistémologique’ as we tend to associate creativity with the inner and unregulated properties of individuals. At first glance, Robert K. Merton’s posthumously published book, The Travels and Adventures of Serendipity, confirms this view with regard to science.5 On the basis of a lot of historical material, Merton, one of the founders of the scientific study of the science system, together with his co-author Elinor Barber, describes how the gene- ration of scientific knowledge is characterized by non-linear processes, wherein surprise, deviation and luck are of paramount importance. Such processes can be handled only by exceptionally creative individuals. With the title of the book, the two authors refer to an ancient Persian fairy tale, ‘The Three Princes of Serendip’, to explain the processes of scientific discovery. During their travels, these three princes unintentionally deviate from their planned route, and as a result they have to deal actively and creatively with the unknown and the unexpected challenges encountered on their way. In this process, the three princes make important discoveries by accident. Merton and Barber use the travels of the three princes as a metaphor for scientific discoveries. For science, though, pure creativity does not suffice. https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press G E O R G K R Ü C K E N * , L A R S E N G W A L L * * a n d E R I K D E C O R T E † It also needs, as Merton and Barber outline in their book, theoretical expectations and methodological skills in order to contextualize the unknown and the unexpected. In the words of Louis Pasteur: ‘Le hasard ne favorise que les esprits préparés’.6 What do these insights into the winding paths of science mean for the relationship between governance and creativity? Are they really two contradicting requirements for universities, who are expected to demonstrate both more accountability and more knowledge creation? Is this relationship a zero-sum game, in which more governance necessarily means less creativity and vice versa, or can we develop sensible syntheses https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press S3 Introduction between the two requirements? How should external rules and regulations be formulated in order to facilitate and not impede creativity? There are certainly no clear-cut answers to these questions, but they are eminent for the future of academia. One approach to address these questions is to allow for what organizational researchers call ‘slack’ in universities.7 In most research, slack has a bad reputation as it implies wasting of resources within organizations. However, business firms increasingly make use of slack and deliberately do not use all of their resources efficiently by allowing their members to engage in activities that do not serve a clear- cut purpose, and in this manner foster creativity and develop innovative solutions. However, for universities the opposite seems to be true. Increasing third-party competition, externally stimulated research networks, performance indicators and short-term incentive schemes are supposedly straightforward and lead towards a direction that hardly fosters creativity in research and teaching. Therefore, more slack would lessen the pressure on universities and their members by allowing for deviations and winding paths, being well aware of the fact that resources will not always be used efficiently and at times will even be wasted. Allowing for creativity by such indirect means implies a high degree of trust between the various governance actors that is not always given. The discussion of appropriate forms of university governance with regard to creativity expresses an erosion of trust. This situation requires serious answers from the academic field. One approach to address these questions is to allow for what organizational researchers call ‘slack’ in universities.7 In most research, slack has a bad reputation as it implies wasting of resources within organizations. G E O R G K R Ü C K E N * , L A R S E N G W A L L * * a n d E R I K D E C O R T E † However, business firms increasingly make use of slack and deliberately do not use all of their resources efficiently by allowing their members to engage in activities that do not serve a clear- cut purpose, and in this manner foster creativity and develop innovative solutions. Against the above backdrop, the HERCulES group within Academia Europaea took the initiative for a conference on the topic ‘University Governance: Impeding or Facilitating Creativity?’. It was realized in collaboration with the International Centre for Higher Education Research Kassel (INCHER-Kassel) at the University of Kassel, Germany and by means of generous support from, and in collaboration with, the German Volkswagen Foundation (VolkswagenStiftung). This special issue of European Review is based on papers presented at the conference that took place on 29 and 30 September 2016, at the Royal Palace of Herrenhausen, Hannover. It includes a broad variety of academic researchers dealing with issues of governance and creativity in universities as well as practitioners representing the field of higher education leadership. The first contribution is the opening address by Wilhelm Krull, Secretary General of the Volkswagen Foundation, where he provides a background for the conference and sets the scene. He is followed by Ivar Bleiklie, who presents three different per- spectives on the relation between university governance and creativity, i.e. with reference to the Medieval University, the Humboldt University and the present time managerial university. In relation to the latter, Michael Power discusses the recent requirements in the United Kingdom that universities should demonstrate the social and economic impact of their research. Performance management is also the topic of the contribution by Peter Scott, who elaborates on its relationship to new modes of learning, more open curricula and more distributed patterns of research. In a second group of contributions, the paper by Francisco Michavila and Jorge Martinez analyses the relationship between autonomy and excellence in a group of countries. This in turn is closely related to the discussion of Jean-Claude Thoenig and Catherine Paradeise on higher education institutions as strategic actors and that of https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press Georg Krücken et al. S4 Jetta Frost and Fabian Hattke on ways to balance managerial and collegial gover- nance and thereby create viable university commons. Similarly, Gerhard Casper in his contribution makes a plea for accountable leadership as well as flexibility on all levels in a university. References 1. I. Bleiklie, J. Enders and B. Lepori (Eds) (2017) Managing Universities Policy and Organizational Change from a Western European Comparative Perspective (Palgrave Studies in Global Higher Education, Basingstoke: Palgrave Macmillan); and J. Frost, F. Hattke and M. Reihlen (Eds) (2016) Multi-level Governance in Universities: Strategy, Structure, Control (New York: Springer). 2. G. Krücken and F. Meier (2006) Turning the university into an organizational actor. In: G. Drori, J. Meyer and H. Hwang (Eds.), Globalization and Organization (Oxford: Oxford University Press), pp. 241–257. 3. See for example, R. Whitley, J. Gläser and L. Engwall (Eds) (2010) Reconfiguring Knowledge Production: Changing Authority Relationship in the Sciences and their Consequences for Intellectual Innovation (Oxford: Oxford University Press); and R. Whitley and J. Gläser (Eds) (2014) Organisational Transformation and Scientific Change: The Impact of Institutional Restructuring on Universities and Intellectual Innovation, Research in the Sociology of Organizations (Bingley: Emerald Group). 4. G. Bachelard (1938) La Formation de l’esprit scientifique: Contribution à une psychanalyse de la connaissance objective (Paris: J. Vrin). 5. R.K. Merton and E. Barber (2004) The Travels and Adventures of Serendipity: A Study in Sociological Semantics and the Sociology of Science (Princeton, NJ: Princeton University Press). 6. L. Pasteur and L.-P. Vallery-Radot (1939) Oeuvres de Pasteur. Tome 7, Mélanges scientifiques et littéraires (Paris: Masson & Cie), p. 129. 7. R.M. Cyert and J.G. March (1963) A Behavioral Theory of the Firm (New Jersey: Prentice-Hall). G E O R G K R Ü C K E N * , L A R S E N G W A L L * * a n d E R I K D E C O R T E † In a third group of contributions, Sandra Ohly provides a review of creativity research and discusses implications of this body of knowledge for universities, while Gili Drori presents governance models for academic creativity. They are followed by Jan De Groof, who argues that creativity among universities is likely to increase with a larger differentiation among them, and Lauritz B. Holm-Nielsen, who elaborates on the implications of universities moving from being local to global actors. Together, the contributions in this Special Issue demonstrate that modern uni- versity governance may not always facilitate the creativity that is expected to be the hallmark of universities. In contrast, it may hamper creativity through too short-term perspectives. It therefore appears important for the future to find ways to handle the accountability of universities in a way that provides opportunities for them to con- tribute to a long-term creativity. About the Authors Georg Krücken is Director of the International Centre for Higher Education Research Kassel (INCHER-Kassel) and Professor of Sociology and Higher https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press S5 Introduction Education Research at the University of Kassel. After undergraduate and graduate studies in sociology, philosophy, and political sciences at Bielefeld University and the University of Bologna, Krücken received his PhD in sociology from Bielefeld University in 1996, where he worked as an associate professor until 2006. From 1999 to 2001 he was a visiting scholar at the Department of Sociology at Stanford University. He taught as a guest professor at the Institute for Science Studies, University of Vienna, and at the Centre de Sociologie des Organisations, Sciences Po, Paris. Before coming to Kassel he held the endowed Chair for Science Organization, Higher Education and Science Management at the University of Speyer. His research interests include science studies, organizational studies, the management of higher education, and neo-institutional theory. Lars Engwall is professor emeritus of management at Uppsala University, Sweden. His research has been particularly directed toward the production and diffusion of management knowledge. Among his recent publications are From Books to MOOCs? (2016, ed. with Erik De Corte and Ulrich Teichler, Portland Press), Defining Management (2016 with Matthias Kipping and Behlül Üsdiken, Routledge) and Corporate Governance in Action (ed. 2018, Routledge). He is an elected member of a number of learned societies and has received honorary degrees from Åbo Akademi University and the Stockholm School of Economics. Erik De Corte is Emeritus Professor (of Educational Psychology) in the Faculty of Psychology and Educational Sciences at the University of Leuven, Belgium, where he chaired, from August 1994 until July 1998, the Department of Educational Sciences. His major research interest is to contribute to the development of theories of learning from instruction and the design of powerful learning environments, focusing on learning, teaching, and assessment of thinking and problem solving. He was the first President (1985–1989) of the European Association for Research on Learning and Instruction (EARLI). In 2000 and 2003, respectively, he received doctorates honoris causa of the University of Johannesburg, and the University of the Free State, Bloemfontein, South Africa. During the academic year 2005–2006 he stayed as a Fellow at the Center for Advanced Study in Behavioral Sciences at Stanford, USA. (http://perswww.kuleuven.be/ ~ u0004455) https://doi.org/10.1017/S1062798717000485 Published online by Cambridge University Press
https://openalex.org/W4309309856
https://www.qeios.com/read/U81D5K/pdf
English
null
Review of: "[Case Study] Mumps pneumonia in adults – a forgotten entity"
null
2,022
cc-by
156
Qeios, CC-BY 4.0 · Review, November 16, 2022 Qeios ID: U81D5K · https://doi.org/10.32388/U81D5K Review of: "Mumps pneumonia in adults – a forgotten entity" Muhammad Suleman Rana Muhammad Suleman Rana Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. The article is interesting and well written. In this article authors discussed very important and rare implications of Mumps virus. Mumps rarely cause pneumonia in Patients, however, the findings and investigation reported here are the suggestive of this rare cause as all pneumonia causing viral agents are rollout. It will be more clear if the author rollout the bacterial agents causing pneumonia. There are minor grammatic errors in the text and needs to improve the English language of the manuscript draft before acceptance and publication. This study will helpful for the understanding of complications caused by Mumps virus and patients management as well. Qeios ID: U81D5K · https://doi.org/10.32388/U81D5K 1/1
https://openalex.org/W1812467058
https://dash.harvard.edu/bitstream/1/33459441/1/nihms571993.pdf
English
null
Clinical reappraisal of the Composite International Diagnostic Interview Screening Scales (CIDI‐SC) in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS)
International Journal of Methods in Psychiatric Research
2,013
cc-by
17,547
Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Open Access Policy Articles, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#OAP Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33459441 Clinical reappraisal of the Composite International Diagnostic Interview Screening Scales (CIDI-SC) in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS) Clinical reappraisal of the Composite International Diagnostic Interview Screening Scales (CIDI-SC) in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS) Citation Kessler, Ronald C., Patcho N. Santiago, Lisa J. Colpe, Catherine L. Dempsey, Michael B. First, Steven G. Heeringa, Murray B. Stein, et al. 2013. “Clinical Reappraisal of the Composite International Diagnostic Interview Screening Scales (CIDI-SC) in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS).” International Journal of Methods in Psychiatric Research 22 (4) (December): 303–321. doi:10.1002/mpr.1398. Published Version doi:10.1002/mpr.1398 NIH Public Access Author Manuscript NIH-PA Author Manuscript Published in final edited form as: Int J Methods Psychiatr Res. 2013 December ; 22(4): 303–321. doi:10.1002/mpr.1398. Additional Contributions: The Army STARRS Team consists of Co-Principal Investigators: Robert J. Ursano, MD (Uniformed Services University of the Health Sciences) and Murray B. Stein, MD, MPH (University of California San Diego and VA San Diego Healthcare System); Site Principal Investigators: Steven Heeringa, PhD (University of Michigan) and Ronald C. Kessler, PhD (Harvard Medical School); NIMH collaborating scientists: Lisa J. Colpe, PhD, MPH and Michael Schoenbaum, PhD; Army liaisons/ consultants: COL Steven Cersovsky, MD, MPH (USAPHC) and Kenneth Cox, MD, MPH (USAPHC). Other team members: Pablo A. Aliaga, MA (Uniformed Services University of the Health Sciences); COL David M. Benedek, MD (Uniformed Services University of the Health Sciences); Susan Borja, PhD (National Institute of Mental Health); Gregory G. Brown, PhD (University of California San Diego); Laura Campbell-Sills, PhD (University of California San Diego); Catherine L. Dempsey, PhD, MPH (Uniformed Services University of the Health Sciences); Richard Frank, PhD (Harvard Medical School); Carol S. Fullerton, PhD (Uniformed Services University of the Health Sciences); Nancy Gebler, MA (University of Michigan); Joel Gelernter, MD (Yale University); Robert K. Gifford, PhD (Uniformed Services University of the Health Sciences); Stephen E. Gilman, ScD (Harvard School of Public Health); Marjan G. Holloway, PhD (Uniformed Services University of the Health Sciences); Paul E. Hurwitz, MPH (Uniformed Services University of the Health Sciences); Sonia Jain, PhD (University of California San Diego); Tzu-Cheg Kao, PhD (Uniformed Services University of the Health Sciences); Karestan C. Koenen, PhD (Columbia University); Lisa Lewandowski-Romps, PhD (University of Michigan); Holly Herberman Mash, PhD (Uniformed Services University of the Health Sciences); James E. McCarroll, PhD, MPH (Uniformed Services University of the Health Sciences); Katie A. McLaughlin, PhD (Harvard Medical School); James A. Naifeh, PhD (Uniformed Services University of the Health Sciences); Matthew K. Nock, PhD (Harvard University); Rema Raman, PhD (University of California San Diego); Nancy A. Sampson, BA (Harvard Medical School); LCDR Patcho N. Santiago, MD, MPH (Uniformed Services University of the Health Sciences); Michaelle Scanlon, MBA (National Institute of Mental Health); Jordan Smoller, MD, ScD (Harvard Medical School); Nadia Solovieff, PhD (Harvard Medical School); Michael L. Thomas, PhD (University of California San Diego); Christina Wassel, PhD (University of Pittsburgh); and Alan M. Zaslavsky, PhD (Harvard Medical School). Declaration of interest statement: In the past five years Kessler has been a consultant for Eli Lilly & Company, Glaxo, Inc., Integrated Benefits Institute, Ortho-McNeil Janssen Scientific Affairs, Pfizer Inc., Sanofi-Aventis Groupe, Shire US Inc., and Transcept Pharmaceuticals Inc. and has served on advisory boards for Johnson & Johnson. Kessler has had research support for his epidemiological studies over this time period from Eli Lilly & Company, EPI-Q, GlaxoSmithKline, Ortho-McNeil Janssen Scientific Affairs, Sanofi-Aventis Groupe, Shire US, Inc., and Walgreens Co. Kessler owns a 25% share in DataStat, Inc. First received consultation fees from the Henry M. Jackson Foundation for the Advancement of Military Medicine, the sponsor of the study. Stein has in the last three years been a consultant for Healthcare Management Technologies and had research support for pharmacological imaging studies from Janssen. The remaining authors report nothing to disclose. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility NIH-PA Author Manuscript Clinical reappraisal of the Composite International Diagnostic Interview Screening Scales (CIDI-SC) in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS) r Manuscript NIH-PA Author Manuscript Ronald C. Kessler1, Patcho N. Santiago2, Lisa J. Colpe3, Catherine L. Dempsey2, Michael B. First4, Steven G. Heeringa5, Murray B. Stein6, Carol S. Fullerton2, Michael J. Gruber1, James A. Naifeh2, Matthew K. Nock7, Nancy A. Sampson1, Michael Schoenbaum3, Alan M. Zaslavsky1, and Robert J. Ursano2 on behalf of the Army STARRS Collaborators 1Department of Health Care Policy, Harvard Medical School, Boston, Massachusetts, USA NIH-PA Author Manuscript 2Center for the Study of Traumatic Stress, Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA enter for the Study of Traumatic Stress, Department of Psychiatry, Uniformed Services niversity of the Health Sciences, Bethesda, Maryland, USA 3National Institute of Mental Health, Bethesda, Maryland, USA 4Department of Psychiatry, Columbia University, New York, New York and New York State Psychiatric Institute, New York, New York, USA epartment of Psychiatry, Columbia University, New York, New York and New York State ychiatric Institute, New York, New York, USA 5University of Michigan, Institute for Social Research, Ann Arbor, Michigan, USA Correspondence: Ronald C. Kessler, PhD, Department of Health Care Policy, Harvard Medical School, 180 Longwood Avenue, Boston, MA 02115; kessler@hcp.med.harvard.edu; 617-432-3587 (voice); 617-432-3588 (fax). Additional Contributions: The Army STARRS Team consists of Co-Principal Investigators: Robert J. Ursano, MD (Uniformed Services University of the Health Sciences) and Murray B. Stein, MD, MPH (University of California San Diego and VA San Diego Healthcare System); Site Principal Investigators: Steven Heeringa, PhD (University of Michigan) and Ronald C. Kessler, PhD (Harvard Medical School); NIMH collaborating scientists: Lisa J. Colpe, PhD, MPH and Michael Schoenbaum, PhD; Army liaisons/ consultants: COL Steven Cersovsky, MD, MPH (USAPHC) and Kenneth Cox, MD, MPH (USAPHC). Other team members: Pablo A. Aliaga, MA (Uniformed Services University of the Health Sciences); COL David M. Benedek, MD (Uniformed Services University of the Health Sciences); Susan Borja, PhD (National Institute of Mental Health); Gregory G. Brown, PhD (University of California San Diego); Laura Campbell-Sills, PhD (University of California San Diego); Catherine L. Dempsey, PhD, MPH (Uniformed Services University of the Health Sciences); Richard Frank, PhD (Harvard Medical School); Carol S. Fullerton, PhD (Uniformed Services University of the Health Sciences); Nancy Gebler, MA (University of Michigan); Joel Gelernter, MD (Yale University); Robert K. Gifford, PhD (Uniformed Services University of the Health Sciences); Stephen E. Gilman, ScD (Harvard School of Public Health); Marjan G. Holloway, PhD (Uniformed Services University of the Health Sciences); Paul E. Correspondence: Ronald C. Kessler, PhD, Department of Health Care Policy, Harvard Medical School, 180 Longwood Avenue, Boston, MA 02115; kessler@hcp.med.harvard.edu; 617-432-3587 (voice); 617-432-3588 (fax). Abstract A clinical reappraisal study was carried out in conjunction with the Army STARRS All-Army Study (AAS) to evaluate concordance of DSM-IV diagnoses based on the Composite International Diagnostic Interview screening scales (CIDI-SC) and PTSD Checklist (PCL) with diagnoses based on independent clinical reappraisal interviews (Structured Clinical Interview for DSM-IV [SCID]). Diagnoses included: lifetime mania/hypomania, panic disorder, and intermittent explosive disorder; 6-month adult attention-deficit/hyperactivity disorder; and 30-day major depressive episode, generalized anxiety disorder, PTSD, and substance (alcohol or drug) use disorder (abuse or dependence). The sample (n=460) was weighted for over-sampling CIDI- SC/PCL screened positives. Diagnostic thresholds were set to equalize false positives and false negatives. Good individual-level concordance was found between CIDI-SC/PCL and SCID diagnoses at these thresholds (AUC = .69–.79). AUC was considerably higher for continuous than dichotomous screening scale scores (AUC = .80–.90), arguing for substantive analyses using not only dichotomous case designations but also continuous measures of predicted probabilities of clinical diagnoses. NIH-PA Author Manuscript Clinical reappraisal of the Composite International Diagnostic Interview Screening Scales (CIDI-SC) in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS) Hurwitz, MPH (Uniformed Services University of the Health Sciences); Sonia Jain, PhD (University of California San Diego); Tzu-Cheg Kao, PhD (Uniformed Services University of the Health Sciences); Karestan C. Koenen, PhD (Columbia University); Lisa Lewandowski-Romps, PhD (University of Michigan); Holly Herberman Mash, PhD (Uniformed Services University of the Health Sciences); James E. McCarroll, PhD, MPH (Uniformed Services University of the Health Sciences); Katie A. McLaughlin, PhD (Harvard Medical School); James A. Naifeh, PhD (Uniformed Services University of the Health Sciences); Matthew K. Nock, PhD (Harvard University); Rema Raman, PhD (University of California San Diego); Nancy A. Sampson, BA (Harvard Medical School); LCDR Patcho N. Santiago, MD, MPH (Uniformed Services University of the Health Sciences); Michaelle Scanlon, MBA (National Institute of Mental Health); Jordan Smoller, MD, ScD (Harvard Medical School); Nadia Solovieff, PhD (Harvard Medical School); Michael L. Thomas, PhD (University of California San Diego); Christina Wassel, PhD (University of Pittsburgh); and Alan M. Zaslavsky, PhD (Harvard Medical School). Declaration of interest statement: In the past five years Kessler has been a consultant for Eli Lilly & Company, Glaxo, Inc., Integrated Benefits Institute, Ortho-McNeil Janssen Scientific Affairs, Pfizer Inc., Sanofi-Aventis Groupe, Shire US Inc., and Transcept Pharmaceuticals Inc. and has served on advisory boards for Johnson & Johnson. Kessler has had research support for his epidemiological studies over this time period from Eli Lilly & Company, EPI-Q, GlaxoSmithKline, Ortho-McNeil Janssen Scientific Affairs, Sanofi-Aventis Groupe, Shire US, Inc., and Walgreens Co. Kessler owns a 25% share in DataStat, Inc. First received consultation fees from the Henry M. Jackson Foundation for the Advancement of Military Medicine, the sponsor of the study. Stein has in the last three years been a consultant for Healthcare Management Technologies and had research support for pharmacological imaging studies from Janssen. The remaining authors report nothing to disclose. NIH-PA Author Manuscript NIH-PA Author Manuscript Page 2 Page 2 NIH-PA Author Manuscript Kessler et al. 6Departments of Psychiatry and Family and Preventive Medicine, University of California San Diego, La Jolla, California and VA San Diego Healthcare System, San Diego, California, USA 7Department of Psychology, Harvard University, Cambridge, Massachusetts, USA 6Departments of Psychiatry and Family and Preventive Medicine, University of California San Diego, La Jolla, California and VA San Diego Healthcare System, San Diego, California, USA 7Department of Psychology, Harvard University, Cambridge, Massachusetts, USA 7Department of Psychology, Harvard University, Cambridge, Massachusetts, USA Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Keywords Composite International Diagnostic Interview (CIDI); CIDI Screening Scales (CIDI-SC); diagnostic concordance; PTSD Checklist (PCL); screening scales; validity As described in more detail earlier in this issue (Kessler et al., this issue) and elsewhere (Ursano et al., under review), the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS; www.armystarrs.org) is a multi-component epidemiological and neurobiological study of risk and resilience factors for suicidality and its psychopathological correlates in the U.S. Army. The literature on risk and resilience factors for suicidality makes it clear that mental disorders are powerful risk factors (Nock et al., 2008; Nock et al., in press). As a result, a wide range of mental disorders were assessed in the Army STARRS surveys. However, due to the size and logistical complexities of these surveys, which are described earlier in this issue (Heeringa et al., this issue), it was impossible to administer an in-depth psychiatric diagnostic interview to participants. Instead, mental disorders were assessed with short self-administered screening scales. NIH-PA Author Manuscript NIH-PA Author Manuscript A number of screening scales exist to assess such disorders as attention-deficit hyperactivity disorder (Kessler et al., 2005a), bipolar disorder (Hirschfeld et al., 2000), generalized anxiety disorder (Spitzer et al., 2006), major depressive episode (Kroenke et al., 2001), and post-traumatic stress disorder (Breslau et al., 1999). Although in some cases these scales were developed originally to assess symptom severity among patients in treatment, they subsequently have been adapted for use either as web-based tools for self-diagnosis (Donker et al., 2009; Farvolden et al., 2003) or as brief evaluations of mental disorders in primary care settings or community surveys (Broadhead et al., 1995; Gaynes et al., 2010; Hunter et Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Page 3 Kessler et al. NIH-PA Author Manuscript al., 2005; Kessler et al., 2012). Clinical reappraisal studies comparing scores on these screening scales with independent clinical diagnoses show that many of these screening scales have good concordance with clinical diagnoses (Kessler and Pennell, in press). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth The screening scales that form the core diagnostic assessment in Army STARRS are the WHO Composite International Diagnostic Interview Screening Scales (CIDI-SC) (Kessler et al., 2012). These were selected largely because they are a coordinated set of short scales that cover a wide range of disorders and have good psychometric properties. Keywords However, another appeal of the CIDI-SC scales is that they are embedded in the WHO Composite International Diagnostic Interview (CIDI) (Kessler and Üstün, 2004), the research diagnostic interview used in most large-scale epidemiological surveys of psychiatric disorders throughout the world (Haro et al., 2006). Use of the CIDI-SC scales in Army STARRS thereby creates a crosswalk to an in-depth diagnostic interview that might be used in more focused follow-up studies of Army STARRS high-risk subsamples. The exception is that we used the Post-Traumatic Stress Disorder (PTSD) Checklist (PCL) (Weathers et al., 1993) to assess PTSD based on the widespread use of this screening scale in previous military studies of PTSD (Barnes et al., 2013; Brown et al., 2012; Jones et al., 2012) coupled with strong evidence for the validity of the PCL in both military and civilian samples (Wilkins et al., 2011). NIH-PA Author Manuscript Although good concordance of DSM-IV (American Psychiatric Association, 1994) diagnoses based on the CIDI-SC (Kessler et al., 2005a; Kessler et al., 2007; Kessler et al., 2006a; Kessler et al., 2012) and PCL (Wilkins et al., 2011) with diagnoses based on independent clinical reappraisal interviews has been reported in a number of studies, this does not guarantee that these screening scales will perform equally well among soldiers in the Army STARRS surveys. As a result, a new clinical reappraisal study was carried out in conjunction with the Army STARRS All-Army Study (AAS) (Ursano et al., under review) to examine the psychometric characteristics of the CIDI-SC and PCL in the context of the field conditions encountered in the Army STARRS surveys. Results of this clinical reappraisal study are presented in the current report. nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. The samples NIH-PA Author Manuscript The All-Army Study (AAS)—As described in more detail previously in this issue (Kessler et al., this issue), the AAS is a cross-sectional survey of active duty Army personnel exclusive of Soldiers in Basic Combat Training administered in quarterly replicates to a total of nearly 50,000 Soldiers during calendar years 2011–2012. Each quarterly AAS replicate consisted of a stratified (by Army Command-location and unit size) probability sample of Army units, excluding units of fewer than 30 Soldiers (less than 2% of all Army personnel). All targeted personnel in these units were ordered to attend an informed consent presentation explaining study purposes, confidentiality procedures, and the voluntary nature of participation before requesting written informed consent for a group self-administered questionnaire (SAQ). Respondents were additionally asked for consent to link their Army and Department of Defense administrative records to their SAQ responses and to participate in future longitudinal follow-up data collections. Identifying information Page 4 Kessler et al. NIH-PA Author Manuscript (name, birthday, SSN for record linkage; telephone number, email, secondary contact information for longitudinal follow-up) was collected from consenting respondents and kept in a separate secure file. These recruitment, consent, and data protection procedures were approved by the Human Subjects Committees of the Uniformed Services University of the Health Sciences for the Henry M. Jackson Foundation (the primary grantee), the Institute for Social Research at the University of Michigan (the organization implementing Army STARRS surveys), and all other collaborating organizations. NIH-PA Author Manuscript NIH-PA The clinical reappraisal study (CRS) was carried out between March 2012 and November 2012. All quarterly AAS replicates over that time period were based on representative samples of Soldiers stationed both in the continental U.S. and elsewhere in the world other than a combat theater, while the Q2-3 2012 replicates also included probability samples of Soldiers stationed in Afghanistan who were surveyed in group-administered sessions while they were passing through Kuwait either leaving for or returning from their mid-tour leave. However, because of logistical issues requiring that the CRS clinical reappraisal interviews be administered within two weeks of the AAS survey, the CRS was implemented exclusively in the continental U.S. among Regular (active component) Army AAS respondents providing consent for administrative data linkage and completing the SAQ. Activated Army Reserve and National Guard respondents were excluded from the CRS due to small numbers. nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. The samples NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth Although, as noted above, all unit members in these replicates were ordered to report to the informed consent session, 19.4% of those in the replicates used for the CRS were absent due to conflicting duty assignments. The vast majority of those attending (99.6%) consented to the survey and 98.8 % of consenters completed the survey. In addition, 71.4% of completers provided successful record linkage. Most incomplete surveys were due to logistical complications (e.g., units either arriving late to survey sessions or having to leave early), although some respondents needed more than the allotted 90 minutes to complete the survey. The survey completion-successful-linkage cooperation rate was 63.9% and the completion-successful-linkage response rate was 51.5 % based on the American Association of Public Opinion Research COOP1 and RR1 calculation methods (American Association for Public Opinion Research, 2009). NIH-PA Author Manuscript NIH-PA Author Manuscript The clinical reappraisal study sample—In order to evaluate the concordance of diagnoses based on the CIDI-SC and PCL in the AAS with independent clinical diagnoses, a sample of AAS respondents was selected to participate in clinical follow-up interviews within two weeks of completing the AAS in selected AAS sessions. As soon as the AAS survey was completed in these sessions, each AAS respondent was classified as threshold, subthreshold or no on each of the eight screening scales considered here. A probability subsample of AAS respondents from the session was then invited to participate in a confidential clinical reappraisal interview with the goal of obtaining a total (i.e., over the entire 9-month interview recruitment period) of 30 CRS interviews with respondents selected at random from those classified as threshold cases on each diagnosis, 10 from among those classified as subthreshold on each diagnosis, and 40 respondents selected at random from those classified as meeting neither threshold nor subthreshold criteria for any diagnosis. CRS respondents with each diagnosis were selected with replacement (i.e., the Kessler et al. Page 5 NIH-PA Author Manuscript same respondent could be selected for more than one diagnosis). The initial sampling fractions varied across disorders due to differences in prevalence among the disorders. These sampling fractions were then modified over sessions in order to achieve a roughly equal distribution of cases within each diagnosis across sessions while meeting the sample quotas. The samples The 460 clinical interviews completed by the end of the CRS is more than the 360 needed (i.e., 30 interviews with threshold CIDI-SC/PCL cases for each of eight disorders plus 10 interviews with CIDI-SC/PCL subthreshold cases for each of these disorders plus 40 respondents screening negative on all eight CIDI-SC/PCL scales) because it was necessary to recruit additional respondents in the later replicates to fill the sample quotas for the least common disorders. NIH-PA Author Manuscript Invitations to participate in the CRS were made through unit points-of-contact who scheduled two-hour time blocks during which respondents were relieved of their usual duty assignments in order to report to the Army STARRS study office on the installation. Once at the study office, an Army STARRS data collection specialist explained the content and purposes of the CRS and obtained written informed consent to participate. Consenting respondents were then assigned to a private room where they were administered the CRS interview telephonically by one of the CRS clinical interviewers, all of whom were located at the Uniformed Services University of the Health Sciences (USUHS) in Bethesda, Maryland. The CRS clinical supervisor (CLD), also located at USUHS, coordinated with Army STARRS data collection specialists at the local AAS installations to schedule these remote CRS telephone interviewers. NIH-PA Author Manuscript nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. An overview of screening scale content A similar attempt to collect information about subthreshold symptoms was made in selecting stem question skip rules for each of the other screening scales. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Aut The CIDI-SC mania-hypomania (MHM) scale focuses on subthreshold hypomania as well as mania and hypomania based on evidence that subthreshold hypomania can be highly impairing (Merikangas et al., 2007). In addition, the questions focus on lifetime rather than 30-day prevalence due to the fact that recent bipolar disorder (BPD) can manifest as either MHM or as MDE. As described in more detail elsewhere (Kessler et al., 2006a; Kessler et al., 2012), the single MHM entry question begins with a vignette describing a hypomanic episode and then asks respondents if they ever had an episode of this sort at any time in their life. A positive response is followed by four questions about the frequency of core MHM symptoms during a typical intense episode of this sort. These symptoms include being much higher, happier, or optimistic than usual; much more irritable than usual; so hyper or wound up that you felt out of control; and having thoughts race through your mind so fast you could hardly keep track of them. Respondents who report that at least one of these symptoms occurrs at least some of the time during a typical intense episode are then administered six additional questions about the inclusion criteria of MHM and are then asked about episode recency to assess 30-day prevalence of MHM. Lifetime rather than 30- day MHM is evaluated here due to the rarity of 30-day MHM in the AAS sample. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Au The CIDI-SC panic disorder (PD) scale includes two entry questions about lifetime atacks of panic, anxiety, or strong fear that came on very suddenly and made you feel very frightened or uneasy; and attacks of heart pounding or chest pain that came on very suddenly and made you feel very frightened or uneasy (Kessler et al., 2012). A positive response to either entry question is followed by one additional question on how often these attacks are triggered (i.e., occur in situations where the respondent has a strong fear – like a fear of snakes or heights – or where the respondent is in real danger – like a car accident) versus untriggered (i.e., occur without provocation “out of the blue”). An overview of screening scale content Screening scales were included in the AAS for eight DSM-IV disorders that have been found in previous general population studies to be significant predictors of suicidality (Nock et al., 2008; Nock et al., in press; Nock et al., 2009). These include two mood disorders (major depressive episode, mania/hypomania), three anxiety disorders (panic disorder with or without agoraphobia, generalized anxiety disorder, PTSD), and three externalizing disorders (adult attention-deficit hyperactivity disorder, intermittent explosive disorder, substance use disorder). NIH-PA Author Manuscript NIH-PA Author Manuscript Symptom questions in most CIDI-SC scales ask respondents about the frequency of particular symptoms over the 30 days before interview using the response options all or almost all of the time, most of the time, some of the time, a little of the time, and none of the time. Each CIDI-SC scale has an embedded skip logic whereby all respondents are administered one or more entry questions and then either skipped if they fail to endorse these questions or continue to a series of follow-up questions if they endorse the entry question(s). This approach was designed to reduce overall scale administration time and respondent burden while minimizing the number of true positives incorrectly skipped out by the entry questions. Respondents who fail to endorse any of the entry questions are asked a total of 46 questions across all eight scales combined, while respondents who endorse every single question are asked an additional 82 questions. The CIDI-SC major depressive episode (MDE) scale begins with four entry questions that ask about being sad, depressed, or discouraged; having little or no interest or pleasure in Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Page 6 Kessler et al. Page 6 NIH-PA Author Manuscript things; and feeling down on yourself, no good, or worthless (Kessler et al., 2012). Respondents who report that at least one of these symptoms occurred at least some of the time in the past 30 days are administered 10 additional questions to assess the inclusion criteria of MDE. The some of the time threshold, while low for a DSM-IV diagnosis of MDE (which requires depressive symptoms to last most of the day nearly every day for two weeks or longer), was chosen because we wanted to collect information not only on threshold cases but also on subthreshold manifestions of MDE. An overview of screening scale content Respondents who report ever having untriggered attacks are then administered 13 additional questions to assess the remaining DSM-IV inclusion criteria of PD. Lifetime rather than 30-day PD is evaluated here due to the rarity of 30-day PD in the AAS sample. NIH-PA Author Manuscript NIH-PA Author Manuscript The CIDI-SC generalized anxiety disorder (GAD) scale includes five entry questions about 30-day frequency of being anxious or nervous; worried about a number of different things; more anxious or worried than other people in your same situation; worried about things most other people don’t worry about; and having trouble controlling your worry or anxiety (Kessler et al., 2012). Respondents who report any of these symptoms at least some of the time are administered an additional nine questions to assess the remaining DSM-IV inclusion criteria of GAD along with a final question to assess persistence of symptoms. As Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 7 Page 7 NIH-PA Author Manuscript a minimum duration of six months is required to meet DSM-IV criteria of GAD, the CIDI- SC assesses duration of symptoms, although the concordance data reported here are for symptoms in the 30-days before interview. NIH-PA Author Manuscript NIH-PA A As noted above, PTSD is assessed in the AAS with the PCL. The PCL Civilian version (Weathers et al., 1993) was used in Army STARRS because we covered traumatic experiences both in and out of the line of duty. This is a 17-question scale that assesses the 17 DSM-IV Criterion B-D symptoms of PTSD. Although there are no entry questions in the PCL, AAS respondents are first asked 15 questions about traumatic experiences (TEs) that might have happened to them during deployments and 15 additional questions about TEs that might have happened at to them at any other time in life. Only respondents who report at least one of these 30 TEs are administered the PCL. The PCL questions ask how much respondents were bothered in the past 30 days by symptoms associated with any of the TEs they ever experienced. Response categories are extremely, quite a bit, moderately, a little bit, and not at all. The CIDI-SC adult attention-deficit/hyperactivity disorder (ADHD) scale includes four entry questions found in previous research to provide an optimal short inclusion screen for ADHD in the adult general population (Kessler et al., 2010a). An overview of screening scale content Respondents who report at least two of these symptoms at least some of the time in the past six months then receive an additional 8 questions shown in a number of previous studies to detect adult ADHD with good accuracy (Kessler et al., 2007; Kessler et al., 2010a; Kessler et al., 2009). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A The CIDI-SC intermittent explosive disorder (IED) scale includes one entry question about lifetime attacks of anger when the respondent all of a sudden … lost control and either broke or smashed something worth more than a few dollars, hit or tried to hurt someone, or threatened someone (Kessler et al., 2006b). A positive response is followed by 6 additional questions that assess the remaining DSM-IV inclusion criteria of IED. As the assessment of IED followed the same logic as the assessment of PD, lifetime rather than 30-day IED is evaluated here in parallel with the evaluation of PD. The CIDI-SC assessment of substance use disorder (SUD), finally, begins with 12 entry questions about quantity-frequency of alcohol use, illicit drug use, and prescription drug misuse, where the latter is defined as use either without a doctor’s prescription, more than prescribed, or to get high, buzzed, or numbed out. Prescription drug misuse is included in the assessment based on evidence that it is considerably more common than illicit drug use in the Army (Bray et al., 2010). Respondents who report any of these types of substance use are then administered the four CIDI-SC questions about DSM-IV substance abuse in the 30 days before interview and eight additional questions to screen for substance dependence in the 30 days before interview including five from the Severity of Dependence Scale (Gossop et al., 1995) and three additional CIDI-SC questions. Substance use disorders (i.e., either abuse or dependence) are assessed only once for alcohol and/or drugs combined. NIH-PA Author Manuscript NIH-PA Author Manuscript nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Scoring the screening scales Each screening scale was initially scored continuously by summing values across all items in the scale, assigning respondents who were skipped out after screening questions the Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Page 8 Kessler et al. NIH-PA Author Manuscript lowest possible scores on the remaining items. Receiver operating characteristic curve (ROC) analysis (Margolis et al., 2002) was then used to estimate area under the ROC curve (AUC) for the entire continuous scale and to dichotomize the scale at a point that optimized aggregate concordance between the prevalence estimate based on the SCID and the prevalence estimate based on the CIDI-SC at the designated threshold. This threshold also makes the number of false positives equal the number of false negatives. It is noteworthy, though, that other criteria exist to select diagnostic thresholds and that decisions about which threshold to choose can vary depending on the criterion used. For example, if we had wanted to use the screening scales in a primary care setting to select patients for more in-depth evaluation, we might have lowered the threshold to the point where the vast majority of SCID cases were detected. Or if we were using the screening scales to select patients for a clinical intervention, we might have raised the threshold to the point where the vast majority of screened positives consisted of SCID cases. If the relative importance of minimizing false positives and minimizing false negatives can be specified based on the considerations of such competing criteria, it is possible to minimize this weighted sum of errors in a formal way (Kraemer, 1992). Based on these considerations, a number of alternative thresholds are examined below. NIH-PA Author Manuscript The clinical reappraisal interview The clinical reappraisal interview was a modified Research Version, Non-Patient Edition of the Structured Clinical Interview for DSM-IV (SCID-I) (First et al., 2002) focused on the eight syndromes under study with the variations in recall periods noted above to match the recall periods used in the screening scales. As noted above, these interviews were administered by telephone. Telephone administration is now widely accepted in clinical reappraisal studies based on evidence of comparable validity to in-person administration (Kendler et al., 1992; Rohde et al., 1997; Sobin et al., 1993). A great advantage of telephone administration is that a centralized and closely supervised clinical interview staff can carry out the interviews without the geographic restrictions required for face-to-face clinical assessment. A disadvantage is that people without telephones cannot be included in the assessment. As noted below, though, this difficulty was resolved in the Army STARRS CRS by having pre-designated respondents report to the central Army STARRS research office on their installations, where they were placed in a private room and interviewed remotely by telephone. NIH-PA Author Manuscript NIH-PA Author Manuscript A major impediment to making accurate evaluations of concordance between screening scales and clinical diagnoses is the fact that respondents are inconsistent in their reports over time. Indeed, our own previous experience and that of other researchers shows consistently that respondents in community surveys tend to report less and less as they are interviewed more and more due to respondent fatigue (Bromet et al., 1986). Part of this pattern is a tendency for respondents to endorse a smaller number of diagnostic stem questions in follow-up interviews than in initial interviews (Kessler et al., 1998), leading to the biased perception that initial fully-structured assessments overestimate prevalence compared to clinical reappraisal interviews. Consistent with the approach used in a number of other clinical reappraisal studies (Haro et al., 2006; Kessler et al., 2005b; Kessler et al., 1998), we Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 9 Page 9 NIH-PA Author Manuscript modified the conventional blinded clinical re-interview design in three important ways to address this problem. NIH-PA Author Manuscript NIH-PA A First, we unblinded the clinical interviewers to whether respondents endorsed diagnostic stem questions in the CIDI-SC. Importantly, though, we did not unblind clinical interviewers to whether the respondents who endorsed CIDI-SC diagnostic stem questions went on to meet full diagnostic criteria. The clinical reappraisal interview Second, we rephrased entry questions in the clinical reappraisal interviews to acknowledge prior endorsement of diagnostic stem questions in the CIDI-SC/PCL in order to minimize the problem of false negative diagnostic stem responses in the SCID. For example, rather than repeating a question about presence-absence of 30-day depressed mood in the SCID to respondents who reported 30-day depressed mood in the CIDI-SC, SCID interviews began the assessment of major depression with a declarative sentence: “In your earlier survey you reported feeling sad or depressed most of the time over the past 30 days. The next questions ask more about those feelings.” Third, in order to guarantee that this partial unblinding did not bias clinical interviewers in the direction of rating all stem-positive respondents as cases, we enriched the clinical reappraisal sample to include a higher proportion of respondents than in the sample who endorsed CIDI-SC/PCL diagnostic stem questions but did not meet full CIDI-SC/PCL diagnostic criteria. This third feature of the design actually makes the interviewer task more difficult than it would be in a standard clinical reappraisal study in which there is an over- sample of respondents classified as meeting full diagnostic criteria but not of respondents meeting partial criteria. NIH-PA Author Manuscript nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Clinical interviewer training and quality control The SCID interviews were administered by 14 trained clinical interviewers. These included four doctoral-level psychologists, seven MA-level psychologists, and three MSW-level clinical social workers. Half of the interviewers had a decade or more of clinical experiences (10–21 years), while the other half had 3–9 years of clinical experience (two with 3 years of experience and one each with 5, 6, 7, 8, and 9 years of experience). The 32-hour SCID interviewer training program began with a 16-hour centralized group training session taking place over a full weekend that was taught by one of the developers of the SCID (MBF) with the assistance of an experienced SCID supervisor (CLD). Training then continued with biweekly individual and group training sessions with homework assignments totaling 32 hours. The training was carried out at USUHS using a modification of the standard SCID training protocol tailored to the diagnoses assessed by the screening scales. In addition to completing this training, each clinical interviewer was required to pass a proficiency test before they began production interviewing based on trainer and supervisor ratings of three practice interviews using a modified version of the SCID Interviewing Skills Evaluation Form created specifically for this study. NIH-PA Author Manuscript NIH-PA Author Manuscript All SCID interviews were audio-recorded with permission of respondents and responses recorded on a hard copy interview. The supervisor reviewed the tape recordings of the first five interviews carried out by each interviewer and a minimum of 10% of all subsequent Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 10 Page 10 NIH-PA Author Manuscript interviews carried out by each interviewer. The supervisor also reviewed all hard copy interviews completed by all interviewers and reviewed tape recordings of all interviews in which concerns were raised by the hard copy reviews. The symptom-level hard-copy clinical ratings were double-entered into a computerized data file after supervisor review and approval. Each interviewer had a weekly one-on-one feedback meeting with the supervisor and participated in a biweekly group calibration meeting with the supervisor and trainer to prevent rater drift. Diagnoses were made without diagnostic hierarchy rules but with organic exclusions. Analysis methods Weighting—The CRS sample was weighted to adjust for over-sampling respondents screened as threshold or subthreshold using a weighting method that adjusted for the fact that sampling was made with replacement. This is important because a number of the statistics used to describe scale characteristics are biased when differential selection of screened positives and negatives is not taken into account. Analysis of screening scale operating characteristics—As noted above in the description of screening scale scoring, a summary continuous screening scale score was created for each diagnosis by summing scores across the screening scale items. Receiver operating characteristic curve (ROC) analysis (Margolis et al., 2002) was then used to estimate area under the ROC curve (AUC) for the entire scale. Each continuous screening scale was then dichotomized at a threshold that equalized the (weighted) number of false positives and false negatives, thereby maximizing concordance between prevalence estimates based on the SCID and the screening scales. The McNemar χ2 test was used to evaluate the significance of differences between screening scale and SCID prevalence estimates at this threshold. A range of other thresholds was then selected so that SCID prevalence estimates increased monotonically across screening scale strata but did not differ significantly within strata using the logic of stratum-specific likelihood ratio analysis (Pepe, 2003). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH- Screening scale operating characteristics were then evaluated for each of these thresholds. Individual-level concordance was evaluated using AUC and Cohen’s κ (Cohen, 1960). NIH-PA Author Manuscript NIH-PA Author Manuscript Individual level concordance was evaluated using AUC and Cohen s κ (Cohen, 1960). Although κ is the traditional measure used in psychiatric research, κ is not emphasized here because it varies across populations that differ in prevalence even when sensitivity (SN; the percent of true cases correctly classified) and specificity (SP; the percent of true non-cases correctly classified) are constant (Cook, 1998). AUC, in comparison, is a function of SN and SP, which are considered the fundamental parameters of agreement (Kraemer, 1992). AUC equals (SN + SP)/2 when the screen is dichotomous. AUC scores between 0.5 and 1.0 are often interpreted in parallel with κ as slight (AUC = .50–.59; κ = 0.0–.19), fair (AUC = .6–. 69; κ = .2–.39), moderate (AUC = .7–.79; κ = .4–.59), substantial (AUC = .8–.89; κ = .6–. 79), and almost perfect (AUC = .9+; κ = .8+) (Landis and Koch, 1977). Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Analysis methods We also report total classification accuracy (TCA), the proportion of all respondents whose CIDI-SC and SCID classifications are consistent. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 11 NIH-PA Author Manuscript In addition, we report disaggregated measures of operating characteristics, including SN and SP, positive predictive value (PPV; the proportion of screened positives confirmed by the SCID), negative predictive value (NPV; the proportion of screened negatives confirmed as non-cases by the SCID), likelihood ratio positive (LR+; [SN/(100-SP)]), and likelihood ratio negative (LR−; [(100-SN)/SP)]). LR+ and LR− assess relative proportions of screened positives vs. screened negatives confirmed as cases (LR+) or non-cases (LR−). LR+ values greater than or equal to 5 and LR− values less than or equal to 0.2 are generally considered useful, while LR+ values greater than or equal to 10 and LR− values less than or equal to 0.1 are considered sufficient to rule in/out diagnoses (Haynes et al., 2006). Significance tests were based on Taylor series design-based standard errors to adjust for data weighting (Wolter, 1985). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth Multiple imputation of predicted probabilities of DSM-IV/SCID diagnoses—As noted above in the subsection on scoring the screening scales, each screening scale was originally scored continuously and then dichotomized. However, it is not necessary to dichotomize screening scales to make them useful. This is true even in clinical applications, where simple dichotomous scoring rules can be refined by using polychotomous rules that collapse screening scale scores into strata based on analysis of data in a clinical reappraisal study such that the observed prevalence of the clinical outcome differs significantly across strata but not within strata (Guyatt and Rennie, 2001). Designations of patients into multiple risk strata can be useful for clinical purposes when no sharp distinction between cases and non-cases exists in the screening scale (e.g., borderline hypertension). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth An extension of this approach can be used in epidemiological surveys to classify respondents into multiple risk strata based on screening scale scores and to assign predicted probabilities of clinical diagnoses to respondents in each stratum based on the results of a clinical reappraisal survey. nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Analysis methods It is also possible to ignore the construction of strata in this approach when a monotonic association exists throughout the scale range between a screening scale and probability of a diagnosis, in which case regression analysis can be used to generate predicted probabilities of clinical diagnoses for each respondent in a large sample based on regression coefficients estimated in a smaller clinical reappraisal subsample. These predicted probabilities can then be used either as continuous variables or as the basis for making dichotomous distinctions using any of several different methods discussed elsewhere (Kessler et al., 2010b; Kessler and Pennell, in press). NIH-PA Author Manuscript NIH-PA Author Manuscript The creation of continuous scores of this sort is only useful, though, when significant monotonic associations exist between screening scale scores and probabilities of having the clinical diagnosis. We demonstrate below that such associations exist between screening scale scores and diagnoses based on the SCID in the Army STARRS data by comparing AUC for the continuous versions of the screening scales with AUC based on various dichotomous versions of the scales. Given that these monotonic associations exist, we used the method of multiple imputation (MI) (Rubin, 1987) to assign predicted probabilities of SCID diagnoses based on screening scale scores to all respondents in the Army STARRS surveys. MI is a two-phase method designed to impute missing values of particular variables to respondents who have information on variables strongly related to the variable(s) with Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 12 Page 12 NIH-PA Author Manuscript missing values in such a way as to maximize the use of all available data in examining multivariate associations. NIH-PA Author Manuscript NIH-PA Aut The first phase of MI develops prediction equations based on any of several different complex search methods (Schafer, 2003; White et al., 2011) to estimate multivariate associations of predictors with the variables to be imputed in the subset of respondents with complete data and to use those equations to generate predicted values (imputations) for the missing variables in the remainder of the sample. Analysis methods In order to address the fact that imputed values are less precise than observed values, this first phase uses pseudo-replication (i.e., estimation of a new set of coefficients based on the same model from pseudo-samples selected with replacement from the actual sample of people with complete data) to generate multiple imputations for each missing value. The second phase of MI, in which the multiple imputations are used in substantive analysis, then uses each set of imputed values to carry out the substantive analysis separately and then combines the coefficient values across these replications to adjust standard errors of estimates for the fact that some of the data used in the analyses were imputed rather than observed. Importantly, the first phase of MI allows the inclusion not only of a screening scale (in this case, the CIDI-SC or PCL) designed to provide a proxy measure for the unmeasured variable of interest (in this case, DSM-IV/SCID diagnoses), but also other variables that might be used in second-phase analyses as predictors or consequences of the imputed variable. This is important because the use of only the CIDI-SC or PCL to impute clinical diagnoses would lead to under-estimation of the associations of predictors and consequences of clinical diagnoses with the components of the clinical diagnoses that are not predicted by the CIDI-SC or PCL scores (Collins et al., 2001). As a result, the multiply-imputed predicted probabilities of DSM-IV/SCID diagnoses in Army STARRS were based on complex multivariate equations that included the complete set of CIDI-SC/PCL scores to impute each clinical diagnosis (to adjust for comorbidities among clinical disorders) along with a wide range of substantive correlates included in the AAS and Army/DoD administrative data systems. We produced 20 imputations for each respondent in Army STARRS, a number at the high end of the number recommended in applying MI (Graham et al., 2007). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Concordance of screening scale scores with DSM-IV/ SCID diagnoses Differences in prevalence estimates based on the dichotomized screening scales and SCID are insignificant for all disorders at optimal screening scale thresholds for estimating prevalence (χ21 = 0.0–0.6, p = .89−.43). (Table 1) This is not surprising, of course, as the thresholds were selected to make CIDI-SC prevalence as similar as possible to SCID prevalence. But this is no guarantee of good concordance at the individual level. Individual- level diagnostic concordance at these thresholds is moderate for seven diagnoses (AUC = . 70–79) and fair for the other diagnosis (ADHD; AUC = .69). Total classification accuracy is in the range 86.0–95.9%. The screening scale estimate of 30-day prevalence of any of the seven disorders assessed for 30-day prevalence (the exception being mania/hypomania, Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 13 NIH-PA Author Manuscript which was only assessed over the entire lifetime), like most of the individual disorders, has moderate concordance with the estimate based on the SCID (AUC = .78). Operating characteristics of the tests The proportions of SCID cases detected (SN) at the optimal screening scale diagnostic thresholds for estimating SCID prevalence are in the range 42.8–66.8% and the proportions of screening scale cases confirmed by the SCID (PPV) at these thresholds are in the range 37.1–65.3% (68.3% for any 30-day disorder). (Table 2) The proportions of SCID non-cases classified correctly (SP) are 90.9–97.8% and the proportions of screening scale non-cases confirmed as non-cases by the SCID (NPV) are 91.5–97.8%. Lower SN and PPV than SP and NPV are expected for thresholds designed to estimate prevalence without bias when only a minority of respondents has a disorder. LR+ is generally considered more informative than SN in such cases (Haynes et al., 2006). LR+ is in the definitive range (i.e., greater than 10.0) at these thresholds for six of the eight disorders and in the informative range (i.e., greater than 5.0) for the others (7.3 for IED; 7.8 for ADHD) and for any 30-day disorder (8.5), indicating that screened positives at these thresholds are much more likely than screened negatives to be confirmed as cases in the clinical reappraisal interviews. LR− values, in comparison, are in a range that would not be considered useful in screening out true non-cases (0.4–0.6). NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. ternative optimization rules in selecting screening scale diagnostic thresholds As noted above in the section on analysis methods, the most useful thresholds for screening scales differ depending on the uses to which the screening scales are put. As Army STARRS is an epidemiological study rather than a clinical study, we place a premium on accurate estimation of SCID prevalence. But in a clinical study, where screening scales might be used for case-finding to select people for additional assessment and treatment, it might make more sense to lower the threshold to capture as large a proportion of clinical cases as feasible within the constraints of the cost-benefit ratio of screening and treatment. To investigate the implications of using such a rule in setting screening scale thresholds, we compared scale operating characteristics when the threshold was selected to detect 80% of DSM-IV/SCID cases (i.e., SN = 80.0%). This change leads to a lowering of screening scale thresholds for all disorders because SN is consistently lower than 80% at the optimal threshold for estimating SCID prevalence. And this, in turn, leads to substantial increases in screening scale prevalence (2.5–7.0 times the prevalence estimates based on the optimal threshold for estimating SCID prevalence) for all disorders other than PD and IED (where CIDI-SC prevalence estimates increase to 1.2–1.3 times the optimal for estimating SCID prevalence) and to correspondingly large reductions in PPV. (Table 4) While PPV at the optimal threshold for estimating SCID prevalence averages 51.6% (i.e., 51.6% of screened positives are true clinical cases, with a range of 37.1–65.3%), average PPV drops to 30.0% (range: 11.9–56.0%) when thresholds are selected so that SN exceed 80%. This means that it would require an average of about three SCID interviews to detect each clinical case among the screened positives at the lower threshold compared to roughly two at the higher threshold. Clinical intervention cost- effectiveness calculations would be needed to determine whether this additional expense of case-finding could be justified based on the human costs (i.e., quality of life, morbidity, mortality) of an untreated case, the costs of treatment, and the likely effectiveness of treatment in reducing human costs. From the perspective of epidemiological research, lowering the thresholds below the optimal for estimating prevalence might still be desirable even though such an anticonservative change introduces upward bias in prevalence estimates, as it is possible that lowering thresholds will lead to greater proportional increases in SN than in (100-SP), in which case LR+ will increase. The implications of modifying diagnostic thresholds The proportions of screened positives confirmed as SCID cases (PPV) could be increased by raising the screening scale diagnostic thresholds beyond the optimal for estimating prevalence. However, this increase in PPV would be obtained at the expense of decreasing SN and creating downwardly biased (conservative) prevalence estimates. The value of making such a change in threshold while still attempting to approximate clinical prevalence can be evaluated by examining relative changes in PPV vs. SN associated with modest increases in screening scale thresholds around the optimal thresholds for estimating SCID prevalence. When we make these small increases in threshold we see that the increases in PPV are much less than the decreases in SN for four disorders (MDE, GAD, ADHD, SUD) (proportional SC decreases of 20%, 7%, 25%, and 31%, respectively; proportional PPV increases of 2%, 0%, 18%, and 4%, respectively). (Table 3) In addition, PPV actually decreases slightly for the other four disorders due to respondents with CIDI-SC scores just above the optimal thresholds for estimating SCID prevalence of these disorders having high SCID prevalence. These results argue against small changes to increase the screening scale thresholds in the service of making diagnoses more conservative while still maintaining estimates that approximate the SCID prevalence estimates. NIH-PA Author Manuscript NIH-PA Author Manuscript We also examined the implications of making small changes in the thresholds in the other direction to increase the proportions of clinical cases screening positive by lowering the screening scale thresholds. Such changes increase SN by definition. This is desirable for purposes of guaranteeing comprehensive detection in treatment samples when PPV does not decrease more than SN increases. However, such anticonservative changes can lead to upward bias in prevalence estimates as well as to reductions in LR+ when the proportional increases in SN are lower than the proportional decreases in SP. An analysis of these Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 14 NIH-PA Author Manuscript changes associated with modest decreases in screening scale thresholds shows that LR+ consistently decreases when modest changes are made to decrease thresholds. (Table 3) These results argue against making the screening scale thresholds less conservative while still maintaining estimates that approximate SCID prevalence. ternative optimization rules in selecting screening scale diagnostic thresholds However, LR+ decreases consistently when the screening scale thresholds are lowered, arguing against making these thresholds less conservative for purposes of epidemiological analysis of the Army STARRS data. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Autho NIH-PA Author Manuscript NIH-PA Author Manuscript Another goal of screening might be to select screening scale thresholds to have a minimum proportion of screened positives confirmed in clinical interviews (i.e., high PPV). For example, minimum PPV might be set at 50% to guarantee that the majority of screened positives are true clinical cases or at 80% to guarantee that the vast majority of screened Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 15 Page 15 positives are true clinical cases. However, this will lead to a reduction in SN that might make the true cases detected unrepresentative of all true cases. If minimum PPV is set at 50%, the thresholds selected to maximize estimation of SCID prevalence meet the PPV criterion in five of eight cases, the exceptions being MHM (PPV = 45.8%), GAD (PPV = 45.6%), and ADHD (PPV = 37.1%). In the case of MHM, the threshold can be raised to increase PPV to 71.1%, but this leads to a dramatic reduction in estimated prevalence (from 4.9% to 0.7%) and in SN (from 43.5% to 9.7%). (Table 5) While more than two-thirds of the small fraction of respondents defined as positive for MHM in the CIDI-SC are SCID cases, the exclusion of the vast majority of SCID cases of MHM from this small fraction (100-SN = 90.3% of SCID cases not detected) means that the proportion of SCID cases among the screened negatives is nearly as high as the proportion among screened negatives (LR− = 0.9), arguing against making the screening scale thresholds this conservative for purposes of epidemiological analysis of the Army STARRS data. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Au In the case of GAD, raising the CIDI-SC threshold to make PPV exceed 50% leads to halving both estimated prevalence (from 6.6% to 3.2%) and SN (from 43.9% to 23.6%) in the service of only a relatively modest increase in PPV (from 45.6% to 50.2%) compared to when the threshold is set to maximize estimation of SCID prevalence. ternative optimization rules in selecting screening scale diagnostic thresholds It is difficult to argue for a threshold that decreases SN so dramatically for such a modest increase in PPV. The situation is similar but less dramatic for ADHD, where a change in the CIDI-SC threshold that increased PPV by roughly 50% (from 37.1% to 55.9%) decreased estimated prevalence by 70% (from 8.2% to 2.4%) and SN by 55% (from 42.8% to 19.3%). Selecting thresholds to have even higher PPV (a minimum of 80%) for disorders where screening scale PPV is greater than 50% at the optimal threshold for estimating SCID prevalence consistently has the same negative effects in that the proportional increases in PPV (in the range 47–59%) are much less than the proportional decreases in prevalence (84–91%), resulting in extremely low levels of SN (7.3–13.4%). These results argue against using such restrictive thresholds for purposes of epidemiological analysis of the Army STARRS data. NIH-PA Author Manuscript NIH-PA Author Manuscript nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. DISCUSSION Previous research has shown that CIDI-SC operating characteristics are equivalent to or better than those of alternative screening scales in samples of the general population (Kessler et al., 2005a; Kessler et al., 2006a; Kessler et al., 2012) and that the PCL has very good concordance with clinical diagnoses of PTSD in samples of both the military and the general population (Wilkins et al., 2011) We nonetheless carried out an independent clinical reappraisal study of these screening scales in Army STARRS due to the fact that the operating characteristics of the same screening scale can differ substantially across surveys depending on such fundamental survey conditions as auspices, level of confidentiality (e.g., complete anonymity versus deidentification), mode of data collection, and situational factors, such as constraint on the amount of time available to complete the survey (Kessler and Pennell, in press). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Au It is not surprising in light of the challenging survey conditions in Army STARRS -- including group-administration in settings with suboptimal physical facilities (e.g., sitting on folding chairs in full field gear in temporary data collection locations) -- that we found that the CIDI-SC and PCL AUCs are somewhat lower than in previous psychometric studies of these scales. Individual-level concordance of diagnoses based on the CIDI-SC and PCL with diagnoses based on independent SCID clinical reappraisal interviews in the AAS is for most part moderate (AUC = .70–.79; κ = .4–.6), whereas most previous evaluations found concordance of the CIDI-SC scales and the PCL with SCID diagnoses to be substantial (AUC = .80–.89; κ = .6–.8). However, the administrative conditions of the screening scales in most previous studies that carried out clinical reappraisals were much better than in Army STARRS, including self-administration in primary care waiting rooms (Kessler et al., 2012), face-to-face interviewer administration in household surveys (Kessler et al., 2006a), and interviewer administration over the telephone with health plan subscribers (Kessler et al., 2005a). NIH-PA Author Manuscript NIH-PA Author Manuscript Perhaps the more striking result in light of the challenging Army STARRS field conditions is that the positive CIDI-SC/PCL operating characteristics for dichotomous versions of the scales designed to optimize aggregate concordance with SCID prevalence estimates are generally quite good. Continuous versus dichotomous diagnostic classification As noted above in the section on analysis methods, we calculated ROC curves for the entire screening scale distributions. (Figure 1) AUC was calculated for each of these curves and compared to the AUC of the dichotomous version of the same screening scale. AUC was found to be substantially higher for the continuous than dichotomous scoring rule for each of the eight screening scales (Range: .80–.90 continuous versus .69–.79 dichotomous; Inter- quartile range: .85–.87 continuous versus .70–.78 dichotomous). (Table 6) This suggests that meaningful variation in SCID prevalence exists at other places on the screening scale ranges than the optimal diagnostic threshold for estimating SCID prevalence. The important implication of this finding for our purposes is that continuous screening scale scores defining predicted probabilities of clinical diagnoses might be more useful than dichotomous diagnostic classifications based on the screening scales for purposes of epidemiological analysis. We consequently calculated both continuous (predicted probability of having a DSM-IV/SCID diagnosis) and dichotomous versions of each screening scale for use in analysis of the Army STARRS data. The continuous versions were produced using the MI method. Importantly, not only the screening scale scores but also a wide range of other NIH-PA Author Manuscript NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 16 Page 16 significant correlates of the DSM-IV/SCID diagnoses were used in the first-phase MI analysis in order to minimize bias in subsequent substantive analyses that will use these variables as correlates of predicted probabilities of DSM-IV/SCID disorders. NIH-PA Author Manuscript nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. DISCUSSION LR+ values for six of the eight disorders are in the range 11.5–27.9, all of which are well above the 10.0 value generally considered sufficient to rule in diagnoses (Haynes et al., 2006), while the 7.3–7.8 LR+ values for the other two diagnoses and the 8.5 LR+ value for any 30-day disorder are well above the 5.0 value considered useful in ruling in diagnoses. However, these good LR+ values are accompanied by LR− values generally considered not to be useful in screening out true negatives (0.4–0.6); that is, to contain proportions of true negative that are not strikingly different from the proportions found among screened positives. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 17 Page 17 NIH-PA Author Manuscript As discussed in more detail elsewhere (Kessler et al., 2012), the definitions of screened positives and screened negatives could be purified for clinical purposes by selecting thresholds at the tails of the distributions that have operating characteristics deemed useful for clinical purposes. For example, an upper threshold of a screening scale could be selected to have a minimum PPV of 0.5 in order to make sure that at least 50% of screened positives are SCID cases. As we saw, though, this desirable feature of that threshold would generally mean that a substantial proportion of SCID cases are missed. Alternatively, the upper threshold of a screening scale could be set at a minimum SN of .80 to make sure that the vast majority of SCID cases are picked up by the screen, but this desirable feature of that threshold would mean that only a small proportion of screened positives have SCID diagnoses. In a similar way, a lower threshold of a screening could be purified by requiring NPV to be, say, at least 1 – p/5, where p = SCID prevalence of the disorder, thereby guaranteeing that the proportion of SCID cases among patients screening negative is no more than 20% as high as the prevalence of the disorder in the sample, but this desirable feature of that threshold might mean that a substantial proportion of true non-cases are excluded from this ruled-out group. nt J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. DISCUSSION NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth It is also possible to select multiple thresholds at upper and lower tails both to maximize the positives (i.e., definitive screen-ins and/or screen-outs) and minimize the negatives (i.e., minimizing the numbers of false positives and/or false negatives) and leave one or more intermediate strata that define those with high-but-not-definitively-high scores, low-but-not- definitively-low scores, and uninformative intermediate scores. We noted earlier that such polychotomous scoring rules are fairly common in screening scales developed for clnical practice (Guyatt and Rennie, 2001). Indeed, CIDI-SC polychotomous thresholds have been developed for exactly this reason to facilitate the use of these scales in primary care screening (Kessler et al., 2012). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Aut However, a more useful approach for purposes of epidemiological analysis of the screening scales considered here is likely to be retention of the entire screening scale range given that AUCs of continuous versions of the screening scales are higher than AUCs of dichotomized versions of the scales at their unbiased thresholds. Based on this observation, we are using MI to assign predicted probabilities of DSM-IV/SCID diagnoses to all Army STARRS respondents who completed the screening scales. We are addressing the uncertainty of inference from prediction equations using imputed rather than observed values by estimating 20 MI estimates of the predicted probability of having each clinical diagnosis for each respondent. The practical use of this approach is illustrated in a more detailed methodological exposition published previously in this journal (Kessler and Üstün, 2004) as well as in a number of subsequent substantive reports that used this approach to estimate the prevalence and correlates of several different DSM-IV/SCID disorders in other psychiatric epidemiological studies (Fayyad et al., 2007; Huang et al., 2009; Kessler et al., 2005a). However, second-phase of MI analysis can be computationally intensive even after the first- phase multiple imputations, as each model has to be estimated 20 separate times rather than once and the coefficients in these 20 replicates then need to be combined to calculate adjusted standard errors. Acknowledgments Funding/Support: Army STARRS was sponsored by the Department of the Army and funded under cooperative agreement number U01MH087981 with the U.S. Department of Health and Human Services, National Institutes of Health, National Institute of Mental Health (NIH/NIMH). The contents are solely the responsibility of the authors and do not necessarily represent the views of the Department of Health and Human Services, NIMH, the Department of the Army, or the Department of Defense. Role of the Sponsors: As a cooperative agreement, scientists employed by NIMH (Colpe and Schoenbaum) and Army liaisons/consultants (COL Steven Cersovsky, MD, MPH USAPHC and Kenneth Cox, MD, MPH USAPHC) collaborated to develop the study protocol and data collection instruments, supervise data collection, plan and supervise data analyses, interpret results, and prepare reports. Although a draft of this manuscript was submitted to the Army and NIMH for review and comment prior to submission, this was with the understanding that comments would be no more than advisory. DISCUSSION As a result, we also plan to work with dichotomously-scored screening scale measures at the optimal diagnostic thresholds and to investigate the extent to However, a more useful approach for purposes of epidemiological analysis of the screening scales considered here is likely to be retention of the entire screening scale range given that AUCs of continuous versions of the screening scales are higher than AUCs of dichotomized versions of the scales at their unbiased thresholds. Based on this observation, we are using MI to assign predicted probabilities of DSM-IV/SCID diagnoses to all Army STARRS respondents who completed the screening scales. We are addressing the uncertainty of inference from prediction equations using imputed rather than observed values by estimating 20 MI estimates of the predicted probability of having each clinical diagnosis for each respondent. The practical use of this approach is illustrated in a more detailed NIH-PA Author Manuscript NIH-PA Author Manuscript Kessler et al. Page 18 which substantive results differ depending on whether this dichotomous approach is used instead of MI. Dichotomous screening scale scoring will be used in cases where results are relatively insensitive to the more refined estimates using MI. which substantive results differ depending on whether this dichotomous approach is used instead of MI. Dichotomous screening scale scoring will be used in cases where results are relatively insensitive to the more refined estimates using MI. NIH-PA Author Manuscript REFERENCES Page 19 Page 19 NIH-PA Author Manuscript Fayyad J, De Graaf R, Kessler R, Alonso J, Angermeyer M, Demyttenaere K, De Girolamo G, Haro JM, Karam EG, Lara C, Lepine JP, Ormel J, Posada-Villa J, Zaslavsky AM, Jin R. Cross-national prevalence and correlates of adult attention-deficit hyperactivity disorder. British Journal of Psychiatry. 2007; 190:402–409. [PubMed: 17470954] NIH-PA Author Manuscript NIH-PA Au First, MB.; Spitzer, RL.; Gibbon, M.; Williams, JBW. Structured Clinical Interview for DSM-IV Axis I Disorders, Research Version, Non-patient Edition (SCID-I/NP). New York, NY: Biometrics Research, New York State Psychiatric Institute; 2002. Gaynes BN, DeVeaugh-Geiss J, Weir S, Gu H, MacPherson C, Schulberg HC, Culpepper L, Rubinow DR. Feasibility and diagnostic validity of the M-3 checklist: a brief, self-rated screen for depressive, bipolar, anxiety, and post-traumatic stress disorders in primary care. Annals of Family Medicine. 2010; 8(2):160–169. [PubMed: 20212303] Gossop M, Darke S, Griffiths P, Hando J, Powis B, Hall W, Strang J. The Severity of Dependence Scale (SDS): psychometric properties of the SDS in English and Australian samples of heroin, cocaine and amphetamine users. Addiction. 1995; 90(5):607–614. [PubMed: 7795497] Graham JW, Olchowski AE, Gilreath TD. How many imputations are really needed? Some practical clarifications of multiple imputation theory. Prevention Science. 2007; 8(3):206–213. [PubMed: 17549635] Guyatt, G.; Rennie, D. User's Guide to the Medical Literature: A Manual for Evidence-based Clinical Practice. Chicago, IL: AMA Press; 2001. Haro JM, Arbabzadeh-Bouchez S, Brugha TS, de Girolamo G, Guyer ME, Jin R, Lepine JP, Mazzi F, Reneses B, Vilagut G, Sampson NA, Kessler RC. Concordance of the Composite International Diagnostic Interview Version 3.0 (CIDI 3.0) with standardized clinical assessments in the WHO World Mental Health surveys. International Journal of Methods in Psychiatric Research. 2006; 15(4):167–180. [PubMed: 17266013] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A Haynes, RB.; Sackett, DL.; Guyatt, GH.; Tugwell, P. Clinical Epidemiology: How to Do Clinical Practice Research. Third Edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2006. Heeringa SG, Colpe LJ, Fullerton CS, Gebler N, Naifeh JA, Nock MK, Sampson NA, Schoenbaum M, Zaslavsky AM, Stein MB, Ursano RJ, Kessler RC. Field procedures in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS). International Journal of Methods and Psychiatric Research. (this issue). Hirschfeld RM, Williams JB, Spitzer RL, Calabrese JR, Flynn L, Keck PE Jr, Lewis L, McElroy SL, Post RM, Rapport DJ, Russell JM, Sachs GS, Zajecka J. REFERENCES American Association for Public Opinion Research. Standard Definitions: Final Dispositions of Case Codes and Outcome Rates for Surveys. Deerfield, IL: American Association for Public Opinion Research; 2009. NIH-PA Author Manuscript American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). Fourth Edition. Washington, DC: American Psychiatric Association; 1994. Barnes JB, Nickerson A, Adler AB, Litz BT. Perceived military organizational support and peacekeeper distress: A longitudinal investigation. Psychological Services. 2013; 10(2):177–185. [PubMed: 23730963] Bray RM, Pemberton MR, Lane ME, Hourani LL, Mattiko MJ, Babeu LA. Substance use and mental health trends among U.S. military active duty personnel: key findings from the 2008 DoD Health Behavior Survey. Military Medicine. 2010; 175(6):390–399. [PubMed: 20572470] Breslau N, Peterson EL, Kessler RC, Schultz LR. Short screening scale for DSM-IV posttraumatic stress disorder. American Journal of Psychiatry. 1999; 156(6):908–911. [PubMed: 10360131] Broadhead WE, Leon AC, Weissman MM, Barrett JE, Blacklow RS, Gilbert TT, Keller MB, Olfson M, Higgins ES. Development and validation of the SDDS-PC screen for multiple mental disorders in primary care. Archives of Family Medicine. 1995; 4(3):211–219. [PubMed: 7881602] Bromet EJ, Dunn LO, Connell MM, Dew MA, Schulberg HC. Long-term reliability of diagnosing lifetime major depression in a community sample. Archives of General Psychiatry. 1986; 43(5): 435–440. [PubMed: 3964022] NIH-PA Author Manuscript NIH-PA Author Manuscript Brown JM, Williams J, Bray RM, Hourani L. Postdeployment alcohol use aggression, and post- traumatic stress disorder. Military Medicine. 2012; 177(10):1184–1190. [PubMed: 23113445] Cohen J. A coefficient of agreement for nominal scales. Educational and Psychological Measurement. 1960; 20(1):37–46. Collins LM, Schafer JL, Kam CM. A comparison of inclusive and restrictive strategies in modern missing data procedures. Psychological Methods. 2001; 6(4):330–351. [PubMed: 11778676] Cook, RJ. Kappa and its dependence on marginal rates. In: Armitage, P.; Colton, T., editors. The Encyclopedia of Biostatistics. New York, NY: Wiley; 1998. p. 2166-2168. Donker T, van Straten A, Marks I, Cuijpers P. A brief Web-based screening questionnaire for common mental disorders: development and validation. Journal of Medical Internet Research. 2009; 11(3):e19. [PubMed: 19632977] Farvolden P, McBride C, Bagby RM, Ravitz P. A Web-based screening instrument for depression and anxiety disorders in primary care. Journal of Medical Internet Research. 2003; 5(3):e23. [PubMed: 14517114] Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. REFERENCES Development and validation of a screening instrument for bipolar spectrum disorder: the Mood Disorder Questionnaire. American Journal of Psychiatry. 2000; 157(11):1873–1875. [PubMed: 11058490] Huang Y, Kotov R, de Girolamo G, Preti A, Angermeyer M, Benjet C, Demyttenaere K, de Graaf R, Gureje O, Karam AN, Lee S, Lepine JP, Matschinger H, Posada-Villa J, Suliman S, Vilagut G, Kessler RC. DSM-IV personality disorders in the WHO World Mental Health Surveys. British Journal of Psychiatry. 2009; 195(1):46–53. [PubMed: 19567896] NIH-PA Author Manuscript NIH-PA Author Manuscript Hunter EE, Penick EC, Powell BJ, Othmer E, Nickel EJ, Desouza C. Development of scales to screen for eight common psychiatric disorders. Journal of Nervous and Mental Disease. 2005; 193(2): 131–135. [PubMed: 15684916] Jones M, Sundin J, Goodwin L, Hull L, Fear NT, Wessely S, Rona RJ. What explains post-traumatic stress disorder (PTSD) in UK service personnel: deployment or something else? Psychological Medicine. 2012:1–10. Kendler KS, Neale MC, Kessler RC, Heath AC, Eaves LJ. A population-based twin study of major depression in women. The impact of varying definitions of illness. Archives of General Psychiatry. 1992; 49(4):257–266. [PubMed: 1558459] Kessler RC, Adler L, Ames M, Demler O, Faraone S, Hiripi E, Howes MJ, Jin R, Secnik K, Spencer T, Ustun TB, Walters EE. The World Health Organization Adult ADHD Self-Report Scale (ASRS): a short screening scale for use in the general population. Psychological Medicine. 2005a; 35(2):245–256. [PubMed: 15841682] Kessler RC, Adler LA, Gruber MJ, Sarawate CA, Spencer T, Van Brunt DL. Validity of the World Health Organization Adult ADHD Self-Report Scale (ASRS) Screener in a representative sample of health plan members. International Journal of Methods and Psychiatric Research. 2007; 16(2): 52–65. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 20 NIH-PA Author Manuscript Kessler RC, Akiskal HS, Angst J, Guyer M, Hirschfeld RM, Merikangas KR, Stang PE. Validity of the assessment of bipolar spectrum disorders in the WHO CIDI 3.0. Journal of Affective Disorders. 2006a; 96(3):259–269. [PubMed: 16997383] NIH-PA Author Manuscript NIH-PA Aut Kessler RC, Berglund P, Demler O, Jin R, Merikangas KR, Walters EE. Lifetime prevalence and age- of-onset distributions of DSM-IV disorders in the National Comorbidity Survey Replication. Archives of General Psychiatry. 2005b; 62(6):593–602. [PubMed: 15939837] Kessler RC, Calabrese JR, Farley PA, Gruber MJ, Jewell MA, Katon W, Keck PE, Nierenberg AA, Sampson NA, Shear MK, Shillington AC, Stein MB, Thase ME, Wittchen HU. REFERENCES Composite International Diagnostic Interview screening scales for DSM-IV anxiety and mood disorders. Psychological Medicine, e-publication ahead of print. 2012 Kessler RC, Coccaro EF, Fava M, Jaeger S, Jin R, Walters E. The prevalence and correlates of DSM- IV intermittent explosive disorder in the National Comorbidity Survey Replication. Archives of General Psychiatry. 2006b; 63(6):669–678. [PubMed: 16754840] Kessler RC, Colpe LJ, Fullerton CS, Gebler N, Naifeh JA, Nock MK, Sampson NA, Schoenbaum M, Zaslavsky AM, Stein MB, Ursano RJ, Heeringa SG. Design of the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS). International Journal of Methods and Psychiatric Research. (this issue). Kessler RC, Green JG, Adler LA, Barkley RA, Chatterji S, Faraone SV, Finkelman M, Greenhill LL, Gruber MJ, Jewell M, Russo LJ, Sampson NA, Van Brunt DL. Structure and diagnosis of adult attention-deficit/hyperactivity disorder: analysis of expanded symptom criteria from the Adult ADHD Clinical Diagnostic Scale. Archives of General Psychiatry. 2010a; 67(11):1168–1178. [PubMed: 21041618] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Kessler RC, Green JG, Gruber MJ, Sampson NA, Bromet E, Cuitan M, Furukawa TA, Gureje O, Hinkov H, Hu CY, Lara C, Lee S, Mneimneh Z, Myer L, Oakley-Browne M, Posada-Villa J, Sagar R, Viana MC, Zaslavsky AM. Screening for serious mental illness in the general population with the K6 screening scale: results from the WHO World Mental Health (WMH) survey initiative. International Journal of Methods and Psychiatric Research. 2010b; 19(Suppl 1):4–22. Kessler RC, Lane M, Stang PE, Van Brunt DL. The prevalence and workplace costs of adult attention deficit hyperactivity disorder in a large manufacturing firm. Psychological Medicine. 2009; 39(1): 137–147. [PubMed: 18423074] Kessler, RC.; Pennell, B-E. Developing and selecting mental health measures. In: Johnson, TP., editor. Handbook of Health Survey Methods. New York, NY: John Wiley & Sons; (in press) Kessler RC, Üstün TB. The World Mental Health (WMH) Survey Initiative Version of the World Health Organization (WHO) Composite International Diagnostic Interview (CIDI). International Journal of Methods and Psychiatric Research. 2004; 13(2):93–121. Kessler RC, Wittchen H-U, Abelson JM, McGonagle KA, Schwarz N, Kendler KS, Knäuper B, Zhao S. Methodological studies of the Composite International Diagnostic Interview (CIDI) in the US National Comorbidity Survey. International Journal of Methods in Psychiatric Research. 1998; 7(1):33–55. NIH-PA Author Manuscript NIH-PA Author Manuscript Kraemer, HC. Evaluating Medical Tests: Objective and Quantitative Guidelines. Newbury Park, CA: Sage Publications; 1992. Kroenke K, Spitzer RL, Williams JB. REFERENCES The PHQ-9: validity of a brief depression severity measure. Journal of General Internal Medicine. 2001; 16(9):606–613. [PubMed: 11556941] Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977; 33(1):159–174. [PubMed: 843571] Margolis DJ, Bilker W, Boston R, Localio R, Berlin JA. Statistical characteristics of area under the receiver operating characteristic curve for a simple prognostic model using traditional and bootstrapped approaches. Journal of Clinical Epidemiology. 2002; 55(5):518–524. [PubMed: 12007556] Merikangas KR, Akiskal HS, Angst J, Greenberg PE, Hirschfeld RM, Petukhova M, Kessler RC. Lifetime and 12-month prevalence of bipolar spectrum disorder in the National Comorbidity Survey replication. Archives of General Psychiatry. 2007; 64(5):543–552. [PubMed: 17485606] Nock MK, Borges G, Bromet EJ, Cha CB, Kessler RC, Lee S. Suicide and suicidal behavior. Epidemiologic Reviews. 2008; 30(1):133–154. [PubMed: 18653727] Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Page 21 Page 21 NIH-PA Author Manuscript Nock MK, Deming CA, Fullerton CS. Suicide among Soldiers: a review of psychological risk and protective factors. Psychiatry. (in press). NIH-PA Author Manuscript NIH-PA A Nock MK, Hwang I, Sampson N, Kessler RC, Angermeyer M, Beautrais A, Borges G, Bromet E, Bruffaerts R, de Girolamo G, de Graaf R, Florescu S, Gureje O, Haro JM, Hu C, Huang Y, Karam EG, Kawakami N, Kovess V, Levinson D, Posada-Villa J, Sagar R, Tomov T, Viana MC, Williams DR. Cross-national analysis of the associations among mental disorders and suicidal behavior: findings from the WHO World Mental Health Surveys. PLoS Medicine. 2009; 6(8):e1000123. [PubMed: 19668361] Pepe, MS. Statistical Analysis of Medical Tests for Classification and Prediction. New York, NY: Oxford University Press; 2003. Rohde P, Lewinsohn PM, Seeley JR. Comparability of telephone and face-to-face interviews in assessing axis I and II disorders. American Journal of Psychiatry. 1997; 154(11):1593–1598. [PubMed: 9356570] Rubin, DB. Multiple Imputation for Nonresponse in Surveys. New York, NY: John Wiley & Sons; 1987. Schafer JL. Multiple imputation in multivariate problems when the imputation and analysis models differ. Statistica Neerlandica. 2003; 57(1):19–35. Sobin C, Weissman MM, Goldstein RB, Adams P, Wickramaratne P, Warner V, Lish JD. Diagnostic interviewing for family studies: comparing telephone and face-to-face methods for the diagnosis of lifetime psychiatric disorders. Psychiatric Genetics. 1993; 3(4):227–233. Spitzer RL, Kroenke K, Williams JB, Lowe B. A brief measure for assessing generalized anxiety disorder: the GAD-7. Archives of Internal Medicine. 2006; 166(10):1092–1097. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Wolter, KM. Introduction to Variance Estimation. New York, NY: Springer-Verlag; 1985. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. REFERENCES [PubMed: 16717171] NIH-PA Author Manuscript Ursano RJ, Heeringa S, Stein MB, Kessler RC. The Army Study to Assess Risk and Resilience in Servicemembers (STARRS). (under review). Weathers, F.; Litz, B.; Herman, D.; Huska, J.; Keane, T. Annual meeting of the International Society for Traumatic Stress Studies. San Antonio, TX: 1993. The PTSD checklist (PCL): reliability, validity, and diagnostic utility. White IR, Royston P, Wood AM. Multiple imputation using chained equations: Issues and guidance for practice. Statistics in Medicine. 2011; 30(4):377–399. [PubMed: 21225900] Wilkins KC, Lang AJ, Norman SB. Synthesis of the psychometric properties of the PTSD checklist (PCL) military, civilian, and specific versions. Depression and Anxiety. 2011; 28(7):596–606. [PubMed: 21681864] Wolter, KM. Introduction to Variance Estimation. New York, NY: Springer-Verlag; 1985. NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Page 22 Kessler et al. NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1. ROC curves for the associations between continuous screening scales and DSM-IV/ SCID diagnoses (n=460)a aROC = receiver operating characteristic; SN = sensitivity; SP = specificity; DSM-IV = Diagnostic and Statistical Manuel of Mental Disorders, Fourth Edition; SCID = Structured Clinical Interview for DSM-IV. NIH-PA Author Manuscript Figure 1. ROC curves for the associations between continuous screening scales and DSM-IV/ SCID diagnoses (n=460)a Figure 1. ROC curves for the associations between continuous screening scales and DSM-IV/ SCID diagnoses (n=460)a aROC = receiver operating characteristic; SN = sensitivity; SP = specificity; DSM-IV = Diagnostic and Statistical Manuel of Mental Disorders, Fourth Edition; SCID = Structured Clinical Interview for DSM-IV. Figure 1. ROC curves for the associations between continuous screening scales and DSM-IV/ SCID diagnoses (n=460)a aROC = receiver operating characteristic; SN = sensitivity; SP = specificity; DSM-IV = Diagnostic and Statistical Manuel of Mental Disorders, Fourth Edition; SCID = Structured Clinical Interview for DSM-IV. g ( ) aROC = receiver operating characteristic; SN = sensitivity; SP = specificity; DSM-IV = Diagnostic and Statistical Manuel of Mental Disorders, Fourth Edition; SCID = Structured Clinical Interview for DSM-IV. NIH-PA Author Manuscript Page 23 Page 23 Kessler et al. Kessler et al. REFERENCES Table 1 Aggregate (McNemar χ2) and individual-level (AUC, κ, TCA) consistency of DSM-IV diagnoses based on the CIDI screening scales (CIDI-SC) at their optimal (to estimate prevalence) thresholds and on blinded SCID clinical reappraisal interviews (n=460)a Aggregate concordanceb Individual-level concordancec Prevalence estimates CIDI-SC SCID McNemar % (se) % (se) χ21 AUC κ TCA I. Mood disorders Major depressive episode 6.8 (1.0) 6.7 (1.0) 0.0 .78 .55 94.3 Mania/hypomania 4.9 (1.0) 5.2 (0.9) 0.1 .70 .42 94.4 II. Anxiety disorders Panic disorder 5.0 (0.9) 5.1 (0.7) 0.0 .78 .57 95.9 Generalized anxiety disorder 6.6 (0.9) 6.8 (1.0) 0.0 .70 .41 92.6 Post-traumatic stress disorder 6.7 (1.0) 6.4 (0.8) 0.1 .75 .49 93.7 III. Externalizing disorders Adult attention-deficit/hyperactivity disorder 8.2 (1.1) 7.1 (1.1) 0.6 .69 .35 90.8 Intermittent explosive disorder 20.8 (2.3) 20.4 (2.0) 0.1 .79 .57 86.0 Substance use disorder 4.9 (0.4) 5.4 (0.8) 0.1 .73 .47 94.8 IV. Any disorderd 18.9 (1.6) 20.3 (1.9) 0.6 .78 .58 86.6 aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. bThe CIDI-SC prevalence estimates are set at the thresholds designed to maximize concordance with prevalence estimates based on the blinded SCID clinical reappraisal interviews. The McNemar χ2 tests evaluate concordance of these two prevalence estimates. cAUC = Area under the receiver operating characteristic curve; κ = Cohen’s κ; TCA = total classification accuracy. See the text for definitions of these statistics, none three of which provide information about the overnone individual-level concordance between diagnoses based on the CIDI-SC scales and the blinded SCID clinical reappraisal interviews. dAny of the seven disorders other than mania/hypomania, as mania/hypomania were assessed only over the entire lifetime. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc Pag Table 3 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are changed from the optimal for estimating prevalence to either more conservative or more anticonservative thresholds (n=460)a CIDI-SC prevalence estimateb Positive operating characteristicsc Negative operating characteristicsd % (se) SN (se) PPV (se) LR+ SP (se) NPV (se) LR− Major depressive episode Conservative 6.0 (0.8) 49.1 (8.8) 54.9 (8.5) 16.9 97.1 (0.7) 96.4 (1.0) 0.5 Optimal 6.8 (1.0) 58.8 (9.0) 57.5 (6.6) 19.0 96.9 (0.8) 97.0 (0.9) 0.4 Anticonservative 7.5 (1.2) 62.5 (9.2) 55.6 (6.3) 17.4 96.4 (0.9) 97.3 (0.9) 0.4 Mania/hypomania Conservative 2.7 (0.5) 20.9 (5.9) 39.5 (8.3) 12.3 98.3 (0.4) 95.8 (1.0) 0.8 Optimal 4.9 (1.0) 43.5 (8.9) 45.8 (6.3) 15.5 97.2 (0.6) 96.9 (0.8) 0.6 Anticonservative 11.6 (1.4) 72.6 (9.1) 32.3 (6.6) 8.7 91.7 (1.4) 98.4 (0.6) 0.3 Panic disorder Conservative 3.4 (0.7) 37.1 (9.9) 54.8 (8.9) 23.2 98.4 (0.4) 96.7 (0.7) 0.6 Optimal 5.0 (0.9) 58.5 (11.2) 59.5 (7.7) 27.9 97.9 (0.4) 97.8 (0.6) 0.4 Anticonservative 6.4 (0.9) 71.4 (10.4) 57.1 (6.9) 24.6 97.1 (0.5) 98.4 (0.5) 0.3 Generalized anxiety disorder Conservative 6.0 (0.8) 40.8 (4.6) 46.7 (7.7) 10.9 96.6 (0.8) 95.7 (0.7) 0.5 Optimal 6.6 (0.9) 43.9 (4.7) 45.6 (7.4) 11.5 96.2 (0.8) 95.9 (0.7) 0.6 Anticonservative 7.1 (1.0) 44.2 (4.8) 42.6 (7.8) 10.0 95.6 (1.0) 95.9 (0.7) 0.6 Post-traumatic stress disorder Conservative 6.2 (1.0) 46.0 (7.0) 47.5 (7.2) 13.1 96.5 (0.9) 96.3 (0.7) 0.6 Optimal 6.7 (1.0) 53.7 (6.9) 50.8 (7.0) 15.3 96.5 (0.8) 96.8 (0.6) 0.5 Anticonservative 7.7 (1.1) 56.8 (7.2) 47.2 (6.3) 13.2 95.7 (0.9) 97.0 (0.6) 0.4 Adult attention-deficit/hyperactivity disorder Conservative 6.8 (1.1) 31.8 (6.2) 33.4 (6.8) 6.5 95.1 (1.1) 94.8 (1.0) 0.7 Optimal 8.2 (1.1) 42.8 (7.8) 37.1 (7.0) 7.8 94.5 (1.1) 95.6 (1.0) 0.6 Anticonservative 8.8 (1.1) 44.1 (7.9) 35.5 (6.5) 7.2 93.9 (1.1) 95.7 (1.0) 0.5 Intermittent explosive disorder Kessle Table 3 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are changed from the optimal for estimating prevalence to either more conservative or more anticonservative thresholds (n=460)a Kess Table 3 I screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are changed from the optimal for estimating prevalence ti ti ti th h ld ( 460)a Page 25 Page 25 Kessler et al. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc Page 24 Page 24 Kessler et al. NIH-PA Author Manuscript Table 2 CIDI screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc SN (se) PPV (se) LR+ SP (se) NPV (se) LR− I. Mood disorders Major depressive episode 58.8 (9.0) 57.5 (6.6) 19.0 96.9 (0.8) 97.0 (0.9) 0.4 Mania/hypomania 43.5 (8.9) 45.8 (6.3) 15.5 97.2 (0.6) 96.9 (0.8) 0.6 II. Anxiety disorders Panic disorder 58.5 (11.2) 59.5 (7.7) 27.9 97.9 (0.4) 97.8 (0.6) 0.4 Generalized anxiety disorder 43.9 (4.7) 45.6 (7.4) 11.5 96.2 (0.8) 95.9 (0.7) 0.6 Post-traumatic stress disorder 53.7 (6.9) 50.8 (7.0) 15.3 96.5 (0.8) 96.8 (0.6) 0.5 III. Externalizing disorders Adult attention-deficit/hyperactivity disorder 42.8 (7.8) 37.1 (7.0) 7.8 94.5 (1.1) 95.6 (1.0) 0.6 Intermittent explosive disorder 66.8 (6.2) 65.3 (5.2) 7.3 90.9 (1.6) 91.5 (1.6) 0.4 Substance use disorder 47.6 (8.5) 51.6 (8.0) 19.0 97.5 (0.5) 97.0 (0.8) 0.5 IV. Any disorder d 63.7 (4.3) 68.3 (4.0) 8.5 92.5 (1.2) 90.9 (1.6) 0.4 aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. bSN = Sensitivity (the percent of SCID cases detected by the CIDI-SC); PPV = Positive predictive value (the percent of CIDI-SC cases confirmed by the SCID); LR+ = Likelihood ratio positive (the relative proportions of SCID cases among CIDI-SC cases versus non-cases). cSP = Specificity (the percent of SCID non-cases classified as non-cases by the CIDI-SC); NPV = Negative predictive value (the percent of CIDI-SC non-cases confirmed as non-cases by the SCID); LR− = Likelihood ratio negative (the relative proportions of SCID non-cases among CIDI-SC cases versus non-cases). dAny of the seven disorders other than mania/hypomania, as mania/hypomania were assessed only over the entire lifetime. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 0 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Autho NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc gnostic thresholds are changed from the optimal for estimating prevalence NIH-PA Author Manuscript NIH-PA Author Manuscript Table 3 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are to either more conservative or more anticonservative thresholds (n=460)a CIDI-SC prevalence estimateb Positive operating characteristicsc Negative operating characteristicsd % (se) SN (se) PPV (se) LR+ SP (se) NPV (se) LR− Major depressive episode Conservative 6.0 (0.8) 49.1 (8.8) 54.9 (8.5) 16.9 97.1 (0.7) 96.4 (1.0) 0.5 Optimal 6.8 (1.0) 58.8 (9.0) 57.5 (6.6) 19.0 96.9 (0.8) 97.0 (0.9) 0.4 Anticonservative 7.5 (1.2) 62.5 (9.2) 55.6 (6.3) 17.4 96.4 (0.9) 97.3 (0.9) 0.4 Mania/hypomania Conservative 2.7 (0.5) 20.9 (5.9) 39.5 (8.3) 12.3 98.3 (0.4) 95.8 (1.0) 0.8 Optimal 4.9 (1.0) 43.5 (8.9) 45.8 (6.3) 15.5 97.2 (0.6) 96.9 (0.8) 0.6 Anticonservative 11.6 (1.4) 72.6 (9.1) 32.3 (6.6) 8.7 91.7 (1.4) 98.4 (0.6) 0.3 Panic disorder Conservative 3.4 (0.7) 37.1 (9.9) 54.8 (8.9) 23.2 98.4 (0.4) 96.7 (0.7) 0.6 Optimal 5.0 (0.9) 58.5 (11.2) 59.5 (7.7) 27.9 97.9 (0.4) 97.8 (0.6) 0.4 Anticonservative 6.4 (0.9) 71.4 (10.4) 57.1 (6.9) 24.6 97.1 (0.5) 98.4 (0.5) 0.3 Generalized anxiety disorder Conservative 6.0 (0.8) 40.8 (4.6) 46.7 (7.7) 10.9 96.6 (0.8) 95.7 (0.7) 0.5 Optimal 6.6 (0.9) 43.9 (4.7) 45.6 (7.4) 11.5 96.2 (0.8) 95.9 (0.7) 0.6 Anticonservative 7.1 (1.0) 44.2 (4.8) 42.6 (7.8) 10.0 95.6 (1.0) 95.9 (0.7) 0.6 Post-traumatic stress disorder Conservative 6.2 (1.0) 46.0 (7.0) 47.5 (7.2) 13.1 96.5 (0.9) 96.3 (0.7) 0.6 Optimal 6.7 (1.0) 53.7 (6.9) 50.8 (7.0) 15.3 96.5 (0.8) 96.8 (0.6) 0.5 Anticonservative 7.7 (1.1) 56.8 (7.2) 47.2 (6.3) 13.2 95.7 (0.9) 97.0 (0.6) 0.4 Adult attention-deficit/hyperactivity disorder Conservative 6.8 (1.1) 31.8 (6.2) 33.4 (6.8) 6.5 95.1 (1.1) 94.8 (1.0) 0.7 Optimal 8.2 (1.1) 42.8 (7.8) 37.1 (7.0) 7.8 94.5 (1.1) 95.6 (1.0) 0.6 Anticonservative 8.8 (1.1) 44.1 (7.9) 35.5 (6.5) 7.2 93.9 (1.1) 95.7 (1.0) 0.5 Intermittent explosive disorder Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. NIH-PA Author Manuscript NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Page 26 Page 26 Kessler et al. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc Page Table 4 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are changed from (i) the optimal for estimating prevalence to (ii) having high SN (i.e., detecting at least 80% of DSM-IV/SCID cases) (n=460)a CIDI-SC prevalence estimateb Positive operating characteristicsc Negative operating characteristicsd % (se) SN (se) PPV (se) LR+ SP (se) NPV (se) LR− Major depressive episode Optimal for prevalence 6.8 (1.0) 58.8 (9.0) 57.5 (6.6) 19.0 96.9 (0.8) 97.0 (0.9) 0.4 High SN 25.2 (2.1) 80.2 (11.8) 21.3 (3.1) 3.8 78.7 (2.1) 98.2 (1.2) 0.3 Mania/hypomania Optimal for prevalence 4.9 (1.0) 43.5 (8.9) 45.8 (6.3) 15.5 97.2 (0.6) 96.9 (0.8) 0.6 High SN 20.5 (2.2) 82.7 (8.4) 20.8 (4.1) 4.8 82.9 (2.1) 98.9 (0.6) 0.2 Panic disorder Optimal for prevalence 5.0 (0.9) 58.5 (11.2) 59.5 (7.7) 27.9 97.9 (0.4) 97.8 (0.6) 0.4 High SNe 6.4 (0.9) 71.4 (10.4) 57.1 (6.9) 24.6 97.1 (0.5) 98.4 (0.5) 0.3 Generalized anxiety disorder Optimal for prevalence 6.6 (0.9) 43.9 (4.7) 45.6 (7.4) 11.5 96.2 (0.8) 95.9 (0.7) 0.6 High SN 19.1 (2.1) 80.6 (5.2) 28.9 (5.3) 5.5 85.4 (2.3) 98.4 (0.5) 0.2 Post-traumatic stress disorder Optimal for prevalence 6.7 (1.0) 53.7 (6.9) 50.8 (7.0) 15.3 96.5 (0.8) 96.8 (0.6) 0.5 High SN 43.5 (4.0) 81.2 (6.6) 11.9 (2.0) 2.0 59.0 (4.2) 97.9 (0.8) 0.3 Adult attention-deficit/hyperactivity disorder Optimal for prevalence 8.2 (1.1) 42.8 (7.8) 37.1 (7.0) 7.8 94.5 (1.1) 95.6 (1.0) 0.6 High SN 40.3 (2.9) 84.3 (7.5) 14.9 (2.5) 2.3 63.1 (3.1) 98.1 (1.0) 0.2 Intermittent explosive disorder Optimal for prevalence 20.8 (2.3) 66.8 (6.2) 65.3 (5.2) 7.3 90.9 (1.6) 91.5 (1.6) 0.4 High SNe 26.7 (3.3) 73.5 (7.4) 56.0 (5.7) 5.0 85.3 (2.7) 92.6 (1.9) 0.3 Substance use disorder Optimal for prevalence 4.9 (0.4) 47.6 (8.5) 51.6 (8.0) 19.0 97.5 (0.5) 97.0 (0.8) 0.5 High SNe 12.4 (1.6) 66.8 (9.,4) 28.8 (5.4) 7.1 90.6 (1.7) 98.0 (0.8) 0.4 Page 27 Page 27 Kessler et al. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc NIH-PA Author Manuscript CIDI-SC prevalence estimateb Positive operating characteristicsc Negative operating characteristicsd % (se) SN (se) PPV (se) LR+ SP (se) NPV (se) LR− Conservative 16.8 (1.4) 47.3 (4.5) 57.2 (5.5) 5.2 90.9 (1.6) 87.1 (2.0) 0.6 Optimal 20.8 (2.3) 66.8 (6.2) 65.3 (5.2) 7.3 90.9 (1.6) 91.5 (1.6) 0.4 Anticonservative 26.7 (3.3) 73.5 (7.4) 56.0 (5.7) 5.0 85.3 (2.7) 92.6 (1.9) 0.9 Substance use disorder Conservative 4.1 (0.5) 38.1 (8.3) 49.5 (8.4) 17.3 97.8 (0.4) 96.5 (0.8) 0.6 Optimal 4.9 (0.4) 47.6 (8.5) 51.6 (8.0) 19.0 97.5 (0.5) 97.0 (0.8) 0.5 Anticonservative 6.7 (0.6) 57.3 (9.1) 45.6 (5.6) 14.7 96.1 (0.5) 97.5 (0.8) 0.4 aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. bThe CIDI-SC prevalence estimates are varied by changing the threshold to values both above (conservative) and below (anticonservative) the thresholds designed to maximize concordance with prevalence estimates based on the blinded SCID clinical reappraisal interviews. cSN = Sensitivity (the percent of SCID cases detected by the CIDI-SC); PPV = Positive predictive value (the percent of CIDI-SC cases confirmed by the SCID); LR+ = Likelihood ratio positive (the relative proportions of SCID cases among CIDI-SC cases versus non-cases dSP = Specificity (the percent of SCID non-cases classified as non-cases by the CIDI-SC); NPV = Negative predictive value (the percent of CIDI-SC non-cases confirmed as non-cases by the SCID); LR− = Likelihood ratio negative (the relative proportions of SCID non-cases among CIDI-SC cases versus non-cases. Int J Methods Psychiatr Res. Author manuscript; available NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth Table 4 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are c prevalence to (ii) having high SN (i.e., detecting at least 80% of DSM-IV/SCID cases) (n=460)a CIDI-SC prevalence estimateb Positive operating characteristicsc Negative operating characteristicsd % (se) SN (se) PPV (se) LR+ SP (se) NPV (se) LR− Major depressive episode Optimal for prevalence 6.8 (1.0) 58.8 (9.0) 57.5 (6.6) 19.0 96.9 (0.8) 97.0 (0.9) 0.4 High SN 25.2 (2.1) 80.2 (11.8) 21.3 (3.1) 3.8 78.7 (2.1) 98.2 (1.2) 0.3 Mania/hypomania Optimal for prevalence 4.9 (1.0) 43.5 (8.9) 45.8 (6.3) 15.5 97.2 (0.6) 96.9 (0.8) 0.6 High SN 20.5 (2.2) 82.7 (8.4) 20.8 (4.1) 4.8 82.9 (2.1) 98.9 (0.6) 0.2 Panic disorder Optimal for prevalence 5.0 (0.9) 58.5 (11.2) 59.5 (7.7) 27.9 97.9 (0.4) 97.8 (0.6) 0.4 High SNe 6.4 (0.9) 71.4 (10.4) 57.1 (6.9) 24.6 97.1 (0.5) 98.4 (0.5) 0.3 Generalized anxiety disorder Optimal for prevalence 6.6 (0.9) 43.9 (4.7) 45.6 (7.4) 11.5 96.2 (0.8) 95.9 (0.7) 0.6 High SN 19.1 (2.1) 80.6 (5.2) 28.9 (5.3) 5.5 85.4 (2.3) 98.4 (0.5) 0.2 Post-traumatic stress disorder Optimal for prevalence 6.7 (1.0) 53.7 (6.9) 50.8 (7.0) 15.3 96.5 (0.8) 96.8 (0.6) 0.5 High SN 43.5 (4.0) 81.2 (6.6) 11.9 (2.0) 2.0 59.0 (4.2) 97.9 (0.8) 0.3 Adult attention-deficit/hyperactivity disorder Optimal for prevalence 8.2 (1.1) 42.8 (7.8) 37.1 (7.0) 7.8 94.5 (1.1) 95.6 (1.0) 0.6 High SN 40.3 (2.9) 84.3 (7.5) 14.9 (2.5) 2.3 63.1 (3.1) 98.1 (1.0) 0.2 Intermittent explosive disorder Optimal for prevalence 20.8 (2.3) 66.8 (6.2) 65.3 (5.2) 7.3 90.9 (1.6) 91.5 (1.6) 0.4 High SNe 26.7 (3.3) 73.5 (7.4) 56.0 (5.7) 5.0 85.3 (2.7) 92.6 (1.9) 0.3 Substance use disorder Optimal for prevalence 4.9 (0.4) 47.6 (8.5) 51.6 (8.0) 19.0 97.5 (0.5) 97.0 (0.8) 0.5 High SNe 12.4 (1.6) 66.8 (9.,4) 28.8 (5.4) 7.1 90.6 (1.7) 98.0 (0.8) 0.4 Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. NIH-PA Author Manuscript NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Kessler et al. aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc bThe CIDI-SC prevalence estimates are varied by changing the threshold to have a minimum SN of 80.0% based on the blinded SCID clinical reappraisal interviews. cSN = Sensitivity (the percent of SCID cases detected by the CIDI-SC); PPV = Positive predictive value (the percent of CIDI-SC cases confirmed by the SCID); LR+ = Likelihood ratio positive (the relative proportions of SCID cases among CIDI-SC cases versus non-cases cSP = Specificity (the percent of SCID non-cases classified as non-cases by the CIDI-SC); NPV = Negative predictive value (the percent of CIDI-SC non-cases confirmed as non-cases by the SCID); LR−+ = Likelihood ratio negative (the relative proportions of SCID non-cases among CIDI-SC cases versus non-cases. eAs none of the CIDI-SC thresholds for this disorder had SN as high as 80%, the threshold with the highest SN is reported. NIH-PA Author Manuscript Page 28 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Au NIH-PA Author Manuscript NIH-PA Autho NIH-PA Author Manuscript Kessler et al. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc Page Table 5 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are changed from (i) the optimal for estimating prevalence to (ii) having high PPV (i.e., at least 80% of screened positives having a DSM-IV/SCID diagnosis) (n=460)a CIDI-SC prevalence estimateb Positive operating characteristicsc Negative operating characteristicsd % (se) SN (se) PPV (se) LR+ SP (se) NPV (se) LR− Major depressive episode Optimal for prevalence 6.8 (1.0) 58.8 (9.0) 57.5 (6.6) 19.0 96.9 (0.8) 97.0 (0.9) 0.4 High PPV 0.6 (0.3) 7.3 (4.1) 84.7 (11.5) 73.0 99.9 (0.1) 93.8 (1.0) 0.9 Mania/hypomania Optimal for prevalence 4.9 (1.0) 43.5 (8.9) 45.8 (6.3) 15.5 97.2 (0.6) 96.9 (0.8) 0.6 High PPVe 0.7 (0.2) 9.7 (3.0) 71.1 (12.5) 48.5 99.8 (0.1) 95.3 (1.0) 0.9 Generalized anxiety disorder Optimal for prevalence 6.6 (0.9) 43.9 (4.7) 45.6 (7.4) 11.5 96.2 (0.8) 95.9 (0.7) 0.6 PPV GT 50% 3.2 (0.6) 23.6 (3.9) 50.2 (8.5) 13.9 98.3 (0.5) 94.6 (0.9) 0.8 High PPVe 1.0 (0.3) 10.4 (3.5) 69.1 (12.2) 34.7 99.7 (0.2) 93.8 (0.9) 0.9 Post-traumatic stress disorder Optimal for prevalence 6.7 (1.0) 53.7 (6.9) 50.8 (7.0) 15.3 96.5 (0.8) 96.8 (0.6) 0.5 High PPVe 1.1 (0.3) 13.4 (3.3) 79.5 (10.5) 67.0 79.5 (10.5) 99.8 (0.1) 0.9 Adult attention-deficit/hyperactivity disorder Optimal for prevalence 8.2 (1.1) 42.8 (7.8) 37.1 (7.0) 7.8 94.5 (1.1) 95.6 (1.0) 0.6 PPV GT 50% 2.4 (0.5) 19.3 (3.8) 55.9 (12.0) 16.1 98.8 (0.4) 94.1 (1.0) 0.8 High PPVe 2.0 (0.4) 17.8 (3.7) 63.1 (12.7) 22.3 99.2 (0.3) 94.1 (0.9) 0.8 Substance use disorder Optimal for prevalence 4.9 (0.4) 47.6 (8.5) 51.6 (8.0) 19.0 97.5 (0.5) 97.0 (0.8) 0.5 High PPV 0.6 (0.2) 8.8 (3.6) 81.7 (13.3) 88.0 99.9 (0.1) 95.1 (0.8) 0.9 aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. bThe CIDI-SC prevalence estimates are varied by changing the threshold to have a minimum SN of 80.0% based on the blinded SCID clinical reappraisal interviews. Results are not reported for PD or IED because optimal thresholds for predicting SCID prevalence of these disorders also had the highest values of PPV. Kessler et a Table 5 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are changed from (i) the optimal for estimating prevalence to (ii) having high PPV (i.e., at least 80% of screened positives having a DSM-IV/SCID diagnosis) (n=460)a Kessler et al. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc Page 29 NIH-PA Author Manuscript NIH-PA Author Manuscript Table 5 Variation in CIDI screening scale (CIDI-SC) operating characteristics when diagnostic thresholds are chan prevalence to (ii) having high PPV (i.e., at least 80% of screened positives having a DSM-IV/SCID diagno CIDI-SC prevalence estimateb Positive operating characteristicsc Negative operating characteristicsd % (se) SN (se) PPV (se) LR+ SP (se) NPV (se) LR− Major depressive episode Optimal for prevalence 6.8 (1.0) 58.8 (9.0) 57.5 (6.6) 19.0 96.9 (0.8) 97.0 (0.9) 0.4 High PPV 0.6 (0.3) 7.3 (4.1) 84.7 (11.5) 73.0 99.9 (0.1) 93.8 (1.0) 0.9 Mania/hypomania Optimal for prevalence 4.9 (1.0) 43.5 (8.9) 45.8 (6.3) 15.5 97.2 (0.6) 96.9 (0.8) 0.6 High PPVe 0.7 (0.2) 9.7 (3.0) 71.1 (12.5) 48.5 99.8 (0.1) 95.3 (1.0) 0.9 Generalized anxiety disorder Optimal for prevalence 6.6 (0.9) 43.9 (4.7) 45.6 (7.4) 11.5 96.2 (0.8) 95.9 (0.7) 0.6 PPV GT 50% 3.2 (0.6) 23.6 (3.9) 50.2 (8.5) 13.9 98.3 (0.5) 94.6 (0.9) 0.8 High PPVe 1.0 (0.3) 10.4 (3.5) 69.1 (12.2) 34.7 99.7 (0.2) 93.8 (0.9) 0.9 Post-traumatic stress disorder Optimal for prevalence 6.7 (1.0) 53.7 (6.9) 50.8 (7.0) 15.3 96.5 (0.8) 96.8 (0.6) 0.5 High PPVe 1.1 (0.3) 13.4 (3.3) 79.5 (10.5) 67.0 79.5 (10.5) 99.8 (0.1) 0.9 Adult attention-deficit/hyperactivity disorder Optimal for prevalence 8.2 (1.1) 42.8 (7.8) 37.1 (7.0) 7.8 94.5 (1.1) 95.6 (1.0) 0.6 PPV GT 50% 2.4 (0.5) 19.3 (3.8) 55.9 (12.0) 16.1 98.8 (0.4) 94.1 (1.0) 0.8 High PPVe 2.0 (0.4) 17.8 (3.7) 63.1 (12.7) 22.3 99.2 (0.3) 94.1 (0.9) 0.8 Substance use disorder Optimal for prevalence 4.9 (0.4) 47.6 (8.5) 51.6 (8.0) 19.0 97.5 (0.5) 97.0 (0.8) 0.5 High PPV 0.6 (0.2) 8.8 (3.6) 81.7 (13.3) 88.0 99.9 (0.1) 95.1 (0.8) 0.9 aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. bThe CIDI-SC prevalence estimates are varied by changing the threshold to have a minimum SN of 80.0% based on the blinded SCID clinica because optimal thresholds for predicting SCID prevalence of these disorders also had the highest values of PPV. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. NIH-PA Author Manuscript NIH-PA Author Manuscript Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. Page 30 Page 30 Kessler et al. Kessler et al Table 2 I screening scale (CIDI-SC) operating characteristics at optimal thresholds for estimating DSM-IV/SCID prevalence (n = 460)a Positive operating characteristicsb Negative operating characteristicsc NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript cSN = Sensitivity (the percent of SCID cases detected by the CIDI-SC); PPV = Positive predictive value (the percent of CIDI-SC cases confirmed by the SCID); LR+ = Likelihood ratio positive (the relative proportions of SCID cases among CIDI-SC cases versus non-cases dSP = Specificity (the percent of SCID non-cases classified as non-cases by the CIDI-SC); NPV = Negative predictive value (the percent of CIDI-SC non-cases confirmed as non-cases by the SCID); LR−+ = Likelihood ratio negative (the relative proportions of SCID non-cases among CIDI-SC cases versus non-cases. eAs none of the CIDI-SC thresholds for this disorder had PPV as high as 80%, the threshold with the highest PPV is reported. NIH-PA Author Manuscript NIH-PA Author Manuscript Kessler et al. Page 31 Table 6 Comparison of area under the receiver operating characteristic curve (AUC) based on the dichotomous versions of the CIDI-SC scales as the optimal thresholds for estimating DSM-IV/SCID prevalence and based on the continuous versions of the CIDI-SC scales (n = 460) a NIH-PA Author Manuscript Area under the curve (AUC) Dichotomous Continuous I. Mood disorders Major depressive episode .78 .90 Mania/hypomania .70 .86 II. Anxiety disorders Panic disorder .78 .90 Generalized anxiety disorder .70 .87 Post-traumatic stress disorder .75 .81 III. Externalizing disorders Adult attention deficit/hyperactivity disorder .69 .85 Intermittent explosive disorder .79 .86 Substance use disorder .73 .80 aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. Int J Methods Psychiatr Res. Author manuscript; available in PMC 2014 December 01. aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales I. Mood disorders NIH-PA Author Manuscript NIH-PA NIH-PA Author Manuscript aAnalyses are based on weighted data to adjust for the over-sampling of respondents screening positive on the CIDI-SC scales. NIH-PA Author Manuscript NIH-PA Author Manuscript
https://openalex.org/W4365459350
https://madoc.bib.uni-mannheim.de/64811/1/s11615-023-00463-5.pdf
English
null
No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and Defense Policy After Russia’s Invasion of Ukraine
Politische Vierteljahresschrift
2,023
cc-by
12,301
ERROR: type should be string, got "https://doi.org/10.1007/s11615-023-00463-5\nPolitische Vierteljahresschrift (2023) 64:525–547 CRITICAL PAPER \u0002 Matthias Mader\nDepartment of Politics and Public Administration, University of Konstanz, P.O. Box\n91, 78457 Konstanz, Germany\nE-Mail: matthias.mader@uni-konstanz.de Harald Schoen\nSchool of Social Sciences, University of Mannheim, A 5, 6, 68159 Mannheim, Germany\nE-Mail: harald.schoen@uni-mannheim.de Keywords Russo-Ukraine war · Turning point · Stability and change · Core\npostures · Policy attitudes · Survey data No Zeitenwende (yet): Early Assessment of German\nPublic Opinion Toward Foreign and Defense Policy\nAfter Russia’s Invasion of Ukraine Matthias Mader\n· Harald Schoen Received: 2 October 2022 / Revised: 9 March 2023 / Accepted: 21 March 2023 / Published online: 13\nApril 2023\n© The Author(s) 2023 Received: 2 October 2022 / Revised: 9 March 2023 / Accepted: 21 March 2023 / Published online: 13\nApril 2023\n© Th A h\n( ) 2023 Abstract This paper addresses the question of whether Russia’s invasion of Ukraine\nled to a turning point (Zeitenwende) in public opinion on foreign and defense policy\nin Germany. To this end, we provide a theoretical analysis of how the concept of\nturning point can be applied to public opinion. We identify the durability of the\nchange in attitudes as well as its significance as necessary conditions to speak of\na turning point. In the remainder of the paper, we focus on the argument that changes\nin different types of orientations are significant to different degrees. Change in core\npostures is more significant than change in policy attitudes; change in attitudes\nthematically distant from the Russian invasion is more significant than change in\nattitudes directly related to the event. Empirically, we present a panel data analysis\nof attitude change triggered by the Russian invasion. Analysis of data from several\nwaves of the German Longitudinal Election Study (GLES) panel survey collected\nbefore the invasion (2017–2021) and in two waves after (May and October 2022)\nshows that there were sizable shifts in policy attitudes directly related to the event. Postures remained essentially unchanged, as did thematically distant attitudes. We\nconclude that there has been no turning point at the level of public opinion (yet). Keywords Russo-Ukraine war · Turning point · Stability and change · Core\npostures · Policy attitudes · Survey data K 526 M. Mader, H. Schoen (Noch) Keine Zeitenwende: Eine vorläufige Untersuchung der\nöffentlichen Meinung in Deutschland zur Außen- und\nVerteidigungspolitik nach Russlands Einmarsch in die Ukraine Zusammenfassung\nDer Beitrag untersucht, ob der Einmarsch Russlands in die\nUkraine zu einer Zeitenwende in der öffentlichen Meinung zur Außen- und Ver-\nteidigungspolitik in Deutschland geführt hat. Zu diesem Zweck diskutieren wir\nzunächst theoretisch, wie das Konzept der Zeitenwende auf die öffentliche Mei-\nnung angewendet werden kann. Wir identifizieren die Dauerhaftigkeit des Einstel-\nlungswandels sowie seine Signifikanz als notwendige Bedingungen, um von einer\nZeitenwende zu sprechen. Im weiteren Verlauf des Papiers konzentrieren wir uns\nauf das Argument, dass Veränderungen unterschiedlicher Einstellungstypen unter-\nschiedlich signifikant sind. Veränderungen von Grundhaltungen sind wichtiger als\nsolche spezifischer Sachfrageorientierungen; Veränderungen von Einstellungen, die\nthematisch weiter von der russischen Invasion entfernt sind, sind signifikanter als\nVeränderungen von Einstellungen, die direkt auf das Ereignis bezogen sind. Um em-\npirisch zu prüfen, inwieweit infolge der russischen Invasion Einstellungsänderungen\neingetreten sind, analysieren wir Umfragedaten aus dem GLES-Panel, die vor der\nInvasion (2017-2021) und in zwei Wellen danach (Mai und Oktober 2022) erhoben\nwurden. Die Befunde zeigen, dass es signifikante Verschiebungen spezifischer Sach-\nfrageorientierungen gab, die thematisch eng mit dem Ereignis zusammenhängen. Grundhaltungen zur Außen- und Sicherheitspolitik blieben dagegen im Wesentli-\nchen unverändert, ebenso wie thematisch entfernte Einstellungen. Wir kommen zu\ndem Schluss, dass auf der Ebene der öffentlichen Meinung bisher keine Zeitenwende\nstattgefunden hat. Schlüsselwörter Russisch-Ukrainischer Krieg · Stabilität und Wandel ·\nGrundhaltungen · Sachfrageorientierungen · Umfragedaten Schlüsselwörter Russisch-Ukrainischer Krieg · Stabilität und Wandel ·\nGrundhaltungen · Sachfrageorientierungen · Umfragedaten 1 Introduction The claim that Russia’s invasion of Ukraine in 2022 was a Zeitenwende quickly\nbecame an anchor of public debates in Germany. Chancellor Scholz popularized the\nterm when he used it just days after the invasion began. “[Zeitenwende] means,”\nScholz said, “that the world will not be the same afterwards as it was before”\n(Scholz 2022). Accordingly, the invasion represented a fundamental break with the\n(liberal) international order that required Germany to revise some of its basic foreign\npolicy beliefs and policies. Scholz elaborated on this by announcing, among other\nthings, arms deliveries to Ukraine, a significant increase in military spending, and\nthe strengthening of cooperation with like-minded countries. The following debates\nfocused on what effects the Russian invasion had or should have on the international\norder and German foreign policy, and whether these are correctly described by the\nterm Zeitenwende (e.g., Blumenau 2022; Bunde 2022; Groitl 2022; Fröhlich 2023;\nRisse 2022). K K 527 No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... Less systematic attention has so far been paid to the question of how the German\npopulation reacted to the Russian invasion. Was there a Zeitenwende at the level\nof German public opinion? This question is highly relevant, since democracies are\ncharacterized by a complex interrelationship between citizens and political decision-\nmakers. Changes of course initiated from the top not only gain democratic legiti-\nmacy, but they are also more likely to last if they are broadly in line with public\nopinion. If they are not, and decision-makers fail to persuade the public of their po-\nsition, (announced) course changes may be reversed and those responsible may even\nbe voted out of office. In foreign policy, too, public opinion provides such bound-\naries for decision-makers. Granted, leaders can usually move quite freely within\nthese boundaries and lead public opinion on specific policy issues (Rattinger 1985;\nSaunders 2014). However, if the boundaries are overstepped and public interest ac-\ntivated, foreign policy issues can influence citizens’ voting decisions (Aldrich et al. 2006; Schoen 2011). To avoid jeopardizing their popularity ratings, governments\npay attention to public opinion between elections and may take it into account in\ntheir decisions (Baum and Potter 2015; Mello 2022; Sobel 2001). A comprehensive\nassessment of the Zeitenwende claim or, more generally, of the impact of Russia’s\ninvasion of Ukraine on German foreign policy therefore requires an analysis of\npublic opinion. 1 Introduction Surveys of the German population conducted shortly after the start of the Russian\ninvasion showed that sympathy ratings for Putin had fallen and the perceived threat\nfrom Russia had risen sharply (Graf 2022). There was also an increased willingness\nto defend allies in the event of a Russian attack, although this was still not a majority\nposition (Smith 2022). Increasing military spending, sanctions against Russia, and\narms deliveries to Ukraine met with majority approval (Bunde and Lubbock 2022; de\nVries and Hofmann 2022). These early post-invasion surveys hence stand to some\nextent in contrast to traditional findings of German public opinion as “Venutian”\nrather than “Marsian” (Kagan 2004), as preferring cooperation over confrontation\nand persuasion over coercion (Duffield 1998; Gravelle et al. 2017; Rattinger et al. 2016). They suggest a shift from restraint to a more assertive posture. Against this\nbackdrop, a constraint that may have prevented German policymakers from assuming\nsuch a posture in the past might be loosening. p\np\ng\ng\nHowever, caution is warranted in rashly diagnosing a Zeitenwende at the level\nof German public opinion. Previous major events, such as the terrorist attacks of\nSeptember 11, 2001, and the Iraq war in 2003, influenced public opinion in Germany\nonly with respect to a limited number of issues and for a limited time (e.g., Rattinger\net al. 2016). It is therefore advisable not simply to assert a “new mindset in German\nsociety” (Scholz 2023) but to subject the question to careful empirical analysis. Such an analysis should start with a specification of the key concept. The Zeit-\nenwende claim can be usefully clarified by drawing on existing scholarly work on\nturning points (Abbott 2001) and related concepts (Capoccia and Kelemen 2007;\nBaumgartner et al. 2009). Based on this literature, we argue that a Zeitenwende\nclaim at the level of public opinion requires significant and durable attitude change. This conceptual clarification is useful in guiding systematic analysis and evaluating\nexisting evidence. For example, the shifts in public opinion described above, which\nwere measured shortly after the start of the Russian invasion, cannot speak to the K 528 M. Mader, H. Schoen criterion of durability, and are hence not sufficient evidence for diagnosing a turning\npoint. In this article we take several steps toward answering the question of whether\nRussia’s invasion of Ukraine marked a Zeitenwende at the level of German public\nopinion. 1 Similar concepts have been developed elsewhere, for example critical juncture (Capoccia and Kelemen\n2007) and punctuated equilibrium (Baumgartner et al. 2009), in the literature on institutional change. These\nmay prove particularly useful for scrutinizing the Zeitenwende claim at the institutional level. 1 Introduction First, we elaborate on significance and durability as criteria of turning points\nand specify these for public opinion as the unit of analysis. The main argument is that\nby distinguishing between different types of orientations—policy attitudes vs. core\npostures; attitudes thematically related to Russia’s invasion vs. more removed—we\ncan assess the likelihood and significance of the change that has occurred and, on\nthat basis, make educated guesses about the durability of the change, even though\nnot much time may have passed since the event. Second, we analyze German pub-\nlic opinion data from a panel survey conducted in the framework of the German\nLongitudinal Election Study (e.g., GLES 2021). These data were collected before\n(December 2021 and earlier) and after (May and October 2022) Russia’s invasion of\nUkraine, and the broad set of foreign and defense orientations that were repeatedly\nmeasured in this survey allow a detailed account of German mass belief systems in\nthis domain. Furthermore, the panel data structure allows analysis not only of ag-\ngregate but also of individual-level change, hence providing a much more detailed\naccount of whose opinion changed in what direction. To anticipate the most important result, which will be elaborated upon in the\nfollowing pages: the data suggest that Russia’s invasion of Ukraine was not a historic\nturning point for German public opinion. Significant change was limited to attitudes\ntoward policy issues and actors directly related to the Ukraine war. Neither did\ncore postures change appreciably, nor did attitudes toward thematically more distant\nobjects. In short, systematic analysis of survey data consisting of several pre- and\npost-invasion waves spanning a period of several years and extending into October\n2022 shows that Germans did not see the world much differently after Russia’s\ninvasion of Ukraine than before. 2 Conceptual and Methodological Issues We draw on the concept of turning point (Abbott 2001) to link the popular Zeiten-\nwende claim to previous scholarly work and to guide our analysis of opinion change. Focusing on the analysis of life courses, Abbott defines turning points as short, rad-\nical shifts that set the life course on a new trajectory (Abbott 2001, p. 243).1 We\nfocus here on two aspects of this definition, each necessary but not sufficient to\nspeak of a turning point. First, turning points are characterized by changes of a cer-\ntain significance. It is not enough for Russia’s invasion to change the world—or,\nin our case, public opinion in Germany—in some small, insignificant way; to be\nconsidered a turning point, the effects must be of fundamental importance. Second,\nturning points separate periods of stability. “What makes a turning point a turning K K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 529 point rather than a minor ripple is the passage of sufficient time on ‘the new course’\nsuch that it becomes clear that the direction has indeed been changed” (Abbott 2001,\np. 245). Changes must therefore have a certain durability. If public opinion were\nto quickly return to its pre-invasion level, the event would not qualify as a turning\npoint (for public opinion). These two criteria are simple but also abstract—they need to be specified in order\nto make them accessible to empirical analysis. To specify the criterion of signif-\nicant change in public opinion, we argue that not only the magnitude of change\nmatters—more on that below—but also which type of attitude is affected. To de-\nvelop this argument, we draw on the concept of belief system (Converse 1964). Accordingly, attitudes are organized in associative networks, where a horizontal\nand a vertical dimension can be distinguished (Converse 1964; Peffley and Hurwitz\n1985). The horizontal dimension captures the idea that attitudes refer to different\ntopics, and that associations between two attitudes on the same topic tend to be\nstronger than associations between two attitudes on different topics. Because of this\npattern of association strengths, attitude change is more likely to spread among at-\ntitudes on the same topic and less likely to spread to attitudes on a different topic. 2 Conceptual and Methodological Issues The key idea, in other words, is to consider how far changes that occur\nat one point ripple through the belief system. The further away from the “point of\nimpact” changes occur in both horizontal and vertical (upward) directions, the more\nsignificant the change is overall. So far, we have stressed that significant attitude change is not exclusively related\nto the magnitude of the change—it is also relevant where in the attitude system\nchanges occur. Nevertheless, the magnitude of change should not be ignored. A tiny\nchange, even at the level of postures, is not significant in the sense of the above\ndefinition of turning points. But how to decide whether a given observed change is\nsufficiently large to be considered significant? Ideally, such a threshold could be de-\nrived from theoretical considerations. However, it is not clear to us what these might\nbe. We are not alone in this, as existing research has no useful suggestions to offer\neither. Our threshold must certainly be above Page and Shapiro’s (1992) of six per-\ncentage points, since their only concern was to distinguish genuine attitude change\nfrom random fluctuations (rather than detect significant change).2 We tentatively\nsuggest a threshold of ten percentage points, which corresponds, under reasonable\nassumptions, with a Cohen’s d of 0.20 and hence indicates, by convention, a “weak\neffect” (Cohen 1988).3 While this low threshold may seem undemanding and bias the\nanalysis in favor of finding turning points, we will show that most opinion changes\ntriggered by Russia’s invasion of Ukraine do not even clear this hurdle. Given our\noverall conclusion that there has been no Zeitenwende at the level of German public\nopinion, relying on this low threshold hence represents a conservative strategy. Turning to the criterion of durability, the obvious question is how long the change\nmust persist to be considered “durable.” We could retreat to the position that the\nmore durable the change, the more legitimate it is to speak of a turning point, but\nthat would be quite unsatisfactory. While setting any threshold is arbitrary to some\nextent, we believe it is appropriate to set it in the order of years rather than weeks or\nmonths. If a shorter period were used, short-term fluctuations could be misinterpreted\nas a turning point. 3 Cohen’s d is probably the most widely used effect size measure and is defined as the difference between\ngroup means divided by the standard deviation of either group (Cohen 1988). Conventional thresholds are\nd= 0.20/0.50/0.80 for small/medium/large effects. Accepting d= 0.20 as an absolute lower boundary for\na “significant” change, this implies for the proportion-scaled variables we primarily examine in this paper\na threshold of 10 percentage points, assuming a standard deviation of 0.50 and normally distributed data\n(which is more or less true for the distributions of the variables considered here). 2 Conceptual and Methodological Issues The vertical dimension captures the idea that some elements of the belief system\nare more central, more important to the individual than others, which imposes a hi-\nerarchical structure on the belief system (Peffley and Hurwitz 1985). With respect\nto belief system change, elements at higher levels of the hierarchy are assumed to\nremain stable across situations, whereas elements at lower levels are more variable,\nas they are shaped by an interaction of the former and situational features. Applying these concepts to the foreign policy realm, Hurwitz and Peffley (1987)\nargued that policy attitudes are typically situated at the lowest level of the hierarchy,\nwhereas core postures are located at a higher level. While the former refer to the\nevaluation of specific policy options, the latter refer to general principles to which\npolicy in a particular area should conform. Empirical research has fleshed out this\nidea, showing that citizens in many countries rely on three different postures when\nthinking about the foreign and defense policy domain (e.g., Chittick et al. 1995;\nGravelle et al. 2017; Holsti and Rosenau 1990; Mader 2015; Wittkopf 1990). First,\nthere are postures toward the extent of international involvement, with internation-\nalists favoring an active role of their country in international affairs and isolationists\nfavoring abstention. Second, individuals have principled positions on using military\nforce—some reject it as a tool in international politics altogether, whereas others see\nits use as legitimate and effective. Finally, postures toward cooperation with other\ncountries can be arrayed on a continuum ranging from multilateralists who prefer\ncooperation to unilateralists who prefer their country to act alone. In Europe and,\nin particular, Germany, postures toward the relationship with the United States also\nplay an important role. While Atlanticists prefer their government to steer a close\ncourse to the United States and accept its leadership role in international affairs,\nothers reject these notions to the extent of exhibiting a staunch anti-Americanism\n(Asmus et al. 2005; Katzenstein and Keohane 2007; Mader 2016). Going back to the significance criterion, we use these prior insights on the struc-\nture of belief systems to argue that change in postures is more significant than\nchange in policy attitudes and change in attitudes toward objects unrelated to Rus- K 530 M. Mader, H. Schoen sia’s invasion of Ukraine is more significant than change in attitudes directly related\nto the event. 2 Their threshold is based on statistical not substantive significance. They assume survey-based estimates\nof public opinion to have an average margin of error of +/– three percentage points, so that when comparing\ntwo measurement points, one can only be sure that there is indeed a difference in the population if the\ndifference is six points or more in the sample (Page and Shapiro 1992, p. 45). 2 Their threshold is based on statistical not substantive significance. They assume survey-based estimates\nof public opinion to have an average margin of error of +/– three percentage points, so that when comparing\ntwo measurement points, one can only be sure that there is indeed a difference in the population if the\ndifference is six points or more in the sample (Page and Shapiro 1992, p. 45).\n3 Cohen’s d is probably the most widely used effect size measure and is defined as the difference between\ngroup means divided by the standard deviation of either group (Cohen 1988). Conventional thresholds are\nd= 0.20/0.50/0.80 for small/medium/large effects. Accepting d= 0.20 as an absolute lower boundary for\na “significant” change, this implies for the proportion-scaled variables we primarily examine in this paper\na threshold of 10 percentage points, assuming a standard deviation of 0.50 and normally distributed data\n(which is more or less true for the distributions of the variables considered here). 4 The pre-registration can be viewed here: https://osf.io/ufvg8/. Deviations from the preregistration plan\n(all of which are substantively inconsequential) are discussed in Supplementary Material S6.\n5 The terms “assertiveness” and “Western orientation” are used to present our hypothesis in an efficient\nway. By the former we mean preferences for a strong military a more confrontational foreign policy (which\nin the U.S. context is referred to as hawkishness), while by Western orientation we mean primarily positive\nattitudes toward the U.S. but also a certain tendency toward camp formation, which includes not only the\npositive evaluation of members of one’s own group but also the negative evaluation of foreign groups (such\nas Russia and China). Below, we elaborate on exactly which attitudes we examine and present results in\na disaggregated fashion. 2 Conceptual and Methodological Issues Furthermore, turning points do not require immediate change, but\nmay involve periods of change as long as that period is relatively small compared to\nthe longer periods of stability around it (Abbott 2001: 251–2). Thus, if it takes some\ntime for the public to understand the implications of Russia’s invasion of Ukraine,\nit will not be possible to diagnose whether there has been a turning point for several\nyears. K K 531 No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... In terms of post-invasion data, our data analysis below covers two points in time,\nthree months and eight months after the invasion, respectively. Accordingly, our\nanalysis provides a solid basis for testing whether some initial changes took place\nand whether they survived another five months or disappeared in the meantime. But\nas the preceding discussion has shown, our analysis will still not be sufficient to\nsettle the durability issue. If we find that public opinion has changed significantly,\nthe question is whether this change will endure. If we find that public opinion has\nnot changed significantly, the question is whether this can still happen (and how long\nthe change will endure afterwards). We will return to these issues in the conclusion. 6 We do not claim that this takes into account all possible sources of heterogeneity. Different socialization\nexperiences during and after the Cold War could fuel region- and age-specific responses (e.g., Steinbrecher\n2022); more generally, strong prior attitudes might color how citizens react to international events (e.g.,\nHerrmann 2017). Supplementary Material S5 provides subgroup results for East and West Germans, Sup-\nplementary Material S6 documents heterogeneity in rates of change based on age and prior foreign and\nsecurity policy attitudes. These additional analyses reveal some differences, but they are of small magni-\ntude. Perhaps most notably, the change in support for certain assertive foreign policy measures appears\nto be weaker among East Germans than among West Germans. Overall, however, the additional analyses\nsupport our conclusion of a relatively homogeneous (non-)reaction of German public opinion, which is\ndocumented in detail below. 3 Boundary Conditions, Hypotheses, and Research Questions Next, we briefly describe relevant boundary conditions of the empirical case to\nformulate hypotheses and research questions about attitude change. Due to the mul-\ntitude of aspects that could be mentioned here, such a description invites selection\nbias and ex-post rationalizations. In order to avoid this, we have tied our hands by\npreregistering all hypotheses and research questions with the Open Science Frame-\nwork (OSF) prior to accessing the data.4 In general, the actions of elites and public discourse following Russia’s invasion\nof Ukraine—which significantly shape the information environment in which citi-\nzens form opinions on political issues—are quickly described. The overwhelming\nmajority of German and other Western elites condemned Russia’s actions, called for\nsolidarity with Ukraine and Western unity, emphasized the need for jointly uphold-\ning the principle of territorial integrity and liberal international order, and called\nfor increased national defense efforts; in terms of policy, direct participation in\nthe war by the country’s own forces was ruled out, but significant assistance was\nprovided to Ukraine, including arms deliveries, and strict sanctions were imposed\non Russia (e.g., Adams 2022; Balfour et al. 2022; European Council 2022; NATO\n2022). Assuming that public opinion reflected these mainstream interpretations in\nthe aggregate, we should observe that public opinion after the invasion was more\nassertive in its policy orientations (H1) and more Western oriented (H2).5 Given this\nassumption, we should also expect the public to be more multilateralist (H3) and\nless isolationist (H4) on average than before. Following the idea that belief systems\nare constrained on a vertical and horizontal axis, we furthermore expect that changes\nin postures are smaller than changes in policy attitudes (H5) and changes in policy K K 532 M. Mader, H. Schoen attitudes to change less the more thematically distant they are from the topic of\nRussia’s invasion of Ukraine (H6). While these fundamental positions were largely shared across party lines in Ger-\nmany, there were differences regarding more specific issues. For example, there were\ndifferent levels of condemnation of Russia and different positions on how assertive\nthe new Russia policy should be (Horowitz 2022; Pfaff 2022; Becker et al. 2022;\nNienaber 2022; Feldenkirchen et al. 2022). This variation in elite cues opens the\nroom for heterogenous opinion change along partisan lines (Campbell et al. 1960;\nZaller 1992). Other individual differences may also lead to heterogenous reactions. Political involvement is a likely candidate. 3 Boundary Conditions, Hypotheses, and Research Questions Given its effects on the willingness and\ncapability of citizens to process information (e.g., Fazio 1990), those involved in\npolitics might react differently to events than their fellow citizens who follow events\nless closely. We therefore conduct exploratory subgroup analyses to determine the\nextent to which the diagnosis for aggregate opinion change needs to be differentiated. Focusing on heterogeneity based on partisan loyalties and political involvement, we\ngenerally ask whether attitude changes differed across subgroups (RQ1) and whether\ndifferences led to a convergence or polarization of public opinion (RQ2).6 7 To ensure the validity of inferences from the achieved sample to the population, we replicated quanti-\nties of interest using a survey weight that adjusts for key socio-demographics (age, sex, education, and\nresidence in East or West Germany). The weighted and unweighted results were substantively identical. 4 Research Design, Data, and Measures We analyze attitude change using a multi-wave panel survey. Two panel waves were\nconducted shortly before and after Russia’s invasion of Ukraine (in December 2021\nand May 2022, respectively). Differences in attitudes measured in these two surveys\ncan be attributed to this event, since it was the only event related to foreign and\ndefense policy that occurred in the short inter-wave period. This analysis hence\nallows evaluation of the significance criterion of turning points. There are two extensions to this core element of the research design. First, we also\nconsider pre-invasion data collected at various points in the 2017–2022 period. That\nway we can gauge change in a larger set of attitudes, even if causal attribution of\ndifferences between pre- and post-invasion data becomes more difficult. Principally,\nas the time between datapoints increases, so does the number of potential causes\nof attitude change. While we cannot discard this possibility, we are not aware of\nevents or developments in that period that might have caused attitude change in this\ndomain, and hence we deem it worthwhile to look at these additional data. However,\nto allow the reader to consider this potential inference problem when interpreting K K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 533 Table 1 Item coverage, timing, and sample size of German Longitudinal Election Study (GLES) panel\nwaves\nGLES wave number\n23\n22\n21\n17\n16\n8\n2\nIncrease military spending\nX\nX\n–\nX\n–\n–\n–\nOppose annexation of Crimea\nX\nX\n–\n–\n–\nX\n–\nStrive for good relations with\nPutin\nX\nX\n–\n–\n–\n–\nX\nConfrontational RUS policy\nX\nX\n–\n–\nX\n–\n–\nConfrontational CHI policy\nX\nX\n–\n–\nX\n–\n–\nProtect EU borders\nX\nX\nX\n–\n–\n–\n–\nViews of RUS, USA, CHI\nX\nX\nX\n–\n–\n–\n–\nForeign policy postures\nX\nX\nX\n–\n–\n–\n–\nField time (start)\n10/22\n05/22\n12/21\n07/21\n05/21\n09/17\n02/17\nSample size\n11448\n12115\n12997\n13704\n15073\n13400\n13129\nReported pre-invasion coverage is limited to the most recent pre-invasion time that the items were asked. Additional data are used for selected analyses. 4 Research Design, Data, and Measures Schoen selection of available items is not perfectly suited to the purpose of this specific anal-\nysis, as the Russian invasion of Ukraine and the salient policy issues arising from\nit were unforeseeable, and tailored items in the pre-invasion waves hence unavail-\nable. For example, attitudes toward arms deliveries, sanctions, and compliance with\nalliance commitments were not measured in the waves preceding the invasion and\nthus cannot be included here. Nevertheless, the pool of available items does allow\nanalysis of all types of orientations discussed above. We provide an overview of the\nsurvey items used here and document the original question wording and response\noptions in Supplementary Material S1. Several items refer to policy issues more or less directly related to the Ukraine\nwar. These are (1) increasing German military spending, (2) the preliminary accep-\ntance of Russia’s annexation of Crimea, (3) whether German governments should\nseek good relations with Putin, and (4) whether Germany should rely less on co-\noperation and more on confrontation in dealing with Russia. With regard to issues\nnot directly related to the invasion, questions about a confrontational (rather than\ncooperative) China policy and stronger EU efforts to protect its external borders are\navailable. The former is particularly important for our endeavors, as it is completely\nanalogous to the question about a confrontational Russia policy but differs in top-\nical closeness to Russia’s invasion of Ukraine. These items are hence particularly\nsuited to address hypothesis 6 (i.e., the expectation that the further away attitudes\nare thematically from Russia’s invasion of Ukraine, the less they changed). Attitudes\ntoward these issues were all measured using a five-point Likert-type scale (strongly\ndisagree–strongly agree). We also examine views of foreign actors directly (Russia) or indirectly (U.S. and\nChina) involved in the war, even if their hierarchical position in the belief system is\nsomewhat unclear. In part, they presumably reflect country stereotypes, which should\nbe located at a higher level; at the same time, they strongly depend on contextual\nfeatures—e.g., who occupies the White House strongly influences public opinion of\nthe U.S. (Wike et al. 2021). We therefore assume that they are rather located on the\nlower level of the hierarchy. Views of foreign actors are measured with items from\na battery that asks respondents what they think of different countries and politicians. 4 Research Design, Data, and Measures See Supplementary Material S2 for complete coverage able 1 Item coverage, timing, and sample size of German Longitudinal Election Study (GLES) panel eported pre-invasion coverage is limited to the most recent pre-invasion time that the items were asked. dditional data are used for selected analyses. See Supplementary Material S2 for complete coverage the results, we will indicate which time interval each comparison is based on when\npresenting the results. Second, we consider a second post-invasion datapoint (from\nOctober 2022) to provide a preliminary analysis of the fluidity of attitudes in the post-\ninvasion period. This allows us to gain an impression of whether attitude changes\nin response to the invasion subsequently remained stable at the new level, whether\nchanges continued in a given direction, or whether a trend reversal occurred. The data source is waves of a panel survey conducted in the framework of the\nGerman Longitudinal Election Study (e.g., GLES 2021). Questions on foreign and\ndefense policy were included throughout the 2017–2022 period. Table 1 provides in-\nformation about coverage of key items in different waves and the timing and sample\nsizes of these waves. The sample was recruited from the Respondi AG online access\npanel of pre-recruited persons using quotas that are representative of the German\nonline population. Due to panel mortality on the one hand and sample refreshment\nbetween panel waves on the other, the size and composition of the sample varies\nacross the analyses.7 To maximize statistical power, we conduct all analyses with the\nlargest possible sample, even though this might marginally affect the comparability\nof the findings. The analysis of intra-individual change between waves 21 and 22,\nfor example, is based on about 10,500 observations; in the subgroup analyses, the\nresults for supporters of The Left—currently the smallest party represented in the\nBundestag—are based on about 1000 respondents. In addition to the large number\nof foreign and defense policy questions repeatedly asked of the same individuals,\nthese large sample sizes are a unique feature of this data source. Corresponding to our theoretical discussion, we analyze opinions on different\ntopics and at different hierarchical levels of the belief system. Needless to say, the K K K K 534 M. Mader, H. K 8 Reproduction materials for all results reported in this article are available at Harvard Dataverse: https://\ndoi.org/10.7910/DVN/WY2OFU.\n9 Another option is to treat the scales as metric and look at changes in means. We provide these in Supple-\nmentary Material S2.\n10 The (relative) stability of the postures is not a methodological artifact of using two-item indexes. Sup-\nplementary Material S3 reports the stability of responses to the individual items, which is also high. The\nminor change in views of Russia, however, is partially an artifact of the dichotomized measure. The intra-\nindividual analysis below shows that there was actually significant movement toward more negative views\nof Russia. 4 Research Design, Data, and Measures Respondents could register their views on an 11-point scale (I don’t think anything\nof them at all—I think very highly of them). Foreign policy postures are captured with a set of established indicators (e.g.,\nGraf 2020; Mader 2015; Steinbrecher 2018). For each posture, the responses to two\nitems were aggregated into an additive index. The items are statements that express\nthe respective foreign policy principle at an abstract level, in opposing wording to\navoid approval bias. For the posture toward the use of military force, for example,\nthe statements are “War is sometimes necessary to protect a country’s interests”\nand “The use of military force is never justified” (reverse coded). Responses were\nrecorded using a five-point Likert-type scale (strongly disagree–strongly agree). For\nthe items used for the remaining postures, we refer again to Supplementary Material\nS1. The correspondence between these orientations and the hypotheses is as follows:\nall policy items address the assertiveness dimension, as does the core posture on the\nuse of military force (H1). A Western orientation is expressed in positive views of K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 535 the U.S. and an Atlanticist posture (H2). Hypotheses H3 and H4 can be directly tied\nto the corresponding postures, multilateralism, and isolationism. the U.S. and an Atlanticist posture (H2). Hypotheses H3 and H4 can be directly tied\nto the corresponding postures, multilateralism, and isolationism. 5 Results8 Is there evidence that Russia’s invasion of Ukraine triggered aggregate opinion\nchange in the German population? To answer this question, we look at the percent-\nage of agreement with a given survey item. Responses to the military spending item,\nfor example, were transformed into a dummy variable indicating whether respon-\ndents chose one of the two agreement options (“agree” or “completely agree”) or one\nof the three other substantive options (“completely disagree,” “disagree,” “neither\nagree nor disagree”). Rare refusals to answer were treated as missing values. The\n11-point scales capturing respondents’ views of Russia, the U.S., and China were\ntreated analogously; thus, we report the percentage of respondents who expressed\na positive opinion about each actor (by choosing a response option above the middle\ncategory). Finally, the sum indices capturing postures were also dichotomized above\nthe midpoint of the scale, so that in each case the percentage is reported at which\nrespondents (tend to) agree with the use of military means and a multilateralist, iso-\nlationist, and Atlanticist foreign policy, respectively.9 Fig. 1 reports these data from\nmultiple pre- and post-invasion waves of the panel survey. Data from the last wave\nbefore the invasion are colored solid red; data from the first wave thereafter are col-\nored solid blue; data from waves further away in time are displayed in progressively\nlighter red or blue. Figure 1 shows that German public opinion became more assertive on policy\nissues directly related the Russian invasion of Ukraine. Comparing the two datapoints\nbracketing the invasion, support for higher military spending increased significantly\n(+26 percentage points), as did opposition to the annexation of Crimea (+25) and\nsupport for a more confrontational Russia policy (+16). Striving for good relations\nwith Putin became significantly less popular (–23 points). In contrast, there was\nno or only minor change in all other orientations considered here—core postures,\nattitudes toward policy issues not directly related to the war, and views of Russia,\nthe U.S., and China.10 Considering the second datapoint after the invasion, from October 2022 (in light\nblue), shows subsequent trends. First, there is no evidence of a delayed reaction\nto the invasion. As the war in Ukraine continued through the summer and fall of\n2022, core postures and attitudes toward thematically distant policy issues and actors K K 536 M. Mader, H. Schoen Postures\nViews of actors\nPolicy attitudes\nFig. 1\nStability and change in aggregate opinion. K 5 Results8 Support for a more confrontational China policy\nwas virtually identical before and after the Russian invasion, and the increase in the K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 537 percentage of citizens generally supporting the use of military force (from 18 to 20%)\nis so small as to be negligible. Similarly, favorable views of the U.S. (+1 percentage\npoint) and Atlanticism (+5) were more common after the invasion, but not to the\nextent that one could speak of a significant spread of “Western orientations” (H2);\nmultilateralism became more widespread among the German population (+4) and\nisolationist attitudes declined (–5), but again the magnitude of these changes is too\nsmall to constitute a significant change (H3, H4). Hypotheses H5 and H6 perform much better. We find movement mainly in atti-\ntudes toward political issues directly related to the invasion of Russia, while there is\nlittle change in postures and attitudes toward issues that are thematically distant. As\nnoted above, these hypotheses are key to gauging the significance of belief system\nchange overall, and hence to understanding whether the Russian invasion marked\na turning point for German public opinion. The aggregate-level findings presented\nso far give a clear answer: changes are limited to a small topical area at a low hi-\nerarchical level of citizens’ belief systems; they do not extend beyond that either in\nthe horizontal dimension (to thematically more distant policy issues) nor the vertical\ndimension (to core postures). Based on the criteria we have developed above, there\nis thus insufficient evidence overall for a turning point at the level of German public\nopinion. Before going further, we should revisit the concern that causal attribution is not\nentirely unproblematic in a design of this type. Perhaps the observed patterns of\nstability and change are random or have causes other than the Russian invasion\nof Ukraine. This concern is especially important in cases where the pre-invasion\ndatapoint is further in the past. Arguing against much empirical relevance of this\nobjection is the fact that all orientations considered here—even those that changed\nin the course of the invasion—were stable over a long period before the invasion\n(see Supplementary Material S2). Thus, a long uneventful period was associated\nwith stability in public opinion, while a salient event was associated with (some)\nchange. 5 Results8 Reported are percent of respondents who score above\nthe midpoint of the original scale. December 2021 data in solid red (last wave before Russia’s invasion of\nUkraine), May 2022 data in solid blue (first wave after the invasion); data from more distant waves in in-\ncreasingly transparent red and blue, respectively. See Supplementary Material S2 for data in tabular form. Pre-invasion marker for “Confrontational CHI policy” completely overlaps with post-invasion marker Fig. 1\nStability and change in aggregate opinion. Reported are percent of respondents who score above\nthe midpoint of the original scale. December 2021 data in solid red (last wave before Russia’s invasion of\nUkraine), May 2022 data in solid blue (first wave after the invasion); data from more distant waves in in-\ncreasingly transparent red and blue, respectively. See Supplementary Material S2 for data in tabular form. Pre-invasion marker for “Confrontational CHI policy” completely overlaps with post-invasion marker Fig. 1\nStability and change in aggregate opinion. Reported are percent of respondents who score above\nthe midpoint of the original scale. December 2021 data in solid red (last wave before Russia’s invasion of\nUkraine), May 2022 data in solid blue (first wave after the invasion); data from more distant waves in in-\ncreasingly transparent red and blue, respectively. See Supplementary Material S2 for data in tabular form. Pre-invasion marker for “Confrontational CHI policy” completely overlaps with post-invasion marker remained at pre-invasion levels. Second, the changes in attitudes diagnosed above\nprove stable in some cases, but in others the initially more assertive stance seems\nto be moving back to the conciliatory pre-invasion levels. While greater support\nfor higher defense spending and rejection of recognition of Russia’s annexation of\nCrimea prove durable, there were again more respondents who wanted good relations\nwith Putin and a Russia policy that focuses more on cooperation than confrontation. With respect to hypotheses H1–H4, we can summarize that the direction of the\nchange observed immediately after the invasion was as expected. If we consider the\nmagnitude of change, however, H2–H4 have to be rejected completely and H1 can\nbe only partially confirmed. There was a shift toward greater assertiveness on certain\npolicy issues, but this did not extend to issues further removed from the Ukraine\nwar or to foreign policy postures. 11 Supplementary Material S4 replicates the analysis with the radical cut-off points of no change vs. any\n+/– change, respectively. 12 The latter problem can be addressed by looking at the intra-individual stability of the posture indica-\ntors themselves, where a five-point scale was used. Corresponding findings are shown in Supplementary\nMaterial S4. They confirm the high stability of the postures shown in Table 2. 5 Results8 This pattern encourages us to interpret the differences that appear in the\nbefore/after comparison as actually being reactions to the event. The above diagnosis of extensive opinion stability is based on aggregate findings. It is possible that the stability in the aggregate masks significant, countervailing\nchange at the individual level that cancel each other out when aggregated. To test\nthis possibility, we first make use of the panel data and examine the extent to\nwhich the opinions of individual citizens have changed in reaction to the invasion. We calculated between-wave differences (first post-invasion minus last pre-invasion\ndatapoint) using the original scales and trichotomized these differences. Table 2 con-\ntrasts respondents who exhibit change of +/–1 scale points or less with respondents\nwho exhibit a more than +/–1 scale-point change. We believe these to be reasonable\ncut-off values to discriminate between stability and change. We do not use an even\nmore rigorous method because the change in a single scale point could reflect ran-\ndom variation.11 Furthermore, it should be noted that the comparability of results is\nattenuated by the use of different response scales. The use of an 11-point response K K K 538 M. Mader, H. Schoen Table 2\nIntra-individual stability and change, pre–post invasion comparison\nInterval\n(month/\nyear)\nMore than\n–1 point (%)\n+/–1 point or\nless (%)\nMore than\n+1 point\n(%)\nResponse\nscale\nPolicy attitudes\nIncrease military\nspending\n07/21!05/22\n2\n71\n26\n5 points\nOppose annexation\nof Crimea\n09/17!05/22\n4\n74\n22\n5 points\nStrive for good\nrelations with Putin\n02/17!05/22\n31\n67\n3\n5 points\nConfrontational\nRUS policy\n05/21!05/22\n5\n76\n19\n5 points\nConfrontational\nCHI policy\n05/21!05/22\n8\n82\n10\n5 points\nProtect EU borders\n12/21!05/22\n5\n89\n6\n5 points\nViews of actors\nFavorable view of\nRUS\n12/21!05/22\n38\n55\n7\n11 points\nFavorable view of\nUSA\n12/21!05/22\n16\n66\n17\n11 points\nFavorable view of\nChina\n12/21!05/22\n17\n66\n18\n11 points\nPostures\nUse of military\nforce\n12/21!05/22\n10\n82\n8\nIndex\nMultilateralism\n12/21!05/22\n3\n92\n5\nIndex\nIsolationism\n12/21!05/22\n6\n91\n3\nIndex\nAtlanticism\n12/21!05/22\n3\n93\n4\nIndex\nReported are row percentages. 5 Results8 Due to rounding, row percentages do not always add up to 100% Table 2\nIntra-individual stability and change, pre–post invasion comparison scale is more likely to result in response variability than use of a five-point scale\nfor methodological reasons alone, whereas the construction of indexes to capture\npostures increases the stability in the measure.12 scale is more likely to result in response variability than use of a five-point scale\nfor methodological reasons alone, whereas the construction of indexes to capture\npostures increases the stability in the measure.12 Table 2 shows a similar pattern at the individual level as was shown before at the\naggregate level. Policy attitudes toward issues not directly related to the Ukraine war\nwere essentially stable—regarding the question of how to handle China, for example,\nfour out of five respondents gave the same answer or answered differently by only\none scale point. Those who did indicate a change in opinion did so in roughly equal\nproportions in both directions. A similar result emerges for views of the U.S. and\nthe core postures, although here the absolute stability levels are somewhat different,\npartly for the methodological reasons mentioned above. At this point, it is instructive to take a closer look at the views of foreign actors. More clearly than above, we see a significant net shift toward more negative views K K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 539 Fig. 2\nPost-invasion trajectories of change. Reported are group sizes with different types of reaction to the\ninvasion (increased support vs. no change) and the subsequent post-invasion change within these groups,\nwith the total sample as the percentage base. Results for the third type of reaction in the first interval\n(decreased support) not shown due to small group sizes. See Supplementary Material S4 for complete\nresults Fig. 2\nPost-invasion trajectories of change. Reported are group sizes with different types of reaction to the\ninvasion (increased support vs. no change) and the subsequent post-invasion change within these groups,\nwith the total sample as the percentage base. Results for the third type of reaction in the first interval\n(decreased support) not shown due to small group sizes. See Supplementary Material S4 for complete\nresults of Russia. More than a third of respondents expressed a more negative opinion,\nwhile only seven percent expressed a more positive opinion. By comparison, views\nof the U.S. 5 Results8 and China are far more stable, and among those who changed their\nopinion, there is no clear trend in one direction or the other. Thus, a similar pattern\nemerges here as with political attitudes: changes are limited to views about Russia,\nthe country directly involved in the war. The post-invasion trajectories of individual-level change parallel those of the ag-\ngregate analysis and are therefore documented here only by way of example. Figure 2\nshows for attitudes toward increasing military spending that the shift also endured at\nthe individual level. In October 2022, 24% of respondents still indicated increased\nsupport for higher military spending (relative to pre-invasion levels)—almost as\nmany as in May 2022, when the value was 27%. In contrast, with regard to the\nquestion of what Germany’s policy toward Russia should look like, there was a cer-\ntain trend back toward support for a cooperative policy, as the aggregate analysis\nalready showed. While 20% in May 2022 exhibited change toward a more con-\nfrontational Russia policy compared with before the invasion, only 12% did so in\nOctober 2022. The second step in our analysis is to look at selected subgroups. Even if the\nchanges in the aggregate are not sufficiently large to diagnose a turning point, this\ncould well be the case in subgroups. A follow-up question concerns the convergence\nof public opinion—it is possible that different rates of change led Germans to view\nthe world more similarly after the Russian invasion than before. The subgroup\nanalysis was conducted for all orientations for which data are available and our\nconclusions are based on this overall picture, but we present only a selection of\nresults here. This is done for lack of space, for the sake of clarity, and to avoid\nrepetition—the subgroup differences are quite similar across the different (types of)\norientations, so there is no value added by showing all results here. All results are\nprovided in Supplementary Material S5, alongside regressions that model difference\nvariables as a function of subgroup variables. We find that the differences between the subgroups are by and large small (RQ1). In other words, we find no evidence that there has been a turning point in the\nopinions of at least certain subgroups. This is true in particular for the foreign policy K K 540 M. Mader, H. Schoen Fig. 3\nPre- and post-war opinions, by party group and political interest. K 5 Results8 Reported are percent of respon-\ndents within subgroups who score above the midpoint of the original scale. Pre-invasion data refers to the\nlast available data before the invasion; post-invasion data from 05/2022. The red vertical line is the pre-\ninvasion average, the blue line is the post-invasion average\npostures—their stability is high without exception in all subgroups. Where we do Fig. 3\nPre- and post-war opinions, by party group and political interest. Reported are percent of respon-\ndents within subgroups who score above the midpoint of the original scale. Pre-invasion data refers to the\nlast available data before the invasion; post-invasion data from 05/2022. The red vertical line is the pre-\ninvasion average, the blue line is the post-invasion average Fig. 3\nPre- and post-war opinions, by party group and political interest. Reported are percent of respon-\ndents within subgroups who score above the midpoint of the original scale. Pre-invasion data refers to the\nlast available data before the invasion; post-invasion data from 05/2022. The red vertical line is the pre-\ninvasion average, the blue line is the post-invasion average postures—their stability is high without exception in all subgroups. Where we do\nfind differences, they are gradual rather than in kind, i.e., the changes are going\nin the same direction but at slightly different rates. Specifically, attitudes tended\nto change more among supporters of mainstream parties than among supporters of\nfringe parties and citizens with no party affiliation, and changes were somewhat\nmore pronounced among citizens with high political interest than among citizens\nwith low political interest. The most interesting subgroup results are displayed in Fig. 3 and relate to support\nfor increased military spending and a confrontational Russia policy (top row), and\nto views of Russia and the U.S. (bottom row). They illustrate the general patterns K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 541 described in the previous paragraph well, but they are unrepresentative in terms\nof magnitude of change overall and subgroup differences—change and subgroup\ndifferences are smaller in the other cases. Consider defense spending (top-left plot). There were significant increases in\nsupport for more defense spending in all subgroups, but the rate of change varied\nbetween supporters of the mainstream parties (27–36 percentage points) and those\nof AfD and The Left (13 and 17 points, respectively). 5 Results8 Analogously, increases in\nsupport for a more confrontational Russia policy were also more pronounced among\nthe former than the latter. These patterns are in line with party positioning. While\nthe parties on the political fringes condemned the Russian attack, they did so less\nvehemently and were critical of German arms deliveries to Ukraine. They were also\nquick to provide justifications for Russia’s attack and call for diplomatic solutions,\nwhich at the time would have meant accepting Russian territorial gains, if not\nUkrainian loss of sovereignty (Horowitz 2022; Pfaff 2022). The differences in change among supporters of the established parties are small\nand essentially negligible. Here, partisan disputes over the Russian invasion appar-\nently did not spread to their followers. For example, one might have suspected that\nthe SPD’s supporters moved less, given Chancellor Scholz’s reluctance in delivering\narms to Ukraine (Nienaber 2022). The Greens called for a more decisive approach\nand, in the process, surprisingly quietly moved further away from their pacifist roots\n(Feldenkirchen et al. 2022). Accordingly, one might have expected supporters of\nthe Greens to move the most on policy issues, and also to show a comparatively\nlarge change in their postures toward the use of military force. However, we see no\nsuch differences. The differences in positioning in the day-to-day business of party\npolitics were apparently not sufficiently fundamental to leave a lasting impression\nat the public level. Finally, we can answer the question of whether Russia’s invasion of Ukraine led to\na convergence or polarization of public opinion (RQ2). Since subgroup differences\nin attitude change were generally small, the straightforward answer is that there was\nneither. Only at second glance can trends of polarization be discovered for some\nissues, while for others there is a slight convergence. Views of the U.S. is the only\ncase with an instance of subgroups changing in opposite directions. Supporters of\nthe two fringe parties and citizens with no party affiliation—who were more critical\nof the U.S. from the start—became more critical, whereas the views of supporters\nof the established parties became more positive. As Fig. 3 shows, however, these\nopposing effects were substantively insignificant. In addition, there were some cases\nin which, depending on the distribution of opinion before the war, different rates of\nchange led to polarization (e.g., in military spending) or convergence (e.g., in views\nabout Russia). 5 Results8 Overall, however, these effects are too small to call into question the\ngeneral finding that neither polarization nor convergence occurred. 6 Conclusion Our analysis suggests that Russia’s invasion of Ukraine passed by the German\npublic without leaving deep traces. This diagnosis is based on a simple conceptual K 542 M. Mader, H. Schoen idea and clear empirical findings. Conceptually, we propose that attitude change\nshould be considered significant only if the invasion caused widespread changes\nof a certain magnitude in mass belief systems. We posit that change in postures\nis more significant than change in policy attitudes, and change in attitudes not\ndirectly related to an event is more significant than change in attitudes that are\ndirectly related to it. Empirically, we find that policy attitudes toward war-related\nissues such as military spending and a confrontational Russia policy became more\nassertive, but that neither postures nor attitudes toward issues (and actors) that are\nnot directly related to the invasion changed. This pattern of limited, topical change\nand more general, overarching stability holds regardless of political orientation and\ninterest. Moreover, in the post-invasion period studied, there is nothing to suggest\na continuing shift in orientations that might eventually add up to significant change. In short, if we apply our criteria for significant attitudinal change, we conclude that\nthere has been no such change, and thus no Zeitenwende at the level of German\npublic opinion (yet). The diagnosis of course depends on the criteria used. We have already pointed\nout above that the chosen threshold of ten percentage points for a change that is\n“significant” in terms of magnitude is somewhat arbitrary. However, we also argued\nthat this threshold is set rather low—setting it higher leads even more clearly to the\ndiagnosis made here. Furthermore, conceptual decisions could reasonably be made\ndifferently. For example, we have categorized attitudes toward defense spending as\npolicy attitudes, following convention (Hurwitz and Peffley 1987; Wlezien 1995). If\nthese were interpreted as indicating general postures toward the role of the military\nin foreign policy instead, the results on the lack of opinion change at higher levels of\nGerman belief systems would be more mixed. Concrete political issues can indeed\nbecome powerful symbols, and public attitudes toward these issues can in turn\noccupy central positions in mass belief systems (Sears 1993). Given the importance\nof this issue in the German Zeitenwende debate and in light of the state of the\nGerman armed forces, it is not impossible that military spending will acquire such\nsymbolism. K 6 Conclusion Depending on the normative point of view and whether one views the Ukraine war\nas a turning point for international relations, German public opinion thus appears\nas an anchor of stability in stormy times or as an obstacle to urgent changes in the\nstatus quo of Germany’s strategic posture. To the extent that a fundamental reorien-\ntation has occurred at the level of German policymakers (Bunde 2022), the stability\nat the public level creates—or rather deepens (Oppermann 2019)—a disconnect that\nmay lead to tensions in the future. On the other hand, the electoral incentives from\nstability in public opinion make fundamental policy change a risky, even unrea-\nsonable strategy from a political point of view. Therefore, any pressure for change\nemanating from the international situation could soon be neutralized by domestic,\nelectoral considerations. Our conclusions are of course subject to the proviso that significant changes in\npublic opinion will not still occur. Here lies an obvious weakness of the present\nempirical analysis: our post-invasion data cover only the period until October 2022,\nonly about eight months after the event. We are thus unable to examine the dura-\nbility of change, the second criterion for a turning point that we discussed above. Perhaps significant change in public opinion will yet occur. Triggers could be a sin-\ngle prominent event or a series of smaller events that cumulatively lead to such\nchange. Beyond academic questions about what constitutes an event and whether\nattitude change would then still be a reaction to the Russian invasion, the main issue\nhere is what exactly this other event (sequence) might be and how likely it is to\noccur. In terms of singular evens, candidates might be the Russian use of weapons\nof mass destruction, complete Ukrainian defeat, an extension of the war to other\n(NATO) countries, or severe economic shocks on the home front. We do not want\nto speculate how likely these or other potentially relevant events are, but merely\nnote that we would assume an inverse relationship of the probabilities in which they\noccur and in which they cause a significant shift in attitudes. As for smaller events that could cumulatively lead to significant change, sus-\ntained opinion leadership could convince the public of new foreign and security\npolicy imperatives (Giegerich and Terhalle 2021). 6 Conclusion This reading conflicts, however, with existing evidence of the context\ndependence of these attitudes (Wlezien 1995), and even if we assigned them posture-\nlike status, the large change in this one case would be offset by the evidence of\nstability of the other postures. Even then, a balanced assessment of all the available\nevidence seems to amount to rejecting the thesis of a turning point at the level of\npublic opinion. Beyond the issue of diagnosing a turning point in public opinion, the results\npresented here are in line with more general insights about citizens’ foreign and\ndefense policy attitudes. First, we find no evidence that these opinions are particu-\nlarly fickle or in any way not “real.” While this was the verdict of early observers\nof public opinion on foreign and defense policy, subsequent studies have revised\nthis view (see Holsti 1992). Our findings strike the same chord. The changes in\nopinion that we do find are quite compatible with notions of a rational public (Page\nand Shapiro 1992) that reacts like a thermostat to changes in its information en-\nvironment (Wlezien 1995). We also find exactly the pattern in core postures that\none would theoretically expect—they turn out to be stable across contexts. Taking\nthe theory of posture-based attitude formation seriously (Feldman 1988; Hurwitz K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 543 and Peffley 1987), we should expect changes in policy attitudes to be short lived\nwithout changes in overarching postures. When the salience of the Ukraine war\nfades and other issues capture the citizens’ attention, postures will remain as central\ndeterminants of policy attitudes. Citizens who, under the impressions of the war and\nthe (largely) consensual reactions of the established political parties, were ready to\nsupport assertive policies “against type” may then return to their default, posture-\nbased position. What assertiveness German public opinion did show in the months\nafter the invasion could then turn back into restraint. This brings us to the policy implications. At present, there seems to be no man-\ndate for a fundamental change in Germany’s foreign and security policy orientation. 6 Conclusion We suspect, however, that it will\nbe difficult even for committed policymakers to achieve this—at least in the short\nterm and insofar as it involves changing citizens’ core postures. Such opinion leader-\nship would likely require a sustained elite consensus (among the established parties)\nthat grapples with difficult value trade-offs and defends these positions against other\npressing concerns. Whether such a consensus will emerge among German elites and\nwhether serious attempts will be made to bring the public along remains uncertain K 544 M. Mader, H. Schoen for the time being. It is also uncertain whether such an attempt at opinion leadership\nwould be successful. Research to date says little about the potential of elite commu-\nnication for shaping core postures. Systematic analyses of the malleability of these\norientations—beyond the results presented here—are not yet available and are an\nimportant task for future research. Prudence dictates that no conclusive assessment of the German public’s reaction\nof Russia’s invasion of Ukraine be made on the basis of the evidence presented\nhere. This much, however, is clear: the invasion did not immediately represent\na Zeitenwende for German public opinion on foreign and defense policy. Whether\nthis early assessment will have to be revised and whether turning points occurred in\nother domains, the future and further research will show. Supplementary Information The online version of this article (https://doi.org/10.1007/s11615-023-\n00463-5) contains supplementary material, which is available to authorized users. Funding Open Access funding enabled and organized by Projekt DEAL. Funding Open Access funding enabled and organized by Projekt DEAL. onflict of interest M. Mader and H. Schoen declare that they have no competing interests. Conflict of interest M. Mader and H. Schoen declare that they have no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,\nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as\nyou give appropriate credit to the original author(s) and the source, provide a link to the Creative Com-\nmons licence, and indicate if changes were made. The images or other third party material in this article\nare included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the\nmaterial. 6 Conclusion If material is not included in the article’s Creative Commons licence and your intended use is not\npermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly\nfrom the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4. References Abbott, Andrew. 2001. Time matters: on theory and method. Chicago: University of Chicago Press. Adams, Paul. 2022. Ukraine war: How long can the Western consensus hold? BBC News. https://www. bbc.com/news/world-europe-61664991. Accessed 6 Mar 2023. Adams, Paul. 2022. Ukraine war: How long can the Western consen\nbbc.com/news/world-europe-61664991. Accessed 6 Mar 2023. g\nbbc.com/news/world-europe-61664991. Accessed 6 Mar 2023. Aldrich, John.H., Peter Feaver, Christopher Gelpi, Jason Reifler, and Kristin T. Sharp. 2006. Foreign policy\nand the electoral connection. Annual Review of Political Science 9:477–502. https://doi.org/10.1146/\nannurev.polisci.9.111605.105008. p\nAsmus, Ronald, Philip P. Everts, and Pierangelo Isernia. 2005. The Transatlantic Gap in Public Opinion. In\nTransatlantische Beziehungen. Sicherheit – Wirtschaft – Öffentlichkeit, ed. Thomas Jäger, Alexander\nHöse, and Kai Oppermann, 397–424. Wiesbaden: VS. Balfour, Rosa, Dimitar Bechev, Raluca Csernatoni, Judy Dempsey, Thomas de Waal, Peter Kellner, Stefan\nLehne, Marc Pierini, Gwendolyn Sasse, Sinan Ülgen, and Pierre Vimot. 2022. Russia’s Invasion\nof Ukraine Changes Everything. Carnegie Europe. https://carnegieeurope.eu/strategiceurope/86525. Accessed 6 Mar 2023. Baum, Matthew A., and Philip B.K. Potter. 2015. War and democratic constraint. Princeton: Princeton\nUniversity Press. Baumgartner, Frank R., Christian Breunig, Christoffer Bryan D.Jones Green-Pedersen, Peter B. Mortensen,\nMichiel Nuytemans, and Stefaan Walgrave. 2009. Punctuated equilibrium in comparative perspec-\ntive. American Journal of Political Science 53(3):603–620. https://doi.org/10.1111/j.1540-5907. 2009.00389.x. Becker, Markus, Markus Feldenkirchen, Florian Gathmann, Konstantin von Hammerstein, and Veit\nMedick. 2022. Discontent Grows in Berlin over Chancellor’s Ukraine Response. Spiegel Inter-\nnational. https://www.spiegel.de/international/germany/the-scholz-problem-discontent-grows-in- K K No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... 545 berlin-over-chancellor-s-ukraine-response-a-47dae068-fd09-4890-9ef8-d13c144b10a6. Accessed\n6 Mar 2023. berlin-over-chancellor-s-ukraine-response-a-47dae068-fd09-4890-9ef8-d13c144b10a6. Accessed\n6 Mar 2023. Blumenau, Bernhard. 2022. Breaking with convention? Zeitenwende and the traditional pillars of Germa\nforeign policy. International Affairs 98(6):1895–1913. https://doi.org/10.1093/ia/iiac166. Bunde, Tobias. 2022. Lessons (to be) learned? Germany’s Zeitenwende and European security after the\nRussian invasion of Ukraine. Contemporary Security Policy 43(3):516–530. https://doi.org/10.1080/\n13523260.2022.2092820. Bunde, Tobias, and Tom Lubbock. 2022. How united is the West on Russia? Monkey Cage Blog. https://\nwww.washingtonpost.com/politics/2022/07/05/nato-russia-putin-threat-opinion-surveys-ukraine-\ninvasion/. Accessed 6 Mar 2023. invasion/. Accessed 6 Mar 2023. Campbell, Angus, Philip E. Converse, Warren E. Miller, and Donald E. Stokes. 1960. The American voter. Chicago: University of Chicago Press. Capoccia, Giovanni, and R. Daniel Kelemen. 2007. The study of critical junctures: theory, narrative, and\ncounterfactuals in historical institutionalism. World politics 59(3):341–369. https://doi.org/10.1017/\nS0043887100020852. Chittick, William O., Keith R. Billingsley, and Rick Travis. 1995. A three-dimensional model of Amer-\nican foreign policy beliefs. International Studies Quarterly 39(3):313–331. https://doi.org/10.2307/\n2600923. Cohen, Jacob. 1988. References Statistical power analysis for the behavioral sciences, 2nd edn., New York: Routledge. Converse, Philip E. 1964. The Nature of Belief Systems in Mass Publics. In Ideology and Discontents, ed. David E. Apter, 214–261. New York: Free Press. Cohen, Jacob. 1988. Statistical power analysis for the behavioral sciences, 2nd edn., New York: Routledg ,\np\ny\nf\n,\n,\ng\nConverse, Philip E. 1964. The Nature of Belief Systems in Mass Publics. In Ideology and Discontents, ed. David E. Apter, 214–261. New York: Free Press. p\n,\nDe Vries, Catharine, and Isabell Hoffmann. 2022. Under pressure: The war in Ukraine and European\npublic opinion. Gütersloh: Bertelsmann Stiftung. p\np\ng\nDuffield, John. 1998. World power forsaken: political culture, international institutions, and german secu-\nrity policy after unification. Stanford: Stanford University Press. European Council. 2022. EU response to Russia’s invasion of Ukraine. https://www.consilium.europa.eu/\nen/policies/eu-response-ukraine-invasion/. Accessed 6 Mar 2023. Fazio, Russell H. 1990. Multiple processes by which attitudes guide behavior: the mode model as an\nintegrative framework. Advances in Experimental Social Psychology 23(1):75–109. https://doi.org/\n10.1016/S0065-2601(08)60318-4. Feldenkirchen, Markus, Matthias Gebauer, Kevin Hagen, Christoph Hickmann, Dirk Kurbjuweit, Veit\nMedick, Laura Meyer, Serafin Reiber, Jonas Schaible, Christoph Schult, Marco Schulz, Christian\nTeevs, Gerald Traufetter, and Mascha Wolf. 2022. From peaceniks to hawks? Germany’s greens have\ntransformed in the face of russia’s war. Spiegel international. https://www.spiegel.de/international/\ngermany/from-peaceniks-to-hawks-germany-s-greens-have-transformed-in-the-face-of-russia-s-\nwar-a-19bd95f6-fcbc-497d-8ad9-1a767be205f1. Accessed 6 Mar 2023. Feldman, Stanley. 1988. Structure and consistency in public opinion: the role of core beliefs and value\nAmerican Journal of Political Science 32(2):416–440. https://doi.org/10.2307/2111130. Fröhlich, Manuel. 2023. „Wenn möglich bitte wenden“? Die deutsche Außenpolitik und die Navigation\nder Zeitenwende. Zeitschrift für Politikwissenschaft https://doi.org/10.1007/s41358-022-00338-y. f f\nf\np\ng\ny\nGiegerich, Bastian, and Maximilian Terhalle. 2021. The responsibility to defend: rethinking Germany’s\nstrategic culture. New York: Routledge. LES. 2021. GLES Panel 2016–2021, Wellen 1–15. Köln: GESIS Datenarchiv. https://doi.org/10.4232/1. 13783. ZA6838 Data file Version 5.0.0. GLES. 2021. GLES Panel 2016–2021, Wellen 1–15. Köln: GESIS Datenarchiv. https://doi.org/10.4232/1. 13783. ZA6838 Data file Version 5.0.0. raf Timo. 2020. Unity in the face of threat? Exploring the empirical relationship between strategic\nthreat perceptions and public support for a common European army in Germany. European Secu-\nrity 29(1):55–73. https://doi.org/10.1080/09662839.2019.1704267. Graf Timo. 2020. Unity in the face of threat? Exploring the empirical relationship between strategic\nthreat perceptions and public support for a common European army in Germany. European Secu-\nrity 29(1):55–73. https://doi.org/10.1080/09662839.2019.1704267. y\n( )\np\ng\nGraf Timo. 2022. p\ny\nAccessed 6 Mar 2023. Accessed 6 Mar 2023. Hurwitz, Jon, and Mark Peffley. 1987. How are foreign policy attitudes structured? A hierarchical mode\nAmerican Political Science Review 81(4):1099–1120. https://doi.org/10.2307/1962580. Jon, and Mark Peffley. 1987. How are foreign policy attitudes structured? A hierarchical model. erican Political Science Review 81(4):1099–1120. https://doi.org/10.2307/1962580. Hurwitz, Jon, and Mark Peffley. 1987. How are foreign policy attitudes structured? A hierarchical mode\nAmerican Political Science Review 81(4):1099–1120. https://doi.org/10.2307/1962580. Kagan, Robert. 2004. Of paradise and power: America and Europe in the new world order. New York:\nAlfred A. Knopf. p\nKatzenstein, Peter J., and Robert O. Keohane. 2007. Anti-Americanisms in World Politics. Ithaca: Cornell\nUniversity Press. Mader, Matthias. 2015. Grundhaltungen zur Außen- und Sicherheitspolitik in Deutschland. In Sicherheit-\nspolitik und Streitkräfte im Urteil der Bürger, ed. Heiko Biehl, Harald Schoen, 69–96. Wiesbaden:\nSpringer VS. p\ng\nMader, Matthias. 2016. Öffentliche Meinung zu Auslandseinsätzen der Bundeswehr: Zwischen Antimili-\ntarismus und transatlantischer Orientierung. Wiesbaden: Springer VS. Mello, Patrick A. 2022. Incentives and constraints: a configurational account of European involvement in\nthe Anti-Daesh coalition. European Political Science Review 14(2):226–244. https://doi.org/10.1017/\nS1755773921000333. NATO. 2022. Statement by NATO heads of state and government on russia’s attack on Ukraine. NATO\npress release. https://www.nato.int/cps/en/natohq/official_texts_192489.htm?selectedLocale=en. Ac-\ncessed 6 Mar 2023. Nienaber, Michael. 2022. Germany’s Scholz accused of slow-walking Ukraine weapon supplies. Bloomberg\nEurope edition. https://www.bloomberg.com/news/articles/2022-04-14/germany-s-scholz-accused-\nof-slow-walking-ukraine-weapon-supplies. Accessed 6 Mar 2023. Oppermann, Kai. 2019. Between a rock and a hard place? Navigating domestic and international expecta-\ntions on German foreign policy. German Politics 28(3):482–498. https://doi.org/10.1080/09644008. 2018.1481208. Page, Benjamin I., and Robert Y. Shapiro. 1992. The rational public: fifty years of trends in americans’\npolicy preferences. Chicago: University of Chicago Press. Peffley, Mark A., and Jon Hurwitz. 1985. A hierarchical model of attitude constraint. American Journal\nPolitical Science 29(4):871–890. https://doi.org/10.2307/2111185. Pfaff, Jan. 2022. Die Nato-war-schuld-Linken. taz am Wochenende. https://taz.de/Linke-und-der-\nUkrainekrieg/!5834130/. Accessed 6 Mar 2023. Rattinger, Hans. 1985. Auswirkungen der öffentlichen Meinung auf den sicherheitspolitischen Hand-\nlungsspielraum der Bundesrepublik. In Aktuelle Herausforderungen der repräsentativen Demokratie,\ned. Ulrich Matz, 131–148. Köln: Carl Heymanns. Rattinger, Hans, Harald Schoen, Fabian Endres, Sebastian Jungkunz, Matthias Mader, and Jana Pötzschke. 2016. Old friends in troubled waters. Policy principles, elites, and U.S.-German relations at the citi-\nzen level after the cold war. Baden-Baden: Nomos. zen level after the cold war. Baden-Baden: Nomos Risse, Thomas. 2022. Zeitenwende? Internationale Politik. https://internationalepolitik.de/de/zeitenwende-\n2. Accessed 6 Mar 2023. Saunders, Elizabeth N. 2014. Good democratic leadership in foreign affairs: an elite-centered approach. References Zeitenwende im sicherheits- und verteidigungspolitischen Meinungsbild. Potsdam:\nZMSBw. https://doi.org/10.48727/opus4-560. Gravelle, Timothy B., Jason Reifler, and Thomas J. Scotto. 2017. The structure of foreign policy attitudes\nin transatlantic perspective: comparing the United States, united kingdom, France and Germany. Eu-\nropean Journal of Political Research 56(4):757–776. https://doi.org/10.1111/1475-6765.12197. p\nf\n( )\np\ng\nGroitl, Gerlinde. 2022. Zeitenwende Revisited: Neue Weltordnung nach Russlands Angriff auf die\nUkraine? Zeitschrift für Politik 69(4):393–408. Herrmann, Richard K. 2017. How attachments to the nation shape beliefs about the world: a the-\nory of motivated reasoning. International organization 71(S1):S61–S84. https://doi.org/10.1017/\nS0020818316000382. Holsti, Ole R. 1992. Public opinion and foreign policy: challenges to the almond-Lippmann consensus. International Studies Quarterly 36(4):439–466. https://doi.org/10.2307/2600734. Holsti, Ole R. 1992. Public opinion and foreign policy: challenges to the almond-Lippmann consensus. International Studies Quarterly 36(4):439–466. https://doi.org/10.2307/2600734. K K 546 M. Mader, H. Schoen Holsti, Ole R., and James N. Rosenau. 1990. The structure of foreign policy attitudes among American\nleaders. The Journal of Politics 52(1):94–125. https://doi.org/10.2307/2131421. f\np\ng\nHorowitz, Jason. 2022. Putin’s aggression leaves his right-wing fan club squirming. New York Times. https://www.nytimes.com/2022/02/26/world/europe/russia-putin-matteo-salvini-marine-le-pen.html. Horowitz, Jason. 2022. Putin’s aggression leaves his right-wing fan club squirming. New York Times. https://www.nytimes.com/2022/02/26/world/europe/russia-putin-matteo-salvini-marine-le-pen.html. Accessed 6 Mar 2023 p\ny\nAccessed 6 Mar 2023. In Good democratic leadership: on prudence and judgment in modern democracies, ed. John Kane,\nHaig Patapan, 158–177. Oxford: Oxford University Press. g\np\ny\nSchoen, Harald. 2011. Two indicators, one conclusion. On the public salience of foreign affairs in Germany\nbefore and after reunification. In Issue salience in international politics, ed. Kai Oppermann, Henrike\nViehrig, 23–38. New York: Routledge. Scholz, Olaf. 2022. Policy statement by Olaf Scholz, Chancellor of the Federal Republic of Germany\nand Member of the German Bundestag, 27 February 2022 in Berlin. https://www.bundesregierung. de/breg-en/news/policy-statement-by-olaf-scholz-chancellor-of-the-federal-republic-of-germany-\nand-member-of-the-german-bundestag-27-february-2022-in-berlin-2008378. Accessed\n6\nMar\n2023. Scholz, Olaf. 2023. The global Zeitenwende: how to avoid a new cold war in a multipolar era. Foreig\nAffairs 102(1):22–38. https://www.foreignaffairs.com/node/1129585. Sears, David O. 1993. Symbolic politics: a socio-psychological theory. In Explorations in political ps\nchology, ed. Shanto Iyengar, William McGuire, 113–149. Duke University Press. K K 547 No Zeitenwende (yet): Early Assessment of German Public Opinion Toward Foreign and... Smith, Matthew. 2022. What impact has the Russian invasion of Ukraine had on European attitudes to\nNATO? YouGov Report. https://yougov.co.uk/topics/international/articles-reports/2022/03/16/what-\nimpact-has-russian-invasion-ukraine-had-europ. Accessed 6 Mar 2023. Sobel, Richard. 2001. The impact of public opinion on U.S. Foreign policy since Vietnam. Oxford: Oxford\nUniversity Press. Steinbrecher, Markus. 2018. Einigkeit macht stark! Erklärung von Einstellungen zur Gemeinsamen Sicher-\nheits- und Verteidigungspolitik. In Freiheit oder Sicherheit?, ed. Markus Steinbrecher, Heiko Biehl,\nEvelyn Bytzek, and Ulrich Rosar, 217–251. Wiesbaden: Springer VS. y\ny\np\ng\nSteinbrecher, Markus. 2022. Lebt der Kalte Krieg weiter? In Wahlen und politische Einstellungen in Ost-\nund Westdeutschland. Wahlen und politische Einstellungen, ed. Martin Elff, Kathrin Ackermann, and\nHeiko Giebler, 207–244. Wiesbaden: Springer VS. Wike, Richard, Jacob Poushter, Laura Silver, Janell Fetterolf, and Mara Mordecai. 2021. America’s\nimage abroad rebounds with transition from Trump to Biden. Pew research center. https://www. pewresearch.org/global/2021/06/10/americas-image-abroad-rebounds-with-transition-from-trump-\nto-biden/. Accessed 6 Mar 2023. to-biden/. Accessed 6 Mar 2023. Wittkopf, Eugene R. 1990. Faces of internationalism: public opinion and American foreign policy. Durham: Duke University Press. y\nWlezien, Christopher. 1995. The public as thermostat: dynamics of preferences for spen\nJournal of Political Science 39(4):981–1000. https://doi.org/10.2307/2111666. Wlezien, Christopher. 1995. The public as thermostat: dynamics of preferences for spending. America ristopher. 1995. The public as thermostat: dynamics of preferences for spending. American\nof Political Science 39(4):981–1000. https://doi.org/10.2307/2111666. p\np\ny\np\np\ng\nJournal of Political Science 39(4):981–1000. https://doi.org/10.2307/2111666. Journal of Political Science 39(4):981–1000. https://doi.org/10.2307/2111666. Zaller, John R. 1992. The nature and origins of mass opinion. p\ny\nAccessed 6 Mar 2023. Cambridge: Cambridge University Press. Zaller, John R. 1992. The nature and origins of mass opinion. Cambridge: Cambridge University Press. K K"
https://openalex.org/W3133805570
https://aip.scitation.org/doi/pdf/10.1063/5.0042591
English
null
Electronegative microchannel guided streamer propagation for in-liquid spark breakdown applications
Applied physics letters
2,021
cc-by
4,031
Articles You May Be Interested In Enhanced shock wave generation via pre-breakdown acceleration using water electrolysis in negative streamer pulsed spark discharges Towards a fully kinetic 3D electromagnetic particle-in-cell model of streamer formation and dynamics in high-pressure electronegative gases Phys. Plasmas (September 2011) Bent paths of a positive streamer and a cathode-directed spark leader in diffuse discharges preionized by runaway electrons 24 October 2024 04:05:54 Phys. Plasmas (March 2015) Phys. Plasmas (March 2015) ABSTRACT One of the well-known challenging issues of in-liquid spark breakdown is electrode wear and wear-dependent deposit energy fluctuation, regardless of the electrode materials. This work suggests a method that can reduce the breakdown threshold by an order of magnitude and hence enhances the likelihood of breakdown, regardless of wear. Generally, the negative streamer propagates in a branching way; however, the present experiment indicates that the electronegative microchannel is converged with the streamer propagation and extends the break- down gap distance between the electrodes. Subsequently, the breakdown-possible gap distance was extended by 14.3 times, leading to an enhancement of shockwave intensity by 33%. Such an extension of the breakdown conditions was achievable without any additional source energy input or changing the substance of dielectric electrodes. Thus, the results provide a favorable scheme for energy reduction in high- voltage systems, cost saving for electrode replacement, and enhancement of the propagating shock pressure. 24 October 2024 04:05:54 V C 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0042591 The spark-induced plasma in liquid exhibits its substantial uses in inactivating hazardous organic compounds and toxicity of water,1,2 generating shock waves on MEMS technology such as micropumps,3 well cleaning,4 fabrication of nanomaterials,5 and the extracorporeal shock wave lithotripsy.6 However, one noticeable drawback of the use of underwater spark breakdown is the electrode erosion. A large elec- tric field can be achieved due to the sharp tip of the electrodes; how- ever, the sharp tip anode is quickly eroded by the discharge.5 Also, the limitation posed by high electrical conductivity of water gives rise to a considerable energy loss during the pre-breakdown process, and the electrical energy at the moment of breakdown is only 30%–80% of the initial electrical energy storage.7,8 particular, the negative streamer propagates in a tree-like branching way toward the anode and finally bridges the gap, resembling a fila- ment, which results in the spark breakdown, accompanied by the gen- eration of strong shock waves.12 g The questions arise considering the breakdown mechanism based on the Bubble theory. If the streamer path between the electrodes is filled with a stack of microbubbles, herein named “microchannel” along the propagation path, would it guide the streamer propagation? What type of substance for microchannel is the most suitable for effi- cient streamer propagation? Electronegative microchannel guided streamer propagation for in-liquid spark breakdown applications Cite as: Appl. Phys. Lett. 118, 103905 (2021); doi: 10.1063/5.0042591 Submitted: 31 December 2020 . Accepted: 18 February 2021 . Published Online: 11 March 2021 Cite as: Appl. Phys. Lett. 118, 103905 (2021); doi: 10.1063/5.0042591 Submitted: 31 December 2020 . Accepted: 18 February 2021 . Published Online: 11 March 2021 AFFILIATIONS Department of Mechanical and Aerospace Engineering, Seoul National University, 1 Gwanakro, Gwanakgu, Seoul 08826, South Korea Department of Mechanical and Aerospace Engineering, Seoul National University, 1 Gwanakro, Gwanakgu, Seoul 08826, South Korea Applied Physics Letters ARTICLE scitation.org/journal/apl ABSTRACT In this work, we used the gaseous micro- channel with electronegative behavior that guides and improves the streamer propagation. Via the high-speed photography, the focused streamer propagation along with the microchannel was visualized. Also, the breakdown voltage and shockwave intensity were compared with the microchannel substances. In other words, the strategy for overcoming the electrode erosion is sought that considers extending the stable breakdown gap distance using the electronegative micro- channel, regardless of electrode wear. The experimental methods are composed of (1) the use of electron-attracting behavior of the electro- negative microchannel that enhances the streamer propagation and The fundamental nature of in-liquid spark breakdown lies in the rapid formation of joule-heated microbubbles on the electrode surface under the influence of an applied high voltage, following the “Bubble theory.”9–11 Kolb showed that preexisting microbubbles could acceler- ate the initiation of the breakdown process.10 When the discharge is initiated, the electron avalanche propagates from the microbub- bles.12,13 Under a sufficiently strong electric field, the electron ava- lanche can transform into ionizing waves called the streamer. In Appl. Phys. Lett. 118, 103905 (2021); doi: 10.1063/5.0042591 V C Author(s) 2021 118, 103905-1 118, 103905-1 Applied Physics Letters ARTICLE scitation.org/journal/apl anode reaction ð Þ 2H2O ! O2 þ 4Hþ þ 4e; cathode reaction ð Þ 4Hþ þ 4e ! 2H2: (1) (2) the generation of locally amplified electric field inside the gas bub- ble of the microchannel. (1) Figure 1(a) shows the pulsed-high voltage discharge system, which includes the capacitor module and the spark gap for the trigger release. The electrodes are submerged into the dielectric liquid or the distilled water. The formation of a microchannel is supported by a switched-mode power supply that applies direct current (36 DCV, 0.2A) to the electrodes. Before the breakdown, the applied current through the electrode chemically break downs the water and generates the gaseous oxygen microchannel (from the anode) or the hydrogen microchannel (from the cathode). This microchannel preparation time is 20 s. Here, the breakdown characteristics in pure liquids are also tested, where no microchannel between the electrodes is created. After the preparation time is elapsed, the high voltage is induced to the electrodes for the discharge initiation. The configuration of the water chamber indicates that the anode or cathode is located on the lower side for building up a gaseous microchannel. ABSTRACT Water reacts at the anode to form oxygen or at the cathode to form hydrogen via the following reaction: When the direct current is applied by a power supply, the micro- sized gaseous oxygen bubbles are formed at the lower electrode (anode). Those microbubbles can flow upward due to buoyancy. While the microbubbles flow toward the upper electrode (cathode), new microbubbles are continuously generated at the bottom, forming a thin bubble stream called “microchannel.” The opposite electrode arrangement is for the hydrogen microchannel. The oxygen has elec- tronegative behavior, which attracts the electrons.14 The electronega- tivity of oxygen is 3.44, which is the second-highest value of all existing atoms, whereas that of hydrogen is 2.2.15 The electrode was in the shape of an eroded cone [dimensions shown in Fig. 1(a)], made of molybdenum. We visualized the pressure gradient using a high-speed camera (Hyper vision HPV-X2, Shimadzu Corp.). We used two ND filters (ND  8, Hoya) and a 532 nm bandpass filter. As a light source, a 532 nm laser was used with an optical lens module for generating a collimated beam. We used a high voltage probe (1000:1, 900MX, PINTEK HVP-39pro) for measuring the voltage at the electrodes. The pressure sensor (113B23, PCB Piezotronics) is mounted on the cham- ber 40 mm aside from the electrodes. FIG. 1. (a) Schematic of the experimental setup, (b) calculated electric field inside the microchannel, (c) structure of conventional negative streamers in surrounding liquid, (d) a speculated structure of streamers in the presence of an hydrogen microchannel surrounded by liquid, and (e) a speculated structure of negative streamers in the presence of an electronegative oxygen microchannel surrounded by liquid. Figure 1(b) shows the electric field of the micro-scale area inside the microchannel. A microchannel is in a form of aggregation of microbubbles. We calculated the electric field of microbubble arrange- ment. The diameter of the single bubble inside the microchannel is set to 250 lm, and their electric permittivity is 1 F/m to represent both hydrogen and oxygen. The electric permittivity of the surrounding liq- uid is 80 F/m. The externally applied electric field is E0 ¼ 5.0kV. ABSTRACT The locally amplified electric field exists inside and outside the microchan- nel due to the difference between the permittivity of the gas, Eg, and the liquid, El,16 24 October 2024 04:05:54 Er; bubble ¼ 0; Ez; bubble ¼ 3El 2El þ Eg E0; Er; surround ¼ 3 El=Eg  1   El=Eg þ 2 a3rz r2þz2 ð Þ5=2 E0; Ez; surround ¼ E0 þ El=Eg  1 El=Eg þ 2 a3 2z2  r2 ð Þ r2þz2 ð Þ5=2 E0: (2) (2) The highest amplified electric field exists on the bubble’s inside (Ez,bubble ¼ 1.5E0). This could initiate and accelerate the breakdown process. Several research studies support that the single bubble can be a source for streamer propagation.12,13,17 Thus, such an array of micro- bubbles can form the intensified electric field inside the streamer’s path. Figures 1(c)–1(e) illustrate the methodologies for focusing the negative streamer. Figure 1(c) explains the conventional negative streamer’s tree-like branching propagation. This branching pattern of propagation stems from the fact that the electron avalanche produces a large number of photons, which can induce a new electron avalanche in a branching way.12 FIG. 1. (a) Schematic of the experimental setup, (b) calculated electric field inside the microchannel, (c) structure of conventional negative streamers in surrounding liquid, (d) a speculated structure of streamers in the presence of an hydrogen microchannel surrounded by liquid, and (e) a speculated structure of negative streamers in the presence of an electronegative oxygen microchannel surrounded by liquid. Figure 1(d) shows the negative streamer along with the hydrogen microchannel where the streamer heads to the upward because it starts from the cathode. The locally amplified electric field inside the micro- channel accelerates the streamer propagation. Appl. Phys. Lett. 118, 103905 (2021); doi: 10.1063/5.0042591 V C Author(s) 2021 118, 103905-2 118, 103905-2 Applied Physics Letters scitation.org/journal/apl ARTICLE However, as shown in Fig. 1(e), the streamer in an electronegative (oxygen) microchannel could propagate in a conversing manner. Its electronegative property attracts electrons that comprise the head of the streamer. The electric field produced by this electronegativity effec- tively focuses the streamer head into the most efficient way of the propagation path. through the oxygen microchannel. At 54 ls, the streamer bridged the gap, followed by the breakdown. The shockwave is propagated at a speed of 1550 m/s afterward. When the microchannel is not fully developed to bridge the two electrodes, the breakdown failed. ABSTRACT (a) Pure liquid (water) between the elec- trodes without preparation of the microchannel [setup indicated in Fig. 1(c)], (b) hydrogen as the gas channel between the electrodes [breakdown failed—setup indicated in Fig. 1(d)], and (c) oxygen as the gas channel between the electrodes [setup indicated in Fig. 1(e)]. 24 October 2024 04:05:54 ad þ ln a P   ¼ 14:5 þ ln V Pd   þ 1 2 ln d P   : (4) (4) In general, the breakdown voltage in the gas is lower than in the liquid state. Since the microchannel is partially gaseous, we analyzed the breakdown efficiency of the microchannel compared with the pure gaseous oxygen and pure liquid. Figure 3 shows two breakdown volt- age curves for gaseous oxygen according to Paschen’s law [black dashed line, Eq. (3)] and streamer mechanism [red dotted line, Eq. (4)]. Also shown are the experimental values of breakdown voltages of hydrogen/oxygen microchannels in liquid explained earlier in Fig. 2. The experimental data of the pure liquid breakdown (blue triangles) show that this breakdown voltage was higher in the same order of magnitude as that of pure gaseous oxygen. However, the breakdown voltage of the oxygen microchannel in liquid (black squares) was lower than that of pure gaseous oxygen breakdown, which indicates that the breakdown could take place with much lower energy than gaseous oxygen. Moreover, its tendency followed Paschen’s curve and streamer mechanism. The hydrogen microchannel’s breakdown voltage (red circles) was 2.5–6.0kV higher than that of the oxygen channel at the same gap distance, indicating that the hydrogen microchannel required more energy for the breakdown. Both hydrogen and oxygen gas bubbles inside the microchannel have the same electric permittivity (1 F/m), which makes no significant difference in the locally amplified electric field. Thus, the cause of such discrepancy between the breakdown effi- ciencies of hydrogen and oxygen gas channel is inevitably the electronegativity. FIG. 2. Sequential images of streamer and shock wave propagations with the volt- age of 6.5 kV and 5.7 mm of gap distance. (a) Pure liquid (water) between the elec- trodes without preparation of the microchannel [setup indicated in Fig. 1(c)], (b) hydrogen as the gas channel between the electrodes [breakdown failed—setup indicated in Fig. 1(d)], and (c) oxygen as the gas channel between the electrodes [setup indicated in Fig. 1(e)]. ABSTRACT Spark breakdown could be explained by the two types of mecha- nisms. One is based on Paschen’s law that covers relatively low pres- sures and short gaps (pressuregap< 200 Torrcm).18 This law is represented by Eq. (3) where V is the breakdown threshold (volt) at the maximum gap, A and B are empirical parameters, P is the pressure, and d is the maximum gap distance. The current goes infinite (a break- down) as electrons multiply its number by ionization collision, which is also being supplied by electrons produced from the cathode surface (related to the secondary emission coefficient Ç),19 Figure 2 shows the observation for the breakdown incidences according to the streamer focusing methodologies explained earlier. Figures 2(a) and 2(b) show that the breakdown of the hydrogen chan- nel and of the pure liquid failed at the same gap distance. Although the hydrogen microchannel could locally intensify the electric field, the hydrogen has insufficient electronegativity to overcome the long gap distance. Pure liquid has no higher electronegativity than oxygen or hydrogen, and no microchannel was formed, making it the least effective in terms of breakdown amongst the three cases considered. V ¼ B Pd ð Þ lnA  lnln 1=c þ 1 ð Þ þ ln Pd ð Þ : (3) (3) On the other hand, Fig. 2(c) shows the streamer propagation and the breakdown of the oxygen microchannel. After forming an oxygen microchannel (white thin line), a discharge is initiated at 0 ls. At 24 ls, the negative streamer is initiated from the cathode, propagating Otherwise, one can observe the streamer at high pressure  gap. When the number of electrons reaches the specific value (Ncr  1018), the avalanche-to-streamer transition takes place. The streamer propa- gates toward the anode, and the gap is bridged.20 Experimental obser- vations yield Eq. (4), which represents the condition required for the streamer breakdown.20 A is the Townsend first ionization coefficient, which is involved in electron multiplication (avalanche), and its theo- retical value is a ¼ P A exp (B P d/V).19 Equation (4) is then solved based on the relation between a and V at which a given p and d satisfy the equation. These values are chosen until the equation is satisfied, FIG. 2. Sequential images of streamer and shock wave propagations with the volt- age of 6.5 kV and 5.7 mm of gap distance. ABSTRACT One needs to verify the deposit energy at the extended gap in terms of voltage after the pre-breakdown delay (Vs, PBD). As shown in Appl. Phys. Lett. 118, 103905 (2021); doi: 10.1063/5.0042591 V C Author(s) 2021 118, 103905-3 118, 103905-3 Applied Physics Letters scitation.org/journal/apl FIG. 3. The curves represent breakdown voltages for gas phase oxygen according to Paschen’s law [black dash line, Eq. (3)] and the streamer mechanism [red dotted line, Eq. (4)]. Also, experimental values of breakdown voltage for oxygen, hydrogen gas channels, and pure liquid channel are shown. FIG. 4. The experimental data of (a) voltage after pre-breakdown delay (Vs,PBD) with respect to the gap distance with the voltage of 3.5 kV and (b) peak pressure with respect to the gap distance with the voltage of 3.5 kV. FIG. 3. The curves represent breakdown voltages for gas phase oxygen according to Paschen’s law [black dash line, Eq. (3)] and the streamer mechanism [red dotted line, Eq. (4)]. Also, experimental values of breakdown voltage for oxygen, hydrogen gas channels, and pure liquid channel are shown. Fig. 4(a), Vs, PBD varies according to the gap distance. Here, a decrease in Vs, PBD (definition indicated in Fig. 5) means the lower deposit energy transferred.7 Vs, PBD of pure liquid (blue triangle) was 2.67 kV at the gap of 0.1 mm. Further breakdown at the increased gap distance could not be achieved. Vs, PBD of the hydro- gen microchannel (white circles) decreased exponentially at the extended gap distance. Vs,VPD of the hydrogen microchannel was higher than that of the oxygen microchannel (black squares) in a gap range of 0.1–0.4 mm. However, it decreased more rapidly than the oxygen microchannel after 0.4 mm, and the breakdown was not possible from the gap of 0.7 mm. Vs, PBD of the oxygen microchan- nel also decreased at the extended gap, but more slowly than the hydrogen microchannel, and the breakdown-possible gap reached 2.0 mm. 24 October 2024 04:05:54 However, as shown in Fig. 4(b), as the gap distance is extended, the peak pressure of both microchannels’ breakdown was observed to increase, and the peak pressure reached the range of 0.7–0.8 MPa. In particular, the peak pressure of the hydrogen chan- nel increased most rapidly with respect to the gap. However, over the gap of 0.9 mm, the peak pressure of the oxygen microchannel is measured to be higher than that of the hydrogen microchannel. ABSTRACT The maximum gap distance also increased to about 2.9 times than that of the hydrogen microchannel. This increasing tendency of peak pressure with an extended gap is explained by the joule heat- ing power during the early stage of the discharge. The joule heating is proportional to the spark channel resistance that is inversely pro- portional to the length of the gap distance.4 In the gap distance of 0.1–0.4 mm, the peak pressure of the pure liquid (0.45 MPa) was higher than that of the microchannel. The difference of the acoustic impedance between the gas microchannel and surrounding liquid may attenuate the breakdown pressure. However, further gap dis- tance increases the resistance of a spark channel that increased the breakdown pressure. The hydrogen microchannel has a rapidly growing tendency of peak pressure than the oxygen microchannel, but its breakdown-possible gap is limited at the shorter gap. FIG. 4. The experimental data of (a) voltage after pre-breakdown delay (Vs,PBD) with respect to the gap distance with the voltage of 3.5 kV and (b) peak pressure with respect to the gap distance with the voltage of 3.5 kV. FIG. 4. The experimental data of (a) voltage after pre-breakdown delay (Vs,PBD) with respect to the gap distance with the voltage of 3.5 kV and (b) peak pressure with respect to the gap distance with the voltage of 3.5 kV. As shown in Fig. 5, dielectric matter started to breakdown after the pre-breakdown delay. Followed was the time lag during which a pressure wave or a jump propagates toward the sensor at a sonic speed (1500 m/s). The pre-breakdown delay of the oxygen micro- channel was the shortest amongst other microchannels. A bit lon- ger time was taken for the hydrogen microchannel, and the longest was the pure liquid. Given that the presence of a microchannel accelerates the pre-breakdown process, which reduces the energy consumption,7,8 the short pre-breakdown delay generates the high peak pressure value, which was consistently reported by Lee et al.21 Regardless of these three distinguished gap distances, the oxygen microchannel presented the shortest pre-breakdown delay, which means that the oxygen microchannel is better at accelerating the streamer by its electronegative behavior. The breakdown-available gap distance increased by 14.3 times for 6.5 kV with the aid of oxy- gen microchannel. Appl. Phys. Lett. 118, 103905 (2021); doi: 10.1063/5.0042591 V C Author(s) 2021 118, 103905-4 118, 103905-4 118, 103905-4 Appl. Phys. Lett. ABSTRACT 118, 103905 (2021); doi: 10.1063/5.0042591 Applied Physics Letters scitation.org/journal/apl ARTICLE FIG. 5. The experimental data of voltage (left axis) and pressure (right axis) with respect to time at the voltage of 6.5 kV and gap distance of (a) 0.4 mm, (b) 1.8 mm, and (c) 5.6 mm. practical point of view, this finding could remedy adverse effects of electrode wear and wear-dependent deposit energy fluctuations. The electronegative microchannel as developed here inside the dielectric liquid can be used in various in-liquid spark and plasma applications that include water treatment, MEMS technology such as micropumps, fabrication of nanomaterials, and shockwave lithotripsy, wherein the electrode erosion was a pivotal drawback in extending its wide use. Furthermore, the microchannel could easily be initialized by inducing a direct current before the breakdown. This provides an option for one needing to either mitigate or augment the deposit energy by switching on or off the direct current. The financial support was provided from Baz Biomedic Co. Ltd. as a collaborative research grant contracted through IAAT and IOER at Seoul National University. DATA AVAILABILITY The data that support the findings of this study are available within the article. The additional data that support the findings of this study are available from the corresponding author upon reasonable request. REFERENCES 1B. Sun, Y. Xin, X. Zhu, Z. Gao, Z. Yan, and T. Ohshima, Bioelectrochemistry 120, 112 (2018). 1B. Sun, Y. Xin, X. Zhu, Z. Gao, Z. Yan, and T. Ohshima, Bioelectrochemistry 120, 112 (2018). 2 2X. Wang, D. Zhao, X. Tan, Y. Chen, Z. Chen, and H. Xiao, Chem. Eng. J. 328, 708 (2017). 24 October 2024 04:05:54 2X. Wang, D. Zhao, X. Tan, Y. Chen, Z. Chen, and H. Xiao, Chem. Eng. J. 328, 708 (2017). 3N. D. Taylor, G. Fridman, A. Fridman, and D. Dobrynin, Int. J. Heat Mass Transfer 126, 1104 (2018). 4 4K. J. Chung, S. G. Lee, Y. S. Hwang, and C. Y. Kim, Curr. Appl. Phys. 15, 977 (2015). 5S. Horikoshi and N. Serpone, RSC Adv. 7, 47196 (2017). 6 6P. Gupta, J. Urol. Nephrol. Hepatol. Sci. 2, 50 (2019). 7 7X. D. Li, Y. Liu, S. W. Liu, Z. Y. Li, G. Y. Zhou, H. Li, F. C. Lin, and Y. Pan, Phys. Plasma 23, 103104 (2016). 8 8G. Touya, T. Reess, L. Pecastaing, A. Gibert, and P. Domens, J. Phys. D: Appl. Phys. 39, 5236 (2006). 9H. Fujita, S. Kanazawa, K. Ohtani, A. Komiya, T. Kaneko, and T. Sato, J. Appl. Phys. 116, 213301 (2014). y 10J. Kolb, A. A. Mohamed, R. O. Price, R. J. Swanson, A. Bowman, R. L. Chiavarini, M. Stacey, and K. H. Schoenbach, J. Phys. D: Appl. Phys. 92, 241501 (2008). 11O. Lesaint, J. Phys. D: Appl. Phys. 49, 144001 (2016). 12 11O. Lesaint, J. Phys. D: Appl. Phys. 49, 144001 (2016). 12 12X. D. Li, Y. Liu, G. Y. Zhou, S. W. Liu, Z. Y. Li, and F. C. Lin, J. Phys. D: Appl. Phys. 50, 255301 (2017). 13V. A. Panov, L. M. Vasilyak, V. Y. Pecherkin, S. P. Vetchinin, and E. E. Son, J. Phys. D: Appl. Phys. 946, 012160 (2018). 14 14P. K. Chattaraj and S. Duley, J. Chem. Eng. Data 55, 1882 (2010). 15 15R. M. LoPachin, T. Gavin, D. R. Petersen, and D. S. Barber, Chem. Res. Toxicol. 22, 1499 (2009). FIG. 5. The experimental data of voltage (left axis) and pressure (right axis) with respect to time at the voltage of 6.5 kV and gap distance of (a) 0.4 mm, (b) 1.8 mm, and (c) 5.6 mm. 16J. D. Jackson, Classical Electrodynamics (Wiley, New York, 1999), p. 57. 17 y y 17M. N. Shneider and M. Pekker, J. Appl. Phys. REFERENCES 117, 224902 (2015). 18M. F€ullekrug, Sprites, Elves and Intense Lightning Discharges (Springer Science & Business Media, Berlin, 2006), p. 257. In conclusion, we have shown that the electronegativity of the microchannel and locally amplified electric field inside the microchan- nel effectively guides the streamer propagation, which then lowered the breakdown voltage. The analysis on the electronegative micro- channel has never been attempted by other researchers. From the 19Y. Yang, Plasma Discharge in Liquid: Water Treatment and Applications (CRC Press, Boca Raton, 2017), p. 35. 20M. S. Naidu and V. Kamaraju, High Voltage Engineering (McGraw-Hill, New York, 1995), p. 46. 21K. Lee, K. J. Chung, and Y. S. Hwang, Appl. Phys. Lett. 112, 134101 (2018). 118, 103905-5 Appl. Phys. Lett. 118, 103905 (2021); doi: 10.1063/5.0042591 V C Author(s) 2021 118, 103905-5 118, 103905-5
https://openalex.org/W4382986113
https://digital.csic.es/bitstream/10261/332096/1/Paraburkholderia_phytofirmans_PsJN.pdf
English
null
Paraburkholderia phytofirmans PsJN colonization of rice endosphere triggers an atypical transcriptomic response compared to rice native Burkholderia s.l. endophytes
Scientific reports
2,023
cc-by
12,879
www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Paraburkholderia phytofirmans PsJN colonization of rice endosphere triggers an atypical transcriptomic response compared to rice native Burkholderia s.l. endophytes OPEN Eoghan King 1,3*, Adrian Wallner 1,4, Ludivine Guigard 1, Isabelle Rimbault 1, Hugues Parrinello 2, Agnieszka Klonowska 1, Lionel Moulin 1 & Pierre Czernic 1* The plant microbiome has recently emerged as a reservoir for the development of sustainable alternatives to chemical fertilizers and pesticides. However, the response of plants to beneficial microbes emerges as a critical issue to understand the molecular basis of plant-microbiota interactions. In this study, we combined root colonization, phenotypic and transcriptomic analyses to unravel the commonalities and specificities of the response of rice to closely related Burkholderia s.l. endophytes. In general, these results indicate that a rice-non-native Burkholderia s.l. strain, Paraburkholderia phytofirmans PsJN, is able to colonize the root endosphere while eliciting a markedly different response compared to rice-native Burkholderia s.l. strains. This demonstrates the variability of plant response to microbes from different hosts of origin. The most striking finding of the investigation was that a much more conserved response to the three endophytes used in this study is elicited in leaves compared to roots. In addition, transcriptional regulation of genes related to secondary metabolism, immunity, and phytohormones appear to be markers of strain-specific responses. Future studies need to investigate whether these findings can be extrapolated to other plant models and beneficial microbes to further advance the potential of microbiome-based solutions for crop production. The potential of plant-associated microbes is nowadays appearing as a promising alternative solution to reduce the negative impacts caused using chemical pesticides and fertilizers in agriculture. Indeed, the application of beneficial microbes can enhance plant production by improving nutrient uptake or alleviating the impact of environmental stresses through various ­mechanisms1. Of particular interest is the use of endophytes, which are microbes able to colonize the inner tissues of plants and provide beneficial effects to the plants throughout their life cycle. Among the diversity of microbial endophytes, one particular bacterial strain, Paraburkholderia phytofirmans PsJN, hereafter termed PsJN, has the ability to colonize the endosphere of numerous plant species such as Arabidopsis, potato, tomato, grapevine, maize and ­barley2. PsJN also exhibit multiple beneficial effects to plants, in particular by increasing the tolerance of inoculated plants to biotic and abiotic ­stresses3.if p p y g p In order to identify the molecular mechanisms implicated in the beneficial effects of PsJN, several studies analyzed the transcriptional regulations of PsJN-treated Arabidopsis plants. Scientific Reports | (2023) 13:10696 Results A smaller population of PsJN cells can colonize the rice roots endosphere compared to rice native Burkholderia s.l. endophytes. The capacity of PsJN to colonize several plant species has been previously described, however its ability to colonize rice roots has never been established. To test this, we inocu- lated a DsRed-tagged strain on rice (O. sativa cv Nipponbare) roots of 4-days old plants grown in hydroponic conditions. PsJN colonization was first quantitatively evaluated by estimating the size of the bacterial population associated to rice roots (Fig. 1a). PsJN rapidly colonized rice roots by reaching a median value of population size of 1.28 × ­104 cfu ­g−1. Afterwards, from 1 to 7 dpi, the population size of PsJN massively increases to reach a median value of 6.57 × ­107 cfu ­g−1. Finally from 7 to 14 dpi, the bacterial population size almost doubled to reach a median value of 1.26 × ­108 cfu ­g−1. To estimate the amount of PsJN cells internally colonizing the rice root tissues, we conducted surface disinfection experiments. The size of the endophytic population of PsJN is dramatically (10,000 to 100,000 times) smaller than the root-associated population. Nonetheless, from 7 to 14 dpi, similarly to the total root-associated population, the endophytic population increased by a factor 1.5 by increasing from a median value of 4.07 × ­103 to 6.09 × ­103 cfu ­g−1.l g g We then carried histochemical and fluorescence microscopy analysis to qualitatively describe the colonization of rice roots by PsJN cells. Our results show that PsJN cells can massively colonize the surface of root hairs, the root cap of lateral roots and the surface of the primary root (Fig. 1b,c). The GUS histochemical staining experi- ments showed that the surface of the primary root is largely colonized, as well as the root caps of lateral roots while their surface remain relatively free of bacteria comparatively to the primary root (Fig. 1b). When observing the colonization of primary root epidermis, bacterial aggregates can be observed on the primary roots and lateral root emergences (Fig. 1c). Finally, root cross-section followed by confocal microscopy revealed that PsJN cells can colonize the inner tissues of rice roots. Indeed, fluorescent cells can be found associated to the cell wall of epidermic and cortical cells and most notably, PsJN cells have been observed in the xylem (Fig. 1d). www.nature.com/scientificreports/ transcriptional regulations lead to life cycle shortening through the early up-regulation of flowering control ­genes5. Transcriptional analyses were also carried out in the context of PsJN-induced resistance to ­pathogens6,7, ­salt8 and cold ­tolerance9. However, the interaction between the model endophyte PsJN and rice (Oryza sativa) has never been studied. Furthermore, it appears interesting to compare the plant response to the colonization by broad-spectrum versus rice-natural endophytes to unravel plant–microbe specific interactions. Similar comparative analyses of hosts transcriptomic response have been done to compare the response to symbiont and ­pathogens10–12 and the response to different beneficial strains of Azospirillum isolated from different cultivars of ­rice13. Therefore, we analyzed the response of rice to the inoculation with PsJN. Then, we performed a comparative analysis of rice transcriptomic response to PsJN with the response to two rice-isolated Burkholderia s.l. endophytic strains that we described in a previous ­study14: the rice-endophytic models Paraburkholderia kururiensis ­M13015, hereafter termed Pk, and Burkholderia vietnamiensis TVV75, hereafter termed Bv. Our objective was to unravel common and specific signatures of rice transcriptomic response to these three strains to characterize the specificities of the physiological response of rice plants to a diversity of closely related strains.i p y g p p y y To study the response of rice to PsJN, we first analyzed the colonization of Oryza sativa cultivar Nipponbare rice roots and showed endophytic colonization. The phenotypic analysis showed that PsJN, Pk and Bv have respectively neutral, positive and negative effects on rice productivity. Then, the transcriptomes of leaves and roots of axenic and PsJN-colonized plants were produced to characterize the transcriptional response of rice to PsJN. Finally, we performed a comparative analysis of rice phenotypic and transcriptomic response to PsJN, Bv and Pk. The comparative transcriptomic analysis revealed a more conserved response in leaves than in roots. Also, strain-specific root transcriptomes are enriched in genes implicated in immunity, hormone signaling and sec- ondary metabolism. Finally, surprisingly the transcriptomic responses to the two Paraburkholderia strains were markedly dissimilar, indeed the response of rice to PsJN shared characteristics of the response to both Pk and Bv. Paraburkholderia phytofirmans PsJN colonization of rice endosphere triggers an atypical transcriptomic response compared to rice native Burkholderia s.l. endophytes OPEN For direct beneficial effects, PsJN- triggered root growth promotion by involving hormone signaling, particularly auxin and ­ethylene4. Also, systemic 1Plant Health Institute of Montpellier, IRD, CIRAD, University of Montpellier, l’Institut Agro, Montpellier, France. 2Montpellier GenomiX (MGX), c/o Institut de Génomique Fonctionnelle, Montpellier, France. 3Present address: Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid (UPM)– Instituto Nacional de Investigación y Tecnología Agraria y Alimentación (INIA/CSIC), Campus de Montegancedo, Pozuelo de Alarcón, Madrid, Spain. 4Present address: SFR Condorcet – FR CNRS 3417, University of Reims Champagne-Ardenne, Induced Resistance and Plant Bioprotection (RIBP) – EA 4707, Cedex 2, BP1039, 51687 Reims, France. *email: eoghan.king.pro@gmail.com; pierre.czernic@umontpellier.fr | https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 www.nature.com/scientificreports/ Results For the endophytic compartment the data correspond to 5 biological replicates (b) Colonization of rice primary root picture by PsJN::pINGUS. White bar represents 1 mm. (c) Epifluorescence microscopy pictures of the colonization of rice primary roots at 7 days post-inoculation by PsJN::pIN29 cells. White bars represent 200 μm (d) Confocal microscopy picture of a cross-section of rice primary roots at 7 days post-inoculation by PsJN::pIN29 cells. White bars represent 200 μm. Figure 1. Endophytic colonization of rice roots by PsJN. (a) Population dynamics of DsRed-tagged PsJN associated with rice roots. The data reported are the median of bacterial population size from 9 plants for 1 dpi and 18 plants-conducted in two independent experiments- for 7 and 14 dpi. and 5 plants. For the endophytic compartment the data correspond to 5 biological replicates (b) Colonization of rice primary root picture by PsJN::pINGUS. White bar represents 1 mm. (c) Epifluorescence microscopy pictures of the colonization of rice primary roots at 7 days post-inoculation by PsJN::pIN29 cells. White bars represent 200 μm (d) Confocal microscopy picture of a cross-section of rice primary roots at 7 days post-inoculation by PsJN::pIN29 cells. White bars represent 200 μm. per treatment (control or inoculated), each replicate being a pool of 5 plants. On average 42,782,744 reads were sequenced from each sample of which 69% were uniquely mapped to the rice genome (IRGSP, version1) (Sup- plementary Table S1). The genes with an FDR adjusted p-value inferior to 0.01 were considered significantly differentially expressed. In leaves, 2646 and 2337 DEGs (Supplementary Table S2) were identified as up and down-regulated genes respectively whereas in roots 379 and 609 DEGs, respectively, were identified (Supple- mentary Table S3). y To identify the biological processes transcriptionally regulated in rice during the interaction with PsJN, we performed GO terms and KEGG pathway enrichment analysis. Significantly enriched GO terms in root tran- scriptome encompasses terms related to hormone signaling, metal ion transport (especially iron) and signaling for the up-regulated DEGs and genes implicated in immunity, carbon fixation and secondary metabolism for the down-regulated genes (Fig. 2a, Supplementary Table S4). KEGG pathway enrichment analysis on down- regulated DEGs in roots also pinpoints an enrichment of defense-related genes such as PR1, PBZ1 and ALD1 as well as jasmonic acid (JA) signaling such as JAZ genes. Results These results show by two approaches that PsJN is able to establish endophytically in rice roots in our experimental conditions. y pp p y y p As PsJN was not originally isolated from rice plants, while it colonizes its endosphere in our conditions, it can be considered a non-native endophyte. We wanted to compare rice PsJN-induced phenotypic and transcrip- tional responses to the colonization by Burkholderia s.l. endophytes naturally interacting with rice. We chose Paraburkholderia kururiensis M130, hereafter termed Pk, and Burkholderia vietnamiensis ­TVV75T, hereafter termed Bv, which were isolated from rice roots and previously shown to endophytically colonize rice ­roots14. The colonization pattern of PsJN was then compared to the two model endophytes of rice Pk and Bv using the same protocol. If PsJN appears to colonize slower the root surface at 1 and 7 dpi, it reaches a similar population level than Bv and Pk at 14 dpi (Supplementary Fig. S1). However, PsJN colonized less (10 to 100 times) the root interior at 7 and 14 dpi compared to Bv and Pk. The epifluorescence microscopy pictures of a lateral root emer- gence colonized by Pk and Bv shows different patterns. Indeed, Pk appears to colonize more homogeneously than PsJN, with smaller micro-colonies (Supplementary Fig. S1). On the other hand, the colonization pattern of Bv is different than the Paraburkholderia strains, as we observed patches of cells with high fluorescence intensity and potential intracellular colonization of epidermic cells. Furthermore, the confocal microscopy pictures of root section show that xylem colonizing Pk cells can be observed (Supplementary Fig. S1). Besides, Bv endophytic colonization seems to be restricted to rice root epidermic cells. The transcriptional analysis of rice response to PsJN shows important and dynamic regula- tions of defense, JA signaling and iron homeostasis in roots. To identify differentially expressed genes (DEGs) in leaves and roots following PsJN inoculation, we used RNA-sequencing on roots and leaves at 7 dpi in a hydroponic system (see the Methods section). Total RNA was extracted from three biological replicates https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ Figure 1. Endophytic colonization of rice roots by PsJN. (a) Population dynamics of DsRed-tagged PsJN associated with rice roots. The data reported are the median of bacterial population size from 9 plants for 1 dpi and 18 plants-conducted in two independent experiments- for 7 and 14 dpi. and 5 plants. Results In leaves, a large proportion of enriched GO terms are related to primary metabolism: carbon fixation, photosynthesis and amide metabolism are enriched among the up-regulated genes (Supplementary Fig. S2, Supplementary Table S4). For the down-regulated genes, namely, carboxylic acid metabolism and starch biosynthesis are enriched. Also, GO terms related to developmental pro- cess are significantly enriched for both up and down-regulated DEGs (Supplementary Table S4). i To get a better view of some of the genes highlighted by the RNAseq analyses, we performed a time course experiment based on RT-qPCR (qPCR) analysis of genes implicated in the process highlighted by the GO term and KEGG pathway such as defense, hormones and iron metabolism. We chose defense-related genes PBZ1, ALD1 and WRKY28 mentioned above. Also, the transcription factor bHLH148 and JAZ10 were added for the JA signaling pathway. g g p y Finally, IRO2 and Os10g0195250 were added to exemplify the metal ion transport pathway. All these genes were analyzed in root tissues sampled at 6 h, 1-, 7- and 14-days post inoculation. The Fig. 2b shows through a heatmap the expression kinetics of all those genes. Interestingly, the expression of PBZ1 and ALD1 at 7 dpi Scientific Reports | (2023) 13:10696 | https://doi.org/10.1038/s41598-023-37314-7 www.nature.com/scientificreports/ ure.com/scientificreports/ Figure 2. Root transcriptional response to PsJN colonization. (a) Enrichment analysis of Biological Process GO terms of PsJN-treated rice root transcriptome. Y-axis corresponds to the significantly enriched GO terms (FDR < 0.05 and an enrichment ratio threshold of 2), X-axis corresponds to the adjusted p-value, the enrichment ratio represent by the size of the dots corresponds to the ratio between the proportion of genes related to the given GO term in the transcriptome and the proportion of genes related to the given GO term in the rice genome. Left and right panels respectively correspond to up- and down-regulated DEGs (b) The values of mean log2FoldChange of 7 genes compared to a non-inoculated control over 6 hpi, 1, 7 and 14 dpi were represented as heatmaps. Expression level is color-coded with red indicating up-regulation and blue indicating down- regulation. Only values greater than 0.5 or lower than − 0.5 were represented. Figure 2. Root transcriptional response to PsJN colonization. (a) Enrichment analysis of Biological Process GO terms of PsJN-treated rice root transcriptome. www.nature.com/scientificreports/ confirms by qPCR the down-regulation in response to PsJN detected by RNA-Seq. Additionally, an early up- regulation of PBZ1 was detected at 6 hpi while ALD1 is up-regulated at 14 dpi. For the expression of JA-related genes, we demonstrated a down-regulation of WRKY28 and bHLH148 at all time points. Furthermore, JAZ10 expression undergoes up-regulation at 6 hpi and 14 dpi while being down-regulated at 1 and 7 dpi. This pattern of early induction of expression at 6 hpi followed by a transient down-regulation phase prior to a strong induction of the expression can be observed for the kinetics of iron-related genes IRO2 and Os10g0195250. Rice response to endophytic colonization by PsJN is atypic from the response to rice native Burkholderia s.l. endophytes. Phenotypic analysis of soil-grown rice plants inoculated with PsJN, Bv and Pk showed no significant effect on plant size or shoot biomass (Fig. 3a,b) two months after inoculation while PsJN inoculation significantly decreased the number of tillers (Fig. 3d). On the other hand, PsJN and Bv inocu- lation significantly increased root biomass compared to uninoculated plants (Fig. 3e). However, Pk inoculation was the only condition where a significant increase in the number of seeds was observed (Fig. 3c) whereas Bv- inoculated plants produced significantly lighter grains (Fig. 3f). Additionally, we performed colonization assays of soil-grown rice plants inoculated with the three strains at 15 dpi (Supplementary Fig. S3). This experiment firstly showed that all strains can colonize the rhizosphere and the roots of rice plants grown in soil. In addition, we show that Bv and Pk can consistently colonize the base of the rice stem whereas PsJN was only detected in 2 out of 5 stem samples. Finally, all leaves were colonized by Bv suggesting that this strain can colonize the above- ground parts of rice plants. Figure 3. Analysis of phenotypic traits of rice in response to three Burkholderia s.l. endophytes. Analysis of the phenotypic response of rice to the inoculation by P. phytofirmans (PsJN), P. kururiensis (Pk) and B. vietnamiensis (Bv) or mock-inoculated (Control). Results presented in (a), (b), (d) and (e) correspond to the mean results obtained for 12 plants for 2 months post-inoculation with ­108 bacterial cells one week after sowing. Results Y-axis corresponds to the significantly enriched GO terms (FDR < 0.05 and an enrichment ratio threshold of 2), X-axis corresponds to the adjusted p-value, the enrichment ratio represent by the size of the dots corresponds to the ratio between the proportion of genes related to the given GO term in the transcriptome and the proportion of genes related to the given GO term in the rice genome. Left and right panels respectively correspond to up- and down-regulated DEGs (b) The values of mean log2FoldChange of 7 genes compared to a non-inoculated control over 6 hpi, 1, 7 and 14 dpi were represented as heatmaps. Expression level is color-coded with red indicating up-regulation and blue indicating down- regulation. Only values greater than 0.5 or lower than − 0.5 were represented. Figure 2. Root transcriptional response to PsJN colonization. (a) Enrichment analysis of Biological Process GO terms of PsJN-treated rice root transcriptome. Y-axis corresponds to the significantly enriched GO terms (FDR < 0.05 and an enrichment ratio threshold of 2), X-axis corresponds to the adjusted p-value, the enrichment ratio represent by the size of the dots corresponds to the ratio between the proportion of genes related to the given GO term in the transcriptome and the proportion of genes related to the given GO term in the rice genome. Left and right panels respectively correspond to up- and down-regulated DEGs (b) The values of mean log2FoldChange of 7 genes compared to a non-inoculated control over 6 hpi, 1, 7 and 14 dpi were represented as heatmaps. Expression level is color-coded with red indicating up-regulation and blue indicating down- regulation. Only values greater than 0.5 or lower than − 0.5 were represented. https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Results presented in (c) and (f) correspond respectively to the number of seeds and the average grain weight produced per pot out of 8 pots, each containing 4 plants grown for 4 months post-inoculation with ­108 bacterial cells one week after sowing. Significant levels in variations were determined using Student’s t-test for plant size, shoot biomass and root biomass and Wilcoxon test for number of tillers, yield and grain weight (p < 0.05, p < 0.01 and p < 0.001 for *, ** and *** respectively). Figure 3. Analysis of phenotypic traits of rice in response to three Burkholderia s.l. endophytes. Analysis of the phenotypic response of rice to the inoculation by P. phytofirmans (PsJN), P. kururiensis (Pk) and B. vietnamiensis (Bv) or mock-inoculated (Control). Results presented in (a), (b), (d) and (e) correspond to the mean results obtained for 12 plants for 2 months post-inoculation with ­108 bacterial cells one week after sowing. Results presented in (c) and (f) correspond respectively to the number of seeds and the average grain weight produced per pot out of 8 pots, each containing 4 plants grown for 4 months post-inoculation with ­108 bacterial cells one week after sowing. Significant levels in variations were determined using Student’s t-test for plant size, shoot biomass and root biomass and Wilcoxon test for number of tillers, yield and grain weight (p < 0.05, p < 0.01 and p < 0.001 for *, ** and *** respectively). https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ To describe which physiological processes are commonly and specifically regulated in rice response to endo- phytic colonization, we performed a comparative analysis of the transcriptomic response of rice to PsJN, Pk and Bv. Additionally, the root and leaf tissues were treated and harvested simultaneously with the material used in our previous ­study14 to study respectively the local and systemic transcriptomic response to root colonization. The analysis of leaf transcriptomes of inoculated plants revealed an important conserved response of rice to three Burkholderia s.l endophytes. Indeed, 25% and 28% of up- and down-regulated DEGs respectively are commonly regulated in response to the three Burkholderia s.l (Fig. 4a). Additionally, among all DEGs in leaves, 56% are shared between the response to at least two endophytes. However, a very low proportion of genes (5%) are com- monly regulated following Pk and Bv root colonization strictly. www.nature.com/scientificreports/ Finally, among all DEGs in leaves, approximately 40% are strain-specific with half of them being specifically induced by Pk. In contrast, the comparative analysis of root transcriptomes revealed a very different pattern. Indeed, among all root DEGs, 28% are specifically regulated in response to PsJN (Fig. 4b). Furthermore, in opposition to leaves, a much larger proportion of PsJN-induced DEGs in roots is shared with Bv than with Pk. Indeed, on average between up and down-regulated DEGs, 17% of DEGs are shared in response to PsJN and Bv while only 3% are shared in response to the two Paraburkholderia strains. Finally, a larger proportion of DEGs in common are down-regulated. For example, 14% of all down- regulated DEGs are commonly responsive to the three endophytes while only 5% for the up-regulated DEGs.h g y p p y y p g This analysis reveals an important overlap of PsJN-induced gene regulations with both Pk and Bv-induced regulations in leaves. On the other hand, most of DEGs regulated by both rice-isolated endophytes are also induced following PsJN root colonization. This corroborates with principal component analysis (PCA) of the normalized reads in the transcriptome of leaves, indeed the response to Bv overlaps with the response to PsJN (Supplementary Fig. S4a). On the other hand, PsJN root colonization specifically induced the regulation of the largest proportion of genes (Fig. 4b) leading to a separation of the response of roots to PsJN and Bv on the PCA analysis (Supplementary Fig. S4b). Defining the core transcriptomic response of rice to endophytic colonization. To further describe the physiological processes regulated in response to endophytic colonization, we performed Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the different pool of DEGs shared or not between the response to the three endophytic species (Fig. 4, Supple- mentary Tables S5, S6, S7 and S8). Following this, we conducted a manual functional categorization of the most highly regulated DEGs (|Log2FoldChange|> 1) for the common response to the three endophytes additionally to the enrichment analysis (Fig. 5, Supplementary Table S9). y ( g pp y ) For the commonly up-regulated DEGs in leaves, genes implicated in the response to stresses, photosynthesis and oxidant detoxification are significantly enriched (Supplementary Table S5). www.nature.com/scientificreports/ Among the top up-regulated DEGs we identified genes related to development, mainly genes encoding for transcription factors and cell wall-related metabolic processes, transport, mostly proton pumps (Supplementary Table S9). Also, genes related to immunity were identified among the most highly up-regulated DEGs in leaves. Both regulatory genes like WRKY7, WRKY28 or MKK4 and also antimicrobial compounds encoding genes such as PR2, PAL9 and Cht6 are commonly up-regulated in response to PsJN, Pk and Bv. y g Among the down-regulated DEGs in leaves, the largest proportions correspond to genes related to signaling, mostly kinase-encoding genes, and RNA and DNA metabolism especially genes related to RNA polymerase II and chromatin conformation (Fig. 5). Furthermore, we identified DEGs related to transport, particularly transport- ers associated to the vacuole and related to N metabolism such as AMT3.3 and a Trp/Tyr transporter. Finally, down-regulated defense-related DEGs are mostly putative resistance genes, with 8 out of 9 genes encoding for NB-ARC proteins (Supplementary Table S9). y As previously described, the root common response to the three endophytes is much more restricted in pro- portion compared to leaves. This is particularly the case of the up-regulated DEGs which also reach lower level of relative expression (Fig. 5, Supplementary Table S9). This low proportion of commonly up-regulated DEGs in roots is also associated to no significant GO term or KEGG pathway (Supplementary Table S7). However, among the most highly up-regulated genes, we identified two genes encoding for aminocyclopropane carboxylate oxidase (Os06g0573900 and Os02g0771600), two genes encoding for putative protein kinase (Os09g0551251 and Os04g0633900), two genes related to root development (RSL9 and Os05g0552600) and a putative sugar transporter (Os03g0197100).i p g Additionally, enrichment analysis showed that the KEGG pathway "Glycolysis/Gluconeogenesis" is signifi- cantly enriched among the common down-regulated DEGs in roots (Supplementary Table S8). Also, among the most highly regulated DEGs, the second functional category in proportion is primary metabolism with genes related to carbon metabolism and most particularly four genes implicated in pyruvate metabolism (Os05g0469600, Os03g0432100, Os06g0104900, Os05g0469800) also three genes related to amino acid synthesis (Os05g0244700, Os01g0720700 and ARD3). g g However, genes related to immunity represent the largest category among the most highly down-regulated genes (Fig. 5). www.nature.com/scientificreports/ Particularly, we identified two genes encoding for transcription factors of the WRKY family (namely WRKY62 and WRKY76), three genes implicated in secondary metabolism involved in defense response (CCR1, CCR10 and Os03g0122300), two genes implicated in the response to SA (RH2 and RH3) and finally one gene of the PR family (PR1a). Comparative analysis of transcriptomes reveals important strain‑specific transcriptional regu- lation of primary and secondary metabolism. We then described the transcriptional regulations com- mon to the response to two Burkholderia s.l. strains or specific to the response to each strain. In leaves, this com- parative analysis revealed that the most significantly enriched processes are related to energy production and https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ Figure 4. Comparative analysis of rice transcriptomic response to Burkholderia s.l. endophytes. Venn diagrams show the number of DEGs in response to each Burkholderia s.l. strains in leaves (a) and roots (b) as well as the proportion of genes shared between the responses to these three strains. For each subpart, the upper numbers correspond to the amount of up-regulated DEGs while lower numbers, the down-regulated DEGs. Representative significantly enriched GO terms and KEGG pathways related to each pool of shared or specific DEGs are respectively written in green and red. Figure 4. Comparative analysis of rice transcriptomic response to Burkholderia s.l. endophytes. Venn diagrams show the number of DEGs in response to each Burkholderia s.l. strains in leaves (a) and roots (b) as well as the proportion of genes shared between the responses to these three strains. For each subpart, the upper numbers correspond to the amount of up-regulated DEGs while lower numbers, the down-regulated DEGs. Representative significantly enriched GO terms and KEGG pathways related to each pool of shared or specific DEGs are respectively written in green and red. https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ Figure 5. Common transcriptomic response of rice to Burkholderia s.l. endophytes. Number of common significantly DEGs highly regulated (|Log2FoldChange|> 1) in response to PsJN, Pk and Bv related to functional categories. Figure 5. Common transcriptomic response of rice to Burkholderia s.l. endophytes. Number of common significantly DEGs highly regulated (|Log2FoldChange|> 1) in response to PsJN, Pk and Bv related to functional categories. Figure 5. Common transcriptomic response of rice to Burkholderia s.l. endophytes. Number of common significantly DEGs highly regulated (|Log2FoldChange|> 1) in response to PsJN, Pk and Bv related to functional categories. primary metabolism. www.nature.com/scientificreports/ Particularly, the common transcriptomic response to PsJN and Bv is significantly enriched in genes related to carbon fixation, photosynthesis and carboxylic acid metabolism (Fig. 4a and Supplementary Table S5). On the other hand, the common response to PsJN and Pk is enriched in genes related to oxidative phosphorylation according to both GO terms and KEGG pathways. Additionally, the strain-specific response to PsJN and Pk showed an enrichment in photosynthesis and respiration respectively.i primary metabolism. Particularly, the common transcriptomic response to PsJN and Bv is significantly enriched in genes related to carbon fixation, photosynthesis and carboxylic acid metabolism (Fig. 4a and Supplementary Table S5). On the other hand, the common response to PsJN and Pk is enriched in genes related to oxidative phosphorylation according to both GO terms and KEGG pathways. Additionally, the strain-specific response to PsJN and Pk showed an enrichment in photosynthesis and respiration respectively.i Furthermore, KEGG enrichment analysis showed strain-specific transcriptional regulations of secondary metabolites pathways. For example, the specific response to Pk in leaves, is enriched in genes implicated in the phenylpropanoid and flavonoid synthesis pathways (Fig. 4a). Contrastingly, in response to PsJN, genes implicated in the terpene synthesis are specifically enriched among up-regulated DEGs. No specific pathway is enriched among the common DEGs in response to PsJN and Bv, however the "secondary metabolism" KEGG term is sig- nificantly enriched. Similarly, the root transcriptomic response to Pk, both specific and shared with Bv is enriched for the down-regulation of genes implicated in phenylpropanoid synthesis (Fig. 4b and Supplementary Table S8). Th l b l f l l f k d l d f h d ff This global functional analysis of rice transcriptomic response to PsJN, Pk and Bv led us to focus on the differ- ential expression of genes implicated in secondary metabolism, hormonal signaling and defense in response to the three endophytes. To further deepen our analysis, we compared the expression of the most-highly regulated DEGs (|Log2FoldChange|> 1) implicated in these processes during the interaction with PsJN, Pk and Bv (Figs. 6 and 7). This global functional analysis of rice transcriptomic response to PsJN, Pk and Bv led us to focus on the differ- ential expression of genes implicated in secondary metabolism, hormonal signaling and defense in response to the three endophytes. www.nature.com/scientificreports/ To further deepen our analysis, we compared the expression of the most-highly regulated DEGs (|Log2FoldChange|> 1) implicated in these processes during the interaction with PsJN, Pk and Bv (Figs. 6 and 7). Relatively few genes implicated in secondary metabolism are commonly regulated following endophytic colonization, namely two backbone terpene synthesis are down-regulated (HDS and SPS2), and 4 genes related to h l id h i l d (POX5 1 61 PAL9 d O 03 0762300) l ifi ll i li d Relatively few genes implicated in secondary metabolism are commonly regulated following endophytic colonization, namely two backbone terpene synthesis are down-regulated (HDS and SPS2), and 4 genes related to phenylpropanoid synthesis are regulated (POX5.1, prx61, PAL9 and Os03g0762300) mostly specifically implicated in lignin synthesis except for PAL9 (Fig. 6b and Supplementary Table S10). Furthermore, genes implicated in lignin synthesis appear to be globally down-regulated in roots while almost all genes related to lignin synthesis in leaves are up-regulated in response to root colonization. p g p Particularly, Pk-inoculated plants induced the regulation of 10 and 12 genes implicated in lignin synthe- sis, respectively up-regulated in leaves and down-regulated in roots (Fig. 6a). Consistently with the GO term Scientific Reports | (2023) 13:10696 | https://doi.org/10.1038/s41598-023-37314-7 www.nature.com/scientificreports/ Figure 6. Comparative analysis of secondary metabolites genes expression at 7 dpi in rice roots and leaves in response to Burkholderia s.l. endophytes. Relative expression of DEGs (|Log2FoldChange|> 1) related to secondary metabolism in rice roots (a) and leaves (b) in response to the colonization by P. phytofirmans, P. kururiensis, B. vietnamiensis at 7 days post-inoculation. Expression level is color-coded with red indicating up-regulation and blue indicating down-regulation. Figure 6. Comparative analysis of secondary metabolites genes expression at 7 dpi in rice roots and leaves in response to Burkholderia s.l. endophytes. Relative expression of DEGs (|Log2FoldChange|> 1) related to secondary metabolism in rice roots (a) and leaves (b) in response to the colonization by P. phytofirmans, P. kururiensis, B. vietnamiensis at 7 days post-inoculation. Expression level is color-coded with red indicating up-regulation and blue indicating down-regulation. https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ Figure 7. Comparative analysis of hormone and immunity-re to Burkholderia s.l. endophytes. Relative expression of (a) horm (|Log2FoldChange|> 1) in rice roots in response to the coloniza vietnamiensis at 7 days post-inoculation. Expression level is col blue indicating down-regulation. Figure 7. www.nature.com/scientificreports/ Comparative analysis of hormone and immunity-related genes expression in rice roots in response to Burkholderia s.l. endophytes. Relative expression of (a) hormone-related and (b) immunity-related DEGs (|Log2FoldChange|> 1) in rice roots in response to the colonization by P. phytofirmans, P. kururiensis, B. vietnamiensis at 7 days post-inoculation. Expression level is color-coded with red indicating up-regulation and blue indicating down-regulation. Figure 7. Comparative analysis of hormone and immunity-related genes expression in rice roots in response to Burkholderia s.l. endophytes. Relative expression of (a) hormone-related and (b) immunity-related DEGs (|Log2FoldChange|> 1) in rice roots in response to the colonization by P. phytofirmans, P. kururiensis, B. vietnamiensis at 7 days post-inoculation. Expression level is color-coded with red indicating up-regulation and blue indicating down-regulation. https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ enrichment analysis, rice response to Pk stands out because of the specific up-regulation of genes related to flavonoids synthesis in both leaves and roots. Inversely, PsJN and Bv colonization induced the up-regulation of 3 genes implicated in terpenoids synthesis (Os03g0184550, Os03g0347900 and Os01g0662700) and 2 genes related to alkaloids synthesis (TDC3 and Os11g0438700) in leaves. Also, the colonization of PsJN and Bv induced in roots the regulation of respectively 4 and 3 genes related to terpenoids synthesis while only 1 is down-regulated in response to Pk. Immunity and hormone‑related transcriptional regulations in roots reveals a largely shared response to PsJN and Bv dissimilar of the response to Pk. In roots, the transcriptomic response to the three endophytes is enriched in genes related to defense, stress response and hormonal signaling (Fig. 4, Supplementary Tables S7 and S8). Also, the common response to Bv and PsJN is enriched in genes related to hor- mone signaling, response to stress, signaling and defense. This led us to focus on the regulation of genes related to hormonal signaling (Fig. 7a) and immunity (Fig. 7b) to pinpoint fundamental differences in the response to colonization by PsJN, Pk and Bv. y As previously described, the common response to the three endophytes is characterized by the up-regulation of 2 ACO genes, implicated in ethylene synthesis. Additionally, 13 genes implicated in the ethylene signaling pathways, mostly from the ERF family, are down-regulated by at least one of the three Burkholderia s.l. strains (Fig. 7a). Notably, only Bv colonization induces the down-regulation of DREB genes. Discussion PsJN can colonize the rice root endosphere while not impacting rice yield in contrast to rice‑native endophytes. Unraveling the molecular bases of plants response to various endophytes will help identify the physiological processes implicated in the establishment of plant-endophyte interactions. Based on a previous study analyzing the transcriptional response of rice to two rice-isolated Burkholderia s.l. endophytic ­strains14, we performed a comparative analysis of the phenotypic and transcriptomic responses of Oryza sativa, cultivar Nipponbare, to Paraburkholderia phytofirmans PsJN, a wide spectrum endophyte, with the response to rice-native Burkholderia s.l. endophytes.h p p y The analysis of rice root colonization showed that PsJN can colonize the endosphere of rice roots. Indeed, from 7 to 14 dpi the amount of PsJN cells colonizing rice roots reaches approximately ­108 cfu ­g−1 which is consist- ent with the level of colonization observed for several other plant-rhizobacteria ­models16. On the other hand, this population size of PsJN cells is significantly smaller than for rice-native Burkholderia s.l. endophytes Pk and Bv14. (Supplementary Fig. S1b). This is particularly the case for the early time point at 1 and 7 dpi for the root- associated population and for the endophytic population at both time points when compared to Pk.f Although an important colonization of rice roots by PsJN cells was observed, the different root areas are not homogeneously colonized. Indeed, PsJN cells particularly accumulate in the root hair zone and on lateral root tips (Fig. 1b). This was also observed during the colonization of grapevine by ­PsJN17. Interestingly, these root areas correspond to the most nutrient-rich zones. Particularly, the root hair zone is a hotspot in terms of exudates ­concentration18. Also, the root cap cells could be microbial hotspot for several reasons. First, these cells exude large amount of polysaccharide-based mucilage which could be used as nutrients by PsJN cells, as it was previ- ously observed in ­pea19. Secondly, root cap cells have a shorter life cycle compared to other root ­cells20, which is a consequence of a programmed cell death. This leads to the leakage of the cellular content of which some components could be used as nutrients by bacteria. Finally, PsJN cells were also observed colonizing the cortex and the xylem of rice roots similarly to Pk (Supplementary Fig. www.nature.com/scientificreports/ Also, the up-regulation of RR genes, implicated in cytokinin signaling is common to the response to the three endophytes although different genes are triggered by each strain. While the transcriptional regulation of genes implicated in the eth- ylene and cytokinin signaling appears to be shared among all conditions, only two strains trigger a substantial down-regulation of genes related to JA signaling. Indeed, as revealed by the GO term enrichment analysis, roots colonized by PsJN and Bv commonly induce the down-regulation of JA-related genes: JAZ2, JAZ9, JAZ10 and bHLH148 (Fig. 7a). Additionally, LOX6 and RERJ1 are specifically down-regulated by PsJN.f i Finally, the regulation of defense-related genes probably reveals the most striking differences in the local response to the three endophytes (Fig. 7b). Indeed, while Pk root colonization triggers only the down-regulation of two WRKY genes, 8 additional genes from this family are down-regulated in response to both PsJN and Bv. Furthermore, 5 genes encoding for putative resistance proteins (NB-ARC) are among the most down-regulated genes following PsJN and Bv colonization specifically. On the other hand, the response of rice to Pk is character- ized by the down-regulation of 15 Pathogenesis-Related (PRs) genes. Although, PsJN colonization also induces the down-regulation of chitinase-encoding genes (PR3 family), the regulation of genes from the PR10, PR12 and PR13 families is specific to the response to Pk (Fig. 7b). Discussion Furthermore, a substantial proportion of the genes commonly down- regulated in leaves are implicated in DNA conformation and the RNA polII machinery implicated in gene silenc- ing (Fig. 5, Supplementary Table S9). This supports the fact that chromatin compaction and post-transcriptional regulations are also implicated in the common response of plants to endophytes as previously ­described37. In roots, two genes encoding for ACC oxidase are commonly up-regulated, this enzyme is the last one impli- cated in the synthesis of ­ethylene38. This was also observed in the roots of PsJN-treated Arabidopsis ­plants4. This could be related to the fact that all strains harbor the gene encoding for ACC deaminase which is a well-known plant growth promoting trait. Indeed, this enzyme is able to cleave ACC, an ethylene precursor, and therefore lowers plants stress response and maintains ­growth39. Although ACC deaminase has a much lower affinity towards ACC than ­ACO40, here the microbial population is very high. Therefore, the up-regulation of two ACO- encoding genes could be a way for rice roots to compensate the accumulation of microbial ACC deaminase which possibly led to the down-regulation of the ERF genes (Fig. 7a, Supplementary Table S11). The common response to three endophytes in roots is also characterized by the regulation of genes implicated in signaling, especially membrane receptors such as the gene SHR5 of which an ortholog in sugarcane was shown to be down-regulated in response to endophytic ­colonization41. Also, other putative protein kinase such as WAK21, Os03g0643250, Os07g0251900, Os07g0668500, Os09g0551251, Os04g0633900 as commonly regulated by the three endophytes and could therefore be promising targets for functional assays. The strain‑specific transcriptomic regulations are characterized by pool of genes implicated in secondary metabolism. Following the analysis of the common transcriptomic response of rice to three Burkholderia s.l. endophytes, we performed enrichment analysis to identify physiological processes differen- tially regulated in response to the three strains. Among the pool of genes responsive to one or two strains, most important enriched terms are related to metabolism as describe earlier but also secondary metabolism as revealed by the KEGG pathways enrichment analysis in both roots and leaves. Similarly, strain-dependent regulation of secondary metabolism were previously described in Brassica oleracea in response Paraburkholderia ­species42 and rice in response to Azospirillum ­strains43. Particularly, while all strain triggered the up-regulation of 3 genes related to lignin synthesis, Pk inoculation specifically up-regulates additional genes related to lignin and flavonoid synthesis (Fig. Discussion Also, a number of genes related to metal ion transport are up-regulated in response to PsJN colonization similarly to what was observed in response to Herbasprillum seropedicae33. Furthermore, the rice gene Os10g0195250 was commonly induced by Azospirillum lipoferum and Azospirillum sp. B510 ­inoculation13 and iron ­deficiency34. We could show by qPCR that this gene and IRO2 were particularly highly over-expressed at later time points of the kinetic analysis while being induced as well at 6 hpi. This suggests that iron deficiency response is triggered in roots colonized by diverse rhizobacteria.h y The most striking observation following the comparative analysis of rice transcriptome in response to PsJN, Pk and Bv is that a more conserved transcriptional response is triggered in leaves than in roots (Fig. 4). As relatively few studies analyzed the response of both roots and leaves in response to endophytic colonization, the representativity of this observation must be tempered. We can hypothesize that regulations occurring in roots are more specific in roots to trigger the appropriate response to each strain while transcriptomic regulations occurring in leaves are more related to metabolism and therefore more shared. The enrichment analysis per- formed on leaves transcriptomic data supports this hypothesis. Indeed, a large proportion of enriched terms are related to primary metabolism (Fig. 4a, Supplementary Tables S5 and S6). However, different energy-producing pathways appear to be triggered by Bv and PsJN on one side and Pk on the other. Indeed, while Pk inoculated plants up-regulate genes implicated in respiration, the shared response to Bv and PsJN is enriched in genes related to photosynthesis and carbon fixation. This can be one of the factors explaining the increased root biomass in response to PsJN and Bv (Fig. 3e). However, the up-regulation of genes implicated to photosynthesis, primary metabolism and development are also part of the core transcriptomic response of rice to the three endophytic strains (Fig. 5, Supplementary Table S9). Also, a significant amount of genes regulated in leaves are related to immunity such as WRKY735, WRKY2830 and the gene MKK4, which encodes for a MAPK kinase implicated in rice ­immunity36, is also com- monly up-regulated in the leaves of inoculated plants. In addition, the fact that 9 putative resistance genes and 24 putative protein kinases are transcriptionally regulated may result in a reduced or enhanced susceptibility to leaf pathogens (Supplementary Table S9). Discussion S1a) and previous observations particularly for grapevine ­plantlets21,22.h The analysis of the phenotypic response of rice plants to the inoculation with PsJN, Pk and Bv revealed no significant increase of the aboveground biomass contrarily to what was previously described in response to Pk and ­Bv15,23. This can be related to the growth conditions we used for the phenotypic assay especially in terms of nutrients available in the substrate. However, we measured a significant increase of the number of grains pro- duced by plants inoculated with Pk as previously ­described15. Oppositely, in our experiment plants colonized by Bv produce grains of a lower average weight. This negative impact of Bv colonization on grain weight can be related to the more invasive colonization patterns we previously described in hydroponic ­conditions14. This could be related to a reduced fitness, indeed grain size is correlated with higher germination rate and seedling vigour in ­rice24. Nonetheless, the inoculation of Bv induced a significant increase of rice root biomass. This is also the case for plants inoculated with PsJN who show an increased root biomass and a grain production like https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ the uninoculated plants although PsJN-treated plants had less tillers (Fig. 3). In conclusion, these results sug- gest that in our growth conditions, the endophytic colonization of PsJN, Pk and Bv respectively have a neutral, beneficial and negative impact on rice fitness. Comparative rice transcriptomic response to PsJN and rice‑native Burkholderia s.l. reveals a common response enriched in defense, hormonal and signaling regulation. The analysis of rice Comparative rice transcriptomic response to PsJN and rice‑native Burkholderia s.l. reveals a common response enriched in defense, hormonal and signaling regulation. The analysis of rice transcriptomic response to PsJN revealed an important enrichment in genes related to photosynthesis in leaves (Supplementary Fig. S2, Supplementary Table S4). Coherently, PsJN inoculation can induce an increase photo- synthetic efficiency in Arabidopsis25 and ­switchgrass26. Also, genes related to development and reproduction are significantly enriched among the DEGs in rice leaves following PsJN inoculation similarly to what was observed in Arabidopsis5. In roots, PsJN induced the regulation of genes related to hormones previously described to be modulated during root colonization by rhizobacteria namely ­cytokinin13 and jasmonic ­acid27,28. We confirmed these RNASeq results by qPCR analyses demonstrating the maintained and transient down-regulation expres- sion of JA-responsive genes such as bHLH14829, WRKY2830,31 and JAZ1032 respectively. Defense‑related transcriptional regulations triggered in rice by PsJN endophytic colonization re atypical from the response to the rice‑native Pk and Bv endophytes.  To colonize rice roots are atypical from the response to the rice‑native Pk and Bv endophytes. To colonize rice roots, bacterial cells need to evade plant immunity. Indeed, the perception of conserved microbe-associated molecular patterns (MAMPs) by plant cells usually leads to the activation of MAMPs-triggered immunity (MTI)51. This basal immune response is mainly characterized by the production of Pathogenesis-Related (PR) proteins during microbial colonization of plant tissues. Beneficial and pathogenic microbes have evolved several mechanisms to avoid or suppress this ­response52,53. Therefore, all three endophytes used in this study must dynamically suppress rice root immunity to colonize the tissues. We could show by qPCR (Fig. 2b) that PBZ1 and ALD154 are down- regulated at 7 dpi by PsJN while being respectively up-regulated at 6 hpi and 14 dpi. Interestingly, important variation of expression of PBZ1 and SA-related genes, between different time points post inoculation, were pre- viously described in rice roots inoculated with Pseudomonas putida ­RRF355. Additionally, the functional analysis of the RNA-Seq data revealed that most of the defense-related genes are down-regulated in response to the three Burkholderia s.l. endophytes (Fig. 7b). Interestingly, by comparing the response to three endophytes, two pat- terns emerge. On one hand, the inoculation of Pk induced the down-regulation of genes from a number of PR family. On the other hand, PsJN and Bv induced the down-regulation of several WRKY genes, NB-ARC genes and also induced the down-regulation of several JA-related genes (Fig. 7a). Interestingly, several examples of beneficial microbes suppressing root immunity depending on JA signaling components have been ­described56. For example, JA-deficient rice plants were more susceptible to the colonization by the bacterial endophyte Azo- arcus olearius ­BH7257. Consequently, the analysis of the role of JA in the establishment of the interaction between the three Burkholderia s.l. endophytes and rice could be very interesting to investigate. Regarding these results we could hypothesize that the Pk-induced suppression of root immunity in a JA-independent manner in oppo- sition to the response to PsJN and Bv. The similarities between the responses to PsJN and Bv suggest that the belonging to the genus Paraburkholderia or Burkholderia s.s. or the plant of isolation does not drive the mecha- nism by which the host immune response is suppressed. The characterization of the transcriptomic response of rice to a wider diversity of Burkholderia s.l. would need to be performed to further deepen this hypothesis. Defense‑related transcriptional regulations triggered in rice by PsJN endophytic colonization re atypical from the response to the rice‑native Pk and Bv endophytes.  To colonize rice roots In addition, investigating the role of bacterial proteins and metabolites on the response of the host plant would be particularly interesting. Indeed, we have previously identified several pathways such as Entner-Doudoroff pathway, synthesis of Vitamin B12 and the c-di-GMP signalling to be differentially involved in the colonization of rice roots by Pk and Bv 58. We also identified the putrescine synthesis pathway to be differentially transcrip- tionally regulated between rice isolated Burkholderia s.s. strains in response to rice root ­exudates59. It would be particularly interesting to investigate the response of rice plants to mutant strains lacking such component to relate bacterial functions to host response. Discussion 6, Supplementary Table S10). Among the latest, FNSII and CYP75B4, are genes up-regulated in the leaves of Pk-inoculated plants, implicated in the polymerization of lignin in grasses through tricin ­biosynthesis44,45. Additionally, genes implicated in anthocyanin synthesis such as CHI and F3’H46 are- https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ upregulated in leaves following Pk colonization. This may induce an increased tolerance of plants colonized by Pk to leaf pathogens. In roots, both Pk and Bv induced the down-regulation of a number of genes encoding for peroxidases impli- cated in lignin synthesis (Fig. 6a). The regulation of such genes in response to the colonization by endophytes has been described in several ­studies47. Additionally, two genes encoding for Cinnamoyl-CoA reductase, namely CCR1 and CCR10, are down-regulated in roots colonized by each strain (Fig. 6a). Interestingly, CCR1 participates in root immunity through lignin ­synthesis48. Therefore, we can hypothesize that this down-regulation of genes implicated in lignin synthesis in roots is related to suppression of root immunity to accommodate bacterial endophytes. Oppositely, the fact that plants colonized by endophytic fungi trigger the up-regulation of genes implicated in lignin synthesis led to propose that it was a local response to infection to fungal hyphae whether pathogenic or ­not49,50. www.nature.com/scientificreports/ to replace the DsRed fragment with the gusA gene which was directionally inserted using a T4 DNA ligase (Pro- mega). Both plasmids contain a chloramphenicol resistance gene as well as the DsRed or gusA genes under the control of a constitutive TAC promoter. After 24 h of incubation on selective medium LBm Cm (200 μg ­mL−1) at 28 °C, the most fluorescent colonies were selected (for DsRed constructs). For GusA constructs, potential trans- formants were plated on LBm plates supplemented with 60 µg ­mL−1 X-Gluc and 200 μg ­mL−1 chloramphenicol. Colonies showing deep blue coloration after 24 h of incubation at 28 °C were selected for further procedures. Rice root colonization assays. The roots of PsJN::pIN29-inoculated plants grown in hydroponics were harvested at 1, 7 and 14 dpi. For global colonization, the roots were weighted and grinded in sterile water with a sterile ceramic bead using a FastPrep-24™ 5G at 6 m ­s−1 for 40 s. The solution was then diluted and inoculated on low salt LB (Sigma-Aldrich) selective medium containing 200 μg ­mL−1 of chloramphenicol and incubated at 28 °C for 24 h. Colony forming units (cfu) were then enumerated. In order to measure the size of the endo- phytic population, we followed the protocol described in King et al.14. The inoculated rice roots were surface- disinfected for 1 min using a solution of 1% Chloramine T (Sigma-Aldrich) supplemented with 0.1% Tween 20 and finally rinsed 4 times with sterile water. Controls of disinfection were performed by plating rinsing water on TSA medium (Sigma-Aldrich) overnight. Surface-disinfected roots were then treated as described above. The solution was then diluted and inoculated on low salt LB selective medium containing 200 μg ­mL−1 of chloram- phenicol and incubated at 28 °C for 24 h. Visualization of root‑colonizing bacteria. All microscopic observations of the bacterial colonization were restricted to the primary root to compare the colonization patterns on roots which have been in contact with the bacterial population for the same amount of time. Primary roots were harvested at 7 dpi and mounted between slide and slips cover and directly examined with an Epifluorescence Nikon Eclipse Ni-E microscope. For detection of endophytical behavior, primary roots were harvested at 14 dpi and included in 5.5% agar blocks. www.nature.com/scientificreports/ Cross sections of 90 µm thickness were obtained using a Microm HM 650 V vibrating blade microtome (Thermo Scientific), mounted between slide and cover slip and directly observed with a LSM 880 upright confocal mul- tiphoton microscope (Zeiss). Histochemical localization of β‐glucuronidase activity was performed by vacuum- infiltrating a GUS staining solution containing 50 µg ­mL−1 X-Gluc (Duchefa Biochemie) for 20 min in mock and PsJN::pINGUS-treated root systems in separate containers. The containers were then incubated overnight at 37 °C, then upon blue coloration visualization, roots systems were rinsed three times using a sterile phosphate buffer solution (AMRESCO). Roots were then visualized using a Nikon SMZ1500 stereomicroscope. RNA extraction. For the analysis of roots and leaves transcriptional profiles, plants’ tissues were harvested in triplicate at 7 dpi. Roots and leaves of untreated plants were collected at the same time. Each biological rep- licate consisted of five pooled root system, or 5 pooled last mature leaves harvested from a single hydroponic system. After harvest, samples were snap-frozen in liquid nitrogen and stored at − 80 °C. t Rice roots were homogenized in liquid nitrogen using cooled mortar and pestles. Rice leaves were grinded using a TissueLyser II (Retsch) set to 30 Hz for 30 s. Total RNA extraction using TRI-reagent (Sigma) was per- formed according to manufacturer’s instructions. All samples were treated with DNase I (Ambion) and purified using the RNA Clean & Concentrator kit (Zymo) according to manufacturer’s instructions. The integrity and quality of the total RNA was confirmed using a Nanodrop™ 1000 spectrophotometer (ThermoFisher) and a 2100 BioAnalyzer (Agilent). RNA sequencing and mapping of reads. Quality of RNA was checked by determining the RNA Integ- rity Number (RIN) with a Fragment Analyzer (Agilent). For the library preparation, samples with a RIN value > 6 were used. 12 RNA libraries were prepared using an Illumina TruSeq stranded mRNA sample preparation kit by MGX-Montpellier GenomiX core facility (MGX) France (https://​www.​mgx.​cnrs.​fr/). Library construction, sequencing and quality assessment was performed as described in King et al.14 on an Illumina HiSeq 2500. RNA-Seq reads were aligned to the IRGSP 1.0 version of the rice genome using HISAT2 v2.0.5.162. The number of reads mapped to each gene locus was counted using HTSEq-count v0.6.063. Differential gene expression and gene ontology (GO) term enrichment analysis. DESeq2 v3.764 was used to calculate differential gene expression between non-inoculated and inoculated conditions. Methods l d Plants and bacterial cultivation. Oryza sativa L. ssp. japonica cv Nipponbare was used in this study. For all experiments, seeds were dehusked and sterilized as previously ­described14. Briefly, seeds were sterilized with 70% EtOH and 9.6% NaClO supplemented with 1% Tween 20, then rinsed with sterile distilled water and a 2% thiosulfate solution. Sterilized seeds germinated in sterile distilled water for 24 h and on ­H2O agar plate for 30 h at 28 °C. Homogeneously germinated seeds were finally transferred to sterile magenta boxes (SPL Lifesciences Co. Ltd) holding autoclaved perlite and 200 mL of sterile hydroponic medium. Plants were grown in a growth chamber (16 h light; 8 h dark; 28 °C; 70% humidity).i g y Paraburkholderia phytofirmans ­PsJNT was cultured as follows: Glycerol stocks (20%) of bacterial cells con- served at − 80 °C were plated on low salt LB (Sigma-Aldrich) agar plates and incubated for 72 h at 28 °C. 20 mL Liquid LB low salt medium were then inoculated 16 h under agitation (180 rpm) at 28 °C. 500 µL of overnight cul- ture were inoculated in fresh liquid medium for 2 h. Bacterial cells were then centrifuged for 5 min at 4000 rpm and resuspended in sterile distilled water. Each plantlet was inoculated with ­107 bacterial cells 4 days after sowing in hydroponic system. For greenhouse experiments (28 °C, 60% humidity), homogeneously germinated seeds were inoculated with ­107 bacterial cells when transferred in round small pots (Ø = 5 cm, h = 7 cm). At 15 dpi, homogeneously grown plantlets were transferred to 12 × 12 × 18.7 cm pots (1 per pot). The substrate used was the GO M2 growing media (Jiffy). Bacterial transformation. Paraburkholderia phytofirmans ­PsJNT cells were transformed by electropora- tion with the pIN29 ­plasmid60 and its derivative pINGUS (Supplementary Fig. 3). The latest was obtained by replacing the gene encoding for the DsRed fluorescent protein by the gusA gene from the GusSH ­vector61 which encodes for a β‐glucuronidase. The primers used to amplify the gusA gene were: Fwd:5’GAC​CGC​AAG​ATC​TCT​ CCT​CTAT-3’; Rev:5’CGA​GCG​GCC​GCT​ATC​GAA​TTAA-3’). A BglII + NotI double digestion (NEB) was used https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ All genes having an adjusted p-value inferior to 0.01 were considered as significantly differentially expressed. All func- tional enrichment analysis was performed using g:Profiler (version e95_eg42_p13_f6e58b9) with g:SCS multiple testing correction method applying significance threshold of 0.0165,66. To avoid redundancy between GO terms, we used ­ReviGO67 to reduce the number of enriched GO terms as well as replacing terms related to the exact same DEGs lists by the most representative and understandable semantic expression. Reverse transcription and qPCR. cDNA was synthesized from total RNA using Oligo dT (Invitrogen) and the SuperScript III Reverse Transcriptase (Invitrogen) according to manufacturer instructions. Gene expres- sion measurements were performed using Takyon SYBR 2X qPCR Mastermix Blue (Eurogentec). The gene EF-1α was used as a reference gene to normalize expression results using the ΔΔCt ­method68 with non-inoculated sam- ples as experimental controls. Primers used in this study can be found in Supplementary Table S12. https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ Ethical approval. The seeds and the plant materials of Oryza sativa L. ssp. japonica cv Nipponbare were obtained from the International Rice Research Institute (IRRI) and propagated in our laboratory. All plant experiments involved in this study were carried out in accordance with relevant regulations and guidelines. References & Poupin, M. J. Burkholderia phytofirmans PsJN induces long-term metabolic and transcriptional changes involved in Arabidopsis thaliana salt tolerance. Front. Plant Sci. 6, 466 (2015).i p 8. Pinedo, I., Ledger, T., Greve, M. & Poupin, M. J. Burkholderia phytofirmans PsJN induces long-term metabolic and transcriptiona changes involved in Arabidopsis thaliana salt tolerance. Front. Plant Sci. 6, 466 (2015).i 9. Su, F. et al. Burkholderia phytofirmans PsJN reduces impact of freezing temperatures on photosynthesis in Arabidopsis thaliana Front. Plant Sci. 6, 810 (2015). 9. Su, F. et al. Burkholderia phytofirmans PsJN reduces impact of freezing temperatures on photosynthesis in Arabidopsis thaliana. Front. Plant Sci. 6, 810 (2015). 10 G i il S l C i i i f i l i f i bi l l i i P N l 0. Guimil, S. et al. Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization. Proc. Natl Acad. Sci. 102, 8066–8070 (2005). 1. Giovannetti, M., Mari, A., Novero, M. & Bonfante, P. Early Lotus japonicus root transcriptomic responses to symbiotic and patho- genic fungal exudates. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2015.​00480 (2015). g g p g p ( ) 12. Kelly, S., Mun, T., Stougaard, J., Ben, C. & Andersen, S. U. Distinct Lotus japonicus transcriptomic responses to a spectrum of bacteria ranging from symbiotic to pathogenic. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2018.​01218 (2018). g g g 12. Kelly, S., Mun, T., Stougaard, J., Ben, C. & Andersen, S. U. Distinct Lotus japonicus transcriptomic responses to a spectr b t i i f bi ti t th i F t Pl t S i htt //d i /10 3389/f l 2018 01218 (2018) 12. Kelly, S., Mun, T., Stougaard, J., Ben, C. & Andersen, S. U. Distinct Lotus japonicus transcriptomic responses to a spectrum of bacteria ranging from symbiotic to pathogenic. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2018.​01218 (2018).i bacteria ranging from symbiotic to pathogenic. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2018.​01218 (2018).i g g y g g 3. Drogue, B. et al. Plant root transcriptome profiling reveals a strain-dependent response during Azospirillum-rice cooperation Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2014.​00607 (2014).i 13. Drogue, B. et al. Plant root transcriptome profiling reveals a strain-dependent response du Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2014.​00607 (2014). 14. King, E. et al. Monitoring of rice transcriptional responses to contrasted colonizing patterns of phytobeneficial Burkholderia s.l. reveals a temporal shift in JA systemic response. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2019.​01141 (2019). pt y p p g p 15. References Mattos, K. A. et al. Endophytic colonization of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth. An. Acad. Bras. Ciênc. 80, 477–493 (2008).h y to enhance plant growth. An. Acad. Bras. Ciênc. 80, 477–493 (200 y p g 6. Compant, S., Clément, C. & Sessitsch, A. Plant growth-promoting bacteria in the rhizo- and endosphere of plants: Their role colonization, mechanisms involved and prospects for utilization. Soil Biol. Biochem. 42, 669–678 (2010).i 17. Compant, S. et al. Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: From the rhizosphere to inflorescence tissues. FEMS Microbiol. Ecol. 63, 84–93 (2008).l l 18. Gamalero, E. et al. Colonization pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron microscopy. FEMS Microbiol. Ecol. 48, 79–87 (2004). p gl y yl g py 9. Knee, E. M. et al. Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source. Mol. Plant Microb Interact. 14, 775–784 (2001).h 20. Kumpf, R. P. & Nowack, M. K. The root cap: A short story of life and death. J. Exp. Bot. 66, 5651–5662 (2015). h y p 1. Compant, S. et al. Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN Appl. Environ. Microbiol. 71, 1685–1693 (2005). d l b l d h l d d b h l ( ) pp . Kandel, S., Joubert, P. & Doty, S. Bacterial endophyte colonizati l d l pp 22. Kandel, S., Joubert, P. & Doty, S. Bacterial endophyte colonization and distribution within plants. Microorganisms 5, 77 (2017). l d l d b fi l ff f l h f terial endophyte colonization and distribution within plants. Microorganisms 5, 77 (2017).if 23. Trân Van, V., Berge, O., Ngô Kê, S., Balandreau, J. & Heulin, T. Repeated beneficial effects of rice inoculation with a strain of Bur- kholderia vietnamiensis on early and late yield components in low fertility sulphate acid soils of Vietnam. Plant Soil 218, 273–284 (2000).f 4. Roy, S. K. S., Hamid, A., Miah, M. G. & Hashem, A. Seed size variation and its effects on germination and seedling vigour in rice J. Agron. Crop Sci. 176, 79–82 (1996). g p ( ) 25. Su, F. et al. References 1. Backer, R. et al. Plant growth-promoting rhizobacteria: Context, mechanisms of action, and roadmap to commercialization o biostimulants for sustainable agriculture. Front. Plant Sci. 9, 1473 (2018).i g 2. Mitter, B. et al. Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants. Front. Plant Sci. https://doi.org/10.3389/fpls.2013.00120 (2013). g 2. Mitter, B. et al. Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants. Front. Plant Sci. https://doi.org/10.3389/fpls.2013.00120 (2013). g 2. Mitter, B. et al. Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2013.​00120 (2013).i g p p g p ( ) 3. Esmaeel, Q. et al. Paraburkholderia phytofirmans PsJN-plants interaction: From perception to the induced mechanisms. Front Microbiol. 9, 2093 (2018). 4. Poupin, M. J., Greve, M., Carmona, V. & Pinedo, I. A complex molecular interplay of auxin and ethylene signaling pathways is involved in Arabidopsis growth promotion by Burkholderia phytofirmans PsJN. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2016 00492 (2016).f 5. Poupin, M. J., Timmermann, T., Vega, A., Zuñiga, A. & González, B. Effects of the plant growth-promoting bacterium Burkholderia phytofirmans PsJN throughout the life cycle of Arabidopsis thaliana. PLoS ONE 8, e69435 (2013).f p y fi g y p 6. Su, F. et al. Different Arabidopsis thaliana photosynthetic and defense responses to hemibiotroph distal inoculation of Burkholderia phytofirmans. Photosynth. Res. 134, 201–214 (2017). p y fi g y p 6. Su, F. et al. Different Arabidopsis thaliana photosynthetic and defense responses to hemibiotrophic pathogen induced by local or distal inoculation of Burkholderia phytofirmans. Photosynth. Res. 134, 201–214 (2017). i 6. Su, F. et al. Different Arabidopsis thaliana photosynthetic and defense responses to hemibiotrophic pathogen induced by loc di t l i l ti f B kh ld i h t fi Ph t th R 134 201 214 (2017) 6. Su, F. et al. Different Arabidopsis thaliana photosynthetic and defense responses to hemibiotrophic pathogen induced by local or distal inoculation of Burkholderia phytofirmans. Photosynth. Res. 134, 201–214 (2017).i p y fi y 7. Timmermann, T. et al. Gene networks underlying the early regulation of Paraburkholderia phytofirmans PsJN induced systemic resistance in Arabidopsis. PLoS ONE 14, e0221358 (2019).i resistance in Arabidopsis. PLoS ONE 14, e0221358 (2019). 8. Pinedo, I., Ledger, T., Greve, M. y These sequence data for this study can be found in the European Nucleotide Archive (ENA) database under accession number PRJEB31936: https://​www.​ebi.​ac.​uk/​ena/​brows​er/​view/​PRJEB​31936. y These sequence data for this study can be found in the European Nucleotide Archive (ENA) database under accession number PRJEB31936: https://​www.​ebi.​ac.​uk/​ena/​brows​er/​view/​PRJEB​31936. Received: 10 February 2023; Accepted: 20 June 2023 www.nature.com/scientificreports/ & Vilo, J. g:Profiler: A web-based toolset for functional profiling of gene lists from large-scale experiments. Nucleic Acids Res. 35, W193–W200 (2007).i g p 66. Raudvere, U. et al. g:Profiler: A web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. https://​doi.​org/​10.​1093/​nar/​gkz369 (2019). p g g ( ) 67. Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE 6, e21800 (2011). f ffl h l d l f l fi l l d ( ) ( ) 8. Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 45e–445 (2001). www.nature.com/scientificreports/ Perception of pathogenic or beneficial bacteria and their evasion of host immu the frontline. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2015.​00219 (2015). 53. Trdà, L. et al. Perception of pathogenic or beneficial bacteria and their evasion of host immunity: Pattern recognition receptors in the frontline. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2015.​00219 (2015). 54. Jung, G. Y. et al. A rice gene homologous to Arabidopsis AGD2-LIKE DEFENSE1 participates in disease resistance response against infection with Magnaporthe oryzae. Plant Pathol. J. 32, 357–362 (2016). infection with Magnaporthe oryzae. Plant Pathol. J. 32, 357–362 g p y 5. Kandaswamy, R., Ramasamy, M. K., Palanivel, R. & Balasundaram, U. Impact of Pseudomonas putida RRF3 on the root transcrip- tome of rice plants: Insights into defense response, secondary metabolism and root exudation. J. Biosci. 44, 98 (2019).i 6. Yu, K., Pieterse, C. M. J., Bakker, P. A. H. M. & Berendsen, R. L. Beneficial microbes going underground of root immunity. Plan Cell Environ. https://​doi.​org/​10.​1111/​pce.​13632 (2019). 57. Chen, X., Marszałkowska, M. & Reinhold-Hurek, B. Jasmonic Acid, not salicyclic acid restricts endophytic root colonization of rice. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2019.​01758 (2020).f 58. Wallner, A. et al. Differential genetic strategies of Burkholderia vietnamiensis and Paraburkholderia kururiensis for root coloniza- tion of Oryza sativa subsp. japonica and O. sativa subsp. indica, as revealed by transposon mutagenesis sequencing. Appl. Environ. Microbiol. https://​doi.​org/​10.​1128/​aem.​00642-​22 (2022). g 59. Wallner, A. et al. Comparative genomics and transcriptomic response to root exudates of six rice root-associated Burkhold sensu lato species. Peer Commun. J. 3, e25 (2023). p 60. Vergunst, A. C., Meijer, A. H., Renshaw, S. A. & O’Callaghan, D. Burkholderia cenocepacia creates an intramacrophage replica- tion niche in zebrafish embryos, followed by bacterial dissemination and establishment of systemic infection. Infect. Immun. 78, 1495–1508 (2010). ( ) 61. Pasquali, G., Ouwerkerk, P. B. F. & Memelink, J. Versatile transformation vectors to assay the promoter activity of DNA elements in plants. Gene 149, 373–374 (1994). p 2. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015). ( ) 63. Anders, S., Pyl, P. T. & Huber, W. HTSeq: A Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015). 4. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).ii 5. Reimand, J., Kull, M., Peterson, H., Hansen, J. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 34. Wairich, A. et al. The combined strategy for iron uptake is not exclusive to domesticated rice (Oryza sativa). Sci. Rep. 9, 16144 (2019). 34. Wairich, A. et al. The combined strategy for iron uptake is not exclusive to domesticated rice (Oryza sativa). Sci. Rep. 9, 16144 (2019). 35 Li Q t l O WRKY67 iti l l t bl t d b t i bli ht i t b di t ti ti f PR i i BMC 35. Liu, Q. et al. OsWRKY67 positively regulates blast and bacteria blight resistance by direct activation of PR genes in rice. B Plant Biol. 18, 257 (2018).l ( ) 36. Kishi-Kaboshi, M. et al. A rice fungal MAMP-responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis. Plant J. 63, 599–612 (2010).h 37. Carvalho, T. L. G., Ballesteros, H. G. F., Thiebaut, F., Ferreira, P. C. G. & Hemerly, A. S. Nice to meet you: Genetic, epigenetic and metabolic controls of plant perception of beneficial associative and endophytic diazotrophic bacteria in non-leguminous plants. Plant Mol. Biol. 90, 561–574 (2016). 38. Wang, K.L.-C., Li, H. & Ecker, J. R. Ethylene biosynthesis and 39. Ravanbakhsh, M., Sasidharan, R., Voesenek, L. A. C. J., Kowalchuk, G. A. & Jousset, A. Microbial modulation of plant ethylene signaling: Ecological and evolutionary consequences. Microbiome 6, 52 (2018). Modulation of plant ethylene levels by the bacterial enzyme ACC l S A l l k l d l fi . Glick, B. R. Modulation of plant ethylene levels by the bacterial l l l k l d 40. Glick, B. R. Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase. FEMS Microbiol. Lett. 251, 1–7 (2005). 41. Vinagre, F. et al. SHR5: A novel plant receptor kinase involved in plant–N2-fixing endophytic bacteria association. J. Exp. Bot. 57, 559–569 (2006). 2. Jeon, J.-S. et al. Impact of root-associated strains of three Paraburkholderia species on primary and secondary metabolism o Brassica oleracea. Sci. Rep. 11, 2781 (2021).if p 3. Chamam, A. et al. Plant secondary metabolite profiling evidences strain-dependent effect in the Azospirillum–Oryza sativa asso- ciation. Phytochemistry 87, 65–77 (2013).l y y 4. Lam, P. Y. et al. Disrupting flavone synthase II alters lignin and improves biomass digestibility. Plant Physiol. References Spatio-temporal responses of Arabidopsis leaves in photosynthetic performance and metabolite contents to Burkholderia phytofirmans PsJN. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2016.​00403 (2016). g p 25. Su, F. et al. Spatio-temporal responses of Arabidopsis leaves in photosynthetic performance and met phytofirmans PsJN. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2016.​00403 (2016). i 6. Wang, B., Mei, C. & Seiler, J. R. Early growth promotion and leaf level physiology changes in Burkholderia phytofirmans strain PsJN inoculated switchgrass. Plant Physiol. Biochem. 86, 16–23 (2015).i PsJN inoculated switchgrass. Plant Physiol. Biochem. 86, 16–23 (2i g y 7. Xie, Y. et al. Digital gene expression profiling of the pathogen-resistance mechanism of Oryza sativa 9311 in response to Bacillus amyloliquefaciens FZB42 induction. Biol. Control 110, 89–97 (2017).i i amyloliquefaciens FZB42 induction. Biol. Control 110, 89–97 (201 y q f 8. Rekha, K., Kumar, R. M., Ilango, K., Rex, A. & Usha, B. Transcriptome profiling of rice roots in early response to Bacillus subtilis (RR4) colonization. Botany 96, 749–765 (2018). y 9. Seo, J.-S. et al. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. Plant J. 65, 907–921 (2011). 0. Chujo, T. et al. OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus. Plant Mol. Biol. 82, 23–37 (2013). g 31. Wang, P. et al. OsWRKY28 regulates phosphate and arsenate accumulation, root system architecture and fertility in rice. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2018.​01330 (2018). p g p 2. Uji, Y. et al. Overexpression of OsMYC2 results in the up-regulation of early JA-rresponsive genes and bacterial blight resistance in rice. Plant Cell Physiol. 57, 1814–1827 (2016). y , ( ) 33. Brusamarello-Santos, L. C. C. et al. Modulation of defence and iron homeostasis genes in rice roots by the diazotrophic endophyte Herbaspirillum seropedicae. Sci. Rep. 9, 10573 (2019). Scientific Reports | (2023) 13:10696 | https://doi.org/10.1038/s41598-023-37314-7 www.nature.com/scientificreports/ 174, 972–985 (2017) 5 L P Y Li H & L C C l ti f t i i bi th i th i i C t h P450 75B4 i i h i 44. Lam, P. Y. et al. Disrupting flavone synthase II alters lignin and improves biomass digestibility. Plant Physiol. 174, 972–985 (2017). 45. Lam, P. Y., Liu, H. & Lo, C. Completion of tricin biosynthesis pathway in rice: Cytochrome P450 75B4 is a unique chrysoeriol 5′-hydroxylase. Plant Physiol. 168, 1527–1536 (2015). l 45. Lam, P. Y., Liu, H. & Lo, C. Completion of tricin biosynthesis pathway in rice: Cytochrome P450 75B4 is a unique chrysoeriol 5′ hydroxylase Plant Physiol 168 1527 1536 (2015) l 45. Lam, P. Y., Liu, H. & Lo, C. Completion of tricin biosyn 5. Lam, P. Y., Liu, H. & Lo, C. Completion of tricin biosynthesis pathway in rice: Cytochrome P450 75B4 is a unique chrysoerio 5′-hydroxylase. Plant Physiol. 168, 1527–1536 (2015). 5′-hydroxylase. Plant Physiol. 168, 1527–1536 (2015). 6. Zheng, J. et al. Determining factors, regulation system, and domestication of anthocyanin biosynthesis in rice leaves. New Phytol 223, 705–721 (2019).i 7. Pinski, A., Betekhtin, A., Hupert-Kocurek, K., Mur, L. A. J. & Hasterok, R. Defining the genetic basis of plant-endophytic bacteria interactions. Int. J. Mol. Sci. 20, 1947 (2019).f 8. Kawasaki, T. et al. Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice. Proc. Natl. Acad. Sci. 103, 230–235 (2006). 9. Dupont, P. et al. Fungal endophyte infection of ryegrass reprograms host metabolism and alters development. New Phytol. 208 1227–1240 (2015). 0. Bajaj, R. et al. Transcriptional responses of soybean roots to colonization with the root endophytic fungus Piriformospora indica reveals altered phenylpropanoid and secondary metabolism. Sci. Rep. 8, 10227 (2018). l Th l ( ) reveals altered phenylpropanoid and secondary metabolism. Sci. Rep. 8, 10227 (2018). 51. Jones, J. D. G. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006). y y Jones, J. D. G. & Dangl, J. L. The plant immune system. Nature 444 51. Jones, J. D. G. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006). gh p y 2. Hacquard, S., Spaepen, S., Garrido-Oter, R. & Schulze-Lefert, P. Interplay between innate immunity and the plant microbiota Annu. Rev. Phytopathol. 55, 565–589 (2017).i y p 53. Trdà, L. et al. Acknowledgementsh g The authors gratefully acknowledge financial support from the CGIAR research program (CRP) RICE as well as from the French national research agency (ANR) funding the BURKADAPT project (ANR-19-CE20-0007). We thank Geneviève Conéjéro (PHIV platform, Cirad Lavalette, Montpellier, France) as well as the MRI imaging facility (member of the national infrastructure France-BioImaging, supported by the ANR-10-INBS-04 grant). EK, AW and LG were supported by fellowships from the French Ministry of Higher Education, Research and Innovation. MGX acknowledges financial support from France Génomique National infrastructure, funded as part of “Investissement d’Avenir” programme managed by Agence Nationale pour la Recherche (contract ANR-10-INBS-09). The authors would like to thank Mathilde Hutin for constructive review of the manuscript. https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 | www.nature.com/scientificreports/ Author contributions E K ib d h d i E.K. contributed to the design of the work, acquisition, analysis, interpretation of data and article writing; A.W. and I.R. contributed to data acquisition; H.P. and L.G. contributed to data analysis; A.K. contributed to the design of the work and performed experiments; P.C. and L.M. contributed to the design of the study and article writing. Competing interests h g The authors declare no competing interests. Additional informationh Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​37314-7. Correspondence and requests for materials should be addressed to E.K. or P.C. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2023 https://doi.org/10.1038/s41598-023-37314-7 Scientific Reports | (2023) 13:10696 |
https://openalex.org/W3003938798
https://www.iiste.org/Journals/index.php/EJBM/article/download/51340/53040
English
null
Future Career in Hospitality Industry Based on Internship Experience: A Principal Component Analysis
null
2,020
cc-by
5,965
European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 Abstract The purpose of this research is to investigate the factors associated with internship programs and the relationships between internship experiences in tourism and hospitality industry and aspect of future career in the same industry. A principal component analysis was used to determine the degree of impact of internship program planning, industry’s involvement and students’ self commitment. Effective planning of the internship program played a positive and influential role in deciding future career in tourism and hospitality industry. The extent of industry involvement during the internship period mediated internship students’ confidence in tourism industry. However, students’ self-commitment have found rather low to join the industry. Keywords: Internship program, Tourism and Hospitality industry, Factor Analysis DOI: 10.7176/EJBM/12-3-22 DOI: 10.7176/EJBM/12-3-22 Publication date: January 31st 2020 DOI: 10.7176/EJBM/12-3-22 Publication date: January 31st 2020 DOI: 10.7176/EJBM/12-3-22 Publication date: January 31st 2020 Publication date: January 31st 2020 Future Career in Hospitality Industry Based on Internship Experience: A Principal Component Analysis Samia Afrin Shetu1 Takrima Sayeda2* 1. Department of Tourism & Hospitality Management, University of Dhaka 2. Department of Tourism & Hospitality Management, University of Dhaka 1. Introduction Experience based knowledge interfaces our theoretical understanding with practical learning. When practical implication backs the theoretical knowledge, the learning remains long lasting. More specifically the service sector like tourism and hospitality needs experience-based practices to make its circle fulfilled. Experiential education has an extended and significant journey in tertiary level education; internship is one of them (Beggs, Ross, & Goodwin, 2008). It is termed as “Windows to the Real World” or “Supervised Work Experienced” or “Bridge to the Real World” (Chen & Shen, 2012). All around the world travel and tourism industry has tremendous effect on economic development. According to World travel and tourism Council (WTTC, 2018), travel and tourism’s direct contribution to GDP all over the world was 21.5 (US$bn) and in Bangladesh it was 5.3 (US$bn). Travel and tourism’s direct contribution to employment all over the world average was 937.5 thousand and Bangladesh average was 1178.4 thousand. This advancement shows that, travel and tourism education, curricula development, training, educational courses, and programs are very much needed to produce quality graduates who will be capable to serve this industry (Williams and Buswell, 2003). Hospitality educationalists think that experiential learning component should be included in the curriculum and it help to develop managerial skill (Petrillose & Montgomery, 1998). However the success of such internship program depends on the satisfaction of the students (Chen & Shen, 2012). Ju, Emenheiser, Clayton, and Reynold (1998) discovered that students’ perception about their internship experience had eight dimensions: professional skills development, leadership development, general knowledge improvement, future career marketing, self- actualization, relationship and supervisor, structure of the program and structure of the academic courses. They also indicated two depended variables – students’ commitment to continue to work in the hospitality sector and their satisfaction level for successfully completing the internship. The industry that has worth more than a multi- billion dollar may face huge risk without highly educated professionals (Williams, 1990). The purpose of the study is to understand the factors in internship program, that can encourage inter students to have future career in tourism and hospitality industry. 2. Literature Review (1996), found that for managing a property a manager trainee should have human relation skill, crisis management skill, operational efficiency skill and knowledge of record-keeping and legal regulations. Finally Dopson and Tas (2004) specified that the curriculum of hospitality schools should not only motivate students to learn essential skills for running business but also should have substantial knowledge to manage human resources. Other scholars claim three major components, such as substantive knowledge, skills, and values are needed during developing a curriculum (Dopson and Tas, 2004; Gursoy and Swanger, 2004, 2005). Many students consider internship as a pathway for their future career and the experiences they get from internship may facilitate their desire to work in the hospitality sector (Tse, 2010). During school, internships help to get realistic expectation of perspective career by letting hospitality students experiencing the actual job circumstances (Lam & Ching, 2007). Before participating in the internship, knowing the responsibilities of interns and the intern supervisors can be the two elements that can lead to a successful internship (Beggs, Ross, & Goodwin, 2008; Young & Hurlic, 2007). This experiential learning have several benefits for the student. Lee (2007) has addressed that experiential learning increases the understanding of how organizations function and the ability to view career expectations realistically. Additionally it increases network of professional contacts, increases skill to take initiative and adjust to change, develops leadership skills, and increases financial management skills (Chen, et al., 2009). Some researchers have found that an effective internship program can retain students as their employees, accelerate their passion and lessen their worries about future career (Ju et al., 1998). Collins (2002) surveyed 113 students’ (senior); 76 industry professionals and 6 university participants with three sets of questionnaire. The finding was that students’ may have the opportunity to get employed in the same organization if they successfully completed the internship program after their graduation. Additionally his findings were backed by the data of 62 organizations who hired their interns after the internship program. Not only that, experiences gathered from internship helps to build confidence among the students in career progress (Ko, 2007). Students from hospitality and tourism usually kept higher expectations but in reality their primary expectations did not meet with their actual satisfaction (Dickerson, 2009). 2. Literature Review It has been proved that student expectations are mainly molded by the prior perception of the internship, that are influenced by some of the factors including: academic theories, sources of information, prior industry experience, training by the university and the willingness of the student (Jiang & Tribe, 2009,Singh & Dutta, 2010). Chen et al. (2009) conducted a study on 632 undergraduate students of Taiwan to develop the emotional personnel satisfaction and he used descriptive statistics and factor analysis to analyze the data. He asserted that, demand of the job; emotional support and social support are the prime factors that can satisfy interns and stimulate their intention to stay in the industry. Many studies had been done to identify the motivating factors for studying tourism and hospitality and the major factors identified as are interest in tourism, parental influence, cultural development, travel capability and the hypothesis that a degree produces low level graduates with the guarantee of ‘quick way to the top’ (Chak-keung Wong & Jing Liu, 2010 & Kim & Park, 2013). Wan & Kong (2011) studied on career perception of undergraduate gaming management student of Morocco, where 81 students of game management were surveyed through questionnaire. Descriptive study, two t-test and variance analysis were done to analyze the data. The finding of the study was that, 63% of the respondent said that teachers and gaming members played a great role in choosing their career in this industry. About 74% of the respondent gave their consent to come back to work in the same industry after graduation. Besides, internship programs help them to get a screening of workplaces, reduce the expectation gap, provide career satisfaction and employee retention (Dickerson, 2009). Structured internship might have a great impact on the performances of the students and can increase the retention rate by decreasing the turnover rate (Siegel, Blackwood & Landy, 2010). Internship advisors supervise the students and play the role of a connector among students, employees and schools to handle any matter (Collins, 2002; Lee & Chao, 2008). So their role is undoubtedly important. However, several studies have do find that, bitter internship experiences influence students unwillingness to work in the hospitality sector(Barron & Maxwell, 1993 & Callan, 1997). Zopiaits (2007) conducted a study on three prominent stakeholders of internship program to investigate different issues. The three stakeholders are students, educators and professionals. He conducted the research in “methodological triangulation”. 2. Literature Review Internship is an outstanding prospect to acquire exchangeable skills and the precise knowledge that is required in today’s workstation (Busby, 2003). Though there are many controversies among the scholars, several studies have considered hospitality curriculum and integrated operational and managerial skills are needed for professional success (Baum, 1991 & Dopson and Nelson, 2003). Li and Kivela (1988) conducted a study on industry practitioners, educators and students of USA. He surveyed human resource directors and managers in 40 food service operations and educators and students from 200 institutions. They asserted that, business communication and managerial skills are the most important factors that they should acquire. Whereas some scholars suggested that working knowledge of product or services were significant for success (Kay and Russette, 2000). Okeiyi et 181 European Journal of Business and Management www.iiste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 al.(1994) advocated that interpersonal relations and managerial skills are more vital for success. Burgidge (1994) studied on student’ perception and preparation for success and came up with finding that revolutionize is compulsory in educational intuitions and training programs. Kay and Russette (2000) specified that six skills are required for hospitality-management competency and these are: identification of customer problems; having passion to serve; upholding professional and moral principles; promoting trust; and adapting change. Moreover, Tas et al. (1996), found that for managing a property a manager trainee should have human relation skill, crisis management skill, operational efficiency skill and knowledge of record-keeping and legal regulations. Finally Dopson and Tas (2004) specified that the curriculum of hospitality schools should not only motivate students to learn essential skills for running business but also should have substantial knowledge to manage human resources. Other scholars claim three major components, such as substantive knowledge, skills, and values are needed during developing a curriculum (Dopson and Tas, 2004; Gursoy and Swanger, 2004, 2005). European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 al.(1994) advocated that interpersonal relations and managerial skills are more vital for success. Burgidge (1994) studied on student’ perception and preparation for success and came up with finding that revolutionize is compulsory in educational intuitions and training programs. Kay and Russette (2000) specified that six skills are required for hospitality-management competency and these are: identification of customer problems; having passion to serve; upholding professional and moral principles; promoting trust; and adapting change. Moreover, Tas et al. 3.2 Sampling and Data Collection A convenience sample of intern students was selected from few hospitality institutions in Dhaka City, who take undergraduate or graduate students as their interns for 3 to 6 months duration. Only female inter students were taken into consideration. From 60 copies of questionnaire, a total of 50 copies were completed and returned. Thus the sample size is 50. 2. Literature Review Thus, an unsuccessful internship program can discourage students to enter into the hospitality sector and the effective one can encourage the students to enter and work up to the advancement. European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 www.iiste.org and positive environment. Additionally government policies are also responsible for widening the interns’ expectation gap. Thus internship programs failed to motivate potential employees (interns) to join the hospitality sector after completing the study. Earlier studies have revealed that experienced hospitality students within the profession are much less devoted to the hospitality industry (Teng, 2008). Pressures from the job is considered as one of the vital reasons for this deviation while some believe that the opportunity to express the emotions must be a part of the job (Ashforth& Humphrey, 1993; Morris & Feldman, 1997). Teng(2008) has also found that work- related stress can possibly affect the work performance of employees, regardless of full time employee or internship posts in hospitality sector. Richardson (2008) conducted an online survey on 86 students from hospitality sector of Australia to find out the attitude and perceptions of current undergraduate tourism and hospitality students. He divided the survey in three sections: demographics; career aspirations; and a multidimensional and multi-item attitude scale. The upsetting finding is that, after graduation about 33.7% (more than one-third) of the respondents opined that they are unwilling to work in the tourism and hospitality industry and the “working experience in the industry” is the main reason behind this refusal. Graduate psychological training on students have found that female students have higher stress scores than males in terms of the impact of emotional, financial, and academic stressors. Several studies concluded that gap between expectation and real experience is the prime reason for interns to leave the hospitality industry. When an internship program can’t fulfill the expectations of an intern, he/she might lose interest to work in tourism and hospitality sector after completing his/her graduation (Waryszak, 1999). Likewise, many hospitality students get less fascinated to pick this sector as their first choice to start career after having exposure to the subject and work experience (Jenkins, 2001). Thus, an unsuccessful internship program can discourage students to enter into the hospitality sector and the effective one can encourage the students to enter and work up to the advancement. Data Analysis The collected data were analyzed with statistical package STATA. The demographic information of the participants and their responses to internship questions were summarized using descriptive statistics. The 28 item perception questions were subjected to the factor analysis using principal component method with varimax rotation to group the related items into components. The factor loadings, Eigenvalues, and percent of variances explained by each component were reported. Items that have factor loadings greater than 0.5 were retained for factor grouping. 3.1 Instrument design and development h i i d l d b The questionnaire was developed based on a comprehensive review of the literature. The three-section questionnaire included 12 questions on Internship Program design, 8 questions included Industry Involvement and 8 questions included on Students’ Self Commitment and 5 questions about students’ overall satisfaction about the internship program including and demographic characteristics their age, marital status, timing and duration of their internship. The items were adopted from other research questionnaires assessing job satisfaction, on the job training, industry involvement and overall satisfaction of the students. In the questionnaire students were asked to reveal their perception on a five-pointLikert scale. Statements are rated on a scale from (1) strongly disagree, (2) disagree, (3) neutral, (4) agree, (5) strongly agree. 2. Literature Review His target population was all the students of Cyprus (with at least one internship experience) and to avoid over-representation, he used stratified random sample. He surveyed 150 hospitality professional who are associated with the internship. Finally, he came up with the findings that hospitality educators merely allocate a placement to their interns, overlooking the other issues: hospitality professionals are unable to provide interns meaningful works; no on-the-job supervisor 182 European Journal of Business and Management www.iiste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 and positive environment. Additionally government policies are also responsible for widening the interns’ expectation gap. Thus internship programs failed to motivate potential employees (interns) to join the hospitality sector after completing the study. Earlier studies have revealed that experienced hospitality students within the profession are much less devoted to the hospitality industry (Teng, 2008). Pressures from the job is considered as one of the vital reasons for this deviation while some believe that the opportunity to express the emotions must be a part of the job (Ashforth& Humphrey, 1993; Morris & Feldman, 1997). Teng(2008) has also found that work- related stress can possibly affect the work performance of employees, regardless of full time employee or internship posts in hospitality sector. Richardson (2008) conducted an online survey on 86 students from hospitality sector of Australia to find out the attitude and perceptions of current undergraduate tourism and hospitality students. He divided the survey in three sections: demographics; career aspirations; and a multidimensional and multi-item attitude scale. The upsetting finding is that, after graduation about 33.7% (more than one-third) of the respondents opined that they are unwilling to work in the tourism and hospitality industry and the “working experience in the industry” is the main reason behind this refusal. Graduate psychological training on students have found that female students have higher stress scores than males in terms of the impact of emotional, financial, and academic stressors. Several studies concluded that gap between expectation and real experience is the prime reason for interns to leave the hospitality industry. When an internship program can’t fulfill the expectations of an intern, he/she might lose interest to work in tourism and hospitality sector after completing his/her graduation (Waryszak, 1999). Likewise, many hospitality students get less fascinated to pick this sector as their first choice to start career after having exposure to the subject and work experience (Jenkins, 2001). 4.1 Results and Discussion European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 iste.org its mean. This explains that the respondents perceived internship program planning as an important factor in their internship and it shows that schools play an important role in the internship experience of the respondents. The second highest factor is Industry Involvement with a mean score of 3.91. Based on the previous results, training obtained the highest score among the sub-factors of industry involvement. This shows that the respondents greatly value the training program that they received from their supervisors. A poor training program may affect the quality of their internship. The involvement of the industry itself affects the internship experience since this is where students apply what they have learned in class and where they would be exposed in the corporate world in which they could learn and develop proper skills appropriate for the industry. Lastly, Students‟ Self-Commitment is the least appreciated factor by the respondents with a mean score of 3.22. The results may show that the students are highly dedicated to learn, but it does not greatly influence their internship experience. A factor analysis was utilized to derive the scope of the internship experience to choose hospitality industry as their future profession developed by Chen & Shen (2012). These 28 items were seen as the attributes that would define students’ future job perception based on their internship experience. The determinants were factor-analyzed using a principal component analysis using orthogonal varimax rotation. Only factors with an Eigenvalue of 1 or higher were taken. The factors were identified and named. The factors for future job determination were: internship planning program, industry involvement and students’ self commitment. All the items with factors loading above 0.5 were included. Three components were extracted in order to determine the factors loadings of the three pre determined factors. The total values present the grouping of the factors that received the highest scores. Percentage of variance shows that internship planning program accounts for 26.40% of the variability in all the assessed factors. This can be related to the results of the factor mean scores as shown earlier, where internship planning program received the highest mean score out of three factors. Conversely, industry involvement and students’ self commitment accounts for 20.86% and 9.59% respectively. 4.1 Results and Discussion 4.1 Results and Discussion Descriptive statistics of the respondents are presented in Table 1. A total of 50 copies of questionnaires were collected. The study solely administered on female interns to observe their perceptions regarding the internship program and their future career in tourism and hospitality sector. The major intern periods for the students were during the semester break (40%). Two –third of the internship duration is in between one to four months. Intern students are mainly coming from public universities (72%). The season behind the influx of public university students is that they have full fledged and independent department for tourism and hospitality management. In case of placement of the interns in the hotels, majority of them are concentrated in front office (30%), after that marketing & sales (28%) and then food & beverages (22%). Table 2 presents the mean scores and standard deviation of the three major factors tested in the study. It shows that Internship Program Planning has the highest factor mean score of 4.04. It also has the smallest standard deviation, with a value of 0.79, which means that its individual sub-factor scores are almost concentrated and very close to 183 European Journal of Business and Management www.iiste.org ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 its mean. This explains that the respondents perceived internship program planning as an important factor in their internship and it shows that schools play an important role in the internship experience of the respondents. The second highest factor is Industry Involvement with a mean score of 3.91. Based on the previous results, training obtained the highest score among the sub-factors of industry involvement. This shows that the respondents greatly value the training program that they received from their supervisors. A poor training program may affect the quality of their internship. The involvement of the industry itself affects the internship experience since this is where students apply what they have learned in class and where they would be exposed in the corporate world in which they could learn and develop proper skills appropriate for the industry. Lastly, Students‟ Self-Commitment is the least appreciated factor by the respondents with a mean score of 3.22. The results may show that the students are highly dedicated to learn, but it does not greatly influence their internship experience. 5. Conclusion The findings show that each of the three factors is significantly different to one another and the respondents see at least one of the factors as the most important in their internship experience. For this reason, the internship program planning is the most important factor for the respondents. Since the schools mostly do the program planning, this shows that schools have the most influence in providing a good internship program to their students. The design of the internship program is also essential for the respondents. Holding career consultations helps them to gain ideas where they would want to have their internship. In terms of the industry’s involvement in the internship experience of the respondents, training is important for them. Majority of them are eager to learn more skills and knowledge during their internship training. They believe that rotation opportunities are essential for their training where they can gain more knowledge and skills by working with the different departments of the establishment. Intern students want to have a training program where they can learn important skills that they can use in their chosen industry. Hence, they do not see their internship as stressful, exhausting and a waste of time. It actually strengthens their abilities and improves their values towards the industry. Overall, the respondents were highly satisfied with their internship experience. It leads them to recommend other people to engage in the tourism and hospitality industry after they had their internship. Few of the respondents considered having a career transition. Majority of them decided to pursue a career in the industry after graduation. But, it is still important to note that 18% of the 50 respondents chose not to join the industry after they had their internship. In some way, their internship experience influences them to turn away from the industry. The following limitations were inherent in the study. First, the study was conducted in tourism and hospitality institutions. To overcome this limitation, future research findings should compare students in both hospitality institutions and universities. Second, the population in this research was limited to northern Dhaka city only. Therefore, the results from the study may not be generalized beyond this population. The cross comparison of students’ internship perception might also be undertaken to reveal whether results differ from city to city. Third, it is administered only on female interns. Had there been male students, the results might have different. 4.1 Results and Discussion Similar with the results with factor mean scores, industry involvement obtained second highest score and students’ self commitment received the lowest score. The results show that the three factors explained 56.85% of the overall variance. A reliability test was also conducted in the study and it shows that the obtained Cronbach’s alpha value is 0.9304, which is a relatively high value since a Cronbach’s alpha value of 0.75 is usually the standard minimum requirement to be able to conclude good scale reliability. Therefore, it is concluded and justified that the questionnaire containing 33 scale questions is a reliable instrument in measuring the factors that influence students’ satisfaction with their internship experience and their willingness to stay in the tourism and hospitality industry. 5. Conclusion In addition, 184 European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 European Journal of Business and Management www.iiste.org the relationship of internship experience and employees’ willingness to stay working in the hospitality industry requires more research. References Ashforth, B. E., & Humphrey, R. H. (1993), “Emotional labor in service roles: The influence of identity”, The Academy of Management Review, 18(1), 88-115. Barron, P., & Maxwell, G. (1993), “Hospitality management students’ image of the hospitality industry”, International Journal of Hospitality Management, 5(5), 5-8. Baum, T. (1991), “The US and the UK: comparing expectations of management trainees”, Cornell Hotel & Restaurant Administration Quarterly, Vol. 32 No. 2, pp. 79-84. Beggs, B., Ross, C. M. & Goodwin, B. (2008), “A Comparison of Student and Practitioner Perspectives of the Travel and Tourism Internship”, Journal of Hospitality, Leisure, Sport & Tourism Education, Vol. 7, No. 1. ISSN: 1473-8376. Berta, D. (2003), “Study shows students eager to work in hospitality”, Nation’s Restaurant News, 24th March, 16. Burgidge, D.J. (1994), “Student perception of preparation for success: a view from Europe”, Journal of Hospitality and Tourism Educators, Vol. 6 No. 4, pp. 45-50. ( ) y g p y Burgidge, D.J. (1994), “Student perception of preparation for success: a view from Europe”, Journal of Hospitality and Tourism Educators, Vol. 6 No. 4, pp. 45-50. pp Busby, G. (2003), “Tourism degree internships: a longitudinal study”, Journal of Vocational Education and Training, 55(3), 319-334. g Busby, G. (2005), “Work experience and industrial links”, In: D. Airey, & J. Tribe (Eds.), An International Handbook of Tourism Education (pp. 93-107). Oxford Elsevier. Callan, R. (1997), “Supervised work experience in Europe: a profile of UK undergraduate perc International journal of Contemporary Hospitality Management, 9(1), 35-39. Carey, M.L. and Franklin, J.C. (1991), “Industry output and job growth continues to slow into the next Monthly Labor Review, Vol. 114 No. 11, p. 45. y p Chak-keung Wong, S., & Jing Liu, G. (2010), “Will parental influences affect career choice? Evidence from hospitality and tourism management students in China”, International Journal of Contemporary Hospitality Management, 22(1), 82–102. Chen, et. al, (2009), “Job demand, emotional awareness, and Job satisfaction in internships: the moderating effect of social support”, Social Behavior and Personality, 37(10), 1429-1440 © Society for Personality Research (Inc.) Retrieved from, DOI 10.2224/sbp.2009.37.10.1429 ( ) p Chen, T. & Shen, C. (2012), “Today’s interns tomorrow’s practitioner?-The influence of internship programs on students’ career development in the Hospitality Industry”, Journal of Hospitality, Leisure, Sport & Tourism Education,11 29-40. Christou, E. (2002), “Revisiting competencies for hospitality management: contemporary views of the stakeholders”, Journal of Hospitality and Tourism Education, Vol. 14 No. 1, pp. 25-32. Collins, A. Jenkin, A. K. (2001), “Making a career of it? Hospitality students’ Future perspectives: an Anglo-Dutch study”, References (2009), “Tourism jobs-short lived professions: Student attitudes towards tourism c China”, Journal of Hospitality, Leisure, Sports and Tourism Education (Pre-2012), 8(1), 4. Ju, J., Emenheiser, D. A., Clayton, H. R., & Reynolds, J. (1998), “Korean students’ perceptions of the effectiveness of their internship experiences in the hospitality industry in Korea”, Asia Pacific Journal of Tourism Research, 3(1), 37-44. ( ) and Russette, J. (2000), “Hospitality-management competencies”, Cornell Hotel & Restaurant dministration Quarterly, Vol. 41 No. 2, pp. 52-63. Kim, H. B., & Park, E. J. (2013), “The role of social experience in undergraduates' career perceptions through internships”, Journal of Hospitality, Leisure, Sport & Tourism Education, 12(1), 70–78. Knutson, B.J. and Patton, M.E. (1992), “How prepared am I to succeed in the hospitality industry? What the students are telling us”, Hospitality and Tourism Educator, Vol.4 No.3, pp.38-43. Lam, T., & Ching, L. (2007), “An exploratory study of an internship program: The case of Hong Kong students,” International Journal of Hospitality Management, 26(2), 336-351. Li, L. and Kivela, J.J. (1988), “Different perceptions between hotel managers and students regarding levels of competency demonstrated by hospitality degree graduates”, Australian Journal of Hospitality Management, Vol. 5 No. 2, pp. 47-54. g , pp Morris, J. A., & Feldman, D. C. (1997), “Managing emotions in the workplace”, Journal of Managerial Issues, 9(3), 257-274. Okeiyi, E., Finley, D. and Postel, R.T. (1994), “Food and beverage management competencies: educator, industry, and student perspectives”, Hospitality and Tourism Educator, Vol. 6 No. 4, pp. 37-40. p p p y pp Petrillose, M. J., & Montgomery, T. (1998), “An exploratory study of internship practices in hospitality education and industry’s perceptions of the importance of internships in hospitality curriculum,” Journal of Hospitality and Tourism Education, 9(4), 46-51. Purcell, K. (1993), “Equal opportunities in the hospitality industry: custom and credentials”, International Journal of Hospitality Management, Vol. 12 No. 2, pp. 127-40. Richardson, S. (2008), “Undergraduate tourism and hospitality students’ attitudes toward a career in the industry: a preliminary investigation”, Journal of Business education, 6(1), 5-12. Siegel, P. H., Blackwood, B. J., & Landy, S. D. (2010), “Tax professional internships and subsequent pro performance”, American Journal of Business Education, 3(5), 51-56. p , f , ( ), Singh, A., & Dutta, K. (2010), “Hospitality internship placements: Analysis for United Kingdom and India,” Journal of Services Research, 10(1), 85. f , ( ), Tas, R.F. References B. (2002), “Gateway to the real world, industrial training: dilemmas and problems”, Tourism Management, 23, 93-96. son, J. P. (2009), “The realistic preview may not yield career satisfaction”, International journal Hospitality Management, 28, 297-299. Dopson, L.R. and Nelson, A.A. (2003), “Future of hotel education: required program content areas for graduates of US hospitality programs beyond the year 2000 – Part II”, Journal of Hospitality and Tourism Education, Vol. 15 No. 3, pp. 11-17. pp Dopson, L.R. and Tas, R.F. (2004). “A practical approach to curriculum development: a case study”, Jo Hospitality and Tourism Education, Vol. 16 No. 1, pp. 39-46. p y pp Farkas, D. (1993), “Trained in vain?”, Restaurant Hospitality, Vol. 68. Feiertag, H. (1998), “College students can be bright prospects for prospecting”, Hotel & Motel Management, Vol. 213 No. 11, p. 20. Gursoy, D. and Swanger, N. (2004), “An industry-driven model of hospitality curriculum for programs housed in accredited colleges of business”, Journal of Hospitality and Tourism Education, Vol. 16 No. 4, pp. 13-20. oy, D. and Swanger, N. (2004), “An industry-driven model of hospitality curriculum for programs housed accredited colleges of business”, Journal of Hospitality and Tourism Education, Vol. 16 No. 4, pp. 13-2 Gursoy, D. and Swanger, N. (2004), An industry-driven model of hospitality curriculum for programs housed in accredited colleges of business”, Journal of Hospitality and Tourism Education, Vol. 16 No. 4, pp. 13-20. Gursoy, D. and Swanger, N. (2005), “An industry-driven model of hospitality curriculum for programs housed in accredited colleges of business – Part II”, Journal of Hospitality and Tourism Education, Vol. 17 No. 2, pp. 46-56. Gursoy, D. and Swanger, N. (2005), “An industry-driven model of hospitality curriculum for programs housed in accredited colleges of business – Part II”, Journal of Hospitality and Tourism Education, Vol. 17 No. 2, pp. 46-56. Gursoy, D. and Swanger, N. (2005), “An industry-driven model of hospitality curriculum for programs housed in accredited colleges of business – Part II”, Journal of Hospitality and Tourism Education, Vol. 17 No. 2, pp. 46-56. Jenkin, A. K. (2001), “Making a career of it? Hospitality students’ Future perspectives: an Anglo-Dutch study”, 185 European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 www.iiste.org iste.org Vol.12, No.3, 2020 International journal of Contemporary Hospitality Management, 13(1), 13-20. International journal of Contemporary Hospitality Management, 13(1), 13-20. Jiang, B., & Tribe, J. European Journal of Business and Management ISSN 2222-1905 (Paper) ISSN 2222-2839 (Online) Vol.12, No.3, 2020 References (1988), “Teaching future managers”, Cornell Hotel & Restaurant Administration Quarterly, Vol. 29 No. 2, pp. 41-3. Tas, R.F., LaBrecque, S.V. and Clayton, H.R. (1996), “Property-management competencies for management trainees”, Cornell Hotel & Restaurant Administration Quarterly,Vol.37 No.4, pp. 90-6. Teng, C. C. (2008), “The effects of personality traits and attitudes on student uptake in hospitality employment”, International Journal of Hospitality Management, 27(1), 76-86. Tse, T. S. M. (2010), “What do hospitality students find important about internships?”, Journal of Teaching in Travel & Tourism, 10(3), 251-264. Y. K., P., & Kong, W. H. (2011), “Career perceptions of undergraduate gaming management students Journal of Teaching in Travel & Tourism, 11(4), 367–391. f g ( ) Waryszak, R. Z. (1990), “Student’s expectations from there cooperative education placements in the hospitality industry: an international perspective”, Journal of Business education, 41(1), 33-40. West, A. J., & Jameson, S. M. (1990), “Supervised work experience in graduate employment”, Journal of European Industrial Training, 14(3), 29-32. Williams, A.G. (1990), “So...what's wrong with hospitality education?”, Florida International University Hospitality Review, 8(1), 72-82. Williams, C. and Buswell, J. (2003), “Service Quality in Leisure and Tourism”, Cambridge, MA: CABI Publishing. World Travel & Tourism Council 2018. Retrieved from https://www.wttc.org/economic-impact/country- analysis/country-reports/ on 22th June, 2019 at 9:20 pm. World Travel & Tourism Council 2018. Retrieved from https://www.wttc.org/economic-impact/country- analysis/country-reports/ on 22th June, 2019 at 9:20 pm. y y p p Yıldırım, A. (2002), “Turizm eğitimi [Tourism education]”, Ankara: Aren Ajans. Young, A. M., & Hurlic, D. (2007), “Gender enactment at work: The importance of gender and gender-related behavior to person-organizational fit and career decisions”, Journal of Managerial Psychology, 22(2), 168- 187. Zopiatis, A. (2007), “Hospitality internships in Cyprus: a genuine academic experience or a continuing frustration?,” International journal of Contemporary Hospitality Management, 19(1), 65-77. 186 iste.org Table 1 Description Statistics of the Internship Students (n=50) Variable Frequency Percentage Age 19-24 25-29 30-35 >35 21 17 10 2 42 34 20 4 Internship time After Graduation During Semester Job Semester Break Others 13 10 4 20 3 26 20 8 40 6 Origin of Intern Students Public Universities Private Universities Govt. References Entity 36 11 3 72 22 6 Internship Duration 1-4 months 5-8 months 9-12 months >12 months 33 10 1 5 66 20 2 10 Placement Area Food & Beverage Front Office Marketing & Sales Finance Housekeeping Human Resource Travel Agent Tour Operator 11 15 14 2 4 4 4 3 22 30 28 4 8 8 8 6 Table 2: Factor Mean Scores and Standard Deviation Factors influencing Mean Rank Standard Deviation Internship Program Planning 4.04 1 0.79 Industry Involvement 3.91 2 0.845 Students’ self commitment 3.22 3 0.97 187 www.iiste.org g Table 3: Principal Component Analysis of Factors influencing Internship Students’ Job Selection Component Initial Eigenvalues Rotation sums of squared Loadings Eigenvalues % of Varaince Cumulative % Total % of Variance Cumulative % 1 12.0914 0.4318 0.4318 7.39215 0.2640 0.2640 2 2.69555 0.0963 0.5281 5.84077 0.2086 0.4726 3 2.11669 0.0756 0.6037 2.684 0.0959 0.5685 4 1.46181 0.0522 0.6559 5 1.2851 0.0459 0.7018 6 1.07833 0.0385 0.7403 7 1.02243 0.0365 0.8058 8 0.812209 0.0290 0.8318 9 0.728588 0.0260 0.8557 10 0.667755 0.0238 0.8762 11 0.574304 0.0205 0.8942 12 0.50361 0.0180 0.9100 13 0.444159 0.0159 0.9243 14 0.398862 0.0142 0.9379 15 0.381034 0.0136 0.9482 16 0.289038 0.0103 0.9576 17 0.262624 0.0094 0.9660 18 0.234127 0.0084 0.9576 19 0.203202 0.0073 0.9660 20 0.162909 0.0058 0.9732 21 0.37104 0.0049 0.9790 22 0.103705 0.0037 0.9839 23 0.0777832 0.0028 0.9876 24 0.0731755 0.0026 0.9904 25 0.0700491 0.0025 0.9930 26 0.0544179 0.0019 0.9955 27 0.0479013 0.0017 0.999 28 0.0227345 0.0008 1.0000 Table 3: Principal Component Analysis of Factors influencing Internship Students’ Job Selection 188
https://openalex.org/W4375760840
https://comserva.publikasiindonesia.id/index.php/comserva/article/download/671/855
Indonesian
null
Penerapan Model Pembelajaran Kooperatif Tipe STAD Untuk Meningkatkan Hasil Belajar Prakarya dan Kewirausahaan (Rekayasa) Peserta Didik
COMSERVA
2,022
cc-by-sa
5,449
e-ISSN: 2798-5210 p-ISSN: 2798-5652 Penerapan Model Pembelajaran Kooperatif Tipe STAD Untuk Meningkatkan Hasil Belajar Prakarya dan Kewirausahaan (Rekayasa) Peserta Didik Application of STAD Type Cooperative Learning Model to Improve Student Craft and Entrepreneurship (Engineering) Learning Outcomes Syarifah Maisar MAN Nagan Raya Aceh, Indonesia Syarifah Maisar MAN Nagan Raya Aceh, Indonesia *Email: syarifahmaisar@gmail.com *Correspondence: Syarifah Maisar *Email: syarifahmaisar@gmail.com *Correspondence: Syarifah Maisar DOI: 10.36418/comserva.v2i5.671 Histori Artikel Diajukan : 07-09-2022 Diterima : 18-09-2022 Diterbitkan : 29-09-2022 Kajian Pemeliharaan Bangunan Gedung Bandara Pattimura Kota Ambon ABSTRAK Tujuan penelitian (1) Untuk meningkatkan hasil belajar prakarya dan kewirausahaan (Rekayasa) peserta didik kelas XI IPS 1 MAN Nagan Raya melalui model pemmbelajaran kooperatif tipe STAD (2) Untuk meningkatkan aktivitas guru dan pesera didik kelas XI IPS 1 MAN Nagan Raya dalam proses belajar mengajar dengan menggunakan model pembelajaran kooperatif tipe STAD. Dalam penelitian ini, yang menjadi subjek penelitian adalah seluruh peserta didik Kelas XI IPS 1 MAN Nagan Raya tahun pelajaran 2022 /2023 dengan jumlah Peserta Didik 29 orang peserta didik. Metode pengumpulan data menggunakan tes akhir, tes wawancara, observasi, respon Peserta Didik dan catatan lapangan. Data yang dikumpulkan meliputi data aktivitas guru, aktivitas peserta didik, hasil belajar Peserta Didik dan respon Peserta Didik. Hasil penelitian menunjukkan (1) Hasil belajar Peserta Didik mengalami peningkatan dari siklus pertama dengan nilai rata- rata 67,93% meningkat menjadi 76,20% pada siklus kedua, (2) Aktivitas guru mengalami peningkatan, siklus pertama nilai presentase 79,04% menjadi 90,47% pada siklus kedua sedangkan aktivitas peserta didik mengalami peningkatan, siklus pertama dengan nilai persentase 65,62% menjadi 84,37% pada siklus kedua. Kata Kunci: Hasil Belajar; Kooperatif Tipe STAD; Rekayasa PENDAHULUAN Seiring dengan perkembangan zaman dan teknologi, tuntutan untuk menghasilkan sumber daya manusia yang berkompeten serta mampu bersaing di dunia kerja semakin meningkat (Wijaya et al., 2016). Hal ini tentunya harus diikuti dengan peningkatan mutu pendidikan dengan cara mengoptimalkan segala unsur yang ada di dalamnya (Kholili & Fajaruddin, 2020). Sampai saat ini persoalan pendidikan yang di hadapi bangsa indonesia adalah masih rendahnya kualitas pendidikan (Nurhuda, 2022). Pembelajaran di MAN Nagan Raya yang terjadi saat ini masih menerapkan teori pembelajaran konvensional, dimana pembelajaran ini akan menenggelamkan interaktivitas, daya serap, dan minat peserta didik terhadap materi pembelajaran. Pada periode tingkat MA, idealnya para peserta didik sudah memiliki pola pikir sendiri dalam usaha memecahkan masalah – masalah yang kompleks dan abstrak (Rumawan et al., 2017). Kemampuan berpikir para peserta didik berkembang sedemikian rupa sehingga mereka dengan mudah dapat membayangkan banyak alternatif pemecahan masalah (Pantiwati, 2017). Kapasitas berpikir secara logis dan abstrak mereka berkembang sehingga mereka mampu berpikir multi – dimensi (Afrianti, 2017). Menurut (Wahyuni et al., 2016) para peserta didik tidak lagi menerima informasi apa adanya, tetapi mereka akan memproses informasi itu serta mengadaptasikannya dengan pemikiran mereka sendiri. Sehingga pembelajaran yang menerapkan metode ceramah tidak efektif lagi digunakan untuk peserta didik tingkat MA (Mahmudah, 2016). Demikian juga halnya yang dialami oleh Peserta didik kelas XI IPS 1 MAN Nagan Raya, berdasarkan data awal yang peneliti peroleh selama ini, hasil belajar Peserta didik kelas XI IPS 1 materi sebelumnya belum sesuai dengan yang diharapkan. Rata–rata nilai tes prasiklus adalah 60,4, sementara itu KKM untuk Kompetensi Dasar tersebut yaitu 75. Dari 29 peserta didik hanya 8 orang (27,5%) yang mencapai ketuntasan belajar. Hal ini berati terdapat 21 Peserta didik yang tidak mencapai nilai tuntas (72,4%). Prakarya dan Kewirausahaan ini merupakan mata pelajaran baru yang terdapat pada kurikulum 2013. Tujuan utama dari mata pelajaran ini adalah untuk menjawab persoalan praktis dalam kehidupan manusia termasuk didalamnya kebutuhan komersial atau industry (Rumawan et al., 2017). Karena tergolong pelajaran yang baru, para guru cenderung belum menemukan metode yang cocok digunakan untuk pelajaran ini (Harta et al., 2017). Selama ini guru cenderung memilih menerapkan pendekatan konvensional yaitu metode ceramah sebagai pilihan utama dalam proses pembelajaran. Langkah – langkah pembelajaran atau urutan sajian materi dalam pembelajaran Prakarya dan Kewirausahaan yang biasa dilakukan adalah pembelajaran di awali penjelasan singkat materi oleh guru, peserta didik diajarkan teori, pemberian contoh soal, kemudian diakhiri dengan latihan soal (Fitriani, 2019). ABSTRACT Research objectives (1) To improve the learning outcomes of crafts and entrepreneurship (Engineering) students of class XI IPS 1 MAN Nagan Raya through a STAD type cooperative learning model (2) To increase the activities of teachers and students of class XI IPS 1 MAN Nagan Raya in the teaching and learning process using the STAD type cooperative learning model. In this study, the subject of the study was all students of Class XI IPS 1 MAN Nagan Raya for the 2022/2023 academic year with a total of 29 students. The data collection method uses final tests, interview tests, observations, Learner responses and field notes. The data collected includes data on teacher activities, student activities, student learning outcomes and Student responses. The results showed (1) Student learning outcomes increased from the first cycle with an average score of 67.93% increased to 76.20% in the second cycle, (2) Teacher activity increased, the first cycle percentage value was 79.04% to 90.47% in the second cycle while student activity increased, the first cycle with a percentage value of 65.62% to 84.37% in the second cycle. 515 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Keywords: Learning Outcomes; Cooperative Type STAD; Engineering COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) PENDAHULUAN Dalam pembelajaran ini konsep yang diterima peserta didik hampir semuanya berasal dari kata guru. Konsekwensinya, bila diberikan soal yang berbeda dengan soal latihan maka peserta didik cenderung membuat kesalahan (Ichsan, 2016). Salah satu alternatif metode pembelajaran yang dapat dikembangkan untuk memenuhi tuntutan tersebut adalah metode pembelajaran kooperatif tipe STAD dimana metode tersebut dapat membangkitkan motivasi dan semangat belajar peserta didik agar lebih mandiri, aktif dan kreatif dalam proses pembelajaran (Murtihapsari et al., 2021). COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 516 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Berdasarkan uraian di atas, maka muncul permasalahan apakah ada peningkatan hasil belajar peserta didik dengan menerapkan model pembelajaran kooperatif tipe STAD (Student Team Achievement Division) dalam pembelajaran Prakarya dan Kewirausahaan (Rekayasa) di kelas XI IPS 1 MAN Nagan Raya. Untuk mendapatkan jawaban permasalahan tersebut penulis ingin melakukan suatu penelitian dengan judul “Penerapan Model Pembelajaran Kooperatif Tipe STAD Untuk Meningkatkan Hasil Belajar Prakarya dan Kewirausahaan (Rekayasa) Peserta Didik Kelas XI IPS 1 MAN Nagan Raya Tahun Pelajaran 2022/2023.“ Subjek Penelitian Subjek penelitian adalah peserta didik kelas XI IPS 1 MAN Nagan Raya, semester ganjil tahun pelajaran 2022 /2023. Kelompok peserta didik ini umunya berasal dari keluarga ekonomi menengah ke bawah, di dominasi oleh peserta didik perempuan, sehingga lebih cenderung mudah diberdayakan untuk melakukan pembelajaran yang baik. Prosedur atau Langkah-Langkah Tindakan Metode Pengumpulan Data 1. Test Akhir Tes yang dilakukan yaitu untuk mengetahui serta menganalisis dan merefleksi terhadap hasil belajar peserta didik. Prosedur atau Langkah-Langkah Tindakan Penelitian ini menggunakan desain PTK dengan langkah utama pelaksanaan terdiri tahap: (1) perencanaan tindakan, (2) pelaksanaan tindakan, (3) observasi dan pengumpulan data, (4) analisis data dan refleksi (Mulyatiningsih, 2015). Prosedur penilitian dimulai dari pelaksanaan bimbingan teknis PTK pada tanggal 11 s.d 13 Agustus 2022. Dilanjutkan dengan penyempurnaan proposal dan rencana tindakan yang dilakukan di Madarasah di mana penulis bertugas, yakni MAN Nagan Raya. Setelah mendapat izin dari kepala Madrasah, penelitian dilanjutkan dengan pelaksanan tindakan dalam proses belajar mengajar di kelas XI IPS 1. Lokasi dan Waktu Penelitian Penelitian dilaksanakan di MAN Nagan Raya. Pelaksanan penelitian diperkirakan mencakup pembelajaran 1 kompetensi dasar dengan alokasi waktu (8 Jam Pelajaran atau 4 x Pertemuan) pada rentang waktu 20 Agustus s.d 10 September 2022. Deskripsi Hasil Tindakan Siklus 1 Deskripsi Hasil Tindakan Siklus 1 Pelaksanaan penelitian ini dilaksanakan di kelas XI IPS 1 MAN Nagan Raya sebanyak 29 peserta didik. Pengumpulan data pada penelitian ini disajikan melalui siklus-siklus yang direncanakan dan dibahas sesuai dengan pembahasan yang telah direncanakan dan dirumuskan. Siklus I (2 x pertemuan) dengan materi perencanaan usaha produk sistem teknik, sistem produksi usaha sistem teknik dan menghitung titik impas sedangkan siklus II dengan materi strategi promosi usaha sistem teknik dan laporan kegiatan usaha kerajinan dari bahan limbah berbentuk bangun datar. Penelitian ini hanya dapat dilaksanakan dalam 2 siklus, Tiap siklus terdiri dari 2 x pertemuan (4 jam pelajaran atau 4 x 45 menit) karena Singkatnya waktu yang diberikan untuk menyelesaikan laporan, Siklus 1 dilaksanakan pada tanggal 20 s.d 27 Agustus 2022 sedangkan siklus 2 dilaksanakan pada tanggal 03 s.d 10 September 2022. 2. Wawancara uk mengetahui aktivitas guru dan peserta didik dianalisis dengan menggunakan persentase.  % 100 x maksimal Skor skor Jumlah P Persentase  Sumber: Sudijono, 2004:43  % 100 x maksimal Skor skor Jumlah P Persentase  Sumber: Sudijono, 2004:43 Kriteria taraf keberhasilan tindakan aktivitas guru dan peserta didik yaitu: E: tidak baik dengan skor 1 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 2. Wawancara Penulis melakukan tanya jawab langsung dengan guru kolaborator untuk rnengetahui secara mendalam tentang pemahaman dan kesulitan-kesulitan yang dihadapi oleh peserta didik dalam belajar Prakarya dan Kewirausahaan (rekayasa) Observasi yaitu mengadakan pengamatan, pencatatan secara sistematik mengenai hal-hal yang perlu di selidiki dan bertujuan untuk memperkuat penjelasan serta keterangan, karena dilakukan secara langsung terhadap yang diselidiki (Hamid, 2012). Observasi yaitu mengadakan pengamatan, pencatatan secara sistematik mengenai hal-hal yang perlu di selidiki dan bertujuan untuk memperkuat penjelasan serta keterangan, karena dilakukan secara langsung terhadap yang diselidiki (Hamid, 2012). 4 Respon Peserta didik Untuk mengetahui respon peserta didik pada penerapan model pembelajaran kooperatif Tipe STAD pada pembelajaran Prakarya dan Kewirausahaan (Rekayasa). 5 C t t L Untuk mengetahui respon peserta didik pada penerapan model pembelajaran kooperatif Tipe STAD pada pembelajaran Prakarya dan Kewirausahaan (Rekayasa). COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 517 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Merupakan cerita secara tertulis tentang hal-hal yang terjadi selama penelitian ini berlangsung, meliputi aktivitas peserta didik dan peneliti selama pembelajaran berlangsung. Analisis Data Langkah yang harus ditempuh setelah pengumpulan data yaitu analisis data yang meliputi: 1. Analisis Tes Hasil Belajar (THB) Langkah yang harus ditempuh setelah pengumpulan data yaitu analisis data yang m 1 A li i T H il B l j (THB) Langkah yang harus ditempuh setelah pengumpulan data yaitu analisis data yang meliputi: 1. Analisis Tes Hasil Belajar (THB) 1. Analisis Tes Hasil Belajar (THB) 1. Analisis Tes Hasil Belajar (THB) Adapun kriteria hasil adalah jika ≥ 85% peserta didik mendapat skor 75% pada tes akhir setiap tindakan (Maidiyah, 2008 :26) dengan menggunakan rumus sebagai berikut: Adapun kriteria hasil adalah jika ≥ 85% peserta didik mendapat skor 75% pada tes akhir setiap tindakan (Maidiyah, 2008 :26) dengan menggunakan rumus sebagai berikut:  % 100 75 x siswa seluruh Jumlah skor mendapat yang siswa Jumlah P Persentase   isis Aktivitas Guru dan Peserta didik 2. Analisis Aktivitas Guru dan Peserta didik 2. Analisis Aktivitas Guru dan Peserta didik 2. Analisis Aktivitas Guru dan Peserta didik 2. Analisis Aktivitas Guru dan Peserta didik Untuk mengetahui aktivitas guru dan peserta didik dianalisis dengan menggunakan persentase. Untuk mengetahui aktivitas guru dan peserta didik dianalisis dengan menggunakan persentase. ngetahui aktivitas guru dan peserta didik dianalisis dengan menggunakan persentase. Perencanaan Siklus I Pada awal siklus ini, dimulai terlebih dahulu diinformasikan pada peserta didik tentang maksud, bentuk dan tujuan dari penelitian ini. Karena semuanya perlu sosialisasi yang baik dan terarah (Yulia, 2022). Adapun yang dilakukan dalam tahap ini adalah: 1. Guru menyiapkan Rencana Pelaksanaan Pembelajaran (RPP) 2. Guru memberikan motivasi kepada peserta didik. 3. Guru menyampaikan cakupan materi pelajaran. 3. Guru menyampaikan cakupan materi pelajaran. 4. Guru menjelaskan tujuan pembelajaran yang ingin dicapai pada siklus I. 518 COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 5. Guru menjelaskan materi tentang wirausaha produk rekayasa sistem teknik serta menjelaskan manfaat mempelajari materi tersebut dalam kehidupan sehari-hari. 5. Guru menjelaskan materi tentang wirausaha produk rekayasa sistem teknik serta menjelaskan manfaat mempelajari materi tersebut dalam kehidupan sehari-hari. 5. Guru menjelaskan materi tentang wirausaha produk rekayasa sistem teknik serta menjelaskan manfaat mempelajari materi tersebut dalam kehidupan sehari-hari. 5. Guru menjelaskan materi tentang wirausaha produk rekayasa sistem teknik serta menjelaskan manfaat mempelajari materi tersebut dalam kehidupan sehari-hari. 6. Guru membimbing peserta didik yaitu peserta didik dibagi dalam 5 kelompok, dimana 2 kelompok terdiri dari 5 peserta didik dan 3 kelompok terdiri dari 6 - 7 peserta didik yang kemampuan peserta didik tiap kelompok juga bervariasi dari peserta didik yang berkemampuan rendah, sedang dan tinggi. 7. Guru membimbing peserta didik dalam mengecek pemahaman dengan memberi umpan balik yaitu dengan meminta peserta didik untuk mempresentasikan hasil kerja kelompok tentang materi perencanaan dan sistem produksi usaha sistem teknik. 8. Guru memberikan kesempatan kepada peserta didik lebih lanjut yaitu membimbing membuat rangkuman, memberikan tugas rumah. 9. Selanjutnya guru bersama pengamat akan melakukan refleksi tentang apa yang telah dilakukan oleh guru maupun peserta didik dan apa yang dialami ketika proses pembelajaran berlangsung, seta bagaimana dampak dari tindakan yang telah diterapkan guru terhadap suasana belajar dan hasil belajar peserta didik. Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) Pelaksanaan Siklus I Kegiatan ini dilaksanakan pada hari sabtu tanggal 20 Agustus 2022 pada jam ketiga sampai keempat pukul 09.30 Wib sampai pukul 11.00 Wib. Pemberian tindakan dilaksanakan berdasarkan RPP yang telah disusun sebelumnya. Pelaksanaan tindakan dilakukan melalui 1 tahapan yang dilaksanakan selama 2 jam tatap muka (90 menit). Jumlah peserta didik yang hadir pada saat pelaksanaan tindakan adalah sebanyak 29 orang. 1. Tahap Awal Guru masuk ke kelas dengan mengucapkan salam, lalu mengecek kehadiran siswa, menyampaikan tujuan dan mempersiapkan peserta didik. Guru mempersiapkan peserta didik untuk belajar dan mengingatkan kembali materi sebelumnya tentang wirausaha produk rekayasa. Selanjutnya memotivasi peserta didik dengan menanyakan manfaat mempelajari kewirausahaan, menjelaskan cakupan materi pelajaran, dan memberi pengarahan kepada peserta didik tentang proses pembelajaran dengan menerapapkan pembelajaran kooperatif tipe STAD. 2. Tahap Inti Fase memberikan informasi awal tentang wirausaha produk rekayasa sistem teknik. Guru membimbing siswa dalam pembentukan kelompok terdiri dari 6 – 7 orang, guru membagi LKPD kepada peserta didik, guru memberi kesempatan kepada peserta didik untuk membaca LKPD dan bertanya jika ada hal – hal yang belum dipahami, guru Meminta beberapa orang peserta didik untuk menyebutkan pengertian rekayasa dan sistem tekhnik. Fase membimbing peserta didik, pada fase ini guru menerapkan model pembelajaran kooperatif tipe STAD, guru membentuk 5 kelompok belajar dengan anggota tiap kelompok ada yang 5 peserta didik dan ada yang 6-7 peserta didik. Hal ini dikarenakan jumlah peserta didik kelas XI IPS 1 sebanyak 29 orang. Setiap kelompok terdiri dari peserta didik berkemampuan tinggi, sedang dan rendah. Guru memberi tugas yang sama kepada tiap kelompok untuk mendiskusikan materi perencanaan usaha produk sistem teknik dan sistem produksi usaha sistem tekhnik. 2. Tahap Inti Fase memberikan informasi awal tentang wirausaha produk rekayasa sistem teknik. Guru membimbing siswa dalam pembentukan kelompok terdiri dari 6 – 7 orang, guru membagi LKPD kepada peserta didik, guru memberi kesempatan kepada peserta didik untuk membaca LKPD dan bertanya jika ada hal – hal yang belum dipahami, guru Meminta beberapa orang peserta didik untuk menyebutkan pengertian rekayasa dan sistem tekhnik. Fase membimbing peserta didik, pada fase ini guru menerapkan model pembelajaran kooperatif tipe STAD, guru membentuk 5 kelompok belajar dengan anggota tiap kelompok ada yang 5 peserta didik dan ada yang 6-7 peserta didik. Hal ini dikarenakan jumlah peserta didik kelas XI IPS 1 sebanyak 29 orang. Setiap kelompok terdiri dari peserta didik berkemampuan tinggi, sedang dan rendah. Pelaksanaan Siklus I Guru memberi tugas yang sama kepada tiap kelompok untuk mendiskusikan materi perencanaan usaha produk sistem teknik dan sistem produksi usaha sistem tekhnik. Fase mengecek pemahaman peserta didik dengan memberi umpan balik. Fase ini guru meminta masing-masing kelompok untuk mempresentasikan hasil kerja kelompok ke depan kelas dan kelompok lain diminta untuk menanggapinya, serta guru memberikan penguatan terhadap hasil COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 519 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City diskusi. Selanjutnya guru memberikan contoh soal perencanaan usaha produk sistem teknik dan sistem produksi usaha sistem tekhnik. diskusi. Selanjutnya guru memberikan contoh soal perencanaan usaha produk sistem teknik dan sistem produksi usaha sistem tekhnik. p 3. Tahap Akhir Pada tahap akhir, guru membimbing peserta didik untuk membuat rangkuman dan memberikan penghargaan untuk kelompok yang terbaik. p Pada tahap akhir, guru membimbing peserta didik untuk membuat rangkuman dan memberikan penghargaan untuk kelompok yang terbaik. Pada akhir siklus, siklus I guru memberikan tes terhadap peserta didik, dengan memberikan 10 buah soal dalam bentuk pilihan ganda yang telah disiapkan oleh guru kepada peserta didik untuk diisi pada lembaran jawaban yang dsisediakan, yang hasilnya dapat dilihat pada tabel 1.berikut : Tabel 1. Hasil Evaluasi Peserta Didik Siklus I Tabel 1. Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) a. Tahap Awal a. Tahap Awal Pada saat guru masuk semua peserta didik suadah berada di kelas karena pada pertemuan sebelumnya sudah diberitahukan bahwa akan dilaksanakan lanjutan kerja kelompok, peserta didik cukup antusias untuk mengikuti pembelajaran. Fase menyampaikan tujuan dan mempersiapkan peserta didik, guru mempersiapkan peserta didik untuk belajar dan mengingatkan kembali materi sebelumnya tentang sistem produksi usaha sistem tekhnik, selanjutnya memotivasi peserta didik, serta menjelaskan secara singkat cakupan materi pelajaran, menyampaikan tujuan pembelajaran dan memberi pengarahan kepada peserta didik bagaimana proses pembelajaran dengan menggunakan model pembelajaran kooperatif tipe STAD. a. Tahap Awal Pada saat guru masuk semua peserta didik suadah berada di kelas karena pada pertemuan sebelumnya sudah diberitahukan bahwa akan dilaksanakan lanjutan kerja kelompok, peserta didik cukup antusias untuk mengikuti pembelajaran. Fase menyampaikan tujuan dan mempersiapkan peserta didik, guru mempersiapkan Pada saat guru masuk semua peserta didik suadah berada di kelas karena pada pertemuan sebelumnya sudah diberitahukan bahwa akan dilaksanakan lanjutan kerja kelompok, peserta didik cukup antusias untuk mengikuti pembelajaran. Fase menyampaikan tujuan dan mempersiapkan peserta didik, guru mempersiapkan peserta didik untuk belajar dan mengingatkan kembali materi sebelumnya tentang sistem produksi usaha sistem tekhnik, selanjutnya memotivasi peserta didik, serta menjelaskan secara singkat cakupan materi pelajaran, menyampaikan tujuan pembelajaran dan memberi pengarahan kepada peserta didik bagaimana proses pembelajaran dengan menggunakan model pembelajaran kooperatif tipe STAD. Fase menyampaikan tujuan dan mempersiapkan peserta didik, guru mempersiapkan peserta didik untuk belajar dan mengingatkan kembali materi sebelumnya tentang sistem produksi usaha sistem tekhnik, selanjutnya memotivasi peserta didik, serta menjelaskan secara singkat cakupan materi pelajaran, menyampaikan tujuan pembelajaran dan memberi pengarahan kepada peserta didik bagaimana proses pembelajaran dengan menggunakan model pembelajaran kooperatif tipe STAD. b. b. Tahap Inti Fase memberikan informasi awal tentang strategi promosi sistem tekhnik. Guru membimbing siswa dalam pembentukan kelompok terdiri dari 6 – 7 orang, guru membagi LKPD kepada peserta didik, guru memberi kesempatan kepada peserta didik untuk membaca LKPD dan bertanya jika ada hal – hal yang belum dipahami, guru Meminta beberapa orang peserta didik untuk menyebutkan pengertian sistem tekhnik . Fase membimbing peserta didik, pada fase ini guru menerapkan model pembelajaran kooperatif tipe STAD, guru membentuk 5 kelompok belajar dengan anggota tiap kelompok ada yang 5 peserta didik dan ada yang 6 - 7 peserta didik. Hal ini dikarenakan jumlah peserta didik kelas XI IPS 1 sebanyak 29 orang. Setiap kelompok terdiri dari peserta didik berkemampuan tinggi, sedang dan rendah. 1. Perencanaan Siklus II 1. Perencanaan Siklus II Adapun upaya yang d Adapun upaya yang dilakukan untuk meningkatkan hasil belajar peserta didik pada siklus II yaitu: a. Guru menyiapkan Rencana Pelaksanaan Pembelajaran (RPP) tentang materi strategi promosi sistem teknik dan Laporan kegiatan pembuatan produk sistem tekhnik. Adapun upaya yang dilakukan untuk meningkatkan hasil belajar peserta didik pada a. Guru menyiapkan Rencana Pelaksanaan Pembelajaran (RPP) tentang materi strategi prom sistem teknik dan Laporan kegiatan pembuatan produk sistem tekhnik. b. Mengupayakan pembelajaran lebih berpusat pada pemahaman peserta didik. Mengupayakan pembelajaran lebih berpusat pada pemahaman peserta didik. c. Guru mengarahkan peserta didik untuk berperan aktif dalam melaksanakan Diskusi. c. Guru mengarahkan peserta didik untuk berperan aktif dalam melaksanakan Diskus d. Guru lebih mengoptimalkan dalam mengelola model pembelajaran kooperatif tipe ST Dari hasil refleksi pada siklus pertama, guru peneliti melakukan perbaikan agar terlaksananya proses pembelajaran lebih meningkat yang tergambar pada siklus II yang mencakup langkah-langkah berikut: Pelaksanaan Siklus I Hasil Evaluasi Peserta Didik Siklus I No Nama Peserta didik Jenis Kelamin Nilai Tuntas Tidak Tuntas 1 Abil Azhar L 80  2 Ahmad Taqiyyuddin L 50  3 Alfia Akmalia P 80  4 Ansarullah L 80  5 Cut Fitri L 60  6 Cut safrida P 60  7 Eka Wati L 80  8 Fera Wati P 90  9 Fitria Ramadhani P 60  10 Laila Dewi P 60  11 Lisda Salwa P 80  12 Mardiana P 60  13 Muhajirin L 80  14 Muhammad Jaihari L 80  15 Nadrira Rifatul M P 80  16 Naufal Al Hanif L 80  17 Novina Riani P 50  18 Nur Kalimah P 50  19 Nur Sariyanti P 60  20 Putri Juana P 60  21 Rahmad Dani K L 60  22 Rahmad Fauzi L 70  23 Raudhatul Fitri Z P 70  24 Rosmawar P 80  25 Ruslan L 80  26 Siti Barorah P 60  27 Yulia Anggun S P 60  28 Zainal Abidin L 50  29 Zulfan Mulia L 60  COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 520 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Jumlah Nilai 1970 Rata-rata Kelas 67,93 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Jumlah Nilai 1970 Rata-rata Kelas 67,93 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Rata-rata Kelas Deskripsi Tindakan Siklus II 1. Perencanaan Siklus II 2. Pelaksanaan Siklus II Kegiatan ini dilaksanakan pada hari Sabtu tanggal 03 september 2022 dan 10 september 2022 pada jam ketiga s.d jam keempat pukul 09.30 WIB sampai pukul 11.00 WIB. Pelaksanaan tindakan pada siklus II dilaksanakan berdasarkan RPP yang telah disusun sebelumnya. Pelaksanaan tindakan dilakukan dalam beberapa tahap yang dilaksanakan selama 90 menit. Jumlah peserta didik yang hadir pada pelaksanaan tindakan sebanyak 29 Peserta didik. Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) Uraian Hasil Siklus I dan II Uraian Hasil Siklus I dan II Berdasarkan hasil yang dicapai pada 4 kali pertemuan dan 2 kali tes selama penelitian 2 siklus, maka diperoleh hasil dengan uraian dalam tabel sebagai berikut: a. Tahap Awal Guru memberi tugas yang sama kepada tiap kelompok untuk mendiskusikan materi strategi promosi sistem tekhnik dan laporan kegiatan pembuatan produk sistem tekhnik. COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 521 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Fase mengecek pemahaman peserta didik dengan memberi umpan balik. Fase ini guru meminta masing-masing kelompok untuk mempresentasikan hasil kerja kelompok ke depan kelas dan kelompok lain diminta untuk menanggapinya, serta guru memberikan penguatan terhadap hasil diskusi. Selanjutnya guru memberikan contoh soal strategi promosi sistem tekhnik dan laporan kegiatan pembuatan produk sistem tekhnik. c. Tahap Akhir Fase memberikan kesempatan membimbing peserta didik, fase ini guru membimbing peserta didik untuk membuat rangkuman, memberikan penghargaan untuk kelompok yang terbaik dan memberikan tugas rumah. c. Tahap Akhir Fase memberikan kesempatan membimbing peserta didik, fase ini guru membimbing peserta didik untuk membuat rangkuman, memberikan penghargaan untuk kelompok yang terbaik dan memberikan tugas rumah. Pada akhir siklus siklus II guru melakukan tes terhadap peserta didik, dengan memberikan 10 buah soal yang telah disiapkan oleh guru kepada peserta didik untuk diisi pada lembaran jawaban yang disediakan, yang hasilnya dapat dilihat pada tabel 2. berikut: Tabel 2. Hasil evaluasi peserta didik siklus II No Nama Peserta didik Jenis Kelamin Nilai Tuntas Tidak Tuntas 1 Abil Azhar L 80  2 Ahmad Taqiyyuddin L 100  3 Alfia Akmalia P 80  4 Ansarullah L 80  5 Cut Fitri L 80  6 Cut safrida P 90  7 Eka Wati L 90  8 Fera Wati P 80  9 Fitria Ramadhani P 60  10 Laila Dewi P 80  11 Lisda Salwa P 80  12 Mardiana P 90  13 Muhajirin L 80  14 Muhammad Jaihari L 80  16 Nadrira Rifatul M P 80  17 Naufal Al Hanif L 100  18 Novina Riani P 60  19 Nur Kalimah P 60  20 Nur Sariyanti P 100  21 Putri Juana P 100  22 Rahmad Dani K L 60  23 Rahmad Fauzi L 60  24 Raudhatul Fitri Z P 60  25 Rosmawar P 90  26 Ruslan L 60  27 Siti Barorah P 70  Tabel 2. Hasil evaluasi peserta didik siklus II COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. a. Tahap Awal 2 (05) September 2022 - (515-526) 522 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City 28 Yulia Anggun S P 80  29 Zainal Abidin L 80  Jumlah Nilai 2210 Rata-rata kelas 76,20 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City 28 Yulia Anggun S P 80  29 Zainal Abidin L 80  Jumlah Nilai 2210 Rata-rata kelas 76,20 SIKLUS II 1. Pada pertemuan 1 guru mengalami kesulitan dalam mengorganisasikan peserta didik hal ini disebabkan karena guru harus memberikan pemahaman kepada peserta didik tentang metode kooperatif tipe STAD, peserta didik belum terbiasa belajar dengan metode tersebut. 1. Pada pertemuan 2 peserta didik semakin antusias dan termotivasi belajar karena tertarik dengan metode baru yang diterapkan oleh guru, dan mereka terlibat langsung dalam proses belajar mengajar. 1. Pada pertemuan 2 peserta didik semakin antusias dan termotivasi belajar karena tertarik dengan metode baru yang diterapkan oleh guru, dan mereka terlibat langsung dalam proses belajar mengajar. 2. Peserta didik sudah semakin aktif dalam mengikuti pelajaran, aktif membuat pertanyaan kepada kelompok lain dan mencoba menjawab pertanyaan yang diajukan oleh peserta didik anggota kelompok lain. 2. Ketika membimbing peserta didik guru cenderung memberikan jawaban langsung atas jawaban peserta didik, tanpa mengalihkan pertanyaan kepada peserta didik anggota kelompok lain. 3. Nilai pada siklus I menunjukkan hasil rata- rata yang dicapai peserta didik masih berada dibawah KKM 3. Guru sudah mulai bisa untuk menjadi fasilitator dalam proses belajar mengajar dan menjadi mediator bagi peserta didik tiap-tiap kelompok. 4. Nilai rata-rata yang dicapai peserta didik telah mencapai KKM. 5. Hasil pengamatan dan wawancara langsung, 90 % peserta didik senang dengan metode kooperatif tipe STAD. Analisis Siklus I dan II Setelah melalui uraian hasil, maka dapat di ketahui analisis hasil siklus 1 dan 2 seperti yang tertulis dalam table berikut: Setelah melalui uraian hasil, maka dapat di ketahui analisis hasil siklus 1 dan 2 seperti yang tertulis dalam table berikut: Tabel 4. Analisis Hasil Siklus I dan II NO Kategori Penilaian Siklus I Siklus II 1 Penilaian Proses Para peserta didik menganggap pembelajaran dengan metode kooperatif tipe STAD seperti metode diskusi biasa seperti yang Peserta didik mulai memahami perbedaan metode ini dengan metode diskusi biasa, dan mereka merasa tertarik dan Tabel 4. Analisis Hasil Siklus I dan II Tabel 4. Analisis Hasil Siklus I dan II COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 523 COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City sering dipraktekkan selama ini minat belajar pun semakin meningkat 2 Penilaian Hasil Nilai Total: 1970 Rata – rata: 67,93 Nilai Total: 2210 Rata – rata: 76,20 3 Portofolio Penilaian berupa rangkuman pembelajaran Umumnya peserta didik dapat menyelesaikan soal tentang sistem tekhnik tepat pada waktunya. 4 Minat Peserta didik Berdasarkan hasil wawancara kepada peserta didik, mereka menyukai belajar dengan metode kooperatif tipe STAD. Peserta didik menginginkan belajar menggunakan metode yang sama, bahkan mereka menginginkan metode lain untuk diterapkan oleh guru untuk pembelajaran berikutnya. Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) SIMPULAN Dari hasil analisis data dan pembahasan hasil penelitian yang telah peneliti lakukan, maka dapat ditarik kesimpulan antara lain, 1) Penerapan model pembelajaran kooperatif tipe STAD dapat meningkatkan hasil belajar peserta didik, pada siklus pertama ketuntasan belajar peserta didik sebesar 41,37 % meningkat menjadi 72,41 % pada siklus kedua. 2) Aktivitas guru pada siklus pertama dengan persentase 79,04 % dan mengalami peningkatan sebesar 11 % menjadi 90,47 % pada siklus kedua sedangkan aktivitas peserta didik pada siklus pertama sebesar 65,62 % dan mengalami peningkatan sebesar 19 % menjadi 84,37 % pada siklus kedua dan tergolong dalam kategori sangat baik. COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 524 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City DAFTAR PUSTAKA Afrianti, N. (2017). Profil kecerdasan sosial siswa SMA di Kota Bandung dan implikasinya terhadap penyelenggaraan layanan bimbingan konseling. Jurnal Ilmiah Psikologi Terapan, 5(1), 40–59. Fitriani, E. (2019). Pengembangan Instrument Assessment Hots (High Order Thinking Skill) pada Mata PelajaraN IPS Terintegrasi Nilai-Nilai Pembangunan Karakter Kelas V SD/MI dI Bandar Lampung. UIN Raden Intan Lampung. Hamid, M. (2012). Upaya Meningkatkan Hasil Belajar Siswa Pada Materi Kegiatan Ekonomi Masyarakat Di Kelas Vii Smp Swasta Nujumush Shaghirah Aceh Utara Dengan Model Pembelajaran Kooperatif Tipe NHT (Numbered-Heads-Together). Lentera: Jurnal Ilmiah Sains Dan Teknologi, 145783. Harta, I. K., Nurhayata, I. G., & Krisnawati, L. (2017). Pengembangan Prototipe Egg Boiler Sebagai Media Pembelajaran Prakarya Dan Kewirausahaan Untuk Materi Teknologi Tepat Guna Kelas Xi Mia Sma Negeri 4 Singaraja Tahun Ajaran 2016/2017. Jurnal Pendidikan Teknologi Dan Kejuruan, 14(2). Ichsan, M. (2016). Perbedaan Peningkatan Kemampuan Pemecahan Masalah dan Belief Siswa antara Siswa yang Diberi PMR dengan PBM di SD N 1 Trienggadeng Kabupaten Pidie Jaya Provinsi Aceh. UNIMED. Kholili, A. N., & Fajaruddin, S. (2020). Manajemen strategik peningkatan mutu lembaga pendidikan Muhammadiyah di Kabupaten Gunungkidul. Jurnal Akuntabilitas Manajemen Pendidikan, 8(1), 53–69. Mahmudah, M. (2016). Urgensi Diantara Dualisme Metode Pembelajaran Ceramah Dalam Kegiatan Belajar Mengajar Untuk Siswa MI/SD. Cakrawala: Jurnal Studi Islam, 11(1), 116–129. Mulyatiningsih, E. (2015). Metode Penelitian Tindakan Kelas. Modul Pelatiihan Pendidikan Profesi Guru: Fakultas Teknik, Universitas Negeri Yogyakarta. Universitas Negeri Yogyakarta. Murtihapsari, M., Patandean, S. N., & Yogaswara, R. (2021). Ulasan: Keberhasilan Belajar Siswa Melalui Penerapan Pembelajaran Kooperatif Tipe STAD. Orbital: Jurnal Pendidikan Kimia, 5(1), 38–48. Nurhuda, H. (2022). Masalah-Masalah Pendidikan Nasional; Faktor-Faktor Dan Solusi Yang Ditawarkan. Dirasah: Jurnal Pemikiran Dan Pendidikan Dasar Islam, 5(2), 127–137. Pantiwati, Y. (2017). Kemampuan literasi dan teknik asesmen literasi. Research Report. Rumawan, M., Santo Gitakarma, M., & Nugraha, N. P. (2017). Penerapan Metode Demonstrasi Untuk Meningkatkan Hasil Belajar Prakarya dan Kewirausahaan Siswa Kelas XI MIA 4 SMA Negeri 3 COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 2 (05) September 2022 - (515-526) 525 Samsul Bahri Keledar, Fauzan A. Sangadji, Imran Oppier Study of The Maintenance of the Pattimura Airport Building, Ambon City Singaraja. Jurnal Pendidikan Teknik Elektro Undiksha, 6(2), 86–95. Wahyuni, F. T., Sujadi, I., & Subanti, S. (2016). Proses berpikir reflektif siswa kelas VII SMP Negeri 3 Polanharjo Klaten dalam pemecahan masalah pecahan. Jurnal Pembelajaran Matematika, 4(4). Wijaya, E. Y., Sudjimat, D. A., Nyoto, A., & Malang, U. N. (2016). Transformasi pendidikan abad 21 sebagai tuntutan pengembangan sumber daya manusia di era global. Prosiding Seminar Nasional Pendidikan Matematika, 1(26), 263–278. Yulia, E. (2022). Meningkatkan Hasil Belajar Siswa Pada Materi Organ Gerak Hewan Dan Manusia Melalui Problem Based Learning Pada Siswa Kelas V SD Negeri 12 Bireuen. JEMAS: Jurnal Edukasi Matematika Dan Sains, 3(1), 30–32. © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY SA) license (https://creativecommons.org/licenses/by-sa/4.0/). 526
https://openalex.org/W2898436590
https://www.dora.lib4ri.ch/empa/islandora/object/empa%3A18055/datastream/PDF/Fiore-2018-Peroxy_acetyl_nitrate_%28PAN%29_measurements-%28published_version%29.pdf
English
null
Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships
Atmospheric chemistry and physics
2,018
cc-by
17,477
Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships Fiore (amfiore@ldeo.columbia.edu) Received: 27 January 2018 – Discussion started: 26 February 2018 Revised: 28 August 2018 – Accepted: 1 October 2018 – Published: 25 October 2018 Received: 27 January 2018 – Discussion started: 26 February 2018 Revised: 28 August 2018 – Accepted: 1 October 2018 – Published: 25 October 2018 Atmos. Chem. Phys., 18, 15345–15361, 2018 https://doi.org/10.5194/acp-18-15345-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships Stevenson11, Sophie Szopa24, Christoph Zellweger23, and Guang Zeng25 1Department of Earth and Environmental Science, Columbia University, Palisades, NY 10964, USA 2Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA 3Department of Atmospheric Science, Colorado State University, Fort Collins, CO 80521, USA y y y, , , 3Department of Atmospheric Science, Colorado State University, Fort Collins, CO 80521, USA 4Institute for Atmospheric and Climate Science, ETH Zürich, Switzerland 5Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, UK 6School of STEM, University of Washington, Bothell, WA 98011, USA 6School of STEM, University of Washington, Bothell, WA 98011, USA Department of Atmospheric Science, University of Washington, Seattle, WA 98195, USA 7Department of Atmospheric Science, University of Washington, Seattle, WA 98195, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA 8Lawrence Livermore National Laboratory, Livermore, CA 94550, USA Department of Meteorology, University of Reading, Reading, RG6 6BB, UK 9Department of Meteorology, University of Reading, Reading, RG6 6BB, UK 10European Commission, Joint Research Centre, Ispra, 21027, Italy 10European Commission, Joint Research Centre, Ispra, 21027, Italy 11School of GeoSciences, The University of Edinburgh, Edinburgh, EH9 3FF, UK 11School of GeoSciences, The University of Edinburgh, Edinburgh, EH9 3FF, UK 12Atmospheric Chemistry and Dynamics Laboratory, NASA GSFC, Greenbelt, MD 20720, USA 12Atmospheric Chemistry and Dynamics Laboratory, NASA GSFC, Greenbelt, MD 2072 Federal Environment Agency (UBA), Schauinsland, 79254, Oberried, Germany g y y 14Meteorological Observatory Hohenpeissenberg, German Meteorological Service (D 14Meteorological Observatory Hohenpeissenberg, German Meteorological Service (DWD), Hohenpeissenberg, Germany g y p g g p Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, U p g g g y 16Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration, Princeton, NJ 08540, USA 17 16Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration 16Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration, Princeton, NJ 08540, USA 1 Centre for Research in Earth and Space Science, York University, Toronto, M3J 1P3, Canada p , y, , , 18School of Earth and Environmental Sciences, Seoul National University, Seoul, 08826, Republic of Korea 19II4 5 7 German Environment Agency (UBA) Zugspitze 82475 Germany p y 18School of Earth and Environmental Sciences, Seoul National University, Seoul, 08826, Republic of Korea 19II4.5.7, German Environment Agency (UBA), Zugspitze, 82475, Germany 20 School of Earth and Environmental Sciences, Seoul National University, Seoul, 08826, Republic 18School of Earth and Environmental Sciences, Seoul National University, Seoul 19II4.5.7, German Environment Agency (UBA), Zugspitze, 82475, Germany 20Met Office, Exeter, EX1 3PB, UK School of Earth and Environmental Sciences, Seoul National University, Seo II4.5.7, German Environment Agency (UBA), Zugspitze, 82475, Germany 21Jülich Supercomputing Centre, Forschungszentrum Jülich, 52425 Jülich, Germany 22 22Nicholas School of the Environment, Duke University, Durham, NC 27708, USA 23 Laboratory for Air Pollution/Environmental Technology, Empa – Swiss Federal Laboratories for aterials Science and Technology Dübendorf 8600 Switzerland 23Laboratory for Air Pollution/Environmental Technology, Empa – Swiss Federal Laboratorie Materials Science and Technology, Dübendorf, 8600, Switzerland 23Laboratory for Air Pollution/Environmental Technology, Empa – Swiss Federal Laboratories Materials Science and Technology, Dübendorf, 8600, Switzerland 24Laboratoire des Sciences du Climat et de l’Environnement, Institut Pierre Simon Laplace, CEA/CNRS/UVSQ, Gif-sur-Yvette, France CEA/CNRS/UVSQ, Gif-sur-Yvette, France 25National Institute of Water and Atmospheric Research, Wellington, 6021, New Zealand anow at: Advanced Study Program, National Center for Atmospheric Research, Boulder, CO, USA 25National Institute of Water and Atmospheric Research, Wellington, 6021, New Zealand a Ad d S d P N i l C f A h i R h B ld CO USA y g p bnow at: DWD, Research Center Human Biometeorology, Freiburg, Germany bnow at: DWD, Research Center Human Biometeorology, Freiburg, Germany cnow at: Air Quality Research Division, Environment and Climate Change Canada, Toronto, M3H Correspondence: Arlene M. Peroxy acetyl nitrate (PAN) measurements at northern midlatitude mountain sites in April: a constraint on continental source–receptor relationships Arlene M. Fiore1,2, Emily V. Fischer3, George P. Milly2, Shubha Pandey Deolal4, Oliver Wild5, Daniel A. Jaffe6,7, Johannes Staehelin4, Olivia E. Clifton1,2,a, Dan Bergmann8, William Collins9, Frank Dentener10, Ruth M. Doherty11, Bryan N. Duncan12, Bernd Fischer13, Stefan Gilge14,b, Peter G. Hess15, Larry W. Horowitz16, Alexandru Lupu17,c, Ian A. MacKenzie11, Rokjin Park18, Ludwig Ries19, Michael G. Sanderson20, Martin G. Schultz21, Drew T. Shindell22, Martin Steinbacher23, David S. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15346 Abstract. Abundance-based model evaluations with obser- vations provide critical tests for the simulated mean state in models of intercontinental pollution transport, and under cer- tain conditions may also offer constraints on model responses to emission changes. We compile multiyear measurements of peroxy acetyl nitrate (PAN) available from five mountain- top sites and apply them in a proof-of-concept approach that exploits an ensemble of global chemical transport models (HTAP1) to identify an observational “emergent constraint”. In April, when the signal from anthropogenic emissions on PAN is strongest, simulated PAN at northern midlatitude mountaintops correlates strongly with PAN source–receptor relationships (the response to 20 % reductions in precursor emissions within northern midlatitude continents; hereafter, SRRs). This finding implies that PAN measurements can pro- vide constraints on PAN SRRs by limiting the SRR range to that spanned by the subset of models simulating PAN within the observed range. In some cases, regional anthropogenic volatile organic compound (AVOC) emissions, tracers of transport from different source regions, and SRRs for ozone also correlate with PAN SRRs. Given the large observed in- terannual variability in the limited available datasets, estab- lishing strong constraints will require matching meteorology in the models to the PAN measurements. Application of this evaluation approach to the chemistry–climate models used to project changes in atmospheric composition will require routine, long-term mountaintop PAN measurements to dis- cern both the climatological SRR signal and its interannual variability. tinents, and (2) O3 can be produced in transit from the export and subsequent chemical evolution of PAN and other pre- cursors. Below, we examine the extent to which springtime PAN observations at northern midlatitude mountaintop sites can be used to constrain the spread in multi-model estimates of source–receptor relationships (SRRs), where the sources are continental-scale regions and the receptors are the moun- taintop sites, for both PAN and O3. p Observations during several aircraft field campaigns in the eastern Pacific and at mountaintop sites in the western US and North Atlantic document efficient O3 production in the lower troposphere following subsidence of PAN-containing air masses (Fischer et al., 2010; Heald et al., 2003; Hudman et al., 2004; Kotchenruther et al., 2001a, b; Val Martin et al., 2008; Zhang et al., 2008). A. M. Fiore et al.: A constraint on continental source–receptor relationships When PAN decomposes in low- NOx regions of the atmosphere, the NOx released can pro- duce O3 up to 8 times more efficiently than in polluted (high- NOx) regions (Liang et al., 1998; Liu et al., 1987) and thus increase global O3 abundances (Moxim et al., 1996; Wang and Jacob, 1998), as O3 formation is NOx limited in most of the free troposphere (Chameides et al., 1992). The life- time of PAN against thermal decomposition is about 1 h at 20 ◦C, and it approximately doubles for every 4 ◦C decrease in temperature, leading to a lifetime of at least a month in the mid-troposphere during spring. This strong temperature de- pendence implies that a warmer climate will decrease PAN export from polluted continental boundary layers, although a rise in temperature-sensitive biogenic precursor emissions may temper this response (e.g., Doherty et al., 2013). Future projections of atmospheric composition under global change scenarios will thus benefit from a thorough understanding of the role PAN plays in transporting oxidized reactive nitrogen and thereby altering ozone production throughout the tropo- sphere. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15347 free-tropospheric O3 abundance over western North Amer- ica. Over East Asia, Lin et al. (2010) found that the export of PAN produced from European anthropogenic emission changes and subsequent downwind O3 formation contributed 20 % of the spatially averaged response of surface O3 levels, and up to 50 % of the O3 response at mountain sites. 2014), we hypothesize that PAN measurements may offer much-needed constraints for discriminating across model es- timates of intercontinental transport of PAN, and possibly O3. The number of models contributing to the HTAP1 study, which was designed to maximize comparability across in- dividual model estimates of ozone responses to changes in precursor emissions within northern midlatitude continental- scale source regions, offers an opportunity to evaluate this hypothesis. p 3 p In addition to the direct influence of PAN on interconti- nental O3 transport, PAN may serve as a sensitive diagnos- tic of model uncertainties in O3 production chemistry and transport (Emmerson and Evans, 2009; Kuhn et al., 1998). Prior analysis of measurements and global model simulations suggests that PAN abundances at high-altitude sites may be more sensitive than O3 itself to changes in precursor emis- sions (Fiore et al., 2011; Fischer et al., 2011; Jaffe et al., 2007). We interpret this stronger sensitivity of PAN than O3 to changes in precursor emissions as reflecting buffering of O3 by changes to O3 losses that compensate for changes in production, whereas PAN loss pathways are far less sensi- tive to changes in precursor emissions. PAN loss pathways include thermal decomposition (which dominates below ap- proximately 7 km), photolysis in the upper troposphere, and dry deposition within the boundary layer (Kirchner et al., 1999; Roberts, 2007; Turnipseed et al., 2006). All of the HTAP1 models include PAN formation, but the chemical mechanisms and kinetic rate coefficients differ, with likely implications for long-range transport (Emmerson and Evans, 2009; Knote et al., 2015). A prior multi-model study found that even with the same emissions, PAN differs widely across models, reflecting differences in simulated photochemistry (Emmons et al., 2015). While the absence of direct emis- sions and its low background make PAN a useful tracer of photochemistry, we note that O3 typically responds more strongly to changes in NOx emissions, while PAN responds more strongly to changes in VOC emissions in many regions (Fischer et al., 2014; see their Fig. 4). A. M. Fiore et al.: A constraint on continental source–receptor relationships We describe the HTAP1 model simulations, mountaintop measurements, and our strategy to sample the models at these sites (Sect. 2) before illustrating our rationale for selecting the month of April for our analysis (Sect. 3). We then borrow from the emergent constraint approach in climate science to show that correlations between simulated total PAN and SRRs for PAN are sufficiently strong to permit PAN mea- surements at mountaintop sites (one in each of the three ma- jor northern midlatitude source regions) to narrow the wide inter-model spread in estimates of PAN origin (Sect. 4). We further examine inter-model relationships among the simu- lated PAN SRRs at these three mountaintop sites and re- gional precursor emissions, and with a proxy for model trans- port (Sect. 5). Finally, we assess the relationship between PAN and O3 SRRs (Sect. 6) and conclude with a summary and recommendations for future work based on our proof- of-concept analysis (Sect. 7). 1 Introduction Peroxy acetyl nitrate (PAN) is produced alongside ozone (O3) from photochemical reactions involving precursor emis- sions of nitrogen oxides (NOx) and non-methane volatile or- ganic compounds (VOCs). Once ventilated from a source re- gion to the free troposphere where it is more stable at colder temperatures, PAN can be efficiently transported throughout the hemisphere (Singh, 1987; Singh and Hanst, 1981). When a PAN-containing free-tropospheric air mass subsides, PAN thermally decomposes to release NOx and can thus facili- tate O3 formation far downwind (Wild et al., 1996; Schultz et al., 1999; Jaeglé et al., 2003; Kotchenruther et al., 2001a; Hudman et al., 2004). Both PAN and O3 distributions over any northern midlatitude region reflect the combined influ- ence of production from sources within the region and trans- port from outside that region. At northern midlatitudes, the intercontinental influence from anthropogenic emissions on surface O3 levels is largest during spring (e.g., HTAP 2010) and occurs via at least two pathways: (1) O3 can be produced within a polluted continental boundary layer, ventilated to the free troposphere and efficiently transported to other con- To better distinguish among disparate estimates for inter- continental O3 transport in the published literature, the Task Force on Hemispheric Transport of Air Pollution (HTAP) or- ganized an international global modeling study, referred to here as HTAP1. The HTAP1 study identified a range of a factor of 2 across individual model estimates of surface O3 response to changes in anthropogenic precursor emissions from continental-scale, northern midlatitude source regions (HTAP, 2007, 2010; Fiore et al., 2009; Wild et al., 2012). The HTAP1 models do not distinguish between intercontinental O3 transport occurring due to O3 produced from PAN chem- istry and direct transport of O3 formed in a remote boundary layer, but other work indicates that both pathways contribute. Jaegle et al. (2003) find that 28 % of the O3 in the Pacific Northwest free troposphere between 0 and 6 km is associ- ated with PAN-to-NOx conversion, consistent with Jiang et al. (2016), who found that PAN produced from East Asian emissions and exported to the free troposphere contributes 35 % and 25 % in spring and summer, respectively, to the www.atmos-chem-phys.net/18/15345/2018/ www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 2.1 HTAP1 model simulations Fiore et al.: A constraint on continental source–receptor relationships 15348 15348 A. M. Fiore et al.: A constraint on continental source–receptor relationships Table 1. Models contributing to the HTAP1 simulations (SR1, SR6xx, and COfromXX) used in this study. Model Resolution (lat × long × layer) Institute Model contact SR1 SR6xx COfrom xx Plotting symbol CAMCHEM-3311m13 2.5◦× 2◦× 30 NCAR, USA Peter Hess X X X Filled circle FRSGCUCI-v01 2.81◦× 2.81◦× 37 Lancaster Univ., UK Oliver Wild X X X Filled upward triangle GEMAQ-v1p0 2◦× 2◦× 28 York Univ., Canada Alex Lupu X X X Filled downward triangle GEOSChem-v07 2.5◦× 2◦× 30 Harvard Univ., USA Rokjin Park X X X Filled diamond GISS-PUCCINI- modelE 5◦× 4◦× 23 NASA GISS, USA Drew Shindell X X X Filled square GMI-v02f 2.5◦× 2◦× 42 NASA GSFC, USA Bryan Duncan X X X Open circle LMDZ3-INCA1 3.75◦× 2◦× 19 CEA, France Sophie Szopa X X Open upward triangle LLNL-IMPACT-T5a 2.5◦× 2◦× 48 LLNL, USA Dan Bergmann X Open downward triangle MOZARTGFDL-v2 1.88◦× 1.88◦× 28 NOAA GFDL, USA Arlene Fiore X X X Open diamond MOZECH-v16 1.88◦× 1.88◦× 28 FZ Jülich, Germany Martin Schultz X X X Open square STOC-HadAM3-v01 5◦× 5◦× 19 University of Edinburgh, UK Ruth Doherty, David Stevenson X X X Plus sign STOCHEM-v02 3.75 × 2.5◦× 20 Met Office, Hadley Center, UK Bill Collins, Michael Sanderson X X TM5-JRC-cy2-ipcc-v1 1◦× 1◦× 25 JRC, Italy Frank Dentener X X X Filled right-facing triangle UM-CAM-v01 3.75◦× 2.5◦× 19 University of Cambridge, UK, and NIWA, New Zealand Guang Zeng X X X Filled left-facing triangle ble 1. Models contributing to the HTAP1 simulations (SR1, SR6xx, and COfromXX) used in this study. denote the tracers emitted from EA, NA, and EU, respec- tively (Table 2; see also Doherty et al., 2013, and Shindell et al., 2008). For HTAP1, each model used its own emissions invento- ries (see Table A1 of Fiore et al., 2009); Fiore et al. (2009) provide emission totals within each HTAP1 source region for all (their Table A2) and anthropogenic (their Table A3) emissions of NOx, non-methane volatile organic compounds (NMVOCs), and CO. The relative inter-model spread in re- gional anthropogenic emissions is smallest for NOx emis- sions in EU and NA (< 10 %) and largest for VOCs from EU (58 %) (Fiore et al., 2009). 2.1 HTAP1 model simulations We use monthly mean PAN mixing ratios for the year 2001 simulated by 14 global chemistry transport models (Table 1); the temporal resolution for three-dimensional chemical fields archived from the HTAP1 models is limited to monthly. We use four HTAP1 source–receptor (SR) simulations (Ta- ble 2): a base case (SR1) and three perturbation simula- tions in which anthropogenic O3 precursor emissions (NOx, VOCs, carbon monoxide, and aerosols) are reduced simul- taneously by 20 % within East Asia (EA, SR6EA), Europe and northern Africa (EU, SR6EU), and North America (NA, SR6NA). We calculate PAN SRRs by differencing the per- turbation and base simulations (SR1-SR6XX), for which XX refers to the region in which emissions of PAN precursors were decreased by 20 %. A challenge in discriminating among model estimates of O3 produced from different source regions is the lack of di- rect observational constraints on SRRs. For example, Fiore et al. (2009) did not find any relationship across models be- tween their biases against surface O3 observations and the strength of their response to emission changes. In the absence of an observable quantity to constrain these relationships, one approach is to identify an “emergent constraint” (Borod- ina et al., 2017), whereby an unobservable quantity corre- lates strongly across a multi-model ensemble with an ob- served variable. The inter-model range of the non-observable quantity is then narrowed by limiting it to the range encom- passed by the models closest to the observed variable. This approach has gained traction for narrowing the spread across future climate projections (e.g., Hall and Qu, 2006; Cox et al., 2018). Given that PAN both directly facilitates interconti- nental ozone transport and can serve as a proxy for ozone for- mation chemistry, and that prior work indicates a large sig- nature of PAN originating from the European boundary layer during spring at Jungfraujoch (Pandey Deolal et al., 2013, Of the models in Table 1, 11 used 2001 meteorological fields. Two models are chemistry-transport models coupled directly to a general circulation model forced by observed sea surface temperatures (STOC-HadAM3 and STOCHEM) and one model incorporates chemistry directly into a general cir- culation model (UM-CAM). We include these models as our evaluation compiles PAN measurements across several years (Sect. 2.2). The individual model specifications and emis- sions are described in Tables 1 and 2 of Fiore et al. (2009). www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. 2.2 Multiyear PAN measurements at mountaintop sites and model sampling (2010, 2011) 121.687◦W 30 Aug–7 Oct 2008, https://digital.lib.washington.edu/researchworks/ 26 Mar–20 May 2009, (last access: 22 October 2018) 23 Mar–25 May 2010 Hohenpeissenberg 47.80◦N, 985 m Jan 2003–Dec 2008 http://ds.data.jma.go.jp/gmd/wdcgg/cgi-bin/wdcgg 11.02◦E (last access: 22 October 2018) Gilge et al. (2010) Jungfraujoch 46.55◦N, 3580 m Apr 1997–May 1998, Balzani Lööv et al. (2008); 7.98◦E Aug 30 2005–16 Sep 2005, Carpenter et al. (2000); Throughout (2005); Zellweger et al. (2000, 2003) but not continuous Zugspitze 47.42◦N, 2960 m May 2004–Dec 2008 https://gaw.kishou.go.jp 10.98◦E (last access: 16 October 2018) Schauinsland 47.92◦N, 1205m Jan 1995–Dec 2010 https://www.umweltbundesamt.de 7.92◦E (last access: 22 October 2018) A. M. Fiore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships Table 2. Simulations from HTAP1 used in this study. Simulation Description SR1 Base case (see Sect. 2.1 for details). SR6EA SR1 but with anthropogenic emissions of all O3 precursors (NOx + CO + NMVOC) and aerosols within EA decreased by 20 %. SR6EU SR1 but with 20 % emissions reductions within the EU region SR6NA SR1 but with 20 % emissions reductions within the NA region. COfromEA Idealized tracer simulation in which all models use identical CO emissions, emitted within the EA region, with a 50-day e-folding lifetime. COfromEU Same as COfromEA but for the EU region. COfromNA Same as COfromEA but for the NA region. 15349 Table 2. Simulations from HTAP1 used in this study. Table 3. Mountaintop sites with multiple years of PAN observations used in this study. Site Location Elevation Measurement period (s) Reference (s) Mount Bachelor 43.979◦N, 2763 m 3 Apr–18 Jun 2008, Fischer et al. (2010, 2011) 121.687◦W 30 Aug–7 Oct 2008, https://digital.lib.washington.edu/researchworks/ 26 Mar–20 May 2009, (last access: 22 October 2018) 23 Mar–25 May 2010 Hohenpeissenberg 47.80◦N, 985 m Jan 2003–Dec 2008 http://ds.data.jma.go.jp/gmd/wdcgg/cgi-bin/wdcgg 11.02◦E (last access: 22 October 2018) Gilge et al. (2010) Jungfraujoch 46.55◦N, 3580 m Apr 1997–May 1998, Balzani Lööv et al. (2008); 7.98◦E Aug 30 2005–16 Sep 2005, Carpenter et al. (2000); Throughout (2005); Zellweger et al. (2000, 2003) but not continuous Zugspitze 47.42◦N, 2960 m May 2004–Dec 2008 https://gaw.kishou.go.jp 10.98◦E (last access: 16 October 2018) Schauinsland 47.92◦N, 1205m Jan 1995–Dec 2010 https://www.umweltbundesamt.de 7.92◦E (last access: 22 October 2018) Table 3. Mountaintop sites with multiple years of PAN observations used in this study. at Mount Bachelor (Fischer et al., 2010). 2.2 Multiyear PAN measurements at mountaintop sites and model sampling To evaluate the HTAP1 models, we compiled April mean climatologies of lower-tropospheric PAN measurements from northern midlatitude mountain observatories (Table 3). Given the large interannual variability in PAN abundances, we require at least 2 years of observations in April. PAN observations from Mount Bachelor (USA), Jungfraujoch (Switzerland), and Zugspitze (Schneefernerhaus), Hohen- peissenberg, and Schauinsland (all in Germany) meet these criteria. Taken together, these mountaintop measurements span 15 years, from 1995 to 2010 (Table 3), although only one site (Schauinsland) overlaps with the HTAP1 simulation year of 2001. To separate the role of inter-model differences in trans- port from the combined impacts of inter-model differences in emissions and chemistry on simulated PAN at the mountain- top sites, we analyze an additional set of idealized tracer sim- ulations available from 11 models (COfromXX in Table 1, for which XX is the source region). In these simulations, a set of tagged carbon-monoxide-like tracers are emitted, each from a single HTAP1 source region with a 50-day lifetime, and with identical emissions across models. Biomass burning emissions for the CO tracers are from GFED (van der Werf et al., 2006, 2010) and other emissions are from the RETRO project (Schultz et al., 2007, 2008). We refer to these tracers as “COfromEA”, “COfromNA”, and “COfromEU”, which PAN was measured at all five mountain sites using gas chromatography with electron capture detection (ECD). A custom system using a Shimadzu Mini-2 ECD was employed www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15349 Table 2. Simulations from HTAP1 used in this study. Simulation Description SR1 Base case (see Sect. 2.1 for details). SR6EA SR1 but with anthropogenic emissions of all O3 precursors (NOx + CO + NMVOC) and aerosols within EA decreased by 20 %. SR6EU SR1 but with 20 % emissions reductions within the EU region SR6NA SR1 but with 20 % emissions reductions within the NA region. COfromEA Idealized tracer simulation in which all models use identical CO emissions, emitted within the EA region, with a 50-day e-folding lifetime. COfromEU Same as COfromEA but for the EU region. COfromNA Same as COfromEA but for the NA region. Table 3. Mountaintop sites with multiple years of PAN observations used in this study. Site Location Elevation Measurement period (s) Reference (s) Mount Bachelor 43.979◦N, 2763 m 3 Apr–18 Jun 2008, Fischer et al. 2.2 Multiyear PAN measurements at mountaintop sites and model sampling The commercially available Meteorologie Consult (GmbH) system was used at the European sites (Zellweger et al., 2000). Calibrations generate PAN from the photolysis of excess acetone and NO in air (Warneck and Zerbach, 1992; Volz-Thomas et al., 2002). Reported detection limits are ∼20 ppt for PAN mea- surements at Mount Bachelor and ∼50 ppt for the European sites, with total uncertainties of < 10 % (Fischer et al., 2010; Zellweger et al., 2003). For comparison with the observations, we sample each model on its native grid (Table 1) at the horizontal grid cell containing the latitude and longitude of each mountain site. Orography at these mountain sites is poorly resolved at the relatively coarse HTAP1 model horizontal resolutions. This mismatch requires us to apply some approximations for ver- tical sampling. We convert the station altitude to an approxi- mate pressure level by assuming a mean tropospheric temper- ature of 260 K and a corresponding atmospheric scale height of 7.6 km. We then use monthly mean pressure fields from each model to linearly interpolate PAN based on the pres- sures of the two model grid cells that vertically bound the station pressure. While different sampling strategies may al- ter the exact value of simulated PAN and its comparison to observations, our primary interest is in the inter-model differ- ences. Although the Zugspitze and Hohenpeissenberg sites fall within the same horizontal grid cell in the HTAP1 mod- els, the station altitudes differ, so we consider the two sites separately. We include all available data at these sites without filter- ing for upslope winds or any other criteria. At Mount Bach- elor, the cleanest of the five mountaintop sites (Supplement Fig. S1), Fischer et al. (2010) have shown that PAN mix- ing ratios are not primarily controlled by diurnal wind pat- terns, which lead to variations an order of magnitude smaller than the total observed range in measured PAN. When mea- surements fall below the detection limit, we include half of the detection limit. This assumption should not affect our conclusions as mountaintop sites generally sample free- tropospheric air at night (e.g., Weiss-Penzias et al., 2004), but PAN values below the detection limit typically occur due to deposition in a shallow nocturnal boundary layer. Given that we seek constraints on intercontinental trans- port from the three major midlatitude source regions, we con- duct a more in-depth analysis at the highest-altitude Euro- Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15350 pean site (Jungfraujoch), the most likely of the available sites to measure PAN transported among continents in the free tro- posphere, as well as at Mount Bachelor in North America. At Jungfraujoch, we also evaluate SRRs in the models with an estimate of PAN originating in the European boundary layer based on an analysis of 20-day back trajectories (Pandey De- olal et al., 2013). We conduct a proof-of-concept analysis at Mount Waliguan in Asia (36.28◦N, 100.90◦E, 3816 m) to assess the potential for future PAN measurements at this site to narrow the inter-model range in SRRs. Short-term mea- surements have previously been collected at this site (Xue et al., 2011). While aircraft and satellite observations have ad- vanced the understanding of the chemistry and dynamics of individual PAN plumes using models that archived chemi- cal fields at high temporal frequencies (e.g., Alvarado et al., 2010; Payne et al., 2014; Emmons et al., 2015), their limited temporal coverage is not well suited for comparison with the HTAP1 monthly mean PAN mixing ratios. across the models. The multi-model mean falls in the range of the measurements at four of the sites but is higher than ob- served in any year at Mount Bachelor. The model rankings show some consistency across the different sites, suggest- ing systematic model differences that can be narrowed with a limited set of observational constraints, especially for mod- els that rank similarly across the sites on all three continents (Fig. 3). For example, CAMCHEM and GEMAQ are consis- tently at the higher end of the range while GISS-PUCCINI and LLNL-IMPACT are at the low end. The two mod- els falling closest to the observed 2001 value at Schauins- land (MOZECH and MOZARTGFDL) fall into the observed range at either Mount Bachelor or Jungfraujoch; we analyze these two sites further in the following sections. The longest observational dataset at Schauinsland varies by over a factor of 3 across years, consistent with large in- terannual variability found in prior analyses at mountaintop sites (Zellweger et al., 2003; Fischer et al., 2011; Pandey De- olal et al., 2013, 2014). All but one of the models (LLNL- IMPACT) fall within the wide range of observed interan- nual variability at Schauinsland, underscoring the tenuous nature of conclusions regarding model performance drawn from short observational records unless the modeled and ob- served meteorological years match. A. M. Fiore et al.: A constraint on continental source–receptor relationships Future work to coor- dinate consistent time periods between measurements and models would provide tighter constraints than are possible with our proof-of-concept analysis described in the follow- ing sections. 3 Modeled and measured lower tropospheric PAN at northern midlatitudes in April Our goal is to assess the potential for mountaintop PAN mea- surements to discriminate among model estimates of PAN and O3 produced by regional anthropogenic emissions and transported to the mountaintop sites. We thus focus our anal- ysis on April when measured PAN reaches its seasonal max- imum (Penkett and Brice, 1986; Singh and Salas, 1989; Bottenheim et al., 1994; Schmitt and Volz-Thomas, 1997; Fig. S1) and when the HTAP1 models indicate that the pro- duction of PAN from the EA, EU, and NA source regions dominates total simulated PAN (Fig. 1). April thus offers the strongest possible signal of the influence of anthropogenic emissions from these three northern midlatitude source re- gions in the mountaintop measurements. 4 Exploring emergent constraints on model SRRs from measured total PAN The range of the PAN SRRs across the HTAP1 models at Jungfraujoch, Mount Bachelor, and Mount Waliguan is wide for all three source regions, spanning a factor of 5 or more in several cases (Fig. 4). The key to a successful emer- gent constraint analysis is for this range in inter-model PAN SRRs, our unobservable quantity, to correlate with the total PAN simulated at the mountaintop site, our observable vari- able. The strongest correlations emerge for PAN originating in the region where the mountain is located, but some in- tercontinental SRR pairs also show significant correlations (p ≤0.05) with total simulated PAN (Fig. 4). Figure 2 shows the spatial distribution of the HTAP1 model ensemble mean PAN mixing ratios at 650 hPa (∼3 km), the level sampled by the highest-altitude sites on which we focus the majority of our analysis. PAN mixing ratios in April generally increase with latitude, as expected from the strong thermal dependence of the PAN lifetime, al- though some of the highest mixing ratios are simulated over the Asian source region. The multi-model spread in lower- tropospheric PAN, represented by the coefficient of variation (standard deviation over the 14 models divided by the model ensemble mean) is within ±45 % across much of the North- ern Hemisphere (Fig. 2). The large inter-model spread over much of Europe in Fig. 2b implies that observational con- straints in this region would be particularly valuable. We illustrate here how PAN measurements can be used to narrow the inter-model range in the SRR pairs. For the sites with significant correlations, the range across years (i.e., red vertical lines in Fig. 4) bound the April mean values observed at Jungfraujoch and Mount Bachelor. The models falling in this range are highlighted in red. We select these models to narrow the range in SRRs, indicated by the red horizontal dashed lines extending from the bounding models (red sym- bols) to the ordinate axis. Figure 4 shows that the constraint from total measured PAN narrows the inter-model range in SRRs for PAN by at least half, revealing some models as out- Observed and modeled PAN mixing ratios at the north- ern midlatitude mountain sites are compared in Fig. 3 (see Fig. S1 for a comparison extended throughout the year). We consider the measured range across years to bound the “plausible” portion of the wide range in simulated total PAN Atmos. Chem. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships Multi-model ensemble (n = 14; Table 1) average PAN mixing ratios (ppt; a) and relative standard deviation (the absolute standard deviation across the models divided by the ensemble mean; b) at 650 hPa in April; relative standard deviations are masked out (white) for regions where multi-model mean PAN falls below 100 ppt. The models were sampled at 650 hPa by vertically interpolating among the bounding grid cells and then re-gridded horizontally to a common 1◦×1◦grid. White lines denote the HTAP1 source regions: North America (NA), Europe and North Africa (EU), and East Asia (EA) from left to right. White circles indicate the five mountain sites with multiyear PAN observations used in our analysis (note that Zugspitze and Hohenpeissenberg are too close to differentiate on the map; see Table 3). Mount Waliguan in Asia, where we lack multiyear measurements but conduct model analysis, is denoted by the white square. 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 25 100 175 250 325 400 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 0.30 0.40 0.50 0.60 0.70 (a) Multi-model mean PAN (ppt) (b) Multi-model relative standard deviation April 650 hPa (b) Multi-model relative standard deviation Figure 2. Multi-model ensemble (n = 14; Table 1) average PAN mixing ratios (ppt; a) and relative standard deviation (the absolute standard deviation across the models divided by the ensemble mean; b) at 650 hPa in April; relative standard deviations are masked out (white) for regions where multi-model mean PAN falls below 100 ppt. The models were sampled at 650 hPa by vertically interpolating among the bounding grid cells and then re-gridded horizontally to a common 1◦×1◦grid. White lines denote the HTAP1 source regions: North America (NA), Europe and North Africa (EU), and East Asia (EA) from left to right. White circles indicate the five mountain sites with multiyear PAN observations used in our analysis (note that Zugspitze and Hohenpeissenberg are too close to differentiate on the map; see Table 3). Mount Waliguan in Asia, where we lack multiyear measurements but conduct model analysis, is denoted by the white square. liers. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15351 0 2 4 6 8 10 12 Month 0 50 100 150 200 250 PAN (ppt) 0 2 4 6 8 10 12 Month 0 100 200 300 400 PAN (ppt) (a) Mount Bachelor (b) Jungfraujoch Figure 1. Multi-model monthly mean total PAN mixing ratios (black circles and solid lines) at Mount Bachelor (a) and Jungfraujoch (b). We take the difference between the base simulation (SR1) and one in which emissions are decreased by 20 % and then multiply the difference by 5 to estimate a 100 % contribution associated with anthropogenic precursor emissions from Europe (green), North America (red), and East Asia (blue). The sum of the anthropogenic contribution from these three regions is shown (dashed black) for comparison with total simulated PAN. 0 2 4 6 8 10 12 Month 0 50 100 150 200 250 PAN (ppt) 0 2 4 6 8 10 12 Month 0 100 200 300 400 PAN (ppt) (a) Mount Bachelor (b) Jungfraujoch 12 0 2 4 6 8 10 12 Month 0 100 200 300 400 PAN (ppt) (b) Jungfraujoch (b) Jungfraujoch Figure 1. Multi-model monthly mean total PAN mixing ratios (black circles and solid lines) at Mount Bachelor (a) and Jungfraujoch (b). We take the difference between the base simulation (SR1) and one in which emissions are decreased by 20 % and then multiply the difference by 5 to estimate a 100 % contribution associated with anthropogenic precursor emissions from Europe (green), North America (red), and East Asia (blue). The sum of the anthropogenic contribution from these three regions is shown (dashed black) for comparison with total simulated PAN. 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 25 100 175 250 325 400 10o N 20o N 30o N 40o N 50o N 60o N 70o N 80o N 180o 120o W 60o W 0o 60o E 120o E 180o 0.30 0.40 0.50 0.60 0.70 (a) Multi-model mean PAN (ppt) (b) Multi-model relative standard deviation April 650 hPa Figure 2. A. M. Fiore et al.: A constraint on continental source–receptor relationships Other models simulate SRRs within the observationally constrained range (between the dashed red horizontal lines) despite falling outside the observed range for total PAN, pos- sibly indicating a role for inter-model differences in non- anthropogenic sources of PAN or in the relative contributions from the individual midlatitude source regions, which we in- vestigate further in the next section. Given the year-to-year variability in total PAN, stronger constraints could be placed in future work in which the model meteorology corresponds to the same year as the measurements. region. The horizontal blue dashed lines indicate the bounds obtained from this trajectory-based approach to estimating PAN from EU. The models falling in these bounds overlap with those constrained by the total PAN measurements, lend- ing some confidence that these two independent approaches (one using total PAN and the correlated inter-model spread in SRRs; the other using back trajectories to estimate SRRs) yield useful constraints on the influence of the EU source re- gion on PAN measured at Jungfraujoch. We note that for consistency with the model SRRs in Fig. 4, which are the responses to 20 % emission reduc- tions in the source region, we divide the Pandey Deolal et al. (2013) EU SRRs by 5 to scale back from their estimated “full contribution” (100 %). This linear scaling of the PAN response between 20 % and 100 % may incur errors due to nonlinear chemistry. With an additional simulation in which the FRSGCUCI model sets European anthropogenic emis- sions of NOx, CO, and VOCs to zero (a 100 % perturbation), we estimate this error to be ∼10 %. For intercontinental re- gions, this error reduces to < 3 %. Earlier work shows that At Jungfraujoch, we additionally consider PAN SRRs for the EU source region with those estimated previously using back-trajectory analysis (Pandey Deolal et al., 2013). While Pandey Deolal et al. (2013) also attribute trajectories to NA and EA, fewer than 15 % and 4 % of trajectories are attributed to those regions compared to 25 %–50 % from EU (range across years; see Fig. 1 of Pandey Deolal et al., 2013). Com- bining these low frequencies with the inevitable growth in uncertainty as trajectories lengthen, we have the most confi- dence in the Pandey Deolal et al. (2013) estimates for the EU 4 Exploring emergent constraints on model SRRs from measured total PAN Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ A. M. Fiore et al.: A constraint on continental source–receptor relationships ore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships 15352 Total PAN MBO ZUG JFJ HOH SCH MTW 0 500 1000 1500 2000 ppt Figure 3. April mean PAN abundances (ppt) simulated (black sym- bols, one per model as defined in Table 1; blue circles offset to the left show multi-model mean values) and measured (red circles offset to the right of the model values) at northern midlatitude mountain- top sites: Mount Bachelor (MBO), Zugspitze (ZUG), Jungfraujoch (JFJ), Hohenpeissenberg (HOH), Schauinsland (SCH), and Mount Waliguan (MTW). The observed year 2001 April mean, which cor- responds to the meteorological year used by most of the models at Schauinsland, is shown in blue to the right of the models. Total PAN MBO ZUG JFJ HOH SCH MTW 0 500 1000 1500 2000 ppt differences in regional AVOC emissions in contributing to the inter-model range in PAN SRRs. Differences in model transport (e.g., Arnold et al., 2015; Orbe et al., 2017) may also contribute to the inter-model dif- ferences in PAN SRRs. Our analysis of the HTAP1 ideal- ized CO tracers, however, reveals little correlation between inter-model differences in these idealized tracers (which have identical regional emissions and lifetimes applied in all of the models) and in the PAN SRRs sampled at these sites. Although we do not find any clear overall correlation, differ- ences in the idealized CO tracers explain some of the scatter in Fig. 5. For example, at Jungfraujoch for EU AVOC emis- sions of 22 Tg C a−1, the lowest model (GISS-PUCCINI) has one of the smallest values for the COfromEU tracer, whereas the highest model (STOC-HadAM3) has the largest value of COfromEU. Figure 3. April mean PAN abundances (ppt) simulated (black sym- bols, one per model as defined in Table 1; blue circles offset to the left show multi-model mean values) and measured (red circles offset to the right of the model values) at northern midlatitude mountain- top sites: Mount Bachelor (MBO), Zugspitze (ZUG), Jungfraujoch (JFJ), Hohenpeissenberg (HOH), Schauinsland (SCH), and Mount Waliguan (MTW). The observed year 2001 April mean, which cor- responds to the meteorological year used by most of the models at Schauinsland, is shown in blue to the right of the models. In light of the dependence of inter-model differences in PAN attributed to EU and NA during April and the corre- sponding regional AVOC emissions, we illustrate how one could extend our emergent constraints in Fig. A. M. Fiore et al.: A constraint on continental source–receptor relationships 4 (horizontal dashed red lines) to the regional AVOC emission estimates shown in Fig. 5. A major caveat underlying this analysis is the mismatch between meteorological years for the models and measurements as discussed above, and the underlying assumption that the relationships in Fig. 5 can exclusively be attributed to differences in the AVOC emissions (as opposed to chemistry or transport). The observationally constrained SRRs between PAN from NA and total PAN measured at Jungfraujoch and Mount Bachelor can be used to narrow the range of NA AVOC emissions to 12–18 Tg C a−1 (the low end is ruled out by the constraint imposed by PAN from NA at Jungfraujoch; the high end is ruled out by PAN from NA at Mount Bachelor). Similarly, the range for EU AVOC emis- sions would narrow to 16–25 Tg C a−1. the smaller nonlinearity in PAN for intercontinental versus regional source–receptor pairs also holds for ozone (Fiore et al., 2009; Wu et al., 2009; Wild et al., 2012) and demon- strates approximate linearity between the simulated tropo- spheric ozone burden and ±50 % of present-day global NOx emissions (Stevenson et al., 2006). 5 Factors contributing to the inter-model range in PAN SRRs We consider next the importance that various models as- cribe to a given source region relative to another source re- gion. We first correlate the ratios of PAN from two differ- ent source regions with the total PAN simulated by the indi- vidual models in April. We find little relationship, with the exception of Mount Bachelor, where the observational con- straint implies that more PAN originating from EA should be present at Mount Bachelor than PAN originating from NA (Fig. 6a). We interpret this as indicating that models with higher total PAN at Mount Bachelor are overestimating North American influence at this mountain site (which sam- ples free-tropospheric air). This interpretation is supported by the idealized CO tracer simulations (with identical re- gional emissions and the same lifetime applied in all the models), which suggest that some of the variance in the ratio of PAN from NA to EA at Mount Bachelor is due to differ- ences in transport from the two regions (Fig. 6b). We empha- size that these transport differences do not simply reflect the use of different meteorology in the models (Fig. 6b). We investigate the role of inter-model differences in re- gional emissions of PAN precursors versus transport in con- tributing to inter-model differences in the PAN response to continental-scale emission changes at the three mountaintop sites shown in Fig. 4. At each site, we examine the corre- lation across models between simulated PAN SRRs and re- gional anthropogenic emissions of VOCs (AVOCs; Fig. 5) or NOx (ANOx). The relationships for the EA SRRs are not sig- nificant, even at Mount Waliguan. We find, however, that the inter-model range in regional AVOC emissions explains as much as 64 % of the variation in PAN attributed to EU emis- sions, and at least 25 % of the variance in PAN attributed to the NA region (Fig. 5). In contrast to AVOCs, we find lit- tle relationship between the range in simulated PAN SRRs at the mountain sites and the model spread in regional ANOx emissions. Fischer et al. (2014) have previously shown that PAN abundances respond more strongly to changes in emis- sions of VOCs than of NOx. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships At Jungfraujoch, the HTAP1 multi-model mean attributes much of the PAN to emissions from the EU and NA source regions during April (Fig. 1). The ratio of PAN attributed to EU to PAN attributed to NA at Jungfraujoch, however, varies from approximately 0.5 to 2 across the individual HTAP1 models (Fig. S2). In contrast to our findings at Mount Bachelor, this ratio at Jungfraujoch depends most strongly on the ratio of ANOx emissions in the EU to NA regions (r = 0.6) and more the relative importance of emissions within the source region, where the measurement site is located, versus the upwind intercontinental source region of PAN (Fig. S2). At Mount Bachelor, the HTAP1 multi-model mean SRRs from NA, EA, and EU are roughly equal in April (Fig. 1). The differences across the HTAP models in the relative importance of the NA : EA source regions of PAN (which range from about 0.5 to 2.5) correlate roughly equally with the ratio of the NA : EA CO transport tracers and with the ratio of the NA : EA AVOC emissions (Spearman’s rank correlation coefficient (r) = 0.6 for both cases); we find no relationship with the ratio of the NA : EA ANOx emissions (Figs. 6b and S2, upper left). At Jungfraujoch, the HTAP1 multi-model mean attributes much of the PAN to emissions from the EU and NA source regions during April (Fig. 1). The ratio of PAN attributed to EU to PAN attributed to NA at Jungfraujoch, however, varies from approximately 0.5 to 2 across the individual HTAP1 models (Fig. S2). In contrast to our findings at Mount Bachelor, this ratio at Jungfraujoch depends most strongly on the ratio of ANOx emissions in the EU to NA regions (r = 0.6) and more We repeat this correlation analysis of inter-model differ- ences in ratios of ANOx emissions, AVOC emissions, or the idealized CO tracers of transport from a region, but for the ratio of PAN SRRs from two intercontinental regions. At Mount Bachelor, the EU and EA source regions con- tribute similar amounts to multi-model mean PAN during April (Fig. 1). Across the individual models, however, the ratio of the EU to EA source regions on PAN at Mount Bach- elor varies from less than half to a factor of 2 (Fig. S3). A. M. Fiore et al.: A constraint on continental source–receptor relationships We find that the ratio of PAN attributed to the EU versus EA source regions at Mount Bachelor correlates strongly across the models with the ratio of the AVOC emissions in the re- spective source regions (r = 0.8; Fig. S3). In contrast, the 5 Factors contributing to the inter-model range in PAN SRRs Our analysis supports that ear- lier finding and furthermore highlights a key role for model By comparing NA : EA at Mount Bachelor, EU : NA at Jungfraujoch, and EA : EU at Mount Waliguan, we examine www.atmos-chem-phys.net/18/15345/2018/ www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships For source–receptor pairs with significant correlations (p ≤0.05), models falling within the observed range (across years) are colored red, and horizontal red dashed lines extend to the ordinate, representing the emergent constraint (narrower range resulting from selecting only those models falling in the observed range of total PAN). At Jungfraujoch, the range (across years) in PAN attributed to the EU source region by back-trajectory analysis (Pandey Deolal et al., 2013) is indicated by horizontal dashed blue lines. Individual models are denoted by the symbols defined in Table 1. g j Mount Bachelor, vertical red lines bound the observed range in total PAN. For source–receptor pairs with significant correlations (p ≤0.05), models falling within the observed range (across years) are colored red, and horizontal red dashed lines extend to the ordinate, representing the emergent constraint (narrower range resulting from selecting only those models falling in the observed range of total PAN). At Jungfraujoch, the range (across years) in PAN attributed to the EU source region by back-trajectory analysis (Pandey Deolal et al., 2013) is indicated by horizontal dashed blue lines. Individual models are denoted by the symbols defined in Table 1. weakly on the ratio of EU : NA AVOC emissions (r = 0.5; Fig. S2). The correlation is even weaker between the ratio of PAN SRRs for these two regions with inter-model dif- ferences in transport as diagnosed with the CO tracers from EU to NA (r = 0.4). At Mount Waliguan, the strongest rela- tionship is found for the ratio of AVOC emissions (r = 0.5; Fig. S2). the relative importance of emissions within the source region, where the measurement site is located, versus the upwind intercontinental source region of PAN (Fig. S2). At Mount Bachelor, the HTAP1 multi-model mean SRRs from NA, EA, and EU are roughly equal in April (Fig. 1). The differences across the HTAP models in the relative importance of the NA : EA source regions of PAN (which range from about 0.5 to 2.5) correlate roughly equally with the ratio of the NA : EA CO transport tracers and with the ratio of the NA : EA AVOC emissions (Spearman’s rank correlation coefficient (r) = 0.6 for both cases); we find no relationship with the ratio of the NA : EA ANOx emissions (Figs. 6b and S2, upper left). A. M. Fiore et al.: A constraint on continental source–receptor relationships ore et al.: A constraint on continental source–receptor relationships 15353 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.58 p=0.05 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.85 p=0.01 100 200 300 400 500 600 700 Total PAN (ppt) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.77 p=0.01 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from NA (ppt) r=0.97 p=0.00 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from EU (ppt) r=0.76 p=0.01 100 200 300 400 500 Total PAN (ppt) 0 5 10 15 20 PAN from EA (ppt) r=0.22 p=0.46 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 PAN from NA (ppt) r=0.45 p=0.14 100 200 300 400 500 600 700 Total PAN (ppt) 0 10 20 30 40 PAN from EU (ppt) r=0.22 p=0.46 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 PAN from EA (ppt) r=0.59 p=0.05 Jungfraujoch Mount Bachelor Mount Waliguan Figure 4. Simulated total PAN versus source–receptor relationships (SRRs) at each of three northern midlatitude sites. For Jungfraujoch and Mount Bachelor, vertical red lines bound the observed range in total PAN. For source–receptor pairs with significant correlations (p ≤0.05), models falling within the observed range (across years) are colored red, and horizontal red dashed lines extend to the ordinate, representing the emergent constraint (narrower range resulting from selecting only those models falling in the observed range of total PAN). At Jungfraujoch, the range (across years) in PAN attributed to the EU source region by back-trajectory analysis (Pandey Deolal et al., 2013) is indicated by horizontal dashed blue lines. Individual models are denoted by the symbols defined in Table 1. A. M. Fiore et al.: A constraint on continental source–receptor relationships 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.85 p=0.01 Jungfraujoch 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.58 p=0.05 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.85 p=0.01 100 200 300 400 500 Total PAN (ppt) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.77 p=0.01 Jungfraujoch 100 200 300 400 500 600 700 Total PAN (ppt) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.77 p=0.01 300 400 500 Total PAN (ppt) Total PAN (ppt) 0 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from EU (ppt) r=0.76 p=0.01 100 200 300 400 500 Total PAN (ppt) 0 5 10 15 20 PAN from EA (ppt) r=0.22 p=0.46 Mount Bachelor Mount Bachelor 100 200 300 400 500 Total PAN (ppt) 0 10 20 30 PAN from NA (ppt) r=0.97 p=0.00 Mount Waliguan 100 200 300 400 500 600 700 Total PAN (ppt) 0 10 20 30 40 PAN from EU (ppt) r=0.22 p=0.46 f ( ) Mount Waliguan 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 PAN from NA (ppt) r=0.45 p=0.14 100 200 300 400 500 600 700 Total PAN (ppt) 0 10 20 30 40 PAN from EU (ppt) r=0.22 p=0.46 100 0 20 40 60 PAN from EA (ppt) Mount Waliguan 100 200 300 400 500 600 700 Total PAN (ppt) 0 5 10 15 20 PAN from NA (ppt) r=0.45 p=0.14 100 200 300 400 500 600 700 Total PAN (ppt) 0 20 40 60 PAN from EA (ppt) r=0.59 p=0.05 total PAN versus source–receptor relationships (SRRs) at each of three northern midlatitude sites. For Jungfra or relationships (SRRs) at each of three northern midlati Figure 4. Simulated total PAN versus source receptor relationships (SRRs) at each of three northern midlatitude sites. For Jungfraujoch and Mount Bachelor, vertical red lines bound the observed range in total PAN. A. M. Fiore et al.: A constraint on continental source–receptor relationships 20 25 Tg C a -1) 0 10 20 30 40 EU AVOC (Tg C a -1) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.80 p=0.01 5 10 15 20 25 EA AVOC (Tg C a -1) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.21 p=0.48 (b) (c) Jungfraujoch 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.49 p=0.10 0 10 20 30 40 EU AVOC (Tg C a -1) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.80 p=0.01 5 10 15 20 25 EA AVOC (Tg C a -1) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.21 p=0.48 (a) (b) (c) Jungfraujoch 5 10 15 20 25 NA AVOC (Tg C a -1) 0 10 20 30 PAN from NA (ppt) r=0.52 p=0.08 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 PAN from EU (ppt) r=0.56 p=0.06 5 10 15 2 EA AVOC (Tg C a 0 5 10 15 20 PAN from EA (ppt) r=0.33 p=0.27 (d) (e) (f) Mount Bachelor 5 20 25 (Tg C a -1) 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 PAN from EU (ppt) r=0.56 p=0.06 5 10 15 20 25 EA AVOC (Tg C a -1) 0 5 10 15 20 PAN from EA (ppt) r=0.33 p=0.27 (e) (f) Mount Bachelor Mount Bachelor 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 PAN from NA (ppt) r=0.58 p=0.05 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 40 PAN from EU (ppt) r=0.60 p=0.05 5 10 EA A 0 20 40 60 PAN from EA (ppt) r=-0.20 p=0.50 (g) (h) (i) Mount Waliguan 20 25 C a -1) 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 40 PAN from EU (ppt) r=0.60 p=0.05 5 10 15 20 25 EA AVOC (Tg C a -1) 0 20 40 60 PAN from EA (ppt) r=-0.20 p=0.50 (h) (i) Mount Waliguan he difference between the SR1 and SR6xx simulations in Table 1 for PAN (ppt) at Jungfraujoch (a, b, c), Figure 5. A. M. Fiore et al.: A constraint on continental source–receptor relationships SRRs diagnosed as the difference between the SR1 and SR6xx simulations in Table 1 for PAN (ppt) at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i) in each HTAP1 model (see Table 1 for the symbol assigned to each model) versus the annual emission of anthropogenic VOCs (AVOCs; Tg C a−1) within the NA (a, d, g), EU (b, e, h), and EA (c, f, i) source regions. The Spearman’s rank correlation coefficient (more robust to outliers than the traditional Pearson coefficient) and associated p value are shown in each panel. The horizontal red lines correspond to the values identified with the red symbols in Fig. 4. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15354 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 25 30 PAN from NA (ppt) r=0.49 p=0.10 0 10 20 30 40 EU AVOC (Tg C a -1) 0 20 40 60 80 100 120 PAN from EU (ppt) r=0.80 p=0.01 5 10 15 20 25 EA AVOC (Tg C a -1) 0 2 4 6 8 10 12 PAN from EA (ppt) r=0.21 p=0.48 5 10 15 20 25 NA AVOC (Tg C a -1) 0 10 20 30 PAN from NA (ppt) r=0.52 p=0.08 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 PAN from EU (ppt) r=0.56 p=0.06 5 10 15 20 25 EA AVOC (Tg C a -1) 0 5 10 15 20 PAN from EA (ppt) r=0.33 p=0.27 5 10 15 20 25 NA AVOC (Tg C a -1) 0 5 10 15 20 PAN from NA (ppt) r=0.58 p=0.05 0 10 20 30 40 EU AVOC (Tg C a -1) 0 10 20 30 40 PAN from EU (ppt) r=0.60 p=0.05 5 10 15 20 25 EA AVOC (Tg C a -1) 0 20 40 60 PAN from EA (ppt) r=-0.20 p=0.50 (a) (d) (g) (b) (e) (h) (c) (f) (i) Jungfraujoch Mount Bachelor Mount Waliguan Figure 5. SRRs diagnosed as the difference between the SR1 and SR6xx simulations in Table 1 for PAN (ppt) at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i) in each HTAP1 model (see Table 1 for the symbol assigned to each model) versus the annual emission of anthropogenic VOCs (AVOCs; Tg C a−1) within the NA (a, d, g), EU (b, e, h), and EA (c, f, i) source regions. The Spearman’s rank correlation coefficient (more robust to outliers than the traditional Pearson coefficient) and associated p value are shown in each panel. The horizontal red lines correspond to the values identified with the red symbols in Fig. 4. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15355 (a) 100 200 300 400 500 Total PAN (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.66 p=0.03 (b) 0.2 0.4 0.6 0.8 1.0 COFROMNA/COFROMEA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.62 p=0.05 Figure 6. Ratio of the PAN response to 20 % emission reductions within NA to that from EA plotted against (a) total PAN and (b) the ratio of idealized tracers of model transport emitted from NA to that from EA (COfromNA/COfromEA; see Table 2) at Mount Bachelor as simulated by the HTAP1 models. Each symbol in (a) represents a model as defined in Table 1; the range of observed total PAN at Mount Bachelor is indicated by the black vertical lines. The col- ored symbols in (b) represent the meteorological fields used in the simulation: blue triangles for GEOS winds, red circles for NCEP, black diamonds for ECMWF, cyan upside-down triangles for the Canadian Meteorological Centre, and green squares for general cir- culation models forced by observed sea surface temperatures and sea ice. Both panels show Spearman’s rank correlation coefficients and p values, as well as a black dashed horizontal line at 1 to sep- arate the models suggesting a higher NA influence (above) versus higher EA influence (below) on PAN SRRs. (a) 100 200 300 400 500 Total PAN (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.66 p=0.03 (b) 0.2 0.4 0.6 0.8 1.0 COFROMNA/COFROMEA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ratio of NA/EA PAN r=0.62 p=0.05 sions. Model differences in transport as diagnosed by the idealized regional CO tracers correlate more with O3 SRRs than for PAN for all source–receptor pairs, though the cor- relations remain weak except for COfromEU with O3 SRRs at Jungfraujoch. Overall, this analysis supports earlier find- ings that PAN is more sensitive to changes in emissions (and subsequent chemistry), particularly for VOC precursors, than O3. The correlations between SRRs for PAN and O3 could re- flect a role for PAN transport in contributing to O3 production over the receptor region, or may instead reflect coproduction of PAN and O3 from oxidation of regional precursor emis- sions followed by transport in the same air mass. In the lat- ter case, PAN serves as a proxy for O3 transport, whereas in the former case PAN serves as the actual pathway by which O3 is transported. A. M. Fiore et al.: A constraint on continental source–receptor relationships We do not have model diagnostics that al- low us to distinguish between these two roles for PAN. The correlations between PAN and O3 SRRs, however, suggest that long-term PAN measurements contain signals relevant for constraining the relative importance of regional vs. in- tercontinental emissions on both PAN and O3. We examine the strength of these signals by correlating the O3 SRRs at each site with total PAN as simulated at each site. Relation- ships are far weaker than for the PAN SRRs and total PAN shown in Fig. 4, but correlations are significant between total PAN at Jungfraujoch for O3 from EU (r = 0.67; p = 0.03) and at Mount Bachelor for O3 from NA (r = 0.61; p = 0.04; Fig. S4). Figure 6. Ratio of the PAN response to 20 % emission reductions Figure 6. Ratio of the PAN response to 20 % emission reductions within NA to that from EA plotted against (a) total PAN and (b) the ratio of idealized tracers of model transport emitted from NA to that from EA (COfromNA/COfromEA; see Table 2) at Mount Bachelor as simulated by the HTAP1 models. Each symbol in (a) represents a model as defined in Table 1; the range of observed total PAN at Mount Bachelor is indicated by the black vertical lines. The col- ored symbols in (b) represent the meteorological fields used in the simulation: blue triangles for GEOS winds, red circles for NCEP, black diamonds for ECMWF, cyan upside-down triangles for the Canadian Meteorological Centre, and green squares for general cir- culation models forced by observed sea surface temperatures and sea ice. Both panels show Spearman’s rank correlation coefficients and p values, as well as a black dashed horizontal line at 1 to sep- arate the models suggesting a higher NA influence (above) versus higher EA influence (below) on PAN SRRs. is relevant for interpreting O3 SRRs by correlating PAN and O3 SRRs at the three mountaintop sites (Fig. 7). Rela- tionships vary across the individual source–receptor pairs, with the inter-model variability in PAN explaining 16 %– 60 % of the inter-model differences in O3 at the mountain sites. The strongest relationships occur for the influence of regional sources at Mount Bachelor (from NA) and Jungfrau- joch (from EU). At Mount Waliguan, the EU and EA source– receptor relationships for PAN and O3 are of similar strength (r = 0.7). A. M. Fiore et al.: A constraint on continental source–receptor relationships Intercontinental source–receptor pairs for O3 and PAN at Mount Bachelor and Mount Waliguan are also sig- nificant to within 90 %, with variability in the PAN attributed to intercontinental source regions explaining 25 %–35 % and 30 %–45 %, respectively, of the variability in the correspond- ing O3 SRRs. 6 Linking PAN and O3 SRRs ratio of EU : EA anthropogenic emission influence on PAN at Mount Bachelor shows little correlation with the respec- tive regional NOx emissions used in the models, or with the differences in the simulated transport tracers (r = 0.3 for both cases). As at Mount Bachelor, the model spread in the contribution to total simulated PAN from the EA versus NA source regions at both Jungfraujoch and Mount Waliguan de- pends most on the regional AVOC ratios (r = 0.8 and 0.6, re- spectively; Fig. S3), with little correlation with inter-model differences in NA : EA ANOx emissions. Some correlation also emerges between the NA : EA SRRs for PAN and the NA : EA transport tracers (r = 0.6 at both sites; Fig. S3). Fi- nally, we do not find any obvious link between PAN SRRs and the choice of meteorological fields (the individual sym- bols in Figs. S2 and S3). We address here the extent to which observational constraints on PAN SRRs might also serve to narrow the range of un- certainty in the inter-model spread in intercontinental SRRs for O3 (e.g., Fiore et al., 2009). We expect some common- ality between the sensitivity of PAN and O3 to changes in precursor emissions because (1) both species are pro- duced from chemical reactions involving NOx and VOCs and (2) PAN serves as a NOx reservoir, which upon decomposi- tion releases NOx that can then produce O3 far downwind of the region where the PAN (and O3) precursors were origi- nally emitted. Furthermore, earlier analysis of HTAP1 ozone continental-scale SRRs also identified a correlation with the model AVOC emissions, particularly over EU (Fiore et al., 2009). We assess the extent to which the inter-model range in source region influence on mountaintop PAN levels in April Atmos. Chem. Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ 7 Conclusions and recommendations Our proof-of-concept approach applies the HTAP1 multi- model ensemble to identify a strong inter-model correlation between PAN source–receptor relationships (SRRs; defined as the difference in simulations with 20 % emission reduc- tions separately within each of the northern midlatitude con- tinents) and simulated total PAN at mountaintop sites during April. Our findings imply promise for developing emergent constraints (e.g., Hall and Qu, 2006; Borodina et al., 2017; Cox et al., 2018) from more routine PAN measurements to narrow uncertainty in wide-ranging model estimates of PAN SRRs, quantities that are not directly observable yet relevant to air quality policy (e.g., HTAP, 2010). Inter-model correla- tions of the responses of PAN versus O3 to perturbations in regional anthropogenic emissions (Figs. 7 and 8) imply that constraints on PAN SRRs are relevant for lowering uncer- tainty in O3 SRR estimates. This connection between PAN and O3 likely reflects the dual role of PAN as both a pathway for O3 transport (by producing O3 upon its decomposition following transport), and as a proxy for O3 transport (as it is produced alongside O3 in the polluted continental boundary layer). We expand the correlation analysis of ozone and PAN SRRs from the free troposphere sampled at the mountain- top sites to large-scale SRRs in surface air over the HTAP1 continental regions. Of the significant relationships in Fig. 7 (p < 0.10), six out of seven also emerge as significant in Fig. 8. We infer that conclusions drawn from a limited num- ber of mountaintop sites regarding PAN SRRs and their rela- tionship to ozone SRRs are relevant, at least according to the models, on much broader scales. We repeat the analysis in Fig. 5 but for O3 to consider the influence of the three source regions on the three mountain- top sites (nine total source–receptor pairs) but find little rela- tionship between the model spread in the simulated O3 SRRs and in the magnitude of the regional AVOCs or ANOx emis- Establishing the strongest constraints possible on simu- lated SRRs for PAN and O3 will require (1) measurements www.atmos-chem-phys.net/18/15345/2018/ A. M. Fiore et al.: A constraint on continental source–receptor relationships 30 0 20 40 60 80 100 120 PAN from EU (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 O3 from EU (ppb) r=0.77 p=0.01 2 4 6 8 10 12 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from EA (ppb) r=0.40 p=0.18 (b) (c) Jungfraujoch 5 10 15 20 25 30 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 O3 from NA (ppb) r=0.46 p=0.13 0 20 40 60 80 100 120 PAN from EU (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 O3 from EU (ppb) r=0.77 p=0.01 2 4 6 8 10 12 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from EA (ppb) r=0.40 p=0.18 (a) (b) (c) Jungfraujoch 15 20 25 30 35 rom NA (ppt) 5 10 15 20 25 30 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.59 p=0.05 0 5 10 15 20 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EA (ppb) r=0.51 p=0.09 (e) (f) Mount Bachelor 20 0 5 10 15 20 25 30 35 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.72 p=0.02 0 10 20 30 40 50 60 PAN from EA (ppt) 0.0 0.5 1.0 1.5 2.0 O3 from EA (ppb) r=0.67 p=0.03 (h) (i) Mount Waliguan Figure 7. SRRs for O3 versus PAN at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations (Table 2) available from 12 models, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Each model thus contributes one point (symbols defined in Table 1) in each panel. Spearman’s (rank) correlation coefficient and p values are also shown. Waliguan would provide constraints on PAN SRRs, partic- ularly for PAN originating in EA (Fig. 4). Additional work could systematically examine over 60 stations at altitudes above 2500 m in the Tropospheric Ozone Assessment Report (TOAR) database (Schultz et al., 2017). and simulations with chemical transport models that coincide and (2) a sufficiently long measurement record to build a cli- matology suitable for evaluating chemistry–climate models that generate their own meteorology. Repeated sampling for the month of April may be sufficient to provide constraints on model responses to changes in anthropogenic emissions. A. M. Fiore et al.: A constraint on continental source–receptor relationships PAN measurements over multiple seasons are necessary to evaluate model responses of PAN to climate change (e.g., by changing temperature and weather-sensitive precursor emis- sions) and the resulting influence on atmospheric O3 and ox- idizing capacity (e.g., Doherty et al., 2013). For example, changes in meteorology and biomass burning (Fischer et al., 2011; Zhu et al., 2015) such as those driven by El Niño– Southern Oscillation (Koumoutsaris et al., 2008), as well as biogenic and lightning sources (Payne et al., 2017) vary from year to year and are expected to change as climate warms. and simulations with chemical transport models that coincide and (2) a sufficiently long measurement record to build a cli- matology suitable for evaluating chemistry–climate models that generate their own meteorology. Repeated sampling for the month of April may be sufficient to provide constraints on model responses to changes in anthropogenic emissions. PAN measurements over multiple seasons are necessary to evaluate model responses of PAN to climate change (e.g., by changing temperature and weather-sensitive precursor emis- sions) and the resulting influence on atmospheric O3 and ox- idizing capacity (e.g., Doherty et al., 2013). For example, changes in meteorology and biomass burning (Fischer et al., 2011; Zhu et al., 2015) such as those driven by El Niño– Southern Oscillation (Koumoutsaris et al., 2008), as well as biogenic and lightning sources (Payne et al., 2017) vary from year to year and are expected to change as climate warms. We recommend archiving daily model fields for future ap- plications of this multi-model emergent constraint approach to SRRs. Access to daily model fields permits (1) a more rigorous process-oriented evaluation of specific events (e.g., Fischer et al., 2010; Alvarado et al., 2010; Arnold et al., 2015) and (2) comparison with satellite-derived tropospheric PAN columns, which show promise for documenting PAN distributions, particularly in the upper troposphere, and their temporal variability and spatial patterns across the globe (e.g., Fadnavis et al., 2014; Jiang et al., 2016; Payne et al., 2014, 2017; Zhu et al., 2015, 2017). We also suggest archiv- ing daily tracers tagged by emission region to isolate the role of model differences in transport during individual events. In addition, Lin et al. A. M. Fiore et al.: A constraint on continental source–receptor relationships A. M. Fiore et al.: A constraint on continental source–receptor relationships 5 10 15 20 25 30 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 O3 from NA (ppb) r=0.46 p=0.13 0 20 40 60 80 100 120 PAN from EU (ppt) 0.0 0.5 1.0 1.5 2.0 2.5 O3 from EU (ppb) r=0.77 p=0.01 2 4 6 8 10 12 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from EA (ppb) r=0.40 p=0.18 0 5 10 15 20 25 30 35 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from NA (ppb) r=0.67 p=0.03 5 10 15 20 25 30 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.59 p=0.05 0 5 10 15 20 PAN from EA (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EA (ppb) r=0.51 p=0.09 0 5 10 15 20 PAN from NA (ppt) 0.0 0.2 0.4 0.6 0.8 O3 from NA (ppb) r=0.56 p=0.06 0 5 10 15 20 25 30 35 PAN from EU (ppt) 0.0 0.2 0.4 0.6 0.8 1.0 O3 from EU (ppb) r=0.72 p=0.02 0 10 20 30 40 50 60 PAN from EA (ppt) 0.0 0.5 1.0 1.5 2.0 O3 from EA (ppb) r=0.67 p=0.03 (a) (d) (g) (b) (e) (h) (c) (f) (i) Jungfraujoch Mount Bachelor Mount Waliguan Figure 7. SRRs for O3 versus PAN at Jungfraujoch (a, b, c), Mount Bachelor (d, e, f), and Mount Waliguan (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations (Table 2) available from 12 models, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Each model thus contributes one point (symbols defined in Table 1) in each panel. Spearman’s (rank) correlation coefficient and p values are also shown. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 15356 A. M. Fiore et al.: A constraint on continental source–receptor relationships EU mean 0 5 10 15 PAN (SR1-SR6NA) 0.0 0.2 0.4 0.6 0.8 O3 (SR1-SR6NA) r=0.28 p=0.33 EU mean 0 20 40 60 80 100 120 PAN (SR1-SR6EU) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6EU) r=0.62 p=0.03 O3 (SR1 SR6EA) (a) (b) EU mean 0 2 4 6 8 10 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6EA) r=0.61 p=0.04 (c) PAN (SR1 SR6NA) PAN (SR1 SR6EU) NA mean 0 20 40 60 PAN (SR1-SR6NA) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6NA) r=0.49 p=0.09 NA mean 0 5 10 15 20 PAN (SR1-SR6EU) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EU) r=0.62 p=0.03 0 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EA) (d) (e) (f) ) ( ) ( ) mean 40 60 R1-SR6NA) r=0.49 p=0.09 NA mean 0 5 10 15 20 PAN (SR1-SR6EU) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EU) r=0.62 p=0.03 NA mean 0 2 4 6 8 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EA) r=0.56 p=0.05 (e) (f) EA mean 0 2 4 6 8 10 PAN (SR1-SR6NA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6NA) r=0.56 p=0.05 EA mean 0 5 10 15 20 25 30 PAN (SR1-SR6EU) 0.0 0.2 0.4 0.6 O3 (SR1-SR6EU) r=0.70 p=0.02 EA mean 0 10 20 30 40 PAN (SR1-SR6EA 0.0 0.5 1.0 1.5 O3 (SR1-SR6EA) r= p= (g) (h) (i) 10 56 05 EA mean 0 5 10 15 20 25 30 PAN (SR1-SR6EU) 0.0 0.2 0.4 0.6 O3 (SR1-SR6EU) r=0.70 p=0.02 EA mean 0 10 20 30 40 50 60 PAN (SR1-SR6EA) 0.0 0.5 1.0 1.5 O3 (SR1-SR6EA) r=0.31 p=0.29 (h) (i) Figure 8. SRRs for O3 versus PAN in surface air over each of the HTAP1 northern midlatitude continental regions: EU (a, b, c), NA (d, e, f), and EA (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Spearman’s (rank) correlation coefficient and p values are also shown. Symbols denote individual models as defined in Table 1. STOC-HadAM3-v01 is excluded here as an outlier that artificially raised the correlation significance. ture model simulations (and multi-model ensembles) can be assessed. tracers can better isolate free-tropospheric air from surface air masses when comparing coarse-resolution models with high-altitude measurements. A. M. Fiore et al.: A constraint on continental source–receptor relationships By focusing on April, our analysis largely minimizes com- plexities introduced by inter-model differences in biogenic, fire, and lightning sources that further complicate disentan- gling summertime discrepancies in simulated PAN and O3 (e.g., Arnold et al., 2015; Emmons et al., 2015) and re- stricts inter-model differences to those associated with an- thropogenic emissions and the subsequent chemistry and transport. Nevertheless, we find a wide range in inter-model SRR relationships that reflects uncertainties in emissions and different model representations of VOC chemistry, including PAN yields from VOCs (Fig. 5; Emmerson and Evans, 2009; Fischer et al., 2014; Arnold et al., 2015; Emmons et al., 2015; Knote et al., 2015). Future multi-model efforts could seek to separately parse the influence of differences in total anthro- pogenic VOC emissions, the mix of emitted VOC species and their reactivity, and the chemical production of PAN and O3. Documenting these aspects of model configuration would help to establish benchmarks for inter-model differences in simulated total PAN, O3, and their SRRs, against which fu- Data availability. Upon publication, the data used to generate the figures can be found in a CSU digital repository that we established for this paper (https://hdl.handle.net/10217/185610). A. M. Fiore et al.: A constraint on continental source–receptor relationships (2017) have demonstrated that applying a filtering technique based on daily idealized CO regional We identified only five multiyear datasets at mountain sites, four of which are located near each other in Europe, and only one of which continues at present (Schauinsland). Our analysis suggests that future measurements at Mount www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15357 EU mean 0 5 10 15 PAN (SR1-SR6NA) 0.0 0.2 0.4 0.6 0.8 O3 (SR1-SR6NA) r=0.28 p=0.33 EU mean 0 20 40 60 80 100 120 PAN (SR1-SR6EU) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6EU) r=0.62 p=0.03 EU mean 0 2 4 6 8 10 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6EA) r=0.61 p=0.04 NA mean 0 20 40 60 PAN (SR1-SR6NA) 0.0 0.5 1.0 1.5 2.0 O3 (SR1-SR6NA) r=0.49 p=0.09 NA mean 0 5 10 15 20 PAN (SR1-SR6EU) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EU) r=0.62 p=0.03 NA mean 0 2 4 6 8 PAN (SR1-SR6EA) 0.0 0.1 0.2 0.3 0.4 0.5 O3 (SR1-SR6EA) r=0.56 p=0.05 EA mean 0 2 4 6 8 10 PAN (SR1-SR6NA) 0.0 0.1 0.2 0.3 0.4 O3 (SR1-SR6NA) r=0.56 p=0.05 EA mean 0 5 10 15 20 25 30 PAN (SR1-SR6EU) 0.0 0.2 0.4 0.6 O3 (SR1-SR6EU) r=0.70 p=0.02 EA mean 0 10 20 30 40 50 60 PAN (SR1-SR6EA) 0.0 0.5 1.0 1.5 O3 (SR1-SR6EA) r=0.31 p=0.29 (a) (d) (g) (b) (e) (h) (c) (f) (i) Figure 8. SRRs for O3 versus PAN in surface air over each of the HTAP1 northern midlatitude continental regions: EU (a, b, c), NA (d, e, f), and EA (g, h, i), obtained by subtracting the SR6XX from the SR1 simulations, for which XX denotes the NA (a, d, g), EU (b, e, h), or EA (c, f, i) source region. Spearman’s (rank) correlation coefficient and p values are also shown. Symbols denote individual models as defined in Table 1. STOC-HadAM3-v01 is excluded here as an outlier that artificially raised the correlation significance. The Supplement related to this article is available online at: https://doi.org/10.5194/acp-18-15345-2018- supplement Author contributions. AMF and EVF conceived the analysis with guidance from SPD, OW, DAJ, and JS. EVF compiled monthly mean PAN measurements from EVF, DAJ, SPD, JS, BF, SG, LR, MGSa, MS, and CZ. AMF led the model evaluation, emergent con- straint analysis, and writing. AMF, GPM, and OEC prepared the figures. SPD and JS contributed trajectory analysis at Jungfrau- joch. OW contributed to linearity analysis. AMF, OW, DB, WC, FD, RMD, BND, PGH, LWH, AL, IAM, RP, MGSa, MGSc, DTS, DSS, SS, and GZ contributed HTAP Phase 1 model simulations. All References Emmons, L. K., Arnold, S. R., Monks, S. A., Huijnen, V., Tilmes, S., Law, K. S., Thomas, J. L., Raut, J.-C., Bouarar, I., Tur- quety, S., Long, Y., Duncan, B., Steenrod, S., Strode, S., Flem- ming, J., Mao, J., Langner, J., Thompson, A. M., Tarasick, D., Apel, E. C., Blake, D. R., Cohen, R. C., Dibb, J., Diskin, G. S., Fried, A., Hall, S. R., Huey, L. G., Weinheimer, A. J., Wisthaler, A., Mikoviny, T., Nowak, J., Peischl, J., Roberts, J. M., Ryerson, T., Warneke, C., and Helmig, D.: The POLARCAT Model Intercomparison Project (POLMIP): overview and eval- uation with observations, Atmos. Chem. Phys., 15, 6721–6744, https://doi.org/10.5194/acp-15-6721-2015, 2015. Alvarado, M. J., Logan, J. A., Mao, J., Apel, E., Riemer, D., Blake, D., Cohen, R. C., Min, K.-E., Perring, A. E., Browne, E. C., Wooldridge, P. J., Diskin, G. S., Sachse, G. W., Fuel- berg, H., Sessions, W. R., Harrigan, D. L., Huey, G., Liao, J., Case-Hanks, A., Jimenez, J. L., Cubison, M. J., Vay, S. A., Wein- heimer, A. J., Knapp, D. J., Montzka, D. D., Flocke, F. M., Pol- lack, I. B., Wennberg, P. O., Kurten, A., Crounse, J., Clair, J. M. St., Wisthaler, A., Mikoviny, T., Yantosca, R. M., Carouge, C. C., and Le Sager, P.: Nitrogen oxides and PAN in plumes from boreal fires during ARCTAS-B and their impact on ozone: an integrated analysis of aircraft and satellite observations, At- mos. Chem. Phys., 10, 9739–9760, https://doi.org/10.5194/acp- 10-9739-2010, 2010. Fadnavis, S., Schultz, M. G., Semeniuk, K., Mahajan, A. S., Poz- zoli, L., Sonbawne, S., Ghude, S. D., Kiefer, M., and Eck- ert, E.: Trends in peroxyacetyl nitrate (PAN) in the upper tro- posphere and lower stratosphere over southern Asia during the summer monsoon season: regional impacts, Atmos. Chem. Phys., 14, 12725–12743, https://doi.org/10.5194/acp-14-12725- 2014, 2014. Arnold, S. R., Emmons, L. K., Monks, S. A., Law, K. S., Rid- ley, D. A., Turquety, S., Tilmes, S., Thomas, J. L., Bouarar, I., Flemming, J., Huijnen, V., Mao, J., Duncan, B. N., Steen- rod, S., Yoshida, Y., Langner, J., and Long, Y.: Biomass burn- ing influence on high-latitude tropospheric ozone and reac- tive nitrogen in summer 2008: a multi-model analysis based on POLMIP simulations, Atmos. Chem. Phys., 15, 6047–6068, https://doi.org/10.5194/acp-15-6047-2015, 2015. Fiore, A. M., Dentener, F. J., Wild, O., Cuvelier, C., Schultz, M. G., Hess, P., Textor, C., Schulz, M., Doherty, R. M., Horowitz, L. W., MacKenzie, I. A., Sanderson, M. Competing interests. The authors declare that they have no conflict of interest. Competing interests. The authors declare that they have no conflict of interest. Bottenheim, J. W., Sirois, A., Brice, K. A., and Gallant, A. J.: Five years of continuous observations of PAN and ozone at a rural location in eastern Canada, J. Geophys. Res.-Atmos., 99, 5333– 5352, 10.1029/93JD02716, 1994. Special issue statement. This article is part of the special issue “Global and regional assessment of intercontinental transport of air pollution: results from HTAP, AQMEII and MICS”. It is not associ- ated with a conference. Carpenter, L. J., Green, T. J., Mills, G. P., Bauguitte, S., Penkett, S. A., Zanis, P., Schuepbach, E., Schmidbauer, N., Monks, P. S., and Zellweger, C: Oxidized nitrogen and ozone production efficien- cies in the springtime free troposphere over the Alps, J. Geophys. Res., 105, 14547–14559, 2000. Acknowledgements. We thank Mathew Evans (York University, UK) and Terry Keating (U.S. EPA) for useful discussions and the two anonymous referees for their constructive comments. Arlene M. Fiore and Bryan N. Duncan acknowledge NASA MAP (NNX14AM38G). David S. Stevenson acknowledges NERC (grants NE/K001329/1 and NE/N003411/1) and the ARCHER UK National Supercomputing Service (http://www.archer.ac.uk, last access: 17 October 2018). Drew T. Shindell acknowledges the NASA High-End Computing Program through the NASA Center for Climate Simulation at GSFC for computational resources. Michael G. Sanderson was supported by the Met Office Hadley Centre Climate Programme funded by BEIS and Defra. This is Lamont contribution number 8251. Chameides, W. L., Fehsenfeld, F. C., Rodgers, M. O., Cardelino, C., Martinez, J., Parrish, D., Lonnerman, W., Lawson, D. R., Ras- mussen, R. A., Zimmerman, P., Greenberg, J., Middleton, P., and Wang, T.: Ozone precursor relationships in the ambient atmo- sphere, J. Geophys. Res., 97, 6037–6055, 1992. Cox, P. M., Huntingford, C., and Williamson, M. S., Emer- gent constraint on equilibrium climate sensitivity from global temperature variability: Nature, 553, 319–323, https://doi.org/10.1038/nature25450, 2018. Doherty, R. M., Wild, O., Shindell, D. T., Zeng, G., MacKenzie, I. A., Collins, W. J., Fiore, A. M., Stevenson, D. S., Dentener, F. J., Schultz, M. G., Hess, P., Derwent, R. G., and Keating, T. J.: Impacts of climate change on surface ozone and intercontinental ozone pollution: A multi-model study, J. Geophys. Res.-Atmos., 118, 3744–3763, https://doi.org/10.1002/jgrd.50266, 2013. Edited by: Kathy Law Reviewed by: two anonymous referees Edited by: Kathy Law Reviewed by: two anonymous referees Edited by: Kathy Law Reviewed by: two anonymous referees Emmerson, K. M. and Evans, M. Competing interests. The authors declare that they have no conflict of interest. J.: Comparison of tropospheric gas-phase chemistry schemes for use within global models, At- mos. Chem. Phys., 9, 1831–1845, https://doi.org/10.5194/acp-9- 1831-2009, 2009. www.atmos-chem-phys.net/18/15345/2018/ www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15358 Alpine site Jungfraujoch (3580 m asl), J. Geophys. Res., 113, D22305, https://doi.org/10.1029/2007JD009751, 2008. Alpine site Jungfraujoch (3580 m asl), J. Geophys. Res., 113, D22305, https://doi.org/10.1029/2007JD009751, 2008. authors helped to shape the paper content by editing prior versions of the paper. Borodina, A., Fischer, E. M., and Knutti, R.: Emergent Constraints in Climate Projections: A Case Study of Changes in High- Latitude Temperature Variability, J. Climate, 30, 3655–3670, https://doi.org/10.1175/JCLI-D-16-0662.1, 2017. A. M. Fiore et al.: A constraint on continental source–receptor relationships 15359 Jonson, J. E., Kaminski, J. W., Keating, T. J., Lupu, A., Marmer, E., Montanaro, V., Park, R. J., Pitari, G., Pringle, K. J., Pyle, J. A., Schroeder, S., Vivanco, M. G., Wind, P., Wojcik, G., Wu, S., and Zuber, A.: Multimodel estimates of intercontinental source- receptor relationships for ozone pollution, J. Geophys. Res., 114, D04301, https://doi.org/10.1029/2008jd010816, 2009. Jonson, J. E., Kaminski, J. W., Keating, T. J., Lupu, A., Marmer, E., Montanaro, V., Park, R. J., Pitari, G., Pringle, K. J., Pyle, J. Jaffe, D., Thornton, J., Wolfe, G., Reidmiller, D., Fischer, E. V., Ja- cob, D. J., Cohen, R., Singh, H., Weinheimer, A., and Flocke, F.: Can we Detect an Influence over North America From Increas- ing Asian NOx Emissions?, EOS Trans. AGU, 88(52), Fall Meet. Suppl., Abstract A51E-04, 2007. Jiang, Z., Worden, J. R., Payne, V. H., Zhu, L., Fischer, E., Walker, T., and Jones, D. B. A.: Ozone export from East Asia: The role of PAN, J. Geophys. Res.-Atmos., 121, 6555–6563, https://doi.org/10.1002/2016JD024952, 2016. Fiore, A. M., Levy II, H., and Jaffe, D. A.: North American isoprene influence on intercontinental ozone pollution, Atmos. Chem. Phys., 11, 1697–1710, https://doi.org/10.5194/acp-11- 1697-2011, 2011. Kirchner, F., Mayer-Figge, A., Zabel, F., and Becker, K. H.: Ther- mal stability of peroxynitrates, Int. J. Chem. Kinet., 31, 127–144, 1999. Fischer, E. V., Jaffe, D. A., Reidmiller, D. R., and Jaegle, L.: Me- teorological controls on observed peroxyacetyl nitrate (PAN) at Mount Bachelor during the spring of 2008, J. Geophys. Res., 115, D03302, https://doi.org/10.1029/2009JD012776, 2010. Knote, C., Tuccella, P., Curci, G., Emmons, L., Orlando, J. J., Madronich, S., Baro, R., Jimenez-Guerrero, P., Luecken, D., Hogrefe, C., Forkel, R. , Werhahn, J., Hirtl, M., Perez, J., San Jose, R., Giordano, L., Brunner, D., Yahya, K., and Zhang, Y.: Influence of the choice of gas-phase mechanism on predictions of key gaseous pollutants during the AQMEII phase-2 intercom- parison, Atmos. Environ., 115, 553–568, 2015. Fischer, E. V., Jaffe, D. A., and Weatherhead, E. C.: Free tro- pospheric peroxyacetyl nitrate (PAN) and ozone at Mount Bachelor: potential causes of variability and timescale for trend detection, Atmos. Chem. Phys., 11, 5641–5654, https://doi.org/10.5194/acp-11-5641-2011, 2011. Kotchenruther, R. A., Jaffe, D. A., and Jaeglé, L.: Ozone potochem- istry and the role of PAN in the springtime northeastern Pacific Troposphere: Results from the PHOBEA Campaign, J. Geophys. Res., 106, 28731–28741, 2001a. Fischer, E. V., Jacob, D. J., Yantosca, R. M., Sulprizio, M. P., Millet, D. A. M. Fiore et al.: A constraint on continental source–receptor relationships B., Mao, J., Paulot, F., Singh, H. B., Roiger, A., Ries, L., Talbot, R. W., Dzepina, K., and Pandey Deolal, S.: Atmospheric peroxyacetyl nitrate (PAN): a global budget and source attribution, Atmos. Chem. Phys., 14, 2679–2698, https://doi.org/10.5194/acp-14-2679-2014, 2014. Kotchenruther, R. A., Jaffe, D. A., Beine, H. J., Anderson,T., Bot- tenheim, J. W., Harris, J. M., Blake, D., and Schmitt, R.: Obser- vations of ozone and related species in the Northeast Pacific dur- ing the PHOBEA Campaigns: 2. Airborne observations, J. Geo- phys. Res., 106, 7463–7483, 2001b. Gilge, S., Plass-Duelmer, C., Fricke, W., Kaiser, A., Ries, L., Buch- mann, B., and Steinbacher, M.: Ozone, carbon monoxide and nitrogen oxides time series at four alpine GAW mountain sta- tions in central Europe, Atmos. Chem. Phys., 10, 12295–12316, https://doi.org/10.5194/acp-10-12295-2010, 2010. Koumoutsaris, S., Bey, I., Generoso, S., and Thouret, V.: Influence of El Niño-Southern Oscillation on the interannual variability of tropospheric ozone in the northern midlatitudes, J. Geophys. Res., 113, D19301, https://doi.org/10.1029/2007jd009753, 2008. Hall, A. and Qu, X., Using the current seasonal cycle to constrain snow albedo feedback in future climate change, Geophys. Res. Lett., 33, L03502, https://doi.org/10.1029/2005GL025127, 2006. Kuhn, M., Builtjes, P. J. H., Poppe, D., Simpson, D., Stockwell, W. R., Andersson-Sköld, Y., Baart, A., Das, M., Fiedler, F., Hov, Ø., Kirchner, F., Makar, P. A., Milford, J. B., Roemer, M. G. M., Ruhnke, R., Strand, A., Vogel, B., and Vogel, H.: Intercompari- son of the gas-phase chemistry in several chemistry and transport models, Atmos. Environ., 32, 693–709, 1998. p g Heald, C. L., Jacob, D. J., Fiore, A. M., Emmons, L. K., Gille, J. C., Deeter, M. N., Warner, J., Edwards, D. P., Crawford, J. H., Hamlin, A. J., Sachse, G. W., Browell, E. V., Avery, M. A., Vay, S. A., Westberg, D. J., Blake, D. R., Singh, H. B., Sandholm, S. T., Talbot, R. W., and Fuelberg, H. E.: Asian outflow and trans- Pacific transport of carbon monoxide and ozone pollution: An integrated satellite, aircraft, and model perspective, J. Geophys. Res., 108, 4804, https://doi.org/10.1029/2003jd003507, 2003. Liang, J., Horowitz, L. W., Jacob, D. J., Wang, Y., Fiore, A. M., Logan, J. A., Gardner, G. M., and Munger, J. W.: Seasonal vari- ations of reactive nitrogen species and ozone over the United States and export fluxes to the global atmosphere, J. Geophys. Res., 103, 13435–413450, 1998. References G., Shindell, D. T., Stevenson, D. S., Szopa, S., Van Dingenen, R., Zeng, G., Ather- ton, C., Bergmann, D., Bey, I., Carmichael, G., Collins, W. J., Duncan, B. N., Faluvegi, G., Folberth, G., Gauss, M., Gong, S., Hauglustaine, D., Holloway, T., Isaksen, I. S. A., Jacob, D. J., Balzani Lööv, J. M., Henne, S., Legreid, G., Staehelin, J., Reimann, S., Prévôt, A. S. H., Steinbacher, M., and Vollmer, M. K.: Esti- mation of background concentrations of trace gases at the Swiss www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15360 Res., 101, 12621–12638, https://doi.org/10.1029/96jd00338, 1996. Surface Ozone Observations, Elem. Sci. Anth., 5, 58, https://doi.org/10.1525/elementa.244, 2017. Orbe, C., Waugh, D. W., Yang, H., Lamarque, J.-F., Tilmes, S., and Kinnison, D. E.: Tropospheric transport differ- ences between models using the same large-scale me- teorological fields, Geophys. Res. Lett., 44, 1068–1078, https://doi.org/10.1002/2016GL071339, 2007. Shindell, D. T., Chin, M., Dentener, F., Doherty, R. M., Faluvegi, G., Fiore, A. M., Hess, P., Koch, D. M., MacKenzie, I. A., Sanderson, M. G., Schultz, M. G., Schulz, M., Stevenson, D. S., Teich, H., Textor, C., Wild, O., Bergmann, D. J., Bey, I., Bian, H., Cuvelier, C., Duncan, B. N., Folberth, G., Horowitz, L. W., Jonson, J., Kaminski, J. W., Marmer, E., Park, R., Pringle, K. J., Schroeder, S., Szopa, S., Takemura, T., Zeng, G., Keat- ing, T. J., and Zuber, A.: A multi-model assessment of pollu- tion transport to the Arctic, Atmos. Chem. Phys., 8, 5353–5372, https://doi.org/10.5194/acp-8-5353-2008, 2008. g y Bian, H., Cuvelier, C., Duncan, B. N., Folberth, G., Horowitz, L. W., Jonson, J., Kaminski, J. W., Marmer, E., Park, R., Pringle, Pandey Deolal, S., Staehelin, J., Brunner, D., Cui, J., Steinbacher, M., Zellweger, C., Henne, S., and Vollmer, M. K.: Transport of PAN and NOy from different source regions to the Swiss high alpine site Jungfraujoch, Atmos. Environ., 64, 103–115, https://doi.org/10.1016/j.atmosenv.2012.08.021, 2013. Singh, H. B.: Reactive nitrogen in the troposphere, Environ. Sci. Technol., 21, 320–327, 1987. Pandey Deolal, S., Henne, S., Ries, L., Gilge, S., Weers, U., Stein- bacher, M., Staehelin, J., and Peter, T.: Analysis of elevated springtime levels of Peroxyacetyl nitrate (PAN) at the high Alpine research sites Jungfraujoch and Zugspitze, Atmos. Chem. Phys., 14, 12553–12571, https://doi.org/10.5194/acp-14-12553- 2014, 2014. Singh, H. B. and Hanst, P. L.: Peroxyacetyl nitrate (PAN) in the un- polluted atmosphere: An important reservoir for nitrogen oxides, Geophys. Res. Lett., 8, 941–944, 1981. Singh, H. B. and Salas, L. J.: Measurements of peroxyacetyl ni- trate (pan) and peroxypropionyl nitrate (ppn) at selected ur- ban, rural and remote sites, Atmos. Environ., 23, 231–238, https://doi.org/10.1016/0004-6981(89)90115-7, 1989. Payne, V. H., Alvarado, M. J., Cady-Pereira, K. E., Worden, J. R., Kulawik, S. S., and Fischer, E. V.: Satellite obser- vations of peroxyacetyl nitrate from the Aura Tropospheric Emission Spectrometer, Atmos. Meas. Tech., 7, 3737–3749, https://doi.org/10.5194/amt-7-3737-2014, 2014. Stevenson, D. S., Dentener, F. J., Schultz, M. G., Ellingsen, K., van Noije, T. P. C., Wild, O., Zeng, G., Amann, M., Ather- ton, C. A. M. Fiore et al.: A constraint on continental source–receptor relationships S., Bell, N., Bergmann, D. J., Bey, I., Butler, T., Co- fala, J., Collins, W. J., Derwent, R. G., Doherty, R. M., Drevet, J., Eskes, H. J., Fiore, A. M, Gauss, M., Hauglustaine, D. A., Horowitz, L. W., Isaksen, I. S. A, Krol, M. C., Lamarque, J.-F., Lawrence, M. G., Montanaro, V., Müller, J.-F., Pitari, G., Prather, M. J., Pyle, J. A., Rast, S., Rodriguez, J. M., Sanderson, M. G., Savage, N. H., Shindell, D. T., Strahan, S. E., Sudo, K., and Szopa, S.: Multimodel ensemble simulations of present-day and near-future tropospheric ozone, J. Geophys. Res., 111, D08301, https://doi.org/10.1029/2005JD006338, 2006. Payne, V. H., Fischer, E. V., Worden, J. R., Jiang, Z., Zhu, L., Kurosu, T. P., and Kulawik, S. S.: Spatial variability in tropo- spheric peroxyacetyl nitrate in the tropics from infrared satellite observations in 2005 and 2006, Atmos. Chem. Phys., 17, 6341– 6351, https://doi.org/10.5194/acp-17-6341-2017, 2017. J., Eskes, H. J., Fiore, A. M, Gauss, M., Hauglustaine, D. A., Penkett, S. A. and Brice, K. A.: The spring maximum in photo- oxidants in the Northern Hemisphere troposphere, Nature, 319, 655–657, 1986. M. J., Pyle, J. A., Rast, S., Rodriguez, J. M., Sanderson, M. G., Savage, N. H., Shindell, D. T., Strahan, S. E., Sudo, K., and Szopa, S.: Multimodel ensemble simulations of present-day and near-future tropospheric ozone, J. Geophys. Res., 111, D08301, https://doi.org/10.1029/2005JD006338, 2006. Roberts, J. M.: PAN and Related Compounds, in: Volatile Organic Compounds in the Atmosphere, edited by: Koppmann, R., Blackwell Publishing, 221–268, https://doi.org/10.1002/9780470988657.ch6, 2007. Turnipseed, A. A., Huey, L. G., Nemitz, E., Stickel, R., Higgs, J., Tanner, D. J., Slusher, D. L., Sparks, J. P., Flocke, F., and Guen- ther, A.: Eddy covariance fluxes of peroxyacetyl nitrates (PANs) and NOy to a coniferous forest, J. Geophys. Res.-Atmos., 111, D09304, https://doi.org/10.1029/2005jd006631, 2006. Schmitt, R. and Volz-Thomas, A.: Climatology of Ozone, PAN, CO, and NMHC in the Free Troposphere Over the Southern North Atlantic, J. Atmos. Chem., 28, 245–262, https://doi.org/10.1023/A:1005801515531, 1997. Val Martin, M., Honrath, R. E., Owen, R. C., and Lapina, K.: Large-scale impacts of anthropogenic pollution and bo- real wildfires on the nitrogen oxide levels over the cen- tral North Atlantic region, J. Geophys. Res., 113, D17308, https://doi.org/10.1029/2007JD009689, 2008. A. M. Fiore et al.: A constraint on continental source–receptor relationships HTAP: Task Force on Hemispheric Transport of Air Pollution 2007 Interim Report, United Nations Economic Commission for Eu- rope, New York and Geneva, 2007. Lin, M., Holloway, T., Carmichael, G. R., and Fiore, A. M.: Quan- tifying pollution inflow and outflow over East Asia in spring with regional and global models, Atmos. Chem. Phys., 10, 4221– 4239, https://doi.org/10.5194/acp-10-4221-2010, 2010. HTAP: Hemispheric transport of air pollution 2010 part a: ozone and particulate matter, Air Pollution Studies No. 17, UNITED NATIONS, New York, 2010. Lin, M., Horowitz, L. W., Payton, R., Fiore, A. M., and Tonnesen, G.: US surface ozone trends and extremes from 1980 to 2014: quantifying the roles of rising Asian emissions, domestic con- trols, wildfires, and climate, Atmos. Chem. Phys., 17, 2943– 2970, https://doi.org/10.5194/acp-17-2943-2017, 2017. Hudman, R. C., Jacob, D. J., Cooper, O. R., Evans, M. J., Heald, C. L., Park, R. J., Fehsenfeld, F., Flocke, F., Holloway, J., Hübler, G., Kita, K., Koike, M., Kondo, Y., Neuman, A., Nowak, J., Olt- mans, S., Parrish, D., Roberts, J. M., and Ryerson, T.: Ozone production in transpacific Asian pollution plumes and implica- tions for ozone air quality in California, J. Geophys. Res., 109, D23S10, https://doi.org/10.1029/2004jd004974, 2004. Liu, S. C., Trainer, M., Fehsenfeld, F. C., Parrish, D. D., Williams, E. J., Fahey, D. W., Hubler, G., and Murphy, P. C.: Ozone pro- duction in the Rural Troposphere and the Implications for Re- gional and Global Ozone Distributions, J. Geophys. Res., 92, 4191–4207, 1987. Jaeglé, L., Jaffe, D. A., Price, H. U., Weiss-Penzias, P., Palmer, P. I., Evans, M. J., Jacob, D. J., and Bey, I.: Sources and budgets for CO and O3 in the northeastern Pacific during the spring of 2001: Results from the PHOBEA-II Experiment, J. Geophys. Res., 108, D20, 8802, https://doi.org/10.1029/2002JD003121, 2003. Moxim, W. J., Levy II, H., and Kasibhatla, P. S.: Simulated global tropospheric PAN: Its transport and impact on NOx, J. Geophys. www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018 A. M. Fiore et al.: A constraint on continental source–receptor relationships 15361 Zellweger, C., Ammann, M., Buchmann, B., Hofer, P., Lu- gauer, M., Rüttimann, R., Streit, N., Weingartner, E., and Bal- tensperger, U.: Summertime NOy speciation at the Jungfrau- joch, 3580 m asl, Switzerland, J. Geophys. Res., 105, 6655– 6667, https://doi.org/10.1029/1999JD901126, 2000. Wang, Y. and Jacob, D. J.: Anthropogenic forcing on tropospheric ozone and OH since preindustrial times, J. Geophys. Res., 103, 31123–131135, 1998. Warneck, P. and Zerbach, T.: Synthesis of peroxyacetyl nitrate by acetone photolysis, Environ. Sci. Technol., 26, 74–79, 1992. Zellweger, C., Forrer, J., Hofer, P., Nyeki, S., Schwarzenbach, B., Weingartner, E., Ammann, M., and Baltensperger, U.: Par- titioning of reactive nitrogen (NOy) and dependence on me- teorological conditions in the lower free troposphere, Atmos. Chem. Phys., 3, 779–796, https://doi.org/10.5194/acp-3-779- 2003, 2003. Weiss-Penzias, P., Jaffe, D. A., Jaeglé, L., and Liang, Q.: In- fluence of long-range-transported pollution on the annual and diurnal cycles of carbon monoxide and ozone at Cheeka Peak Observatory, J. Geophys. Res.-Atmos., 109, D23S14, https://doi.org/10.1029/2004JD004505, 2004. Wild, O., Law, K., McKenna, D., Bandy, B., Penkett, S., and Pyle, J.: Photochemical trajectory modeling studies of the North At- lantic region during August 1993, J. Geophys. Res.-Atmos., 101, 29269–29288, 1996. Zhang, L., Jacob, D. J., Boersma, K. F., Jaffe, D. A., Olson, J. R., Bowman, K. W., Worden, J. R., Thompson, A. M., Avery, M. A., Cohen, R. C., Dibb, J. E., Flock, F. M., Fuelberg, H. E., Huey, L. G., McMillan, W. W., Singh, H. B., and Weinheimer, A. J.: Transpacific transport of ozone pollution and the effect of recent Asian emission increases on air quality in North Amer- ica: an integrated analysis using satellite, aircraft, ozonesonde, and surface observations, Atmos. Chem. Phys., 8, 6117–6136, https://doi.org/10.5194/acp-8-6117-2008, 2008. Wild, O., Fiore, A. M., Shindell, D. T., Doherty, R. M., Collins, W. J., Dentener, F. J., Schultz, M. G., Gong, S., MacKenzie, I. A., Zeng, G., Hess, P., Duncan, B. N., Bergmann, D. J., Szopa, S., Jonson, J. E., Keating, T. J., and Zuber, A.: Modelling fu- ture changes in surface ozone: a parameterized approach, At- mos. Chem. Phys., 12, 2037–2054, https://doi.org/10.5194/acp- 12-2037-2012, 2012. Zhu, L., Fischer, E. V., Payne, V. H., Worden, J. R., and Jiang, Z.: TES observations of the interannual variabil- ity of PAN over Northern Eurasia and the relationship to springtime fires, Geophys. Res. Lett., 42, 7230–7237, https://doi.org/10.1002/2015GL065328, 2015. Wu, S., Duncan, B. N., Jacob, D. J., Fiore, A. A. M. Fiore et al.: A constraint on continental source–receptor relationships Schultz, M., Rast, S., van het Bolscher, M., Pulles, T., Brand, R., Pereira, J., Mota, B., Spessa, A., Dalsoren, S., van Noije, T., and Szopa, S.: Emission data sets and methodologies for estimat- ing emissions, Hamburg, Max Planck Institute for Meteorology, 2007. van der Werf, G. R., Randerson, J. T., Giglio, L., Collatz, G. J., Kasibhatla, P. S., and Arellano Jr., A. F.: Interannual variability in global biomass burning emissions from 1997 to 2004, Atmos. Chem. Phys., 6, 3423–3441, https://doi.org/10.5194/acp-6-3423- 2006, 2006. Schultz, M. G., Jacob, D. J., Wang, Y., Logan, J. A., Atlas, E. L., Blake, D. R., Blake, N. J., Bradshaw, J. D., Browell, E. G., Fenn, M. A., Flocke, F., Gregory, G. L., Heikes, B. G., Sachse, G. W., Sandholm, G. W., Shetter, R. E., Singh, H. B., and Talbot, R. W.: On the origin of tropospheric ozone and NOx over the tropical South Pacific, J. Geophys. Res., 104, 5829–5843, 1999. van der Werf, G. R., Randerson, J. T., Giglio, L., Collatz, G. J., Mu, M., Kasibhatla, P. S., Morton, D. C., DeFries, R. S., Jin, Y., and van Leeuwen, T. T.: Global fire emissions and the contribution of deforestation, savanna, forest, agricultural, and peat fires (1997–2009), Atmos. Chem. Phys., 10, 11707–11735, https://doi.org/10.5194/acp-10-11707-2010, 2010. Schultz, M. G., Heil, A., Hoelzemann, J. J., Spessa, A., Thon- icke, K., Goldammer, J. G., Held, A. C., Pereira, J. M. C., and van het Bolscher, M.: Global wildland fire emissions from 1960 to 2000, Global Biogeochem. Cy., 22, GB2002, https://doi.org/10.1029/2007GB003031, 2008. Volz-Thomas, A., Xueref, I., and Schmitt, R.: An automatic gas chromatograph and calibration system for ambient measure- ments of PAN and PPN, Environ. Sci. Pollut. R., 4, 72–76, 2002. p g Schultz, M. G., Schröder, S., Lyapina, O., Cooper, O., Gal- bally, I., Petropavlovskikh, I., et al.: Tropospheric Ozone Assessment Report: Database and Metrics Data of Global Atmos. Chem. Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ A. M. Fiore et al.: A constraint on continental source–receptor relationships M., and Wild, O.: Chemical nonlinearities in relating intercontinental ozone pollution to anthropogenic emissions, Geophys. Res. Lett., 36, L05806, https://doi.org/10.1029/2008gl036607, 2009. Zhu, L., Payne, V. H., Walker, T. W., Worden, J. R., Jiang, Z., Kulawik, S. S., and Fischer, E. V.: PAN in the eastern Pacific free troposphere: A satellite view of the sources, seasonality, interannual variability, and timeline for trend detection, J. Geophys. Res.-Atmos., 122, 3614–3629, https://doi.org/10.1002/2016JD025868, 2017. Xue, L. K., Wang, T., Zhang, J. M., Zhang, X. C., Deliger, Poon, C. N., Ding, A. J., Zhou, X. H., Wu, W. S., Tang, J., Zhang, Q. Z., and Wang, W. X.: Source of surface ozone and reactive nitrogen speciation at Mount Waliguan in western China: New insights from the 2006 summer study, J. Geophys. Res., 116, D07306, https://doi.org/10.1029/2010JD014735, 2011. Atmos. Chem. Phys., 18, 15345–15361, 2018 www.atmos-chem-phys.net/18/15345/2018/ Atmos. Chem. Phys., 18, 15345–15361, 2018
https://openalex.org/W2922479144
https://europepmc.org/articles/pmc6371428?pdf=render
English
null
Cancer-associated fibroblasts induce epithelial–mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling
BMC cancer
2,019
cc-by
10,156
Goulet et al. BMC Cancer (2019) 19:137 https://doi.org/10.1186/s12885-019-5353-6 Goulet et al. BMC Cancer (2019) 19:137 https://doi.org/10.1186/s12885-019-5353-6 Open Access Abstract Keywords: CAFs, IL-6, EMT, Bladder cancer Keywords: CAFs, IL-6, EMT, Bladder cancer Keywords: CAFs, IL-6, EMT, Bladder cancer * Correspondence: stephane.bolduc@fmed.ulaval.ca 1Centre de recherche en organogénèse expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec-Université Laval Research Center, QC, Québec, Canada 2Department of Surgery, Faculty of Medicine, Laval University, QC, Quebec, Canada Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cancer-associated fibroblasts induce epithelial–mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling Cassandra Ringuette Goulet1,2,3, Audrey Champagne2,3, Geneviève Bernard1,2, Dominique Vandal1, Stéphane Chabaud1,2, Frédéric Pouliot2,3 and Stéphane Bolduc1,2,4* Abstract Background: Cancer-associated fibroblasts (CAFs), activated by tumour cells, are the predominant type of stromal cells in cancer tissue and play an important role in interacting with neoplastic cells to promote cancer progression. Epithelial-mesenchymal transition (EMT) is a key feature of metastatic cells. However, the mechanism by which CAFs induce EMT program in bladder cancer cells remains unclear. Methods: To investigate the role of CAFs in bladder cancer progression, healthy primary bladder fibroblasts (HFs) were induced into CAFs (iCAFs) by bladder cancer-derived exosomes. Effect of conditioned medium from iCAFs (CM iCAF) on EMT markers expression of non-invasive RT4 bladder cancer cell line was determined by qPCR and Western blot. IL6 expression in iCAFs was evaluated by ELISA and Western blot. RT4 cell proliferation, migration and invasion were assessed in CM iCAF +/−anti-IL6 neutralizing antibody using cyQUANT assay, scratch test and transwell chamber respectively. We investigated IL6 expression relevance for bladder cancer progression by querying gene expression datasets of human bladder cancer specimens from TCGA and GEO genomic data platforms. Results: Cancer exosome-treated HFs showed CAFs characteristics with high expression levels of αSMA and FAP. We showed that the CM iCAF induces the upregulation of mesenchymal markers, such as N-cadherin and vimentin, while repressing epithelial markers E-cadherin and p-ß-catenin expression in non-invasive RT4 cells. Moreover, EMT transcription factors SNAIL1, TWIST1 and ZEB1 were upregulated in CM iCAF-cultured RT4 cells compared to control. We also showed that the IL-6 cytokine was highly expressed by CAFs, and its receptor IL-6R was found on RT4 bladder cancer cells. The culture of RT4 bladder cancer cells with CM iCAF resulted in markedly promoted cell growth, migration and invasion. Importantly, inhibition of CAFs-secreted IL-6 by neutralizing antibody significantly reversed the IL-6-induced EMT phenotype, suggesting that this cytokine is necessary for CAF-induced EMT in the progression of human bladder cancer. Finally, we observed that IL6 expression is up-regulated in aggressive bladder cancer and correlate with CAF marker ACTA2. Conclusions: We conclude that CAFs promote aggressive phenotypes of non-invasive bladder cancer cells through an EMT induced by the secretion of IL-6. * Correspondence: stephane.bolduc@fmed.ulaval.ca 1 1Centre de recherche en organogénèse expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec-Université Laval Research Center, QC, Québec, Canada 2 2Department of Surgery, Faculty of Medicine, Laval University, QC, Quebec, Canada Full list of author information is available at the end of the article Full list of author information is available at the end of the article Background inducing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which dimerizes and translocates into the nucleus to regulate target gene tran- scription. A number of studies have highlighted the role of IL-6 and STAT3 in promoting tumor metastasis as their overexpression and/or hyper-activation have been re- ported in several human cancers [14–16]. Moreover, the level of IL-6 in blood of patients has been suggested as a prognostic marker [17]. Also, studies have shown that IL-6 contributes to cancer’s drug resistance [18]. IL-6 is overexpressed in bladder cancer tissues compared to non-malignant tissues at both mRNA and protein levels and elevated IL-6 levels correlated with higher clinical stage, higher recurrence rate after curative treatment, and reduced survival rate [19]. Bladder cancer is the 9th most commonly diagnosed can- cer and is ranked 13th for cancer deaths in the overall population worldwide. Of all newly diagnosed cases, 75% present as non-muscle-invasive bladder cancer (NMIBC) disease, while 25% present as muscle-invasive bladder can- cer (MIBC) disease with 10–15% of cases that are already metastatic [1, 2]. Of all solid cancers, bladder cancer has the highest rate of recurrence. After first line of treatment, 50 to 70% of patients with NMIBC will experience disease recurrence within 5 years and 10 to 30% of them will see their cancer progress to an invasive form [3–5]. p g [ ] The complex process of tumor metastasis consists of multiple steps during which cancer cells spread from primary tumor to other organs. Metastasis involves the epithelial–mesenchymal transition (EMT) process by which cancer cells transit between adherent epithelial and mobile mesenchymal states facilitating cancer cells dissemination. Epithelial cells loss E-cadherin expres- sion, cell-cell adhesion and apico-basal polarization to gain vimentin expression and motility [6]. EMT is regu- lated by several transcription factors including snail homolog 1 (SNAIL1), twist basic helix–loop–helix tran- scription factor 1 (TWIST1) and zinc-finger E-box- binding homeobox 1 (ZEB1) [6]. Cadherin switch from E-cadherin to N-cadherin is a key step occurring during the EMT process [7]. This down-regulation of the E-cadherin and also the phosphorylation of GSK3β, an effector of Wnt signaling pathway, are associated with the release of β-catenin, which then translocates to the nucleus and activates the Wnt signaling pathway, known to be involved in metastasis formation. However, the pre- cise molecular events that initiate this complex EMT process in bladder cancer are poorly understood. Background Increas- ing evidence suggests that the tumor microenvironment (TME) plays an important role in promoting EMT in tumor cells. Fibroblasts, the predominant stromal cell type in the TME, are activated by tumor cells into cancer-associated fibroblasts (CAFs) through the secre- tion of paracrine growth factors [8]. CAFs display a specific subset of markers, including α-smooth muscle actin (α-SMA; coded by ACTA2 gene), fibroblast-activating protein (FAP), fibroblast-specific protein-1 (FSP1) and tenascin C [9, 10]. Previous studies suggest that CAFs play a pivotal role in establishing a metastatic niche and promoting tumor cell proliferation, invasion and metas- tasis by secretion of chemokines and cytokines in the microenvironment [9, 11, 12]. However, it is still un- clear by which mechanisms CAFs affect the metastatic potential of bladder cancer cells. Although there is evidence suggesting that CAFs and IL-6 may be a critical factor in metastatic spreading, their role in EMT of bladder cancer cells remains unclear. Therefore, we designed this study to understand how CAFs may be promoting EMT in bladder cancer cells. Our results suggest that iCAFs induce EMT-related changes in cancer cells predominantly via the secretion of IL-6. We showed that the exposition of bladder cancer cells to the CAF conditioned medium (CM iCAF) signifi- cantly induced the expression of N-cadherin, vimentin, SNAIL1, TWIST1 and ZEB1 while repressing E-cadherin and phospho-ß-catenin expression. In addition, the CM iCAF significantly enhanced cancer cell proliferation, mi- gration and invasion. We also observed that IL6 expres- sion is up-regulated in aggressive bladder cancer tissues, correlates with CAF marker ACTA2 and is associated with a poor prognosis. These results suggest an important role of IL-6 in mediating EMT and metastatic spreading of bladder cancer cells. Goulet et al. BMC Cancer (2019) 19:137 Goulet et al. BMC Cancer (2019) 19:137 Page 2 of 13 Page 2 of 13 Ethics statement Bladder biopsies from paediatric patients undergoing non-oncologic urologic surgery were obtained at the CHU de Québec Research Center in accordance with the institutional review board. All patients provided their formal, informed and written consent, each agreeing to supply a biopsy for this study. Immunoblot Equal amounts of proteins (10 μg) were loaded into 12% polyacrylamide gels, resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked for 30 min with 5% non-fat milk and 0.05% Tween 20 in PBS, and then incubated with primary antibodies overnight at 4 °C followed by 45 min at RT with HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). The protein expression was detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). Bands were imaged by using the Fusion Fx7 imager (Vilber Lourmat, France) and analyzed with the ImageJ software (NIH, Bethesda, MD). The following antibodies were used: αSMA (1/5000; Abcam, San Francisco, CA), FAP (1/1000, Novus Biologi- cals, Littleton, CO), tubulin (1/1000; Novus Biologicals, Littleton, CO), E-cadherin (1/3000; R&D Systems, Minne- apolis, MN), N-cadherin (1/3000; Millipore, Burlington, MA), vimentin (1/1000; Abcam, San Francisco, CA), phospho-β-Catenin (1/1000; Cell Signaling, Danvers, MA), total β-catenin (1/1000; Abcam, San Francisco, CA), pGSK3β (1/500; Cell Signaling, Danvers, MA), GSK3β (1/750; Cell Signaling, Danvers, MA), SNAIL1 (1/2000; Invitrogen, Carlsbad, CA), TWIST1 (1/1500; LifeSpan BioSciences, Seattle, WA), ZEB1 (1/1000; Abcam, San Francisco, CA), IL-6R (1/1000; Abcam, San Francisco, CA), pSTAT3 (1/1.000, Cell Signaling, Danvers, MA), STAT3 (1/1000; Cell Signaling, Danvers, MA) and β-actin (1/5000; Abcam, San Francisco, CA), pAKT (1/ 1000; Cell Signaling, Danvers, MA), AKT (1/1000; Cell Signaling, Danvers, MA). Cell isolation and culture Healthy primary bladder fibroblasts (HFs) were isolated from two different human bladder biopsies as previ- ously described [8, 20]. Briefly, the stroma was sepa- rated from the urothelium after incubation overnight at 4 ° C in HEPES buffer with 500 μg/mL thermolysin (Sigma- Aldrich, Saint-Louis, MO). Fibroblasts were enzymatically dissociated from the extracellular matrix by treating the stroma with 0.125 U/mL collagenase H (Roche, Missisauga, Canada) for 3 h at 37 °C under gentle agitation. Then, IL-6 is a pleiotropic cytokine that modulates a variety of physiological events including metabolism, inflammation and immune response [13]. Activation of classic signalling requires binding of the IL-6 to its receptor (IL-6R) Goulet et al. BMC Cancer (2019) 19:137 Goulet et al. BMC Cancer (2019) 19:137 Page 3 of 13 Page 3 of 13 fibroblasts were cultured in Dulbecco-Vogt modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Burlington, Canada) and antibiotics (100 U/ml penicillin and 25 μg/ml gen- tamicin; Sigma-Aldrich, Saint-Louis, MO). The results from the two HF populations were pooled together to minimize excessive data spread due to inter-individual variations. The RT4 bladder cancer cell line was ob- tained from the ATCC (HTB-2™) and cultured in DMEM containing 10% FBS and antibiotics. We se- lected the RT4 cells for our studies as a representative example of non-invasive bladder cancer cells [20]. Cells were cultured fewer than 5 passages after purchasing them for all the experiments and tested for mycoplasma contamination. Proteins were collected in RIPA buffer containing protease inhibitors COmplete (Roche, Missi- sauga, Canada), quantified using the BCA kit and store at −80 C until their use. until they reached a confluency of approximately 80%, the cell culture medium was thus collected and centrifuged at 1200 g for 10 min. RT4 bladder cancer cells were cultured in freshly collected conditioned mediums (CM). Exosomes production and isolation Exosomes were isolated as previously described [8]. Briefly, to remove any residual bovine exosomes from the FBS, serum was treated with the FBS Exosome Depletion Kit (Norgen Biotek Corp., ON, Canada) according to manufacturer’s instructions. Bladder cancer cells were cul- tured in DMEM containing 10% exosome-depleted FBS for 48 h. Then, the conditioned medium was centrifuged at 2000 g for 30 min to remove cells and debris, and the supernatant was mixed with 0.5 volume of the Total Exo- somes Isolation Reagent (Invitrogen, Carlsbad, CA). Sam- ples were mixed thoroughly by vortexing and incubated at 4 C overnight. Then, they were centrifuged at 10,000 g for 60 min at 4 C. Exosomes, contained in the pellet, were re-suspended in phosphate buffered saline (PBS). The pro- tein concentration was measured by using the BCA kit (Pierce™, ThermoFisher, Waltham, MA). Elisa HFs were co-cultured with freshly isolated exosomes (1 mg/ml) for 48 h in DMEM 10% exosome-free FBS. Recombinant human transforming growth factor β1 (rhTGFβ1; 4 ng/mL, Peprotech, Rocky Hill, NJ) was used as a positive control. Then, the supernatant was aliquoted and store at −80 C until the ELISA assay, while proteins were collected in RIPA buffer containing protease inhibi- tors COmplete (Roche, Missisauga, Canada), quantified with the BCA kit and store at −80 C until the Western blots. RNAs were isolated using 1 mL of TRIzol Reagent (Invitrogen Corporation, Carlsbad, CA) and stored at − 80 °C until the RT-PCR assay. IL-6 protein expression was quantified by using an ELISA according to the manufacturer’s protocol (Duoset; R&D Systems). The plates were read at 450 nm using a Spectra- Max Plus spectrometer with SoftmaxPro Version 4.7.1. RT-qPCR RNA was extracted using the TRIzol reagent according to the manufacturer’s instructions. RNA quality was assessed on a Bioanalyzer using the Agilent RNA 600 Nano Kit. The concentration and purity of the RNA was determined using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA). One μg of RNA was used to reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). qPCR was performed in triplicate using Cell migration assay RT4 cells were first seeded at a concentration of 4 × 104 cells/100 μl in both chambers of an Ibidi-silicone insert (Ibidi, Martinsried, Germany). This insert allows for the formation of a well-defined edge without physically scratching or wounding the cell monolayer. Cells were cultured for 24 h in DMEM containing 10% FBS to form a confluent monolayer. Then, cells were grown in serum-free DMEM and Mitomycin C (5 μg/ml, Sigma #M4287) to inhibit cell proliferation for 12 h prior to careful removal of the insert. Cells were incubated in the CM from HFs, HFs + TGFß or iCAFs for 24 h in presence or absence of an anti-IL-6 antibody (1 μg/mL). The migra- tion was visualized at the indicated times (0, 6, and 12 h) under an inverted microscope (TE2000, Nikon). Migration distances were measured using the ImageJ analysis soft- ware (National Institutes of Health, Bethesda, MD). Preparation of the conditioned medium p HFs, HFs + TGFß or induced CAFs (iCAFs) were cul- tured in DMEM containing 10% FBS and antibiotics Goulet et al. BMC Cancer (2019) 19:137 Page 4 of 13 Page 4 of 13 the DyNAmo HS SYBR Green qPCR Kit (ThermoFisher Scientific, Waltham, MA) and Lightcycler® 480 system (Roche, Mississauga, Canada), following the manufac- turer’s instructions. The β-2-microglobulin transcript was used as an endogenous control for normalization. the DyNAmo HS SYBR Green qPCR Kit (ThermoFisher Scientific, Waltham, MA) and Lightcycler® 480 system (Roche, Mississauga, Canada), following the manufac- turer’s instructions. The β-2-microglobulin transcript was used as an endogenous control for normalization. RT4 cells were treated with the CM from HFs, HFs + TGFß or iCAFs for 24 h in presence or absence of mi- tomycin C (0,5 μg/mL, Sigma-Aldrich, Saint-Louis, MO). Plates were washed with PBS to remove any deb- ris and cell viability was assessed by fluorometric quan- tification of DNA using CyQUANT Proliferation Assay Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. RT4 cells were treated with the CM from HFs, HFs + TGFß or iCAFs for 24 h in presence or absence of mi- tomycin C (0,5 μg/mL, Sigma-Aldrich, Saint-Louis, MO). Plates were washed with PBS to remove any deb- ris and cell viability was assessed by fluorometric quan- tification of DNA using CyQUANT Proliferation Assay Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Proliferation assay RT4 cells were seeded in 96-well plates at a density of 3 × 103 cells/well and allowed to attach for 12 h. Then, RT4 were treated with the CM from HFs, HFs + TGFß or iCAFs for 24 h in presence or absence of an anti-IL-6 antibody (1 μg/mL). Cell proliferation was assessed by fluorometric quantification of DNA using CyQUANT Proliferation Assay Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Genomic data processing and tumor purity analysis Clinicopathological profiles and genomic data from TCGA [21] and GSE13507 [22] were downloaded re- spectively on GDC (Genomic Data Common; https:// portal.gdc.cancer.gov/) and GEO (Gene Expression Omnibus; https://www.ncbi.nlm.nih.gov/geo/) data por- tal (n = 412 and n = 272, respectively). The expression of IL6 in human bladder cancer specimens was analyzed using GraphPad Prism 7.0 in bladder cancer patients linked with their clinical parameters and follow-up data informa- tion. Tumor purity of the TCGA datasets was obtained using the ABSOLUTE algorithm (https://confluence.broa- dinstitute.org/display/CGATools/ABSOLUTE (2013)). We applied a Pearson’s correlation to test for the association be- tween tumor sample purity and mRNA expression of IL6 and ACTA2 expression. Transwell invasion assays Invasion assays were performed using transwell 24-well plates with 8-μm diameter filters (Corning, NY, USA). Fil- ters were precoated with 40 μL of purified Type I bovine collagen gel (2.5 mg/mL, Sigma-Aldrich, Saint-Louis, MO) and incubated for 4 h at 37 °C. Approximately 1 × 105 cells in 200 μL of serum-free DMEM + Mitomycin C (5 μg/ml) with or without an anti-IL-6 antibody (1 μg/ml) were placed in the upper chamber and 500 μL of the CM from HFs, HFs + TGFß or iCAFs was added in the lower cham- ber. The plates were incubated for 24 h. Then, cells on the upper side of the filters were removed with a cotton swab, and the filters were washed with PBS. Cells were fixed in methanol for 15 min and nuclei were stained with DAPI. The relative cell migration was determined by the number of migrated cells in 10 randomly selected fields. Statistical analysis Graphical representation of data and statistical analysis was performed with the GraphPad Prism v.7 Software (San Diego, CA, USA). The results are expressed as mean ± standard error (SD) and were interpreted using one- or two-way analysis of variance (ANOVA). mRNA expres- sion distribution was analyzed using the non-parametric Mann-Whitney U test. Survival analyses were determined by Kaplan-Meier method, where the difference was evalu- ated by the Log-rank test. Differences between the groups were considered significant at P < 0.05. Results Healthy bladder primary fibroblasts treated with bladder cancer-derived exosomes exhibit characteristics of CAFs To induce the activation of healthy bladder primary fibro- blasts (HFs) into CAFs (iCAFs), we cultured HFs with 1 mg/mL of bladder cancer-derived exosomes for 48 h [20]. HFs + TGFß was used as positive control for activated fi- broblasts [8]. iCAFs were positive for αSMA staining and exhibited a spindle-shape morphology, similar to CAFs (Fig. 1a). Moreover, iCAFs presented higher levels of αSMA and FAP proteins expression (Fig. 1b). iCAFs induce EMT program in bladder cancer cells As EMT plays a pivotal role in tumor metastasis, we investigated the effects of iCAFs on cancer cells EMT, the CM iCAF was collected and used to grow the bladder iCAFs activate the STAT3 signalling pathway in bladder cancer cells via IL-6 secretion cancer cell line RT4 (Fig. 2a). We examined changes of EMT phenotype induces by CM iCAF by measuring the protein expression of epithelial markers E-cadherin, phos- pho-ß-catenin and phospho-GSKß and of mesenchymal markers N-cadherin and vimentin. Results showed that RT4 cells cultured with CM iCAF had decreased expression of E-cadherin, phospho-ß-catenin and phospho-GSK3ß, while the expression of N-cadherin and vimentin was in- creased (Fig. 2b). To investigate whether the EMT pro- gramming was activated by the CAF secretome in RT4 cells, the expression of EMT-related transcription factors (EMT-TFs) SNAIL1, TWIST1 and ZEB1 was measured by qPCR. The results showed that SNAI1 and ZEB1 expres- sion levels were highly upregulated, while TWIST1 expres- sion levels were slightly increased in RT4 cells cultured with CM iCAF (Fig. 2c). These results were also confirmed at protein level (Fig. 2d). These results suggested that the CAF secretome enhances the aggressive behaviour of bladder cancer cells by inducing an EMT phenotype through well-known EMT-TFs. iCAFs activate the STAT3 signalling pathway in bladder cancer cells via IL-6 secretion cancer cell line RT4 (Fig. 2a). We examined changes of EMT phenotype induces by CM iCAF by measuring the protein expression of epithelial markers E-cadherin, phos- pho-ß-catenin and phospho-GSKß and of mesenchymal markers N-cadherin and vimentin. Results showed that RT4 cells cultured with CM iCAF had decreased expression of E-cadherin, phospho-ß-catenin and phospho-GSK3ß, while the expression of N-cadherin and vimentin was in- creased (Fig. 2b). To investigate whether the EMT pro- gramming was activated by the CAF secretome in RT4 cells, the expression of EMT-related transcription factors (EMT-TFs) SNAIL1, TWIST1 and ZEB1 was measured by qPCR. The results showed that SNAI1 and ZEB1 expres- sion levels were highly upregulated, while TWIST1 expres- sion levels were slightly increased in RT4 cells cultured with CM iCAF (Fig. 2c). These results were also confirmed at protein level (Fig. 2d). These results suggested that the CAF secretome enhances the aggressive behaviour of bladder cancer cells by inducing an EMT phenotype through well-known EMT-TFs. The pro-inflammatory cytokine interleukin-6 (IL-6) has been shown as an important EMT inducer in breast and lung cancers [14, 15]. Therefore, we quantified the IL-6 mRNA expression in HFs, HFs + TGFß and iCAF cells as well as the IL-6 protein secretion in supernatants. Our results showed that iCAFs expressed more IL6 mRNA and secreted significantly more IL-6 protein than HFs (Fig. 3a, b). Mitomycin C sensitivity assay Mitomycin C sensitivity assay RT4 cells were seeded in 96-well plates at a density of 3 × 103 cells/well and allowed to attach for 12 h. Then, Goulet et al. BMC Cancer (2019) 19:137 Page 5 of 13 Fig. 1 Healthy vesical primary fibroblasts (HFs) treated with bladder cancer-derived exosomes exhibit characteristics of cancer-associated fibroblasts (CAFs). HFs were treated with 1 mg/mL of bladder cancer cells-derived exosomes for 48 h to induce their activation in CAFs (iCAFs). HFs treated with TGFβ1 (4 ng/mL) served as a positive control. a. Cells were examined by immunocytochemistry for the expression of αSMA (α-smooth muscle actin, green). Nuclei were stained with DAPI (blue). Scale bar = 100 μm. b. The protein expression of αSMA, fibroblast-activating protein (FAP) was determined by western blot. The tubulin was used as loading control. The graph shows mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ****P < 0.0001, n = 3 Fig. 1 Healthy vesical primary fibroblasts (HFs) treated with bladder cancer-derived exosomes exhibit characteristics of cancer-associated fibroblasts (CAFs). HFs were treated with 1 mg/mL of bladder cancer cells-derived exosomes for 48 h to induce their activation in CAFs (iCAFs). HFs treated with TGFβ1 (4 ng/mL) served as a positive control. a. Cells were examined by immunocytochemistry for the expression of αSMA (α-smooth muscle actin, green). Nuclei were stained with DAPI (blue). Scale bar = 100 μm. b. The protein expression of αSMA, fibroblast-activating protein (FAP) was determined by western blot. The tubulin was used as loading control. The graph shows mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ****P < 0.0001, n = 3 iCAFs activate the STAT3 signalling pathway in bladder cancer cells via IL-6 secretion In order to determine if RT4 cells could interact with iCAFs-secreted IL-6, we evaluated the ex- pression of the IL-6R on RT4 cells. The IL-6R was highly expressed in RT4 cells compared to iCAFs, indicating that IL-6 could induce responses in RT4 cells (Fig. 3c). The canonical IL-6 signal transduction pathway is initi- ated by the cytokine binding to the IL-6R and the subse- quent phosphorylation of STAT3. To investigate the effect of the CM iCAF on the activation of IL-6 signaling pathway in bladder cancer cells, we compared the ex- pression levels of phosphorylated STAT3 (pSTAT3) and Page 6 of 13 Goulet et al. BMC Cancer (2019) 19:137 a b c Fig. 2 The conditioned medium (CM) from CAFs induces epithelial-mesenchymal transition (EMT) programming in RT4 bladder cancer cells. a. Experimental design. b. The expression of epithelial markers E-cadherin, phospho-ß-catenin (control: total ß-catenin) and phospho-GSK3ß (control: GSK3ß total) and of mesenchymal markers N-cadherin and vimentin was analyzed by Western blotting. The ß-actin was used as loading control. c. The expression of EMT-related transcription factors (EMT-TFs) was determined by qPCR or d. Western blotting. The graphs show mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = non-significant, n = 3–5 a a b b c c Fig. 2 The conditioned medium (CM) from CAFs induces epithelial-mesenchymal transition (EMT) programming in RT4 bladder cancer cells. a. Experimental design. b. The expression of epithelial markers E-cadherin, phospho-ß-catenin (control: total ß-catenin) and phospho-GSK3ß (control: GSK3ß total) and of mesenchymal markers N-cadherin and vimentin was analyzed by Western blotting. The ß-actin was used as loading control. c. The expression of EMT-related transcription factors (EMT-TFs) was determined by qPCR or d. Western blotting. The graphs show mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = non-significant, n = 3–5 phosphorylated AKT (pAKT) in RT4 cells cultured with CM iCAF to control media (CM HF and CM HF + TGFß). The results showed that CM iCAF significantly increased pSTAT3 and pAKT in RT4 cells compared to CM HF, while total STAT3 and AKT expression remained un- changed (Fig. 3d). iCAFs activate the STAT3 signalling pathway in bladder cancer cells via IL-6 secretion with or without the anti-IL-6 neutralizing antibody. When RT4 cells were treated with CM iCAF, the prolifer- ation rate increased by 21.2% after 24 h (Fig. 4a). To test the specificity of the IL-6 stimulation, we added an anti-IL-6 antibody (x-IL-6) to the culture medium to block IL-6-induced proliferation. The activity of the IL-6 on cell proliferation of RT4 cells was reversed by the anti-IL-6 antibody (Fig. 4a). The migration assay was used to examine the effect of the iCAFs-secreted IL-6 on RT4 cells. As shown in Fig. 4b, the CM iCAF increased the migration activity of RT4 cells significantly. When RT4 cells were incubated with the CM iCAF and prolifer- ation inhibitor, the migration rate after 6 h and 12 h was increased of 36.4 and 23.7%, respectively (Fig. 4b). To examine the effect of the iCAFs-secreted IL-6 on the iCAFs-secreted IL-6 promotes the proliferation, migration and invasion of bladder cancer cells The tumor metastatic cascade is characterized by the ac- tivation of EMT process in cancer cells, where loss of cell–cell junctions and cell polarity lead to the acquisi- tion of migratory and invasive properties. Therefore, we investigated the role of iCAFs-secreted IL-6 on cell growth and motility of RT4 cells treated with CM iCAF Page 7 of 13 Goulet et al. BMC Cancer (2019) 19:137 Fig. 3 iCAFs express IL-6 and induce the activation of the STAT3 signaling pathway in RT4 bladder cancer cells. a. Interleukin-6 (IL-6) mRNA expression levels in HFs, HFs + TGFß and iCAFs cells were determined by qPCR. b. The expression of the IL-6 protein was measured in supernatants from HFs, HFs + TGFß and iCAFs cells using enzyme-linked immunosorbent assay (ELISA). c. The presence of the IL-6 receptor (IL-6R) in iCAFs and RT4 cells was detected by Western blotting. d. The activation of the signal transducer and activator of transcription 3 (STAT3) and AKT signaling pathway in RT4 cells cultured in conditionned medium (CM) was evaluated by Western blotting. The graphs show mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P = 0.001, n.s. = non-significant, n = 3–5 Fig. 3 iCAFs express IL-6 and induce the activation of the STAT3 signaling pathway in RT4 bladder cancer cells. a. Interleukin-6 (IL-6) mRNA expression levels in HFs, HFs + TGFß and iCAFs cells were determined by qPCR. b. The expression of the IL-6 protein was measured in supernatants from HFs, HFs + TGFß and iCAFs cells using enzyme-linked immunosorbent assay (ELISA). c. The presence of the IL-6 receptor (IL-6R) in iCAFs and RT4 cells was detected by Western blotting. d. The activation of the signal transducer and activator of transcription 3 (STAT3) and AKT signaling pathway in RT4 cells cultured in conditionned medium (CM) was evaluated by Western blotting. The graphs show mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P = 0.001, n.s. = non-significant, n = 3–5 significantly reduced after the addition of the anti-IL-6 antibody to CM iCAF compared to the CM iCAF alone (12.4% vs 12.3%). IL6 expression is up-regulated in aggressive bladder cancer, correlates with the CAF marker ACTA2 and stromal compartment and is associated with poor clinical outcome We analyzed IL6 and ACTA2 expression in the publicly available TCGA and GSE13507 gene expression profil- ing datasets of human bladder cancer samples [21, 22]. The stratification of patients according to tumor sta- ging, grading and invasiveness revealed that IL6 expres- sion was found up-regulated in stage III/IV compared to stage I/II (****P < 0.0001), in high grade compared to low grade (****P < 0.0001) and in invasive compared to superficial bladder cancer (****P < 0.0001) (Fig. 5a-c). Similar results were obtained with CAF marker ACTA2 expression, suggesting an activation of fibroblasts in ag- gressive bladder cancer (Fig. 5d-f). The expression levels of IL6 and ACTA2 were positively correlated (r = 0.4543; ****P < 0.0001; Fig. 5g). Moreover, Kaplan–Meier analysis of the cancer specific survival showed a significant correl- ation between patients with high versus low IL6/ACTA2 expression (Fig. 5h, HR = 2.738, 95% CI = 1.056–7.099, iCAFs-secreted IL-6 promotes the proliferation, migration and invasion of bladder cancer cells Thus, cell death induced by mitomycin C was attenuated by the CM iCAF but was not mediated by the iCAFs-secreted IL-6. invasion of RT4 cells, a transwell assay was used where artificial extracellular matrix (ECM) was precoated on the upper side of all filters. We found that the number of cells that invaded through the ECM was raised by 51.5% when RT4 cells were cultured in the CM iCAF compared to the CM HF (Fig. 4c). In addition, when RT4 cells were treated with the CM iCAF in combination with the anti-IL-6 antibody (1 μg/ml), the IL-6-induced cell migration and invasion was markedly inhibited (Fig. 4b, c). These data imply that the iCAFs-secreted IL-6 is clearly implicated in the proliferation, migration and in- vasion abilites of RT4 cells. IL6 expression is up-regulated in aggressive bladder cancer, correlates with the CAF marker ACTA2 and stromal compartment and is associated with poor clinical outcome The CM iCAF increases cancer cell survival in response to the chemotherapeutic drug mitomycin C RT4 cells were cultured in the CM from HFs, HFs + TGFß or iCAFs with or without the anti-IL-6 antibody (1 μg/mL; x-IL-6). a. The proliferation of the RT4 cells was evaluated using the CyQUANT Kit. b. The migration of RT4 cells was determined by using Ibidi-silicone insert. Insert margins were marked by white vertical lines on optical micrographs. After 6 h and 12 h, the percentage of wound closure was evaluated by measuring migration distances (spaces between the white vertical lines). c. The invasion of RT4 cells was measured by transwell assays. Cells that had migrated through the membrane were fixed and nuclei were stained with DAPI. Representative photographs of migratory cells on the membrane are shown. The relative cell migration was determined by the number of migrated cells in 10 randomly selected fields. d. The sensitivity of RT4 cells to mitomycin C (0.5 μg/mL; Mito C) was evaluated using The CyQUANT Kit. RFU means relative fluorescence unit. All values are averages of three independent replicates. The graphs show mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = non-significant, n = 5–25 *P = 0.0471). Tumor purity is the proportion of cancer cells within a tumor. Despite some selection for inclusion, TCGA tissue samples may retain a heterogeneous mix of cell types, causing varying levels of tumor purity. To inde- pendently assess the origin of IL6 and ACTA2 expression, we correlated bladder cancer tumor purity scores gener- ated using the ABSOLUTE algorithm with the corre- sponding IL6 and ACTA2 expression. To this end, TCGA samples with matching RNAseq expression and ABSOLUTE-based tumor purity estimates (N = 399) were analyzed. We observed a highly negative correl- ation between the tumor purity and IL6 mRNA (r = − 0.6274; ****P < 0.0001. Additional file 1) or ACTC2 mRNA (r = −0.4888; ****P < 0.0001. Additional file 2), indicating that higher average IL6 and ACTA2 expression correspond with higher stromal component of the tumor. We next investigated whether ACTA2/IL6 mRNA co-expression in bladder cancer tumors correlated with clinicopatho- logical features. For these analyses, bladder cancer pa- tients were stratified based on low and high quartiles of ACTA2/IL6 co-expression levels in tumors. The CM iCAF increases cancer cell survival in response to the chemotherapeutic drug mitomycin C Chemotherapeutic agents such as mitomycin C have long been used to treat bladder tumors, but development of drug resistance remains a substantial problem [1, 23]. To investigate how CAFs might influence the sensitivity of bladder cancer cells to mitomycin C, RT4 cells were cul- tured in the CM from HFs, HFs + TGFß or iCAFs with 0.5 μg/mL mitomycin C. As expected, the viability of RT4 cells was significantly decreased by 72.9% after the addition of mitomycin C for 12 h, demonstrating the ef- fectiveness of the drug (Fig. 4d). When cultured with the CM iCAF, RT4 cells showed reduced cell death (12.3%) as compared with the CM HF. However, cell death was not Goulet et al. BMC Cancer (2019) 19:137 Page 8 of 13 a c b d Fig. 4 CAFs-derived IL-6 enhances survival and promotes migration and invasion in RT4 bladder cancer cells. RT4 cells were cultured in the CM from HFs, HFs + TGFß or iCAFs with or without the anti-IL-6 antibody (1 μg/mL; x-IL-6). a. The proliferation of the RT4 cells was evaluated using the CyQUANT Kit. b. The migration of RT4 cells was determined by using Ibidi-silicone insert. Insert margins were marked by white vertical lines on optical micrographs. After 6 h and 12 h, the percentage of wound closure was evaluated by measuring migration distances (spaces between the white vertical lines). c. The invasion of RT4 cells was measured by transwell assays. Cells that had migrated through the membrane were fixed and nuclei were stained with DAPI. Representative photographs of migratory cells on the membrane are shown. The relative cell migration was determined by the number of migrated cells in 10 randomly selected fields. d. The sensitivity of RT4 cells to mitomycin C (0.5 μg/mL; Mito C) was evaluated using The CyQUANT Kit. RFU means relative fluorescence unit. All values are averages of three independent replicates. The graphs show mean +/−SD. The difference between groups was analyzed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = non-significant, n = 5–25 a c a c b b d d d Fig. 4 CAFs-derived IL-6 enhances survival and promotes migration and invasion in RT4 bladder cancer cells. The CM iCAF increases cancer cell survival in response to the chemotherapeutic drug mitomycin C As shown in Table 1, no significant association was observed be- tween ACTA2/IL6 mRNA co-expression and patient age, metastatic invasion, cancer recurrence or progres- sion. A correlation was seen between high ACTA2/IL6 co-expression and the tumor grade (*P = 0.0179) and lymph node metastasis (*P = 0.0281). A strong associ- ation was observed between high ACTA2/IL6 mRNA co-expression and tumor invasiveness (****P < 0.0001), correlating with our in vitro findings. Goulet et al. BMC Cancer (2019) 19:137 Page 9 of 13 b c d e f g h Fig. 5 IL6 mRNA expression is up-regulated in aggressive bladder cancer patient tumor specimens, correlates with CAF marker ACTA2 expression, and is associated with poor prognosis. Boxplots of IL6 (a-c) and ATCA2 (d-f) mRNA expression with respect to tumor stage, grade or invasiveness for TCGA (a-b, d-e, g) and GSE13507 (c, f, h) bladder cancer data. Number of tissues in each group is shown in brackets. The difference between groups was analyzed by Mann-Whitney test. ****P < 0.0001. g. Spearman’s correlation between ACTA2 and IL6 gene expression (r = 0.4543; ****P < 0.0001). h. Kaplan-Meier cancer specific survival curves according to lower versus higher half of ACTA2/IL6 mRNA co-expression level in bladder cancer patients (HR = 2.738; *P = 0.0471). Non-muscle invasive bladder cancer (NMIBC); muscle invasive bladder cancer (MIBC) b c b c d e f d g h h g Fig. 5 IL6 mRNA expression is up-regulated in aggressive bladder cancer patient tumor specimens, correlates with CAF marker ACTA2 expression, and is associated with poor prognosis. Boxplots of IL6 (a-c) and ATCA2 (d-f) mRNA expression with respect to tumor stage, grade or invasiveness for TCGA (a-b, d-e, g) and GSE13507 (c, f, h) bladder cancer data. Number of tissues in each group is shown in brackets. The difference between groups was analyzed by Mann-Whitney test. ****P < 0.0001. g. Spearman’s correlation between ACTA2 and IL6 gene expression (r = 0.4543; ****P < 0.0001). h. Kaplan-Meier cancer specific survival curves according to lower versus higher half of ACTA2/IL6 mRNA co-expression level in bladder cancer patients (HR = 2.738; *P = 0.0471). Non-muscle invasive bladder cancer (NMIBC); muscle invasive bladder cancer (MIBC) Discussion Similarly, IL-6 have been found to be increased in fibroblasts cultured with the CM from lung cancer [18]. Moreover, RT4 ex- press its receptor IL-6R, indicating that RT4 cells were suitable to respond to the iCAF-secreted IL-6 cytokine. Initiation of metastasis requires EMT, which is charac- terized by enhanced capability of active locomotion of cancer cells. We found that the iCAF-secreted IL-6 could promote cell growth, migration and invasion sig- nificantly. To our knowledge, this is the first study that has shown that iCAF-secreted IL-6 was enough to trans- form a non-invasive cell line (RT4) into invasive-like cells. To determine the potential of targeting IL-6 signaling in bladder cancer patients, we performed in silico ana- lysis using IL-6 expression data acquired from public database. Remarkably, we demonstrated that IL-6 was expressed at higher levels in bladder cancer stages III/IV than in stages I/II. Moreover, high IL-6 expression was preferentially associated with high grade MIBC. These results are somewhat consistent with the analysis of An- drews et al., in which a small cohort of human bladder cancer plasma samples revealed IL-6 were associated with cancer stage, metastasis and disease specific sur- vival [44]. Therefore, IL-6 expression might be related to a more malignant phenotype. In addition, IL-6 correlates with CAF marker ACTA2 and negatively correlates with tumor purity, suggesting that IL-6 is produced primarily by CAFs and not tumor cells. Nonetheless, our analysis revealed a correlation between high IL6/ATCA2 mRNA levels and poor cancer specific survival. the accumulation and nuclear translocation of β-catenin. In the nucleus, β-catenin binds to the lymphoid-enhancing factor/T-cell factor (LEF/TCF), to initiate the transcription of EMT genes [36–38]. Therefore, the increase expression of phosphorylated GSK3β and decreased expression of the p-β-catenin in RT4 cells cultured in the CM iCAF suggests a decrease in the β-catenin degradation, confirmed by the total-β-catenin Western blotting. Moreover, EMT tran- scription factors SNAI1, TWIST1 and ZEB1 were upregu- lated in the CM iCAF-cultured RT4 cells. the accumulation and nuclear translocation of β-catenin. In the nucleus, β-catenin binds to the lymphoid-enhancing factor/T-cell factor (LEF/TCF), to initiate the transcription of EMT genes [36–38]. Therefore, the increase expression of phosphorylated GSK3β and decreased expression of the p-β-catenin in RT4 cells cultured in the CM iCAF suggests a decrease in the β-catenin degradation, confirmed by the total-β-catenin Western blotting. Discussion of mechanisms [31–35]. However, their role in EMT of bladder cancer cells remains poorly defined. EMT plays a crucial role in metastasis dissemination and is correlated with poor prognosis in cancer patients [24–26]. However, the precise molecular events that initiate this complex process in bladder cancer are still poorly understood. A mounting body of evidence sug- gests that dynamic interplay between cancer cells and their microenvironment contributes to metastasis [27–30]. CAFs represent the major component of the TME and have been reported to support tumor progression by a variety Thus, we induced the activation of HFs into iCAFs in order to examine their role in bladder cancer cell EMT activation. We found that iCAFs promotes the upregula- tion of mesenchymal markers, such as N-cadherin and vimentin, while repressing epithelial markers E-cadherin and p-ß-catenin expression in bladder cancer cells. In presence of Wnt ligands, the glycogen synthase kinase 3 beta (GSK3β) is inhibited by phosphorylation, leading to Goulet et al. BMC Cancer (2019) 19:137 Page 10 of 13 Page 10 of 13 Table 1 Correlation between clinicopathological features and ACTA2/IL6 mRNA co-expression in bladder cancer patients Features No. of patients ACTA2/IL6 expression p value Low/Low High/High Age (years) < 65 53 28 (52,8%) 25 (47,2%) 0.6950 > 65 51 24 (47,1%) 27 (52,9%) Gender Male 85 48 (56,5%) 37 (43,5%) 0.0058** Female 19 4 (21,1%) 15 (78,9%) Invasiveness Superficial 67 43 (64,2%) 24 (35,8%) 0.0001**** Invasive 37 9 (24,3%) 28 (75,7%) Grade Low 75 43 (57,3%) 32 (42,7%) 0.0179* High 29 9 (31,0%) 20 (69,0%) Lymph node metastasis N0 92 50 (54,3%) 42 (45,7%) 0.0281* N1, N2 11 2 (18,2%) 9 (81,8%) Metastatic invasion M0 99 50 (50,5%) 49 (49,5%) 1.000 M1 5 2 (40,0%) 3 (60,0%) Recurrence (non-invasive cancer only) No 36 26 (72,2%) 10 (27,8%) 0.2016 Yes 31 17 (54,8%) 14 (45,2%) Progression No 63 34 (54,0%) 29 (46,0%) 0.3254 Yes 41 18 (43,9%) 23 (56,1%) Fisher’s exact test with 50% lower or higher level of mRNA expression between CAFs and IL-6 secretion in bladder cancer has not been shown. These studies drove us to investigate the role of IL-6 in bladder cancer. We examined the ex- pression of IL-6 by iCAFs and its receptors on the blad- der cancer cell line RT4. We found that IL-6 was upregulated in iCAFs compared to HFs. Competing interests Frédéric Pouliot has received speaker’s bureau honoraria from Sanofi, Genzyme, Amgen, Astellas, and Janssen; he is a consultant/advisory board member of Sanofi, Abbvie, Astellas, Janssen, Genzyme and Roche. No potential conflicts of interest were disclosed by the other authors. Availability of data and materials TCGA and GSE13507 datasets are available on GDC (Genomic Data Common; https://portal.gdc.cancer.gov/) and GEO (Gene Expression Omnibus; https:// www.ncbi.nlm.nih.gov/geo/) data portal. Authors’ contributions CRG designed and performed the experiments and analyzed the data. GB and DV performed the experiments. AC analysed TCGA and GSE13507 datasets. CRG wrote the manuscript and SC and AC revised it. SB and FP supervised the study and revised the manuscript. All authors have read and approved the final manuscript. Publisher’s Note Additional file 1: Exosomes characterization. A. TEM micrographs showing morphology of exosomes immunoprecipitated with anti-CD9 mAb from bladder cancer cells. Exosomes were stained with 2% uracyl acetate after being placed on carbon-coated TEM grid. B. NanoSight ana- lysis show three repeated measures of exosomes isolate according to their size. (PDF 945 kb) Additional file 2: Correlation of tumor purity scores obtained by using the ABSOLUTE algorithm with mean IL6 (A) and ACTA2 (B) expression. (PDF 163 kb) Additional file 1: Exosomes characterization. A. TEM micrographs showing morphology of exosomes immunoprecipitated with anti-CD9 mAb from bladder cancer cells. Exosomes were stained with 2% uracyl acetate after being placed on carbon-coated TEM grid. B. NanoSight ana- lysis show three repeated measures of exosomes isolate according to their size. (PDF 945 kb) Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conclusion Identify factors involved in the dynamic interaction be- tween tumor cells TME is much-needed to improve can- cer therapy. Herein, we show how one of the mediators of such an interaction, namely IL-6, mainly secreted by CAFs, can support tumor progression and how it can be antagonized by a neutralizing antibody to its receptor, which significantly reduces proliferation, migration and invasion in bladder cancer cells. These results highlight a prominent role for CAFs in bladder cancer and pro- vide a framework for further studies to develop relevant TME-based anti-cancer therapy. Discussion OPB-31121 and OPB-51602 are orally administrated STAT3 inhibi- tors, which are capable of binding to the SH2 domain of STAT3, and disrupt STAT3 dimerization and DNA bind- ing activity, and are currently under evaluation in clinical trials (NCT00657176, NCT01406574, NCT01423903 and NCT01184807). A combination of these two drugs could be used to more efficiently target some of the downstream mediators of IL-6 signaling. approaches, such as IL-6 signaling inhibition, particularly in bladder cancer. IL-6 signal through multimeric com- plexes that includes the gp130 receptor/IL-6Rα chain and ultimately triggers a signaling cascade that is mediated by STAT3 pathway. STAT3 activation was demonstrated to drive the proliferation, survival, invasiveness, and metasta- sis of tumor cells, while strongly suppressing the antitu- mour immune response [16]. Moreover, STAT3 activation was associated with bladder cancer cell growth and sur- vival [46]. In our study, we observed that iCAFs activates the STAT3 signaling pathway in RT4 cells. OPB-31121 and OPB-51602 are orally administrated STAT3 inhibi- tors, which are capable of binding to the SH2 domain of STAT3, and disrupt STAT3 dimerization and DNA bind- ing activity, and are currently under evaluation in clinical trials (NCT00657176, NCT01406574, NCT01423903 and NCT01184807). A combination of these two drugs could be used to more efficiently target some of the downstream mediators of IL-6 signaling. Consent for publication Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations CAF: Cancer-associated fibroblast; CM: Conditionned medium; DMEM: Dulbecco-Vogt modified Eagle’s media; EMT: Epithelial-mesenchymal transition; FAP: Fibroblast associated protein; FBS: Fetal bovine serum; FDA: Food and Drug Administration; GSK3β: Glycogen synthase kinase 3 beta; HF: Healthy fibroblast; iCAF: Induced cancer-associated fibroblast; IL- 6: Interleukin 6; IL-6R: IL-6 receptor; LEF: Lymphoid-enhancing factor; MIBC: Muscle-invasive bladder cancer; NMIBC: Non-muscle-invasive bladder cancer; pSTAT3: Phosphorylated STAT3; STAT3: Signal transducer and activator of transcription 3; TCF: T-cell factor; TME: Tumor microenvironment; α-SMA: Alpha smooth muscle actin Funding This work was supported by a Bladder Cancer Canada Grant (FP and CRG) and a Ferring Innovation Grant (SB). CRG and AC are recipient of an FRQS Doctoral Research Award from the Fonds de recherche du Québec – Santé (FRQS). FP is a Research Scholar of the FRQS. SB is the recipient of Canadian Urological Association Scholarship Funds and Canadian Institute for Health Research Grant #258229. The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Our study has some limitations. The TME consists of cancer cells, fibroblasts, endothelial cells, and immune cells, all of which contribute to the tumor secretome. Al- though our findings were focused on the interaction be- tween tumor cells and CAFs, IL-6 is a multifunctional cytokine known to be secreted by and to influence mul- tiple cell types in the TME, from immune to endothelial cells, and therefore, it is important to keep in mind the potential influence of these cells in our system [19, 47]. Thus, this strongly indicates that neutralization of IL-6 signaling could have even more potent antitumor effects. Acknowledgments W h k S l L We thank Solange Landreville, Bastien Paré and Isabelle Lorthois for their critical comments and experimental input. This work was supported by a Bladder Cancer Canada Grant (FP and CRG) and a Ferring Grant (SB). CRG and AC are recipient of a Doctoral Research Award from the Fonds de recherche du Québec - Santé (FRQS). FP is a Research Scholar of the FRQS. SB is the recipient of Canadian Urological Association Scholarship Funds and Canadian Institutes of Health Research Grant #258229. Discussion Moreover, EMT tran- scription factors SNAI1, TWIST1 and ZEB1 were upregu- lated in the CM iCAF-cultured RT4 cells. Taken together, our results reveal that IL-6 secreted from iCAFs promotes malignant behavior through the ac- tivation of the EMT program in bladder cancer cells. These results provide a mechanistic explanation for the role of IL-6 in the bladder cancer microenvironment, as well as the correlation observed between high IL-6 levels and metastatic potential in bladder cancer patients. Over- all, our findings may serve as an attractive therapeutic tar- get for human bladder cancer driven by IL-6 signaling. In this study, we demonstrated that IL-6 blockade reverses the effect of CAFs on tumor progression. Therefore, since IL-6 blocking antibodies are already approved by the Food and Drug Administration (FDA), such an approach to im- pede CAFs functions may be a clinical promising strategy. In solid tumour model, siltuximab, an IL-6 antibody, has demonstrated antitumor efficacy against ovarian, prostate, and lung cancers [16]. Despite these preclinical data, there remains a dearth of clinical trials investigating targeted The extent to which specific proteins secreted by CAFs contribute directly to tumor progression is un- clear. Previous studies have showed that IL-6 is involved in tumor progression and metastasis in various types of cancer, including lung cancer [39], pancreatic cancer [40], liver cancer [41], gastric cancer [42] and colon can- cer [43]. In bladder cancer, clinical findings showed that patients have higher serum IL-6 levels than the healthy controls and that higher IL-6 levels are associated with poorer prognosis [44, 45]. However, the association Goulet et al. BMC Cancer (2019) 19:137 Page 11 of 13 Page 11 of 13 Goulet et al. BMC Cancer (2019) 19:137 Page 11 of 13 approaches, such as IL-6 signaling inhibition, particularly in bladder cancer. IL-6 signal through multimeric com- plexes that includes the gp130 receptor/IL-6Rα chain and ultimately triggers a signaling cascade that is mediated by STAT3 pathway. STAT3 activation was demonstrated to drive the proliferation, survival, invasiveness, and metasta- sis of tumor cells, while strongly suppressing the antitu- mour immune response [16]. Moreover, STAT3 activation was associated with bladder cancer cell growth and sur- vival [46]. In our study, we observed that iCAFs activates the STAT3 signaling pathway in RT4 cells. Ethics approval and consent to participate Bladder biopsies from paediatric patients undergoing non-oncologic urologic surgery were obtained at the CHU de Québec-Université Laval Research Cen- ter in accordance with the institutional review board. All legal guardians of donors provided their formal, informed and written consent, each agreeing to supply a biopsy for this study. References Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6:392–401. 9. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6:392–401. 10. Öhlund D, Elyada E, Tuveson D. Fibroblast heterogeneity in the cancer 9. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6:392–401. 10. Öhlund D, Elyada E, Tuveson D. Fibroblast heterogeneity in the cancer wound J Exp Med 2014;211:1503–23 , g ; 10. Öhlund D, Elyada E, Tuveson D. Fibroblast heterogeneity in the cancer wound. J Exp Med. 2014;211:1503–23. 33. Giannoni E, Bianchini F, Masieri L, Serni S, Torre E, Calorini L, et al. Reciprocal activation of prostate Cancer cells and Cancer-associated fibroblasts stimulates epithelial-mesenchymal transition and Cancer Stemness. Cancer Res. 2010;70:6945–56. 11. Tao L, Huang G, Song H, Chen Y, Chen L. Cancer associated fibroblasts: an essential role in the tumor microenvironment (Review). Oncol Lett 2017;1–10. 12. Augsten M. Cancer-associated fibroblasts as another polarized cell type of the tumor microenvironment. Front Oncol Frontiers. 2014;4:1–8. 34. Zhuang J, Lu Q, Shen B, Huang X, Shen L, Zheng X, et al. TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT. Sci Rep. 2015;5:11924–13. 13. Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. 2014;6 a016295–5. 35. Karagiannis GS, Poutahidis T, Erdman SE, Kirsch R, Riddell RH, Diamandis EP. Cancer-associated fibroblasts drive the progression of metastasis through both paracrine and mechanical pressure on Cancer tissue. Mol Cancer Res. 2012;10:1403–18. 14. Abana CO, Bingham BS, Cho JH, Graves AJ, Koyama T, Pilarski RT, et al. IL-6 variant is associated with metastasis in breast cancer patients. Coleman WB, editor. PLoS One. 2017;12:e0181725–15. 15. Shang G-S, Liu L, Qin Y-W. IL-6 and TNF-α promote metastasis of lung cancer by inducing epithelial-mesenchymal transition. Oncol Lett. 2017;13: 4657–60. 36. Verheyen EM, Gottardi CJ. Regulation of Wnt/β-catenin signaling by protein kinases. Dev Dyn. 2010;239:34–44. 37. Stamos JL, Weis WI. The B-catenin destruction complex. Cold Spring Harbor Perspectives in Biology Cold Spring Harbor Lab. 2013;5 a007898–8. 16. Johnson DE, O'Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018;15:234–48. 38. Jamieson C, Sharma M, Henderson BR. Wnt signaling from membrane to nucleus: β-catenin caught in a loop. Int J Biochem Cell Biol Elsevier Ltd. 2012;44:847–50. 17. References Chang CH, Hsiao CF, Yeh YM, Chang GC, Tsai YH, Chen YM, Huang MS, Chen HL, Li YJ, Yang PC, Chen CJ, Hsiung CA, Su WC. Circulating interleukin-6 level is a prognostic marker for survival in advanced nonsmall cell lung cancer patients treated with chemotherapy. Int J Cancer. 2013;132(9):1977–85. 39. Wang L, Cao L, Wang H, Liu B, Zhang Q, Meng Z, Wu X, Zhou Q, Xu K. Cancer- associated fibroblasts enhance metastatic potential of lung cancer cells through IL-6/STAT3 signaling pathway. Oncotarget. 2017;8(44):76116–28. 18. Shintani Y, Fujiwara A, Kimura T, Kawamura T, Funaki S, Minami M, Okumura M. IL-6 secreted from Cancer-associated fibroblasts mediates Chemoresistance in NSCLC by increasing epithelial-mesenchymal transition signaling. J Thorac Oncol. 2016;11(9):1482–92. 40. Wu YS, Chung I, Wong WF, Masamune A, Sim MS, Looi CY. Paracrine IL-6 signaling mediates the effects of pancreatic stellate cells on epithelial- mesenchymal transition via Stat3/Nrf2 pathway in pancreatic cancer cells. Biochim Biophys Acta Gen Subj. 2017;1861(2):296–306. 19. Chen MF, Lin PY, Wu CF, Chen WC, Wu CT. IL-6 expression regulates Tumorigenicity and correlates with prognosis in bladder Cancer. PLoS One. 2013;8:e61901–10. 41. Xiong S, Wang R, Chen Q, Luo J, Wang J, Zhao Z, Li Y, Wang Y, Wang X, Cheng B. Cancer-associated fibroblasts promote stem cell-like properties of hepatocellular carcinoma cells through IL-6/STAT3/notch signaling. Am J Cancer Res. 2018;8(2):302–16. 20. Ringuette-Goulet C, Bernard G, Chabaud S, Couture A, Langlois A, Neveu B, et al. Tissue-engineered human 3D model of bladder cancer for invasion study and drug discovery. Biomaterials. 2017;145:233–41. 42. Wu X, Tao P, Zhou Q, Li J, Yu Z, Wang X, Li J, Li C, Yan M, Zhu Z, Liu B, Su L. IL-6 secreted by cancer-associated fibroblasts promotes epithelial- mesenchymal transition and metastasis of gastric cancer via JAK2/STAT3 signaling pathway. Oncotarget. 2017;8(13):20741–50. 21. Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD, Hinoue T, Laird PW, Hoadley KA, Akbani R, Castro MAA, Gibb EA, Kanchi RS, Gordenin DA, Shukla SA, Sanchez-Vega F, Hansel DE, Czerniak BA, Reuter VE, Su X, de Sa Carvalho B, Chagas V, Mungall KL, Sadeghi S, Pedamallu CS, Lu Y, Klimczak LJ, Zhang J, Choo C, Ojesina AI, Bullman S, Leraas KM, Lichtenberg TM, Wu CJ, Schultz N, Getz G, Meyerson M, Mills GB, McConkey DJ, Research Network TCGA, Weinstein JN, Kwiatkowski DJ, Lerner SP. Comprehensive molecular characterization of muscle-invasive bladder Cancer. Cell. 2017;171(3):540–556.e25. 43. Author details 1 1Centre de recherche en organogénèse expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec-Université Laval Research Center, QC, Québec, Canada. 2Department of Surgery, Faculty of Medicine, Laval University, QC, Quebec, Canada. 3Oncology Division, CHU de Québec Research Center, QC, Quebec, Canada. 4Centre de recherche du CHU de Québec-Université Laval, Centre de recherche en organogénèse Additional file 2: Correlation of tumor purity scores obtained by using the ABSOLUTE algorithm with mean IL6 (A) and ACTA2 (B) expression. (PDF 163 kb) Page 12 of 13 Page 12 of 13 Page 12 of 13 Goulet et al. BMC Cancer (2019) 19:137 Goulet et al. BMC Cancer (2019) 19:137 expérimentale de l’Université Laval/LOEX, 1401, 18e rue, Quebec city, Québec G1J 1Z4, Canada. gene classifier in primary non-muscle invasive bladder cancer. Mol Cancer. 2010;9:3. gene classifier in primary non-muscle invasive bladder cancer. Mol Cancer. 2010;9:3. 23. Chou R, Selph SS, Buckley DI, Gustafson KS, Griffin JC, Grusing SE, et al. Treatment of muscle-invasive bladder cancer: A systematic review. Cancer. 2016;122:842–51. Received: 30 May 2018 Accepted: 5 February 2019 Received: 30 May 2018 Accepted: 5 February 2019 Received: 30 May 2018 Accepted: 5 February 2019 Received: 30 May 2018 Accepted: 5 February 2019 24. Breyer J, Gierth M, Shalekenov S, Aziz A, Schäfer J, Burger M, et al. Epithelial– mesenchymal transformation markers E-cadherin and survivin predict progression of stage pTa urothelial bladder carcinoma. World J Urol. 2016; 34:709–16. References 1. Pugashetti N, Alibhai SMH, Yap SA. Non-muscle-invasive bladder Cancer: review of diagnosis and management. J Curr Clin Care. 2015;5:40–50. 25. Imani S, Hosseinifard H, Cheng J, Wei C, Fu J. Prognostic value of EMT- inducing transcription factors (EMT-TFs) in metastatic breast Cancer: A systematic review and Meta- analysis. Sci. Rep Nat Publ Group. 2016;6:1–10. review of diagnosis and management. J Curr Clin Care. 2015;5:40–5 2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016; 66:7–30. 26. Wan T, Zhang T, Si X, Zhou Y. Overexpression of EMT-inducing transcription factors as a potential poor prognostic factor for hepatocellular carcinoma in Asian populations: A meta-analysis. Oncotarget. 2017;8:59500–8. 3. Kamat AM, M Hahn N, A Efstathiou J, P Lerner S, Malmstrom P-U, Choi W, et al. Bladder cancer. The lancet. Elsevier Ltd. 2016;388:2796–810. 4. Sanli O, Dobruch J, Knowles MA, Burger M, Alemozaffar M, Nielsen ME, et al. Bladder cancer. Nature Publishing Group Macmillan Publishers Limited. 2017;3:1–19. 27. Catalano V, Turdo A, Di Franco S, Dieli F, Todaro M, Stassi G. Tumor and its microenvironment: A synergistic interplay. Semin Cancer Biol Elsevier Ltd; 2013; 23:522–532. 5. Mari A, Campi R, Tellini R, Gandaglia G, Albisinni S, Abufaraj M, et al. Patterns and predictors of recurrence after open radical cystectomy for bladder cancer: a comprehensive review of the literature. World. J Urol Springer Berlin Heidelberg. 2018;36:157–70. 28. Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013;19:1423–37. 29. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer. 2008;9:239–52. 6. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J. Clin. Invest. American society for. Clinical Investig. 2009;119:1420–8. 30. Dvorak HF, Weaver VM, Tlsty TD, Bergers G. Tumor microenvironment and progression. Leong S, editor. J Surg Oncol. 2011;103:468–74. 7. Bryan RT, Tselepis C. Review articles cadherin switching and bladder Cancer. J Urol Elsevier Inc. 2010;184:423–31. 31. Kharaishvili G, Simkova D, Bouchalova K, Gachechiladze M, Narsia N, Bouchal J. The role of cancer-associated fibroblasts, solid stress and other microenvironmental factors in tumor progression and therapy resistance. 2014;14:1–8. 8. Ringuette-Goulet C, Bernard G, Tremblay S, Chabaud S, Bolduc S, Pouliot F. Exosomes induce fibroblast differentiation into Cancer-associated fibroblasts through TGFβ signaling. Molecular Cancer Research. American association for Cancer Research. 2018:1–25. 32. Yamaguchi H, Sakai R. Direct interaction between carcinoma cells and Cancer associated fibroblasts for the regulation of Cancer invasion. Cancers. 2015;7:2054–62. 9. 47. Kato T, Noma K, Ohara T, Kashima H, Katsura Y, Sato H, Komoto S, Katsube R, Ninomiya T, Tazawa H, Shirakawa Y, Fujiwara T. Cancer-associated fibroblasts affect Intratumoral CD8+ and FoxP3+ T cells via IL6 in the tumor microenvironment. Clin Cancer Res. 2018;24(19):4820–33. 45. Kumari N, Agrawal U, Mishra AK, Kumar A, Vasudeva P, Mohanty NK, Saxena S. Predictive role of serum and urinary cytokines in invasion and recurrence of bladder cancer. Tumour Biol. 2017;39(4):1010428317697552. 46. Chen CL, Cen L, Kohout J, Hutzen B, Chan C, Hsieh FC, Loy A, Huang V, Cheng G, Lin J. Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival. Mol Cancer. 2008;7:78. References Zhu L, Cheng X, Ding Y, Shi J, Jin H, Wang H, Wu Y, Ye J, Lu Y, Wang TC, Yang CS, Tu SP. Bone marrow-derived myofibroblasts promote colon tumorigenesis through the IL6/JAK2/STAT3 pathway. Cancer Lett 2014 1;343(1):80–9. 44. Andrews B, Shariat SF, Kim JH, Wheeler TM, Slawin KM, Lerner SP. Preoperative plasma levels of interleukin-6 and its soluble receptor predict disease recurrence and survival of patients with bladder cancer. J Urol. 2002; 167(3):1475–81. 22. Kim WJ, Kim EJ, Kim SK, Kim YJ, Ha YS, Jeong P, Kim MJ, Yun SJ, Lee KM, Moon SK, Lee SC, Cha EJ, Bae SC. Predictive value of progression-related 22. Kim WJ, Kim EJ, Kim SK, Kim YJ, Ha YS, Jeong P, Kim MJ, Yun SJ, Lee KM, Moon SK, Lee SC, Cha EJ, Bae SC. Predictive value of progression-related Page 13 of 13 Page 13 of 13 Goulet et al. BMC Cancer (2019) 19:137 Goulet et al. BMC Cancer (2019) 19:137
https://openalex.org/W4306725895
https://sciendo.com/pdf/10.2478/abm-2022-0021
English
null
Long noncoding and micro-RNA expression in a model of articular chondrocyte degeneration induced by stromal cell-derived factor-1
Asian biomedicine/Asian Biomedicine (Research, Reviews and News)
2,022
cc-by
7,299
Long noncoding and micro-RNA expression in a model of articular chondrocyte degeneration induced by stromal cell-derived factor-1 Guoliang Wang1,2 , Lu He1 , Yaoyu Xiang1 , Di Jia1 , Yanlin Li1,* *Correspondence to: Yanlin Li, Department of Sports Medicine, First Affiliated Hospital of Kunming Medical University, No. 295, Xichang Road, Kunming, Yunnan 650032, China, email: 852387873@qq.com 1Department of Sports Medicine, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, China 2Kunming Medical University, Kunming, Yunnan 650032, China Open Access. © 2022 Wang et al., published by Sciendo.   This work is licensed under the Creative Commons Attribution 4.0 International License. *Correspondence to: Yanlin Li, Department of Sports Medicine, First Affiliated Hospital of Kunming Medical University, No. 295, Xichang Road, Kunming, Yunnan 650032, China, email: 852387873@qq.com 1Department of Sports Medicine, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, China 2Kunming Medical University, Kunming, Yunnan 650032, China DOI 10.2478/abm-2022-0021 — Asian Biomed (Res Rev News) 2022; 16(4):169–179 Abstract Background: Gene regulatory network analysis has found that long noncoding ribonucleic acids (lncRNAs) are strongly associated with the pathogenesis of osteoarthritis. Objectives: To determine the differential expression of lncRNAs and microRNAs (miRNAs) in normal chondrocytes and those from a model of articular chondrocyte degeneration. Methods: Chondrocytes were cultured from cartilage obtained from patients diagnosed with osteoarthritis of the knee. Stromal cell-derived factor-1 (SDF-1) was used to induce their degeneration. Total RNA was extracted, analyzed, amplified, labeled, and hybridized on a chip to determine expression. The set of enriched differentially expressed miRNAs was analyzed by gene ontology and the Kyoto Encyclopedia of Genes and Genomes to describe the functional properties of the key biological processes and pathways. We conducted a bioinformatics analysis using Cytoscape to elucidate the interactions between miRNAs and proteins. Results: We found that the expression of 186 lncRNAs was significantly different in the model of chondrocyte degeneration, in which 88 lncRNAs were upregulated, and 98 were downregulated. Expression of 684 miRNAs was significantly different. Analysis of the protein–protein interaction (PPI) network indicated that the genes for CXCL10, ISG15, MYC, MX1, OASL, IFIT1, RSAD2, MX2, IFI44L, and BST2 are the top 10 core genes, identifying the most important functional modules to elucidate the differential expression of miRNAs. Conclusions: These data may provide new insights into the molecular mechanisms of chondrocyte degeneration in osteoarthritis, and the identification of lncRNAs and miRNAs may provide potential targets for the differential diagnosis and therapy of osteoarthritis. Keywords: chondrocytes; high-throughput nucleotide sequencing; microRNAs; osteoarthritis; RNA, long Osteoarthritis is a chronic and progressive multifactorial disease characterized by subchondral bone destruction, reduced numbers of chondrocytes, and degradation of the cartilaginous matrix [1–3]. Long noncoding ribonucleic acids (lncRNAs) and microRNAs (miRNAs) play important roles in mediating gene regulatory pathways in the pathogenesis of osteoarthri­ tis and other diseases, including acute early phase spinal cord injury [4–6]. Aberrant expression of lncRNAs and miRNAs is associated with the development of osteoarthritis and may play regulatory roles in its pathogenesis [7–9]. About 4700 Open Access. © 2022 Wang et al., published by Sciendo. This work is licensed under the Creative Commons Attribution 4.0 International License. 170    Wang et al. 170 lncRNAs are expressed aberrantly in cartilage from patients with osteoarthritis compared with normal cartilage from control patients [10]. Materials and osteoarthritis cartilage modeling The cell culture medium in the 2 groups was high-glucose DMEM containing 10% fetal bovine serum and penicillin–streptomycin. In the experimental group, 100 ng/mL SDF-1 (R&D Systems) was All cartilage tissue was obtained from patients diagnosed with knee osteoarthritis who underwent total knee replacement at the First Affiliated Hospital of Kunming Medical University (March 2018 to March 2019). The cartilaginous tissue remai­ ning on the surface of the tibial plateau and femoral condyle after osteotomy was collected during the surgery. The carti­ lage tissue specimen donors were informed of the research in writing and provided their documented consent before the specimens were collected. The present study was approved by the ethics committee of the First Affiliated Hospital of Kunming Medical University (2018 Lun Shen L No. 21), and the study protocols were in compliance with relevant national regulations and laws, including the ethical principles of the China Food and Drug Administration Good Clinical Practice for Medical Devices and People’s Republic of China Regula­ tions for the Management of Medical Institutions (promulga­ ted by the Order No. 149 of the State Council on February 26, 1994; and revised in accordance with the Decision of the State Council on Amending Some Administrative Regulations on February 6, 2016), and international medical ethics docu­ ments, including the International Conference on Harmoni­ sation Good Clinical Practice (ICH-GCP) and the Declara­ tion of Helsinki and its contemporary (2013) revisions. After a diagnosis of osteoarthritis in accordance with the criteria described by Altman et al. [24], 10 patients (4 male and 6 female) underwent artificial knee arthroplasty due to osteoar­ thritis. The patients were aged from 55 to 75 years and had a gross visual grade of cartilage degeneration score of 0 or 1 point, where 0 points indicated a smooth articular surface and usual color, and 1 point indicated a rough articular surface, small cracks, and dark color [25]. Patients with liver or kidney disease, connective tissue disease, endocrine disease, serious cardiovascular disease, and tumors were excluded. The carti­ lage tissue was trimmed to dimensions 2 mm × 2 mm × 1 mm under aseptic conditions. Ten pieces (100 pieces in total from the 10 different patients) of cartilage tissue were placed sepa­ rately into preprepared high-glucose Dulbecco’s modified Eagle’s medium (DMEM) for digestion and culture. Abstract lncRNAs play an important regulatory role in the processes of joint synovial inflammation, cartilage matrix syn­ thesis and metabolism, angiogenesis, chondrocyte autophagy, apoptosis, and other factors associated with osteoarthritis [11–13]. Materials and osteoarthritis cartilage modeling All cartilage tissue was obtained from patients diagnosed with knee osteoarthritis who underwent total knee replacement at the First Affiliated Hospital of Kunming Medical University (March 2018 to March 2019). The cartilaginous tissue remai­ ning on the surface of the tibial plateau and femoral condyle after osteotomy was collected during the surgery. The carti­ lage tissue specimen donors were informed of the research in writing and provided their documented consent before the specimens were collected. The present study was approved by the ethics committee of the First Affiliated Hospital of Kunming Medical University (2018 Lun Shen L No. 21), and the study protocols were in compliance with relevant national regulations and laws, including the ethical principles of the China Food and Drug Administration Good Clinical Practice for Medical Devices and People’s Republic of China Regula­ tions for the Management of Medical Institutions (promulga­ ted by the Order No. 149 of the State Council on February 26, 1994; and revised in accordance with the Decision of the State Council on Amending Some Administrative Regulations on February 6, 2016), and international medical ethics docu­ ments, including the International Conference on Harmoni­ sation Good Clinical Practice (ICH-GCP) and the Declara­ tion of Helsinki and its contemporary (2013) revisions. After a diagnosis of osteoarthritis in accordance with the criteria described by Altman et al. [24], 10 patients (4 male and 6 female) underwent artificial knee arthroplasty due to osteoar­ thritis. The patients were aged from 55 to 75 years and had a gross visual grade of cartilage degeneration score of 0 or 1 point, where 0 points indicated a smooth articular surface and usual color, and 1 point indicated a rough articular surface, small cracks, and dark color [25]. Patients with liver or kidney disease, connective tissue disease, endocrine disease, serious cardiovascular disease, and tumors were excluded. The carti­ lage tissue was trimmed to dimensions 2 mm × 2 mm × 1 mm under aseptic conditions. Ten pieces (100 pieces in total from the 10 different patients) of cartilage tissue were placed sepa­ rately into preprepared high-glucose Dulbecco’s modified Eagle’s medium (DMEM) for digestion and culture. Chond­ rocytes from the first-generation culture were divided equally without special selection into an experimental and control group (n = 3 each). The density of autologous chondrocytes in the carrier was about 1.6–2.0 × 105 cells/cm2. Data collection An Axon 4000B fluorescent scanner (Molecular Probes) was used to scan hybridized microarray slides and convert the scanning signal into a digital signal, and the low quality and weak signal data points were excluded. Fold change (FC) ³2 was the cutoff criterion. A Student t test was performed to cal­ culate the scanning signal values of the 2 groups and to obtain the log2 (ratio) and P value for each probe. When the ratio of the intensity of the hybridization signal between the experi­ mental group and the control group was ³2, the expression was defined as upregulated; otherwise, the expression was defined as downregulated. The miRNA screening conditions were FC ³2 and P < 0.05 or log2 (ratio) ³0.8. Materials and osteoarthritis cartilage modeling lncRNAs and miRNAs in articular chondrocytes  Asian Biomed (Res Rev News) 2022; 16(4):169–179 171 specific target regions according to protocols specified by the manufacturer. added to the chondrocytes [17], and the control group was untreated. The chondrocytes in the 2 groups were cultured under the same conditions for 48 h using an improved method [19, 20], although a vehicle control was not used to conserve resources. RNA extraction Total RNA samples were extracted using an RNeasy Mini Kit (catalog No. 74106; Qiagen). The extraction was performed in accordance with the standard operating procedure hand­ book provided by the manufacturer. The extracted total RNA was examined qualitatively using a Bioanalyzer 2100 system (Agilent Technologies) and quantified using a Qubit 3.0 Fluo­ rometer (Life Technologies) and NanoDrop One spectropho­ tometer (Thermo Fisher Scientific). RNA amplification and labeling Gene ontology (GO) analysis was performed to describe the functional properties of differentially expressed miRNAs. GO analysis included molecular function (MF), biological process (BP), and cellular component (CC). KEGG signaling pathway analysis was performed to describe the biological pathways of differentially expressed miRNAs. Total RNA was amplified and labeled using a Low-Input QuickAmp WT Labeling Kit (catalog No. 5190-2943; Agilent Technologies) according to the kit instructions; the labeled complementary RNA was purified using an RNeasy Mini Kit (catalog No. 74106; Qiagen). Materials and osteoarthritis cartilage modeling Chond­ rocytes from the first-generation culture were divided equally without special selection into an experimental and control group (n = 3 each). The density of autologous chondrocytes in the carrier was about 1.6–2.0 × 105 cells/cm2. The cell culture medium in the 2 groups was high-glucose DMEM containing 10% fetal bovine serum and penicillin–streptomycin. In the experimental group, 100 ng/mL SDF-1 (R&D Systems) was Stromal cell-derived factor-1 (SDF-1) is found at sig­ nificantly higher levels in the synovial fluid of patients with osteoarthritis and has strong effects to induce car­ tilage matrix degradation. The SDF-1/chemokine (CXC motif) receptor 4 (CXCR4) signaling pathway plays a key role in the pathological process of cartilage degeneration in animal models and increases interleukin (IL)-6 production by human synovial fibroblasts [14–17]. Synovial tissue of the knee joints in patients with osteoarthritis can produce SDF-1 at a higher concentration than the synovial tissue of healthy knee joints. SDF-1 can interact with CXCR4- specific receptors on the surface of cartilage to activate the SDF-1/CXCR4 signaling pathway, which activates the ext­ racellular signal-regulating enzyme (Erk) and related kinase (p38 mitogen-activated protein (MAP) kinase) signaling pathways, promoting the release of matrix metalloproteina­ ses (MMP) from the cartilage matrix, which degrade the type II collagen and aggrecan substrates in the cartilage matrix, ultimately degenerating the articular cartilage and inducing osteoarthritis [18–20]. lncRNA-H19 stimulates osteogenic differentiation of bone marrow mesenchymal stem cells by regulating SDP-1 expression via miRNA-149 [21]. miRNA- 126-silenced mice showed that miRNA-126 can regulate the expression of SDF-1 in endothelial cells [22]. miRNA- 141-3p regulator of SDF-1 in bone marrow stromal cells may play an important role in the age-dependent patho­ physiology of the murine and human bone marrow niche [23]. These studies indicate that the expression of SDP-1 in tissues and cells may be regulated by a multifaceted network of lncRNA and miRNA. Here, we examined the expression of miRNAs and lncRNAs in an SDF-1-induced model of chondrocyte dege­ neration. Subsequently, we used microarrays to analyze the differential expression of the identified miRNAs and lncRNAs. We also analyzed the differential expression of lncRNAs in terms of transcript length distribution, classifi­ cation, and exon number. Bioinformatics analysis was used to clarify the interaction between differentially expressed lncRNAs and miRNAs. A gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed to identify the critical biological processes and pathways. Hybridization The NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche) for permutation hybridization was used to enrich To elucidate the interactions between differentially expressed miRNAs, a database of interacting genes was searched, and Figure 1. Morphology of chondrocytes in the 2 groups (×100). A. Chondrocytes in the experimental group were cultured with SDF-1 for 48 h. B. Morphology of chondrocytes in the control group. SDF-1, stromal cell-derived factor-1. Figure 1. Morphology of chondrocytes in the 2 groups (×100). A. Chondrocytes in the experimental group were cultured with SDF-1 for 48 h. B. Morphology of chondrocytes in the control group. SDF-1, stromal cell-derived factor-1. 172    Wang et al. 172    Wang et al. 172 Figure 2. Analysis of miRNAs and lncRNAs. A. Heatmap of the differentially expressed (DE) miRNAs. B. Scatter plot of the DE miRNAs. C. Volcano plot of the DE miRNAs. D. Heatmap of the DE lncRNAs. E. Scatter plot of the DE lncRNAs. F. Volcano plot of the DE lncRNAs. In the heatmap, red represents upregulated miRNAs or lncRNAs, and green represents downregulated miRNAs or lncRNAs. In the scatter plot, the X and Y values are th li d i l l h l l Th d d li t FC li ith d f lt h f 2 0 R d i t Figure 2. Analysis of miRNAs and lncRNAs. A. Heatmap of the differentially expressed (DE) miRNAs. B. Scatter plot of the DE miRNAs. C. Volcano plot of the DE miRNAs. D. Heatmap of the DE lncRNAs. E. Scatter plot of the DE lncRNAs. F. Volcano plot of the DE lncRNAs. In the heatmap, red represents upregulated miRNAs or lncRNAs, and green represents downregulated miRNAs or lncRNAs. In the scatter plot, the X and Y values are the average normalized signal values, shown on a log2 scale. The red and green lines were set as FC lines with a default change of 2.0. Red points (FC >2) indicate upregulated miRNAs or lncRNAs, and blue points (FC ≤2) indicate downregulated miRNAs or lncRNAs. In the volcano plot, the X-axis is the FC (log2), and the Y-axis is P (–log10). Red points (FC >2) indicate upregulated miRNAs or lncRNAs, and blue points (FC ≤2) indicate downregulated miRNAs or lncRNAs. DE, differentially expressed; FC, fold change; FPKM, fragments per kilobase of transcript per million mapped fragments; lncRNAs, long noncoding ribonucleic acids; miRNAs, microRNAs; SDF1, stromal cell-derived factor-1 treated articular chondrocytes. Morphological changes in cell culture Chondrocytes from the first-generation culture of osteoar­ thritis tissue were cultured with SDF-1 for 48 h. The chon­ drocytes in the experimental group were irregular and long spindle–shaped, with a low refractive index and fuzzy struc­ ture in living cells, while the chondrocytes in the control group were spindle-shaped or oval, with an intact nucleus, high refractive index, and clear structure in living cells (Figures 1A and B). Hybridization Normal indicates articular chondrocytes untreated with SDF-1. lncRNAs and miRNAs in articular chondrocytes  Asian Biomed (Res Rev News) 2022; 16(4):169–179 173 Table 1. Top 10 most upregulated and most downregulated lncRNAs in chondrocytes from the SDF-1-induced model of articular chondrocyte degeneration Upregulated lncRNAs Downregulated lncRNAs lncRNA ID P FC lncRNA ID P FC NONHSAT094312.2 5.51E–05 10.27 NONHSAT246243.1 4.01E–06 9.44 NONHSAT060379.2 3.42E–17 8.19 NONHSAT217441.1 3.97E–07 8.57 NONHSAT207507.1 1.60E–16 8.12 NONHSAT238505.1 6.22E–05 7.07 NONHSAT166467.1 5.44E–07 7.77 NONHSAT258030.1 5.57E–05 6.86 NONHSAT198879.1 3.39E–06 7.75 NONHSAT176410.1 4.59E–06 6.81 NONHSAT248596.1 1.21E–11 7.61 NONHSAT022132.2 0.000138 6.68 NONHSAT152279.1 2.89E–06 7.41 NONHSAT119402.2 2.11E–06 6.59 NONHSAT000091.2 9.96E–05 7.21 NONHSAT022138.2 0.000169 6.58 ENST00000559458 4.66E–06 7.05 NONHSAT229871.1 5.25E–05 6.53 NONHSAT038052.2 1.00E–06 6.87 NONHSAT225394.1 4.07E–05 6.23 FC, fold change; SDF-1, stromal cell-derived factor-1. Figure 3. Expression signatures of dysregulated lncRNAs in SDF-1-induced articular chondrocyte degeneration. A. Length distribution showed that dysregulated lncRNAs were mainly concentrated between 700 bp and 3000 bp. B. Differential lncRNAs were classified according to their genomic architecture. Table 1. Top 10 most upregulated and most downregulated lncRNAs in chondrocytes from the SDF-1-induced model of articular chondrocyte degeneration Upregulated lncRNAs Downregulated lncRNAs lncRNA ID P FC lncRNA ID P FC NONHSAT094312.2 5.51E–05 10.27 NONHSAT246243.1 4.01E–06 9.44 NONHSAT060379.2 3.42E–17 8.19 NONHSAT217441.1 3.97E–07 8.57 NONHSAT207507.1 1.60E–16 8.12 NONHSAT238505.1 6.22E–05 7.07 NONHSAT166467.1 5.44E–07 7.77 NONHSAT258030.1 5.57E–05 6.86 NONHSAT198879.1 3.39E–06 7.75 NONHSAT176410.1 4.59E–06 6.81 NONHSAT248596.1 1.21E–11 7.61 NONHSAT022132.2 0.000138 6.68 NONHSAT152279.1 2.89E–06 7.41 NONHSAT119402.2 2.11E–06 6.59 NONHSAT000091.2 9.96E–05 7.21 NONHSAT022138.2 0.000169 6.58 ENST00000559458 4.66E–06 7.05 NONHSAT229871.1 5.25E–05 6.53 NONHSAT038052.2 1.00E–06 6.87 NONHSAT225394.1 4.07E–05 6.23 FC, fold change; SDF-1, stromal cell-derived factor-1. upregulated and most downregulated lncRNAs in chondrocytes from the SDF-1-induced model of articular chondrocyte FC, fold change; SDF-1, stromal cell-derived factor-1. DF-1-induced articular chondrocyte degeneration. A. Length distribution showed n 700 bp and 3000 bp B Differential lncRNAs were classified according to their Figure 3. Expression signatures of dysregulated lncRNAs in SDF-1-induced articular chondrocyte degeneration. A. Length distribution showed that dysregulated lncRNAs were mainly concentrated between 700 bp and 3000 bp. B. Differential lncRNAs were classified according to their genomic architecture. Figure 3. Expression signatures of dysregulated lncRNAs in SDF-1-induced articular chondrocyte degeneration. A. Length distribution showed that dysregulated lncRNAs were mainly concentrated between 700 bp and 3000 bp. B. Differential lncRNAs were classified according to their genomic architecture. differentially expressed miRNAs with a FC threshold >2 and P < 0.05 were regarded as significant. Hybridization Cytoscape visualization was used to integrate biological models with biological graphics visualization tools for mole­ cular interaction networks [26]. Differentially expressed miRNAs with FC >4 and P < 0.05 were identified, and the STRING online tool [27] was used to analyze differentially expressed miRNAs with a combined protein–protein interac­ tion (PPI) score >0.4 as the cutoff value. Cytoscape visualization was used to integrate biological models with biological graphics visualization tools for mole­ cular interaction networks [26]. Differentially expressed miRNAs with FC >4 and P < 0.05 were identified, and the STRING online tool [27] was used to analyze differentially expressed miRNAs with a combined protein–protein interac­ tion (PPI) score >0.4 as the cutoff value. Statistical analysis PPI network core (top 10) genes in the SDF-1-induced model of articular chondrocyte degeneration Protein Degree Eccentricity Edge count CXCL10 16 4 16 ISG15 12 4 12 MYC 11 5 11 MX1 10 5 10 OASL 10 5 10 IFIT1 10 5 10 RSAD2 10 5 10 MX2 10 5 10 IFI44L 10 5 10 BST2 8 5 8 Table 2. PPI network core (top 10) genes in the SDF-1-induced model of articular chondrocyte degeneration (2) the antisense group, (3) the bidirectional group, (4) the intronic group, and (5) the intergenic group (Figure 3B). Difference visualization lncRNA analysis revealed a total of 52,741 lncRNAs with changed expression. Further analysis showed that of these, the expression of 186 lncRNAs was changed significantly; 88 were upregulated, and 98 were downregulated. A total of 119,205 miRNAs had changed their level of expression, and the expression of 684 miRNAs had changed significantly. The heatmap, scatter plot, and volcano plot of the differenti­ ally expressed miRNAs and lncRNAs are shown in Figure 2 (miRNAs, A–C; lncRNAs, D–F). Gene ontology and KEGG analyses The GO analysis showed that the signaling pathways of miRNAs and their target genes are enriched in receptor regula­ tion activities (MF), secondary lysosomes (cell components), lipopolysaccharide regulatory signaling pathways (biological processes), type I interferon signaling pathways, and ionic transmembrane transporter activity regulation (Figure  4). Pathway analysis indicated that miRNAs and their target genes are enriched in cytokine–cytokine receptor interactions, osteoclast differentiation, the nuclear factor k-light-chain- enhancer of activated B cells (NF-kB) signaling pathway, the transforming growth factor (TGF-b) signaling pathway, and the ion signaling pathway, as shown in Figure 5. BST2, bone marrow stromal tetherin antigen 2; CXCL10, interferon γ ­inducible CXC 10 kDa chemokine chemotactic for monocytes and T-lym­ phocytes; IFI44L, interferon-induced protein 44 like protein; IFIT1, inter­ feron-induced with tetratricopeptide repeats 1 protein; ISG15, interferon- α-stimulated gene 15-kDa protein; MX1, interferon inducible myxovirus resistance protein MxA p78; MX2, second interferon-induced myxovirus resistance 2 protein MxB p78; MYC, v-myc myelocytomatosis viral oncoge­ ne homolog protein; OASL, p59 2′,5′-oligoadenylate synthetase-like prote­ in; PPI, protein−protein interaction; RSAD2, radical S-adenosyl methionine domain containing 2 protein; SDF-1, stromal cell-derived factor-1. BST2, bone marrow stromal tetherin antigen 2; CXCL10, interferon γ ­inducible CXC 10 kDa chemokine chemotactic for monocytes and T-lym­ phocytes; IFI44L, interferon-induced protein 44 like protein; IFIT1, inter­ feron-induced with tetratricopeptide repeats 1 protein; ISG15, interferon- α-stimulated gene 15-kDa protein; MX1, interferon inducible myxovirus resistance protein MxA p78; MX2, second interferon-induced myxovirus resistance 2 protein MxB p78; MYC, v-myc myelocytomatosis viral oncoge­ ne homolog protein; OASL, p59 2′,5′-oligoadenylate synthetase-like prote­ in; PPI, protein−protein interaction; RSAD2, radical S-adenosyl methionine domain containing 2 protein; SDF-1, stromal cell-derived factor-1. PPI network construction The PPI network identified genes for 10 proteins, ­interferon-g (IFN-g) inducible CXC 10  kDa chemokine ­chemotactic for monocytes and T-lymphocytes (CXCL10), ­interferon-a-stimulated gene 15-kDa protein (ISG15), v-myc myelocytomatosis viral oncogene homolog protein (MYC), interferon inducible myxovirus resistance protein MxA p78 (MX1), p59 2¢,5¢-oligoadenylate synthetase-like protein (OASL), interferon-induced with tetratricopeptide repeats 1 protein (IFIT1), radical S-adenosyl methionine domain con­ taining 2 protein (RSAD2), second interferon-induced myxo­ virus resistance 2 protein MxB p78 (MX2), interferon-induced protein 44 like protein (IFI44L), and bone marrow stromal tetherin antigen 2 (BST2), that have a higher possibility of being involved in the mechanism of chondrocyte degeneration (Table 2) and showed a network of upregulated and downre­ gulated genes (Figure 6). Statistical analysis Data were analyzed using SPSS Statistics for Windows (version 17.0; SPSS). Differentially expressed (DE) levels of miRNAs and lncRNAs were compared using paired-sam­ ple t tests. Student t tests were to compare values between the groups. The differentially expressed lncRNAs and 174    Wang et al. 174    Wang et al. Figure 4. GO analysis of differentially expressed genes. GO annotations of mRNAs with top 30 enrichment scores. The circles represent biological processes; the triangles represent cell components; and the squares represent MF. GO, gene ontogeny; MF, molecular functions. Figure 4. GO analysis of differentially expressed genes. GO annotations of mRNAs with top 30 enrichment scores. The circles represent biological processes; the triangles represent cell components; and the squares represent MF. GO, gene ontogeny; MF, molecular functions. Figure 4. GO analysis of differentially expressed genes. GO annotations of mRNAs with top 30 enrichment scores. The circles represent biological processes; the triangles represent cell components; and the squares represent MF. GO, gene ontogeny; MF, molecular functions. Figure 5. KEGG signaling pathway analysis of differentially expressed genes. Top 30 for KEGG enrichment. KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure 5. KEGG signaling pathway analysis of differentially expressed genes. Top 30 for KEGG enrichment. KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure 5. KEGG signaling pathway analysis of differentially expressed genes. Top 30 for KEGG enrichment. KEGG, Kyoto Encyclopedia of Genes and Genomes lncRNAs and miRNAs in articular chondrocytes  Asian Biomed (Res Rev News) 2022; 16(4):169–179 175 Table 2. PPI network core (top 10) genes in the SDF-1-induced model of articular chondrocyte degeneration Protein Degree Eccentricity Edge count CXCL10 16 4 16 ISG15 12 4 12 MYC 11 5 11 MX1 10 5 10 OASL 10 5 10 IFIT1 10 5 10 RSAD2 10 5 10 MX2 10 5 10 IFI44L 10 5 10 BST2 8 5 8 BST2, bone marrow stromal tetherin antigen 2; CXCL10, interferon γ ­inducible CXC 10 kDa chemokine chemotactic for monocytes and T-lym­ phocytes; IFI44L, interferon-induced protein 44 like protein; IFIT1, inter­ feron-induced with tetratricopeptide repeats 1 protein; ISG15, interferon- α-stimulated gene 15-kDa protein; MX1, interferon inducible myxovirus resistance protein MxA p78; MX2, second interferon-induced myxovirus resistance 2 protein MxB p78; MYC, v-myc myelocytomatosis viral oncoge­ ne homolog protein; OASL, p59 2′,5′-oligoadenylate synthetase-like prote­ in; PPI, protein−protein interaction; RSAD2, radical S-adenosyl methionine domain containing 2 protein; SDF-1, stromal cell-derived factor-1. Table 2. Discussion Similarly, reducing ceRNA levels upregulate target gene expression, which may ultimately affect cellular biologi­ cal processes [30]. type II collagen and aggrecan by articular chondrocytes, but also stimulates articular chondrocytes to secrete protease that degrades cartilage matrix components, inhibits the expression of type I and type II collagen by articular chondrocytes, and promotes the degeneration of articular chondrocytes [39]. TNF-a also plays an important role in osteoarthritis cartilage degeneration. The mechanism of action of TNF-a is similar to that of IL-1 and includes promoting the generation of MMP and inhibiting the synthesis of cartilage matrix. TNF inhibits the expression of type II collagen and connexin genes through the MAP kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 and NF-kB pathways, which in turn interfe­ res with the synthesis and reconstruction of articular cartilage [40]. The NF-kB transcription factor regulates gene expres­ sion, and the NF-kB signaling pathway is activated in articular cartilage and synovial cells in osteoarthritis [41]. NF-kB regu­ lates the response to joint injury and inflammation by regula­ ting cytokines, including IL-1b and TNF-a [39–46]. Receptor regulatory factors such as Toll-like receptors (TLRs) are evolutionarily conserved molecules that promote immune responses by recognizing molecular patterns related to microorganisms. During infection, TLR signaling is neces­ sary for the proper activation of the immune response [31]. TLRs produce large amounts of interleukin (IL)-1b and tumor necrosis factor (TNF)-a inflammatory factors by activating the NK-kB inflammatory signaling pathway. Liu et al. [32] found that the expression of TLR-2, NF-kB, MMP-13, and related inflammatory factors was significantly upregulated with the severity of osteoarthritis lesions, suggesting that the TLR-2/ NF-kB signaling pathway may be involved in the occurrence of osteoarthritis. The PPI network revealed genes for 10 proteins that have a high possibility of being associated with the patholo­ gical process of chondrocyte degeneration: CXCL10, ISG15, MYC, MX1, OASL, IFIT1, RSAD2, MX2, IFI44L, and BST2. Chemokines, mainly CXC and CC, and their corres­ ponding receptors are expressed in human chondrocytes, and their expression is increased in osteoarthritis articular carti­ lage [47]. Chemokines are involved in cartilage destruction by inducing the expression of related enzymes, mainly N-acetyl- b-d-glucosidase (NAG) and MMP. NAG is the main lysoso­ mal glycosidase in osteoarthritis synovial fluid and catalyzes the hydrolysis of glucosamine polysaccharides, causing carti­ lage destruction [48]. Discussion The top 10 most upregulated and most downregulated lncRNAs in the experimental group are shown in Table 1. Horizontal comparisons based on the transcript structure of the lncRNAs were performed, including the transcript length distribution, classification, and exon quantity differences. The length of lncRNAs was mainly concentrated at appro­ ximately 1000  bp, and lncRNAs constituted various RNA molecules (Figure 3A). The traditional classification method of lncRNAs is based on the location of the transcript in the genome and includes 5 major categories: (1) the sense group, RNA expression data have been uploaded to the Sequence Read Archive database, and the BioProject ID is PRJNA638147. lncRNAs can compete with competing endogenous RNAs (ceRNA), as miRNA sponges, to play a regulatory role [7]. lncRNAs participate in gene regulation as guides, signals, baits, and scaffolds. The specific regulatory mechanism is mainly divided into 4 aspects, which regulate the degenera­ tion of articular cartilage by regulating transcription factors 176    Wang et al. 176    Wang et al. Figure 6. PPI network analysis of the top 10 differentially expressed genes. Nodes represent genes for the proteins indicated. Lines indicate inter­ actions between genes. Red indicates upregulated genes, and green indicates downregulated genes. PPI, protein−protein interaction. Figure 6. PPI network analysis of the top 10 differentially expressed genes. Nodes represent genes for the proteins indicated. Lines indicate inter­ actions between genes. Red indicates upregulated genes, and green indicates downregulated genes. PPI, protein−protein interaction. and transcription processes and mediating post-transcriptional regulation of miRNA and mRNA, and regulation of nuclear structure [28]. Most research studies have focused on the function of lncRNAs as a “sponge,” in which lncRNA under­ goes an endogenous competition interaction with miRNA that has a shared binding site to inhibit the regulatory effect of the miRNA on target mRNA, thereby affecting protein expression. The more binding sites there are, the stronger the “sponge effect” and the more obvious the inhibitory effect of lncRNA on miRNA. Under normal circumstances, various RNAs (such as lncRNA, miRNA, and mRNA) maintain a balanced state, and when an RNA is abnormally expressed, the balance is disrupted, and this results in disease [29]. Nonco­ ding RNA with a common response element (miRNA response lncRNAs and miRNAs in articular chondrocytes  Asian Biomed (Res Rev News) 2022; 16(4):169–179 177 elements [MRE]) can compete with mRNA endogenously to bind miRNA and inhibit miRNA-mediated negative regulation of mRNA. Discussion When intra-articular hemorrhage occurs, lysosomes release degrading enzymes, and decreased proteoglycan concentration reduces chondrocyte synthesis activity and induces articular cartilage degeneration [33]. A high concentration of SDF-1 can increase its interaction with CXCR4 on the surface of chondro­ cytes and accelerate the degradation of type II collagen through the upregulation of MMPs, also leading to cartilage degenera­ tion [34]. Chondrocytes are nonexcitable cells. However, the multiple ion channels present on the cell membrane are the basis for the cell to carry out various life activities, including trans­ porting ions necessary for cell metabolism, regulating osmotic pressure inside and outside the cell, participating in the forma­ tion of electrical impulses, and mediating in signal transmission to adapt organisms to environmental conditions [35, 36]. In osteoarthritis, the cartilage surface is activated by a variety of chemokines, releasing enzymes that mediate the destruction of the cartilage matrix [48, 49]. Kostopoulou et al. [50] and Tardif et al. [51] found that an osteoarthritis-related miRNA can inhibit MMP-13. Pathway analysis showed that miRNAs and their target genes were enriched in cytokine–cytokine receptor interac­ tion, osteoclast differentiation, NF-kB signaling pathway, TGF-b signaling pathway, and Ca2+ signaling. Cytokines regu­ late the balance of anabolic and catabolic metabolism of car­ tilage matrix. They are divided into catabolic cytokines and anabolic cytokines according to their roles in the regulation of metabolism. The balance and imbalance between them are root causes of the degradation and destruction of the cartilage matrix in osteoarthritis. Cytokines, including TNF-a, IL-1, IL-6, IL-2, and IFN-g, are involved in this pathway. These cytokines penetrate the synovium to induce an inflammatory response. In addition, they can activate synovial cells and sti­ mulate the release of MMPs into the synovial fluid, leading to cartilage degradation [37, 38]. MYC is not strongly expressed in normal chondrocyte nuclei, but is scattered in apoptotic chondrocyte nuclei. The degree of articular chondrocyte apoptosis in osteoarthritis is positively correlated with the degree of cartilage degene­ ration, and MYC participates in the process of chondrocyte apoptosis. The mechanism of MYC causing apoptosis may be due to an imbalance in the normal cell cycle, which inhibits cell growth [52]. 178    Wang et al. 178 178 Author contributions. GW and YL contributed to the concept and design of the study. LH contributed to the chondrocyte collection. GW and YL principally interpreted the data and wrote the original draft of the manuscript. All authors contri­ buted toward revising the manuscript critically for important intellectual content, read and approved the final version sub­ mitted for publication, and take responsibility for the state­ ments made in the published article. Author contributions. GW and YL contributed to the concept and design of the study. LH contributed to the chondrocyte collection. GW and YL principally interpreted the data and wrote the original draft of the manuscript. All authors contri­ buted toward revising the manuscript critically for important intellectual content, read and approved the final version sub­ mitted for publication, and take responsibility for the state­ ments made in the published article. [5] Stefani G, Slack FJ. Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol. 2008; 9:219–30. [6] Shi Z, Ning G, Zhang B, Yuan S, Zhou H, Pan B, et al. Signatures of altered long noncoding RNAs and messenger RNAs expression in the early acute phase of spinal cord injury. J Cell Physiol. 2019; 234:8918–27. [7] Chen G, Wang Z, Wang D, Qiu C, Liu M, Chen X, et al. LncRNA­ Disease: a database for long-non-coding RNA-associated diseases. Nucleic Acids Res. 2013; 41(Database issue):D983–6. [8] Bao Z, Yang Z, Huang Z, Zhou Y, Cui Q, Dong D. LncRNADisease 2.0: an updated database of long non-coding RNA-associated diseases. Nucleic Acids Res. 2019; 47(D1):D1034–7. Acknowledgments. A preliminary version of the present article has been published as a PREPRINT on Research Square doi: 10.21203/rs.3.rs-36488/v1. The present study was suppor­ ted by: (1) the ­National Natural Science Foundation of China (No. 81960409, No. 81760403); (2) Yunnan Province Clini­ cal Center for Bone and Joint Diseases (No. ZX2019-03-04); (3) The Yunnan Province Medical Leaders Talent Project (No. L-201601); (4) Expert workstation project of Shiyi Chen (No. 2018IC102); and (5) Joint special fund project for applied basic research of Kunming Medical University, Department of Science and Technology of Yunnan Province (grant No. 202001AY070001-043). Guoliang Wang was a Ph.D. candidate at Kunming Medical University during the time of the study. Xiao Yang contributed to the chondrocyte collection experiment. [9] Kopańska M, Szala D, Czech J, Gabło N, Gargasz K, Trzeciak M, et al. Discussion In-depth studies of cytokine interactions, osteoarthri­ tis signaling pathways, and miRNAs related to lncRNAs are required to investigate the relationship between lncRNAs and miRNAs, to elucidate the molecular mechanism of osteochon­ drocyte degeneration, and provide a new basis and targets for the effective diagnosis and treatment of osteoarthritis. Currently, the most studied cytokines that promote chon­ drocyte catabolism are IL-1 and TNF-a. IL-1 not only inhi­ bits the synthesis of the characteristic matrix components MiRNA expression in the cartilage of patients with osteoarthritis. J Orthop Surg Res. 2017; 12:51. doi: 10.1186/s13018- 017-0542-y. Erratum in: J Orthop Surg Res. 2017; 12:90. [10] Fu M, Huang G, Zhang Z, Liu J, Zhang Z, Huang Z, et al. Expression profile of long noncoding RNAs in cartilage from knee osteoarthritis patients. Osteoarthritis Cartilage. 2015; 23:423–32. [11] Sun H, Peng G, Ning X, Wang J, Yang H, Deng J. Emerging roles of long noncoding RNA in chondrogenesis, osteogenesis, and osteoarthritis. Am J Transl Res. 2019; 11:16–30. [12] Hu J, Wang Z, Shan Y, Pan Y, Ma J, Jia L. Long non-coding RNA HOTAIR promotes osteoarthritis progression via miR-17-5p/ FUT2/b-catenin axis. Cell Death Dis. 2018; 9:711. doi: 10.1038/ s41419-018-0746-z [13] Cen X, Huang X-Q, Sun W-T, Liu Q, Liu J. Long noncoding RNAs: a new regulatory code in osteoarthritis. Am J Transl Res. 2017; 9:4747–55. Conflict of interest statement. Each author has completed and submitted an International Committee of Medical Journal Editors Disclosure Form. Apart from the funding listed in the acknowledgments, none of the authors have any competing relationship, activity, or interest to disclose. [14] He Z, Jia M, Yu Y, Yuan C, Wang J. Roles of SDF-1/CXCR4 axis in cartilage endplate stem cells mediated promotion of nucleus pulposus cells proliferation. Biochem Biophys Res Commun. 2018; 506:94–101. [15] Chen H-T, Tsou H-K, Hsu C-J, Tsai C-H, Kao C-H, Fong Y-C, Tang C-H. Stromal cell-derived factor-1/CXCR4 promotes IL-6 production in human synovial fibroblasts. J Cell Biochem. 2011; 112:1219–27. Availability of data and materials. RNA-seq data have been uploaded to Sequence Read Archive (SRA) database, and the BioProject ID is PRJNA638147. Other data are presented in the published article and further details are available from the corresponding author on reasonable request. [16] Dong Y, Liu H, Zhang X, Xu F, Qin L, Cheng P, et al. Inhibition of SDF-1a/CXCR4 signalling in subchondral bone attenuates post-traumatic osteoarthritis. Int J Mol Sci. 2016; 17:943. doi: 10.3390/ijms17060943 [17] Wei L, Sun X, Kanbe K, Wang Z, Sun C, Terek R, Chen Q. Chondrocyte death induced by pathological concentration of chemokine stromal cell-derived factor-1. J Rheumatol. 2006; 33:1818–26. [1] Jamshidi A, Pelletier JP, Martel-Pelletier J. Machine-learning-based patient-specific prediction models for knee osteoarthritis. Nat Rev Rheumatol. 2019; 15:49–60. References [39] Kobayashi M, Squires GR, Mousa A, Tanzer M, Zukor DJ, Antoniou J, et al. Role of interleukin-1 and tumor necrosis factor a in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum. 2005; 52:128–35. [23] Periyasamy-Thandavan S, Burke J, Mendhe B, Kondrikova G, Kolhe R, Hunter M, et al. MicroRNA-141-3p negatively modulates SDF-1 expression in age-dependent pathophysiology of human and murine bone marrow stromal cells. J Gerontol A Biol Sci Med Sci. 2019; 74:1368–74. [40] Séguin CA, Bernier SM. TNFa suppresses link protein and type II collagen expression in chondrocytes: role of MEK1/2 and NF-κB signaling pathways. J Cell Physiol. 2003; 197:356–69. [24] Altman RD. The classification of osteoarthritis. J Rheumatol Suppl. 1995; 43:42–3. [41] Li M, Guan H. Noncoding RNAs regulating NF-κB signaling. In: Song E, editor. The long and short non-coding RNAs in cancer biology. Singapore: Springer; 2016, p. 317–36. (Cohen IR, Lajtha NSA, Lambris JD, Paoletti R, series editors. Adv Exp Med Biol., vol. 927). [25] Charalambous CP. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II: correlation of morphology with biochemical and metabolic data. In: Banasz­ kiewicz P, Kader D, editors. Classic papers in orthopaedics. London: Springer-Verlag; 2014, p. 385–7. [42] Adli M. IKKa and IKKb each function to regulate NF-κB activation in the TNF-induced/canonical pathway. PLoS One. 2010; 5:e9428. doi: 10.1371/journal.pone.0009428 [26] Smoot ME, Ono K, Ruscheinski J, Wang P-L, Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics. 2011; 27:431–2. [43] Shi J, Zhang C, Yi Z, Lan C. Explore the variation of MMP3, JNK, p38 MAPKs, and autophagy at the early stage of osteoarthritis. IUBMB Life. 2016; 68:293–302. [27] Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, et al. The STRING database in 2021: customizable protein–protein networks, and functional characterization of user-uploaded gene/ measurement sets. Nucleic Acids Res. 2021; 49(D1):D605–12. [44] Jilani AA, Mackworth-Young CG. The role of citrullinated protein antibodies in predicting erosive disease in rheumatoid arthritis: a systematic literature review and meta-analysis. Int J Rheumatol. 2015; 2015:728610. doi: 10.1155/2015/728610 [28] Pearson MJ, Philp AM, Heward JA, Roux BT, Walsh DA, Davis ET, et al. Long intergenic noncoding RNAs mediate the human chondrocyte inflammatory response and are differentially expressed in osteoarthritis cartilage. Arthritis Rheumatol. 2016; 68:845–56. [45] Liang Y, Chen S, Yang Y, Lan C, Zhang G, Ji Z, Lin H. References [18] Wang K, Li Y, Han R, Cai G, He C, Wang G, Jia D. T140 blocks the SDF-1/CXCR4 signaling pathway and prevents cartilage degeneration in an osteoarthritis disease model. PLoS One. 2017; 12:e0176048. doi: 10.1371/journal.pone.0176048 [1] Jamshidi A, Pelletier JP, Martel-Pelletier J. Machine-learning-based patient-specific prediction models for knee osteoarthritis. Nat Rev Rheumatol. 2019; 15:49–60. [1] Jamshidi A, Pelletier JP, Martel-Pelletier J. Machine-learning-based patient-specific prediction models for knee osteoarthritis. Nat Rev Rheumatol. 2019; 15:49–60. [2] Nguyen US, Zhang Y, Zhu Y, Niu J, Zhang B, Felson DT. Increasing prevalence of knee pain and symptomatic knee osteoar­ thritis: survey and cohort data. Ann Intern Med. 2011; 155:725–32. [2] Nguyen US, Zhang Y, Zhu Y, Niu J, Zhang B, Felson DT. Increasing prevalence of knee pain and symptomatic knee osteoar­ thritis: survey and cohort data. Ann Intern Med. 2011; 155:725–32. [19] Kanbe K, Takagishi K, Chen Q. Stimulation of matrix metallo­ protease 3 release from human chondrocytes by the interaction of stromal cell-derived factor 1 and CXC chemokine receptor 4. Arthritis Rheum. 2002; 46:130–7. [3] Aguiar GC, Queiroz-Junior CM, Sitta GL, Amaral FA, Teixeira MM, Caliari MV, Ferreira AJ. Mefenamic acid decreases inflammation but not joint lesions in experimental osteoarthritis. Int J Exp Pathol. 2016; 97:438–46. [20] Kanbe K, Takemura T, Takeuchi K, Chen Q, Takagishi K, Inoue K. Synovectomy reduces stromal-cell-derived factor-1 (SDF-1) which is involved in the destruction of cartilage in osteoarthritis and rheumatoid arthritis. J Bone Joint Surg Br. 2004; 86:296–300. [4] Jones IA, Togashi R, Wilson ML, Heckmann N, Vangsness CT Jr. Intra-articular treatment options for knee osteoarthritis. Nat Rev Rheumatol. 2019; 15:77–90. [21] Li G, Yun X, Ye K, Zhao H, An J, Zhang X, et al. Long non-coding RNA-H19 stimulates osteogenic differentiation of bone marrow 179  Asian Biomed (Res Rev News) 2022; 16(4):169–179 lncRNAs and miRNAs in articular chondrocytes mesenchymal stem cells via the microRNA-149/SDF-1 axis. J Cell Mol Med. 2020; 24:4944–55. mesenchymal stem cells via the microRNA-149/SDF-1 axis. J Cell Mol Med. 2020; 24:4944–55. of osteoarthritis. Mediators Inflamm. 2014; 2014:561459. doi: 10.1155/2014/561459 [38] Choy E. Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford). 2012; 51(Suppl 5):v3–11. [22] van Solingen C, de Boer HC, Bijkerk R, Monge M, van Oeveren- Rietdijk AM, Seghers L, et al. MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+/Lin− progenitor cells in ischaemia. Cardiovasc Res. 2011; 92:449–55. References Vasoactive intestinal peptide alleviates osteoarthritis effectively via inhibiting NF-κB signaling pathway. J Biomed Sci. 2018; 25:25. doi: 10.1186/ s12929-018-0410-z [29] Li Y-F, Li S-H, Liu Y, Luo Y-T. Long noncoding RNA CIR promotes chondrocyte extracellular matrix degradation in osteoarthritis by acting as a sponge for Mir-27b. Cell Physiol Biochem. 2017; 43:602–10. [46] Chang SH, Mori D, Kobayashi H, Mori Y, Nakamoto H, Okada K, et al. Excessive mechanical loading promotes osteoarthritis through the gremlin-1-NF-κB pathway. Nat Commun. 2019; 10:1442. doi: 10.1038/s41467-019-09491-5 [30] Zhang G, Wu Y, Xu D, Yan X. Long noncoding RNA UFC1 promotes proliferation of chondrocyte in osteoarthritis by acting as a sponge for miR-34a. DNA Cell Biol. 2016; 35:691–5. [47] Mazzetti I, Magagnoli G, Paoletti S, Uguccioni M, Olivotto E, Vitellozzi R, et al. A role for chemokines in the induction of chondrocyte phenotype modulation. Arthritis Rheum. 2004; 50:112–22. [31] Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, Mazmanian SK. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science. 2011; 332(6032):974–7. [48] Mahon OR, Kelly DJ, McCarthy GM, Dunne A. Osteoarthritis- associated basic calcium phosphate crystals alter immune cell metabolism and promote M1 macrophage polarization. Osteoar­ thritis Cartilage. 2020; 28:603–12. [32] Liu Y-X, Wang G-D, Wang X, Zhang Y-L, Zhang T-L. Effects of TLR-2/NF-κB signaling pathway on the occurrence of degenerative knee osteoarthritis: an in vivo and in vitro study. Oncotarget. 2017; 8:38602–17. [49] Grieshaber-Bouyer R, Kämmerer T, Rosshirt N, Nees TA, Koniezke P, Tripel E, et al. Divergent mononuclear cell participation and cytokine release profiles define hip and knee osteoarthritis. J Clin Med. 2019; 8:1631. doi: 10.3390/jcm8101631 [33] Parkinson-Lawrence EJ, Shandala T, Prodoehl M, Plew R, Borlace GN, Brooks DA. Lysosomal storage disease: revealing lysosomal function and physiology. Physiology (Bethesda). 2010; 25:102–15. [50] Kostopoulou F, Malizos KN, Papathanasiou I, Tsezou A. MicroRNA-33a regulates cholesterol synthesis and cholesterol efflux-related genes in osteoarthritic chondrocytes. Arthritis Res Ther. 2015; 17:42. doi: 10.1186/s13075-015-0556-y [34] Li P, Deng J, Wei X, Jayasuriya CT, Zhou J, Chen Q, et al. Blockade of hypoxia-induced CXCR4 with AMD3100 inhibits production of OA-associated catabolic mediators IL-1b and MMP-13. Mol Med Rep. 2016; 14:1475–82. [51] Tardif G, Hum D, Pelletier JP, Duval N, Martel-Pelletier J. Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes. BMC Musculoskelet Disord. 2009; 10:148. References doi: 10.1186/1471-2474- 10-148 [35] Wilkins RJ, Browning JA, Ellory JC. Surviving in a matrix: membrane transport in articular chondrocytes. J Membr Biol. 2000; 177:95–108. [52] Yatsugi N, Tsukazaki T, Osaki M, Koji T, Yamashita S, Shindo H. Apoptosis of articular chondrocytes in rheumatoid arthritis and osteoarthritis: correlation of apoptosis with degree of cartilage destruction and expression of apoptosis-related proteins of p53 and c-myc. J Orthop Sci. 2000; 5:150–6. [36] Mow VC, Guo XE. Mechano-electrochemical properties of articular cartilage: their inhomogeneities and anisotropies. Annu Rev Biomed Eng. 2002; 4:175–209. [37] Wojdasiewicz P, Poniatowski ŁA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis [37] Wojdasiewicz P, Poniatowski ŁA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis
https://openalex.org/W2901087969
https://globalizationandhealth.biomedcentral.com/track/pdf/10.1186/s12992-018-0431-0.pdf
English
null
Organizational capacities of national pharmacovigilance centres in Africa: assessment of resource elements associated with successful and unsuccessful pharmacovigilance experiences
Globalization and health
2,018
cc-by
13,402
Ampadu et al. Globalization and Health (2018) 14:109 https://doi.org/10.1186/s12992-018-0431-0 Ampadu et al. Globalization and Health (2018) 14:109 https://doi.org/10.1186/s12992-018-0431-0 RESEARCH Open Access Organizational capacities of national pharmacovigilance centres in Africa: assessment of resource elements associated with successful and unsuccessful pharmacovigilance experiences H. Hilda Ampadu1,2*, Jarno Hoekman3, Daniel Arhinful4, Marilyn Amoama-Dapaah1, Hubert G. M. Leufkens2 and Alex N. O. Dodoo1 Organizational capacities of national pharmacovigilance centres in Africa: assessment of resource elements associated with successful and unsuccessful pharmacovigilance experiences H. Hilda Ampadu1,2*, Jarno Hoekman3, Daniel Arhinful4, Marilyn Amoama-Dapaah1, Hubert G. M. Leufkens2 and Alex N. O. Dodoo1 H. Hilda Ampadu1,2*, Jarno Hoekman3, Daniel Arhinful4, Marilyn Amoama-Dapaah1, Hubert G. M Alex N. O. Dodoo1 RESEARCH Open Access * Correspondence: hilda.ampadu@acc-afro.org 1 * Correspondence: hilda.ampadu@acc-afro.org 1 * Correspondence: hilda.ampadu@acc-afro.org 1The African Collaborating Centre for Pharmacovigilance, 1 Vigilance Place, Mango Tree Avenue, Asylum Down, Accra, Ghana 2WHO Collaborating Centre for Pharmaceutical Policy and Regulation, Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3508 TB Utrecht, the Netherlands * Correspondence: hilda.ampadu@acc-afro.org 1The African Collaborating Centre for Pharmacovigilance, 1 Vigilance Place, Mango Tree Avenue, Asylum Down, Accra, Ghana 2WHO Collaborating Centre for Pharmaceutical Policy and Regulation, Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3508 TB Utrecht, the Netherlands Abstract Background: National pharmacovigilance centres (national centres) are gradually gaining visibility as part of the healthcare delivery system in Africa. As does happen in high-income countries, it is assumed that national centres can play a central coordinating role in their national pharmacovigilance (PV) systems. However, there are no studies that have investigated whether national centres in Africa have sufficient organizational capacity to deliver on this mandate and previous studies have reported challenges such as lack of funding, political will and adequate human resources. We conducted interviews with strategic leaders in national centres in 18 African countries, to examine how they link the capacity of their organization to the outcomes of activities coordinated by their centres. Strategic leaders were asked to describe three situations in which activities conducted by their centre were deemed successful and unsuccessful. We analyzed these experiences for common themes and examined whether strategic leaders attributed particular types of resources and relationships with stakeholders to successful or unsuccessful activities. Results: We found that strategic leaders most often attributed successful experiences to the acquisition of political (e.g. legal mandate) or technical (e.g. active surveillance database) resources, while unsuccessful experiences were often attributed to the lack of financial and human resources. Stakeholders that were most often mentioned in association with successful experiences were national government and development partners, whereas national government and public health programmes (PHPs) were often mentioned in unsuccessful experiences. All 18 centres, regardless of maturity of their PV systems had similar challenges. Conclusions: The study concludes that national centres in Africa are faced with 3 core challenges: (1) over-reliance on development partners, (2) seeming indifference of national governments to provide support after national centres have gained membership of the World Health Organization (WHO) Programme for International Drug Monitoring (PIDM) and (3) engaging public health programmes in a sustainable way. Keywords: National pharmacovigilance centres, Organizational capacity, Resource elements, Stakeholders, Outcomes, National governments, Development partners, Public health Programmes * Correspondence: hilda.ampadu@acc-afro.org 1The African Collaborating Centre for Pharmacovigilance, 1 Vigilance Place, Mango Tree Avenue, Asylum Down, Accra, Ghana 2WHO Collaborating Centre for Pharmaceutical Policy and Regulation, Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3508 TB Utrecht, the Netherlands Full list of author information is available at the end of the article © The Author(s). Abstract 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. National PV centres and PV initiatives National PV centres The WHO defines a national centre as a single, government-designated centre within a country with the clinical and scientific expertise to collect, collate, analyse and give advice on all aspects related to drug safety [16].The functions [16, 17] of a national centre include, but are not limited to: There is a widespread expectation among several stakeholders including the WHO that national PV sys- tems need to be driven by a national centre [7]. However, previous studies have noted that most national centres in sub-Saharan Africa are currently not the cen- tral coordinating bodies of PV efforts in their respective countries [8]. A study by Maigetter et al. [9] revealed that in many countries in Africa, PV functions are not conducted within a separate organization but lumped to- gether with other regulatory functions such as medicines registration, licensing of premises and inspections. The national centre is sometimes a desk in the national med- icines regulatory authority (NMRA) with one or two people assigned to carry out all its functions [1, 8, 10]. It is therefore not surprising that the few studies on the features of pharmacovigilance in Africa have arrived at the same conclusion that PV activities performed by African national centres are limited and beset with sev- eral challenges of which overcoming a lack of resources is one of the most prominent [1, 11, 12]. This is very dif- ferent from the situation in developed countries where the national centre is an integral part of the public health system and plays a key role in implementing the national PV agenda [13].  Coordinating of pharmacovigilance activities nationwide;  Creating awareness on pharmacovigilance among health professionals, healthcare providers, marketing authorization holders and the public;  Post-marketing surveillance of regulated products;  Establishing and maintaining a functional national database on ADRs and other medicine related problems to identify unknown or poorly specified adverse effects;  Leading national and international collaboration on safety issues y  Contributing to the fight against counterfeit medicines It is obvious from the above that national centres in Africa have a broad mandate and thus require adequate resources to undertake these tasks and to coordinate their national PV systems. The available evidence how- ever suggests that the PV landscape in many African countries is dominated by fragmented PV initiatives and programmes rather than a well-coordinated national PV system [18]. Background (MAHs) to have a Qualified Person for Pharmacovigi- lance (QPPV) in line with the Public Health Act of Ghana (Act 851, 2012; Part Seven) [14]. The Pharmacy and Poisons Board of Kenya has been designated as a Regional Centre of Regulatory Excellence (RCORE) in pharmacovigilance by the African Union through the Af- rican Medicines Regulatory Harmonization (AMRH) programme [15]. Despite this attention, our knowledge on the role and experiences of national centres in Africa is limited especially as it relates to the organizational capacity (resources and relationships) they need to de- liver on their mandate. We fill this knowledge gap by providing insight into the activities of national centres that were deemed successful and unsuccessful by the strategic leaders of the centre and by assessing whether the attribution of success or failure is associated with particular types of resources or stakeholders. g The last years have witnessed increasing efforts in low and middle income countries to establish formal national pharmacovigilance centres (national centres) with several of these in sub-Saharan Africa [1, 2]. Pharmacovigilance (PV) became an important discipline in the 1960s follow- ing the thalidomide tragedy [3]. The realization that the tragedy could have been prevented if countries collected and shared data on medicine safety led the World Health Organization (WHO) decision-making body i.e. the World Health Assembly to issue a resolution inviting “Member States to arrange for a systematic collection of information on serious adverse drug reactions (ADR) observed during the development of a drug and, in particular, after its re- lease for general use” ([4], pp 14). In response to this call, national governments around the world established na- tional pharmacovigilance centres to coordinate medicine safety surveillance efforts. Over the years these centres have become key organizations involved in initiating, building and sustaining efforts for safety surveillance [5]. Particularly in high income countries, national centres now function as central nodes for national PV efforts and they contribute to building national PV systems by collab- orating with other stakeholders be they local, national or international [2, 5, 6]. National PV centres and PV initiatives National PV centres However, there is also evidence that the development of PV systems has become a key priority in certain coun- tries which has led to successes. For instance, the Ghana Food and Drugs Authority has implemented legal provi- sions mandating Marketing Authorisation Holders © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 17 Page 2 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 Data collection I t i Interviews were conducted between September 2015 and April 2016. Sixteen interviews were conducted face- to-face and two via phone calls and followed up by emails. The lead investigator had preliminary meetings with participants, explained the research aims and sought verbal consent. Each participant was subse- quently interviewed once, with interview duration ran- ging between 15 and 25 min. The Ghana Health Service Ethics Review Committee’s Standard Operating Proced- ure, mentions that ethical review is not needed for studies documenting “public behaviour” of professionals working in a public organization [22]. Accordingly, we did not seek ethical approval for this study but con- formed with ethical guidance on anonymization of quotes to prevent statements that could be traced back to individuals. p p g g The execution of PHPs resulted in increased access to medicines in African countries but at the same time led to a realisation that safety monitoring systems were largely absent in these countries. This led to calls from the WHO for collaboration among stakeholders to en- sure that these countries develop pharmacovigilance sys- tems to protect their populations from medicinal product associated harms [21]. Typically, NMRAs were tasked to collaborate with these PHPs to ensure safety monitoring. As part of this endeavour, several nations in Africa established national centres. The increased fund- ing for PHPs thus was instrumental in the establishment of some national centres in Africa. Most of the estab- lished national centres were positioned as individual de- partments in the NMRA and most still reside within the Ministry of Health [9]. National centres rely on the na- tional government to provide resources for operations, making the national government their most important stakeholder [11]. National centres are also dependent on healthcare professionals, the pharmaceutical industry, academia, PHPs, intergovernmental organizations and development partners who may provide resources to achieve outcomes. Public health programmes rely on spontaneous ADR reporting as the bedrock for collect- ing safety data on the products used in these pro- grammes and collaborate with the national centres by submitting ADRs directly to the national centres. Some- times, the national centres also contribute to joint mass drug administration campaigns like deworming of school children with the PHPs through collection and monitor- ing of ADRs for the safety of patients. Two pilot interviews led to minor tweaks of the inter- view protocol and are included in the final data analysis. Methods often, in collaboration with external development part- ners. Global health initiatives such as the U.S. President’s Emergency Plan for AIDS Relief (PEPFAR) and the US Presidents Malaria Initiative, Global Fund Against HIV/ AIDS, TB and Malaria (Global Fund), The Bill and Me- linda Gates Foundation’s Malaria Eradication and the adoption of the millennium development goals by the United Nations in the 2000s provided funding for several African countries to combat priority diseases [12, 19, 20]. To qualify to receive this funding, national govern- ments, specifically the Ministries of Health, were tasked to establish formal disease control programmes also known as public health programmes in collaboration with WHO. These programmes were placed under the disease control department of the Ministries of Health and include well known programmes such as the National AIDS/HIV Control Programme, National Tuberculosis Control Programme, National Malaria Control Programmes, the Expanded Programme on Immunization and the lesser known programmes such as the Neglected Tropical Diseases programme. Typically, the programme adminis- trators will draft joint work plans with the development partners providing the funding. This was a qualitative, investigator-administered, semi- structured interview study of strategic leaders in 18 out of 36 national centres in Africa to provide insight into the resource elements, relationships and outcomes they associate with successful and unsuccessful pharmacovigi- lance experiences. PV initiatives and programmes On the African continent, PV activities are often under- taken within public health programmes (PHP) that are executed by the Ministry of Health either alone, or more Page 3 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Page 3 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Selection The participants were purposely selected taking into consideration language (English, French and Portuguese) and region (Central, East, West and Southern Africa) representing sub-Saharan Africa as seen in Fig. 1. To be included in the study, individuals needed to be a current or immediate past employee of a national centre and to be employed in a decision-making role. Sixteen of those interviewed are/were the heads of the national centre in their respective countries. Data collection I t i An interview guide is provided in Additional file 1. In short, participants were asked to describe pharmacovigi- lance experiences defined as an activity in which the na- tional centre was involved and that had an impact on the delivery of the mandate of the centre as defined in “National PV centres” section. The interviewer asked for three successful and unsuccessful experiences defined as experiences that had a positive or negative impact on mandate delivery, respectively. For each situation the interviewer also asked for reasons why the experience was deemed successful or unsuccessful and asked follow-up questions when needed. We subsequently analysed these situations to examine how the strategic leaders attributed positive or negative impact to: Ampadu et al. Globalization and Health (2018) 14:109 Page 4 of 17 Fig. 1 Countries, regions and languages of participants Fig. 1 Countries, regions and languages of participants a) various types of resources (e.g. financial, technical, human, social, political resources) they acquired and how they used them in programme and process management; Coding a) various types of resources (e.g. financial, technical, human, social, political resources) they acquired and how they used them in programme and process management; a) various types of resources (e.g. financial, technical, human, social, political resources) they acquired and how they used them in programme and process management; Interviews were transcribed verbatim by an experienced co-author (DA). Upon compilation, a total of 18 *3 = 54 successful and 54 unsuccessful experiences were derived. Each experience was subsequently coded for mentioned relationships with stakeholders, mentioned acquired re- sources and mentioned outcomes. For instance: if a na- tional centre described an experience where they were able to lobby the Ministry of Health/Minister of Health to present a case in Parliament to get a law passed for Marketing Authorization Holders to be held responsible for the safety of their products on the market, the ex- perience was coded as a relationship with the Ministry of Health/Minister of Health, the acquired resource was legal backing and the function was post-marketing surveil- lance of regulated products. Conversely, a negative experi- ence was defined as any national centre relationship with a stakeholder that did not result in the attainment of a b) creation and maintenance of relationships with different types of stakeholders (e.g. national government, development partners, intergovernmental organizations, industry, academia, public health programmes) Thus, a successful experience was defined as national centre relationship with a stakeholder that resulted in the attainment of a resource facilitating the centre to deliver on its mandate. Conversely a negative experi- ence was defined as any national centre relationship with a stakeholder that did not result in the attainment of a resource hindering the centre to deliver on its mandate. Page 5 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Page 5 of 17 Ampadu et al. Globalization and Health (2018) 14:109 resource. An example is if a national centre was not able to embark on a nationwide training of healthcare workers on ADR reporting because it doesn’t have a budget alloca- tion for such an activity from the national government. The stakeholder mentioned in this case was national gov- ernment, the resource not provided was financial re- sources and the function not delivered was creating awareness on pharmacovigilance. Analysis y The coded interview data was tabulated using frequency ta- bles. Successful and unsuccessful experiences were assessed for frequently mentioned combinations of resources, stake- holders and functions. The combinations of resources, stakeholder and functions that strategic leaders attributed to success or failure were described as themes with verba- tim quotes from the participants. National centres in Africa are at varying levels of maturation thus we also compared experiences within country-groupings using the grouping system developed by Management Sciences for Health (MSH) [6]. According to this, Angola, Burkina Faso, Cam- eroun, Cape Verde, Eritrea, Liberia, Mauritius and Niger are in group 1 - countries with minimal or no capacity for PV. Rwanda, Congo-DRC, Ethiopia, Kenya, Mozambique, Senegal, Sierra Leone and Zimbabwe are in group 2- coun- tries with basic organizational structures. Group 3 coun- tries are countries with the capacity to collect and evaluate safety data based on legal and organizational structure; none of the countries interviewed were in group 3. Namibia and Nigeria are in group 4 - countries that have basic structures for both passive and active surveillance ac- tivities. Statistical analysis was not performed. An initial coding of 9 transcribed texts was done manually per participant by the lead investigator (HHA) and reviewed by two authors (JH, AD). For each experi- ence, resources mentioned were assigned to one of 5 re- source categories, stakeholders associated with the acquisition of these resources were assigned to one of 6 stakeholder groups and functions fulfilled or not fulfilled were assigned to one of 6 groups. Definitions for each resource and stakeholder groups are provided in Table 1, whereas the six functions of national centres are men- tioned in “National PV centres” section. We only consid- ered one dominant resource and stakeholder per experience. In 12 experiences, participants did not men- tion the stakeholders associated with the resources. Upon completion 108 resources and 96 stakeholders were coded for the combination of successful and un- successful experiences. The list of generated codes was compared to the remaining 9 transcribed texts, but no new categories or themes emerged. Results Of the 18 participants, there were 8 females and 10 males. Fifteen were pharmacists and 3 were physicians. All the 18 national centres interviewed (except one) were departments under the NMRA. Table 1 Definitions of resources and relationships used in the study Table 1 Definitions of resources and relationships used in the study Type of resource Definition Financial resources Funding or financial capital Technical resources Materials and infrastructure (e.g. computers, reporting infrastructure) Political resources Law, policy and other legislative instruments Human resources Staff and human expertise Social resources Relationships including collaborations, partnerships and networks Type of stakeholder Definition National government The National Regulatory Agency and the Ministry of Health Development partners Organizations that work with a variety of in-country partners to improve the lives of poor and vulnerable people in developing countries Inter-governmental organizations Organizations comprising mainly of sovereign states Public health programmes Organizations responsible for health services to improve and protect community health Academia Organizations concerned with the pursuit of education, research and scholarship Industry Organizations who market and sell pharmaceutical products Table 2 provides an overview of the MSH country group- ings and the different types of successful and unsuccessful experiences mentioned by participants and the coded re- sources based on each experience. Figure 2 depicts the dominant stakeholder groups mentioned in association with these resources. Of the 108 experiences collected, par- ticipants most often discussed experiences related to the ac- quisition of technical resources (16/54) such as reporting infrastructure, testing laboratories, phones and vehicles, and political resources (13/54) such as legal mandate, decentralization and political support as successful. Finan- cial resources (15/54) such as grants and dedicated budgets as well as human resources (13/54) such as staffing, capacity building, knowledge were most often described as unsuccessful. Stakeholders that were most often mentioned in experiences by participants were national government (50/108), development partners (16/108) and public health programmes (16/108). The resources and stakeholders as- sociated with these experiences are elaborated on below starting from the most frequently mentioned. Experiences involving technical resources The interviewees mainly made reference to technical re- sources that facilitated ADR reporting. For instance, par- ticipants mentioned that having access to online Page 6 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Table 2 MSH country groupings, experiences and resources MSH Group 1- Countries with minimal or no capacity for PV Country Successful experiences MSH Group 1- Countries with minimal or no capacity for PV Country Successful experiences Successful resources assigned Unsuccessful experiences Unsuccessful resources assigned Angola • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • ADR reports received through positive collaboration with HIV and Malaria Programmes • Funds received through collaboration with development partners • Political resource • Social resource • Financial resource • No PV law to enforce regulations • No dedicated budget for PV • No reporting tools • Political resource • Financial resource • Technical resource Burkina Faso • Regulatory framework implemented by government • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Establishment of national technical committees with tools for PV work • Political resource • Political resource • Technical resource • No properly recognized National Regulatory Authority • No dedicated budget for PV • No tools to embark on active monitoring • Political resource • Financial resource • Technical resource Cameroon • Funds received through collaboration with development partners • Continuous receipt of PV literature through established relationship with development partners • PV Decree signed by head of state and minister of health • Financial resource • Social resource • Political resource • No dedicated budget for PV • Untrained PV staff • No internet to submit ADR data to VigiFlow • Financial resource • Human resource • Technical resource Cape Verde • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Improved reporting infrastructure through TV and radio campaigns • Dissemination of ADR data through publication in peer review journals for Portuguese speaking countries • Political resource • Technical resource • Technical resource • No PV law to enforce regulations • Inadequate reporting infrastructure • No dedicated budget for PV • Political resource • Technical resource • Financial resource Eritrea • Funds received through collaboration with development partners • Trained PV staff • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Financial resource • Human resource • Political resource • No PV law to mandate reporting by industry • Low AEFI reporting due to poor collaboration with EPI • Pharma industry does not monitor the safety of their products • Political resource • Technical resource • Political resource Liberia • Trained PV staff • Incorporation of PV into curriculum of educational institutions due to effective collaboration with Academia • Availability of tools for active monitoring of drugs from international donors • Human resource • Social resource • Technical resource • No dedicated budget for PV • Inadequate human resource for PV activities • No PV law to enforce regulations • Financial resource • Human resource • Political resource Mauritius • Full membership in the PIDM due to positive collaboration with WHO • Improved reporting infrastructure through collaboration with PHPs • Technical support received through collaboration with development partners and PHPs • Social resource • Technical resource • Social resource • Inadequate reporting infrastructure • No dedicated budget for PV • No PV law to enforce regulations • Technical resource • Financial resource • Political resource Niger • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Attending trainings with the Head of the NRA, facilitation of travel by Head of NRA • Tools available to embark on district inspections • Political resource • Political resource • Technical resource • Inadequate human resource for PV activities • Untrained PV staff • No dedicated budget for PV • Human resource • Human resource • Financial resource MSH Group 2- Countries with basic organizational structures for PV Country Successful experiences Successful resources assigned Unsuccessful experiences Unsuccessful resources assigned Congo-DRC • Technical support received through collaboration with development partners and PHPs • Introduction of android smartphones to communicate effectively with health practitioners • More trained human resource from Implementation of Drug Therapeutic Committees(DTC) • Social resource • Technical resource • Human resource • Inadequate reporting infrastructure • Untrained PV staff • No dedicated budget for PV • Technical resource • Human resource • Financial resource Successful resources assigned Unsuccessful experiences Unsuccessful resources assigned • Political resource • Social resource • Financial resource • No PV law to enforce regulations • No dedicated budget for PV • No reporting tools • Political resource • Financial resource • Technical resource • Political resource • Political resource • Technical resource • No properly recognized National Regulatory Authority • No dedicated budget for PV • No tools to embark on active monitoring • Political resource • Financial resource • Technical resource • Financial resource • Social resource • Political resource • No dedicated budget for PV • Untrained PV staff • No internet to submit ADR data to VigiFlow • Financial resource • Human resource • Technical resource • Political resource • Technical resource • Technical resource • No PV law to enforce regulations • Inadequate reporting infrastructure • No dedicated budget for PV • Political resource • Technical resource • Financial resource • Financial resource • Human resource • Political resource • No PV law to mandate reporting by industry • Low AEFI reporting due to poor collaboration with EPI • Pharma industry does not monitor the safety of their products • Political resource • Technical resource • Political resource • Human resource • Social resource • Technical resource • No dedicated budget for PV • Inadequate human resource for PV activities • No PV law to enforce regulations • Financial resource • Human resource • Political resource • Social resource • Technical resource • Social resource • Inadequate reporting infrastructure • No dedicated budget for PV • No PV law to enforce regulations • Technical resource • Financial resource • Political resource • Political resource • Political resource • Technical resource • Inadequate human resource for PV activities • Untrained PV staff • No dedicated budget for PV • Human resource • Human resource • Financial resource PV Successful resources assigned Unsuccessful experiences Unsuccessful resources assigned e • Social resource • Technical resource • Human resource • Inadequate reporting infrastructure • Untrained PV staff • No dedicated budget for PV • Technical resource • Human resource • Financial resource Successful resources assigned Unsuccessful experiences gola • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • ADR reports received through positive collaboration with HIV and Malaria Programmes • Funds received through collaboration with development partners rkina Faso • Regulatory framework implemented by government • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Establishment of national technical committees with tools for PV work ola • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • ADR reports received through positive collaboration with HIV and Malaria Programmes • Funds received through collaboration with development partners na Faso • Regulatory framework implemented by government • Deployment of PV focal persons to various regions • ADR reports received through positive collaboration with HIV and Malaria Programmes • Political resource • Political resource • Technical resource Cameroon • Funds received through collaboration with development partners • Continuous receipt of PV literature through established relationship with development partners • PV Decree signed by head of state and minister of health • Financial resource • Social resource • Political resource Cape Verde • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Improved reporting infrastructure through TV and radio campaigns • Dissemination of ADR data through publication in peer review journals for Portuguese speaking countries • Political resource • Technical resource • Technical resource Eritrea • Funds received through collaboration with development partners • Trained PV staff • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Financial resource • Human resource • Political resource Liberia • Trained PV staff • Incorporation of PV into curriculum of educational institutions due to effective collaboration with Academia • Availability of tools for active monitoring of drugs from international donors • Human resource • Social resource • Technical resource Mauritius • Full membership in the PIDM due to positive collaboration with WHO • Improved reporting infrastructure through collaboration with PHPs • Technical support received through collaboration with development partners and PHPs • Social resource • Technical resource • Social resource Niger • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Attending trainings with the Head of the NRA, facilitation of travel by Head of NRA • Tools available to embark on district inspections • Political resource • Political resource • Technical resource h b l f Successful resources assigned Unsuccessful experiences Congo-DRC • Technical support received through collaboration with development partners and PHPs • Introduction of android smartphones to communicate effectively with health practitioners • More trained human resource from Implementation of Drug Therapeutic Committees(DTC) • • • Congo-DRC • Technical support received through collaboration with development partners and PHPs • Introduction of android smartphones to communicate effectively with health practitioners • More trained human resource from Implementation of Drug Therapeutic Committees(DTC) • Social resource • Technical resource • Human resource Page 7 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Table 2 MSH country groupings, experiences and resources (Continued) Ethiopia • Trained PV staff • Introduced PV into national curriculum, to train more human resource for PV • Fulltime MSH employee placed at the national centre to help with PV activities • Human resource • Human resource • Human resource • Lack of accredited laboratories • More human resources are needed to deliver on mandate • Poor AEFI reporting infrastructure • Technical resource • Human resource • Technical resource Kenya • Two ministers of state took part in the launch of the PV system. Successful resources assigned Unsuccessful experiences • Launch of online pharmacovigilance electronic reporting system • Funds provided through joint post market surveillance with PHPs • Political resource • Technical resource • Financial resource • More human resources are needed to deliver on mandate • Inadequate reporting infrastructure • No PV law to enforce regulations • Human resource • Technical resource • Political resource Mozambique • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Funds for training received through collaboration with WHO • Availability of legal instruments to promote PV • Political resource • Financial resource • Political resource • Untrained PV staff • No dedicated budget for PV • Poor collaboration with PHPs • Human resource • Financial resource • Social resource Rwanda • Trained PV staff • Implemented performance based evaluations for district hospitals • Collaboration with AMRH and EAC-PV harmonization to promote PV activities • Human resource • Technical resource • Social resource • Inadequate human resource for PV activities • No dedicated budget for PV • Poor collaboration with PHPs • Human resource • Financial resource • Social resource Senegal • Trained PV staff • Tools available for data analysis and data sharing • Funds for training received through collaboration with NMCP • Human resource • Technical resource • Financial resource • No PV staff with data management expertise • No PV representatives in the regions of the country, only the capital region • No dedicated budget for PV • Human resource • Political resource • Financial resource Sierra Leone • Adjustment of malaria treatment due to strong collaboration with NMCP • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Introduced PV into national curriculum, to train more human resource for PV • Social resource • Political resource • Human resource • No dedicated budget for PV • Inadequate reporting infrastructure • No PV law to enforce regulations • Financial resource • Political resource • Political resource Zimbabwe • Donor funding available for PV related projects • Guidance documents and publications available for PV work • AEFI Surveillance systems established since 2001 • Financial resource • Technical resource • Technical resource • No internet (Wi-Fi) services to submit data to VigiBase • Inability to generate own funds • Inadequate human resource for PV activities • Technical resource • Financial resource • Human resource G 4 C i i h b i f i d i ill Sierra Leone • Adjustment of malaria treatment due to strong collaboration with NMCP • Deployment of PV focal persons to various regions of the country, thus decentralizing PV • Introduced PV into national curriculum, to train more human resource for PV • Social resource • Political resource • Human resource Zimbabwe • Donor funding available for PV related projects • Guidance documents and publications available for PV work • AEFI Surveillance systems established since 2001 • Financial resource • Technical resource • Technical resource • Inadequate human resource for PV activities Group 4- Countries with basic structures for passive and active surveillance Country Successful resources Unsuccessful resources Namibia • Ministry of Health gave the mandate to setup the national centre • Active surveillance tools available for safety monitoring • Implemented patient reporting system • Political resource • Technical resource • Technical resource • Inadequate human resource for PV activities • No dedicated budget for PV • Inadequate spontaneous reporting infrastructure • Human resource • Financial resource • Technical resource Nigeria • Active surveillance tools available for safety monitoring • Funds for training received through collaboration with PHPs • Guidance documents and publications available for PV work • Technical resource • Financial resource • Technical resource • No online reporting infrastructure • Inadequate human resource for PV activities • No PV law to enforce regulations • Technical resource • Human resource • Political resource Group 1: Countries with minimal or no capacity for PV Group 2: Countries with basic organizational structures Group 3: Countries have the capacity to collect and evaluate safety data based on legal and organizational structures Group 4: Countries that have basic structures for both passive and active surveillance activities Successful resources • Political resource • Technical resource • Technical resource • Inadequate human resource for PV activities • No dedicated budget for PV • Inadequate spontaneous reporting infrastructure • Human resource • Financial resource • Technical resource • Technical resource • Financial resource • Technical resource • No online reporting infrastructure • Inadequate human resource for PV activities • No PV law to enforce regulations • Technical resource • Human resource • Political resource • No PV law to enforce regulations p g Group 3: Countries have the capacity to collect and evaluate safety data based on legal and organizational structures Group 4: Countries that have basic structures for both passive and active surveillance activities Ampadu et al. Globalization and Health (2018) 14:109 Page 8 of 17 Fig. 2 Stakeholders mentioned in the provision of resources Fig. 2 Stakeholders mentioned in the provision of resources day to day work from national governments and costly ones from PHPs or development partners. reporting systems made data readily available and had other benefits. “Launching of the online reporting system has helped, it minimizes the paperwork and it is less tedious than the manual reporting”. (Participant 8). “I have ICSRs, but can’t enter into VigiFlow because we don’t have internet connection all the time”. (Participant 3). “I have ICSRs, but can’t enter into VigiFlow because we don’t have internet connection all the time”. (Participant 3). “I have ICSRs, but can’t enter into VigiFlow because we don’t have internet connection all the time”. (Participant 3). Reference was also made to technical resources for day-to-day operations. For instance, having vehicles aided post-market surveillance and in mentioning the benefits of acquiring smartphones, a participant mentioned National governments were more often associated with unsuccessful acquisition of technical resources and de- velopment partners the most successful acquisition of technical resources. Participants indicated that they work closely with de- velopment partners in their day to day work whether in the provision of tools needed for their work or in the provision of other technical resources. “We found that doctors have a problem managing serious ADRs in the field. Our smartphone application allows us (national centre) to communicate with doctors in real time”. (Participant 5). “MSH was instrumental in setting up the national centre. They provided technical resources and then later the national centre was incorporated into the structure of the ministry”. (Participant 12). Successful resources In discussing inability to acquire technical resources, lack of data analysis tools, internet, data management in- frastructure and accredited laboratories were emphasized. “We have only one national laboratory; we are not able to test samples to verify if they are standard or counterfeit when ADRs are reported to us”. National government was lauded for providing space in the national regulatory authority for the national centre and setting up technical committees. (Participant 7). “The government has set up national commission with tools to validate ADR reports, they have the authority to withdraw or suspend any medicine from the country”. (Participant 2). “The government has set up national commission with tools to validate ADR reports, they have the authority to withdraw or suspend any medicine from the country”. Experiences involving political resources Political resources such as launching of the pharmacov- igilance system by the Minister of Health was used to champion pharmacovigilance to other health profes- sionals and the public. Political support sometimes man- ifested in the Ministers of Health accompanying national centre personnel on awareness creation campaigns which helped legitimize the national centre as an organization in the healthcare system. “Vaccine surveillance system is not in place at all at the national centre and the extended programme for immunization, we are currently working on the establishment of such a vaccine surveillance system”. (Participant 6). Experiences in which legal mandates were utilized to withdraw harmful products, decentralize PV activities and mandate reporting by industry were also mentioned. Regarding common successful experiences, three out of the 18 strategic leaders interviewed indicated they had a legal framework or law that specifically mentions pharmacovigilance and a participant described how empowering it can be: Experiences in which legal mandates were utilized to withdraw harmful products, decentralize PV activities and mandate reporting by industry were also mentioned. It was mentioned that PHPs sometimes only provided disease-specific resources. For example, a vaccine sur- veillance system can only fulfil a specific need of a na- tional centre’s mandate and may not be useful for other purposes which leads to national centres having silo sur- veillance systems as the interviews revealed. Further, it was mentioned that development partners provided technical resources based on their programme objec- tives. Participants expressed that they tie their work plans to development partners’ agenda even when their needs were different. Regarding common successful experiences, three out of the 18 strategic leaders interviewed indicated they had a legal framework or law that specifically mentions pharmacovigilance and a participant described how empowering it can be: “The national centre was set up under the NRA with legal framework, guidelines, staff, advisory committee and reporting systems through consultation with all stakeholders”. (Participant, 18). Other common successful experiences related to decentralization which seeks to bring pharmacovigi- lance closer to the patient. Six of the countries inter- viewed had embarked on decentralization initiatives by establishing regional or zonal centres, sometimes by using Drug Therapeutics Committees (DTCs) in re- gional public hospitals as was the case in Congo-DRC and Eritrea or by having regional focal persons as was the case in Angola, Cape Verde, Mozambique and Si- erra Leone. Experiences involving political resources “Working with development partners is sometimes difficult because they decide what level to tie their resources and sometimes the resources are not specific for our needs”. (Participant, 9). Stakeholders Participants expected to acquire basic technical re- sources such as computers and internet needed for their (Participant 2). Page 9 of 17 Page 9 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 A recurring unsuccessful acquisition of technical re- sources associated with public health programmes was the inability to deploy mutual surveillance systems be- tween the programmes and the centre to enable efficient data sharing. Experiences involving political resources Experiences involving financial resources There were 23 experiences (8 successful, 15 unsuccess- ful) mentioning financial resources (Table 2). The dom- inant stakeholder groups associated with financial resources were development partners (8), national gov- ernment (6) and public health programmes (5). Most of the national centres interviewed were not income-generating and got their funding from projects and/or from government budgets. Fourteen of the eight- een countries stated they did not have dedicated budget for PV activities. Successfully attained financial resources were used to acquire other resources, mainly technical and human re- source. Participants discussed buying equipment for day-to-day operations (e.g. computers) and sending na- tional centre personnel to international meetings. “To start pharmacovigilance, the government adopted two regulatory frameworks; one formed the regulatory authority and the second formed the national centre. These two documents helped start pharmacovigilance activities in the country”. (Participant 2). Experiences describing lack of financial resources fo- cused mainly on irregularity of funding and lack of au- tonomy of national centres to generate their own revenue. The interviews revealed that the lack of a stable financial resource stream manifested itself in sev- eral ways: firstly, the national centre was not able to undertake key activities such as ICSR collection. Secondly, they are unable to embark on important ini- tiatives such as active monitoring and lastly, national centre personnel are unable to acquire much needed training necessary for their work. Five of the strategic leaders who indicated they were successful in acquiring financial resources also indicated they were unsuccess- ful in acquiring financial resources usually because some of their efforts didn’t yield results. The inability to generate own revenues was considered particularly problematic when it increased dependency on the government: Most participants had challenges with the acquisition of political resources from the national government. “In the absence of strong regulatory laws, our country has become a dumping ground of fake products. The current law does not specify pharmacovigilance activities making it difficult to prosecute offenders”. (Participant 9). MSH country groupings It was expected that countries in group 4 would discuss more sophisticated technical resources, however the interviews revealed that countries with different levels of maturation of their PV system discussed similar technical resources. In discussing unsuccessful acquisi- tion of technical resources, two countries in group 4 with basic structures for both passive and active surveillance activities were for instance struggling with online reporting: “With the support of the national government, we introduced pharmacovigilance ambassadors in all 4 regions of our country and this has helped increase ICSR reporting”. (Participant 17). Unsuccessful experiences when discussing political resources centered on lack of legislations, inability to amend existing Health Bills to include PV and inabil- ity to mandate reporting by industry. Five of the countries interviewed had processes in place to imple- ment laws. “The issue of reporting online for instance; for some strange reason we haven’t been able to do something as simple as that”. (Participant 14). “Pharmacovigilance is not developed in my country because the processes to implement PV law started in 2003 and is ongoing as of 2015”. (Participant 1). At least one country in each group mentioned success- ful acquisition of technical resources from development partners. Countries in groups 1 and 2 appear to work more closely with the Global Fund whereas countries in group 4 work with a more varied group of development partners (e.g. John Snow Incorporated (JSI) and United States Pharmacopeia (USP). Participants stated they have had to improvise in the absence of specific PV laws by relying on PV statements in the national regulatory authority laws as legal backing for their work. Page 10 of 17 Page 10 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 “We have the regulatory authority act which states to ensure safety of products; it sets the pace that this is the intention of government to eventually enact a PV law”. (Participant 14). Irrespective of level of maturation of the PV system, in- terviewees referred to the absence of specific pharmacov- igilance laws when discussing unsuccessful acquisition of political resources. Moreover, none of the countries had autonomous centres. It was unexpected that some coun- tries in group 4 are still working with acts that reference pharmacovigilance and not PV-specific laws. “I came to this meeting with my Director. She is 2nd to the Minister of Health and she facilitated everything”. (Participant 13). Stakeholders As expected, almost all the political resources were as- sociated with national government. Participants empha- sized that only national governments can provide national centres with legitimacy. Successful acquisition of political resources from national government and the accompanying legitimacy was considered an enabling condition which allowed the national centre to mobilise other resources and have stable operations. However, several participants mentioned not having full political backing as an unsuccessful experience. The interviews revealed that in a considerable number of cases, na- tional governments provided initial political resources by enacting policies which aided national centres to be- come members of the WHO Programme for Inter- national Drug Monitoring (PIDM) but failed to continue with this. This also required the national gov- ernment to launch the national pharmacovigilance sys- tem. It is important to note that many successful experiences to do with the acquisition of political re- sources focus on early stages of the PV system develop- ment when legal systems were still being built and new policies being implemented. “We are not an autonomous agency. The whole idea of our national regulatory agency set up was to remove government bureaucracy so that we can do drug regulation without all those levels of reporting to slow us down”. (Participant 14). Experiences involving human resources Experiences involving human resources Human resource was mentioned 22 out of 108 times, most often (13/22) in relation to unsuccessful experi- ences (Table 2). The stakeholder groups mentioned in association with human resource were national govern- ment (11/20), intergovernmental organizations (4/20) and development partners (3/20) (Fig. 2). “We receive donor funding for PV projects. 50% of our staff are funded by donor projects”. (Participant 18). “We got financial support from United States Pharmacopeia (USP) and United States Agency for International Development (USAID) to conduct minilabs for malaria and post market surveillance for HIV.” (Participant 8). “We got financial support from United States Pharmacopeia (USP) and United States Agency for International Development (USAID) to conduct minilabs for malaria and post market surveillance for HIV.” (Participant 8). Successful experiences in acquiring human resources were about using experts from Drug and Therapeutic Committees (DTCs) to do PV work, having regional focal persons and incorporating PV into the curriculum of health disciplines. Fear of losing funding, partners not delivering prom- ised funds and funding tied to partners’ goals were some of the concerns expressed by participants in discussing inability to acquire financial resources. Adequate staffing appears to be a challenge for most national centres. In some cases, national centres had to rely on personnel from other departments to offer sup- port in addition to their regular duties (4/13) and, due to competing priorities, PV activities were compromised. “Now we are working well with Global Fund but if tomorrow there is no commitment between Global Fund and the country, our activities will be let down. This is a fear I have.” (Participant, 5). “Now we are working well with Global Fund but if tomorrow there is no commitment between Global Fund and the country, our activities will be let down. This is a fear I have.” (Participant, 5). “I have no time to do PV. In the Direction of Pharmacy (national regulatory authority), we have only 6 personnel for all the work and I have other activities to do”. (Participant 13). Discussions on difficulties with acquiring financial re- sources from national government centred around the unpredictability of funding which hindered planning and forecasting and general inadequate funding to support day to day operations. Moreover, participants emphasized the high personnel turnover at national centres (3/13), such as national centre personnel leaving to go work with development partners, industry and academia because these offer stable work environments. MSH country groupings Participants in groups 1 and 2 discussed the lack of fi- nancial resources from national government for basic operations whilst participants in group 4 appear to have stable funding streams. “I don’t belong to the group who discuss budget, it’s the director (of the NRA), I can propose activities, but the director decides whether we do it or not”. (Participant 3). “Our funding previously was from donors but now we have funding from government and it is based on our activity plan”. (Participant 12). MSH country groupings Countries in group 4 spoke of receiving varied resources from government whilst countries in groups 1 and 2 spoke mainly of political support they have received. “We are totally dependent on the Ministry; we do not generate our own income hence we are limited in the number of activities we can undertake”. (Participant 9). “I came to this meeting with my Director. She is 2nd to the Minister of Health and she facilitated everything”. (Participant 13). Page 11 of 17 Page 11 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 “The national centre does not have the autonomy to submit its own budget to the national regulatory authority”. (Participant 16). Stakeholders Participants in groups 1 and 2 discussed acquiring fi- nancial resources from PHPs and development partners to embark on awareness creation and training. National governments (6/13) and development partners (5/13) were mentioned most in association with unsuccessful acquisition of financial resources by all groups as seen in Fig. 2. Development partners appeared to play a key role in the provision of financial resources (8/21) but many partici- pants (5/8) mentioned that they are not always able to acquire funding from them. This might be explained by the fact that national centres have typically enjoyed fi- nancial resources from development partners which has become part of their resource acquisition strategy. p q gy Some participants elaborated on successful acquisition of financial resources from development partners Experiences involving human resources “(Financial) resources are not very predictable. It takes a lot of efforts to have a budget and still the budget is not enough for our priority activities”. (Participant, 4). “If you train 10 people today, one or two years later only 2 will still be working, the rest disappear to the other organizations”. (Participant 5). National government was not mentioned in associ- ation with the successful acquisition of financial re- sources because participants had tacit expectations that funding for national centres activities is an action that governments should routinely undertake. Politics appears to play an important role in the sus- tainability of national centre personnel as most strategic positions at the national regulatory authority are Page 12 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 occupied by political appointees thus affecting who is nominated as head of the national centre. Whilst partici- pants did not state this explicitly, 3 participants provided strong hints. benefits from belonging to regional partnerships such as the East African Community (EAC). “The EAC harmonization provides us with various expertise from the different countries, for instance we are the lead in Pharmacovigilance whilst other functions such as medicines registration are performed by different countries”. (Participant 8). “The EAC harmonization provides us with various expertise from the different countries, for instance we are the lead in Pharmacovigilance whilst other functions such as medicines registration are performed by different countries”. (Participant 8). “In Africa most issues are politicized; there have been changes in the system that has weakened the progress we have made in (PV) so far”. (Participant 9). Stakeholders Lack of adequate human resources both in personnel and expertise was a common theme amongst all three groups. Participants in groups 1 and 2 mentioned not having enough personnel to perform day to day duties whilst par- ticipants in group 4 mentioned not having adequate ex- pertise to do active surveillance. Successful acquisition of human resources by groups 1 and 2 were mostly about using the DTCs to augment their operations. National government was associated most with unsuccess- ful experiences in discussing the inability to acquire hu- man resources (11/13). Participants mentioned challenges such as unavailability of skilled expertise. The interviews revealed that some national centres have collected ICSR data but due to a lack of data analysis expertise have not been able to make decisions out of this data. Participants in groups 1 and 2 also mentioned aca- demia as helping augment human resources by incorp- orating PV into the curriculum of healthcare disciplines. “We use the WHO Method (for causality assessment) but we cannot analyse the data with VigiFlow. We need training”. (Participant, 16). National centres are tasked with monitoring the safety of products sold by MAHs. However, the MAH personnel tend be more knowledgeable in PV than na- tional centre personnel. There have been instances where national centres have received documentations from MAHs and have had to rely on the MAHs to ex- plain what the national centre needs to do with such documentation. Experiences involving social resources l d Social resources were mentioned 9 out of 108 times and mainly in association with successful experiences (8/9) (Table 2). The stakeholder groups associated with social resource were public health programmes (6/9), intergov- ernmental organizations (2/9) and Academia (1/9). The interviews revealed that national centres con- stantly seek resources from various stakeholders thus be- ing able to build linkages is key to their survival. Social resources such as collaborations, building partnerships, establishing trust-based relations and networking there- fore emerged as a separate theme in successful and un- successful experiences. “MAHs sometimes know more about pharmacovigilance than you who is the regulator. It has been a challenge to build the capacity of the national centre staff to regulate the MAHs”. (Participant 14). National centres discussed experiences in which they have been able to build mutually respectful trust-based relationships with some organizations which became in- strumental in safety monitoring efforts: Successes in acquiring human resource were mainly associated with development partners (3 experiences), intergovernmental organizations (3 experiences) and academia (2 experiences). Development partners helped with creation of DTCs, staff augmentation and training. “Through our strong collaboration with the malaria programme, we embarked on joint monitoring and with the evidence collected we switched our first line of malaria drug from Artesunate+Amodiaquine to Artemether-Lumefantrine”. (Participant17). “With help from MSH we implemented DTCs in general hospitals to advice the national centre”. (Participant, 5). “We have a full-time MSH staff placed at the national centre. She is supported by MSH”. (Participant 7). “With help from MSH we implemented DTCs in general hospitals to advice the national centre”. “With help from MSH we implemented DTCs in general hospitals to advice the national centre”. (Participant, 5). “We have a full-time MSH staff placed at the national centre. She is supported by MSH”. (Participant 7). Discussion “We have good collaborations with malaria, tuberculosis and HIV programmes; majority of our ADRs are from the three programmes. Every quarter we share a report with the programmes, so they can appreciate their contributions”. (Participant, 8). This paper examined the organizational capacity ele- ments (resources and relationships) that strategic leaders in national centres in Africa typically associate with suc- cessful and unsuccessful experiences in order to provide insight into the types of resources and relationships na- tional centres need in order to deliver on their mandate. A key finding is that national centres in Africa appear not to be the central coordinating bodies of PV in their various countries but rather conduct a large part of their activities in project-like settings in close collaboration with public health programmes, development partners, intergovernmental organizations and academia. Moreover, national centres experience difficulties in acquiring different types of resources, particularly from national governments, which has made them reliant on external stakeholders, particularly development partners. The difficulties appear to restrict the abilities of national centres to undertake post-market surveillance of the safety and quality of products marketed in the country and the ability to generate the necessary data for evidence-based decision making. Successful acquisition of social resources from aca- demia were about working with the universities to in- corporate PV in the curricula of healthcare disciplines. “We have developed a framework with the universities to incorporate PV into the teaching of medicine, pharmacy and nursing”. (Participant, 7). Finally, a participant indicated that they are encour- aged by invitations to conferences and meetings by intergovernmental organizations for the knowledge shar- ing benefits it produces. “I am here in Accra on invitation of WHO-CC attend- ing a conference. If I get copies of these presentations, we will use them to work better when we go back to my country”. (Participant, 3). Resource deficiencies have been previously cited as a barrier to the successful delivery of national centres’ mandate [6, 9, 10, 21, 23]. In a publication in the WHO’s World Medicines Situation series, Pal et al. [8] showed that most national centres in developing countries were severely understaffed and under-resourced with their PV agenda being very much donor-driven. Discussion Subsequently, a 2012 assessment of 9 African countries by the USAID-SIAPS programme revealed that regulatory in- frastructure for PV is weak with only 41% having a PV national policy, 30% with legislations for ICSR reporting, 28% having legal provisions that required MAHs to re- port ICSRs and only 17% requiring MAHs to conduct post-marketing surveillance activities. These publications showed that national centres in developing countries “We have a full-time MSH staff placed at the national centre. She is supported by MSH”. (Participant 7). Networking with other national centres were also dis- cussed by some participants as beneficial in exchanging knowledge and best practices. Further, PIDM membership guarantees access to publications and advisory support from the WHO, Uppsala Monitoring Centre and the WHO Collaborating Centres in Ghana and Morocco. Intergovernmental organizations were mentioned in relation to capacity-building guidelines and other policy documentations development and human resource Page 13 of 17 Page 13 of 17 Page 13 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 Community harmonization for resource sharing. Countries in groups 1 and 2 appear to hinge their opera- tions on what resources partners can provide. Community harmonization for resource sharing. Countries in groups 1 and 2 appear to hinge their opera- tions on what resources partners can provide. Stakeholders Public health programmes were most often associated with successful acquisition of social resources (4/6) Fig. 2. The interviews revealed that PHPs tend to be well-resourced and use medicines or vaccines in their operations thus making them a key stakeholder to na- tional centres. Some PHPs initiated pharmacovigilance activities in some countries. “We don’t have funds from the Ministry, sometimes we get support from Global Fund or MSH and it’s not fixed so we are not sure how to plan”. (Participant 1). While countries in group 4 did not specifically discuss so- cial resources, they appear to have been able to build long term trust-based relationships with some organizations: “In 2009 the immunization programme embarked on MenAfriVac vaccination campaign. Our country took advantage of this to start some pharmacovigilance activities”. (Participant 13). “MSH is still giving us technical support for active surveillance as we requested from them but not for routine activities”. (Participant 12). By virtue of the huge doses of medications adminis- tered in public health programmes they tend to be a gold mine for ICSR data. MSH country groupings All three groups discussed the same social resources such as building better relationships with partners, ensuring ef- ficient collaborations and linkages with other national centres. For example, the national centre in Cape Verde (group 1) has taken the lead to get all Portuguese speaking countries in Africa to form a partnership for resource mobilization. As of November 2015, Mozambique (group 2) and Angola (group 1) were on board according to the interviews. Another example is Kenya (group 2) and Rwanda (group 1) who are members of the East African Page 14 of 17 Page 14 of 17 Ampadu et al. Globalization and Health (2018) 14:109 have limited organizational capacity. A recent review of pharmacovigilance in resource limited countries (Olson et al. [10]) showed that national centres are still charac- terized by a lack of capacity to collect data. A study by Ampadu et al. [24] on the features of national centres in Africa showed that with the low numbers of ICSRs re- ported to VigiBase®® most national centres have insuffi- cient data to provide locally-relevant evidence on the benefits and risks of medicinal products. exceptions. In view of the important role expected by na- tional centres of their governments, it is important for the national centre and other stakeholders to continue advo- cating to these national governments for long-term re- sources for their national centres in order to fulfil their expected role of providing the needed safety surveillance infrastructure in their countries. have limited organizational capacity. A recent review of pharmacovigilance in resource limited countries (Olson et al. [10]) showed that national centres are still charac- terized by a lack of capacity to collect data. A study by Ampadu et al. [24] on the features of national centres in Africa showed that with the low numbers of ICSRs re- ported to VigiBase®® most national centres have insuffi- cient data to provide locally-relevant evidence on the benefits and risks of medicinal products. The third core challenge facing national centres is how to engage all PHPs in a sustainable way. The interview data showed that in nearly all countries, national centres are successful in engaging some but not all PHPs. Estab- lishing trust-based relationships with PHPs require ad- equate human and technical resources most of which are limited in national centres. MSH country groupings Public health pro- grammes are the main providers of data for national centres in Africa [24, 25] hence successful collaboration with them will provide not just the needed data but also associated resources. It is however, difficult to see how this can be done sustainably if national centres rely on these programmes for their resources. Collaboration be- tween national centres and PHPs is accepted as ex- tremely important and beneficial to both organizations and the WHO strongly encourages this as stated in the WHO manual “Pharmacovigilance in Public Health Pro- grammes” [26]. To encourage efficient collaboration with PHPs it would be important to research and provide guidance on the factors underlying successful collabor- ation between national centres and individual PHPs. Our study goes beyond these studies to distinguish be- tween the various resource elements that centres need to deliver and by associating these resource elements with relevant stakeholders in the PV system. This enables a more nuanced examination of the fundamental require- ments for sustainable PV in Africa and the organizational capacity needed by African national centres to deliver on their mandate. Our findings are generalizable in terms of geographic context, language, MSH country groupings and year of joining PIDM (Table 3). There is a bit of over-representation of relatively recently established na- tional centres in group 1–2 systems. Our sampling strat- egy and the resulting findings are thus particularly pertinent for relatively new centres in the systems with limited capacity for PV. Based on our study, we found 3 core challenges that affect the organizational performance of national centres in Africa. The first challenge is over-reliance on development partners. Pharmacovigilance in most countries started and/or have been facilitated by technical and financial support from development partners, usually the Global Fund, MSH through USAID or the Bill and Melinda Gates Foundation. This has led to a situation whereby national centres align their planning activities with those of the funding partners. Whilst this has been useful in several cases, it has also left national centres vulnerable. Changes of priorities by the development partners have often led to near-cessation of PV activities. Countries are also unable to undertake long-term planning due to uncertainties and volatility of financial support from partners. The fight against counterfeit medicines was not men- tioned in any of the described experiences. MSH country groupings This is sur- prising given that it is a known and ongoing problem in low and middle-income countries [27, 28]. In an article by WHO, it was estimated that one in 10 medicines in low-income countries are counterfeit and likely respon- sible for the deaths of tens of thousands of children from diseases such as malaria and pneumonia every year [29]. Several researchers have concluded that to combat this problem regulators will need sustained political will, fi- nancial support, tools and technical capacity to enforce quality standards in manufacturing, supply and distribu- tion and a coordinated action from the police, customs officials, and Marketing Authorization Holders [30]. National centres could play a role in this but our ana- lysis did not reveal activities focused on counterfeit medicines as a key priority. To address this problem an effective PV programme with enforcement power is needed. Further, it is also surprising that in only a limited number of experiences industry and academia were mentioned as stakeholders. One of the reasons for this might be that there is little industry and academic activity as pertains to pharmacovigilance in the systems under study. The second challenge is the seeming indifference of na- tional governments to provide support after national cen- tres have gained membership of the PIDM. National governments tend to provide some political and modest technical support by designating national centres and launching them publicly. Occasionally, national govern- ments have passed subsidiary legislation to help the work of the national centre. However, in several cases this sup- port seems to evaporate once countries become members of the PIDM leaving national centres bereft of resources. This is reflected in the data published by Ampadu et al. [24] where most national centres in Africa appear to do the barest minimum to gain membership of the PIDM by sending 20 ICSRs to VigiBase®. Thereafter, national cen- tres activities seem to slow down spectacularly with few We provide a number of recommendations based on our findings and discussions. First, to further strengthen Page 15 of 17 Ampadu et al. MSH country groupings Globalization and Health (2018) 14:109 d d PV t i b S h Af i it i i i ti h i l i ll b ti b t Table 3 National pharmacovigilance centres in Africa (Full PIDM members) [6, 13] Country National regulatory authority/national PV centre Year of joining the PIDM Included in this study MSH country group Angola Direcao Nacional de Medicamentos e Equipmentos 2013 □ Group 1 Benin Direction de la Pharmacie et des explorations diagnostics 2011 Burkina Faso Direction Générale de la Pharmacie, du Médicament et des Laboratoires 2010 Cameroon Direction de la Pharmacie, du Médicament et des Laboratoires 2010 □ Cape Verde Agência de Regulação e Supervisão dos Produtos Farmacêuticos e Alimentares 2012 □ Eritrea National Medicine and Food Administration 2012 □ Liberia Liberia Medicines and Health Products Regulatory Authority 2013 □ Madagascar Direction de la Phamacie, des Laboratoires et de la Médecine Traditionnelle 2009 Mauritius Pharmacy Board, Ministry of Health and Quality of Life 2014 □ Niger Direction de la Pharmacie, des Laboratoires et de la Pharmacopée Traditionnelle 2012 □ Sudan National Medicines and Poisons Board 2009 Swaziland Pharmaceutical Services Department 2015 Botswana Drug Regulatory Services, Ministry of Health and Wellness 2009 Group 2 Congo, Democratic Republic Direction de la Pharmacie et du Médicament. 2010 □ Côte d’Ivoire Direction de la Pharmacie et du Médicament. Competing interests Competing interests The authors declare that they have no competing interests. Funding No external sources of funding were used for the conduct of this study, or for the writing, correction, and submission of this article. 10. Olsson S, Pal SN, Dodoo A. Pharmacovigilance in resource-limited countries. Expert Rev Clin Pharmacol. 2015;8(4):449–60. Authors’ contributions HHA and JH conceived and designed the study. HHA conducted the data collection and analysis. DA and MAD helped with the transcription and data analysis. JH and ANOD supervised the study. HHA, JH, ANOD and HGML contributed to interpretation of the results and writing of the manuscript. All authors approved the final version of the manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conclusions This study concludes that national centres in Africa are faced with 3 core challenges. The first is over-reliance on development partners. The second challenge is the seeming indifference of national governments to provide support after national centres have gained membership of the WHO Programme for International Drug Moni- toring (PIDM) and the last core challenge facing na- tional centres in Africa is how to engage all public health programmes in a sustainable way. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Abbreviations ADR: Adverse drug reaction; AMRH: African Medicines Regulatory Harmonization programme; DTC: Drug Therapeutic Committee; FDA: Food and Drug Authority; HCF: Health Care Facility; HCW: Health Care Worker; ICSR: Individual Case Safety Reports; IPAT: Indicator-based Pharmacovigilance Assessment Tool; JSI: John Snow Incorporated; KNUST: Kwame Nkrumah University of Science and Technology; MAH: Market Authorization Holder; MoH: Ministry of Health; MSH: Management Sciences for Health; PIDM: WHO Programme for International Drug Monitoring; PRS: Patient reporting system; PV: Pharmacovigilance; RCORE: Regional Centre of Regulatory Excellence; SPSS: Statistical Package for the Social Sciences; SSA: Sub-Saharan Africa; USAID: US Agency for International Development; USP: United States Pharmacopeia; WHO: World Health Organization 3. Janarthanan VV, Ramakrishnan G, Krishnamurthy S, Sahar AI. Pharmacist as pharmacovigilance practitioner. Indian J Pharm Pract. 2015;8(1):2–6. 4. Venulet J, Helling-Borda M. WHOs international drug monitoring the formative years, 19681975: preparatory, pilot and early operational phases. Drug Saf. 2010;33(7):1–23. 5. Chakrabarty M, Thawani V. Starting a pharmacovigilance center: actions for implementation. J Pharmacol Pharmacother. 2011;2(4):295–9. 6. Management Sciences for Health. Assessment of pharmacovigilance systems and their performance. Arlington: 2011; MSH. http://apps.who.int/ medicinedocs/en/d/Js19152en. Accessed 14 July 2017. 7. World Health Organization. Minimum requirements for a functional pharmacovigilance system. 2010. http://www.who.int/medicines/areas/ quality_safety/safety_efficacy/PV_Minimum_Requirements_2010_2_en.pdf. Accessed 14 July 2017. MSH country groupings Finally, academic and research institutions could go be- yond incorporating PV in their curricula to embarking on PV research and developing tools and techniques relevant for safety surveillance in their respective national context. They could do this in collaboration with national centres. This will contribute to the development of innovative and pragmatic pharmacovigilance approaches [32] that are highly needed for SSA countries. References Ol S 1. Olsson S, Pal SN, Stergachis A, Couper M. Pharmacovigilance activities in 55 low- and middle-income countries: a questionnaire-based analysis. Drug Saf. 2010;33(8):689–703. 1. Olsson S, Pal SN, Stergachis A, Couper M. Pharmacovigilance activities in 55 low- and middle-income countries: a questionnaire-based analysis. Drug Saf. 2010;33(8):689–703. Additional file 1: Interview Questionnaire. (DOCX 17 kb) 2. Lu Y, Hernandez P, Abegunde D, Edejer T. The world medicines situation 2011 - medicine expenditures. World Health Organ. 2011;3:1–34. 2. Lu Y, Hernandez P, Abegunde D, Edejer T. The world medicines situation 2011 - medicine expenditures. World Health Organ. 2011;3:1–34. Acknowledgements 8. Pal SN, Dodoo A, Mantel A, Olsson S. The world medicines situation. Geneva: WHO; 2011. http://apps.who.int/medicinedocs/documents/ s18771en/s18771en.pdf.1. Accessed 16 July 2017. We thank Prince Narkortu Teye and Lawrencia Abrafi Osei for assisting in data analysis. 9. Maigetter K, Pollock AM, Kadam A, Ward K, Weiss MG. Pharmacovigilance in India, Uganda and South Africa with reference to WHO’s minimum requirements. Int J Health Policy Manag. 2015;4(5):295–305. Author details 1The African Collaborating Centre for Pharmacovigilance, 1 Vigilance Place, Mango Tree Avenue, Asylum Down, Accra, Ghana. 2WHO Collaborating Centre for Pharmaceutical Policy and Regulation, Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3508 TB Utrecht, the Netherlands. 3Innovation Studies Group, Copernicus Institute for Sustainable Development, Faculty of Geosciences, Utrecht University, 3508 TB Utrecht, the Netherlands. 4Noguchi Memorial Institute for Medical Research, University of Ghana, P. O Box LG 25, Accra, Ghana. Received: 13 July 2018 Accepted: 2 November 2018 Received: 13 July 2018 Accepted: 2 November 2018 MSH country groupings 2010 Ethiopia Food, Medicine and Health Care Administration and Control of Ethiopia 2008 □ Guinea Direction Nationale de la Pharmacie et du Laboratoire 2013 Kenya Pharmacy and Poisons Board 2010 □ Mali Direction de la Pharmacie et des Médicaments 2011 Mozambique Departamento Farmacêutico 2005 □ Rwanda Department of Pharmaceutical Services 2013 □ Senegal Direction de la Pharmacie et du Médicament 2009 □ Sierra Leone Pharmacy Board of Sierra Leone 2008 □ Togo Direction des Pharmacies, des Laboratoires et des Equipements Technique 2008 Zambia Zambia Medicines Regulatory Agency 2010 Zimbabwe Medicines Control Agency Zimbabwe 1998 □ Ghana Food and Drugs Authority 2001 Group 3 Tanzania, United Republic Tanzania Food and Drugs Authority 1993 Namibia Namibia Medicines Regulatory Council 2009 □ Group 4 Nigeria National Agency for Food and Drug Administration and Control 2005 □ South Africa Medicines Control Council 1992 Uganda National Drugs Authority 2008 Egypt Egyptian Drug Authority 2002 N/A Morocco Direction du Me’dicament et de la Pharmacie 1992 N/A Tunisia Direction de la Pharmacie et du Médicament 1993 N/A N/A: These countries are not included in the MSH groupings □: Country included in the study innovative approaches involving collaboration between development partners, public health programmes, academia and industry could be explored as has also been suggested by Pirmohammed et al. [21]. Such collaborative approaches might also help in preventing a situation where national centres become overly dependent on a single stakeholder. Second, it is important and expand PV systems in sub-Saharan Africa it is im- portant to develop approaches that allow for sustainable financial and technical resources for national centres as these resources have been identified by strategic leaders as key impediments to the functioning of national centres. National governments will remain the key expected provider of these resources; however, Ampadu et al. Globalization and Health (2018) 14:109 Page 16 of 17 Page 16 of 17 Page 16 of 17 that international organizations like WHO and the Global Fund earmark a certain percentage of funds for medicines and vaccines to be set aside solely for safety surveillance and the maintenance of the safety surveillance and quality infrastructure. Third, mandatory QPPV programmes as required in Ghana and other legally enforceable instru- ments put responsibility on surveillance and the provision of safety data on the pharmaceutical industry who should be a main provider of safety data to national centres [31]. Ethics approval and consent to participate Per Page 43, Appendix B, Number 2 of the Ghana Health Service Ethics Review Committee’s Standard Operating Procedure, ethical review is not needed for a research that uses interviews to document “public behaviour” of professionals working in a public organization and not of patients. All quotes were anonymised to prevent statements being traced back to individuals. Consent for publication Not applicable. Availability of data and materials 11. Isah AO, Pal SN, Olsson S, Dodoo A, Bencheikh RS. Specific features of medicines safety and pharmacovigilance in Africa. Ther Adv Drug Saf. 2012; 3(1):25–34. The datasets used and/or analysed during the current study are available from the corresponding author on request. Page 17 of 17 Ampadu et al. Globalization and Health (2018) 14:109 Ampadu et al. Globalization and Health (2018) 14:109 12. Stergachis A, Bartlein RJK, Dodoo A, Nwokike J, Kachur SP. A situational analysis of pharmacovigilance plans in the Global Fund Malaria and U.S. President’s Malaria Initiative proposals. Malar J. 2010;9(1):148. p p 13. Schäublin M. Pharmacovigiance: the spontaneous reporting system in Switzerland. Ther Umsch. 2015;72(11/12):743–8. 13. Schäublin M. Pharmacovigiance: the spontaneo Switzerland. Ther Umsch. 2015;72(11/12):743–8. 13. Schäublin M. Pharmacovigiance: the spontaneous reporting s Switzerland. Ther Umsch. 2015;72(11/12):743–8. 14. The Parliament of the Republic of Ghana. Public Health Act, 2012; 2012. p. 1– 194. http://extwprlegs1.fao.org/docs/pdf/gha136559.pdf. Accessed 14 July 2017 15. NEPAD, PATH. Understanding the role of Regional Centres of Regulatory Excellence in strengthening medicines regulation in Africa. 2014. http:// www.nepad.org/resource/understanding-role-regional-centres-regulatory- excellence-strengthening-medicines. Accessed 14 Apr 2017. 16. World Health Organization. The Importance of Pharmacovigilance - Safety Monitoring of medicinal products. 2002. http://apps.who.int/medicinedocs/ pdf/s4893e/s4893e.pdf. Accessed 14 Apr 2017. 17. NAFDAC. Nigerian National Pharmacovigilance Policy and Implementation framework. 2012. https://drive.google.com/file/d/ 0B1DAmtM1BcbMX3lqemU4bEtxVW8/view. Accessed 16 July 2017. 18. World Health Organisation. Pharmacovigilance in public health programmes. Geneva; WHO. http://www.who.int/medicines/areas/quality_ safety/safety_efficacy/pharmpubhealth/en/. Accessed 3 Mar 2017. 19. Miller V, Nwokike J, Stergachis A. Pharmacovigilance and global HIV / AIDS. Curr Opin HIV AIDS. 2012; https://journals.lww.com/co-hivandaids/Fulltext/ 2012/07000/Pharmacovigilance_and_global_HIV_AIDS.4.aspx. Accessed 16 July 2017. 20. Adjei A, Narh-Bana S, Amu A, Kukula V, Nagai RA, Owusu-Agyei S, et al. Treatment outcomes in a safety observational study of dihydroartemisinin/ piperaquine (Eurartesim®) in the treatment of uncomplicated malaria at public health facilities in four African countries. Malar J. 2016;15(1):1–10. 21. Pirmohamed M, Atuah KN, Dodoo ANO, Winstanley P. Pharmacovigilance in developing countries. BMJ. 2007;335(7618):462. 22. GHS-ERC. Ghana Health Service- Ethics Review Committee: Standard Operating Procedures 2015. https://www.ghanahealthservice.org/ downloads/Standard_Operating_Procedures.pdf. Accessed 16 June 2018. 23. Moscou K, Kohler JC. Matching safety to access: global actors and pharmacogovernance in Kenya- a case study. Glob Health. 2017;13(1):20. 24. Ampadu HH, Hoekman J, de Bruin ML, Pal SN, Olsson S, Sartori D, et al. Adverse drug reaction reporting in Africa and a comparison of individual case safety report characteristics between Africa and the rest of the world: analyses of spontaneous reports in VigiBase®. Drug Saf. 32. Dodoo ANO. Active safety surveillance in Africa: pragmatism and agility. Drug Saf. 2018;41:731–3. Availability of data and materials 2016;39(4):335–45. 25. Dodoo A, Pal SN, Falzon D, Xueref S. Pharmacovigilance for tuberculosis does not feature prominently in grant applications to the Global Fund to Fight AIDS, tuberculosis and malaria. Drug Saf. 2010;33:924. 26. Health Organization W. Pharmacovigilance in public health programmes [internet]. World Health Organization; 2010. http://www.who.int/medicines/ areas/quality_safety/safety_efficacy/pharmpubhealth/en/. Accessed 16 July 2017. 27. Jones M. Gains and losses for Tropical Doctor. 2002. http://journals.sagepub. com/doi/pdf/10.1177/004947550203200101. Accessed 06 July 2017. 28. Gautam CS, Utreja A, Singal GL. Spurious and counterfeit drugs: a growing industry in the developing world spurious and counterfeit drugs: a growing industry in the developing world. Postgrad Med J. 2009; http://pmj.bmj. com/content/85/1003/251.abstract. Accessed 06 July 2017. 29. Pisani E, WHO Secretariat. WHO Global Surveillance and Monitoring System for substandard and falsified medical products. World Health Organization; 2017. http://www.who.int/medicines/regulation/ssffc/publications/GSMS_ Report.pdf?ua=1. Accessed 16 July 2017. 30. Newton PN, Green MD, Fernández FM. Impact of poor-quality medicines in the ‘developing’ world. Trends Pharmacol Sci. 2010. http://www.ncbi.nlm. nih.gov/pmc/articles/PMC2845817. Accessed 16 July 2017. 31. De Bie S, Ferrajolo C, Straus SMJM, Verhamme KMC, Bonhoeffer J, Wong ICK, et al. Pediatric drug safety surveillance in FDA-AERS: a description of adverse events from GRiP project. PLoS One. 2015. https://doi.org/10.1371/ journal.pone.0130399. Accessed 16 July 2017. 32. Dodoo ANO. Active safety surveillance in Africa: pragmatism and agility. Drug Saf. 2018;41:731–3.
https://openalex.org/W2166272159
https://europepmc.org/articles/pmc3045390?pdf=render
English
null
Long-term (5 year) safety of bronchial thermoplasty: Asthma Intervention Research (AIR) trial
BMC pulmonary medicine
2,011
cc-by
8,248
© 2011 Thomson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Long-term (5 year) safety of bronchial thermoplasty: Asthma Intervention Research (AIR) trial Neil C Thomson1*, Adalberto S Rubin2, Robert M Niven3, Paul A Corris4, Hans Christian Siersted5, Ronald Olivenstein6, Ian D Pavord7, David McCormack8, Michel Laviolette9, Narinder S Shargill10, Gerard Cox11, the AIR Trial Study Group Abstract Background: Bronchial thermoplasty (BT) is a bronchoscopic procedure that improves asthma control by reducing excess airway smooth muscle. Treated patients have been followed out to 5 years to evaluate long-term safety of this procedure. Methods: Patients enrolled in the Asthma Intervention Research Trial were on inhaled corticosteroids ≥200 μg beclomethasone or equivalent + long-acting-beta2-agonists and demonstrated worsening of asthma on long- acting-b2-agonist withdrawal. Following initial evaluation at 1 year, subjects were invited to participate in a 4 year safety study. Adverse events (AEs) and spirometry data were used to assess long-term safety out to 5 years post-BT. Results: 45 of 52 treated and 24 of 49 control group subjects participated in long-term follow-up of 5 years and 3 years respectively. The rate of respiratory adverse events (AEs/subject) was stable in years 2 to 5 following BT (1.2, 1.3, 1.2, and 1.1, respectively,). There was no increase in hospitalizations or emergency room visits for respiratory symptoms in Years 2, 3, 4, and 5 compared to Year 1. The FVC and FEV1 values showed no deterioration over the 5 year period in the BT group. Similar results were obtained for the Control group. Conclusions: The absence of clinical complications (based on AE reporting) and the maintenance of stable lung function (no deterioration of FVC and FEV1) over a 5-year period post-BT in this group of patients with moderate to severe asthma support the long-term safety of the procedure out to 5 years. * Correspondence: neil.thomson@glasgow.ac.uk 1Gartnavel General Hospital, University of Glasgow, Glasgow, UK Full list of author information is available at the end of the article Study subjects All patients completing the 12-month follow-up evalua- tions in the AIR Trial (BT group: standard-of-care + BT; Control group: standard-of-care) were invited to participate in a 5 year post-treatment extension study to evaluate longer-term safety under a new protocol (NCT00448812). The exclusion criteria for participation in the extension study were: participation in another clinical trial involving respiratory intervention, or new diagnosis of psychiatric disorder which in the judgment of the investigator could interfere with provision of informed consent, completion of tests, therapy, or follow-up. During Year 1, adverse events were solicited during 12 office visits and 9 telephone contacts over the course of the year, as well as a review of the medical chart. During the longer-term follow-up, adverse events were actively solicited from the subject during the annual evaluation and through a review of the medical chart for the prior year for subjects managed at the investigator’s institute. The recording of adverse events in Year 1 (AIR Trial) differed from the subsequent years (AIR Extension Study) in that in Year 1, multiple symptoms associated with an adverse event were collected as individual adverse events; while in the subsequent years, an adverse event with multiple symptoms was counted as a single adverse event e.g., multiple respiratory symptoms asso- ciated with worsening of asthma were considered as a single event called “asthma (multiple symptoms)” adverse event. The key inclusion criteria to establish eligibility of patients to participate in the original AIR Trial were: 18-65 year old ambulatory adults; stable asthma (no unscheduled visits or change to medications within 6 weeks prior to randomization); requiring inhaled corti- costeroids (ICS): at least 200 μg beclomethasone or equivalent per day, and long-acting b2-agonist (LABA): at least 100 μg salmeterol or equivalent per day; demon- stration of worsening of asthma after 2-week LABA withdrawal; pre-bronchodilator FEV1 ≥60% and ≤85% predicted; methacholine PC20 < 8 mg/ml; and, non- smoker x 1 yr; if former smoker, less than 10 pack-year history. The key exclusion criteria were: history of ≥3 lower respiratory tract infections per year (requiring antibiotics); and, requirement of > 4 puffs/day of a short-acting b2-agonist, excluding for exercise. The pro- tocol for the longer-term extension study was approved by the respective Institutional Review Boards/Ethics Committees at each participating institution prior to Study procedures Subjects in the BT group were evaluated annually; at Year 2, Year 3, Year 4, and Year 5 after their last treat- ment bronchoscopy. Subjects in the Control group were evaluated at Year 2 and Year 3 and then exited from the study. Year 1 data are provided for matched pairs in both the BT and Control groups comprising those sub- jects that enrolled in the longer-term follow-up. Annual evaluations included a physical examination, pre- and post-bronchodilator spirometry, static lung volumes, diffusing capacity, chest x-ray (PA and Lateral), methacholine PC20 (out to Year 3 only), as well as active solicitation of information on any adverse events, emer- gency room visits and hospitalizations for asthma symp- toms, oral corticosteroid pulses for worsening asthma symptoms, and any changes in maintenance asthma medications. Maintenance asthma medication use, oral corticosteroid use, adverse events, emergency room vis- its and hospitalizations were verified through medical record review for about 80% of the subjects whose pri- mary care was under the supervision of the investigator at their respective institution. X-ray observations reported by radiologists at each site were collectively reviewed by an independent pulmonologist to assess clinical relevance of the observations if any. Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Page 2 of 9 obtaining a signed informed consent from the study participants. the percentage of symptom-free days and symptom scores, while fewer puffs of rescue medication were required [9]. Two additional randomized controlled trials, the Research in Severe Asthma (RISA) Trial [10], and the sham-controlled Asthma Intervention Research 2 (AIR2) Trial [11], have provided additional support for the effectiveness of BT. The safety of BT over the post- treatment period out to one year was established in all 3 randomized clinical studies. Because BT is a novel treat- ment that alters the amount of airway smooth muscle, and asthma is a chronic disease that is associated with structural changes in the airway wall, it is important to know whether this treatment is associated with any longer term adverse outcomes. Longer-term safety data out to 5 years are available for a small cohort of BT treated subjects from the first clinical study of patients with mild to moderate asthma [12,13]. We now describe the safety profile of BT out to 5 years post-treatment from patients with moderate to severe asthma from the AIR Trial [9]. the percentage of symptom-free days and symptom scores, while fewer puffs of rescue medication were required [9]. Two additional randomized controlled trials, the Research in Severe Asthma (RISA) Trial [10], and the sham-controlled Asthma Intervention Research 2 (AIR2) Trial [11], have provided additional support for the effectiveness of BT. The safety of BT over the post- treatment period out to one year was established in all 3 randomized clinical studies. Because BT is a novel treat- ment that alters the amount of airway smooth muscle, and asthma is a chronic disease that is associated with structural changes in the airway wall, it is important to know whether this treatment is associated with any longer term adverse outcomes. Longer-term safety data out to 5 years are available for a small cohort of BT treated subjects from the first clinical study of patients with mild to moderate asthma [12,13]. We now describe the safety profile of BT out to 5 years post-treatment from patients with moderate to severe asthma from the AIR Trial [9]. Background provides an additional option for managing patients with severe asthma. BT provides therapeutic benefit by reducing the amount of excess smooth muscle in the airways, with the resultant effect of reducing broncho- constriction in response to asthma triggers. Results from the Asthma Intervention Research (AIR) Trial, the first randomized clinical trial of BT which compared BT plus standard-of-care therapy (inhaled corticosteroids (ICS) and long-acting-b2-agonists (LABA)) to standard-of-care alone, demonstrated that the mean rate of mild exacer- bations, as compared with baseline, was reduced in the BT group but was unchanged in the control group. Furthermore at 12 months, when subjects were on ICS alone, there were significantly greater improvements in the BT group than in the control group in the morning peak expiratory flow, scores on the AQLQ and ACQ, Asthma continues to be a major health concern world- wide, with over 23 million people in the United States who suffer with this disease [1]. Approximately 5-10% of these patients are characterized as having severe persis- tent asthma based on continued presence of asthma symptoms despite treatment with current state-of-the- art medications [2]. Poorly controlled asthma impacts the patient’s quality of life, increases healthcare utiliza- tion, and imposes both a social as well as an economic burden [3-8]. The recent approval of the Alair® Bronchial Thermo- plasty System for delivering bronchial thermoplasty (BT) * Correspondence: neil.thomson@glasgow.ac.uk 1Gartnavel General Hospital, University of Glasgow, Glasgow, UK Full list of author information is available at the end of the article Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Adverse Events There were no incidences of pneumothorax, intubation, mechanical ventilation, cardiac arrhythmias, or death as a result of BT treatment over the 5 year follow-up. Table 1 Number of Subjects Evaluated Annually out to 5 Years Subjects Completing 1 Year Follow-up in AIR Trial Subjects Enrolling for Longer-Term Follow-up Subjects Completing Follow-up Year 2 Year 3 Year 4 Year 5 BT 52 45 41a 41b 43 42c Control 49 24 23d 21e - - a: 4 subjects missed visit. b: 2 subjects missed visit; 1 subject withdrew consent; 1 subject Lost-to- Follow-up. c: 1 subject missed visit. d: 1 subject missed visit. e: 2 subjects withdrew consent after completing Year 2 evaluation, and 1 subject withdrew consent mid-Year 3. Control group subjects exited from study after completing Year 3 evaluation. Table 1 Number of Subjects Evaluated Annually out to 5 Years Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Page 3 of 9 Respiratory adverse events reported during the course of the 5 year follow-up are summarized in Table 3. Dur- ing Year 1, the rate of respiratory adverse events in both the BT and Control groups was higher as a result of the method of recording the adverse events whereby multi- ple symptoms associated with an adverse event were recorded as separate adverse events. In subsequent years (Year 2 to Year 5), an adverse event with multiple symp- toms was recorded as a single adverse event. The rate of respiratory adverse events in the BT group (AEs/subject) remained stable in years 2 to 5 following BT. A repeated measures analysis for the “asthma (multiple symptoms)” adverse events which reflects worsening asthma control showed no deterioration over time from Year 2 to Year 5 (p = 0.47). During Year 2 and Year 3, when data for the Control group was collected, the respiratory adverse event rate between the BT and Control groups was not significantly different. Fisher’s Exact test was used to compare proportion of subjects with respiratory hospitalizations and emergency room visits in the BT and Control groups during Years 1, 2 and 3. Trends in the percent of subjects with hospi- talizations or emergency room visits for respiratory symptoms across Years 1 to 5 were investigated using a repeated measures logistic regression (generalized esti- mating equation), modeling the percent of subjects reporting the event. Maintenance medications ICS dose: Change from Baseline to each follow-up year in ICS dose was analyzed with a Signed Rank test. Statistical Analyses Demographics Group means were compared using Student’s t-test. Hospitalizations and Emergency Room Visits for respira- tory symptoms: The respective number of subjects com- pleting each annual follow-up visit was used to calculate the proportion of subjects with hospitalizations or emer- gency room visits for respiratory symptoms in each year. Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Results Demographics and Clinical Characteristics Forty five (45) of the 52 subjects in the BT group (87%), and 24 of the 49 subjects in the Control group (49%) who completed the Year 1 evaluation opted to partici- pate in the extension study. The 7 subjects in the BT group and 25 subjects in the Control group who declined to participate in the long-term follow-up did so for personal reasons, and not due to mortality. The numbers of subjects enrolling for the longer-term fol- low-up and those completing scheduled annual evalua- tions are summarized in Table 1. g y Respiratory adverse events occurring at a by-subject incidence rate of ≥3.0% in any of the years are given in Table 4. For the majority of respiratory adverse events, the incidence rates were stable during each year from Year 2 to Year 5 in the BT group, and from Year 2 and Year 3 in the Control group. The respiratory adverse events were typical of asthma. One subject in the BT group who had undergone the procedure was diagnosed with a lung abscess in the previously treated left upper lobe at 14 months (Year 2), and was resolved with surgi- cal resection. The subject had undergone BT unevent- fully and had completed the 12 month follow-up with normal spirometric values and good asthma control (Post-BD FEV1 at baseline was 2.27L, and at 12 months was 2.33L). Histological examination of the dissected lung did not reveal an obstruction or any other poten- tially contributory abnormality in the airways as a result of the treatment. The abscess was considered secondary to an infection. At the time of exit from the study at 5 years, the post-BD FEV1 for this subject was 1.78L compared to baseline value of 2.27L. The baseline demographic information and clinical characteristics (at time of entry into the AIR Trial) for subjects participating in the extension study are pro- vided in Table 2. The groups were well matched with no statistically significant differences between them for any given parameter. Healthcare Utilization Events Subjects) 3 (3) 2 (2) Lung Function Measures Morning PEF (L/min) 368.4 ± 99.7 394.1 ± 111.7 Pre-Bronchodilator FEV1 (% predicted) 72.5 ± 10.9 74.9 ± 8.9 Post-Bronchodilator FEV1 (% predicted) 84.4 ± 13.8 86.1 ± 9.5 Diffusion Capacity (mL/min/mm Hg) 15.7 ± 10.7 15.9 ± 11.7 Total Lung Capacity (L) 6.0 ± 1.2 5.9 ± 1.3 Residual Volume (L) 2.1 ± 0.7 2.0 ± 0.7 Methacholine PC20 (mg/ml) 0.25 0.28 Geometric mean (range) (0.2, 0.4) (0.1, 0.6) Definition of abbreviations: BT = Bronchial Thermoplasty; LABA = Long-Acting b2-Agonist; PEF = Peak Expiratory Flow Rate; FEV1 = Forced Expiratory Volume in 1 second. Table 2 Baseline Demographics and Clinical Characteristics c: Patient reported. BT versus Control: All parameters, Not significant (Student’s t-test of the mean). in Years 2, 3, 4, and 5 compared to Year 1 (Table 5) (p = 0.55; repeated measures analysis). The number of ER visits for respiratory symptoms in the Control group in Years 2 and 3 were comparable to the BT group (p = 0.41 and p = 1.00, respectively; Fisher’s Exact test). Oral Corticosteroid Use for Asthma Symptoms in Years 2, 3, 4, and 5 compared to Year 1 (Table 5) (p = 0.55; repeated measures analysis). The number of ER visits for respiratory symptoms in the Control group in Years 2 and 3 were comparable to the BT group (p = 0.41 and p = 1.00, respectively; Fisher’s Exact test). Oral Corticosteroid Use for Asthma Symptoms The frequency of OCS usage for worsening of asthma symp- toms is shown in Table 6. Neither the rate of OCS usage nor the proportion of subjects requiring OCS pulses showed any worsening over the 5 year period in the BT group. OCS The frequency of OCS usage for worsening of asthma symp- toms is shown in Table 6. Neither the rate of OCS usage nor the proportion of subjects requiring OCS pulses showed any worsening over the 5 year period in the BT group. Healthcare Utilization Events During Year 1 and Year 2, more subjects in the BT group required hospitalizations for respiratory symp- toms than the Control group, but these differences were not significant. There was one hospitalization for respiratory symptoms in the Control group in Year 3. Over the course of the 5 year post-BT follow-up, the number of hospitalizations, and the proportion of sub- jects experiencing hospitalizations for respiratory symp- toms did not get worse compared to Year 1 after BT (Table 5) (p = 0.16; repeated measures analysis for pro- portion of subjects). Similarly, the number of emergency room (ER) visits for respiratory symptoms and the pro- portion of subjects experiencing ER visits for respiratory symptoms remained comparable and did not get worse Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Page 4 of 9 Page 4 of 9 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Table 2 Baseline Demographics and Clinical Characteristics BT (n = 45) Control (n = 24) Age (yrs) 40.0 ± 11.2 40.8 ± 12.1 Gender Male 19 (42%) Male 9 (38%) Female 26 (58%) Female 15 (63%) Race White 41 (91%) White 22 (92%) Black 3 (7%) Black 2 (8%) Asian 1 (2%) Asian 0 (0%) Height (cm) 166.1 ± 9.6 164.8 ± 7.7 Weight (kg) 76.3 ± 23.3 77.7 ± 16.9 Inhaled Corticosteroid Dose (μg)a 1305 ± 880 1141 ± 1053 LABA Dose (μg)b 109 ± 34 100 ± 15 Symptom-Free Days (%) 33.3 ± 34.3 46.1 ± 41.0 Asthma Control Questionnaire (ACQ) Score 1.3 ± 0.6 1.2 ± 0.7 Asthma Quality of Life Questionnaire (AQLQ) Score 5.6 ± 0.9 5.6 ± 0.9 Rescue Medication Use (No. of puffs/7days) 10.6 ± 14.7 5.5 ± 10.4 Emergency Room Visits for Respiratory Symptoms in prior 12 monthsc No. Events (No. Subjects) 3 (3) 0 (0) Hospitalizations for Respiratory Symptoms in prior 12 monthsc No. Events (No. Healthcare Utilization Events OCS Table 3 Summary of Respiratory Adverse Events Year 1 Year 2 Year 3 Year 4 Year 5 Number of subjects BT 45a 45 43 43 42 Control 24a 24 21 – b – b Number of subjects reporting (Percent of subjects) BT 38 (84%) 24 (53%) 24 (56%) 23 (53%) 22 (52%) Control 18 (75%) 13 (54%) 12 (57%) – – Events per subject BT 4.5 1.2 1.3 1.2 1.1 Control 3.1 1.2 1.3 – – a: Year 1 data only for subjects who enrolled for longer-term follow-up. b: Control group subjects exited from Study after Year 3 evaluations. Table 3 Summary of Respiratory Adverse Events Thomson et al. Table 3 Summary of Respiratory Adverse Events BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Page 5 of 9 Page 5 of 9 Table 4 Subjects with Respiratory Adverse Events (All events reported at ≥3.0% in any year) Year 1a Year 2 Year 3 Year 4b Year 5b Adverse Event BT (n = 45) Control (n = 24) BT (n = 45) Control (n = 24) BT (n = 43) Control (n = 21) BT (n = 43) BT (n = 42) Dyspnea 19 (42.2%) 12 (50.0%) 4 (8.9%) 3 (12.5%) 4 (9.3%) 3 (14.3%) 4 (9.3%) 4 (9.5%) Cough 17 (37.8%) 7 (29.2%) 4 (8.9%) 1 (4.2%) 2 (4.7%) 3 (14.3%) 3 (7.0%) 2 (4.8%) Wheeze 14 (31.1%) 4 (16.7%) 2 (4.4%) 1 (4.2%) 3 (7.0%) 1 (4.8%) 3 (7.0%) 2 (4.8%) Nasal congestion 13 (28.9%) 5 (20.8%) 2 (4.4%) 0 0 0 0 1 (2.4%) Upper Respiratory Tract Infection 10 (22.2%) 2 (8.3%) 11 (24.4%) 4 (16.7%) 8 (18.6%) 4 (19.1%) 8 (18.6%) 4 (9.5%) Productive cough 9 (20.0%) 5 (20.8%) 2 (4.4%) 1 (4.2%) 2 (4.7%) 0 0 1 (2.4%) Chest discomfort 8 (17.8%) 3 (12.5%) 2 (4.4%) 2 (8.3%) 3 (7.0%) 1 (4.8%) 1 (2.3%) 2 (4.8%) Nasopharyngitis 6 (13.3%) 0 1 (2.2%) 0 0 0 1 (2.3%) 1 (2.4%) Nocturnal Dyspnea 6 (13.3%) 2 (8.3%) 0 0 0 0 0 0 Respiratory Tract Infectionc 5 (11.1%) 5 (20.8%) 3 (6.7%) 2 (8.3%) 5 (11.6%) 1 (4.8%) 5 (11.6%) 4 (9.5%) Pharyngolaryngeal pain 5 (11.1%) 3 (12.5%) 0 0 0 0 0 0 Respiratory Tract congestion 4 (8.9%) 2 (8.3%) 0 0 0 0 0 0 Discolored sputum 4 (8.9%) 0 3 (6.7%) 0 0 0 0 0 Rhinitis 2 (4.4%) 0 0 0 1 (2.3%) 0 0 2 (4.8%) Bronchitisd 1 (2.2%) 0 1 (2.2%) 1 (4.2%) 1 (2.3%) 2 (9.5%) 1 (2.3%) 1 (2.4%) Pharyngitis 1 (2.2%) 1 (4.2%) 0 0 0 0 0 0 Pleuritic Pain 1 (2.2%) 1 (4.2%) 0 0 0 0 0 0 Rhinorrhea 1 (2.2%) 1 (4.2%) 0 0 1 (2.3%) 0 0 0 Asthma (multiple symptoms)e 0 0 4 (8.9%) 2 (8.3%) 7 (16.3%) 1 (4.8%) 7 (16.3%) 6 (14.3%) Sinusitis 0 0 1 (2.2%) 1 (4.2%) 2 (4.7%) 0 2 (4.7%) 2 (4.8%) Nasal polyps 0 0 1 (2.2%) 0 0 0 2 (4.7%) 0 Pneumonia 0 0 0 0 1 (2.3%) 1 (4.8%) 0 0 a: Year 1 data only for subjects who enrolled for longer-term follow-up. Table 3 Summary of Respiratory Adverse Events Adverse events solicited from patient during multiple office visits in Year 1. In subsequent years, adverse events solicited only at annual follow-up visit. b: Control group subjects exited from Study after Year 3 evaluations. c: Includes adverse events reported as “Respiratory Tract Infection” and “Lower Respiratory Tract Infection”. d: Includes adverse events reported as “Bronchitis” and “Tracheobronchitis”. e: Asthma - In Year 1, all symptoms were collected as individual adverse events; in subsequent years, multiple symptoms of asthma exacerbation were considered as a single adverse event. th Respiratory Adverse Events (All events reported at ≥3.0% in any year) a: Year 1 data only for subjects who enrolled for longer-term follow-up. Adverse events solicited from patient during multiple office visits in Year 1. In subsequent years, adverse events solicited only at annual follow-up visit. b: Control group subjects exited from Study after Year 3 evaluations. c: Includes adverse events reported as “Respiratory Tract Infection” and “Lower Respiratory Tract Infection”. usage for asthma symptoms was comparable between the BT and Control groups during Years 1, 2 and 3. number of subjects had been adjusted to reflect their current level of asthma control. Compared to their base- line pre-BT usage, over the course of the 5 years, an average of 57% of BT subjects reported a decrease in their LABA use, 40% of subjects reported no change in a: Control group subjects exited from Study after Year 3 evaluations. b: p value from Fisher’s Exact test Maintenance Asthma Medication Use During the review of maintenance asthma medications at each annual visit, the medication dosages for a Table 5 Summary of Healthcare Utilization Events Hospitalizations Percent of Subjects [95% CI] (Number of Events) Emergency Room Visits Percent of Subjects [95% CI] (Number of Events) Total Number of Subjects BT Control p-valueb BT Control p-valueb Year 1 BT = 45 Control = 24 6.7% [0.0, 14.0] (3) 0 0.55 4.4% [0.0, 10.5] (2) 0 0.54 Year 2 BT = 45 Control = 24 6.7% [0.0, 14.0] (3) 0 0.55 6.7% [0.0, 14.0] (3) 12.5% [0.0, 25.7] (3) 0.41 Year 3 BT = 43 Control = 21 2.3% [0.0, 6.8] (3) 4.8% [0.0, 13.9] (1) 1.00 4.7% [0.0, 10.9] (3) 4.8% [0.0, 13.9] (3) 1.00 Year 4a BT = 43 Control = 0 2.3% [0.0, 6.8] (1) – 9.3% [0.6, 18.0] (6) – Year 5a BT = 42 Control = 0 2.4% [0.0, 7.0] (1) – 4.8% [0.0, 11.2] (2) – Pulmonary Function Tests Pulmonary function tests were performed when subjects were taking only ICS as their maintenance asthma medi- cation (either after a 2 week withdrawal of LABAs for subjects that were on ICS+LABA or for subjects on just ICS). The mean post-bronchodilator FEV1 and FVC values over time are presented graphically in Figure 1, respectively. Both measures of lung function (FEV1 and FVC) remained stable and showed no deterioration over the 5 year period post-BT. Similarly, total lung capacity and residual volumes remained stable out to 5 years in the BT group. There were small improvements over baseline in Diffusion Capacity in both the BT and Control groups over the course of the study. a: Year 1 data only for subjects who enrolled for longer-term follow-up. b: Control group subjects exited from Study after Year 3 evaluations. c: Includes adverse events reported as “Asthma”, “Exacerbations of asthma”, “Wheeze”, “Dyspnea”, and “Respiratory infection”. a: Year 1 data only for subjects who enrolled for longer-term follow-up. b: Control group subjects exited from Study after Year 3 evaluations. c: Includes adverse events reported as “Asthma”, “Exacerbations of asthma”, “Wheeze”, “Dyspnea”, and “Respiratory infection”. There was an improvement over baseline in the metha- choline PC20 doubling in the BT group compared to the Control group in each year out to Year 3. The differences between groups were statistically significant in Year 2 and Year 3, but not in Year 1 (methacholine PC20 dou- blings BT versus Control: Year 1: 1.53 ± 2.29 vs 1.00 ± 2.46, p = 0.378; Year 2: 1.21 ± 2.99 vs -0.47 ± 2.31, p = 0.024; Year 3: 1.31 ± 2.96 vs -0.44 ± 2.27, p = 0.025). their LABA use, and 3% reported an increase in their LABA use. In the Control group, over the course of the 3 years, an average of 54% of Control subjects reported a decrease in their LABA use, 43% of subjects reported no change in their LABA use, and 3% reported an increase in their LABA use. In the same period an aver- age of 49% of subjects in the BT group and 47% of sub- jects in the Control group were no longer taking LABA as controller medication. their LABA use, and 3% reported an increase in their LABA use. Pulmonary Function Tests In the Control group, over the course of the 3 years, an average of 54% of Control subjects reported a decrease in their LABA use, 43% of subjects reported no change in their LABA use, and 3% reported an increase in their LABA use. In the same period an aver- age of 49% of subjects in the BT group and 47% of sub- jects in the Control group were no longer taking LABA as controller medication. Table 5 Summary of Healthcare Utilization Events Table 5 Summary of Healthcare Utilization Events Table 5 Summary of Healthcare Utilization Events Control group subjects exited from Study after Year 3 evaluations. Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Page 6 of 9 Table 6 Use of Oral Corticosteroid Pulses for Asthma Symptoms Oral Corticosteroid Pulses: Events/Subject/Year (Percent of Subjects) Total Number of Subjects BT Control Year 1a BT = 45 Control = 24 0.60 (24.5%)c 0.42 (20.8%)c Year 2 BT = 45 Control = 24 0.49 (24.5%) 0.54 (33.3%) Year 3 BT = 43 Control = 21 0.33 (25.6%) 0.52 (23.8%) Year 4b BT = 43 Control = 0 0.63 (27.9%) – Year 5b BT = 42 Control = 0 0.62 (30.9%) – a: Year 1 data only for subjects who enrolled for longer-term follow-up. b: Control group subjects exited from Study after Year 3 evaluations. c: Includes adverse events reported as “Asthma”, “Exacerbations of asthma”, “Wheeze”, “Dyspnea”, and “Respiratory infection”. Table 6 Use of Oral Corticosteroid Pulses for Asthma Symptoms that typically occur in patients with moderate and severe asthma. Discussion The recently approved Alair System for bronchial ther- moplasty provides a new treatment option for patients with severe asthma that remain symptomatic despite taking inhaled corticosteroids and long-acting-b2- agonists, the current standard-of-care medications. Three randomized clinical trials in patients with differ- ing severity of asthma have demonstrated the safety of this device-based treatment out to one year [9-11]. In the AIR Trial, at 12 months post-treatment, while patients were on ICS alone, these benefits included sig- nificantly greater improvements in the BT group than in the Control group in mild exacerbation rates, morning peak expiratory flow, scores on the AQLQ and ACQ, the percentage of symptom-free days and symptom scores, while fewer puffs of rescue medication were required [9]. Each of these studies has also reported on the short-term (treatment period) and long-term (out to 12 months) adverse event profile associated with the bronchial thermoplasty. The reported increase in respiratory adverse events in the short-term represented the further aggravation of the worsening of asthma symptoms that is associated with bronchoscopy in patients with asthma [14]. During the long-term post- treatment period out to 12 months, fewer subjects in the BT group reported respiratory adverse events [9-11]. Data are now presented for the safety of this treatment over a 5 year period. The mean reduction from baseline in ICS dose for BT subjects was 182 μg/day (p = 0.09), 135 μg/day (p = 0.32), 150 μg/day (p = 0.25), 151 μg/day (p = 0.23), and 194 μg/day (p = 0.16) at Years 1, 2, 3, 4, and 5 respec- tively (p-values from a Signed Rank test). At Year 3, the mean reduction in the ICS daily dose in the Control group was 112 μg/day. The reduction in ICS was not significantly different between the BT group and the Control group at years 2 and 3 (p = 0.93 for Year 2, and p = 0.92 for Year 3), years when data were available for both the BT and Control groups. At 3 years the propor- tion of subjects in the BT group with changes from baseline in their maintenance ICS dose was 27% with a decrease, 56% with no change, and 17% with an increase. The corresponding numbers for the Control group were 29%, 52% and 19% respectively. Review of Serial (Annual) Chest X-rays Review of Serial (Annual) Chest X-rays 18 of the 45 BT subjects (40%), and 9 of the 24 Control subjects (37%) had finding(s) noted on chest x-rays. Findings ranged from air trapping, pleural thickening, increased density/consolidation, hyperinflation, nodules/ granuloma, increased vascular markings, and bronchial wall thickening. None of the findings noted in either group were clinically significant structural changes. The findings noted were either pre-existing, minor and tran- sient, or consistent with acute inter-current illnesses Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Page 7 of 9 Page 7 of 9 Figure 1 FEV1 and FVC over Time. Post-bronchodilator FEV1 and FVC over time. Data represent group mean values for each year. Note that participation of the Control subjects in the study was terminated after Year 3 evaluation. Figure 1 FEV1 and FVC over Time. Post-bronchodilator FEV1 and FVC over time. Data represent group mean values for each year. Note that participation of the Control subjects in the study was terminated after Year 3 evaluation. BT is a novel treatment that alters the amount of air- way smooth muscle, and asthma is chronic disease that is associated with structural changes in the airway wall, it was important to know whether this treatment is asso- ciated with any longer term adverse outcomes. The parti- cipation of patients originally enrolled in the AIR Trial in this additional 4 year follow-up study provides data to support the longer-term safety of BT out to at least 5 years. The absence of serious events indicates that the integrity of the airways is not compromised over the long term as a result of BT treatment. This is supported by the observation of no deterioration of baseline FEV1 or FVC over this period, and the ability of the airways to respond to bronchodilator administration. While the rate of decline in lung function can vary in asthma, with some subjects demonstrating equivalent declines to those with- out airways disease, and others suffer an accelerated decline [15-17], it is reassuring that following BT, there was no significant loss of lung function over a 5 year per- iod. The rate of occurrence of respiratory related adverse events remained low and stable between Year 2 and Year 5 (1.1 to 1.3 events/subject/year) and comparable to the Control group for Year 2 and Year 3 (1.2 and 1.3 events/ subject/year, respectively). Review of Serial (Annual) Chest X-rays The frequency and method of collecting adverse events in this study was different in Year 1 compared to the other 4 years, resulting in a higher rate of respiratory adverse events in both the BT and Control groups in Year 1. During Year 1, the solicita- tion of adverse events was more frequent (12 office visits and 9 telephone contacts) compared to subsequent years when adverse events were solicited once a year and could be subject to recall bias. Also important is the fact that during Year 1, multiple symptoms for a given adverse event were recorded as individual adverse events as com- pared to the subsequent years when multiple related symptoms were recorded as a single event. Because of these methodological issues, comparisons of adverse event rates within each group for Year 1 and subsequent years are not appropriate. The lower rates of respiratory adverse events in Years 2 to 5 compared to Year 1 may reflect some underreporting as a result of recall bias at the yearly solicitation of adverse events. Every effort was made to carefully solicit adverse events from study sub- jects during annual evaluations and augmented with a thorough review of medical records at participating insti- tutions. Review of medical charts is lacking for instances where a subject may have been treated for adverse event(s) at a different institution. Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Page 8 of 9 Page 8 of 9 Nevertheless a high proportion of patients who received BT (86%) participated in long-term follow-up, which provides support for the generalizability of the safety data. Thirdly, the solicitation of adverse events on a yearly basis has the potential of under reporting due to recall bias. However, the occurrence of major events such as severe exacerbations (steroid pulses for asthma symptoms), emergency room visits, and hospitalizations is less frequent and less likely to be forgotten and there- fore not reported. Medical charts were reviewed to ver- ify steroid pulses prescribed for asthma symptoms (severe exacerbations) and reported adverse events or to identify adverse events that may not have been reported by the subject, unless the subject had presented at a dif- ferent medical facility for an adverse event during that year. Review of Serial (Annual) Chest X-rays Finally, the study was not blinded and this may add bias to the eliciting of adverse events. Despite these limitations, over the 5 year follow-up of BT treated sub- jects, the absence of unexpected respiratory adverse events, no increases in hospitalizations or emergency room visits for respiratory symptoms, and maintenance of stable lung function demonstrated safety of BT out to 5 years. Measures such as ER Visits and hospitalizations for respiratory symptoms are generally considered to be important measures of safety, especially if an interven- tion results in an increase in the rate of one or more of these events. Consistent with the low rate of respiratory adverse events is the stable incidence of healthcare utili- zation events (hospitalizations and emergency room vis- its for respiratory symptoms). There was no worsening of the proportion of subjects in the BT group experien- cing hospitalizations or ER visits for respiratory symp- toms beyond Year 1 out to 5 years. The rates of ER visits and the proportion of subjects with ER visits in the Control group were comparable to the BT group during Years 1, 2, and 3. Longer-term safety of BT is supported further by our findings of no deterioration in measurements of static and dynamic lung volumes or diffusing capacity over the 5 years post-treatment. In addition airway responsiveness to methacholine remained stable in the BT group out to the last mea- surement performed at year 3 post-treatment. Stable lung function measures in this group of patients with moderate to severe asthma support the previously reported conclusion based on lung function measures [12,13] and high resolution CT [13] that BT-treated air- ways do not develop late scarring that could result in narrowing of the airways [12]. Conclusions This report provides long-term safety data on BT in patients in whom BT was performed by trained opera- tors, and patients were carefully observed especially in the first year. The absence of clinical complications based on adverse event reporting, healthcare utilization events, and the maintenance of stable lung function (no deterioration of FEV1) over a 5-year period post-BT in patients with moderate to severe asthma suggest long- term safety of the procedure out to 5 years. Although x-rays are not sensitive enough for the pur- pose of demonstrating structural abnormalities, a review of the serial x-rays obtained annually did not reveal any clinically significant findings. While the use of high resolution computed tomography (HCRT) would have been more informative, it was not performed in this study. Bronchial hyperresponsiveness to methacholine improved in the thermoplasty group in years 2 and 3, but not in year 1. This is an interesting result in favour of a long-term efficacy of the procedure. However, the lack of follow-up of the control group on years 4 and 5 and the observational nature of the data limits the rele- vance of this finding. Abbreviations AE Ad E AE: Adverse Event; ACQ: Asthma Control Questionnaire; AIR: Asthma Intervention Research; AIR2: Asthma Intervention Research 2; AQLQ: Asthma Quality of Life Questionnaire; BT: Bronchial thermoplasty; FEV1: Forced Expiratory Volume in 1 second; FVC: Forced Vital Capacity; HRCT: High resolution computed tomography; ICS: Inhaled corticosteroid; L: Liters; LABA: Long-Acting-β2-Agonist; OCS: Oral corticosteroid; PC20: Provocative Concentration causing 25% drop in FEV1; PEF: Peak Expiratory Flow; RISA: Research in Severe Asthma; SD: Standard deviation There are several limitations to the study. Firstly, not all patients enrolled in the Asthma Intervention Research Trial participated in long-term follow-up, although none of the reasons for non-participation was due to mortality. Secondly, the follow-up period was longer in patients who received BT (at 5 years) com- pared to the Control group (3 years). Subjects who had been randomized to the Control group in the predeces- sor AIR Trial may have opted to pursue other treatment options, and for those that agreed to the longer-term follow up, it was deemed to be unethical [18] to require them to withhold other alternative treatment options (including new experimental treatments) for a period of 5 years in order to avoid confounding of data. Acknowledgements Members of the AIR Trial Study Group were as follows: Co-Investigators and Study Coordinators – Brazil: Santa Casa: P.G. Cardoso, M. Cavalcanti, P.R.D. Soares, S. Zelmanovitz; Canada: McMaster University: P. Nair, S. Goodwin, S. Keogh, M. Kjarsgard; Montreal Chest Institute: J. Bourbeau, F. Houghton, R. Mangaser; London Health Science Centre: N. Patterson, S. Metha, J. Howard, L. MacBean; Laval University: S. Martel, L-P. Boulet, S. Savord, L. Morel, L. Trepanier; Denmark: Odense University Hospital: F. Rasmussen, H.M. Christensen; United Kingdom: University of Glasgow: S. Bicknell, R. Chaudhuri, E. Hothersall, J. Lafferty, J. Jarvis; University of Manchester: C. Prys-Picard, G. Fletcher, A. Fletcher; Newcastle University: B. Higgins, T. Small, B. Foggo, L. Mackay, S. Parker; University of Leicester: M. Berry, D Shaw, P. Haldar, A. Charalambou, M. Hopkin, A. Raj, N. Yousaf, N. Goodman. Page 9 of 9 Page 9 of 9 Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 Authors’ contributions IDP, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. DM, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. ML, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. NSS, PhD.: Contributed to the execution of the study, acquisition and interpretation of the data, writing and revision of the manuscript, and gave final approval of the version to be published. Had full access to the data and will vouch for the integrity of data analysis, and the accuracy and completeness of the reported data. 15. Sherrill D, Guerra S, Bobadilla A, Barbee R: The role of concomitant respiratory diseases on the rate of decline in FEV1 among adult asthmatics. Chest 2003, 21(1):95-10. 15. Sherrill D, Guerra S, Bobadilla A, Barbee R: The role of concomitant respiratory diseases on the rate of decline in FEV1 among adult asthmatics. Chest 2003, 21(1):95-10. 16. Lange P, Parner J, Vestbo J, Schnohr P, Jensen G: A 15-Year Follow-Up Study of Ventilatory Function in Adults with Asthma. N Engl J Med 1998, 339(17):1194-200. 16. Lange P, Parner J, Vestbo J, Schnohr P, Jensen G: A 15-Year Follow-Up Study of Ventilatory Function in Adults with Asthma. N Engl J Med 1998, 339(17):1194-200. 17. James AL, Palmer LJ, Kicic E, et al: Decline in Lung Function in the Busselton Health Study: The Effects of Asthma and Cigarette Smoking. Am J Respir Crit Care Med 2005, 171(2):109-14. 17. James AL, Palmer LJ, Kicic E, et al: Decline in Lung Function in the Busselton Health Study: The Effects of Asthma and Cigarette Smoking. Am J Respir Crit Care Med 2005, 171(2):109-14. 18. Rothman KJ, Michels KB: The continuing unethical use of placebo controls. New Engl J Med 1994, 331:394-398. 18. Rothman KJ, Michels KB: The continuing unethical use of placebo controls. New Engl J Med 1994, 331:394-398. Competing interests Neil C. Thomson, Adalberto S. Rubin, Robert M. Niven, Paul A. Corris, Hans Christian Siersted, Ronald Olivenstein, Ian D. Pavord, David McCormack, Michel Laviolette, Gerard Cox all received industry-sponsored grant funding from Asthmatx, the manufacturers of the Alair® System, for participating in clinical trials. Narinder S Shargill is an employee of Asthmatx. Received: 29 October 2010 Accepted: 11 February 2011 Published: 11 February 2011 Received: 29 October 2010 Accepted: 11 February 2011 Published: 11 February 2011 Author details 1 6. Serra-Battles J, Plaza V, Morejon E, Comella A, Brugues 1J: Costs of asthma according to the degree of severity. Eur Respir J 1998, 12:1322-1326. 1Gartnavel General Hospital, University of Glasgow, Glasgow, UK. 2Irmandade Santa Casa de Misericórdia da Porto Alegre, Brazil. 3University Hospital of South Manchester and University of Manchester, Manchester, UK. 4Department of Respiratory Medicine, Freeman Hospital, Newcastle University, Newcastle, UK. 5Odense University Hospital, Odense, Denmark. 6Montreal Chest Institute, McGill University, Montreal, Canada. 7Glenfield General Hospital, University Hospitals of Leicester NHS Trust, Leicester, UK. 8London Health Sciences Center, Ontario, Canada. 9Laval Hospital, Laval University, Quebec, Canada. 10Asthmatx, Inc., Sunnyvale, CA, US. 11St. Joseph’s Healthcare, McMaster University, Hamilton, Canada. 6. Serra-Battles J, Plaza V, Morejon E, Comella A, Brugues 1J: Costs of asthma according to the degree of severity. Eur Respir J 1998, 12:1322-1326. 7. Antonicelli L, Bucca C, Neri M, et al: Asthma severity and medical resource utilization. Eur Respir J 2004, 23:723-729. 8. Godard P, Chanez P, Siraudin L, Nicoloyannis N, Duru G: Costs of asthma l t d ith it E R i J 2002 19 61 67 7. Antonicelli L, Bucca C, Neri M, et al: Asthma severity and medical resource utilization. Eur Respir J 2004, 23:723-729. 8. Godard P, Chanez P, Siraudin L, Nicoloyannis N, Duru G: Costs of asthma are correlated with severity. Eur Respir J 2002, 19:61-67. 9. Cox G, Thomson NC, Sperb-Rubin A, the AIR Trial Study Group, et al: Asthma Control During the Year after Bronchial Thermoplasty. New Engl J Med 2007, 356:1327-1337. 9. Cox G, Thomson NC, Sperb-Rubin A, the AIR Trial Study G , , p , y p, Asthma Control During the Year after Bronchial Thermoplasty. New Engl J Med 2007, 356:1327-1337. 10. Pavord ID, Cox G, Thomson NC, the RISA Trial Study Group, et al: Safety and Efficacy of Bronchial Thermoplasty in Symptomatic, Severe Asthma. Am J Respir Crit Care Med 2007, 176:1185-1191. Pre-publication history y The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2466/11/8/prepub GC, M.B.: Contributed to the acquisition and interpretation of the data, writing and revision of the manuscript, and gave final approval of the version to be published. Had full access to the data and will vouch for the integrity of data analysis, and the accuracy and completeness of the reported data. The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2466/11/8/prepub doi:10.1186/1471-2466-11-8 Cite this article as: Thomson et al.: Long-term (5 year) safety of bronchial thermoplasty: Asthma Intervention Research (AIR) trial. BMC Pulmonary Medicine 2011 11:8. Thomson et al. BMC Pulmonary Medicine 2011, 11:8 http://www.biomedcentral.com/1471-2466/11/8 The database for this study was managed by, and all statistical analyses were performed by B. Armstrong, MS and J. Quiring, PhD (QST Consultations, Ltd., Allendale, MI). Data and Safety Monitoring Board – W. Busse, MD, R. Schellenberg, MD, Scott Berry, PhD, and A.S. Slutsky, MD (Chair) The authors thank Michael Wechsler, MD (Brigham & Women’s Hospital, Boston, MA) for his critical review of this manuscript prior to submission. Supported by: Asthmatx, Inc., Sunnyvale, CA The database for this study was managed by, and all statistical analyses were performed by B. Armstrong, MS and J. Quiring, PhD (QST Consultations, Ltd., Allendale, MI). The database for this study was managed by, and all statistical analyses were performed by B. Armstrong, MS and J. Quiring, PhD (QST Consultations, Ltd., Allendale, MI). Severe Asthma Research Program. J Allergy Clin Immunol 2007, 119:405-413. 3. Fuhlbrigge A, Adams R, Guilbert T, et al: The burden of asthma in the United States. Am J Respir Crit Care Med 2002, 166:1044-1049. Data and Safety Monitoring Board – W. Busse, MD, R. Schellenberg, MD, Scott Berry, PhD, and A.S. Slutsky, MD (Chair) Data and Safety Monitoring Board – W. Busse, MD, R. Schellenberg, MD Scott Berry, PhD, and A.S. Slutsky, MD (Chair) 4. Schatz M, Zeiger R, Mosen D, Vollmer W: Asthma-specific quality of life and subsequent asthma emergency hospital care. Am J Manag Care 2008, 14:206-211. The authors thank Michael Wechsler, MD (Brigham & Women’s Hospital, Boston, MA) for his critical review of this manuscript prior to submission. Boston, MA) for his critical review of this manuscript prior to submission Supported by: Asthmatx, Inc., Sunnyvale, CA Supported by: Asthmatx, Inc., Sunnyvale, CA 5. Vollmer W, Markson L, O’Connor E, Frazier E, Berger M, Buist AS: Association of Asthma Control with Health Care Utilization - A Prospective Evaluation. Am J Resp Crit Care Med 2002, 165:195-199. Received: 29 October 2010 Accepted: 11 February 2011 Published: 11 February 2011 2. Moore W, Bleecker E, Curran-Everett D, et al: Characterization of the severe asthma phenotype by the National Heart, Lung, and Blood Institute’s Authors’ contributions 11. Castro M, Rubin AS, Laviolette M, for the AIR2 Trial Study Group, et al: Effectiveness and Safety of Bronchial Thermoplasty in the Treatment of Severe Asthma: A Multicenter, Randomized, Double-blind, Sham- controlled Clinical Trial. Am J Respir Crit Care Med 2010, 181:116-124. NCT, M.D.: Contributed to the acquisition and interpretation of the data, writing and revision of the manuscript, and gave final approval of the version to be published. Had full access to the data and will vouch for the integrity of data analysis, and the accuracy and completeness of the reported data. controlled Clinical Trial. Am J Respir Crit Care Med 2010, 181:116-12 12. Cox G, Miller J, Goodwin S, Fitzgerald JM, et al: Long-Term Follow-up of Bronchial Thermoplasty for Asthma: Safety Results at 5 Years. Am J Respir Crit Care Med 2008, 177:A567. ASR, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. ASR, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. ASR, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. RMN, M.D.: Contributed to the acquisition and interpretation of the data, writing and revision of the manuscript, and gave final approval of the version to be published. 13. Cox G, Laviolette M, Rubin A, Thomson N: Long Term Safety of Bronchial Thermoplasty (BT): 3 Year Data from Multiple Studies. Am J Respir Crit Care Med 2009, 179:A2780. RMN, M.D.: Contributed to the acquisition and interpretation of the data, writing and revision of the manuscript, and gave final approval of the version to be published. 14. Moore W, Murphy J, Calhoun W, Castro M, Chung F, Erzurum S, Jarjour N, Wenzel S, Peters S, Bleecker E, NHLBI Severe Asthma Research Program (SARP): Safety of investigative bronchoscopy in severe asthma. ATS 2006 Annual Meeting, San Diego, CA 2006. PAC, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. HCS, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. RO, M.D.: Contributed to the acquisition and interpretation of the data, and gave final approval of the version to be published. References l 1. National Health Interview Survey, National Center for Health Statistics. CDC 2008 [http://www.cdc.gov/nchs/fastats/asthma.htm], Accessed 2/10/10. CDC 2008 [http://www.cdc.gov/nchs/fastats/asthma.htm], Accessed 2/10/10. 2. Moore W, Bleecker E, Curran-Everett D, et al: Characterization of the severe asthma phenotype by the National Heart, Lung, and Blood Institute’s
https://openalex.org/W3116664924
https://www.sciendo.com/pdf/10.1515/remav-2020-0031
English
null
Value-Based Management for Real Estate Developers’ Activities
Real Estate Management and Valuation
2,020
cc-by
10,828
JEL Classification: C58, R38, R51. Citation: Kowalski, M. J., & Kazak, J. K. (2020). Value-based management for real estate developers’ activities. Real Estate Management and Valuation, 28(4), 48-62. DOI: https://doi.org/10.1515/remav-2020-0031 DOI: https://doi.org/10.1515/remav-2020-0031 VALUE-BASED MANAGEMENT FOR REAL ESTATE DEVELOPERS’ ACTIVITIES Michał J. Kowalski Faculty of Computer Science and Management Wrocław University of Science and Technology, Poland e-mail: michal.kowalski@pwr.edu.pl Jan K. Kazak Institute of Spatial Management Wrocław University of Environmental and Life Sciences, Poland e-mail: jan.kazak@upwr.edu.pl Jan K. Kazak Institute of Spatial Management Wrocław University of Environmental and Life Sciences, Poland e-mail: jan.kazak@upwr.edu.pl www.degruyter.com/view/j/remav www.degruyter.com/view/j/remav Abstract Real estate development investments are characterized by a high value of projects, which in the event of irregularities in their implementation may result in significant losses for the economy. The lack of tools enabling ongoing control of real estate developers may result in disruptions in the operation of business entities on the real estate market, affecting the proper functioning of many stakeholders. The article proposes a method of measuring value for real estate companies. Accounting principles that regulate financial statements specify that they cannot be used directly to measure the value of a developer. The article proposes examples of corrections to financial statements supporting value measurement. When calculating value management measures, (i) adjustments excluding the impact of asset valuations, (ii) adjustments of settlement negative EVA of the investment phase, (iii) adjustments of advance payment of NPV of the project, and (iv) adjustments of excluding the impact of interest on foreign capital should be made. Examples of using these adjustments in a short-term housing project and a long-term commercial project were presented. The impact of the proposed adjustments on the comparison of formal financial statements and value measures for a large developer listed on the WSE was also discussed. Key words: value based management, real estate market, economic value added. accounting law. In the case of the real estate sector, the moment of recognizing the financial profit of the implemented project is, in many cases, significantly postponed. Accounting policies ensure that financial profits are presented in a manner that is consistent with the conservatism principle, which, however, can be challenging for properly measuring value and implementing value management tools. One way to shorten the time between costs and revenues is to divide investment into separate stages. This approach allows part of the implemented investment project to be settled and balanced earlier. However, this solution is used for large investments consisting of many separate future real estates. This does not change the fact that, for each stage of the investment, there is still a deferral in the time of settlement enabling sustainable monitoring of the enterprise's processes. accounting law. In the case of the real estate sector, the moment of recognizing the financial profit of the implemented project is, in many cases, significantly postponed. Accounting policies ensure that financial profits are presented in a manner that is consistent with the conservatism principle, which, however, can be challenging for properly measuring value and implementing value management tools. One way to shorten the time between costs and revenues is to divide investment into separate stages. This approach allows part of the implemented investment project to be settled and balanced earlier. However, this solution is used for large investments consisting of many separate future real estates. This does not change the fact that, for each stage of the investment, there is still a deferral in the time of settlement enabling sustainable monitoring of the enterprise's processes. g g p p The implementation of the idea of value-based management assumes ongoing monitoring of the expenses incurred by the enterprise and the value that has been generated as a result of the company's operations on the market. The implementation of such a management system could have a positive impact on increasing the level of economic security in the real estate sector, by providing decision- makers with instruments to measure the economic situation of enterprises. The need to develop security mechanisms is confirmed by bankruptcy situations on the real estate market. Such situations do not happen often, but even an individual case may result in significant losses that affect many market stakeholders, such as future property owners, subcontractors or entities providing resources. Under Polish conditions, the bankruptcy of a company in 2014 resulted in financial irregularities of over PLN 770 million (Dzyuma-Zaremba, 2015), which confirms the need to improve the system of control over management processes in entities of the real estate sector. g p The aim of this work was to analyze selected conditions of value-based management in a real estate development company and to propose a method for measuring the economic situation of the company using the concept of management through value. The impact of the applied revenue and cost accounting principles on value measurement was analyzed. The analysis is conducted in terms of providing measurement methods that will be consistent with the logic of value creation, which will ensure that measurement results can be used in management systems, including primarily incentive systems that are the basis for building relationships between owners and managers. The authors have developed examples of adjustments that can be useful in measuring values using value-based management. The developed approach to monitoring the economic situation of the enterprise was presented on the real values of two investments of a Polish enterprise implementing a project of a building complex including, in the first case, 189 apartments and, in the second case, 5 commercial estates. 1. Introduction Striving to build the value of invested capital remains the goal of modern enterprises in all kinds of industries. Many years of research on value management systems have enabled he development of numerous tools supporting the measurement and growth of enterprises to build shareholder value. However, their application requires the adaptation of measures which are aimed at taking into account the specificity of enterprises in various sectors of the economy in which they are applied. On the one hand, the use of this type of procedures is to provide a measurement of value appropriate to the individual business logic; on the other, it should not go against disturb the principles of objectivity and adequacy of measurement. q y The real estate sector is among the sectors that could benefit from the mentioned experiences from value management research. The functioning of real estate enterprises is naturally based on project- oriented activities. However, the project-based approach has to be connected, in this case, with REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 48 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 48 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 48 www.degruyter.com/view/j/remav It is worth emphasizing that specific sectors of the economy create their own specific approach to calculating VBM measures, for example Geyser and Liebenberg (2003) for agribusinesses or Kim (2006) for healthcare companies. ( ) p Research on the use of VBM measures was carried out among companies in the real estate sector. Nappi-Choulet (2009) investigated the association between economic value added (EVA) and market value added (MVA) as proxies for the value generated by French listed companies and the proportion of real estate in their asset portfolio. The empirical results show that an increase in the proportion of real estate assets (over total assets) is negatively associated with EVA, but only for firms in service industries exhibiting low real estate intensity. The obtained results can be considered surprising. An increase in the value of assets should be treated as an investment by investors and effectively cause an increase in value. The reasons for the phenomenon observed by Nappi-Choulet can be seen in the accounting principles used in the real estate area, which the authors themselves emphasize. g p p p Moreover, Parle et al. (2017) examine the economic performance of real estate property developers listed on the Australian Stock Exchange using a performance measure construct derived from the EVA model. Researchers have empirically demonstrated that most property companies did not generate sufficient annual profit to cover their cost of capital. Similarly, they see the reasons for these conclusions in the EVA measurement used. They point to the problems that can be caused by the accounting principles used by developers in measuring value, in particular AASB 116 Property, Plant & Equipment and AASB 102 Inventories. Further, Parle recommends that EVA measurement principles be revised, and assets undervalued in the financial statements be revised (further adjustment to the EVA model is recommended to allow for any revaluation of real estate implemented in the other comprehensive income sections of the financial statements). This modification to EVA would provide a more appropriate metric for assessing the performance efficiency of property firms. Unfortunately, the authors did not indicate detailed rules for their application. Similarly, applications in the field of measuring VBM measures among companies operating in the real estate industry were published in previous works by Ooi and Liow (2002) researching Singapore Property Companies. publications on EVA, which appeared in the years 1994–2007 in key corporate finance and management magazines. publications on EVA, which appeared in the years 1994–2007 in key corporate finance and management magazines. g g Undoubtedly, the key to the use of indicators is their mathematical and repeatedly empirically verified relationship with market value (MV) and market value added (MVA). Income valuation based on discounting future EVAs is synonymous with valuation based on discounting future cash flows (Velez-Pareja & Tham, 2003, Stancu, 2017). Thus, both approaches can be used to estimate MV or MVA. Nevertheless, the discussion about the empirical relationship between EVA and MVA seems endless, EVA-type measures have had a key impact on managing the performance appraisal of enterprises. Lehn and Makhija (1997) proved that EVA has the strongest correlation with rate on return among all measures of business activity. Similarly, Stewart (1994), de Medeiros (2005), Worthington and West (2004) showed EVA to better explain the behavior of share prices than other economic measures and that the rate of return on shares is better correlated with EVA than with profit or FOCF (Free Operation Cash Flow). In 2019, Obaidat (2019), based on empirical research conducted among companies listed on Amman Stock Exchange (ASE), recognized EVA as an enhancement tool to the existing traditional accounting performance measures, not as a substitute for them. However, there are a lot of studies indicating VBM measures not to explain the rate of return on shares, including EVA. Fernandez (2003) performed correlation analysis between EVA and MVA on 582 American companies. The study showed that a negative correlation was identified in 210 cases. p y g Measuring values itself and using EVA-type measures brings many conceptual problems. There is a consensus in the literature on the subject that data contained in financial statements are insufficient to measure value or even that the provisions of accounting law do not allow it. Stewart (1994) has suggested 164 possible adjustments to arrive at the adjusted date from financial statements for the calculation of EVA. The most popular areas of adjustment relate to investments, accounting for goodwill, asset revaluations, deferred income taxes, and pension (Worthington and West, 2004). However, Sharma (2010,) based on studies on value measures, claims that there is a need to harmonize some adjustments that are required to be incorporated in the calculation of EVA. 2. Literature review The beginnings of value-based management date back to the 1980s. From that moment, the ability to build value becomes the perspective of assessing the effectiveness of enterprises, which has been continuously dominant to this day (Kowalski, 2012). The point of view of the owner, who expects to multiply his assets by investing, takes on crucial importance in assessing economic efficiency (Rappaport, 1995). Capital and its cost are becoming a key economic category, accounting profits are beginning to be compared with the cost of capital invested (Copeland, 2000). Capital markets and investors are starting to make use of conclusions drawn in the groundbreaking work of Modiglianini and Miller (1958) and Sharpe (1964), on the cost of capital and assessing the ability of companies to build value when looking for investment opportunities. The ease of moving capital causes its constant migration (Kowalski & Biliński, 2018). As a result, companies compete globally for capital and investors. Value becomes the basis for the assessment of management board activities by owners and corporate governance (Irala, 2005), and value management measures are permanently inscribed in managers' incentive systems (Biddle, 1999). g y ( ) Years of research and practical verification have led to the definition of measures for the ongoing assessment of the ability of enterprises to build value. Myers (1996) even writes about the "war of measures" associated with the flourishing trend of value-based management, whose participants try to assure that the measure they propose creates value and is the best to measure the achievements of company. Concepts such as economic value added (EVA) or return on invested capital (ROIC) (Stewart, 1991) permanently become an element of measuring performance. The measures themselves have been the subject of several studies. For example, Sharma et al. (2010) identified and analyzed 112 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 49 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 49 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav Among their conclusions, they stated that by measuring value based on data contained in financial statements, we can understand the true economic performance of real estate. The necessity to look for methods to improve value measurement for real estate companies therefore seems necessary. REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 50 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 50 www.degruyter.com/view/j/remav 3. Methodological approach for value-based management in real estate sector The aim of the article is to present and discuss proposals for measuring value in a real estate enterprise which conducts development activities. The proposed method is to eliminate or reduce the difficulties in measuring the value associated with the impact of the conservatism principle on the shape of financial statements for this type of enterprise. The concept of economic value added as the most popular VBM measure was adopted as the starting point. Then, taking into account the conditions of accounting law and regulations affecting the shape of developer reports, as well as discussions with the management boards of these enterprises, corrections were proposed to individual components of economic added value. The presented solutions result from actual experience and many years of value measurements in a large capital group on the Warsaw Stock Exchange dealing in development activities. The adjustments presented in this work are the result of discussions regarding measuring the value held with both, the management of a company and representatives of shareholders. The basic measurement of economic value added is made using the equation (Steward 1994): 𝐸𝑉𝐴ൌ𝑁𝑂𝑃𝐴𝑇െ 𝐼𝐶 ൈ𝑊𝐴𝐶𝐶 (1) (1) 𝐸𝑉𝐴ൌ𝑁𝑂𝑃𝐴𝑇െ 𝐼𝐶 ൈ𝑊𝐴𝐶𝐶 (1) or EVA = NOPAT- CoC (2) or 𝐸𝑉𝐴ൌሺ𝑅𝑂𝐼𝐶െ𝑊𝐴𝐶𝐶ሻൈ𝐼𝐶 (3) or (2) or 𝐸𝑉𝐴ൌሺ𝑅𝑂𝐼𝐶െ𝑊𝐴𝐶𝐶ሻൈ𝐼𝐶 (3) where: EVA – Economic Value Added, NOPAT – Net Operating Profit After Tax, WACC – Weight Average Cost of Capital, CoC – Cost of capital = IC x WACC, ROIC – Return On Invested Capital, IC – Invested Capital. – Cost of capital = IC x WACC, – Return On Invested Capital, Risk associated with the possibility of changing future results, for example resulting from uncertainty about the prices obtained or meeting the project deadlines, is, in principle, reflected in the EVA measurement itself. In the EVA measurement, actual profits are compared to expected profits, and the expected profits depend directly on the risk of the business and are reflected in the cost of capital. p Steward and others point to the need to adjust the measurement by making adjustments to the financial statements at both the level of result measurement and the capital employed. The full formula should therefore read as: 𝐸𝑉𝐴ൌሺ𝑁𝑂𝑃𝐴𝑇 േ𝐴𝐷𝐽ሻെሺ𝐼𝐶 േ𝐴𝐷𝐽ሻൈ𝑊𝐴𝐶𝐶 േ𝐴𝐷𝐽 (4) (4) 𝐸𝑉𝐴ൌ𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑎𝑙 𝐸𝑉𝐴 േ 𝑙𝑎𝑛𝑑 𝑏𝑎𝑛𝑘 𝐸𝑉𝐴 േ𝑜𝑛𝑒െ𝑜𝑓𝑓 𝐸𝑉𝐴 𝐸𝑉𝐴ൌሺ𝑅𝑂𝐼𝐶𝑟െ𝑊𝐴𝐶𝐶ሻ ൈ𝐼𝐶 𝐸𝑉𝐴ൌሺ𝑅𝑂𝐼𝐶𝑟െ𝑊𝐴𝐶𝐶ሻ ൈ𝐼𝐶 (5) 𝐸𝑉𝐴ൌሺ𝑅𝑂𝐼𝐶𝑟െ𝑊𝐴𝐶𝐶ሻ ൈ𝐼𝐶 (5) Parle et al. further indicate that the adjustment proposed is to accommodate the revaluation amount for the real estate assets by adding this to the NOPAT figure. This modification to the EVA would provide a more appropriate metric for assessing the performance efficiency of property firms. In this research we propose that the revaluation adjustment should not only concern the sphere of the result, but also exclude the impact of revaluations on the capital employed. The proposed correction could thus take the form of: 𝐸𝑉𝐴ൌሺ𝑁𝑂𝑃𝐴𝑇 േ𝐴𝐷𝐽𝑟ሻെሺ𝐼𝐶 േ𝐴𝐷𝐽𝑟ሻ ൈ𝑊𝐴𝐶𝐶 (6) (6) where ADJr means excluding from the EVA account the impact of the revaluation amount for the real estate assets, both regarding the current result (NOPAT) and cumulative impact on the balance sheet and invested capital. where ADJr means excluding from the EVA account the impact of the revaluation amount for the real estate assets, both regarding the current result (NOPAT) and cumulative impact on the balance sheet and invested capital. 1 The introduction in 2009 of IFRIC 15 (Agreements for the Construction of Real Estate) by the International Financial Reporting Interpretations Committee (IFRIC 15 Real Estate Contracts, IFRIC 15) indicated guidelines as to whether a given contract implemented by a developer falls under the scope of IAS 11 Construction Contracts or IAS 18 Revenue. This distinction is of fundamental importance for the presentation of developers’ results in financial statements and the moment of recognizing revenues from signed contracts. If IAS 11 applies, revenue is recognized on a percentage-of-completion basis provided that reliable estimates of construction progress and future costs can be made. In turn, if the agreement, in which the ability of buyers As indicated in the literature, development activities may require the application of standard corrections to financial statements. The application of corrections resulting from good will accounting or deferred tax exemptions are necessary, and can be implemented according to procedures widely described in the literature (Stewart, 1994; Worthington & West, 2004; Kyriazis &Anastassis, 2007). Regardless of different approaches described in the literature mentioned above, additional corrections resulting from the specifics of real estate development activities and accounting solutions used for developers are introduced in this study. We propose to make the following corrections: p y p p g 1) Exclusion of the impact of asset revaluations. p y p p g 1) Exclusion of the impact of asset revaluations. 2) Introduction of an investment account for short-term projects, e.g. housing, including activation and settlement during the negative EVA of the investment phase and advance payment during the EVA of the project together with its progress. 2) Introduction of an investment account for short-term projects, e.g. housing, including activation and settlement during the negative EVA of the investment phase and advance payment during the EVA of the project together with its progress. 3) Introduction of an investment account for long-term projects, e.g. commercial projects (office, commercial, hotel) including activation and settlement in time of negative EVA investments. 3) Introduction of an investment account for long-term projects, e.g. commercial projects (office, commercial, hotel) including activation and settlement in time of negative EVA investments. 4) Exclusion of the impact of interest on foreign capital in the valuation of assets and the cost of goods sold. 4) Exclusion of the impact of interest on foreign capital in the valuation of assets and the cost of goods sold. where: ADJ – adjustments regarding financial statements. We propose dividing EVA into three components when measuring the value, i.e.: operational EVA, land bank EVA and one-off EVA. The character of real estate development activity means that the reported value drivers can rely on fluctuations. For example, the purchase of land for new investments can abruptly increase the capital employed. Moreover, this land may, for a long time, burden the measurement of value at the expense of capital without generating any result or even burdening the result with its maintenance costs. The land may require preparation or concern future investment plans. On the other hand, the developer result may include one-off events related to the sale of an investment, e.g. an investment project (office or commercial) or a land bank or part of it. Observations and experiments carried out show that it is worth separating the measurement of value resulting from current operations from the cost of capital generated by the land bank as well as the value realized on one-off transactions usually related to the sale of assets. Therefore, we suggest that reporting for the real estate company could be carried out according to the following formula: 𝐸𝑉𝐴ൌ𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑎𝑙 𝐸𝑉𝐴 േ 𝑙𝑎𝑛𝑑 𝑏𝑎𝑛𝑘 𝐸𝑉𝐴 േ𝑜𝑛𝑒െ𝑜𝑓𝑓 𝐸𝑉𝐴 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 51 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 51 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav 3.1. Exclusion of the impact of asset revaluations The first proposed correction realizes the postulate that the calculation of the value should approximate the cash flow approach. The accounting principles adopted by most developers mean that most of them measure assets (land, owned commercial real estate) to fair value at each moment of the balance sheet valuation. In the financial statements of most developers maintained according to IFRS, we find items such as "the result of revaluation of real estate value". The impact of these items on the financial result can be significant. Even though the presentation of the value of real estate at fair value should be considered as the right approach, and the result of the revaluation carries a high cognitive value for the recipient of the financial statements, providing information about the impact of external macroeconomic factors connected with the real estate market on the situation of the enterprise, this presentation may, however, hamper the assessment of operational activity and self value measurement. This aspect is probably most often emphasized in the literature on the subject. Ooi and Liow (2002) indicate that fair value measurement is the reason why, in most cases, the developers surveyed in the market reached negative EVA values. Parle et al. (2017) propose the introduction of the so-called ROICr in the EVA calculation formula, where the lower case “r” is intended to signify the inclusion of revaluation amounts. The modified model would thus appear in the following terms: We propose, for the purposes of measuring the value, the introduction of appropriate adjustments for: p p g pp p j 1) the withdrawal of negative EVAs in the investment phase [1], 2) recognizing the EVA of the project, since its commencement by introducing advances on planned EVA of the project [2]. recognizing the EVA of the project, since its commencement by introducing advances on th planned EVA of the project [2]. The proposed correction will show the economic value generated by the project proportionally throughout the implementation period. The correction will be effective from the month or quarter in which, according to the provisions contained in the contract, it begins work related to the implementation of the project, to the month or quarter in which the investment was settled, i.e. the right to premises was issued to the last buyers. The correction will concern [1] the withdrawal of negative results in the investment phase and the cost of capital employed in the investment phase. Then, the withdrawal of negative EVAs will be accounted for during the periods of sale of the premises, in proportion to the percentage of the premises transferred. At the same time, we suggest introducing an additional modification [2] to recognize current EVA advances from the project since its commencement. The basis for determining the amount of the advance should be the planned NPV of the project. Subsequently, the adjustment would be withdrawn at the project sales stage in proportion to the percent of premises sold and accounting profits recognized. Thus, in the sales phase, project profits would be adjusted by [1] activated negative EVA in the investment phase and [2] advances on the NPV of the project recognized in the investment phase. It should be noted that all adjustments made are charged to invested capital. In turn, their settlement causes their decrease from capital. As a result, the proposed correction mechanism does not change the NPV of the project, but only changes the distribution of residual profits obtained over time. p Applying the correction should reduce the risk of unexpected events in the project. For this reason, we suggest that the basis for calculating the correction are the financial plans approved by the institution financing the project. real estate occurs at the time of the acceptance of the premises by the buyer and after the seller has performed all contractual services to which the developer was contractually obliged as part of the sale (Dylag and Kucharczyk, 2011). As a result, the developer recognizes revenues from the sale of constructed premises at the moment: real estate occurs at the time of the acceptance of the premises by the buyer and after the seller has performed all contractual services to which the developer was contractually obliged as part of the sale (Dylag and Kucharczyk, 2011). As a result, the developer recognizes revenues from the sale of constructed premises at the moment: p 1) obtaining permission to use the object, p 1) obtaining permission to use the object, p 1) obtaining permission to use the object, 2) obtaining payments from a customer in the amount usually less than 80% of the value of the premises sold, 2) obtaining payments from a customer in the amount usually less than 80% of the value of the premises sold, 3) handing over the premises for use to the buyer. 3) handing over the premises for use to the buyer. Until revenue is recognized in the income statement, the amounts of advances paid by buyers are recognized in the financial statements as liabilities. Until revenue is recognized in the income statement, the amounts of advances paid by buyers are recognized in the financial statements as liabilities. Such accounting policies mean that value measurement based on data from financial statements may not properly reflect the logic of creating value in a development enterprise. Measurement made on the basis of financial statements would show the destruction of value for an example of a housing project, since the acquisition of land for the project throughout the entire investment phase. The value would not be revealed until the project is completed and the premises are owned by the buyers. In accordance with the superior conservatism principle for financial statements, activities related to the organization of the project, obtaining permits for its implementation, or project implementation remain not reflected in the financial profits, although they undoubtedly have the potential of value. p g y y p The purpose of the proposed adjustment is to take the value of effects from implemented short- term investments, for example housing projects into account when measuring. to influence the project is limited, it is, according to IAS / IFRS, a contract for the sale of goods and is subject to the regulations of IAS 18. In principle, revenues in this case will be recognized in the developer's income statement after the risk and control related to the goods are transferred to the buyer. 3.2. Investment account for short-term projects The practice of applying the regulations introduced by the accounting law indicates1 that recognition of revenue from the sale of goods related to the transfer of risk and benefits arising from the sale of REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 52 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav 𝐸𝑉𝐴𝑗ൌሺ𝑁𝑂𝑃𝐴𝑇𝑗ሻെቌ𝐼𝐶𝑗൅ ෍𝐴𝐷𝐽௞ ௡௘௚ா௏஺ ௞ୀ௝ିଵ ௞ୀ଴ ൅ ෍𝐴𝐷𝐽௞ ௔௣ா௏஺ ௞ୀ௝ିଵ ௞ୀ଴ ቍൈ𝑊𝐴𝐶𝐶൅𝐴𝐷𝐽௝ ௡௘௚ா௏஺൅ 𝐴𝐷𝐽௝ ௔௣ா௏஺ (7) (7) where: – investment period, j – investment period, ADJknegEVA – correction made for activating negative EVA in the investment phase and its subsequent settlement in the sales phase, ADJkapEVA – correction made for recognizing advanced payments on EVA in the investment phase and its subsequent settlement in the sales phase, while it should be noted that: j p ADJknegEVA – correction made for activating negative EVA in the investment phase and its subsequent settlement in the sales phase, ADJkapEVA – correction made for recognizing advanced payments on EVA in the investment h d it b t ttl t i th l h j p ADJknegEVA – correction made for activating negative EVA in the investment phase and its subsequent settlement in the sales phase, ADJkapEVA – correction made for recognizing advanced payments on EVA in the investment phase and its subsequent settlement in the sales phase, while it should be noted that: 1) ADJknegEVA and ADJkapEVA in the investment phase are positive values and increase the calculation based on data from financial statements, 1) ADJknegEVA and ADJkapEVA in the investment phase are positive values and increase the calculation based on data from financial statements, 2) ADJknegEVA and ADJkapEVA in the sales phase are negative values and reduce the calculation based on data from the financial statements, 2) ADJknegEVA and ADJkapEVA in the sales phase are negative values and reduce the calculation based on data from the financial statements, 3) in the investment phase 𝐴𝐷𝐽௝ ௡௘௚ா௏஺ൌሺ𝑁𝑂𝑃𝐴𝑇𝑗ሻ൅ሺ𝐼𝐶𝑗൅ ∑ 𝐴𝐷𝐽௞ ௡௘௚ா௏஺ ௞ୀ௝ିଵ ௞ୀ଴ ൅ ∑ 𝐴𝐷𝐽௞ ௔௣ா௏஺ሻ ൈ𝑊𝐴𝐶𝐶 ௞ୀ௝ିଵ ௞ୀ଴ , therefore 𝐸𝑉𝐴𝑗ൌ 𝐴𝐷𝐽௝ ௔௣ா௏஺. 3) in the investment phase 𝐴𝐷𝐽௝ ௡௘௚ா௏஺ൌሺ𝑁𝑂𝑃𝐴𝑇𝑗ሻ൅ሺ𝐼𝐶𝑗൅ ∑ 𝐴𝐷𝐽௞ ௡௘௚ா௏஺ ௞ୀ௝ିଵ ௞ୀ଴ ൅ ∑ 𝐴𝐷𝐽௞ ௔௣ா௏஺ሻ ൈ𝑊𝐴𝐶𝐶 ௞ୀ௝ିଵ ௞ୀ଴ , therefore 𝐸𝑉𝐴𝑗ൌ 𝐴𝐷𝐽௝ ௔௣ா௏஺. 3.4. Exclusion of the inflow of interest on foreign capital An additional challenge from the point of view of measuring value are the solutions arising from accounting law in the scope of including interest expenses in the costs. In measuring the value, financial costs are replaced by the cost of capital employed. In order not to include the cost of financing twice, it seems reasonable to exclude these costs from the operating result and omit them from the measurement of the cost of capital included in inventory interest. 3.3. Investment account for long-term projects Long-term projects regarding the construction of such facilities as shopping centers, offices and hotels are burdened with similar recording problems that make measuring the value difficult. As in the case of housing investments, the investment phase means negative results and capital involvement. Then, during the project operation phase, the developer obtains revenues from the project operation and possibly the result on the sale of the object to the financial investor. For this type of projects, a correction analogous to that presented for housing projects, the withdrawal of negative EVAs in the investment phase and their subsequent settlement in the assumed period of business operation to be applied in the measurement of value. If the project is sold, the unadjusted value of the correction increases the investment cost. The total value of the advance should constitute a certain percent of the NPV of the project, which would additionally hedge the risk of adverse deviations from the plan. The adoption of such procedures for estimating the total value is intended to protect the owners' interests against excessive optimism of managers when assessing projects and their value potential. Therefore, the formula for calculating EVA is: REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 53 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav 4.1. Example of short-term project – housing investment A housing project consisting of 189 apartments with a total usable area of 8,870 m2 was chosen as an example. The duration of the project implementation was 2.5 years, i.e. 10 quarters, with the investment phase lasting 6 quarters. The last period of the project was the exploitation phase, in which premises were being sold. Table 1 presents the financial data of the project presented in the form as in financial statements. During the investment phase, the company increases the assets involved in the project to some point along with an increase in external debt that finances them. At the same time, since the third quarter, the level of debt has been stabilizing, and the company is implementing the project with funds paid by future buyers of the premises. In the investment phase, the company presents losses at the level of the operating result, does not generate revenues, and in accordance with the provisions of accounting law, not all costs incurred can be activated. With the start of the exploitation phase, operating income and profit appear in operating income. Table 2 presents the measurement of value using the EVA indicator based on financial data from the reports presented in Table 1. WACC used by the analyzed company is the result of a calculation, which takes into account the financing structure, cost of equity estimated on the basis of the CAPM model and the cost of debt. In this case, the weighted average cost of capital is approved by the supervisory board and remains unchanged throughout the year. The calculations show that, in the REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 54 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 54 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 54 Table 2 Value measurement based on financial statements Value measurement based on financial statements Indicator Q0 Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 EVA [m PLN] -0.4 -0.6 -0.8 -1.0 -1.1 7.2 2.7 0.2 -0.1 -0.4 NOPAT [m PLN] -0.3 -0.4 -0.4 -0.4 -0.4 7.9 2.9 0.3 0.0 -0.3 COC [m PLN] -0.1 -0.2 -0.4 -0.5 -0.7 -0.8 -0.2 -0.1 -0.1 -0.1 IC [m PLN] 8.2 14.9 23.9 35.4 43.6 50.8 16.2 5.8 4.3 8.2 Source: own study. Source: own study. In Table 3 the EVA calculation after making the proposed correction is presented. Negative EVAs incurred in the investment phase are activated, and additionally, an advance on the planned value to be worked out as a result of the entire project is recognized. Both adjustments are settled in the exploitation phase in proportion to the sales of premises and revenues generated. The EVA ADJ values obtained show similar values in all quarters of the implemented project; they show significantly smaller fluctuations in relation to the EVA parameter calculated on the basis of financial data. Table 3 initial phase, the company destroys the value, achieving negative EVA values. The company engages capital and reports negative earnings before interest and taxes (EBIT). The increase in value is only reported in the exploitation phase when the company begins to realize revenues and reduces the capital employed. initial phase, the company destroys the value, achieving negative EVA values. The company engages capital and reports negative earnings before interest and taxes (EBIT). The increase in value is only reported in the exploitation phase when the company begins to realize revenues and reduces the capital employed. Table 1 Table 1 Characteristics of the housing project Indicator [m PLN] Q0 Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Revenues 0.0 0.0 0.0 0.0 0.0 0.0 35.3 13.4 1.7 0.0 EBIT 0.0 -0.3 -0.4 -0.4 -0.4 -0.4 7.9 2.9 0.3 0.0 Assets 8.2 14.9 23.9 35.4 43.6 50.8 16.2 5.8 4.3 4.3 Debt 0.0 3.8 10.5 13.9 14.9 9.2 0.0 0.0 0.0 0.0 Source: own study. Table 2 Value measurement based on financial statements Indicator Q0 Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 EVA [m PLN] -0.4 -0.6 -0.8 -1.0 -1.1 7.2 2.7 0.2 -0.1 -0.4 NOPAT [m PLN] -0.3 -0.4 -0.4 -0.4 -0.4 7.9 2.9 0.3 0.0 -0.3 COC [m PLN] -0.1 -0.2 -0.4 -0.5 -0.7 -0.8 -0.2 -0.1 -0.1 -0.1 IC [m PLN] 8.2 14.9 23.9 35.4 43.6 50.8 16.2 5.8 4.3 8.2 Source: own study. Table 1 Characteristics of the housing project Indicator [m PLN] Q0 Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Revenues 0.0 0.0 0.0 0.0 0.0 0.0 35.3 13.4 1.7 0.0 EBIT 0.0 -0.3 -0.4 -0.4 -0.4 -0.4 7.9 2.9 0.3 0.0 Assets 8.2 14.9 23.9 35.4 43.6 50.8 16.2 5.8 4.3 4.3 Debt 0.0 3.8 10.5 13.9 14.9 9.2 0.0 0.0 0.0 0.0 Source: own study. Characteristics of the housing project Source: own study. Table 3 Table 3 Measuring the value after taking into account the proposed corrections Indicator Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 EVA ADJ [mPLN] 0.9 0.9 0.9 0.9 0.9 1.0 0.4 -0.1 -0.1 NOPAT [mPLN] -0.3 -0.4 -0.4 -0.4 -0.4 7.9 2.9 0.3 0.0 COC [mPLN] -0.1 -0.2 -0.4 -0.6 -0.8 -0.9 -0.3 -0.1 -0.1 IC ADJ [mPLN] 8.2 16.3 26.8 40.0 50.2 59.4 18.8 6.1 4.3 IC [mPLN] 8.2 14.9 23.9 35.4 43.6 50.8 16.2 5.8 4.3 ADJ [mPLN] 0.0 1.3 2.9 4.6 6.5 8.6 2.6 0.3 0.0 ADJ [mPLN] 1.3 1.6 1.7 1.9 2.1 -6.0 -2.3 -0.3 0.0 ADJ negative [mPLN] 0.4 0.7 0.8 1.0 1.2 -2.9 -1.1 -0.1 0.0 ADJ advances payment [mPLN] 0.9 0.9 0.9 0.9 0.9 -3.2 -1.2 -0.2 0.0 Ta Measuring the value after taking into account the proposed corrections Comparison of results Indicator [mPLN] Total Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 EVA 6.1 -0.4 -0.6 -0.8 -1.0 -1.1 7.2 2.7 0.2 -0.1 EVA ADJ 5.7 0.9 0.9 0.9 0.9 0.9 1.0 0.4 -0.1 -0.1 Difference -1.3 -1.5 -1.7 -1.9 -2.0 6.1 2.3 0.3 0.0 PV difference 0.0 -1.3 -1.5 -1.6 -1.7 -1.8 5.6 2.1 0.3 0.0 Source: own study. Source: own study. Source: own study. The results of measuring value on the basis of financial statements were presented graphically in Figure 1, taking into account the adjustment regarding the activation of negative EVA in the investment phase and as well as both the adjustment regarding the activation of negative EVA in the investment phase and the adjustment being an advance on the value generated as a result of the project. The correction regarding the activation of negative EVA in the investment phase means that value destruction is not reported in the investment phase, but there are still significant fluctuations in the reported value in the sales phase of the premises. It is only by applying both corrections that one can reduce fluctuations in value and reporting value increases more or less consistently since the beginning of the project. g g p j Fig. 1. A comparison of the effects of introducing the proposed corrections. Source: own study. ‐ 2,0 ‐ 1,0  0,0  1,0  2,0  3,0  4,0  5,0  6,0  7,0  8,0 Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 EVA EVA ADJ negative EVA ADJ negative + advances payment g g p j Fig. 1. A comparison of the effects of introducing the proposed corrections. Source: own study. 4.2. Example of long-term project – commercial investment ‐ 2,0 ‐ 1,0  0,0  1,0  2,0  3,0  4,0  5,0  6,0  7,0  8,0 Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 EVA EVA ADJ negative EVA ADJ negative + advances payment Q3 Q2 Q4 Q5 Fig. 1. A comparison of the effects of introducing the proposed corrections. Source: own study. 4.2. Example of long-term project – commercial investment Fig. 1. A comparison of the effects of introducing the proposed corrections. Source: own study 4.2. Example of long-term project – commercial investment 4.2. Example of long-term project – commercial investment The second presented example concerns the implementation of an investment involving the construction of an office building. Table 5 presents key financial data. The investment phase lasted 4 quarters. Source: own study. Source: own study. Table 4 lists the EVA values obtained - before adjustments and EVA ADJ including the proposed adjustments and differences between them. Note that the current value of both parameters is identical, i.e. the present value of the difference between EVA and EVA ADJ is equal to zero. Therefore, the proposed correction does not affect the assessment of investment effectiveness; it changes the presentation in time in order to better reflect the economic sense when assessing the ability to build value by a company. REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 55 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 55 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav During it, the company recorded negative results and engaged capital. At the same time, the first revaluation of the investment value was recorded in Q4, which resulted in the realization of the financial profit. Similar verification of the book value of the building, both increasing and decreasing, took place in the following quarters. Then, from the Q5 quarter, the office building operation phase is implemented - its gradual commercialization, which results in positive operating results. In the 12th quarter, the office building is sold to a financial investor. The sale is carried out at book value, and therefore the company does not recognize the result on this account. Table 5 presents key financial data. Table 6 presents the results of economic value added calculation based on accounting data. The investment phase is reported as value destruction. Property revaluations result in fluctuations in economic value added. REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 56 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300 5289 vol. 28, no. 4, 2020 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav Table 6 Table 7 presents the effect of calculating the economic value added using the proposed adjustments. Negative results and the cost of capital in the investment phase have been activated and are subject to settlement in subsequent periods. Accounting revaluations of real estate values do not affect the measurement of value; they are excluded from the result and, at the same time, they correct the capital employed. The result on the sale of real estate appears in the last quarter, when the sale is actually carried out and the invested assets are converted into cash. A comparison of the obtained results is presented in Table 8. Table 7 Table 7 Measuring the value after taking into account the proposed corrections Indicator Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 Q12 EVA [mPLN] 0.0 0.0 0.0 0.0 -0.3 0.0 0.3 1.1 0.3 0.3 0.3 3.6 NOPAT [mPLN] -0.4 -0.4 -0.3 4.6 0.1 0.9 0.7 0.4 0.7 0.7 0.7 0.9 COC [mPLN] -0.1 -0.1 -0.1 -0.2 -0.3 -0.3 -0.3 -0.3 -0.3 -0.3 -0.4 -0.4 IC [mPLN] 5.2 6.5 9.9 13.6 17.7 19.3 23.3 22.7 21.0 21.8 23.5 24.6 IC balance sheet [mPLN] 5.2 6.1 8.9 12.2 20.7 22.3 26.9 26.4 23.7 24.6 26.4 27.7 ADJ [mPLN] 0.4 1.0 1.4 -2.9 -3.0 -3.6 -3.7 -2.7 -2.8 -2.9 -3.0 ADJ [mPLN] 0.4 0.5 0.5 -4.4 -0.1 -0.6 -0.1 1.0 -0.1 -0.1 -0.1 3.0 ADJ negative [mPLN] 0.4 0.5 0.5 0.2 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.9 ADJ revaluation [mPLN] -4.5 0.0 -0.5 0.0 1.1 0.0 0.0 0.0 3.9 Source: own study. Measuring the value after taking into account the proposed corrections Source: own study. Table 5 Characteristics of the commercial project Indicator [m PLN] Q0 Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 Q12 EBIT 0.0 -0.4 -0.4 -0.3 4.6 0.1 0.9 0.7 0.4 0.7 0.7 0.7 0.9 including asset revaluation 0.0 0.0 0.0 0.0 -4.5 0.0 -0.5 0.0 1.1 0.0 0.0 0.0 0.0 Assets 5.2 6.1 8.9 12.2 20.7 22.3 26.9 26.4 23.7 24.6 26.4 27.7 0.0 Debt 0.0 1.1 4.2 7.6 12.4 14.1 18.0 17.6 16.0 16.3 16.7 17.9 0.0 Source: own study. Table 6 Value measurement based on financial statements Indicator Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 Q12 EVA [mPLN] -0.4 -0.5 -0.5 4.4 -0.2 0.5 0.3 0.0 0.4 0.3 0.3 0.5 NOPAT [mPLN] -0.4 -0.4 -0.3 4.6 0.1 0.9 0.7 0.4 0.7 0.7 0.7 0.9 COC [mPLN] -0.1 -0.1 -0.1 -0.2 -0.3 -0.3 -0.4 -0.4 -0.4 -0.4 -0.4 -0.4 IC [mPLN] 5.2 6.1 8.9 12.2 20.7 22.3 26.9 26.4 23.7 24.6 26.4 27.7 Source: own study. Table 5 Characteristics of the commercial project 4.3 Application of the model in a Polish real estate development company The last element of the presented case study is the presentation of the financial results of the selected real estate development company from the perspective of ten years of experience in measuring and reporting value. The presented data relate to the historical data of the capital group listed on the REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 57 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 57 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav Polish Stock Exchange. The company uses management based on value-based management, and therefore measures and evaluates the results from this point of view. The measurement is made taking into account the corrections indicated in the previous paragraphs. Table 9 presents the results of the formal financial statements. We are observing the gradual development of the company and the increasing scale of operations. After 4 initial years of negative operating results and increasing the state of assets, the company began to gradually increase the scale of operations. During the last year of activity, in connection with the use of market position, a significant increase in revenues while maintaining the level of costs is observed. As a result, we can see a 67% increase in revenues and a doubling of operating results without an increase in the assets involved. Table 8 Comparison of results Indicator [m PLN] Total Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 Q12 EVA 5.2 -0.4 -0.5 -0.5 4.4 -0.2 0.5 0.3 0.0 0.4 0.3 0.3 0.5 EVA ADJ 5.5 0.0 0.0 0.0 0.0 -0.3 0.0 0.3 1.1 0.3 0.3 0.3 3.6 difference -0.4 -0.5 -0.5 4.4 0.1 0.5 0.1 -1.0 0.1 0.1 0.1 -3.1 PV difference 0.0 -0.4 -0.5 -0.4 4.1 0.1 0.5 0.0 -0.9 0.1 0.1 0.1 -2.6 Source: own study. Table 9 Real estate development company - financial data Indicator [kPLN] 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 Net Sales 1 028.0 614.2 279.9 200.3 129.7 94.0 30.0 51.1 17.5 7.1 Operating Profit 213.4 82.1 35.6 25.0 8.3 0.5 -6.6 -3.9 -1.0 -2.3 Total Assets 1 811.8 1 946.1 1 695.9 1 327.5 1 153.4 992.6 818.7 799.8 613.2 295.9 Total Financial Debt 731.5 753.3 708.1 508.2 402.3 212.7 135.3 270.4 242.1 119.1 Net Earnings - TTM 165.7 48.2 53.2 25.2 20.9 9.8 -9.5 0.7 1.1 -33.8 Operating Margin - TTM 20.8% 13.4% 12.7% 12.5% 6.4% 0.6% -21.9% -7.7% -5.7% -31.9% ROA 9.1% 2.5% 3.1% 1.9% 1.8% 1.0% -1.2% 0.1% 0.2% -11.4% Source: own study. Table 10 presents the results in the value reporting system. It should be noted that the measures of l t d b k d i t ti ti iti l ti t th b k f d ti Table 8 Table 8 Comparison of results Indicator [m PLN] Total Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 Q12 EVA 5.2 -0.4 -0.5 -0.5 4.4 -0.2 0.5 0.3 0.0 0.4 0.3 0.3 0.5 EVA ADJ 5.5 0.0 0.0 0.0 0.0 -0.3 0.0 0.3 1.1 0.3 0.3 0.3 3.6 difference -0.4 -0.5 -0.5 4.4 0.1 0.5 0.1 -1.0 0.1 0.1 0.1 -3.1 PV difference 0.0 -0.4 -0.5 -0.4 4.1 0.1 0.5 0.0 -0.9 0.1 0.1 0.1 -2.6 Source: own study. Comparison of results Source: own study. Table 10 Fig. 2. Value measure of real estate development company. Source: own study. ‐ 50,0  0,0  50,0  100,0  150,0  200,0  250,0 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 Operating Profit EVA Operational EVA Fig. 2. Value measure of real estate development company. Source: own study. The analysis of value measures clearly indicates three phases. The first phase is up until 2012 when the company was in the phase of value destruction. The financial results achieved did not compensate for the cost of capital employed. At the same time, we see a significant difference between the operational EVA and EVA of the entire enterprise related to the large capital of the land bank and the impact of one-off transactions. In the second phase, from 2013 to 2016, a stable level of value building is generated by the enterprise. During this period, the difference between value measures and accounting results is clearly marked. The projects launched by the company affect value reporting, while accounting results are only disclosed in later years. The third phase, from 2017, is where there is a significant increase in the result and value associated with the increase in the scale of implemented projects. Table 9 Table 10 presents the results in the value reporting system. It should be noted that the measures of value are reported broken down into operating activities, relating to the bank of owned properties where no operating activity is currently carried out, i.e. development projects are not implemented, and regarding one-off events, i.e. in most cases of transactions related to the sale of real estate. The distribution of value measures over time is different from formal results. The differences are presented graphically in Figure 2. The adopted reporting system takes into account, in the EVA of the current period, the value generated by projects initiated that do not generate revenues and results in the financial statements during this period. As a result, EVA measures are constant in the period 2013- 2016, and EVA increases in recent years are definitely smaller than in the case of formal financial results. The distribution of value measures over time is different from formal results. The differences are presented graphically in Figure 2. The adopted reporting system takes into account, in the EVA of the current period, the value generated by projects initiated that do not generate revenues and results in the financial statements during this period. As a result, EVA measures are constant in the period 2013- 2016, and EVA increases in recent years are definitely smaller than in the case of formal financial results. REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 58 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300 5289 vol. 28, no. 4, 2020 vol. 28, no. 4, 2020 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav Table 10 Real estate development company - measures of value Indicator [kPLN] 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 EVA 145.0 92.0 16.6 11.1 13.4 13.0 -17.7 -26.8 -3.2 -29.5 ROIC 22% 11% 5% 6% 10% 10% -3% 0% 5% -1% Operational EVA 156.6 101.6 29.1 24.8 22.4 26.7 -3.7 -11.8 -8.9 -12.2 Land bank EVA -11.6 -9.5 -17.2 -13.0 -13.0 -13.7 -14.3 -15.0 -15.6 -17.3 EVA One Offs 0.0 -0.1 4.8 -0.6 4.0 0.0 0.3 0.0 21.3 0.0 IC 1 113.7 1 110.2 988.1 852.3 642.3 663.3 416.7 359.0 353.3 331.2 IC Land Bank 165.7 155.1 255.0 199.5 185.6 195.5 203.7 214.7 222.9 247.2 NOPAT 248.2 126.5 51.8 52.0 64.4 63.8 -12.7 0.4 19.3 -2.7 Source: own study. Fig. 2. Value measure of real estate development company. Source: own study. ‐ 50,0  0,0  50,0  100,0  150,0  200,0  250,0 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 Operating Profit EVA Operational EVA Table 10 Real estate development company - measures of value Indicator [kPLN] 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 EVA 145.0 92.0 16.6 11.1 13.4 13.0 -17.7 -26.8 -3.2 -29.5 ROIC 22% 11% 5% 6% 10% 10% -3% 0% 5% -1% Operational EVA 156.6 101.6 29.1 24.8 22.4 26.7 -3.7 -11.8 -8.9 -12.2 Land bank EVA -11.6 -9.5 -17.2 -13.0 -13.0 -13.7 -14.3 -15.0 -15.6 -17.3 EVA One Offs 0.0 -0.1 4.8 -0.6 4.0 0.0 0.3 0.0 21.3 0.0 IC 1 113.7 1 110.2 988.1 852.3 642.3 663.3 416.7 359.0 353.3 331.2 IC Land Bank 165.7 155.1 255.0 199.5 185.6 195.5 203.7 214.7 222.9 247.2 NOPAT 248.2 126.5 51.8 52.0 64.4 63.8 -12.7 0.4 19.3 -2.7 Source: own study. Fig. 2. Value measure of real estate development company. Source: own study. ‐ 50,0  0,0  50,0  100,0  150,0  200,0  250,0 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 Operating Profit EVA Operational EVA Table 10 Real estate development company - measures of value Indicator [kPLN] 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 EVA 145.0 92.0 16.6 11.1 13.4 13.0 -17.7 -26.8 -3.2 -29.5 ROIC 22% 11% 5% 6% 10% 10% -3% 0% 5% -1% Operational EVA 156.6 101.6 29.1 24.8 22.4 26.7 -3.7 -11.8 -8.9 -12.2 Land bank EVA -11.6 -9.5 -17.2 -13.0 -13.0 -13.7 -14.3 -15.0 -15.6 -17.3 EVA One Offs 0.0 -0.1 4.8 -0.6 4.0 0.0 0.3 0.0 21.3 0.0 IC 1 113.7 1 110.2 988.1 852.3 642.3 663.3 416.7 359.0 353.3 331.2 IC Land Bank 165.7 155.1 255.0 199.5 185.6 195.5 203.7 214.7 222.9 247.2 NOPAT 248.2 126.5 51.8 52.0 64.4 63.8 -12.7 0.4 19.3 -2.7 Source: own study. ROIC based on financial statements is very difficult or even impossible. The use of standard corrections recommended in the literature is not sufficient. Development activities require the introduction of dedicated adjustments tailored to the specifics of these enterprises and the accounting principles they use. In this study, we proposed that the following adjustments should be made when calculating value management measures: g g xcluding the impact of asset revaluations, g g 1) excluding the impact of asset revaluations, 1) excluding the impact of asset revaluations 2) introduction of an investment account for short-term projects, e.g. housing, including activation and settlement during the negative EVA of the investment phase and advance payment during the EVA of the project together with its progress, 2) introduction of an investment account for short-term projects, e.g. housing, including activation and settlement during the negative EVA of the investment phase and advance payment during the EVA of the project together with its progress, y g j g g 3) introduction of an investment account for long-term projects, e.g. commercial projects (office, commercial, hotel) including activation and settlement in time of negative EVA investment phases, 3) introduction of an investment account for long-term projects, e.g. commercial projects (office, commercial, hotel) including activation and settlement in time of negative EVA investment phases, 4) excluding the impact of interest on foreign capital in the valuation of assets and the cost of generating implemented investments. 4) excluding the impact of interest on foreign capital in the valuation of assets and the cost of generating implemented investments. In addition, in our opinion, the measurement of value measures in three streams is important in assessing value potential: (1) operational, (2) regarding the land bank, i.e. non-working capital most often associated with future investments, (3) one-off events most often associated with the sale of real estate. The use of the indicated adjustments allows to reduce difficulties in interpreting financial data in the developers' reports and their connections with the potential for value growth. The findings of the use of value-based management in real estate sector may allow to avoid situations of inappropriate investments which may finally lead to bankruptcy. Going beyond the obtained test results, it could be considered how the application of the proposed solution may influence stability of socio-economic system connected with real estates by increasing transparency on housing market (Cellmer & Trojanek, 2019). The issue of proper control of real estate development process is especially important taking into account pressure of both local (Źróbek, et al., 2014) and national (Stacherzak, et al., 2019) administration systems in order to redevelop land, reflected also in imbalance between supply and demand on real estates (Foryś & Kazak, 2019; Broża, et al., 2020). High level of interest in proceeding redevelopment activity (Bieda, 2017) may affect inaccurate assessment of real estate development and created value (Kazak, et al., 2019; Krajewska & Pawłowski, 2019). p ( j ) The proposed method of measuring value has a wide application for both practice of managing real estate development companies as well as research on the development industry. The measurement of value for internal management needs should take into account the proposed adjustments. The proposed system allows estimating the value of projects initiated but as a result of accepted accounting solutions not disclosed in the financial result. At the same time, the proposed advance payment of values from a project in progress secures the measurement against the typical risk of development activity or excessive optimism of managers. The proposed adjustments can support the ongoing measurement of value for the needs of management, shaping relationships and the assessment of results on the owner - management line, including the building of incentive systems consistent with the concept of value-based management. p g The proposed adjustments may be relevant when analyzing results of developers and assessing their potential to build value from the point of view of investors or other capital market stakeholders. The proposed adjustments are an attempt to quantify the measures of the value of carriers, such as the land bank, advancement of investment projects and the impact of valuation revaluations, on the financial statements used in developer valuations or investment recommendations in this industry. Therefore, the presented approach may constitute an important input to the research on models of forecasting value of real estate development, valuation of company shares as well as applications of this model for investment purposes. Further research on the concept presented in the work may concern the development of algorithms for estimating the value of developers based on the proposed approach with the use of information published in the financial statements. 5. Discussion and conclusions Value measurement is an important element of capital allocation and investor activities. In the case of real estate development operations, accounting solutions resulting from accounting law, including the revaluation of real estate value and recognition of the financial result once, at the time of transferring the ownership of real estate to buyers, mean that even estimating such measures of value as EVA or REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 59 vol. 28, no. 4, 2020 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 EAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 59 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav 6. References Kyriazis, D., & Anastassis, C. (2007). The Validity of the Economic Value Added Approach: An Empirical Application. European Financial Management, 13(1), 71–100. https://doi.org/10.1111/j.1468-036X.2006.00286.x Copeland, T., Koller, T., & Murrin, J. (2000). Valuation: Measuring and managing the value of companies (3rd ed.). John Wiley & Sons. Biddle, G. C., Bowen, R. M., & Wallace, J. S. (1999). Evidence on EVA. Journal of Applied Corporate Finance, 12(2), 69–79. https://doi.org/10.1111/j.1745-6622.1999.tb00008.x Bieda, A. (2017). Urban renewal and the value of real properties. Studia Regionalne i Lokalne, 6 28. Broża, N., Birnbaum, K., Castro, D. G., & Kazak, J. K. (2020). Spatial Absorbency Assessment for Sustainable Land Development. Geomatics and Environmental Engineering, 14(2), 5–18. https://doi.org/10.7494/geom.2020.14.2.5 g g Cellmer, R., & Trojanek, R. (2019). Towards Increasing Residential Market Transparency: Mapping Local Housing Prices and Dynamics. ISPRS International Journal of Geo-Information, 9(1), 2. https://doi.org/10.3390/ijgi9010002 p // g/ / jg de Medeiros, O. R. (2005). Empirical evidence on the relationship between EVA and stock returns in Brazilian firms Available at SSRN 701421 https://doi org/10 2139/ssrn 701421 p // g/ / jg de Medeiros, O. R. (2005). Empirical evidence on the relationship between EVA and stock returns in Brazilian firms. Available at SSRN 701421 https://doi.org/10.2139/ssrn.701421 eiros, O. R. (2005). Empirical evidence on the relationship between EVA and stock returns in Dylag, R., & Kucharczyk, M. (2011). Recognising revenue from the construction of real estate in financial statements of developers in Poland. Accounting and Management Information Systems, 10(1), 25–42. Dzyuma-ZAremba, U., (2015). Gant Development SA – The Effectiveness of Bankruptcy Prediction Models in Case of Sudden Bankruptcy. Case Study. e-Finanse, 11(3), 45–57. p y y ( ) Fernandez, P. (2003). EVA, Economic profit and cash value added do not measure shareholder value creation. Journal of Applied Finance (JAF), 9(3), 74–94. Foryś, I., & Kazak, J. K. (2019). Absorption” or ”Carrying Capacity” of Areas – Assessment Methods on the Example of Detached Housing Real Estate. Real Estate Management and Valuation, 27(2), 5–19. https://doi.org/10.2478/remav-2019-0011 p g Geyser, M., & Liebenberg, I. E. (2003). Creating a new valuation tool for South African agricultural co- operatives. Agrekon, 42(2), 106–115. https://doi.org/10.1080/03031853.2003.9523614 Irala, L. R. (2005). EVA: The right measure of managerial performance. Journal of Accounting and Finance, 19(2), 77–87. Kazak, J. K., Simeunović, N., & Hendricks, A. (2019). Hidden Public Value Identification of Real Estate Management Decisions. Real Estate Management and Valuation, 27(4), 96–104. https://doi.org/10.2478/remav-2019-0039 p g Kim, G. W. (2006). It seems that most, if not all, information used in corrections is available in published financial documents, not directly in the reports, but in comments or supplementary notes. Estimating value measures in this way would allow the calculation of value measures for entire enterprise groups or the entire sector. That opens up an interesting and wide spectrum of further research. REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 60 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 60 vol. 28, no. 4, 2020 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav Obaidat, A. N. (2019). Is economic value added superior to earnings and cash flows in explaining market value added? An empirical study. International journal of business, accounting, & finance, 13(1). Obaidat, A. N. (2019). Is economic value added superior to earnings and cash flows in explaining market value added? An empirical study. International journal of business, accounting, & finance, 13(1). ( ) Ooi, J., & Liow, K. (2002). Real estate corporations: The quest for value. Journal of Property Investment & Finance, 20(1), 23–35. https://doi.org/10.1108/14635780210416246 Parle, G., Joubert, M., & Laing, G. K. (2017). Measuring economic performance of Real Estate Developers in Australia:(A Longitudinal Study). Journal of New Business Ideas and Trends, 15, 1. Rappaport, A. (1995). Creating Shareholder Value: A Guide For Managers And Investors. The Free P A Di i i f Si & S h I N Y k Parle, G., Joubert, M., & Laing, G. K. (2017). Measuring economic performance of Real Estate Developers in Australia:(A Longitudinal Study). Journal of New Business Ideas and Trends, 15, 1. aport, A. (1995). Creating Shareholder Value: A Guide For Managers And Investors. The Fre ress, A Division of Simon & Schuster Inc., New York. pp p ( ) g g Press, A Division of Simon & Schuster Inc., New York. Sharma, A. K., & Kumar, S. (2010). Economic value added (EVA)-literature review and relevan International Journal of Economics and Finance, 2(2), 200–220. https://doi.org/10.5539/ijef.v2n Sharma, A. K., & Kumar, S. (2010). Economic value added (EVA)-literature review and relevant issues. International Journal of Economics and Finance, 2(2), 200–220. https://doi.org/10.5539/ijef.v2n2p200 Sharpe, W. F. (1964). Capital asset prices: A theory of market equilibrium under conditions of risk. The Journal of Finance, 19(3), 425–442. Sharpe, W. F. (1964). Capital asset prices: A theory of market equilibrium under conditions of risk. The Journal of Finance, 19(3), 425–442. f ( ) Stacherzak, A., Hełdak, M., Hájek, L., & Przybyła, K. (2019). State Interventionism in Agricultural Land Turnover in Poland. Sustainability, 11(6), 1534. https://doi.org/10.3390/su11061534 Stancu, I., Obrejabrasoveanu, L., Ciobanu, A., & Stancu, A. T. (2017). Are Company Valuation Models the Same?-A Comparative analysis Between the Discounted Cash Flows (DCF), the Adjusted Net Assets, Value and Price Multiples, the Market Value Added (MVA) and the Residual Income (RI) Models. Economic Computation and Economic Cybernetics Studies and Research, 51(3), 5–20. Stewart, G. B. (1991). The Quest for Value. HarperCollins Publishers. Stewart, G. B. (1994). EVATM: Fact and Fantasy. 6. References EVA and Traditional Accounting Measures: Which Metric is a better predictor of market value of hospitality companies? Journal of Hospitality & Tourism Research (Washington, D.C.), 30(1), 34–49. https://doi.org/10.1177/1096348005284268 ( ) p // g/ / Kowalski, M. J. (2012). Economic Value Added a topical issue for science and practice. Information System Architecture and technology/Library of Informatics of University Level Schools, Wrocław, 179-188 Kowalski, M. J., & Biliński, Ł. (2018). Interdependence Between Value Drivers and Value Migration Processes: Evidence from Warsaw Stock Exchange. Efficiency in Business and Economics. Springer. Krajewska, M., & Pawłowski, K. (2019). Coherent land policy and land value. Geomatics and Environmental Engineering, 13(4), 33–48. https://doi.org/10.7494/geom.2019.13.4.33 Environmental Engineering, 13(4), 33–48. https://doi.org/10.7494/geom.2019.13.4.33 Lehn, K., & Makhija, A. K. (1997). 1997, EVA, accounting profits, and CEO turnover: An empirical examination 1985-1994. Journal of Applied Corporate Finance, 10(2), 90–97. https://doi.org/10.1111/j.1745-6622.1997.tb00139.x Myers, R. (1996). Metric Wars. CFO Magazine, October 1996. Lehn, K., & Makhija, A. K. (1997). 1997, EVA, accounting profits, and CEO turnover: An empirical examination 1985-1994. Journal of Applied Corporate Finance, 10(2), 90–97. https://doi.org/10.1111/j.1745-6622.1997.tb00139.x p g j Myers, R. (1996). Metric Wars. CFO Magazine, October 1996. p g j Myers, R. (1996). Metric Wars. CFO Magazine, October 1996. Modigliani, F., & Miller, M. H. (1958). The Cost of Capital, Corporation Finance and the Theory of Investments. The American Economic Review, 48, 261–297. Nappi-Choulet, I., Missonier-Piera, F., & Cancel, M. (2009). Value creation and the impact of corporate real estate assets: An empirical investigation with French listed companies. Journal of Corporate Real Estate, 11(2), 78–90. https://doi.org/10.1108/14630010910963124 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 61 vol. 28, no. 4, 2020 www.degruyter.com/view/j/remav Journal of Applied Corporate Finance, 7(2), 71–84. https://doi.org/10.1111/j.1745-6622.1994.tb00406.x p g j Velez-Pareja, I., & Tham, J. (2003). Do the RIM (Residual Income Model), EVA and DCF (Discounted Cash Flow) really match? [Online] Available: https://papers.ssrn.com/sol3/papers.cfm?abstract_id=379740 Worthington, A., & West, T. (2004). Australian Evidence Concerning the Information Content of Economic Value-Added. Australian Journal of Management, 29(2), 201–223. https://doi.org/10.1177/031289620402900204 p // g/ / Źróbek, S., Trojanek, M., & Źróbek-Różańska, A. (2014). Local authorities’ activities aimed at attracting private investments: Effects, barriers and opportunities. Actual Problems of Economics, 3(3), 262–268. REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 62 REAL ESTATE MANAGEMENT AND VALUATION, eISSN: 2300-5289 62 vol. 28, no. 4, 2020 vol. 28, no. 4, 2020
https://openalex.org/W97082658
https://europepmc.org/articles/pmc4472801?pdf=render
English
null
Endobronchial streptokinase for airway thrombus: a case series
Critical care
2,015
cc-by
274,729
Introduction To assess cerebral hemodynamics in an experimental sepsis model. Methods The study was a prospective, observational pilot study conducted in our hospital. Consecutive adult patients with severe sepsis, on a mechanical ventilator with an IL-6 blood concentration ≥100 pg/ml in the acute phase, defi ned as being up to the 28th day of illness in the ICU, were entered in this study between June 2011 and December 2012. Subjects were divided into those who were treated with steroids (steroid treatment group) and those who were not (no- steroids group) during the target period, because steroids strongly aff ect IL-6 blood levels. Methods Nineteen juvenile female Hungahib pigs were subjected into control group (n = 9) or septic group (n = 10). Under general anesthesia in animals of the sepsis group, Escherichia coli culture (2.5 × 105/ml; strain: ATCC 25922) was intravenously administrated in a continuously increasing manner as follows: 2 ml in the fi rst 30 minutes, then 4 ml in 30  minutes and afterwards 16  ml/hour for 2  hours (so a total of 9.5 × 106 E. coli within 3 hours). In the control group the anesthesia was maintained for 8  hours, infusion was administered as a similar volume of isotonic saline solution and no other intervention was made. Hemodynamic monitoring of all animals was performed by PiCCo monitoring system. The middle cerebral artery of the pigs was insonated through the transorbital window and cerebral blood fl ow velocity (MCAV) and pulsatility index was registered. Results The subjects were fi ve adult patients in the acute phase of severe sepsis on a mechanical ventilator. Gastrointestinal motility was measured for a total of 62,399 minutes: 31,544 minutes in three subjects in the no-steroids group and 30,855 minutes in two subjects in the steroid treatment group. In the no-steroids group, the bowel sound counts were negatively correlated with IL-6 blood concentration (r  = –0.76, P  <0.01), suggesting that gastrointestinal motility was suppressed as IL-6 blood concentration increased. However, in the steroid treatment group, gastrointestinal motility showed no correlation with IL-6 blood concentration (r = –0.25, P = 0.27). The IL-6 blood concentration appears to have decreased with steroid treatment irrespective of changes in the state of sepsis, whereas bowel sound counts with the monitoring system refl ected the changes in the state of sepsis, resulting in no correlation. MEETING ABSTRACTS MEETING ABSTRACTS P1 but long-term observation and objective evaluation of gastrointestinal motility are diffi cult. We developed a non-invasive monitoring system capable of quantifying and visualizing gastrointestinal motility in real time. In the study gastrointestinal motility was performed in patients with severe sepsis using this developed bowel sound analysis system, and the correlation between bowel sounds and changes over time in blood concentrations of IL-6, which is associated with sepsis severity, was evaluated. P1 Cerebral autoregulation testing in a porcine model of intravenously administrated E. coli induced fulminant sepsis L Molnar1, M Berhes1, L Papp1, N Nemeth2, B Fulesdi1 1Deoec Aneszteziologia, Debrecen, Hungary; 2University of Debrecen, Hungary Critical Care 2015, 19(Suppl 1):P1 (doi: 10.1186/cc14081) P1 Cerebral autoregulation testing in a porcine model of intravenously administrated E. coli induced fulminant sepsis L Molnar1, M Berhes1, L Papp1, N Nemeth2, B Fulesdi1 1Deoec Aneszteziologia, Debrecen, Hungary; 2University of Debrecen, Hungary Critical Care 2015, 19(Suppl 1):P1 (doi: 10.1186/cc14081) Introduction To assess cerebral hemodynamics in an experimental sepsis model. Introduction To assess cerebral hemodynamics in an experimental sepsis model. Results In the septic group, as expected, all animals developed fulminant sepsis and died within 3 to 7 hours two animals in 3 to 4 hours, and three in 6 to 7 hours). In the septic animals the heart rate rose and mean arterial pressure dropped, their ratio increased signifi cantly compared with both the base values (at the 6th hour: P <0.001) and the control group (P  =  0.004). The control animals showed stable condition over the 8-hour anesthesia. MCAV signifi cantly decreased during the development of sepsis (from 23.6 ± 6.6 cm/s to 16.0 ± 3.9 cm/s, P <0.01) and pulsatility indices increased (from 0.68  ±  0.22 to 1.37  ±  0.58, P  <0.01), indicating vasoconstriction of the resistance vessels. A signifi cant relationship was fund between percent change of the MAP and the pulsatility index in septic animals (R2 = 0.32) referring to maintained cerebral autoregulation. Conclusion The new real-time bowel sound analysis system provides a useful method of continuously, quantitatively, and non-invasively evaluating gastrointestinal motility in severe patients. Furthermore, this analysis may predict disease severity in septic patients. Conclusion Cerebral autoregulation is preserved in the pig model of experimentally induced fulminant sepsis. P3 Usefulness of sepsis screening tools and education in recognising the burden of sepsis on hospital wards EJ Galtrey, C Moss, H Cahill Guy’s and St Thomas’ Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P3 (doi: 10.1186/cc14083) P2 New real-time bowel sound analysis may predict disease severity in septic patients J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 35th International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 17-20 March 2015 Publication charges for this supplement were funded by ISICEM. p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 1University of Cambridge, UK; 2Ipswich Hospital, Ipswich, UK Critical Care 2015, 19(Suppl 1):P5 (doi: 10.1186/cc14085) 1University of Cambridge, UK; 2Ipswich Hospital, Ipswich, UK Critical Care 2015, 19(Suppl 1):P5 (doi: 10.1186/cc14085) fi Methods All referrals to our critical care response team with a diagnosis of sepsis over a 3-month period (September to November 2014) were investigated to determine how many had an EPR sepsis alert comprising a prompt for blood cultures, serum lactate measurement, fl uid challenge if hypotensive, and antibiotics within 1 hour. Introduction Severe sepsis results in ~36,800 UK deaths each year [1]. Prior studies demonstrate the benefi t of early recognition and treatment of sepsis in reducing mortality [2]. The Sepsis Six [1] bundle aims to optimise the fi rst hour of sepsis management. We assessed the proportion of emergency department (ED) patients with severe sepsis receiving the Sepsis Six bundle and whether this was improved by a combination of staff education and use of Sepsis Six management stickers in patient notes. l g yp Results Only 25/174 (14%) patients with a diagnosis of sepsis had an EPR sepsis alert. There was no signifi cant diff erence between acute and nonacute ward areas in their likelihood of using the screening tool or alert, in contrast to previous audits of the alerted population which showed that acute areas such as A&E and medical acute admission wards had higher utilisation and bundle completion rates. Methods A closed loop audit was completed in the ED at Ipswich Hospital, UK. Each cycle was 14  days with interventions made in a 4-week period between the two cycles. The interventions consisted of: Sepsis Six management stickers and posters placed in the ED; two training sessions for all ED nurses on sepsis recognition and management; a teaching session for all middle-grade doctors; and a trolley in the ED with equipment required for the Sepsis Six. The notes of all patients with lactate ≥2 mmol/l were retrospectively reviewed. Those with ≥2 systemic infl ammatory response syndrome criteria and a documented suspicion of infection were deemed to have severe sepsis. The times at which these patients had each of the Sepsis Six completed were recorded, as were the fi nal diagnosis and 7/28 day mortality. P5 tool and electronic order set (EPR alert) alongside an education programme to improve delivery of the SSC bundle. Previous audits showed only 43% full bundle compliance in those that were alerted, and this raised concerns regarding the burden of unalerted sepsis. We sought to estimate the number of unalerted sepsis episodes to assess the effi cacy of our screening tool and improve early recognition. p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 Results In Cycle 1, 31/106 patients met the criteria for severe sepsis, compared with 36/120 in Cycle 2. The delivery of the Sepsis Six interventions was highly variable. In Cycle 1 lactate levels and i.v. access had the highest 60-minute completion rates (90.3%, 83.9% respectively). Blood cultures and i.v. fl uid resuscitation were completed for 61.3% and 64.5% of patients within 60 minutes. Only 38.7% of septic patients were given i.v. antibiotics within 60 minutes. In total, 58.9% of patients received antibiotics in accordance with trust guidelines. High fl ow oxygen, catheters and fl uid balance charts had the lowest 60-minute completion rates (35.5%, 6.5%, 6.5% respectively). In Cycle 2, post intervention, there was no signifi cant change in the percentage of patients receiving the Sepsis Six bundle. Methods A closed loop audit was completed in the ED at Ipswich Hospital, UK. Each cycle was 14  days with interventions made in a 4-week period between the two cycles. The interventions consisted of: Sepsis Six management stickers and posters placed in the ED; two training sessions for all ED nurses on sepsis recognition and management; a teaching session for all middle-grade doctors; and a trolley in the ED with equipment required for the Sepsis Six. The notes of all patients with lactate ≥2 mmol/l were retrospectively reviewed. Those with ≥2 systemic infl ammatory response syndrome criteria and a documented suspicion of infection were deemed to have severe sepsis. The times at which these patients had each of the Sepsis Six completed were recorded, as were the fi nal diagnosis and 7/28 day mortality. Conclusion Despite these interventions, most patients still do not receive the full recommended treatment bundle. These fi ndings have prompted a point prevalence audit at ward level, which will examine all patients’ notes for the preceding 24 hours to ascertain if sepsis is truly unrecognised or whether it is simply that our current tool is not a helpful adjunct to care. With national guidelines expected within the year, we will redesign and re-launch our screening tools and education programme to improve awareness and management of this common medical emergency. Continuous versus intermittent temperature measurement in the detection of fever in critically ill patients p g p Conclusion The low rates of Sepsis Six completion require improvement to meet the targets set out by the College of Emergency Medicine. Our results suggest that simple interventions are ineff ective in increasing Sepsis Six completion and thus lend support to the case for integrated interventions such as electronic recording and alert systems. References Introduction An elevated body temperature is one of the four criteria in diagnosing systemic infl ammatory response syndrome (SIRS), and is often used at the bedside to trigger diagnostic investigations for infection. Standard intermittent temperature measurement may, however, delay the detection of an elevated temperature or miss this altogether. The aim of the study is to compare intermittent non- invasive versus continuous invasive body temperature measurement as a tool to detect an elevated body temperature. References References 1. Dellinger R, et al. Crit Care Med. 2013;41:580-637. 2. http://bit.ly/1nzV3Kp. 3. http://bit.ly/12Y3KHq. 4. http://bit.ly/10PYi8G. 1. Dellinger R, et al. Crit Care Med. 2013;41:580-637. 2. http://bit.ly/1nzV3Kp. 3. http://bit.ly/12Y3KHq. 4. http://bit.ly/10PYi8G. i g y y Results In Cycle 1, 31/106 patients met the criteria for severe sepsis, compared with 36/120 in Cycle 2. The delivery of the Sepsis Six interventions was highly variable. In Cycle 1 lactate levels and i.v. access had the highest 60-minute completion rates (90.3%, 83.9% respectively). Blood cultures and i.v. fl uid resuscitation were completed for 61.3% and 64.5% of patients within 60 minutes. Only 38.7% of septic patients were given i.v. antibiotics within 60 minutes. In total, 58.9% of patients received antibiotics in accordance with trust guidelines. High fl ow oxygen, catheters and fl uid balance charts had the lowest 60-minute completion rates (35.5%, 6.5%, 6.5% respectively). In Cycle 2, post intervention, there was no signifi cant change in the percentage of patients receiving the Sepsis Six bundle. P4 Continuous versus intermittent temperature measurement in the detection of fever in critically ill patients A Heyneman, V Bosschem, N Mauws, D Van Der Jeught, E Hoste, J Decruyenaere, J De Waele Ghent University Hospital, Ghent, Belgium Critical Care 2015, 19(Suppl 1):P4 (doi: 10.1186/cc14084) interventions References 1. Daniels et al. Emerg Med J. 2011;28:507-12. 1. Daniels et al. Emerg Med J. 2011;28:507-12. 1. Daniels et al. Emerg Med J. 2011;28:507-12. 2. Kumar et al. Crit Care Med. 2006;34:1589-96. 2. Kumar et al. Crit Care Med. 2006;34:1589-96. New real-time bowel sound analysis may predict disease severity in septic patients J G t 1 K M t d 1 N H ii1 T M i hi1 M Y i 1 D H d 1 p p J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) Introduction Sepsis is defi ned as the presence of infection with systemic signs of infection, and severe sepsis as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion [1]. Since the Surviving Sepsis Campaign (SSC) in 2002, the Health Service Ombudsman for England published recommendations for improving recognition and treatment of sepsis [2], the Royal College of Physicians issued a toolkit for the management of sepsis in the acute medical unit [3], and NHS England released a patient safety alert to support prompt recognition and treatment of sepsis [4]. In 2012 our Trust introduced a sepsis screening Introduction Sepsis is defi ned as the presence of infection with systemic signs of infection, and severe sepsis as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion [1]. Since the Surviving Sepsis Campaign (SSC) in 2002, the Health Service Ombudsman for England published recommendations for improving recognition and treatment of sepsis [2], the Royal College of Physicians issued a toolkit for the management of sepsis in the acute medical unit [3], and NHS England released a patient safety alert to support prompt recognition and treatment of sepsis [4]. In 2012 our Trust introduced a sepsis screening Introduction Healthy bowel function is an important factor when judging the advisability of early enteral nutrition in critically ill patients, © 2015 BioMed Central Ltd © 2015 BioMed Central Ltd © 2015 BioMed Central Ltd S2 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P5 Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia y q p C Joya-Montosa, MD Delgado-Amaya, E Trujillo-García, E Curiel-Balsera Hospital Regional de Málaga, Spain C Joya-Montosa, MD Delgado-Amaya, E Trujillo-García, E Curiel-Balsera Hospital Regional de Málaga, Spain p g g p ritical Care 2015, 19(Suppl 1):P9 (doi: 10.1186/cc14089) Introduction The aim of the study is to evaluate the calibration and discrimination of two specifi c risk scores for community-acquired pneumonia (CAP) in patients with this illness who required ICU admission. Results Severe sepsis criteria were fulfi lled in 31% (n  = 91) of the patients. These were older (median age: 79  years vs. 71  years) and experienced more symptoms (mean: 2.2, SD 0.9 vs. mean: 1.4, SD 0.7) than patients without severe sepsis, while there was no diff erence in C-reactive protein levels (OR per 50 mg/l: 1.07, 95% CI: 0.96 to 1.20). Among individual symptoms, altered mental status (OR: 4.4, 95% CI: 2.2 to 9.0), dyspnea (OR: 3.5, 95% CI: 2.1 to 5.9), and muscle weakness (OR: 2.2, 95% CI: 1.0 to 4.4) were signifi cantly related to severe sepsis. Adjusting for age and sex, altered mental status (adj. OR: 4.1, 95% CI: 2.0 to 8.4) and dyspnea (adj. OR: 3.1, 05% CI: 1.8 to 5.3) remained signifi cant. Conclusion General symptoms, especially altered mental status and dyspnea, appear to be more common in severe sepsis than in milder infections. These symptoms might be utilized as a diagnostic aid for severe sepsis in the clinical setting, complementing vital signs and laboratory tests. Methods A retrospective descriptive study of patients with severe CAP admitted to the ICU between January 2008 and September 2013. We analyzed clinical and epidemiological variables and APACHE II, SAPS III, CURB-65 and Pneumonia Severity Index (PSI) that were recorded in the fi rst 24 hours. We used the Student t test to compare means and the chi-square test for univariate analysis. The standardized mortality ratio (SMR) and Hosmer–Lemershow test were calculated to analyze the calibration and ROC curve analysis for discrimination of diff erent scores. Results We analyzed 111 patients aged 57.5 ± 17.7 years, with 63.1% (70) males. The APACHE II score at admission was 19.8 ± 17.7 and SAPS III was 60.6 ± 16.7. ICU mortality was 29.7% (33). There was association between the four scores and mortality. P9 weakness, gastrointestinal symptoms, localized pain, altered mental status) that were part of the reason the patient sought medical care were registered. Additionally, age, sex, vital signs, C-reactive protein, and blood cultures were registered. Patients that within 48 hours from admission fulfi lled any criteria for severe sepsis were compared with patients that did not. Odds ratios for severe sepsis were computed using univariable as well as multivariable logistic regression, controlling for age and gender as confounders. 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. P6 Systemic symptoms as markers for severity in sepsis J Edman-Wallér1, L Ljungström2, R Andersson3, G Jacobsson2, M Werner1 1Södra Älvsborg Hospital, Borås, Sweden; 2Skaraborg Hospital, Skövde, Sweden; 3Institute of Biomedicine, University of Gothenburg, Sweden Critical Care 2015, 19(Suppl 1):P6 (doi: 10.1186/cc14086) Methods This was a secondary analysis of a prospective study in 25 critically ill patients comparing diff erent measurement techniques. Temperature was measured intermittently with an axillary digital thermometer every 4 hours, and a urinary bladder thermistor catheter was used for continuous temperature measurement; the latter was considered the reference method. Fever (core temperature ≥38.3°C) episodes occurring within 60 minutes after each other were classifi ed as one episode. We compared the fever detection rate of both methods and calculated the diff erence in timing between both techniques. Introduction The objective of this study was to evaluate six general symptoms as markers for severe sepsis in patients with suspected bacterial infections. Severe sepsis is a common cause of death and morbidity. Early detection and treatment is critical for outcome. Clinical presentation varies widely and no single test is able to discriminate severe sepsis from uncomplicated infections or non-infectious emergencies. Apart from local symptoms of infection, the systemic infl ammatory reaction itself may give rise to general symptoms such as muscle weakness and vomiting. f Results Twenty-nine episodes of fever were detected in 10 patients (seven male) with a median APACHE II score of 10 (IQR 3 to 24) and a median SOFA score of 10 (IQR 8 to 11). Median duration of a fever episode was 1 hour 24 minutes (IQR 47 minutes to 2 hours 59 minutes) and median maximum temperature was 38.4°C (IQR 38.3 to 38.7). Median axillary temperature was 0.7°C (IQR 0.3 to 0.9) lower than core temperature. Using intermittent, non-invasive measurement, 27 out of 29 fever episodes (93.1%) remained undetected. Methods We present an observational, consecutive study. Data from ambulance and hospital medical records were analyzed. The survey included 290 patients (mean age: 70.6  years; median age: 74  years; male: 47%) who were admitted to a 550-bed secondary care hospital, receiving intravenous antibiotics for suspected community-acquired infections. General symptoms (fever/shivering, dyspnea, muscle Conclusion Intermittent, non-invasive temperature measurement failed to detect most of the fever episodes as measured by a bladder thermistor catheter and should not be used to screen for elevated body temperature in critically ill patients. Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study g y S Laukemann1, N Kasper1, N Kasper1, A Kutz1, S Felder1, S Haubitz2, B Müller1, P Schuetz1 Figure 1 (abstract P9). 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) Introduction Collection of blood cultures is routinely performed in patients with suspicion of infection in the emergency department (ED) despite a low yield of positive culture results. To increase sensitivity, diff erent clinical prediction rules and blood biomarkers have been put forward. Herein, we validated the performance of diff erent promising clinical prediction rules alone and in combination with novel blood biomarkers to predict blood culture positivity. Methods This is an observational cohort study including consecutive medical patients with suspected infection and collection of ED admission blood cultures. Five clinical prediction rules were calculated and admission concentrations of procalcitonin (PCT), C-reactive protein, neutrophil–lymphocyte count ratio (NLCR), lymphocyte count, white blood cell count, and red blood cell distribution width were measured. True blood culture positivity was assessed by two independent physicians. We used logistic regression models with area under the curve (AUC) to establish associations between clinical prediction rules and blood culture positivity. Figure 1 (abstract P9). y Results Of 1,083 included patients, 106 (9.8%) cultures were positive. Of the clinical prediction rules, the Shapiro rule performed best (AUC 0.733) followed by the Metersky rule (AUC 0.609). The best biomarkers were PCT (AUC 0.796), NLCR (0.692) and lymphocyte count (AUC 0.671). Combination of the Shapiro rule and PCT showed the best combination result (AUC of combined model 0.822). Limiting blood cultures to either the Shapiro rule ≥4 points or PCT >0.11 μg/l would reduce negative sampling by 25.6% while still identifying 100% of positive cultures. Using a Shapiro rule ≥3 points or PCT >0.25 μg/l limit would reduce negative sampling by 42.1% while still identifying 96.2% of positive cultures. Conclusion The four analyzed scores presented good calibration, but discrimination seems better in SAPS III. Given the diffi culty of calculating PSI, and its low discrimination (similar to CURB-65), we prefer to use the CURB-65 score in routine clinical practice. 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. P6 S3 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia The SMR for APACHE II was 0.87 and 0.85 for the SAPS III. Figure 1 shows the ROC curve for the four scores, the best observed discrimination obtained was for SAPS III score (AuC 0.79) and the worst was obtained for CURB-65 score (AuC 0.7). The Hosmer–Lemeshow test showed acceptable calibration for the four predictive systems (P > 0.05). Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study S Laukemann1, N Kasper1, N Kasper1, A Kutz1, S Felder1, S Haubitz2, B Müller1, P Schuetz1 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study Pre-exposure to mechanical ventilation and endotoxin infl uence bacterial growth and immune response during experimental ventilator-associated pneumonia Table 1 (abstract P10) Sternal infection P value Insulin 9/85 0.001 Current smoker 4/272 0.037 COPD 11/197 <0.001 Transfusion >3 19/302 0.001 J Sperber1, A Nyberg1, M Lipcsey2, A Larsson2, J Sjölin2, M Castegren2 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Uppsala University, Uppsala, Sweden Introduction Overproduction of nitric oxide (NO) is correlated with adverse outcomes in sepsis. NO is additionally a central part of the innate immune system defense against pathogens causing ventilator- associated pneumonia (VAP), which can complicate the clinical course during mechanical ventilation (MV). We hypothesized that pre-exposure to MV and systemic infl ammation from endotoxin each would infl uence bacterial growth in lung tissue, based on an altered immune response in experimental pneumonia. We used a porcine Pseudomonas aeruginosa VAP model with ventilatory and infl ammatory pre-exposures before inoculation to evaluate bacterial growth, development of lung damage, total NO production and infl ammatory cytokine response. Conclusion Postoperative deep sternal wound infections have statistical signifi cant correlation with the following parameters: transfusion with >3 red blood cell units, history of COPD, insulin dependence and when the patient is a current smoker. Also there is a tendency for correlation with CBP time >120 minutes (P = 0.056). References 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg 2007;2:23 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 2. Schimmer C, et al. Prevention of sternal dehiscence and infection in high-risk patients: a prospective randomized multicenter trial. Ann Thorac Surg. 2008;86:1897-904. y Methods Three groups of mechanically ventilated pigs were subjected to experimental VAP for 6  hours with intrapulmonary 1  ×  1011 CFU P. aeruginosa at baseline. Two groups were pre-exposed to MV for 24  hours before bacterial inoculation: MV  + Etx (n  = 6, received endotoxin 0.063 μg × kg–1 × hour–1) and MV (n = 6, received saline in equivalent volume). One group, Un (n  = 8), started the experiment unexposed to both MV and endotoxin, directly from the initiation of VAP. Postmortem lung tissue samples rendered bacterial cultures. References 1. Hoenig M, et al. Procalcitonin fails to predict bacteremia in SIRS patients: a cohort study. Int J Clin Pract. 2014;68:1278-81. y 2. Hoeboer SH, et al. Old and new biomarkers for predicting high and low risk microbial infection in critically ill patients with new onset fever: a case for procalcitonin. J Infect. 2012;64:484-93. y 2. Hoeboer SH, et al. Old and new biomarkers for predicting high and low risk microbial infection in critically ill patients with new onset fever: a case for procalcitonin. J Infect. 2012;64:484-93. q y Results A total of 35 patients (3.44%) were complicated by deep sternal wound infections. No statistical correlation was found with age >75, gender, DM, BMI >30, steroids, emergent operation, prolonged ventilation, CBP time >120 minutes, reintubation and NIV. Factors with statistical signifi cant correlation are presented in Table 1. P10 Predisposing factors for deep sternal wound infection after cardiac surgery Predisposing factors for deep sternal wound infection after cardiac surgery F Ampatzidou, M Sileli, A Madesis, K Antoniou, A Baddur, G Kechagioglou, T Asteri, G Drossos General Hospital G. Papanikolaou Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P10 (doi: 10.1186/cc14090) Conclusion Combination of clinical parameters combined in the Shapiro rule together with admission levels of PCT allows reduction of unnecessary blood cultures with minimal false negative rates. References Introduction The aim of our study was to investigate perioperative risk factors associated with deep sternal wound infections in complicated cardiac surgery. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. y Methods A total of 1,017 patients underwent cardiac surgery in a 2-year period. We investigate the correlation between deep sternal S4 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 factors might be useful to know whether or not adult febrile patients have bacteremia. wound infection with the following 14 preoperative characteristics and perioperative parameters: age >75, female gender, diabetes mellitus (DM), insulin dependence, body mass index (BMI) >30, current smokers, COPD, cardiopulmonary bypass time (CBP) >120 minutes, use of steroids, emergency operation, prolonged mechanical ventilation (>48 hours), reintubation, transfusion with more than 3 units of red blood cells, and the postoperative use of non-invasive ventilation (NIV). The chi-square test was used for statistical analysis. wound infection with the following 14 preoperative characteristics and perioperative parameters: age >75, female gender, diabetes mellitus (DM), insulin dependence, body mass index (BMI) >30, current smokers, COPD, cardiopulmonary bypass time (CBP) >120 minutes, use of steroids, emergency operation, prolonged mechanical ventilation (>48 hours), reintubation, transfusion with more than 3 units of red blood cells, and the postoperative use of non-invasive ventilation (NIV). The chi-square test was used for statistical analysis. P11 Risk factors for bacteremia in adult febrile patients in emergency settings g A Mikami1, Y Natori1, F Omata2, S Ishimatsu1 1St Luke’s International Hospital, Tokyo, Japan; 2St Luke’s Life Science Institute, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P11 (doi: 10.1186/cc14091) Results The animals pre-exposed to endotoxin (MV + Etx) displayed higher bacterial growth (CFU × g–1) (P <0.05), lower PaO2/FiO2 (P <0.05) and lower nitrate levels (P <0.01) than the unexposed animals (Un). Plasma TNFα levels were higher in Un than in both pre-exposed groups MV  + Etx and MV (P  <0.01). There were no signifi cant diff erences between the two pre-exposed groups. Critical Care 2015, 19(Suppl 1):P11 (doi: 10.1186/cc14091) Introduction Blood culture is a critical procedure for detecting potentially life-threatening bloodstream infections (BSI). At the same time, early diagnosis and appropriate treatment of BSI are the key factors in order to improve prognosis. The purpose of the current analysis was to identify risk factors for bacteremia in adult febrile patients in emergency settings. g Conclusion Mechanical ventilation for 24  hours with concomitant endotoxin exposure enhances bacterial growth and lung damage during P. aeruginosa VAP, compared with inoculation without any pre- exposure to MV or endotoxin. The greater bacterial clearance in the unexposed animals was associated with higher NO production and higher levels of pro-infl ammatory cytokines. Methods We conducted a retrospective case–control study within a population of adult patients visiting the emergency department at a community hospital (St Luke’s International Hospital, Tokyo, Japan) and who underwent two sets of blood culture testing between 2003 and 2012. Among a total of 13,582 patients, 1,322 (10%) were detected as bacteremia. We included in this study 179 randomly selected patients from the bacteremia group and 321 randomly selected patients from the negative blood culture group to serve as the comparison group. Multivariate logistic regression was used to evaluate the relationship between clinical characteristics factors and bacteremia. Pre-exposure to mechanical ventilation and endotoxin infl uence bacterial growth and immune response during experimental ventilator-associated pneumonia NO production was measured with urinary nitrate levels over 6 hours of VAP. retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 2. Schimmer C, et al. Prevention of sternal dehiscence and infection in high-risk patients: a prospective randomized multicenter trial. Ann Thorac Surg. 2008;86:1897-904. P15 ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign g p p g J Melville, S Ranjan, P Morgan East Surrey Hospital, Redhill, UK Critical Care 2015, 19(Suppl 1):P15 (doi: 10.1186/cc14095) Introduction The aim of this study was to evaluate the eff ect of the Surviving Sepsis Campaigns on mortality rates, before and after the second surviving sepsis publication, and to assess whether patients with sepsis being admitted to the ICU had a lower APACHE II score on admission. Patients with sepsis, who require ICU care, have an extremely poor prognosis. It has been shown that the mortality rates range from 20.7% (severe sepsis) to 45.7% (septic shock) [1]. The surviving sepsis campaign was initiated in 2002. The fi rst, second and third publications were published in 2004, 2008 and 2012 respectively [2]. g Results According to elevation of CT grades, severity of illness was signifi cantly associated with high score (APACHE II: 10.5 to 4.0, 12.8 to 4.2, 16 to 4.2, SOFA: 2.6 to 1.5, 2.9 to 1.9, 6.8 to 3.7, CRP: 17.8 to 10.6, 22.4 to 10.1, 33.3 to 11.9) and also duration of mechanical ventilation and length of hospital stay were longer (duration of mechanical ventilation: 10.9 to 6.6, 11.5 to 6.7, 15.8 to 7.2, length of hospital stay: 23.4 to 10.6, 27.9 to 21.4, 48.7 to 36.2). y Methods A retrospective case note review was performed, looking at a sample of 5,954 patients who were 18 years or older who had been admitted to East Surrey Hospital (ESH) ICU between 1 January 2005 and 31 October 2014. The total number of patients with sepsis was 941. We compared results before and after the second publication of the surviving sepsis campaign, looking at mortality rates, age of patients, admission length prior to ICU transfer, APACHE II score and the length of stay on the ICU. Conclusion Novel classifi cation of CNF based on CT fi ndings showing the extension of fl uid collection is a useful indicator of the disease severity and predicting clinical outcome. These fi ndings may infl uence the strategy for the success of percutaneous catheter drainage. y Results From the beginning of 2005 to the end of 2008, the mortality rates for septic patients was 51.9% compared with 41.3% from the beginning of 2009 to end of October 2014. P15 ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign Fisher’s two-tailed test showed a signifi cant diff erence (P = 0.003) between the mortality before and after the second publication. The median ages before and after 2009 were 63.9 and 64.8 years. The time in hospital before admission to the ICU was greater before 2009 (6.15 days) compared with after 2009 (5.53 days). There was no signifi cant diff erence (Mann–Whitney test) between the APACHE II scores, with the mean and median score the same at 17.6 and 18 for both groups. The mean length of stay was 1 day longer after 2009 (8.07 days compared with 9.07 days). P15 this study. Cervical spaces were subdivided into three components according to the concept of interfascial planes. The extension of acute fl uid collection in cervical spaces was classifi ed into three grades: Grade I, fl uid collection confi ned to one component; Grade II, fl uid collection spreading into two or three components; and Grade III, fl uid collection spreading into four components or mediastinum. We analyzed association with CT grades and severity of illness (SOFA score, APACHE II score, CRP). All patients underwent percutaneous catheter drainage either ultrasonography guided or CT guided. We compared treatment outcome of CNF with CT grades. 1. Estaban A, et al. Crit Care Med. 2007;35:1284-9. 2. Alberti C, et al. Intensive Care Med. 2002;28:108-21. ICU mortality rates in patients with sepsis compared with patients without sepsis p J Melville, S Ranjan, P Morgan East Surrey Hospital, Redhill, UK Critical Care 2015, 19(Suppl 1):P14 (doi: 10.1186/cc14094) Introduction The aim of the study was to evaluate the diff erence in mortality rates between those admitted to the ICU with and without sepsis, and to assess the proportion of patients who had sepsis. Septic patients are one of the key groups of patients admitted to ICUs around the world. Septic patients have an extremely poor prognosis with published mortality rates ranging from 20.7% (severe sepsis) to 45.7% (septic shock) [1]. With septic patients making up roughly 21% of patients admitted to ICUs, it is important to assess whether these rates of mortality hold true to a district general ICU and to assess the extent of the diff erence in prognosis between patients with and without sepsis [2]. g y p y Conclusion Patients with sepsis admitted to ESH ICU had a 20% relative decrease in mortality after the second publication of surviving sepsis guidelines. The original aim of the campaign was to reduce mortality from sepsis by 25% in 5 years [3]. This decrease was not due to a signifi cant diff erence between the sets of patients. The decreased time to admittance to ICU may be due to improved recognition of the need for ICU care. Overall the surviving sepsis campaign has had a signifi cantly benefi cial eff ect on mortality rates in patients with sepsis. References 1. Estaban A, et al. Crit Care Med. 2007;35:1284-9. Methods We performed a retrospective case note review, looking at a sample of 5,954 patients 18 years or older who were admitted to East Surrey Hospital (ESH) ICU, which has an elective admissions rate of 3%, between 1 January 2005 and 31 October 2014. The total number of patients with sepsis was 941 compared with 5,013 without sepsis. We looked at mortality rates, APACHE II scores and length of stay on the unit. 2. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 3. Slade E, et al. Crit Care. 2003;7:1-2. Percutaneous drainage for patients with cervical necrotizing fasciitis with novel CT classifi cation based on extension of fl uid collection along the deep cervical space T Kiguchi, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P13 (doi: 10.1186/cc14093) Percutaneous drainage for patients with cervical necrotizing fasciitis with novel CT classifi cation based on extension of fl uid collection along the deep cervical space T Kiguchi, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P13 (doi: 10.1186/cc14093) Results In a multivariate logistic regression model, a statistically signifi cant independent eff ect was found for body temperature (BT) >38°C (OR = 2.58, 95% CI, 1.76 to 3.79, P <0.001), systolic blood pressure (SBP) <100 mmHg (OR = 1.72, 95% CI, 1.11 to 2.65, P = 0.01), CRP >10 mg/dl (OR = 3.03, 95% CI, 2.05 to 4.49, P <0.001) and PaCO2 <32 mmHg (OR = 2.3, 95% CI, 1.57 to 3.37, P <0.001). Receiver operating characteristic curve analysis revealed an area under the curve value of 0.725 for diff erentiating patients with bacteremia from negative culture. Introduction Cervical necrotizing fasciitis (CNF) is a rapidly evolving and life-threatening condition. Therefore, it is important for physicians to evaluate the severity of illness and to predict clinical outcome exactly in the early phase. We focused on extension of acute fl uid collection along the deep cervical space by CT fi ndings. The purpose of this study was to produce the CT grade and to analyze whether our CT grade is related to the clinical features and the responses to treatment of CNF. Methods Between June 2004 and December 2012, 42 patients diagnosed and treated for CNF in two institutions were included in p Methods Between June 2004 and December 2012, 42 patients diagnosed and treated for CNF in two institutions were included in Conclusion BT >38°C, SBP  <100  mmHg, CRP >10  mg/dl and PaCO2 <32  mmHg are independently associated with bacteremia. These S5 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P17 Methods A prospective study design was used in order to investigate all sepsis patients either presenting to the emergency department or admitted to the ICU of a regional trauma centre. A total of 106 patients were recruited and all patients were considered eligible as per the SIRS and sepsis criteria [1]. The Sepsis-related Organ Failure Assessment score was determined over the fi rst 24 hours to assess organ function. Patients were assigned to groups as follows: sterile SIRS; uncomplicated sepsis; severe sepsis or septic shock as per the criteria. Assignment into groups was blinded and performed by an intensive care specialist independent of the study. Baseline demographics, clinical characteristics and outcomes were collected and surviving patients were sent a SF-12v2 survey at between 6 months and 2  years post hospital discharge. P18 P18 Long-term health-related quality of life in survivors of sepsis: an epidemiological study CE Battle1,2,3, G Davies1, M Vijayakumar3, PA Evans1 1NISCHR HBRU Morriston Hospital, Swansea, UK; 2College of Medicine, Swansea University, Swansea, UK; 3Ed Major Critical Care Unit, Morriston Hospital, Swansea, UK Critical Care 2015, 19(Suppl 1):P18 (doi: 10.1186/cc14098) Independent risk factors for long-term mortality in patients with severe infection Results From the beginning of 2005 to the end of October 2014, mortality rates in septic patients were 44.6% compared with 26.2% in nonseptic patients. Fisher’s two-tailed test showed a signifi cant diff erence (P <0.0001) between the mortality in septic and nonseptic patients. There was a signifi cant diff erence (Mann–Whitney) between APACHE II scores, with median scores of 18 and 13 in septic and nonseptic patients respectively. Septic patients had longer lengths of stay, with the mean and median 8.73 and 3.89  days respectively, compared with 4.90 and 2.5 in nonseptic patients. Septic patients made up 15.8% of all patients admitted to the ICU. J Francisco, I Aragão, T Cardoso J Francisco, I Aragão, T Cardoso Centro Hospitalar do Porto – Hospital Geral de Santo António, Porto, Portugal Critical Care 2015, 19(Suppl 1):P16 (doi: 10.1186/cc14096) Introduction The purpose of this study was to examine long-term mortality, 5 years after severe infection, and to identify independent risk factors associated with it. Methods A prospective cohort study developed at a tertiary care university-affi liated 600-bed hospital including all patients with severe infection admitted into intensive care, medical, surgical, haematology and nephrology wards, over a 1-year period (2008/2009). The outcome of interest was mortality 5  years following hospitalisation and its association with specifi c risk factors was studied through logistic regression. Conclusion Patients with sepsis admitted to ESH ICU made up a signifi cant minority of patients admitted to the ICU. Septic patients had a 70% relative higher mortality rate compared with nonseptic patients. The mortality rate of 44.6% fi ts with previously quoted mortality rates in septic shock. Patients with sepsis had a signifi cantly higher predicted mortality, recorded by their APACHE II score, which was statistically signifi cant. This also meant they needed longer ICU care, with the average length of stay almost doubled. Results There were 1,013 patients included in the study. Hospital mortality rate was 14% (n = 137) and 5-year mortality was 37% (n = 379). Factors independently associated with 5-year mortality were (adjusted odds ratio (95% confi dence interval)): age = 1.04 per year (1.03 to 1.05), cancer = 8.00 (3.06 to 20.88), chronic hepatic disease = 3.06 (1.06 to 8.87), chronic respiratory disease = 2.21 (1.06 to 4.62), haematologic Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S6 Figure 1 (abstract P17). P18 Long-term health-related quality of life in survivors of sepsis: an epidemiological study Figure 1 (abstract P16). disease = 3.40 (1.64 to 7.04), Karnovsky Index <70 = 2.56 (1.63 to 3.71), infection by an ESKAPE pathogen = 1.65 (1.02 to 2.66) and severity of infection (reference is infection without SIRS): sepsis = 1.14 (0.7 to 1.83), severe sepsis = 1.18 (0.73 to 1.93), septic shock = 3.69 (1.78 to 7.65). The fi nal model had a very good discrimination for long-term mortality with an area under the ROC curve of 0.78 (Figure 1).ii disease = 3.40 (1.64 to 7.04), Karnovsky Index <70 = 2.56 (1.63 to 3.71), infection by an ESKAPE pathogen = 1.65 (1.02 to 2.66) and severity of infection (reference is infection without SIRS): sepsis = 1.14 (0.7 to 1.83), severe sepsis = 1.18 (0.73 to 1.93), septic shock = 3.69 (1.78 to 7.65). The fi nal model had a very good discrimination for long-term mortality with an area under the ROC curve of 0.78 (Figure 1). Conclusion The authors identifi ed several factors that were signifi cantly associated with increased long-term mortality in patients with severe infection. This information will help clinicians in the discussion of individual prognosis and clinical decision-making. Introduction Survivors of sepsis report persistent problems that can last years after hospital discharge. The main aim of this study was to investigate long-term health-related quality of life in survivors of SIRS and sepsis compared with Welsh normative data, controlling for age, length of stay and pre-existing conditions. The second aim was to investigate any diff erences in long-term health-related quality of life specifi cally with the patients categorised into three groups: SIRS, uncomplicated sepsis, and severe sepsis/septic shock. Conclusion The authors identifi ed several factors that were signifi cantly associated with increased long-term mortality in patients with severe infection. This information will help clinicians in the discussion of individual prognosis and clinical decision-making. Independent risk factors for long-term mortality in patients with severe infection Median ICU costs per patients and ICU cost per day according to study years. Figure 1 (abstract P16). Figure 1 (abstract P16). Figure 1 (abstract P17). Median ICU costs per patients and ICU cost per day according to study years. intensivists to contribute a high standard of care within a restricted budget. The cost-eff ectiveness analysis should be evaluated in sepsis care cases. Direct intensive care costs of severe sepsis and septic shock patients in Thailand B Khwannimit, R Bhurayanontachai Songklanagarind Hospital, Hat Yai, Thailand Critical Care 2015, 19(Suppl 1):P17 (doi: 10.1186/cc14097) Introduction Costs of severe sepsis care from middle-income countries are lacking. This study investigated direct ICU costs and factors that could aff ect the fi nancial outcomes. fi Methods A prospective cohort study was conducted in the medical ICU of a tertiary referral university teaching hospital in Thailand over a 4-year period. y p Results A total of 897 patients, with 683 (76.1%) having septic shock. Overall ICU mortality was 38.3%. The median (interquartile range) ICU length of stay (LOS) was 4 (2 to 9) days. Community, nosocomial and ICU-acquired infection were documented in 574, 282 and 41 patients, respectively. The median ICU costs were €2,067.2 (986.3 to 4.084.6) per patient and €456.6 (315.3 to 721.8) per day. The ICU costs accounted for 64.7% of the hospital costs. In 2008 to 2011, the ICU costs signifi cantly decreased by 40% from €2,695.7 to €1,617, whereas the daily ICU costs decreased only 3.3% from €463.9 to €448.7 (Figure  1). The average ICU costs of patient with nosocomial and ICU-acquired infection were signifi cantly higher than patients with community-acquired infection. By multivariate logistic regression analysis, age, nosocomial or ICU infection, admission from emergency department, number of organ failures, ICU LOS, and fl uid balance in the fi rst 72 hours were independently associated with total ICU costs.i Results A total of 106 patients were included in the study. A mortality rate of 34% was recorded, leading to a fi nal response rate of 72% by the end of the data collection period. Quality of life was signifi cantly reduced in all patients when compared with local normative data (all P <0.0001). Reductions in the physical components of health-related quality of life were more pronounced in severe sepsis/septic shock patients when compared with uncomplicated sepsis and SIRS patients. Conclusion This is the fi rst observational study to specifi cally focus on the diff erent groups of SIRS and sepsis patients to assess long-term quality of life. Local population norms were used for comparison, rather than wide geographical norms that fail to refl ect the intricacies of a country’s population. Signifi cant reductions in quality of life were found in severe sepsis/septic shock patients compared with in uncomplicated sepsis and SIRS patients, when controlling for age, pre- existing conditions, hospital and ICU length of stay. Reference g Reference P21 P21 Global burden of sepsis: a systematic review C Fleischmann1, A Scherag1, NK Adhikari2, CS Hartog1, T Tsaganos3, P Schlattmann1, DC Angus4, K Reinhart1 1Jena University Hospital, Jena, Germany; 2University of Toronto, ON, Canada; 3University of Athens, Greece; 4University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P21 (doi: 10.1186/cc14101) Introduction Sepsis is a global healthcare challenge. However, comprehensive information on sepsis morbidity and mortality across the world is scarce. We aimed to estimate the global burden of sepsis and to identify knowledge gaps based on available evidence from observational epidemiological studies. g Methods We searched 15 international and national citation databases for population-level estimates on incidence rates of sepsis or severe sepsis per 100,000 person-years and case fatality rates in adult populations using consensus criteria and published in the last 40 years. No language or publication restrictions were applied. Studies were stratifi ed into four subgroups (setting: hospital or ICU for sepsis and severe sepsis) and meta-analyzed using metaprop of the R 3.0.2 package. Heterogeneity of the underlying eff ects across studies was expressed by the estimated τ, the square root of the between-study variance. y Conclusion Patients admitted to the ICU with severe CAP and immunosuppressive therapy have higher mortality, with no diff erences between HCAP and CAP. The delay in intubation as well as bacterial and inappropriate antibiotic treatment are factors that increase mortality. Results The search yielded 1,553 reports from 1979 to 2013, of which 37 met our criteria and 33 provided data for meta-analysis. The included studies were from 15 high-income countries in North America, Europe, Asia, and Australia. For these countries, the population incidence rate was 256 (95% CI, 182 to 360, τ = 0.43) hospital-treated sepsis cases and 151 (95% CI, 94 to 242, τ = 0.98) hospital-treated severe sepsis cases per 100,000 person-years, with large between-study heterogeneity. Restricted to the last decade, the incidence rate was 427 (95% CI, 281 to 648, τ = 0.24) sepsis cases and 331 (95% CI, 207 to 530, τ = 0.59) severe sepsis cases per 100,000 person-years. Hospital mortality was 15% for sepsis and 25% for severe sepsis during this period of time. There were no population-level sepsis incidence estimates from lower income countries. A tentative extrapolation from high-income-country data suggests global estimates of 30.7 million sepsis and 23.8 million severe sepsis cases, with potentially six million deaths each year. Analysis of the mortality rate in patients admitted to the ICU for severe community-acquired pneumonia Conclusion Severe sepsis and septic shock are conditions that consume large amounts of resources. Nonsurvivors had higher average spending than survivors. Patients admitted with septic shock had higher mortality than patients with severe sepsis with high mortality in relation to the prognostic indices adopted. The beginning of the antibiotics was longer in the nonsurvivors. We should adopt measures aimed at recognizing and earlier treatment of sepsis. If we improve our treatment, especially in septic shock, we will prevent deaths and decrement costs. Introduction The aim of the study was to analyze the factors associated with hospital mortality in patients with severe community-acquired pneumonia (CAP) who required ICU admission. Methods An observational, retrospective study of patients with severe CAP admitted to the ICU between January 2008 and September 2013. We analyzed clinical, epidemiological and outcome variables. Quantitative variables were expressed as the mean and standard deviation. Qualitative variables are expressed as the percentage and absolute value. We applied the Mann–Whitney and Fisher’s exact test, as needed, with an alpha error of 5%. P21 p Results We analyzed 111 patients, 57.5 ± 17.7 years old, with 63.1% (70) males and APACHE II score on admission of 19.8 ± 17.7. ICU mortality was 29.7% (33) and in-hospital mortality was 32.4% (36). Ten percent of patients met criteria for medical care-associated pneumonia (HCAP); there were no signifi cant diff erences in mortality between HCAP and CAP (P = 0.075). Patients chronically taking immunosuppressive therapy had a signifi cantly higher mortality compared with the rest of the patients (47.8% vs. 28.4%, P = 0.07). The mortality rate was also higher in patients in whom NIV fail in the fi rst 24  hours (42.9% vs. 17.6% with P = 0.09). Patients who required intubation and mechanical ventilation in the fi rst 24 hours had a higher mortality rate (47.2% vs. 19%, P = 0.002). Regarding the etiology of pneumonia, in 11 patients the viral origin of infection was confi rmed (10 patients had H1N1 pneumonia and one patient CMV pneumonia), with a mortality rate signifi cantly lower than in patients with bacterial pneumonia (3.6% vs. 35.3%, P = 0.06). The use of the right antibiotic therapy at admission was associated with mortality (P = 0.0001). M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil Introduction Sepsis is a high-prevalence disease in ICUs, associated with high mortality and high costs, mainly in developing countries. The aim of this study is to demonstrate the ICU costs, in a private hospital, in patients admitted with severe sepsis and septic shock. p p p Methods A retrospective, observational, single-center study of patients admitted from November 2013 to March 2014 with severe sepsis and septic shock. The records data were taken from the Software Epimed, MV System, and IBM SPSS Statistics 21. The classifi cation was based on the Surviving Sepsis Campaign 2012. We included all 50 beds of an adult ICU, clinical and surgical. All patients older than 18 years with severe sepsis and septic shock were included. We evaluated the costs of patients during their ICU stay, and its relation to clinical presentation (severe sepsis and septic shock), antibiotic start time, permanence of ICU stay, and mortality. Only the fi rst episode per patient was recorded. Results From November 2013 to March 2014 were included 82 patients with criteria for severe sepsis and septic shock. The mean age of patients was 62.5 ± 21.8 years, divided equally between the genres. The overall mortality rate was 34.15%. The SAPS 3 was 56.43, with death probability set to Latin America 38.83%. Patients with severe sepsis had a mortality of 23.2% and those with septic shock had a mortality rate of 58%. The average total cost during ICU admission per patient was US$17,834 and the average daily cost was US$1,641. The daily cost in patients with severe sepsis and septic shock was US$1,263 and US$2,465 (P = 0.002), respectively, and in survivors and nonsurvivors was US$1,189 and US$2,512 (P = 0.001). The length of stay of patients Conclusion Our analyses underline the urgent need to implement global strategies to monitor sepsis morbidity and mortality  – especially in low-income and middle-income countries. For further epidemiological studies, more consistent and standardized methodological approaches are needed to reduce between-study heterogeneity. In particular, further research on sepsis coding using administrative data seems necessary to derive sensitive and specifi c sepsis case identifi cations. g Reference Conclusion The ICU costs of severe sepsis management signifi cantly declined in Thailand. However, the ICU costs were a fi nancial burden accounting for two-thirds of the hospital costs. It is essential for Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. S7 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P19 in the ICU was 11.09 days, being 11.3 days in patients with severe sepsis and 10.7 days in patients with septic shock (P = 0.785). The beginning of the antibiotics in nonsurvivors was 73.7 minutes and in survivors was 64.7 minutes (P = 0.757), with the earliest onset in patients with septic shock than in patients with severe sepsis (38.5 vs. 81.5 minutes, P = 0.141). P19 Analysis of the mortality rate in patients admitted to the ICU for severe community-acquired pneumonia C Joya-Montosa, MD Delgado-Amaya, H Molina-Diaz, E Curiel Balsera Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P19 (doi: 10.1186/cc14099) Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil Critical Care 2015, 19(Suppl 1):P20 (doi: 10.1186/cc14100) M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil P22 P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) Introduction Disparities in the incidence and outcomes of sepsis have been documented in observational studies but little is known about S8 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We included adult (age >18  years) ED patients presenting with severe sepsis/septic shock (sepsis with elevated lactate (>4  mmol/l)) or hypotension) from the prospective clinical ProCESS trial. We studied a subset of patients with microcirculatory videos obtained along with non-infected control patients. Using a sidestream dark-fi eld videomicroscope, we obtained image sequences from the sublingual mucosa and used video stabilization and frame averaging techniques to visualize slowly-moving leukocytes. We quantifi ed the number of rolling and adhered leukocytes present per 1 mm × 1 mm visual fi eld in a standardized 3-second clip. Furthermore, we extracted the total length of vessels candidate for counting of rolling/adhered leukocytes (vessels with an adequate focus). We report sample means with standard deviation and compare them with Student’s t test. how these occur and how we might prevent them. Our objective is to identify disparities by race, language, gender, socioeconomic status, insurance status and geography in acute sepsis care in emergency department (ED) or ICU settings in the published literature. p g p Methods We performed a systematic review of disparities in sepsis care. The search strategy and inclusion and exclusion criteria were defi ned a priori. A medical librarian searched the entire MEDLINE (PubMed), EMBASE and Cinahl databases prior to 2013. One author reviewed all abstracts and a second author reviewed 10% of all abstracts for agreement. Both reviewers independently reviewed each included article using an explicit study review tool. P22 We included studies that met the following inclusion criteria: ED or ICU setting; disparities due to race, language, gender, socioeconomic status, insurance status or geography; process of care measures (antibiotics, lactate, i.v. fl uid resuscitation, central line placement, vasopressor use) or outcome measures (mortality, length of stay, complications, costs). We excluded studies involving organ-specifi c infectious conditions, pediatric populations, case reports, and review articles.i p Results We included a total of 64 patients with severe sepsis/septic shock and 32 non-infected controls. The mean number of adhered leukocytes per fi eld in the sepsis group was 2.1 (SD 2.3) compared with 0.4 (SD 0.8) in the non-infected group (P <0.001). This corresponded to a mean number of adhered leukocytes per unit vessel length of 0.16/mm (SD 0.22) and 0.03/mm (SD 0.06) for sepsis and non-infected groups, respectively (P <0.001). For the rolling leukocytes, we observed a mean number of 27.8 (SD 19.4) in the sepsis group and 12.0 (SD 8.7) in the non-infected group (P <0.001) per fi eld. This corresponded to a mean number of rolling leukocytes per unit vessel length of 2.00/mm (SD 1.67) and 0.75/mm (SD 0.55), respectively (P <0.001). Results We identifi ed 778 abstracts; yielding 31 for inclusion (k = 0.95), 26 of 31 studies were excluded due to quality issues. Five articles met our inclusion criteria. Only one of the studies [1] contained data on process of care measures, showing that central venous monitoring was less likely to occur in older patients. Three studies [2-4] showed that Black patients had a higher incidence of sepsis, a higher hospitalization rate, and higher mortality rate. Plurad and colleagues [5] reported that Asian patients had increased incidence of post-traumatic sepsis. Overall, Black patients with sepsis were younger, had lower socioeconomic status and were more likely to be cared for in urban settings compared with their cohorts. p y Conclusion Our results show a higher number of rolling and adhered leukocytes in patients with severe sepsis/septic shock when compared with non-infected controls. This also applies when taking the total vessel length in the fi eld of view into consideration. This may hold potential as a useful tool in sepsis assessment. Conclusion We found little published data addressing whether disparities due to race, language, gender, socioeconomic status, insurance status or geography exist in the acute care of sepsis. P23 S bli g y Methods This is a prospective observational study in patients scheduled for elective cardiac surgery. Serum samples were drawn prior to surgery, after connection to cardiopulmonary bypass (ischemia), after opening of cross-clamp (reperfusion) and after termination of surgery. The redox status of patients was measured using the bedside point of care RedoxSYS Diagnostic System™ (Luoxis, USA). Simultaneously the antioxidant capacity in serum samples were calculated in all perioperatively obtained serum samples. P24 P24 Time course of redox potential and antioxidant capacity in patients undergoing cardiac surgery C Stoppe1, G Schaelte1, S Kraemer2, C Benstoem2, D Bar-Or3, A Goetzenich2 1RWTH Aachen University, Aachen, Germany; 2RWTH Aachen University, University Hospital, Aachen, Germany; 3Swedish Medical Center, Trauma Research, Engelwood, CO, USA Critical Care 2015, 19(Suppl 1):P24 (doi: 10.1186/cc14104) References Lagu T, Rothberg MB, Nathanson BH, et al. Variation in the care of se 1. Lagu T, Rothberg MB, Nathanson BH, et al. Variation in the care of septic shock: the impact of patient and hospital characteristics. J Crit Care. 2012;27:329-36. 2. Barnato AE, Alexander SL, Linde-zwirble WT, Angus DC. Racial variation in the incidence, care, and outcomes of severe sepsis: analysis of population, patient, and hospital characteristics. Am J Respir Crit Care Med. 2008;177:279-84. 3. Dombrovskiy VY, Martin AA, Sunderram J, Paz HL. Occurrence and outcomes of sepsis: infl uence of race. Crit Care Med. 2007;35:763-8. Introduction Cardiac surgery regularly provokes infl ammation and oxidative stress which contribute to the development of organ failure and mortality of patients. While the assessment of single markers does not refl ect a comprehensive investigation of redox status, the measurement of oxidation–reduction potential (ORP) provides a reliable measure to assess the balance between total prooxidant and antioxidant balance in the blood. The aim of the present study was to investigate the overall redox potential in patients undergoing cardiac surgery. 4. Mayr FB, Yende S, Linde-zwirble WT, et al. Infection rate and acute organ dysfunction risk as explanations for racial diff erences in severe sepsis. JAMA. 2010;303:2495-503. 5. Plurad DS, Lustenberger T, Kilday P, et al. The association of race and survival from sepsis after injury. Am Surg. 2010;76:43-7. 5. Plurad DS, Lustenberger T, Kilday P, et al. The association of race and survival from sepsis after injury. Am Surg. 2010;76:43-7. P22 As sepsis is a leading cause of in-hospital mortality, future research should determine whether such disparities exist. Specifi cally, prospective studies of the process of care in sepsis management may further elucidate additional factors that may contribute to these disparities. R f Fatty acid composition of erythrocytes in multiple organ dysfunction syndrome y y A Osipenko1, A Marochkov2 A Osipenko1, A Marochkov2 1A. Kuleshov Mogilev State University, Mogilev, Belarus; 2Mogilev Regional Hospital, Mogilev, Belarus Critical Care 2015, 19(Suppl 1):P25 (doi: 10.1186/cc14105) Results We found that severe septic patients showed lower CIV activity/ protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. Survivor severe septic patients (n  = 130) showed lower CIV activity/protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. In addition, nonsurvivor severe septic patients (n = 68) showed lower CIV activity/protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. Besides, nonsurvivor severe septic patients showed lower CIV activity/protein quantity than survivor ones at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis.i p , g , Critical Care 2015, 19(Suppl 1):P25 (doi: 10.1186/cc14105) Introduction Change in fatty acid composition of erythrocytes and blood plasma in cases of various pathological conditions is evidence of lipid metabolism disorder and can indicate the reasons for and the degree of these disorders [1]. The aim of this study was to assess the FA composition of plasma and erythrocytes in patients with multiple organ dysfunction syndrome (MODS). g y y Methods The objects of study were 19 people with MODS (37.6  ±  8.3  years) of various etiologies. The blood of 17 healthy volunteers aged 38.4 ± 3.3 years served as control. The FA analysis was conducted using capillary gas–liquid chromatography. Quantitative evaluation of individual FA content was made as a mass percentage of their total (C14:0 to C22:6). Statistical analysis was performed using the Mann–Whitney U test (P <0.05). y p g Conclusion The major fi nding of our work, that represents the largest series of severe septic patients with data on OXPHOS function, was that survivor and nonsurvivor severe septic patients showed lower platelet CIV activity than healthy controls during the fi rst week of severe sepsis diagnosis. y Results Our data indicate that changes in blood plasma FA composition in patients with MODS are mainly caused by activation of lipolysis in fat depots and are accompanied by an increase of monounsaturated fatty acids, a decrease in saturated stearic acid and polyunsaturated fatty acids in the ratio. Sublingual leukocyte activation in patients with severe sepsis or septic shock BK Fabian-Jessing1, MJ Massey1, MR Filbin2, PC Hou3, H Kirkegaard4, HE Wang5, DM Yealy6, JA Kellum6, DC Angus6, NI Shapiro1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3Brigham and Women’s Hospital, Boston, MA, USA; 4Aarhus University Hospital, Aarhus, Denmark; 5University of Alabama at Birmingham, AL, USA; 6University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P23 (doi: 10.1186/cc14103) y Results All patients’ sera (n = 17) demonstrated a signifi cant increase of ORP upon start of myocardial ischemia (141.0 ± 4.8 mV vs. 157.9 ± 4.9 mV; P  =  0.002) and compared with reperfusion (141.0  ±  4.8 mV vs. 158.6  ±  4.9mV; P  <0.001, Figure  1A). In parallel, the antioxidant capacity signifi cantly decreased during surgery (0.505 ± 0.190 μC vs. 0.384 ± 0.120 μC; P = 0.022) corresponding to the increase of oxidative stress (Figure 1B). Introduction The objective of this study was to compare the number of rolling and adhered leukocytes in patients with severe sepsis/septic shock with non-infected controls. Microcirculatory fl ow alterations and endothelial cell dysfunction are elements of sepsis pathophysiology. Traditionally, microcirculatory emphasis has been on red blood cell vessel perfusion. However, assessment of interactions between white blood cells and endothelial cells may be another early diagnostic modality. Introduction The objective of this study was to compare the number of rolling and adhered leukocytes in patients with severe sepsis/septic shock with non-infected controls. Microcirculatory fl ow alterations and endothelial cell dysfunction are elements of sepsis pathophysiology. Traditionally, microcirculatory emphasis has been on red blood cell vessel perfusion. However, assessment of interactions between white blood cells and endothelial cells may be another early diagnostic modality. Conclusion This preliminary study is the fi rst to highlight the time course of overall redox potential and antioxidant capacity in cardiac surgery patients. Further studies are underway to evaluate the clinical signifi cance on outcome in cardiac surgery patients. Critical Care 2015, Volume 19 Suppl 1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S9 Figure 1 (abstract P24). (A), (B) Perioperative time course of oxidative stress and antioxidant capacity. acids. In the test group of patients, as compared with the control group there was an elevated level (27.12 ± 0.78% vs. 25.80 ±0.77%, P <0.05) of saturated palmitic (C16:0) acid combined with the reduced (11.46 ± 0.52% vs. 13.95 ± 1.09%, P <0.001) level of linoleic (C18:2) acid. Reference 1. Kremmyda LS, et al. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155:195-218. P26 Lower platelet mitochondrial function in severe septic patients than in controls L Lorente1, M Martin2, J Blanquer3, J Solé-Violán4, L Labarta5, C Díaz6, A Jiménez1, E López-Gallardo7, J Montoya7, E Ruiz-Pesini7 1Hospital Universitario de Canarias, La Laguna, Tenerife, Spain; 2Hospital Universitario Nuestra Señora Candelaria, Santa Cruz, Tenerife, Spain; 3Hospital Clínico Universitario de Valencia, Spain; 4Hospital Universitario Dr Negrín, Las Palmas de Gran Canaria, Spain; 5Hospital San Jorge, Huesca, Spain; 6Hospital Insular, Las Palmas de Gran Canaria, Spain; 7Universidad de Zaragoza, Spain Critical Care 2015, 19(Suppl 1):P26 (doi: 10.1186/cc14106) Introduction The oxidative phosphorylation system (OXPHOS) in septic patients has been scarcely analyzed in studies of small sample size and the results are apparently inconsistent. Previously, including 96 severe septic patients, we found that nonsurviving severe septic patients showed lower platelet respiratory complex IV (CIV) activity than surviving patients at the moment of severe sepsis diagnosis and during the fi rst week of sepsis diagnosis. However, we did not examine this enzyme activity in normal individuals. Thus, the objective of this study was to compare the CIV activity between severe septic patients and healthy control individuals in a larger series of patients (including 198 severe septic patients). Introduction The oxidative phosphorylation system (OXPHOS) in septic patients has been scarcely analyzed in studies of small sample size and the results are apparently inconsistent. Previously, including 96 severe septic patients, we found that nonsurviving severe septic patients showed lower platelet respiratory complex IV (CIV) activity than surviving patients at the moment of severe sepsis diagnosis and during the fi rst week of sepsis diagnosis. However, we did not examine this enzyme activity in normal individuals. Thus, the objective of this study was to compare the CIV activity between severe septic patients and healthy control individuals in a larger series of patients (including 198 severe septic patients). Figure 1 (abstract P24). (A), (B) Perioperative time course of oxidative stress and antioxidant capacity. Methods This was a prospective, multicenter, observational study in six Spanish ICUs. We obtained blood samples from 198 severe septic patients at days 1, 4 and 8 of the severe sepsis diagnosis and from 96 sex-matched and age-matched healthy control individuals and determined platelet CIV activity/protein quantity. The endpoint of the study was 30-day mortality. Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection J Sole-Violan1, M López-Rodríguez1, E Herrera-Ramos1, J Ruiz-Hernández1, J Horcajada2, L Borderías3, J Blanquer4, J Ferrer1, O Rajas5, J Aspa5, F Rodríguez de Castro1, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital del Mar, Barcelona, Spain; 3Hospital San Jorge, Huesca, Spain; 4Hospital Clínico, Valencia, Spain; 5Hospital de la Princesa, Madrid, Spain Critical Care 2015, 19(Suppl 1):P27 (doi: 10.1186/cc14107) Fatty acid composition of erythrocytes in multiple organ dysfunction syndrome In conditions of increased level of monounsaturated palmitoleic (C16:1) and oleic (C18:1) FA in blood plasma (2.53 ± 0.40% vs. 1.55 ± 0.29%, P <0.001 and 25.18 ± 2.15% vs. 16.55 ± 1.17%, P <0.001, respectively), only the level of palmitoleic (C16:1) acid is increased in erythrocytes (0.56  ±  0.12% vs. 0.16  ±  0.12%, P  <0.001). Despite the high content of oleic (C18:1) acid in blood plasma in case of MODS, in erythrocytes its relative level is not changed as compared with the control group. The disorder of lipid composition constancy in erythrocyte membranes is also manifested by change in the content of saturated palmitic (C16:0) and polyunsaturated linoleic (C18:2) fatty Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection J Sole-Violan1, M López-Rodríguez1, E Herrera-Ramos1, J Ruiz-Hernández1, J Horcajada2, L Borderías3, J Blanquer4, J Ferrer1, O Rajas5, J Aspa5, F Rodríguez de Castro1, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital del Mar, Barcelona, Spain; 3Hospital San Jorge, Huesca, Spain; 4Hospital Clínico, Valencia, Spain; 5Hospital de la Princesa, Madrid, Spain Critical Care 2015, 19(Suppl 1):P27 (doi: 10.1186/cc14107) 1. Kremmyda LS, et al. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155:195-218. Sublingual leukocyte activation in patients with severe sepsis or septic shock Conclusion The changes revealed in fatty acid composition of erythrocytes may indicate systemic modifi cations of cell membranes in MODS. Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort Results Patients who developed sepsis (n  = 18) presented with signifi cantly higher platelet fi brinogen binding at T1 compared with patients who did not get infected (basal: P = 0.0014, upon stimulation: P <0.0035). At T1, ROC AUC for association of basal fi brinogen binding with the occurrence of sepsis was 0.79 (95% CI: 0.68 to 0.89). Elevated basal CD62P expression level was associated with increased 90-day mortality (P  =  0.042, ROC AUC  = 0.78 (0.64 to 0.88)). Kaplan–Meier survival curves illustrated that mortality was signifi cantly higher after stratifi cation based on T1 basal CD62P level (cutoff MFI >31.56, HR = 13.6, P  =  8.23  ×  10–6). Multivariate logistic regression analysis using clinical scores (SOFA, APACHE II, SAPS II, SAPS III) indicated that addition of CD62P level or of bound fi brinogen level signifi cantly improved prediction of mortality (odds ratio 1.078, P = 0.003) and sepsis (odds ratio 1.033, P = 0.0012), respectively. A Tridente, G Clarke, A Walden, A Gordon, P Hutton, J Chiche, P Holloway, G Mills, J Bion, F Stuber, C Garrard, C Hinds, GenOSept Investigators St Helens and Knowsley, Liverpool, UK l l (d ) Introduction Patients admitted to intensive care following surgery for faecal peritonitis present particular challenges in terms of clinical management and risk assessment that require close collaboration between surgical and intensive care teams [1]. We aimed at establishing whether dynamic assessment of trends in selected variables may be associated with outcomes, and therefore inform medical decision-making. g Methods We analysed trends in all 35 variables available for the fi rst week of ICU stay in 977 patients from 102 centres across 17 countries. The primary study outcome was 6-month mortality. Secondary outcomes were ICU, hospital and 28-day mortality. For each trend, Cox proportional hazards (PH) regression analyses, adjusted for age and gender, were performed for each endpoint. Trends found to be signifi cant in these analyses, after Bonferroni correction for multiple testing, were entered into a multivariate Cox PH model, to determine independent associations with mortality.i Conclusion Predisposition to severe infection in selected critically ill medico-surgical adults can be identifi ed on day 1 of admission based on circulating basally activated platelets. Levels of activated platelets may add incremental prognostic information to clinical scoring. Reference 1. de Stoppelaar SF, van ‘t Veer C, van der Poll T. The role of platelets in sepsis. Thromb Haemost. 2014;11:666-77. Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort p y Results The trends over the fi rst 7 days of ICU stay (primary analysis) retained as independently associated with 6-month outcome were worsening thrombocytopaenia (mortality HR = 1.02, 95% CI = 1.01 to 1.03, P <0.001) and changes in renal function (total daily urine output HR = 1.02, 95% CI = 1.01 to 1.03, P <0.001; renal SOFA subscore HR = 0.87, 95% CI = 0.75 to 0.99, P = 0.047), highest recorded level of bilirubin (HR = 0.99, 95% CI = 0.99 to 0.99, P = 0.02) and GCS SOFA subscore (HR = 0.81, 95% CI = 0.68 to 0.98, P = 0.028). Changes in renal function (total daily urine output and renal component of the SOFA score), GCS component of the SOFA score, total SOFA and worsening thrombocytopaenia were also independently associated with secondary outcomes. Dynamic trends over the fi rst 7 days of ICU stay in all other measured laboratory variables, physiological parameters or radiological fi ndings failed to be retained as independently associated with outcome on multivariate analyses. Furthermore, changes in respiratory support, renal replacement therapy and inotropic and/or vasopressor requirements appeared not to be independently associated with any of the primary or secondary outcomes. Secondary post hoc analyses on trends over the fi rst 3 and 5 days corroborated these fi ndings. P29 P29 Elevated basal levels of circulating activated platelets predict ICU-acquired sepsis and mortality: a prospective study N Layios CHU Sart Tilman, Liège, Belgium Critical Care 2015, 19(Suppl 1):P29 (doi: 10.1186/cc14109) P30 Antiplatelet therapy does not infl uence outcome or host response biomarkers during sepsis: a propensity-matched analysis MA Wiewel1, SF De Stoppelaar1, LA Van Vught1, JF Frencken2, AJ Hoogendijk1, PM Klein Klouwenberg2, J Horn1, MJ Bonten2, MJ Schultz1, AH Zwinderman1, OL Cremer2, T Van der Poll1 1Academic Medical Center, University of Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P30 (doi: 10.1186/cc14110) P28 P28 Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort A Tridente, G Clarke, A Walden, A Gordon, P Hutton, J Chiche, P Holloway, G Mills, J Bion, F Stuber, C Garrard, C Hinds, GenOSept Investigators St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P28 (doi: 10.1186/cc14108) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 have previously demonstrated that variants at SFTPA2 infl uence the severity of H1N1pdm infection. We have now studied genetic variants at diff erent genes, some of them previously associated with infections by infl uenza and/or other viruses. The purpose of this study was to analyze the role of genetic variants in the susceptibility and outcome of IVI. Conclusion Only deterioration in renal function, thrombocytopaenia and hyperbilirubinaemia over the fi rst 7  days of ICU stay were consistently associated with mortality at all endpoints. Reference 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. Methods In total, 136 white Spanish patients developed IVI (80.3% of them by H1N1pdm virus). The general population group consisted of 1,466 unrelated healthy volunteers. Patients and controls were analyzed for diff erent polymorphisms at 13 genes (FCGR2A, FCGR3A, FCGR3B, IL1RN, IL6, LTA, TIRAP, TLR1, TLR2, TLR3, TLR4, CCR5, IGHG2). Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection Introduction The role of genetic variability in the susceptibility and outcome of infl uenza virus infection (IVI) remains largely unknown. We Introduction The role of genetic variability in the susceptibility and outcome of infl uenza virus infection (IVI) remains largely unknown. We S10 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 1. de Stoppelaar SF, van ‘t Veer C, van der Poll T. The role of platelets in sepsis. Thromb Haemost. 2014;11:666-77. 9 Elevated basal levels of circulating activated platelets predict ICU-acquired sepsis and mortality: a prospective study N Layios CHU Sart Tilman, Liège, Belgium Critical Care 2015, 19(Suppl 1):P29 (doi: 10.1186/cc14109) IVI was detected in nasopharyngeal swabs using real-time PCR. The Hardy–Weinberg equilibrium was analyzed by Haploview v. 4.2. The comparisons of genotypes distribution based on susceptibility and severity were performed using the chi-squared test or Fisher’s exact test when needed. The relationship between severity in hospitalized patients and genotypes was evaluated by binary logistic regression models. Introduction Platelets are now considered to be immune and infl am- matory agents as well as key cells in coagulation, and as such have been implicated in the pathophysiology of sepsis [1]. Thrombocytopenia is associated with sepsis severity and poor prognosis, and hyperactivated platelets probably contribute to microvascular thrombosis and organ failure. In the present study, we evaluated platelet activation markers as potential predictive markers of sepsis and of mortality among four commonly encountered populations of patients admitted to ICUs. Results No associations were found between the diff erent genetic variants and susceptibility or severity of IVI. Variants at LTA, FCGR2A, IGHG2, TLR3 and CCR5, previously associated with severity of IVI were not replicated in our study. Methods Ninety-nine non-infected ICU patients were prospectively screened at day 1 (T1) and day 3 (T2) of admission after elective cardiac surgery, trauma, acute neurologic dysfunction or prolonged ventilation (>48 hours). A third sample was drawn when infection was diagnosed (Tx). We evaluated platelet activation by measuring the expression of P-selectin (CD62P) and fi brinogen binding on the cell surface before and after stimulation with major platelet agonists (ADP, collagen, and TRAP) through fl ow cytometry. Clinical scores were obtained at admission. Conclusion Our study does not suggest that polymorphisms at LTA, FCGR2A, IGHG2, TLR3 and CCR5 genes are associated with susceptibility or severity of IVI. P32 Methods We performed a prospective observational study in patients admitted with sepsis to the mixed ICUs of two hospitals in the Netherlands between January 2011 and July 2013. Cox proportional hazards regression was used to estimate the eff ect of antiplatelet therapy on mortality. To account for indication bias, a propensity score was constructed, and used to match antiplatelet therapy users to nonusers. Plasma biomarker levels, providing insight into hallmark host responses to sepsis, including activation of endothelial cells and the cytokine network, were determined during the fi rst 4 days after ICU admission. Mitochondrial dysfunction and ischemia in critical illness: an adipose tissue microdialysis study in 203 ICU patients M Theodorakopoulou, S Apollonatou, N Nikitas, D Vassiliadi, A Diamantakis, V Tsagkari, F Frantzeskaki, I Dimopoulou University Hospital of Athens, Greece Critical Care 2015, 19(Suppl 1):P32 (doi: 10.1186/cc14112) Introduction Ischemia and mitochondrial dysfunction have been implicated in critical illness. The potential of MD to diagnose and separate ischemia and mitochondrial dysfunction in ICU patients remains currently unknown. Introduction Ischemia and mitochondrial dysfunction have been implicated in critical illness. The potential of MD to diagnose and separate ischemia and mitochondrial dysfunction in ICU patients remains currently unknown. Results Of 1,070 sepsis patients, 297 (27.8%) were on antiplatelet therapy, including acetylsalicylic acid, clopidogrel and dipyridamole, prior to ICU admission. Antiplatelet users and nonusers diff ered signifi cantly with regard to several baseline characteristics, such as age, gender and cardiovascular disease. Antiplatelet therapy was not related to sepsis severity at presentation, the primary source of infection, causative pathogens, the development of organ failure or shock during ICU stay, or mortality up to 90 days after admission, in either the unmatched or propensity-matched analyses. Antiplatelet therapy did also not modify plasma concentrations of biomarkers.l Methods A retrospective, observational study of 203 mechanically ventilated patients studied over a 6-year period with MD including medical, surgical and trauma patients. Sepsis stages: SIRS (n  = 24), severe sepsis (n = 46) and septic shock (n = 133). Median age 67 years (range: 17 to 92  years). Mortality was 53%. All subjects had a MD catheter placed in femoral adipose tissue upon admission to the ICU. Interstitial fl uid samples were collected six times per day, for 3 consecutive days, and were analyzed for glucose, lactate, pyruvate, and glycerol levels. The lactate to pyruvate (LP) ratio was calculated. Blood lactate was measured. P31 Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery S Ono1, T Ikeda1, T Kubo2, H Tsujimoto2, M Kinoshita2, T Ueno1 1Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan; 2National Defense Medical College, Tokorozawa, Saitama, Japan Critical Care 2015, 19(Suppl 1):P31 (doi: 10.1186/cc14111) Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery Antiplatelet therapy does not infl uence outcome or host response biomarkers during sepsis: a propensity-matched analysis l1 l 1 h 1 k 2 Introduction Sepsis is a life-threatening condition, during which triggering of infl ammatory and coagulation cascades, together with endothelial damage, invariably leads to activation of platelets. Although platelets are essential components of primary hemostasis, uncontrolled platelet activation during sepsis may contribute to organ failure. The aim of this study was to investigate whether chronic antiplatelet therapy impacts on the presentation and outcome of, and the host response to, sepsis. S11 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery g g g y S Ono1, T Ikeda1, T Kubo2, H Tsujimoto2, M Kinoshita2, T Ueno1 1Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan; 2National Defense Medical College, Tokorozawa, Saitama, Japan Critical Care 2015, 19(Suppl 1):P31 (doi: 10.1186/cc14111) Introduction Programmed death-1 (PD-1) has been reported to be an immunoinhibitory receptor expressed by chronically stimulated T cells after T-cell activation. The present study was designed to evaluate the relationship between perioperative PD-1 expression on CD4+ T cells and the incidence of postoperative infectious complications in patients undergoing gastroenterological surgery. Conclusion Bedside subcutaneous adipose tissue MD is possible to diagnose and separate ischemia and mitochondrial dysfunction in general ICU patients. These two conditions are not so common; however, mitochondrial dysfunction seems to be associated with higher mortality rates. Methods This was a prospective observational study. The subjects of this study included 101 patients with gastroenterological disease who underwent elective abdominal surgery via laparotomy at the National Defense Medical College Hospital. Blood samples were taken on the preoperative day (Pre) and the fi rst postoperative day (POD1). We calculated CD4+ T-cell count and PD-1 expression on CD4+ T cells by fl ow cytometer. The occurrence of postoperative infectious complications was defi ned according to a combination of clinical fi ndings and the results of laboratory and other tests. The postoperative infectious complications in this study included incisional surgical site infections (SSIs), organ/space SSIs, enterocolitis, urinary tract infections, and pneumonia. Incisional and organ/space SSIs were diagnosed according to the defi nitions stated in the guidelines issued by the Center for Disease Control and Prevention. P32 Ischemia was defi ned as LP ratio >30 and pyruvate level <70 mmol, while mitochondrial dysfunction was defi ned as LP ratio >30 and pyruvate >70 mmol. y y Conclusion Pre-existing antiplatelet therapy does not infl uence clinical disease severity at presentation, nor the host response or outcome following sepsis. Acknowledgement This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (http://www. ctmm.nl), project MARS (grant 04I-201). py Results Analysis during the course of the 3-day period revealed three distinct patterns: no ischemia/mitochondrial dysfunction (n = 150 or 74%), ischemia (n = 27 or 13%) and mitochondrial dysfunction (n = 26 or 13%). On day 1, median blood lactate was higher in mitochondrial dysfunction (2.2 mmol/l) compared with both ischemia (1.3 mmol/l) and with no ischemia/mitochondrial dysfunction (1.3  mmol/l) (P = 0.004). Again on day 1, median interstitial fl uid lactate was higher in mitochondrial dysfunction (8.4 mmol/l), in comparison with ischemia (1.4  mmol/l) and with the group without ischemia/mitochondrial dysfunction (2.5 mmol/l) (P <0.001). Similar results were obtained with interstitial fl uid glycerol levels (P = 0.009). Median LP ratio was higher in ischemia (LP = 36), and mitochondrial dysfunction (LP = 33) compared with those without ischemia/mitochondrial dysfunction (LP  =  17) (P  <0.001). Median interstitial fl uid glucose was lower in ischemia (2 mmol/l) compared with both mitochondrial dysfunction (4 mmol/l) and with no ischemia/mitochondrial dysfunction (5 mmol/l) (P <0.001). ICU mortality was 77% in mitochondrial dysfunction, 52% in ischemia and 49% in the group without ischemia/mitochondrial dysfunction (P = 0.033). Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Beth Israel Deaconess Medical Center, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P33 (doi: 10.1186/cc14113) Introduction Pyruvate dehydrogenase (PDH) is a key component of aerobic metabolism. Multiple rodent studies have shown that PDH levels are low in sepsis. This leads to a shift to anaerobic metabolism, resulting in increased lactic acid. Alteration in PDH levels during sepsis, however, has never been studied in humans. The aim of this study was to identify whether PDH levels (activity and quantity) were altered in humans in sepsis. Results Postoperative infectious complications occurred in 30 of the 101 patients. CD4+ T-cell count was signifi cantly lower in the patients who developed postoperative infectious complications at POD1 compared with those from the patients who did not. In addition, PD-1 expression on CD4+ T cells was signifi cantly higher at Pre or POD1 in patients who developed postoperative infectious complications. Those results were similar for the incidence of organ/space surgical site infection. Preoperative PD-1 expression on CD4+ T cells tended to be higher in males than in females. We found there was a signifi cant negative correlation between preoperative PD-1 expression on CD4+ T cells and CD4+ T-cell count. Methods We conducted a case–control study at a single urban tertiary care center. We compared PDH levels between sepsis and healthy control subjects by measuring PDH levels in peripheral blood mononuclear cells via a novel assay. We measured PDH levels in control subjects at baseline and in sepsis subjects at 0, 24, 48 and 72 hours. Results There were 39 sepsis (age 67 ± 14 years, M ± SD) and 19 control (age 50 ± 12 years) subjects of similar gender (56% and 63% female, respectively) and race (79% and 68% Caucasian, respectively). PDH levels in the sepsis group were signifi cantly lower than the control Conclusion Perioperative CD4+ T-cell count or PD-1 expression on CD4+ T cells could be an early predictive marker for the development of postoperative infectious complications. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S12 group at all time points (Figures  1 and 2). After controlling for age, gender, race, and assay plate via multivariable linear regression, the eff ect of treatment group remained signifi cant. We were unable to control for comorbid illness, which was exclusively concentrated in the sepsis group. Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Figure 1 (abstract P33). Figure 2 (abstract P33). Figure 2 (abstract P33). Conclusion In the nonsurvivor or the severe patient with sepsis requiring steroid administration, the enhancement of C1INH activity was not observed, and the C1INH quantitative values were low. Further evaluation of the serial change of C1INH and the validity of C1INH replacement therapy in patients with septic shock may lead to a new strategy for management in sepsis. Expression of apolipoproteins L in neutrophils during sepsis I Akl1, C Lelubre1, M Piagnerelli1, P Biston1, P Uzureau1, H Fayyad Kazan2, B Badran3, M Ezzedine3, K Zouaoui Boudjeltia1, L Vanhamme4 1CHU de Charleroi-Hopital Andre Vesale, Montigny-Le-Tilleul, Belgium; 2Institut Jules Bordet, Université Libre de Bruxelles, Belgium; 3Doctoral School of Sciences and Technology, Platform of Research and Environmental Sciences, Beirut, Lebanon; 4Institute of Medicine and Molecular Biology IBMM, Charleroi, Belgium Critical Care 2015, 19(Suppl 1):P35 (doi: 10.1186/cc14115) Figure 2 (abstract P33). Figure 2 (abstract P33). group at all time points (Figures  1 and 2). After controlling for age, gender, race, and assay plate via multivariable linear regression, the eff ect of treatment group remained signifi cant. We were unable to control for comorbid illness, which was exclusively concentrated in the sepsis group.i Introduction Sepsis is characterized by a strong systemic infl ammatory reaction. The pathogenesis is driven by alterations in the immune system and is associated with high neutrophil counts related to a specifi c delay in apoptosis [1]. The apolipoproteins L (ApoLs) family comprises six members in humans (ApoL1 to ApoL6). In light of their deregulated expression in several pathologies, they are likely to be important molecular players of programmed cell death [2]. We analyzed ApoL expression in cohorts of septic and nonseptic ICU patients and healthy volunteers in order to test whether ApoLs could be involved in the neutrophil apoptotic program. Introduction Sepsis is characterized by a strong systemic infl ammatory reaction. The pathogenesis is driven by alterations in the immune system and is associated with high neutrophil counts related to a specifi c delay in apoptosis [1]. The apolipoproteins L (ApoLs) family comprises six members in humans (ApoL1 to ApoL6). In light of their deregulated expression in several pathologies, they are likely to be important molecular players of programmed cell death [2]. Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno We analyzed ApoL expression in cohorts of septic and nonseptic ICU patients and healthy volunteers in order to test whether ApoLs could be involved in the neutrophil apoptotic program. Conclusion PDH levels are signifi cantly lowered in humans during sepsis when compared with healthy controls, even when controlling for age, race and gender. Further research is needed to determine whether this fi nding persists after adjustment for comorbid disease, and whether lower PDH levels are associated with clinical outcomes. Methods By means of magnetic cell sorting, peripheral neutrophils were purifi ed from 20 healthy volunteers and 40 ICU patients with (n = 20) or without sepsis (n = 20). ApoL expression was analyzed at the mRNA and protein levels by real-time PCR and western blot analysis respectively. Apoptosis of purifi ed neutrophils was assessed using fl ow cytometry following 4 and 24  hours of in vitro incubation. We monitored the expression of C-reactive protein (CRP), an infl ammatory marker, and its correlation with ApoL expression in PMNs was studied by linear regression analysis.i Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Conclusion PDH levels are signifi cantly lowered in humans during sepsis when compared with healthy controls, even when controlling for age, race and gender. Further research is needed to determine whether this fi nding persists after adjustment for comorbid disease, and whether lower PDH levels are associated with clinical outcomes. P34 S i l h f C1 i hibit i ti t ith i li i Figure 1 (abstract P33). Figure 2 (abstract P33). Figure 1 (abstract P33). Figure 1 (abstract P33). but also the plasma kallikrein–kinin system, fi brinolytic system and coagulation system. The biologic activities of C1INH can be divided into the regulation of vascular permeability and anti-infl ammatory functions. In recent years, hereditary angioedema (HAE), caused by an inherited defi ciency of C1INH, has been focused. During HAE attacks, vascular permeability was markedly increased, which leads to angioedema. In sepsis, signifi cant endothelial hyperpermeability is similarly observed systemically, but the role of C1INH has not been clarifi ed in the pathogenesis. The serial change of C1INH in patients with sepsis is not clear. The objective of this study was to clarify the serial change in C1INH in patients with sepsis and evaluate the impact of C1INH on their clinical course. Methods We serially examined C1INH activity values (normal range 70 to 130%) and quantitative values (normal range 160 to 330 μg/ml) in patients with sepsis during the period between December 2012 and February 2013. We also analyzed their clinical course: prognosis, volume of infusion, body weight, urine volume, catecholamine administration, and steroid administration. Results The serial change of C1INH was evaluated in fi ve patients with sepsis (three male and two female; four survivors and one nonsurvivor; mean age, 68  ±  11  years). In the nonsurvivor, C1INH activity on admission value was 97.2% (normal range), and quantitative value was 133.1  μg/ml (below normal). In the patient with severe sepsis requiring fl uid resuscitation, catecholamine and steroid administration to maintain hemodynamics, C1INH activity value on admission was 94.4% (normal range), and quantitative value was 126.7 μg/ml (below normal range). His general condition was improved on day 6, and C1INH activity value and quantitative value increased (139.9%; above normal range, 250.1 μg/ml; normal range). In the other three patients with sepsis not requiring steroid administration, C1INH activity value on admission was 130.6 ± 8.7% (above normal range), and quantitative value was 215 ± 26.5 μg/ml (normal range). P34 Serial change of C1 inhibitor in patients with sepsis: a preliminary report T Hirose1, H Ogura1, K Jinkoo1, Y Nakamura1, H Hosotsubo1, T Shimazu1, E Kitano2, M Hatanaka2 1Osaka University Graduate School of Medicine, Suita, Japan; 2Kobe Tokiwa University, Kobe, Japan Critical Care 2015, 19(Suppl 1):P34 (doi: 10.1186/cc14114) Introduction C1 inhibitor (C1INH), belonging to the superfamily of serine protease inhibitors, regulates not only complement system, Results Our results showed a signifi cant downregulation in mRNA expression of ApoL1 (P <0.0001), ApoL2 (P = 0.0009), ApoL3 (P <0.0001) S13 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 correlates inversely with outcome. The aim of the study was to identify phenotypic and functional early markers of T cells and NK cells related to prognosis in the septic patient population. and ApoL6 (P = 0.0003) in purifi ed PMNs from ICU patients as compared with the healthy individuals. This downregulation was also validated at the protein level for ApoL1 and ApoL2, whereas ApoL 6 was upregulated in septic patients. We could not detect ApoL3 protein in any of the cohorts. This was accompanied by a signifi cant delay in PMN apoptosis in septic patients as compared with healthy volunteers (P <0.05) at 4 and 24 hours. We also showed a strong negative correlation in the three mixed groups between CRP and ApoL1 (R = –0.607), ApoL2 (R = –0.651), ApoL3 (R = –0.578) and ApoL6 (R = –0.506). and ApoL6 (P = 0.0003) in purifi ed PMNs from ICU patients as compared with the healthy individuals. This downregulation was also validated at the protein level for ApoL1 and ApoL2, whereas ApoL 6 was upregulated in septic patients. We could not detect ApoL3 protein in any of the cohorts. This was accompanied by a signifi cant delay in PMN apoptosis in septic patients as compared with healthy volunteers (P <0.05) at 4 and 24 hours. We also showed a strong negative correlation in the three mixed groups between CRP and ApoL1 (R = –0.607), ApoL2 (R = –0.651), ApoL3 (R = –0.578) and ApoL6 (R = –0.506). p g p p p p Methods We collected peripheral blood mononuclear cells from 47 patients with severe sepsis or septic shock at ICU admission (T0) and from 50 healthy controls. Reduced responsiveness of blood leukocytes to lipopolysaccharide does not predict nosocomial infections in critically ill patients LA Van Vught, MA Wiewel, AJ Hoogendijk, BP Scicluna, H Belkasim, J Horn, MJ Schultz, T Van der Poll Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P38 (doi: 10.1186/cc14118) Results The condition of the nonseptic patients was signifi cantly less severe than that of the septic patients. The SOFA score of septic patients and the nonseptic patients was 6 (3 to 18) and 2.5 (1 to 8), respectively (median (IQR), P  =  0.02). The overall mortality rate was 29%. After stimulation with PMA, neutrophils isolated from septic patients released 4.08 ± 1.02% of their total DNA, whereas neutrophils from nonseptic patients released 29.06  ±  2.94% (P  <0.0001). Immunofl uorescent staining of released DNA, elastase, and myeloperoxidase also revealed similar results. Neutrophils from nonseptic patients showed eff ective extracellular killing of E. coli through NETs, whereas neutrophils from septic patients did not (P <0.001). Plasma levels of cf-DNA and histones were higher in septic patients than in nonseptic patients (P <0.001). Introduction Critically ill patients show signs of immune suppression, which is considered to increase vulnerability to nosocomial infections. Whole blood stimulation is a frequently used functional test for immune suppression. We here aimed to assess the association between whole blood leukocyte responsiveness to lipopolysaccharide (LPS) and the subsequent occurrence of nosocomial infections in critically ill patients admitted to the ICU. g Conclusion The increase of the immature PMN count and immature/ total PMN ratio confi rmed recruitment of immature neutrophils from the bone marrow into the circulation. The ex vivo generation of NETs is downregulated in neutrophils isolated from patients with sepsis. However, it is unclear whether in vivo NET formation is also impaired during sepsis, so further investigation is necessary. Methods All consecutive critically ill patients admitted to the ICU between April 2012 and June 2013 with two or more systemic infl ammatory response syndrome criteria and an expected length of ICU stay of more than 24 hours were enrolled. Age-matched and gender-matched healthy individuals were included as controls. Blood was drawn the fi rst morning after ICU admission and stimulated ex vivo with 100 ng/ml ultrapure LPS for 3 hours. Tumor necrosis factor (TNF)α, interleukin (IL)-1β and IL-6 were measured in supernatants. Results Seventy-three critically ill patients were included, 10 of whom developed an ICU-acquired infection. Compared with healthy subjects, whole blood leukocytes of patients were less responsive to ex vivo stimulation with LPS, as refl ected by strongly reduced TNFα, IL-1β and IL-6 levels in culture supernatants. P34 Serial change of C1 inhibitor in patients with sepsis: a preliminary report On these subjects we evaluated frequency and absolute numbers of CD4+ and CD8+ T cells and of NK and B lymphocytes, the rates of regulatory CD4+CD25+Foxp3+ T cells (Tregs), the cytotoxic potential of CD4+, CD8+ T cells and of NK cells by evaluation of perforin (PER) and granzyme (GRA) expression and production of eff ector cytokines (namely IL-2, IL-17, IL-4, TNFα, IFNγ) by CD4+, CD8+ T cells and NK cells upon polyclonal stimulation. The markers were compared in patients with diff erent outcome. p p Conclusion The altered apoptotic fate of neutrophils in sepsis was correlated with the modifi cation of the expression profi le of ApoLs, a family of proteins thought to be involved in the apoptotic process. The role of these proteins in the sepsis-associated phenotype of neutrophils remains to be further elucidated. p pf Results Septic patients, compared with healthy donors, were characterized by global lymphopenia; we found increased frequencies of CD4+ T cells producing IL-2 (P = 0.0000000003), increased percentage of CD8 T cells producing IFNγ (P = 0.03), and reduced proportion of CD4+ T cells (P = 0.00007) and NK cells (P = 0.002) producing IFNγ. We also noticed an increased frequency of CD8+ T cells expressing PER (P = 0.00000025) and GRA (P = 0.01); moreover, the proportion of NK cells expressing GRA was also signifi cantly increased (P = 0.000019). To establish the prognostic value of these biological markers, we compared the cytokine expression by lymphocytes in septic patients that survived with those that died (D). We found that CD4+ and CD8+ TNFα-producing T cells were signifi cantly increased in D (P  =  0.01 and P = 0.0001 respectively); similarly the percentage of CD8+ T cells producing IFNγ was more elevated in D (P  =  0.006). The same was observed for IL-17 production by CD4+ T cells (P = 0.03) in D. On the contrary we observed a tendency to the reduction of circulating CD4+CD25+foxp3 (Tregs) in D (P = 0.08). References 1. Görgülü P, et al. Crit Care. 2011;15:R20. g 2. Vanhollebeke B, et al. Cell Mol Life Sci. 2006;63:1937-44. 2. Vanhollebeke B, et al. Cell Mol Life Sci. 2006;63:1937-44. Reduced responsiveness of blood leukocytes to lipopolysaccharide does not predict nosocomial infections in critically ill patients LA Van Vught, MA Wiewel, AJ Hoogendijk, BP Scicluna, H Belkasim, J Horn, MJ Schultz, T Van der Poll Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P38 (doi: 10.1186/cc14118) However, results were not diff erent between patients who did and those who did not develop an ICU- acquired infection (Figure 1). Ex vivo and in vivo generation of neutrophil extracellular traps by neutrophils from septic patients N Takeyama, MH Huq, M Ando, T Goc N Takeyama, MH Huq, M Ando, T Gocho, MH Hashiba, H Miyabe, H Kano, A Tomino, M Tsuda, T Hattori, A Hirakawa Fujita Health University, Aichi, Japan Critical Care 2015, 19(Suppl 1):P36 (doi: 10.1186/cc14116) Introduction The primary aim of this study was to determine the diff erences in ex vivo generation of neutrophil extracellular traps (NETs) by neutrophils from septic and nonseptic patients. We further sought to examine plasma levels of cell-free DNA (cf-DNA) and histones to assess in vivo NET formation. p g Conclusion Septic patients are characterized by a peculiar immunophenotype which includes global lymphopenia and a specifi c pattern of cytokines. Some of the evaluated markers seem to individuate those with worse outcome; in particular, this group showed an infl ammatory phenotype with a higher expression of IFNγ, TNFα, IL- 17 and a tendency to a reduction of Tregs. Methods We isolated neutrophils from consecutive patients with sepsis (n = 17) and without sepsis (n = 18) admitted to the ICU. Neutrophils were activated by incubation with phorbol myristate acetate to induce release of NETs and NET formation was assessed by measuring the extracellular DNA level. Immunolabeling and fl uorescence imaging were also performed. Extracellular killing of bacteria by NETs was studied by co-culture of Escherichia coli and neutrophils in the presence of the phagocytosis inhibitor cytochalasin D. To assess in vivo NET formation, plasma levels of cf-DNA and histones were measured.i Reduced responsiveness of blood leukocytes to lipopolysaccharide does not predict nosocomial infections in critically ill patients LA Van Vught, MA Wiewel, AJ Hoogendijk, BP Scicluna, H Belkasim, J Horn, MJ Schultz, T Van der Poll Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P38 (doi: 10.1186/cc14118) Alarming levels of heat shock proteins 72 and 90α in critically ill children Introduction The endothelium is a complex organ infl uenced by circulating mediators, adjacent cells, physico-chemical factors, and shear stress. During systemic infl ammation and sepsis, excessive and sustained activation of the endothelium result in the loss of its anti- coagulant and anti-adhesive characteristics as well as in a loss of endothelial barrier function. We set up a cell-culture model to study endothelial activation induced by lipopolysaccharide (LPS) or by plasma from septic patients and studied the eff ect of adsorbent-based mediator modulation on endothelial activation. Introduction The endothelium is a complex organ infl uenced by circulating mediators, adjacent cells, physico-chemical factors, and shear stress. During systemic infl ammation and sepsis, excessive and sustained activation of the endothelium result in the loss of its anti- coagulant and anti-adhesive characteristics as well as in a loss of endothelial barrier function. We set up a cell-culture model to study endothelial activation induced by lipopolysaccharide (LPS) or by plasma from septic patients and studied the eff ect of adsorbent-based mediator modulation on endothelial activation. Introduction Extracellular heat shock proteins (HSP) act as inducers of interleukins (IL) and stimulants for immune cells during systemic infl ammatory response syndrome (SIRS). Little is known about the alarming roles of extracellular HSP72 and HSP90α in the acute phase [1] of sepsis (S) or severe sepsis (SS). We determined serum HSP90α, HSP72 and neutrophil CD64 expression, IL-6, IL-8, IL-10, and TNFα in children with S or SS compared with SIRS (brain injury) or healthy children (H). Introduction Extracellular heat shock proteins (HSP) act as inducers of interleukins (IL) and stimulants for immune cells during systemic infl ammatory response syndrome (SIRS). Little is known about the alarming roles of extracellular HSP72 and HSP90α in the acute phase [1] of sepsis (S) or severe sepsis (SS). We determined serum HSP90α, HSP72 and neutrophil CD64 expression, IL-6, IL-8, IL-10, and TNFα in children with S or SS compared with SIRS (brain injury) or healthy children (H). Methods Critically ill children with S (n = 16), SS (n = 15) or SIRS (n = 18) and H (n = 21) were enrolled in the study. ELISA was used to evaluate HSPs, chemiluminescence to measure ILs, and fl ow cytometry to evaluate nCD64 expression (IRB approved). Methods Human whole blood was stimulated with LPS (100 ng/ ml) from Escherichia coli for 4 hours. P40 P40 Alarming levels of heat shock proteins 72 and 90α in critically ill children M Fitrolaki1, H Dimitriou2, M Venihaki2, M Katrinaki2, G Briassoulis1 1University Hospital, Heraklion, Greece; 2University of Crete, Medical School, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P40 (doi: 10.1186/cc14120) Alarming levels of heat shock proteins 72 and 90α in critically ill children The stimulated blood or plasma from septic patients was treated in vitro with 10 vol% polystyrene– divinylbenzene (PS-DVB)-based polymers (CG161, mean pore size 16 nm; CG300, mean pore size 30 nm) or left untreated. After adsorption, the plasma was separated and diluted with cell culture medium. The resulting conditioned medium was used to stimulate human umbilical vein endothelial cells (HUVEC) for 16  hours. HUVEC activation was assessed by the release of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)α, plasminogen activator inhibitor-1 (PAI- 1), as well as the expression of intercellular adhesion molecule (ICAM)- 1 and E-selectin. HUVEC were cultured at a shear stress of 5 dyne/ cm2 using the Ibidi perfusion system. Adhesion of monocytic THP-1 cells to HUVEC was studied after 4 hours of HUVEC stimulation with conditioned media. THP-1 cells were perfused over HUVEC at 1 dyne/ cm2 for 15 minutes, and adhering THP-1 were quantifi ed over time. p j y y Methods Critically ill children with S (n = 16), SS (n = 15) or SIRS (n = 18) and H (n = 21) were enrolled in the study. ELISA was used to evaluate HSPs, chemiluminescence to measure ILs, and fl ow cytometry to evaluate nCD64 expression (IRB approved). p pp Results Patients in both septic groups had elevated HSP90α (P <0.0001), HSP72 (P  <0.05), IL-6 (P  <0.0001), IL-8 (P  <0.02) and IL-10 (P  <0.05) levels compared with H, whereas SS had increased HSP72, IL6 and TNFα compared with SIRS (P <0.05). SIRS patients presented increased HSP90α, IL-6 and IL-8 compared with H (P  <0.05). Both HSPs were dramatically increased among nonsurvivors. In a logistic regression model, only HSP90α was independently associated with mortality (P <0.0001). HSP90α related positively (P <0.001) to nCD64, IL-8, IL-10, CRP, PRISM, PELOD, TISS, and LOS and negatively to HDL (P <0.001) and LDL (P <0.02). HSP72 also related negatively to HDL (P <0.001). i Results The adsorbents CG161 and CG300 substantially decreased levels of TNFα, IL-1β, IL-6, IL-8 and IL-10 in LPS-stimulated blood. TNFα, a key stimulus for HUVEC, was reduced to 12% and 8% of the initial concentration by CG161 and CG300, respectively. Stimulation of HUVEC with the adsorbent-treated plasma resulted in signifi cantly diminished release of IL-6, IL-8, PAI-1 and decreased ICAM-1 and E-selectin expression, indicating reduced HUVEC activation. P37 Specifi c patterns of T-cell cytokines as an early marker of outcome in septic patients A Franci, A Peris, F Liotta, F Annunziato, P Ruggiano, M Ferraro A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P37 (doi: 10.1186/cc14117) A Franci, A Peris, F Liotta, F Annunziato, P Ruggiano, M Ferraro A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P37 (doi: 10.1186/cc14117) Introduction The infl ammatory response of sepsis is developed in two phases, an infl ammatory phase (SIRS) and a phase more variable in frequency and intensity (CARS): this balance has an important eff ect on morbidity and mortality. Lymphopenia aff ects particularly T cells, and Conclusion The extent of reduced LPS responsiveness of blood leukocytes in critically ill patients on the fi rst day after ICU admission does not relate to the subsequent development of ICU-acquired infections. S14 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P38). Similarly reduced responsiveness of whole blood leukocytes to lipopolysaccharide. Figure 1 (abstract P38). Similarly reduced responsiveness of whole blood leukocytes to lipopolysaccharide. mediators with porous polystyrene-based polymers attenuates endothelial activation and reduces monocyte adhesion. mediators with porous polystyrene-based polymers attenuates endothelial activation and reduces monocyte adhesion. 39 ell-culture model to study endothelial activation in sepsis y p T Eichhorn1, S Rauscher2, C Hammer2, B Führer2, M Gröger2, V Weber1 Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome p p g g p Results Nineteen controls, six SIRS patients and 25 severe sepsis patients were studied. The percent expression of HLADR on CD14+ monocytes was signifi cantly diff erent between the three groups showing progressive decrease from controls (mean 90.5  ±  3.8%) to SIRS (mean 61.2 ± 5.9%) to severe sepsis (mean 39.2 ± 5.5%) patients (controls vs. severe sepsis, P <0.001; controls vs. SIRS, P = 0.006; SIRS vs. severe sepsis, P  =  0.03). hsp70 and hsp90 MFI were signifi cantly diff erent between controls (mean 49.5 ± 4.9 and 33.5 ± 3.4 respectively), SIRS (mean 69.9 ± 16.5 and 46.5 ± 5.7 respectively) and severe sepsis patients (mean 33.3  ±  4.5 and 21.7  ±  2.7 respectively) (P  <0.05 for all comparisons). Notably, the hsp level rose from controls to SIRS and fell from SIRS to severe sepsis patients. APACHE score increased signifi cantly (P = 0.023) in septic patients compared with SIRS.if Introduction Heat shock proteins (HSPs) have intracellular cyto pro tec- tive actions, while they act extracellularly as inducers of cytokines and stimulants for immune cells during stress. Their induction constitutes a highly conserved cellular defense mechanism against all kinds of stress. Our objective was to determine the intracellular as well as extracellular levels of HSP72 and HSP90α in patients with severe sepsis (SS) or systemic infl ammatory response syndrome (SIRS) admitted to a general ICU, compared with those of healthy individuals; to correlate their expression with severity of illness. i Conclusion There were a signifi cant diff erence in CD14/HLADR, a marker of immune paralysis, between controls and patients with SIRS or severe sepsis. hsp70 and hsp90 showed an initial stimulation followed by exhaustion as sepsis progressed.i Methods Eighty-two consecutively admitted patients in the ICU (35 SIRS, 47 SS) as well as 35 healthy controls (H) were fi nally enrolled in the study. Patients’ demographic characteristics, laboratory examinations and Acute Physiology and Chronic Health Evaluation (APACHE II) score were recorded on admission. HSP levels were determined intracellularly using four-color fl ow cytometry. Mean fl uorescence intensity (MFI) values for each HSP were measured and analyzed. Extracellular levels of HSPs were determined via ELISA. Acknowledgements This research was co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. P42 P42 Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1, A Pistiki2, T Christodoulopoulou1, M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) P42 Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1, A Pistiki2, T Christodoulopoulou1, M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopo los1 A Pistiki2 T Christodo lopo lo 1 p p , , p , M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) p p p M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) Results Ninety-nine patients were included in the fi nal analysis. Eighteen patients developed severe sepsis or septic shock. They presented with signifi cantly higher levels of intermediate (CD14++/16+) and CD62L– monocytes and lower IL-2 levels at T1 compared with patients who did not get septic. ROC AUC for association of these parameters with the occurrence of sepsis were 0.78 (95% CI: 0.63 to 0.91), 0.72 (0.62 to 0.82) and 0.73 (0.65 to 0.82), respectively. High counts of these monocytic cells were also associated with increased 90- day mortality (P <0.01, ROC AUC = 0.87 (0.77 to 0.95), 0.79 (0.66 to 0.9)). Kaplan–Meier survival curves showed signifi cantly higher mortality after stratifi cation based on these cell counts at T1 (CD14++CD16+: cutoff >236.8 cells/μl, HR = 23.6 (P = 1.24 × 10–5); CD62L–: cutoff >95.4 cells/μl, HR = 6.67 (P = 7.6 × 10–4)). Multivariate logistic regression analysis using Introduction CD14/HLADR is an index of immune suppression. Heat shock proteins (hsp) regulate cell response to oxidative stress. Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome This abstract is part of the study ‘Heat Shock Proteins and Glutamine Alterations Related to Hormonal, Immunological, Infl ammatory and Molecular Response to Sepsis: A Combined Clinical and Experimental Study’. Results HSP expression diff ered signifi cantly between groups (Kruskal–Wallis), both intracellularly (HSP72 lower in SS, P  <0.001), and extracellularly (higher levels of HSP90α (P  <0.001) and HSP72 (P  =  0.003) in SS). HSP72 and HSP90α intracellular expression was inversely correlated to severity of illness, as expressed by APACHE II score (Spearman’s, P = 0.003 and P = 0.025 respectively). Intracellular HSP72 was correlated to mortality when confounding factors were excluded from the analysis (logistic regression, P = 0.05). Extracellular HSP90α levels correlated with prolonged PT (P  =  0.021) and INR (P = 0.008). Finally, in the SIRS group, intracellular levels of HSP90α were higher in nonsurvivors (P <0.001). Alarming levels of heat shock proteins 72 and 90α in critically ill children THP-1 adhesion was substantially decreased when HUVEC were stimulated with CG300-treated plasma as compared with untreated controls.lf Conclusion Extracellular HSP72 and HSP90α are alarmingly elevated in critically ill children, especially in severe sepsis. HSP90α levels are independently associated with mortality, related to CD64, IL-8, IL-10, severity of illness, and outcome. Both HSPs are inversely related to the low LDL/low HDL septic metabolic pattern [2].i Acknowledgements This research has been co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. Investing in knowledge society through the European Social Fund. Conclusion The fl ow model allows to study the eff ect of cytokine modulation on endothelial activation and to assess the interaction of activated endothelial cells with blood cells. Modulation of infl ammatory S15 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P41 Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome K Apostolou1, K Vardas1, E Briassouli1, K Psara1, D Goukos1, E Mageira1, S Nanas1, C Routsi1, G Briassoulis2 1Evangelismos Hospital, National and Kapodistrian University of Athens, Greece; 2University Hospital, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P41 (doi: 10.1186/cc14121) Prospective immune profi ling in critically ill adults: before, during and after severe sepsis and septic shock Introduction Rethinking the host’s defense mechanisms during severe infection has led to the use of fl ow cytometry (FCM) and to the current concept of sepsis-induced immunosuppression. However, organ dysfunctions that develop in the period preceding severe sepsis as a consequence of surgery, trauma or burn might also trigger immune reprogramming predisposing to overwhelming infection. Our aim was to look for correlation of specifi c phenotypes among four commonly encountered populations of patients and the later occurrence of severe sepsis and septic shock. g Conclusion SS is characterized by high levels of extracellular HSPs. Intracellular HSP72 is highly expressed during the acute phase of stress in SIRS, while being downregulated in SS. HSP72 and HSP90α intracellular expression and extracellular level variations correlate with severity of illness and mortality.i Acknowledgements This research was co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. p p Methods In total, 114 non-infected patients were prospectively screened via FCM on days 1 (T1) and 3 (T2) of elective cardiac surgery, trauma, acute neurologic dysfunction and prolonged ventilation (>48 hours). A third sample was drawn when infection was diagnosed (Tx) and 7  days later (Tx  + 7). Exclusion criteria included use of immunosuppressive agent(s). The broad panel of cell-specifi c antibodies focused on B, T lymphocytes (Tregs, Th17, NKT), NK cells, monocytes and neutrophils. Plasmatic levels of IL-2/IL-6/IL-7/TNFα/ IFNγ were also determined.i References To evaluate the %HLA-DR expression on monocytes, the fresh whole blood was stained with anti-CD14-FITC, anti-HLA-DR-PE and CD45- PC5 while staining with anti-CD33-PE, anti-CD45-PC7, anti-hsp70-FITC and anti-hsp90-PE allowed evaluation of the MFI expression of hsps on CD33+ monocytes. Cells were then analyzed using fl ow cytometry. ANOVA with post hoc tests was used to compare CD14/HLADR cell counts and hsp70 and hsp90 levels among the three groups. References 1. Briassouli E, et al. Nutrition. 2014;30:1185-94. 2. Fitrolaki DM, et al. BMC Pediatr. 2013;13:31. References 1. Briassouli E, et al. Nutrition. 2014;30:1185-94. 2. Fitrolaki DM, et al. BMC Pediatr. 2013;13:31. P42 We evaluated the relationship of CD14/HLADR and hsp70/90 in patients with SIRS and severe sepsis versus healthy volunteers. Methods We evaluated 31 patients with SIRS or severe sepsis against a group of sex-matched healthy volunteers. Demographic data were obtained for all patients. APACHE score was calculated upon admission. Blood samples were collected upon diagnosis of SIRS or severe sepsis. S16 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 clinical scores indicated that addition of IL-2 levels at T1 signifi cantly improved prediction of sepsis (OR = 0.834, P = 0.02). that septic patients display a strong correlation between mHLA-DR and mRNA-levels of HLA-DRA in whole blood [1]. mRNA-based HLA- DR monitoring by PCR would improve the clinical usage and facilitate conduction of multicentre studies. The primary focus in this study was to evaluate the correlation between mHLA-DR and HLA-DRA at diff erent time points during sepsis. In addition, we assessed the dynamic expression of both mHLA-DR and HLA-DRA, in relation to sepsis severity. Conclusion Predisposition to sepsis in selected critically ill medico- surgical adults can be identifi ed on day 1 of admission based on high counts of circulating intermediate and CD62L– monocytes and low levels of IL-2 (the latter provide incremental prognostic information). High counts of these specifi c monocytes correlate with higher 90-day mortality. y Methods Study patients (n  = 54) were included at day 1 to 2 after hospital admission if blood cultures turned positive. Repeated sampling at days 1 to 2, 3, 7, 14 and 28 was performed. mHLA-DR was monitored by FCM and HLA-DRA by quantitative RT-PCR. Mixed models for longitudinal data were used after logarithmic transformation to calculate the interactional eff ects of time and severity on HLA-DR expression. Macrophage phenotype in sepsis immunosuppression E Theodorakis, E Diamantaki, C Tsatsanis, D Georgopoulos, K Vaporidi University of Crete, School of Medicine, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P44 (doi: 10.1186/cc14124) Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Introduction Sepsis is followed by profound, yet poorly characterized, innate immune system suppression. P42 While low monocyte HLA-DR expression is observed in septic patients, its clinical signifi cance has not been established [1]. In vitro, repeated LPS stimulation induces a tolerant or M2 macrophage phenotype, characterized by decreased cytokine production [2], which could contribute to sepsis immunosuppression. The present study examines macrophage phenotype in a mouse model and in patients with sepsis immunosuppression. p p pp Methods Sepsis was induced in C57Bl6 mice by cecal ligation and puncture (CLP) followed by intratracheal instillation of Pseudomonas aeruginosa. Bronchoalveolar lavage fl uid (BALF), cells and serum, collected 12 hours after lung infection, were analyzed for bacterial load, cytokine levels and the classical M1 marker, iNOS. Peripheral blood monocytes isolated from septic adult patients admitted to the ICU on the 1st and 7th day after admission were analyzed by fl ow cytometry for the expression of HLA-DR and CD86 (co-stimulatory molecule and M1 marker), and for the M2 markers, CD163 and CD206. Additional blood samples from patients and healthy volunteers were exposed ex vivo to LPS prior to isolation and analysis of monocyte markers. p y y Results CLP-induced sepsis resulted in immunosuppression in mice, indicated by higher BALF bacterial load after infection in CLP than in sham-operated mice, and more severe injury on histology. Serum cytokines TNF and MIP2 were greater in CLP than in sham-operated mice. Although recruitment of CD11c+ alveolar macrophages post infection was threefold greater in CLP than in sham-operated mice, those macrophages expressed 40% lower levels of iNOS. Evidence of sepsis immunosuppression was present in most patients on the 7th day after ICU admission. Low expression of CD86 and/or HLA-DR was observed in 71% of patients, and increased expression of M2 markers in 15% of patients. Upon LPS stimulation the normal decrease in M2 markers was absent in all patients on day 1, and partially restored in 50% of patients on day 7. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Conclusion Sepsis is associated with decreased monocyte expression of M1 markers and increased expression of M2 markers in septic mice and critically ill patients. P42 Therefore, in addition to decreased HLA-DR expression, M2 macrophage polarization appears to be a component of sepsis-induced monocyte dysfunction, and should be considered for immune monitoring and targeted intervention. g g Acknowledgement Supported by GSRT research grant ExcellenceII-4620. R f References 1. Gomez H, et al. Crit Care Med. 2014;42:771. 2. Porta C, et al. Proc Natl Acad Sci U S A. 2009;106:14978. 2. Porta C, et al. Proc Natl Acad Sci U S A. 2009;106:14978. P45 P45 Expression of mRNA levels of HLA-DRA in relation to monocyte HLA-DR: a longitudinal sepsis study SC Cajander, ET Tina, AB Bäckman, AM Magnuson, KS Strålin, BS Söderquist, JK Källman Örebro University, Örebro, Sweden Critical Care 2015, 19(Suppl 1):P45 (doi: 10.1186/cc14125) Expression of mRNA levels of HLA-DRA in relation to monocyte HLA-DR: a longitudinal sepsis study SC Cajander, ET Tina, AB Bäckman, AM Magnuson, KS Strålin, BS Söderquist, JK Källman Örebro University, Örebro, Sweden Critical Care 2015, 19(Suppl 1):P45 (doi: 10.1186/cc14125) Introduction Decreased monocyte surface HLA-DR (mHLA-DR) measured by fl ow cytometry (FCM) is an independent marker of immunosuppression in sepsis. In a previous report we demonstrated S17 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Correlation between mHLA-DR(FCM) and HLA-DRA(PCR) at day 1 to 2 (R = 0.78) and day 14 (R = 0.27). Both HLA-DR markers increased linearly on a log scale over time. The linear association was signifi cantly diff erent between the severe (n = 16) and nonsevere septic patients (n = 38) when measuring either mHLA-DR(FCM) or HLA-DRA(PCR). By pairwise comparison of means between the two severity groups, at every time point, the diff erences between groups were shown to be signifi cant at days 1 to 2 and 3 when monitoring mHLA-DR(FCM) and at days 1 to 2, 3 and 7 for HLA-DRA(PCR) (Figures 1 and 2).l used as a diagnostic criterion of sepsis. There are no published data to defi ne the role of dynamic control of EC in the process of intensive therapy as a prognostic marker and indicator of severity condition in critically ill patients. The aim was to determine the informative value of EC in the development of SIRS as a biomarker of sepsis and indicator of the severity condition and prognosis of outcome in the pathological process. Methods A total of 143 patients were enrolled in this study who were admitted to the ICU and had SIRS. All patients were divided into a septic group – patients with community-acquired pneumonia, complicated by sepsis – and two SIRS groups of noninfectious genesis – patients who had an acute cerebrovascular accident (CVA) and an acute myocardial infarction (AMI). The absolute EC was measured at admission and in the dynamics on days 3 to 5 of stay. HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) y Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) Introduction The aim of the present study was to evaluate the prognostic value of HLA-DR antigen expression in monocytes, in patients with community-acquired infections presenting with fever, as possible markers for the patients’ fi nal outcome. Table 1 (abstract P47). Dynamics of eosinophil count depending on outcome in groups i Methods A total of 81 patients (males = 46; females = 35) presenting with fever >38°C to the emergency room (ER) of the Department of Internal Medicine of the Patras University Hospital were enrolled in the study during a period of 12 months. Sera for monocyte HLA-DR expression were obtained from the patients on admission (day 1) and on days 3, 7 or discharge/death. Results were expressed as percentages of HLA-DR-positive monocytes, calculated by the coexpression of CD14 and HLA-DR antigens in the total CD14+ population. Additionally, the patients were evaluated using the Simplifi ed Acute Physiology Score (SAPS-II), the Sequential Organ Failure Assessment (SOFA) and the Mortality in Emergency Department Sepsis (MEDS) score on the same days while all the indicated clinical, laboratory and imaging procedures as required for fever’s diff erential diagnosis were followed. A questionnaire regarding demographic characteristics, comorbidities, medications used and patients’ survival was also completed. All statistical analyses were performed using SPSS v.21. Table 2 (abstract P47). Informational value of eosinophil count in assessment of disease outcome Results Lower mean HLA-DR monocyte antigen expression percen- tages were signifi cantly correlated to lower Glasgow Scale scores on all days of measurement. HLA-DR expression was signifi cantly negatively correlated to MEDS, SOFA and SAPS-II scores whereas patients who developed sepsis, severe sepsis, septic shock and MODS had signifi cantly lower HLA-DR values compared with the ones who did not. HLA-DR expression on day 1 was lower in patients who would develop SIRS and/or sepsis on days 3 and 7 (P <0.01). Additionally, HLA- DR expression was signifi cantly decreased in nonsurvivors (n  = 33) compared with survivors (n = 48), whereas lower HLA-DR expression was correlated to longest duration of hospital stay at all time points (P <0.01). Conclusion EC may be an additional diagnostic marker which characterizes the nature of SIRS. Eosinopenia associated with prognosis of outcome in critical conditions. P45 y g Conclusion The correlation between fl ow cytometry and PCR-based HLA-DR monitoring is stronger in the early phase of sepsis. However, the linear associations over time, in relation to sepsis severity, display similar results for both HLA-DR markers. HLA-DRA(PCR) as a biomarker could be an alternative approach in monitoring immune status in sepsis but needs to be evaluated in relation to clinically relevant immunosuppression. Reference y y y Results The median EC was 75 cells/mm3 in septic patients on admission, which was signifi cantly lower than in patients with CVA (120 cells/mm3) and AMI (130 cells/mm3). Comparison of EC in septic patients between survivors and those who died showed signifi cant diff erences (Table 1). Receiver operating characteristic (ROC) analysis determined a value less than 80 cells/mm3 as the optimal diagnostic cutoff value with a high level of confi dence in the comparison of septic and noninfectious groups. Area under the ROC curves was 0.94, sensitivity of 80.8%, specifi city of 95.6%, P <0.0001. There was a signifi cant increase of EC in survivors, while the EC did not change signifi cantly among those who died in the dynamics. ROC analysis determined the cutoff values of EC, which indicated a high risk of an adverse outcome in septic patients (Table 2). 1. Cajander S, et al. Crit Care. 2013;17:R223. 1. Cajander S, et al. Crit Care. 2013;17:R223. HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) Conclusion Monocyte HLA-DR appears to be an early indicator for survival and infection progression and therefore it can be used as a predictive marker for the fi nal outcome of patients presenting in ER departments with fever. P48 Prevalence and clinical signifi cance of early endotoxin activity in septic shock patients M Bottiroli1, R Pinciroli1, G Monti2, M Mininni1, G Casella2, R Fumagalli2 1Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy; 2Anestesia Rianimazione 1, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P48 (doi: 10.1186/cc14128) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) Introduction The endotoxin activity (EA) assay is a useful test to risk stratify critically ill patients and assess for Gram-negative (GN) infection. However, the prevalence and signifi cance of early high levels of EA in patients with septic shock (SS) has yet to be elucidated. Introduction The idea of using the eosinophil count (EC) as a diagnostic marker for clarifying the nature of systemic infl ammatory response syndrome (SIRS) belongs to K Abidi, who showed that EC could be Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 S18 pp http://ccforum.com/supplements/19/S1 Figure 1 (abstract P48). patients with systemic infl ammatory response syndrome (SIRS) and organ failure in ICU setting. Methods In total, 76 patients with SIRS and organ failure or who were suspected of sepsis during critical care were included. According to the levels of EAA, all patients were classifi ed into three groups (group L, EAA <0.4; group M, EAA ≥0.4 or EAA <0.6; group H, EAA ≥0.6). In order to evaluate the severity of illness, the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score and catecholamine (CA) index were recorded. Blood samples were obtained to measure EAA levels, infl ammatory markers (procalcitonin, C-reactive protein and white blood cells), serum lactate level as an indicator of tissue hypoxia, and for blood culture. All patients were followed up for 6 months. APACHE II score, SOFA score, CA index, infl ammatory markers, serum lactate levels and blood culture results were examined for diagnosis of sepsis, severe sepsis, septic shock and for prognosis of 30-day mortality. Each value was also compared with EAA levels. Results Patient age was 69 ± 9.9 years (male: n = 48, female: n = 25). The total number of samples was 106 (group L/group M/group H: 35/35/36). Twenty-seven specimens were obtained from nonseptic patients and 83 specimens were obtained from septic patients. APACHE II score was highly correlated with SOFA score (P <0.05). In group H, the APACHE II score was signifi cantly higher (22.2 ± 0.8) than that in group M (18.4 ± 0.87) (P = 0.01). 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) Results A total of 107 consecutive patients were included. The overall median EA was 0.56 (0.44 to 0.71), with 46/107 (43%) patients testing positive for elevated EA (≥0.6). GN species were identifi ed in microbial cultures as the infective etiology in 49/107 (46%) patients, of which 28 (57%) developed bacteremia. GN infections were associated with higher levels of EA compared with other microbial causatives (0.61 (0.52 to 0.77) vs. 0.52 (0.38 to 0.64), P = 0.021). Patients with EA ≥0.6 showed signifi cantly higher lactate levels (2 (1 to 3) vs. 3.8 (1.7 to 6.4), P = 0.01), Sequential Organ Failure Assessment (9 (6 to 12) vs. 10 (8 to 14), P = 0.04) and inotropic score (20 (5 to 50) vs. 50 (16 to 100), P = 0.003) at inclusion. See Figure 1. Introduction Sepsis is a common reason for admission to ICUs throughout the world. During the past two decades, the incidence of sepsis in the USA has tripled and is now the 10th leading cause of death. As sepsis continues to impact negatively on critically ill patients, it is clear that early diagnosis and eff ective management could improve patient morbidity and mortality. Numerous studies have attempted to examine biomarkers and their ability to diagnose and prognosticate septic patients. Despite multiple eff orts, currently there are no reliable markers that can eff ectively improve our clinical eff ectiveness in diagnosing and managing septic patients. The purpose of our systematic review was to evaluate the diagnostic and prognostic value of various biomarkers used in septic patients. g Conclusion Elevated EA is a common fi nding in SS patients. In patients developing SS from a GN infection, higher levels of endotoxin activity could be measured within 24  hours. Furthermore, in our study, EA ≥0.6 identifi ed a subgroup of subjects at greater risk for worse clinical outcomes. We therefore propose use of the EA assay for the early identifi cation and risk stratifi cation of SS patients. Methods A systematic search of the literature was performed with MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials databases using terminology selected for biomarkers (through to and including November 2013). All articles involving neonates and not in English were excluded. Inclusion was agreed on by two independent reviewers of abstracts or full text. Biomarkers in sepsis: a systematic review Biomarkers in sepsis: a systematic review F Morriello1, J Marshall2 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) The SOFA score in group H was signifi cantly higher (9.9  ±  0.5) than that in group M (7.5  ±  0.6) and group L (7.9 ± 0.6) (P = 0.006). EAA levels were signifi cantly increased in septic patients (septic patients: 0.56 ±0.03, nonseptic patients: 0.42 ±0.05) (P  =  0.011) and in the positive blood culture group (positive group: 0.66 ± 0.05, negative group: 0.48 ± 0.03) (P = 0.006). There was no relationship between EAA levels and other infl ammation markers or 30-day mortality. Conclusion In patients with suspected sepsis and positive blood culture, EAA levels were signifi cantly increased and had strong correlation with severity of disease. This result suggests that EAA indicates the state of sepsis regardless of the possibility of infection in patients with SIRS with organ failure. Figure 1 (abstract P48). Figure 1 (abstract P48). Methods We designed a prospective observational study including adult patients with clinically diagnosed SS. EA was measured on arterial blood by a chemiluminescent assay within the fi rst 24 hours from SS diagnosis. The fi nding of an EA value ≥0.6 was used as the cutoff for test positivity, as described elsewhere. In addition, laboratory, microbiological and clinical data were collected at inclusion. In-hospital follow-up was also conducted. Biomarkers in sepsis: a systematic review F Morriello1, J Marshall2 F Morriello1, J Marshall2 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) P53 Association between apoptosis and mortality in severe septic patients In the fully adjusted model, C C C C C d C d h d l L Lorente1, M Martín2, A González-Rivero1, J Ferreres3, J Solé-Violán4, L Labarta5, C Díaz6, A Jiménez1, J Borreguero-León1 1Hospital Universitario de Canarias, La Laguna, Tenerife, Spain; 2Hospital Universitario Nuestra Señora Candelaria, Santa Cruz, Tenerife, Spain; 3Hospital Clínico Universitario de Valencia, Spain; 4Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain; 5Hospital San Jorge, Huesca, Spain; 6Hospital Insular, Las Palmas de Gran Canaria, Spain Critical Care 2015, 19(Suppl 1):P53 (doi: 10.1186/cc14133) Introduction The apoptotic process, in which cells are actively elimi- nated by a programmed pathway, is increased in sepsis. Extrinsic and intrinsic apoptotic death cell pathways activate caspase-3, which leads to cell apoptosis. Cytokeratin 18 (CK-18), a protein present in most epithelial and parenchymal cells, is cleaved by the action of caspases and released into the blood as caspase-cleaved CK (CCCK)-18 during apoptotic death. The novel objectives of this study were to determine whether there are associations between serum caspase-3 levels, serum CK-18 levels and mortality in septic patients. WBC, CRP, LymC, NeuC, NLCR and EoC were entered into the model. Results A total of 1,302 patients were categorized as nonsepsis SIRS (816, 62.7%) and sepsis (486, 37.3%). In the sepsis group, age, APACHE II, SOFA, mortality, length of ICU stay, CRP, NLCR and EoC were higher; LymC was lower than in the nonsepsis SIRS group (P <0.001 for each). Likelihood of sepsis (reference to nonsepsis SIRS) increased 2.62 (2.05 to 3.34), 2.02 (1.42 to 2.88) and 1.88 (1.36 to 2.60) times (OR (95% CI)) by the values of CRP >4.4  mg/dl, LmyC <500/mm3 and NLCR >15.7 respectively in mutually adjusted multivariate logistic regression (P <0.001 for each). y p p Methods A prospective, multicenter, observational study in six Spanish ICUs, including 216 patients with severe sepsis. We collected blood samples at the severe sepsis diagnosis moment to determine serum levels of caspase-3 (to assess the main executor of apoptosis) and CCCK- 18 (to assess the apoptosis level). The endpoint was 30-day mortality. Results We found that nonsurvivor (n = 76) in comparison with survivor (n = 140) septic patients showed higher serum levels of caspase-3 (0.41 ng/ml (0.14 to 0.52) vs. 0.11 ng/ml (0.10 to 0.25); P <0.001) and CCCK- 18 (448 (310 to 723) vs. 319 (236 to 445); P <0.001). C-reactive protein and hemogram parameters for the nonsepsis SIRS and sepsis: what do they mean? p y B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) y gi Results Reduced BChE activity correlated with trauma severity and the resulting systemic infl ammation. Compared with serum levels of routinely measured infl ammatory biomarkers, changes in the BChE activity were detected signifi cantly earlier, suggesting that the BChE activity might serve as an early indicator of systemic infl ammation. y gi Results Reduced BChE activity correlated with trauma severity and the resulting systemic infl ammation. Compared with serum levels of routinely measured infl ammatory biomarkers, changes in the BChE activity were detected signifi cantly earlier, suggesting that the BChE activity might serve as an early indicator of systemic infl ammation. Conclusion Our results suggest that BChE activity, measured using a POCT system, might play an important role in the early diagnosis of trauma-induced systemic infl ammation. Introduction The aim of this study was to investigate the laboratory parameters as an indicator of sepsis. Sepsis is one of the most common reasons of mortality and morbidity in the ICU [1]. Thus, it is important to distinguish sepsis from nonsepsis SIRS. CRP and hemogram parameters may be fast, easy and aff ordable alternatives in distinguishing sepsis from nonsepsis SIRS. Eosinophil count (EoC), lymphocyte count (LymC) and neutrophil–lymphocyte count ratio (NLCR) are used as sepsis indicators [1,2]. Conclusion Our results suggest that BChE activity, measured using a POCT system, might play an important role in the early diagnosis of trauma-induced systemic infl ammation. P53 Association between apoptosis and mortality in severe septic patients Methods A total of 2,777 patients admitted to the ICU of two centers between 2006 and 2013 were evaluated retrospectively. The patients were diagnosed as SIRS(–), nonsepsis SIRS or sepsis at ICU admission by the consensus of two doctors in accordance with 1992 sepsis guidelines [3]. The patients who were under 18 years old, readmitted, immunosuppressive, SIRS(–) and whose laboratory values and outcomes were unknown were excluded. In total, 1,302 patients were divided into two groups as the nonsepsis SIRS group and the sepsis group. The patient’s age, gender, diagnoses (medical, elective and urgent surgery), APACHE II, SOFA, CRP, WBC, neutrophil count (NeuC), LymC, NLCR, EoC, platelet, mean platelet volume, length of ICU stay and mortality were recorded by a third doctor. In the fully adjusted model, WBC, CRP, LymC, NeuC, NLCR and EoC were entered into the model. Results A total of 1,302 patients were categorized as nonsepsis SIRS (816, 62.7%) and sepsis (486, 37.3%). In the sepsis group, age, APACHE II, SOFA, mortality, length of ICU stay, CRP, NLCR and EoC were higher; LymC was lower than in the nonsepsis SIRS group (P <0.001 for each). Likelihood of sepsis (reference to nonsepsis SIRS) increased 2.62 (2.05 to 3.34), 2.02 (1.42 to 2.88) and 1.88 (1.36 to 2.60) times (OR (95% CI)) by the values of CRP >4.4  mg/dl, LmyC <500/mm3 and NLCR >15.7 respectively in mutually adjusted multivariate logistic regression (P <0.001 for each). Methods A total of 2,777 patients admitted to the ICU of two centers between 2006 and 2013 were evaluated retrospectively. The patients were diagnosed as SIRS(–), nonsepsis SIRS or sepsis at ICU admission by the consensus of two doctors in accordance with 1992 sepsis guidelines [3]. The patients who were under 18 years old, readmitted, immunosuppressive, SIRS(–) and whose laboratory values and outcomes were unknown were excluded. In total, 1,302 patients were divided into two groups as the nonsepsis SIRS group and the sepsis group. The patient’s age, gender, diagnoses (medical, elective and urgent surgery), APACHE II, SOFA, CRP, WBC, neutrophil count (NeuC), LymC, NLCR, EoC, platelet, mean platelet volume, length of ICU stay and mortality were recorded by a third doctor. P52 P52 Serum cholinesterase activity as an early indicator of systemic infl ammation References 1. Abidi K, et al. Crit Care. 2008;2:R59. 2. Castelli GP, et al. Minerva Anestesiol. 2006;1-2:69-80. 3. Bone RC, et al. Chest. 1992;101:1644-55. P53 Association between apoptosis and mortality in severe septic patients Multiple logistic regression showed that serum caspase-3 levels >0.25 ng/ml were associated with mortality at 30 days (odds ratio = 6.51; 95% confi dence interval = 3.32 to 12.77; P <0.001), controlling for SOFA score and age. Kaplan–Meier survival analysis showed a higher risk of death in septic patients with serum caspase-3 levels >0.25 ng/ml than in patients with lower levels (hazard ratio = 3.80; 95% CI = 2.35 to 6.15; P <0.001). We found a positive association between serum levels of caspase-3 and CCCK-18 (ρ = 0.32; P <0.001).i Methods A prospective, multicenter, observational study in six Spanish ICUs, including 216 patients with severe sepsis. We collected blood samples at the severe sepsis diagnosis moment to determine serum levels of caspase-3 (to assess the main executor of apoptosis) and CCCK- 18 (to assess the apoptosis level). The endpoint was 30-day mortality. Conclusion CRP, LymC and NLCR may distinguish sepsis from nonsepsis SIRS. Thus, CRP and hemogram parameters may contribute to early diagnosis of sepsis. Results We found that nonsurvivor (n = 76) in comparison with survivor (n = 140) septic patients showed higher serum levels of caspase-3 (0.41 ng/ml (0.14 to 0.52) vs. 0.11 ng/ml (0.10 to 0.25); P <0.001) and CCCK- 18 (448 (310 to 723) vs. 319 (236 to 445); P <0.001). Multiple logistic regression showed that serum caspase-3 levels >0.25 ng/ml were associated with mortality at 30 days (odds ratio = 6.51; 95% confi dence interval = 3.32 to 12.77; P <0.001), controlling for SOFA score and age. Kaplan–Meier survival analysis showed a higher risk of death in septic patients with serum caspase-3 levels >0.25 ng/ml than in patients with lower levels (hazard ratio = 3.80; 95% CI = 2.35 to 6.15; P <0.001). We found a positive association between serum levels of caspase-3 and CCCK-18 (ρ = 0.32; P <0.001). 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) Assessment was based on the biomarker’s ability to diagnose septic patients and its ability to predict mortality.i P49 Usefulness of endotoxin activity assay for early diagnosis of sepsis S Sekine, H Imaizumi, K Masumoto, H Uchino Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P49 (doi: 10.1186/cc14129) Results Of 5,257 articles identifi ed, all abstracts were screened, and 750 full-text articles were selected for review. These included primarily randomized controlled trials, cohort studies and postmortem studies. Of 49 biomarkers examined, 72% of the studies examined procalcitonin. Comparing the serum of septic patients with that of controls, most biomarkers were elevated in septic patients, even though only a few had high sensitivity (>85%) and high specifi city (>80%). It was often Introduction The purpose of this study was to evaluate the diagnostic and prognostic value of the endotoxin activity assay (EAA) of sepsis in S19 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 an important role in the infl ammatory response. Serum cholinesterase (butyrylcholinesterase (BChE)) is the major Ach hydrolyzing enzyme in plasma. The role of this enzyme during infl ammation has not yet been fully understood. Here, we describe a correlation between the BChE activity and the early systemic infl ammatory response upon traumatic injury. diffi cult to compare study group with control group as the control group patients were usually not healthy controls. Conclusion Overall the heterogeneity of studies, small sample size and the lack of true healthy controls infl uenced the ability to use the biomarker for prognostication of a septic patient. Furthermore, the lack of healthy control raises the question of redefi ning selection criteria in order to better study septic patients. Methods We measured BChE activity in patients with traumatic injury admitted to the emergency room using a point-of-care-test (POCT) system. In addition, we measured levels of routine infl ammation biomarkers during the initial treatment period. We used the Injury Severity Score to assess the trauma severity. Data were statistically analyzed using the Friedman test. Correlation analysis was performed using Spearman’s rank correlation test. P  <0.05 was considered statistically signifi cant. P51 C-reactive protein and hemogram parameters for the nonsepsis SIRS and sepsis: what do they mean? P56 P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial A Kutz1, P Hausfater2, M Oppert3, C Alonso4, C Wissmann4, B Mueller1, P Schuetz1 1Kantonsspital Aarau, Switzerland; 2Hôpital Pitié-Salpêtrière and Univ- Paris 06, Paris, France; 3Klinikum Ernst von Bergmann, Potsdam, Germany; 4BRAHMS GmbH, Hennigsdorf, Germany Critical Care 2015, 19(Suppl 1):P56 (doi: 10.1186/cc14136) P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial A Kutz1, P Hausfater2, M Oppert3, C Alonso4, C Wissmann4, B Mueller1, P Schuetz1 P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial Results We included 87 patients (32 Group A, 25 Group B, 30 Group C). The serum concentration of S1P (mean ± SD) in the control group was 484.6  ±  152.6  μg/l and signifi cantly higher compared with Group A 239.4  ±  61.3  μg/l, Group B 248.6  ±  93.7  μg/l and Group C 141.6 ± 46.3 μg/l. We observed a negative correlation between S1P and SOFA score (Pearson r = –0.45, P <0.001, R2 = 0.2). The median SOFA score in our cohort was 6. We divided the cohort into two groups: SOFA score <6; and SOFA score >6. We tested the sensitivity and specifi city of S1P to indicate disease severity by ROC analysis. In our cohort the area under the curve (AUC) for S1P was 0.77 (CI 0.670 to 0.870) and therefore higher when compared with common markers of infl ammation (PCT, IL-6, CRP with AUC of 0.68 (0.560 to 0.796), 0.68 (0.554 to 0.786) and 0.67 (0.571 to 0.794), resp.).i 1Kantonsspital Aarau, Switzerland; 2Hôpital Pitié-Salpêtrière and Univ- Paris 06, Paris, France; 3Klinikum Ernst von Bergmann, Potsdam, Germany; 4BRAHMS GmbH, Hennigsdorf, Germany Critical Care 2015, 19(Suppl 1):P56 (doi: 10.1186/cc14136) Introduction Procalcitonin (PCT) is increasingly the standard in the emergency department (ED) for the diagnostic and prognostic workup of patients with suspected infections. Recently, B·R·A·H·M·S PCT direct, a new high-sensitive point-of-care test, has been developed for fast PCT measurement on capillary or venous blood samples with a measuring range of 0.1 to 10.0 μg/l. Methods This is a prospective, comparative international study conducted in three European EDs. P56 Consecutive patients with suspicion of bacterial infection were included. Duplicate determination of PCT was performed on two distinct B·R·A·H·M·S PCT direct test devices on capillary (fi ngertip) and venous whole blood (EDTA), and compared with the reference method (B·R·A·H·M·S PCT sensitive Kryptor or Elecsys B·R·A·H·M·S PCT, respectively). The diagnostic accuracy was evaluated by correlation and concordance analyses. Conclusion Our fi ndings suggest that S1P is a novel marker for severity of sepsis with severe sepsis and septic shock being associated with low levels of S1P. Moreover, blood concentrations of S1P might play a key role in sepsis pathophysiology. y gy Acknowledgements MSW and AN are equal contributors. Reference Acknowledgements MSW and AN are equal contributors. Reference 1. Maceyka M, et al. Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol. 2012;22:50-60. y y Results A total of 303 patients were included over a 6-month period (60.4% male, median age 65.2 years). The correlation between capillary or venous whole blood and the reference method was excellent: r2 = 0.96 and 0.97, sensitivity 88.1% and 93.0%, specifi city 96.5% and 96.8%, concordance 93% and 95% respectively at a 0.25 μg/l threshold. No signifi cant bias was observed (–0.04 and –0.02 for capillary and venous whole blood) although there were 6.8% and 5.1% outliers, respectively. B·R·A·H·M·S PCT direct had a shorter time to result as compared with the reference method (25 vs. 147 minutes, diff erence 122 minutes, 95% CI = 110 to 134 minutes, P <0.0001). P55 Diagnostic accuracy and clinical relevance of an infl ammatory biomarker panel in early sepsis in adult critical care patients P Bauer, R Kashyap, S League, J Park, D Block, N Baumann, A Algeciras-Schimnich, S Jenkins, C Smith, O Gajic, R Abraham Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P55 (doi: 10.1186/cc14135) Diagnostic accuracy and clinical relevance of an infl ammatory biomarker panel in early sepsis in adult critical care patients Introduction Low awareness, late recognition and delayed treatment of sepsis are still common. CD64 is a marker of the innate immune response upregulated in sepsis. The primary goal of this prospective, double-blind study was to compare the diagnostic accuracy of neutrophil CD64 and other cellular markers, along with C-reactive protein (CRP) and procalcitonin (PCT) levels, in early sepsis. Conclusion This study found a high diagnostic accuracy and a faster time to result of the PCT direct in the ED setting. The B·R·A·H·M·S PCT direct may allow a more widespread use of PCT tests in outpatient clinics and smaller institutions. Methods Adult ICU patients, between 2012 and 2014 were eligible. The eight-color fl ow cytometric biomarker panel included CD64, CD163, HLA DR, CD15 and others. Diagnostic test results were compared with infection as the reference standard and sepsis as the target condition, using receiver operating characteristic curve analyses. Multivariable logistic regression was used to assess the relationship of sets of markers with the probability of sepsis, adjusting for other patient characteristics. Results A total of 219 patients were enrolled, 120 with sepsis, 99 served as controls. APACHE IV (median 70 vs. 57), SOFA (8 vs. 7), ICU (2 vs. 1) and hospital length of stay (6 vs. 4) were higher in the sepsis group. Serum cholinesterase activity as an early indicator of systemic infl ammation l AR Zivkovic, K Schmidt, J Bender, T Brenner, S Hofer Heidelberg University Hospital, Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P52 (doi: 10.1186/cc14132) l AR Zivkovic, K Schmidt, J Bender, T Brenner, S Hofer Heidelberg University Hospital, Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P52 (doi: 10.1186/cc14132) Conclusion The novel fi ndings of our study were that there is an association between serum caspase-3 levels, serum CK-18 levels and mortality in septic patients. There has been reported decreased apoptosis and increased survival in septic rats with the administration of caspase inhibitors; thus, it may be interesting to explore those agents in septic patients. Introduction Early diagnosis of systemic infl ammation, a generalised response to noxious stimuli, is fundamentally important for eff ective and goal-directed therapy. Various infl ammation biomarkers have been used in clinical and experimental practice. However, a defi nitive diagnostic tool for an early detection of systemic infl ammation remains to be identifi ed. Acetylcholine (Ach) has been shown to play Acknowledgements Funded by Grant FIS-PI-14-00220 from Instituto de Salud Carlos III (Madrid, Spain) and co-fi nanced by Fondo Europeo de Desarrollo Regional (FEDER). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S20 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Mortality was not diff erent. After adjustment for APACHE IV, CRP and PCT, CD64 molecules/neutrophil measure remained a signifi cant predictor of sepsis (OR = 1.852 for one-unit increase on the log scale, 95% CI = 1.083 to 3.168, P = 0.02, AUC = 0.90). See Table 1. P54 Sphingosine-1-phosphate is a new biomarker for severity in human sepsis MS Winkler1, A Nierhaus1, E Mudersbach1, M Holzmann1, A Bauer1, L Robbe1, C Zahrte1, G Daum2, S Kluge1, C Zoellner1 1University Medical Center Eppendorf, Hamburg, Germany; 2University Heart Center, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P54 (doi: 10.1186/cc14134) Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Cutoff for Sensitivity Specifi city Measure N AUC sepsis (%) (%) C-reactive protein (mg/l) 208 0.86 43 76.9 76.9 CD64 molecules/neutrophil 196 0.83 1,040.5 76.4 76.7 Procalcitonin (ng/ml) 216 0.82 0.74 73.1 73.2 %CD64+ neutrophils 196 0.81 49.96 74.5 74.4 Conclusion Neutrophil CD64 expression is an accurate predictor of early sepsis. Measure Introduction During sepsis a leading symptom is capillary leakage caused by endothelial damage, followed by multiorgan dysfunction. Sphingosine-1-phosphate (S1P) is a bioactive lipid with multiple functions. Cellular reactions depend on the S1P concentration in the blood and its binding to fi ve specifi c G-protein coupled receptors. S1P-regulated functions include: control of endothelial permeability; lymphocyte migration across microvessels depending on an S1P gradient; and control of vascular tone [1]. This clinical study will address the question of whether S1P blood concentrations are associated with sepsis severity.i p y Methods Following ethical approval we enrolled patients fulfi lling the ACCP/SCCM sepsis criteria into three groups (Group A: sepsis; Group B: severe sepsis; Group C: septic shock). A group of 20 healthy donors served as controls. Serum blood samples, laboratory data and clinical parameters are presented for day 1. The primary outcome variable was serum S1P concentration (μg/l) quantifi ed by mass spectrometry (Agilent®). The SOFA score was used to describe disease severity. Serum procalcitonin level correlates with endotoxin activity in patients with septic shock Serum procalcitonin level correlates with endotoxin activity in patients with septic shock B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) Introduction Endotoxin, a key component on the outer membrane of Gram-negative bacteria, is considered to be the most important toxin Introduction Endotoxin, a key component on the outer membrane of Gram-negative bacteria, is considered to be the most important toxin S21 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Procalcitonin as prognostic marker in severe sepsis of abdominal origin g A Gonzalez-Lisorge1, C Garcia-Palenciano1, G Ercole1, T Sansano-Sanchez1, M Campos Aranda2, F Acosta Villegas1 1Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; 2Universidad de Murcia, Murcia, Spain Critical Care 2015, 19(Suppl 1):P58 (doi: 10.1186/cc14138) Introduction We evaluated the utility of procalcitonin (PCT) as a marker of outcome in severe sepsis of abdominal origin (SSAO). SSAO is one of the most prevalent pathologies in surgical ICUs (sICUs). Mortality from 19 to 70% has been reported. Biomarkers are basic tools for diagnosis, follow-up and outcome of sepsis. One of the most studied in last decades has been PCT. Its levels and kinetics could be useful to evaluate the outcome of septic patients. Conclusion Both biomarkers, procalcitonin and lactate provide diagnostic and prognostic information in ICU patients with sepsis, particularly when looking at biomarker kinetics. An evidence- based protocol incorporating both markers may further improve management of septic patients. p p Methods We studied all patients admitted to a sICU with SSAO, from 2007 to 2008. Data collected were: PCT levels on days 1, 3 and 7, gender, age, APACHE II on admission, positivity of surgical cultures, microorganisms isolated and sepsis origin (community or nosocomial). Results Sixty-nine patients were included. Mortality was 23%. Median age was 64.94  years. Median APACHE II was 16.43 points. At day 1, PCT levels were higher in survivors (S) than in exitus (E) (S: 29.22 ng/ ml vs. E: 14.93 ng/ml, P <0.05). PCT levels were infl uenced by gender (males: 27.74 ng/ml vs. females: 15.04 ng/ml, P <0.05), positive cultures (positive: 25.25 ng/ml vs. negative: 13.49 ng/ml, P <0.05) and isolation of Gram-negative microorganisms (Gram-negative: 27.53 ng/ml vs. Gram-positive: 14.77 ng/ml, P  <0.05). Patients with community- acquired sepsis had higher levels of PCT on admission (37.53 ng/ml vs. 13.29 ng/ml, P <0.02). None of these factors had an infl uence on mortality. On day 3 PCT levels where higher in S (S: 20.65 ng/ml vs. E: 16.23 ng/ml, P <0.05). On day 7 PCT levels were higher in E (S: 3.54 ng/ ml vs. E: 12.88 ng/ml, P <0.05). PCT kinetics was diff erent depending on outcome. E patients presented persistently higher levels, whereas PCT in S decreased over time (P <0.05). PCT on day 7 best identifi ed outcome (AUC ROC 0.768). PCT ≥3.5 ng/ml predicted mortality (sensitivity 55%, specifi city 73%). References involved in the development of septic shock, and procalcitonin (PCT) serum concentration has been strongly associated with the severity of sepsis. The eff ect of elevated endotoxin activity (EA) on PCT serum level, organ function and mortality of patients with septic shock was evaluated on the day of admission to the ICU. 1. Charles PE, et al. BMC Infect Dis. 2008;8:38. 2. Rau BM, et al. Arch Surg. 2007;142:134-42. 3. Dahaba AA, et al. Minerva Anestesiol. 2009;75:447-52. 4. Karlsson S, et al. Crit Care. 2010;14:R205. Methods ICU patients with diagnosis of septic shock were consecutively added to the study group within the fi rst 24 hours. Serum PCT level and whole blood EA was immediately measured in all patients on admission (n = 157). Endotoxemia was defi ned as EA >0.4 EAU. Critical Care 2015, 19(Suppl 1):P59 (doi: 10.1186/cc14139) Introduction Blood lactate level is a routinely used biomarker for management of patients in septic conditions in the ICU. There is a lack of clinical research data comparing lactate with novel sepsis biomarkers, such as procalcitonin, in regard to the diagnostic and prognostic potential. Herein, we investigated the diagnostic and prognostic value of initial lactate and procalcitonin levels and their kinetics within the ICU stay for prediction of positive blood cultures and fatal outcome in a well characterized cohort of sepsis patients in a US critical care setting. Methods This is a retrospective, observational cohort study of adult patients with confi rmed severe sepsis or septic shock and with at least one procalcitonin and lactate measurement on admission to the ICU of Morton Plant Hospital (Clearwater, FL, USA). Logistic regression models were calculated to assess the association of biomarkers with blood culture positivity and fatal ICU outcome with area under the curve (AUC) as a measure of discrimination. Conclusion Septic shock with endotoxemia was associated with biochemical and clinical consequences including a higher PCT level, higher frequency of bacteremia, kidney failure, and death. Results The in-hospital mortality rate of the 1,075 included patients (age 68 years) was 23.8% (95% CI = 21.2 to 26.3%) and 18.4% of patients had positive cultures. In regard to the diagnostic value for bacteremic disease, initial procalcitonin had a higher discriminatory value (AUC 0.71) compared with initial lactate levels (AUC 0.52). In regard to prognosis, initial lactate level was the better mortality predictor (AUC 0.69) compared with procalcitonin (AUC 0.55), although both initial levels were signifi cantly lower compared with APACHE III (P >0.05 for both comparisons). When looking at biomarker kinetics, procalcitonin decrease was more strongly associated with fatal outcome compared with initial levels alone (AUC 0.66), but still lower compared with lactate kinetics (AUC 0.73). Procalcitonin or lactate clearance, or both, for risk assessment in patients with sepsis? Results from a prospective US ICU patient cohort i Results Endotoxemia was present in 61% of patients (group 1, n = 95, age 66 (57 to 75)), and in 39% of patients EA was low (group 2, n = 62, age 63 (55 to 76)). Median EA was 0.57 EAU (0.46 to 0.67) in group 1 and 0.27 EAU (0.17 to 0.36) in group 2 (P <0.001). The PCT level was six times higher in group 1 than in group 2 (19.6 ng/ml vs. 3.1 ng/ml, P <0.001) and was correlated with EA (P <0.001, R = 0.5). Median APACHE II score was 23 points (16 to 29) in group 1 and 19 (16 to 25) in group 2; but observed diff erence was not signifi cant. The severity of clinical status estimated by SOFA score was similar in both groups (10 (7 to 13) in group 1 and 11 (8 to 12) in group 2; NS). Forty-six percent of patients in group 1 and 27% in group 2 required renal replacement therapy (P = 0.01). ICU mortality of patients was 41%. The mortality rate was higher in group 1, compared with group 2, and Kaplan–Meier survival analysis of time to death showed statistical signifi cance between the two groups (P = 0.001, log-rank test). A Gram-negative pathogen as the primary source of infection was identifi ed in 64% of patients in group 1 and in 44% in group 2 (P = 0.004); bacteremia was detected in 26% of cases in group 1 and in 12% in group 2 (P = 0.02). Diagnostic value of procalcitonin and IL-6 for sepsis of older patients and other patients in the emergency department SH Woo College of Medicine, The Catholic University of Korea, Incheon St. Mary’s Hospital, Incheon, South Korea College of Medicine, The Catholic University of Korea, Incheon St. Mary’s Hospital, Incheon, South Korea Critical Care 2015, 19(Suppl 1):P60 (doi: 10.1186/cc14140) Introduction Emergency physicians are able to identify severely ill older patients with SIRS in the emergency department (ED). A previous study showed that elevated procalcitonin (PCT) and IL-6 level is a known marker of sepsis and predicts bacteremia and mortality. We assessed the diagnostic value of PCT and IL-6 in older patients and other patients with SIRS and sepsis in the ED. Methods This retrospective cohort study was conducted from January 2013 to December 2013. We enrolled 122 patients with SIRS, 55 were classifi ed as the older age group (>65 years of age). Measurement of serum PCT, IL-6, and white blood cell count was performed on initial admission to the ED. We analyzed these markers in older patients and other patients groups with sepsis. Results Of the 55 patients in the older group 33 (60%) patients had sepsis, and 40 (59.7%) patients of the other group had sepsis. PCT and IL-6 levels were signifi cantly higher in other patients with sepsis (P <0.001, P <0.001). But PCT and IL-6 levels were not higher in old age i Conclusion PCT on day 7 and PCT kinetics can be useful to predict outcome in SSAO. PCT is higher in community-acquired sepsis, when surgical cultures are positive, in Gram-negative isolations and in males. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S22 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 activity; and enhancement of sensitivity to antibiotics. In the case of severe infectious disease, antibiotics are often supplemented with administration of IVIg. patients with sepsis (P = 0.400, P = 0.169). The area under the receiver operating characteristic curve (AUC) for diagnosis of sepsis according to PCT and IL-6 was 0.823, and 0.772 for the other patients group. The diagnostic sensitivity, specifi city, positive predictive value, and negative predictive value of PCT for sepsis in other patients group were 79.5%, 81.5%, 86.1%, and 73.3% respectively, with a PCT cutoff value of 0.18 ng/ml. The diagnostic sensitivity, specifi city, positive predictive value, and negative predictive value of IL-6 for sepsis in other patients group were 67.5%, 81.5%, 84.4%, and 62.9% respectively, with a IL-6 cutoff value of 74.43 pg/ml. Procalcitonin levels in patients undergoing left ventricular assist device implantation Results The patient APACHE II score were 24.9 ± 8.2, the SOFA score 9.1 ± 3.7, and the survival rate after 28 days 83.4%. The values before IVIg administration were: procalcitonin 36.0  ±  463.3 (median 110) ng/ml, presepsin 4,548 ± 4,250 (median 3,337) pg/ml, CRP 15.6 ± 9.6 (median 14.7)  mg/dl, and IL-6 13,860  ±  47,299 (median 630)  pg/ml. All values were thus elevated. On the days after the completion of IVIg administration and on day 7, the level of almost all mediators (procalcitonin, presepsin, CRP, IL-6) decreased signifi cantly. In patients with suspected severe sepsis and septic shock, presepsin reveals valuable diagnostic capacity to diff erentiate sepsis severity compared with procalcitonin, IL-6, CRP, and WBC. Additionally, presepsin and IL-6 reveal prognostic value with respect to 30 days and 6 months all-cause mortality throughout the fi rst week of ICU treatment [1]. p M Holek, J Kettner, J Franeková, A Jabor M Holek, J Kettner, J Franeková, A Jabor Institute for Clinical and Experimental Medicine, Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P61 (doi: 10.1186/cc14141) Introduction Procalcitonin (PCT) is used for diagnosis of a bacterial infection. Several works described nonspecifi c elevation of PCT after cardiac surgery with cardiopulmonary bypass (CPB) caused by systemic infl ammatory response syndrome (SIRS) with various cutoff values for the presence of the infection (0.47 to 2.47 μg/l). However, in patients undergoing left ventricular assist device (LVAD), implantation data about PCT dynamics are lacking. y gi Conclusion The results of the present study found signifi cant decreases of procalcitonin, presepsin and IL-6 resulting from 3 days of immunoglobulin administration, but evidence is still limited and this needs to be confi rmed in larger studies. Methods PCT levels in 25 patients indicated for LVAD were prospectively assessed before surgery and during the postoperative period (days 1, 2, 14 and 30). Values were compared according to the presence of infectious complications (IC) and non-infectious complications such as acute renal failure (ARF) defi ned as injury by RIFLE criteria or necessity of right ventricular assist device (RVAD). Data were also analyzed using combined endpoints A (ARF, RVAD) and B (IC, ARF, RVAD). Values are presented as median with interquartile range (in μg/l). Diagnostic value of procalcitonin and IL-6 for sepsis of older patients and other patients in the emergency department SH Woo Methods The changes in sepsis markers (procalcitonin, presepsin, interleukin-6, C-reactive protein) followed by IVIg administration were investigated in severe sepsis or septic shock patients. The subjects were 410 patients admitted to an ICU with a diagnosis of severe sepsis or septic shock and from whom informed consent had been obtained for the present study. IVIg was administered intravenously for 3 days (5.0 g/ day) and measurements were undertaken before administration (day 1), on the day after completion of administration (day 4), and on day 7. The items measured were procalcitonin, presepsin, IL-6, and CRP. The eff ect of IVIg administration on these markers was then studied. The IVIg studied was polyethylene glycol-treated human immunoglobulin injection fl uid (2.5 g, 50 ml, one vial). f Conclusion PCT and IL-6 is useful predictive markers for diagnosing sepsis in adult patients (<65 years of age) with SIRS in the ED. But these markers are not useful for identifi cation of sepsis in older patients. Diagnostic and prognostic performance of PATHFAST Presepsin in patients with SIRS and early sepsis E Spanuth1 R Carpio2 R Thomae3 Diagnostic and prognostic performance of PATHFAST Presepsin i patients with SIRS and early sepsis E Spanuth1, R Carpio2, R Thomae3 1DIAneering GmbH, Heidelberg, Germany; 2Hospital Nacional Edgardo Rebagliati Martins-EsSalud, Lima, Peru; 3Mitsubishi Chemical Europe, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P63 (doi: 10.1186/cc14143) p , p , 1DIAneering GmbH, Heidelberg, Germany; 2Hospital Nacional Edgardo Rebagliati Martins-EsSalud, Lima, Peru; 3Mitsubishi Chemical Europe, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P63 (doi: 10.1186/cc14143) Introduction Presepsin (sCD14-ST) serves as a mediator of the response to infectious agents. First evidence suggested that presepsin may be utilized as a sepsis marker. Introduction Presepsin (sCD14-ST) serves as a mediator of the response to infectious agents. First evidence suggested that presepsin may be utilized as a sepsis marker. Methods Presepsin was determined at presentation (T0), after 8, 24 and 72 hours in 123 individuals admitted with signs of SIRS and/ or infection. Primary endpoint was death within 30  days. Presepsin was determined using the POC assay PATHFAST Presepsin (Mitsubishi Chemical, Japan). Conclusion PCT levels in patients undergoing LVAD implantation rise signifi cantly in the fi rst 2  days after surgery. Interestingly, this elevation is much higher than after routine cardiac surgery with CPB. Recent works suggest that PCT concentrations are aff ected by SIRS caused by contact with a nonphysiological surface. In the case of LVAD this immunological stimulation is long lasting and even more potent with additional RVAD or ARF treated with renal replacement therapy. In accordance with this hypothesis, our data show that the ability of PCT to detect infectious complication in LVAD patients is limited and its concentrations more probably correlate with postoperative complications in general. p Results Mean presepsin concentrations of the patient group at presentation and of the control group were 1,945 and 130  pg/ml, respectively (P <0.0001). Baseline presepsin diff ered highly signifi cant between patients with SIRS, sepsis, severe sepsis and septic shock. Table 1 (abstract P63). Presepsin levels during the course of the disease in survivors and nonsurvivors Table 1 (abstract P63). Presepsin levels during the course of the disease in survivors and nonsurvivors PSEP, median (IQR) (pg/ml) T0 T8 hours T24 hours T72 hours Survivors 590 622 574 533 (345 to 1,396) (367 to 1,912) (336 to 1,610) (324 to 1,246) Nonsurvivors 1,763 1,859 1,731 2,056 (705 to 6,616) (1,001 to 5,744) (809 to 4,586) (811 to 5,540) P value 0.0046 0.0005 0.0003 0.0013 P61 P61 Procalcitonin levels in patients undergoing left ventricular assist device implantation M Holek, J Kettner, J Franeková, A Jabor Institute for Clinical and Experimental Medicine, Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P61 (doi: 10.1186/cc14141) Reference 1. Behnes M, Bertsch T, Lepiorz D, et al. Diagnostic and prognostic utility of soluble CD 14 subtype (presepsin) for severe sepsis and septic shock during the fi rst week of intensive care treatment. Crit Care. 2014;18:507. p q g μg Results PCT levels were low before surgery (0.16, 0.10 to 0.35), increased signifi cantly within the fi rst (5.72, 2.18 to 9.75; P <0.001) and second (5.94, 2.54 to 11.99; P <0.001) day after operation and decreased on the 14th (0.27, 0.11 to 0.74) and 30th (0.10, 0.06 to 0.19) day. There was no signifi cant diff erence in PCT values between patients with or without IC as well as with or without RVAD. ARF increased PCT level signifi cantly only 14 days after LVAD implantation (0.68, 0.37 to 1.65 vs. 0.15, 0.11 to 0.34; P = 0.015). Subjects with endpoint A had signifi cantly higher PCT values on the second (19.53, 5.66 to 63.12 vs. 3.95, 2.33 to 8.85; P = 0.033), 14th (0.55, 0.31 to 1.44 vs. 0.15, 0. to 0.34; P = 0.020) and 30th (0.19, 0.11 to 0.29 vs. 0.08, 0.05 to 0.13; P = 0.016) day after operation. Patients with endpoint B had signifi cantly elevated PCT levels 2 (11.99, 3.23 to 24.16 vs. 3.95, 2.54 to 7.39; P = 0.027) and 14 (0.55, 0.28 to 0.90 vs. 0.13, 0.09 to 0.23; P = 0.005) days after surgery. Changes in procalcitonin and presepsin before and after immunoglobulin administration in septic patients Changes in procalcitonin and presepsin before and after immunoglobulin administration in septic patients T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury Y Nakamura, H Ishikura, J Tanaka, T Nishida, M Mizunuma, D Ohta, N Matsumoto, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P64 (doi: 10.1186/cc14144) g p p Methods We analyzed data from 14 trials and 4,211 ARI patients to study associations of admission PCT levels and setting specifi c treatment failure and mortality alone at 30 days. We used multivariable hierarchical logistic regression and conducted sensitivity analyses stratifi ed by clinical settings and ARI diagnoses to assess the results’ consistency. Introduction At the moment, we have few reports about the diagnostic accuracy of procalcitonin (PCT), presepsin (P-SEP) and CD64 as a diagnostic marker of sepsis for patients with acute kidney injury (AKI). This study aimed to clarify which is a more useful diagnostic biomarker for sepsis using PCT, P-SEP and CD64 with or without AKI in ICU patients. Methods This study was a single-center observational retrospective study. Blood samples were collected from 334 patients admitted to our ICU between April 2013 and March 2014. Then, we classifi ed the patients with or without AKI. In this study, we adopted RIFLE criteria for AKI diagnosis. After that, the patients in each group were classifi ed into the sepsis group and the nonsepsis group. We measured PCT, P-SEP and CD64 levels at the time of ICU admission and subsequently investigated the diagnostic accuracy of these biomarkers for detecting sepsis. y Results Overall, 864 patients (20.5%) experienced treatment failure and 252 (6.0%) died. The ability of PCT to diff erentiate patients with and without treatment failure was highest in the emergency department setting (treatment failure; area under the curve (AUC): 0.64 (95% confi dence interval (CI): 0.61, 0.67), adjusted odds ratio (OR): 1.85 (95% CI: 1.61, 2.12), P <0.001 – mortality; AUC: 0.67 (95% CI: 0.63, 0.71), adjusted OR: 1.82 (95% CI: 1.45, 2.29), P <0.001). In lower respiratory tract infections, PCT was a good predictor of identifying patients at risk for mortality (AUC: 0.71 (95% CI: 0.68, 0.74), adjusted OR: 2.13 (95% CI: 1.82, 2.49), P <0.001). In primary care and ICU patients no signifi cant associations of initial PCT levels and outcome were found. See Figure 1. Conclusion Admission PCT levels are associated with setting specifi c treatment failure and provide most prognostic information in ARI in the emergency department setting. p Results In this study we met 225 patients with non-AKI and 109 patients with AKI. Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury We conducted ROC analysis for diagnosing sepsis. In non-AKI patients, the AUC of PCT, P-SEP and CD64 were 0.904 (95% CI: 0.824 to 0.950), 0.892 (95% CI: 0.794 to 0.947) and 0.917 (95% CI: 0.842 to 0.958), respectively. In AKI patients, the AUC were 0.933 (95% CI: 0.859 to 0.970), 0.755 (95% CI: 0.642 to 0.840) and 0.905 (95% CI: 0.803 to 0.957), respectively. P65 Prognostic value of procalcitonin in respiratory tract infections across clinical settings A Kutz, B Mueller, P Schuetz, for the IPD Study Group Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P65 (doi: 10.1186/cc14145) P65 Prognostic value of procalcitonin in respiratory tract infections across clinical settings A Kutz, B Mueller, P Schuetz, for the IPD Study Group Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P65 (doi: 10.1186/cc14145) P64 Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury Y Nakamura, H Ishikura, J Tanaka, T Nishida, M Mizunuma, D Ohta, N Matsumoto, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P64 (doi: 10.1186/cc14144) Introduction Whether the infl ammatory biomarker procalcitonin (PCT) provides prognostic information across clinical settings and diff erent acute respiratory tract infections (ARI) is poorly understood. Herein, we investigated the prognostic value of admission PCT levels to predict adverse clinical outcome in a large ARI population. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion CD64 and PCT were a useful biomarker for detecting sepsis for ICU patients with AKI. Conclusion CD64 and PCT were a useful biomarker for detecting sepsis for ICU patients with AKI. Twenty-four patients died during 30 days. The 30-day mortality was 19.5% in total, ranging from 10 to 32% between the fi rst and the fourth quartile of presepsin concentration. Nonsurvivors showed high presepsin values with increasing tendency during the course of the disease while in surviving patients this tendency was decreasing. See Table 1. Conclusion Presepsin demonstrated a strong relationship with disease severity and outcome. Presepsin provided reliable discrimination between SIRS and sepsis as well as prognosis and early prediction of 30- day mortality already at admission. Presepsin showed close association with the course of the disease. Changes in procalcitonin and presepsin before and after immunoglobulin administration in septic patients T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) g p T Ikeda, S Ono, T Ueno, H Tanaka, S Suda T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) Introduction The potentially envisaged actions of intravenous immunoglobulin (IVIg) on severe infectious disease include: virus or toxin neutralizing action; opsonic eff ect; complement bacteriolytic S23 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin Introduction The objective of this study was to evaluate the neutrophil to lymphocyte count ratio (NLCR) versus procalcitonin (PCT) in diagnosing bacteremia in the emergency department (ED). The NLCR is a biomarker that appears early in the course of the acute infl ammatory response and reaches maximum levels within 4 hours after onset. An elevated NLCR has been shown to correlate to bacteremia at a cutoff level of >10 [1]. It is rapidly analyzed on a full blood cell count at low cost. The lowest recommended cutoff level for PCT is <0.5 ng/ml. E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) Introduction Early diff erentiation of bacterial from candidal bloodstream infections (BSIs) in the presence of sepsis or septic shock is crucial because of the need for appropriate treatment initiation. Clinical data, although limited, suggest a role for procalcitonin (PCT) [1-3]. The aim of this study was to investigate a possible association between the etiology of BSIs and the serum PCT levels. f g Methods We randomly chose 425 patients from a 9-month epidemiologic study on the incidence of community-onset severe sepsis and septic shock in western Sweden 2011 to 2012. In total, 207 had severe sepsis and 218 had sepsis, mean age 71.2 versus 64.2 years; males 51%. Sampling was made on arrival in the ED. The NLCR was analyzed immediately, PCT later on plasma frozen at –80°C. A total of 122/425 patients had bacteremia, 72 (35%) in the severe sepsis group versus 50 (23%) in the sepsis group. Most common fi ndings were Escherichia coli (n = 33), Staphylococcus aureus (n = 24), streptococcal spp. (n = 33) and other enterobacteriacae spp. (n = 17).i y Methods ICU patients with clinical and laboratory signs of sepsis or septic shock, with documented BSIs and with both serum PCT and CRP measurements on the day of the positive blood sample (±1 day), were included. Illness severity was assessed by SOFA score on both admission and BSI day. Demographic, clinical, and laboratory data including PCT and CRP levels, as well as the white blood cell (WBC) count on the day of the BSI were recorded. PCT was measured by an electrochemiluminescence analyzer and CRP by the tholosimetric method (Roche, Switzerland). References Introduction The purpose of the study was to assess the prognosis value of pro-adrenomedullin (pADM), C-reactive protein (CRP) and procalcitonin (PCT), lactate (LT), albumin (ALB), cholesterol (CHOL), white blood cell (WBC) and severity score in patients with severe sepsis or septic shock. 1. Martini A, et al. J Infect. 2010;60:425. 2. Petrikkos GL, et al. Eur J Clin Microbiol Infect Dis. 2005;24:272. 3. Charles PE, et al. Intensive Care Med. 2006;32:1577. P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin Results The NLCR shows signifi cantly higher sensitivity than PCT at recommended cutoff levels for bacteremia. Interestingly, this is true even for all 207 patients with severe sepsis, irrespective of bacteremia or not. Sensitivity fi gures with 95% confi dence interval: bacteremia (n = 122): NLCR 80% (0.73 to 0.87) versus PCT 66% (0.58 to 0.75), P = 0.01; severe sepsis with bacteremia (n = 72): NLCR 85% (0.77 to 0.93) versus PCT 70% (0.59 to 0.81), P = 0.03; and severe sepsis but no bacteremia (n = 135): NLCR 71% (0.65 to 0.77) versus PCT 61% (0.54 to 0.68), P = 0.03. Conclusion The NLCR can be used in the ED as a biomarker for bacteremia as well as severe sepsis and seems to perform as well as or even better than PCT in this setting. Rapid response, low cost and no need for extra sampling make it useful as a screening tool. R f ( , ) Results A total of 64 ICU patients (mean age 58 ± 18 years, 39 males) with BSIs were included. SOFA sore was 9 ± 4 on ICU admission and 8 ± 4 on the day of BSI. In 30 of these patients Candida spp. were isolated in blood culture (candidemia group) whereas the remaining 34 had a bacterial etiology of BSI (bacteremia group). Serum PCT concentrations remained within normal ranges in most patients with candidemia whereas a wide range was observed in patients with bacteremia. Mean values of PCT and CRP levels were higher in the bacterial than in the candidemia BSI group: 18.5 ± 33.2 versus 0.73 ± 1.40 ng/ml, P <0.001 and 17.7 ± 10.3 versus 8.9 ± 8.0 mg/dl, P = 0.001, respectively. There was a signifi cant diff erence in WBC count between the two groups: 19,460  ±  10.174 versus 11,000  ±  5,440, P <0.001 for the bacteremia and candidemia BSI group, respectively. A ROC curve analysis of the predictive ability of PCT showed an AUC of 0.79 (P <0.001). When a cutoff point of 0.40 ng/ml was selected using Youden’s J statistic, a low value of PCT had in our sample a negative predictive value of 0.76 and a likelihood ratio (negative) of 0.30. 1. de Jager et al. Crit Care. 2010;14:R192. 1. de Jager et al. Crit Care. 2010;14:R192. Prognosis value of biomarkers in severe sepsis and septic shock MV De La Torre-Prados1, A García-de la Torre2, R Escobar-Conesa1, Conclusion A low serum PCT value could be considered as a diagnostic marker in distinguishing between BSIs of candidal or bacterial origin in ICU patients with varying severity of sepsis. R f g y Reference P66 Neutrophil to lymphocyte count ratio performs better than procalcitonin as a biomarker for bacteremia and severe sepsis in the emergency department LL Ljungström1, D Karlsson1, A Pernestig2, R Andersson3, G Jacobsson1 1Skaraborg Hospital Skövde, Sweden; 2School of Biosciences, University of Skövde, Sweden; 3Institute of Biosciences, Sahlgrenska Academy at the University of Gothenburg, Sweden Critical Care 2015, 19(Suppl 1):P66 (doi: 10.1186/cc14146) P68f P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) g y Reference . Schuetz P, Briel M, Christ-Crain M, et al. Procalcitonin to guide initiation and duration of antibiotic treatment in acute respiratory infections: an individual patient data meta-analysis. Clin Infect Dis. 2012;55:651-62. Figure 1 (abstract P65). Multivariate regression analysis for estimation of predictive value of PCT levels on admission stratifi ed by adverse events and mortality in diff erent settings and diagnoses. *Treatment failure, ~mortality. ARI, acute respiratory tract infection; ECOPD, exacerbated chronic obstructive pulmonary disease; CAP, community-acquired pneumonia; VAP, ventilator-associated pneumonia; Adj., adjusted; OR, odds ratio; CI, confi dence interval. Figure 1 (abstract P65). Multivariate regression analysis for estimation of predictive value of PCT levels on admission stratifi ed by adverse events and mortality in diff erent settings and diagnoses. *Treatment failure, ~mortality. ARI, acute respiratory tract infection; ECOPD, exacerbated chronic obstructive pulmonary disease; CAP, community-acquired pneumonia; VAP, ventilator-associated pneumonia; Adj., adjusted; OR, odds ratio; CI, confi dence interval. S24 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 diff erences were signifi cant in pADM (2.46 (1.21 to 4.89) vs. 1.68 (0.94 to 3.32) nmol/l; P = 0.012), LT (2.6 (1.6 to 3.94) vs. 1.6 (1.2 to 2.43) mmol/l; P <0.001) and ALB (2 (1.55 to 2.38) vs. 2.22 (1.96 to 2.7) g/dl; P = 0.001). Conclusion The protein pADM, LT and ALB showed good prognosis accuracy when measured on admission of septic patients to the ICU. diff erences were signifi cant in pADM (2.46 (1.21 to 4.89) vs. 1.68 (0.94 to 3.32) nmol/l; P = 0.012), LT (2.6 (1.6 to 3.94) vs. 1.6 (1.2 to 2.43) mmol/l; P <0.001) and ALB (2 (1.55 to 2.38) vs. 2.22 (1.96 to 2.7) g/dl; P = 0.001). Conclusion The protein pADM, LT and ALB showed good prognosis accuracy when measured on admission of septic patients to the ICU. P67 Prognosis value of biomarkers in severe sepsis and septic shock MV De La Torre-Prados1, A García-de la Torre2, R Escobar-Conesa1, J Perez-Vacas1, A Puerto-Morlán1, E Cámara-Sola1, A García-Alcántara1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Puerto Real University Hospital, Puerto Real, Cádiz, Spain Critical Care 2015, 19(Suppl 1):P67 (doi: 10.1186/cc14147) P69 Methods A prospective, observational study in adult patients with severe sepsis or septic shock in a polyvalent ICU. Demographics, severity scores (APACHE II and SOFA) and all of the biomarkers were studied within 24  hours from septic shock onset. Descriptive and comparative statistical analysis was performed using the statistical software packages SPSS v.15 and MedCalc® 9.2.1.0. P69 Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Estimating the duration of a central venous catheter at time of insertion g g Results A total of 2,148 patients were screened, and 85 (4%) fulfi lled entry criteria. The incidence of candidemia in these 85 patients was 8.2%, compared with 0.5% in the remaining 2,063 patients (relative risk 16.9%, 95% confi dence interval (CI) = 6.63 to 43.55). Baseline BDG was positive in 74 patients (87%), with a median number of positive tests of 3 (range 1 to 3) and a median value of 523 pg/ml (range 83 to 6,860). All seven patients with candidemia had positive baseline BDG (median value 523 pg/ml, range 203 to 3,660). The best cutoff of baseline BDG for the diagnosis of candidemia was 522 pg/ml (area under the ROC curve 0.883, 95% CI = 0.769 to 0.997), with sensitivity and specifi city of 86% and 88%, respectively. The cutoff value of 80 pg/ml had sensitivity and specifi city of 73% and 27%, respectively.f MJ Holmberg1, LW Andersen1, A Graver1, SB Wright1, D Yassa1, MD Howell2, MW Donnino1, MN Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2University of Chicago, IL, USA Introduction The aim of this study was to investigate whether clinicians can estimate, at the time of insertion, the length of time a central venous catheter (CVC) will remain in place, and to identify clinical variables which may predict CVC duration. CVC-related bloodstream infection is a known complication among critically ill patients. As infection rates may increase with duration of catheterization, more expensive antimicrobial-coated catheters may be used in patients with anticipated long duration of CVC use. i Conclusion This dynamic prediction rule was able to diff erentiate a group of ICU patients at high risk to develop candidemia, with a relative risk of 16.9. BDG is frequently positive in ICU patients. A cutoff value of 522 pg/ml was able to discriminate between candidemic and noncandidemic patients. A revision of the cutoff value for BDG in the ICU is needed. p g Methods We conducted a single-center, prospective study from January 2012 to November 2012. Clinicians prospectively estimated the anticipated duration of CVC at the time of line placement in an electronic procedure note. We collected demographics, past medical history, type of ICU, vital signs, laboratory values, SOFA score, mechanical ventilation and use of vasopressors at the time of placement. Continuous variables were compared with the Wilcoxon rank-sum test and categorical variables with the Fisher’s exact test. Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database y g S Fernandes1, D Bruno2, J Morgado3, C Calle4, C Ferreira5 1Centro Hospitalar Lisboa Norte CHLN, Lisbon, Portugal; 2Hospital Fernando Fonseca, Lisbon, Portugal; 3Centro Hospitalar de Lisboa Central, Lisbon, Portugal; 4Instituto Português de Oncologia, Lisbon, Portugal; 5National Institute of Legal Medicine and Forensic Sciences, Coimbra, Portugal Critical Care 2015, 19(Suppl 1):P70 (doi: 10.1186/cc14150) Results We enrolled 150 patients; median age was 65 (IQR: 52 to 74), 63 (42%) were female and mortality was 22%. Median time from CVC placement to removal was 5 (IQR: 3 to 8) days. The correlation between estimated CVC time and actual time was low (r = 0.36, P <0.001). Forty- eight (32%) patients had a long CVC duration. Clinician estimate had 46% sensitivity and 76% specifi city for predicting long duration of CVC. Of 30 variables tested, only temperature at the time of insertion was signifi cantly associated with long duration (OR: 1.30, 95% CI: 1.04 to 1.63, P = 0.02). The AUC for this model was 0.59 (95% CI: 0.49 to 0.69). Results We enrolled 150 patients; median age was 65 (IQR: 52 to 74), 63 (42%) were female and mortality was 22%. Median time from CVC placement to removal was 5 (IQR: 3 to 8) days. The correlation between estimated CVC time and actual time was low (r = 0.36, P <0.001). Forty- eight (32%) patients had a long CVC duration. Clinician estimate had 46% sensitivity and 76% specifi city for predicting long duration of CVC. Of 30 variables tested, only temperature at the time of insertion was signifi cantly associated with long duration (OR: 1.30, 95% CI: 1.04 to 1.63, P = 0.02). The AUC for this model was 0.59 (95% CI: 0.49 to 0.69). Conclusion Our results suggest a low correlation between clinician prediction at time of insertion and actual duration of CVC. We did not fi nd any good predictors of long duration of CVC. Given our relatively low sample size, we may have been underpowered. It may not be feasible to identify patients at the time of insertion who may benefi t from antimicrobial-coated catheters. Introduction Bloodstream infections in the ICU are a major trigger of morbidity and mortality. Several risk factors for bacteremia have been previously identifi ed, such as presence of a central venous catheter or invasive ventilation [1,2]. Iron is a key element for bacteria growth, and its metabolism is extensively altered by infl ammation. References following: dialysis, surgery, pancreatitis, and receipt of corticosteroids, other immunosuppressive agents or parenteral nutrition. Diff erent from the original description of the score which considered only the fi rst 7 days of ICU stay, we selected patients who fulfi lled these criteria at any time during the ICU stay. Once a patient fulfi lled these criteria, AFT (anidulafungin 200  mg followed by 100  mg daily) was initiated provided that the patients also presented with any of the following: fever, hypothermia, hypotension, leukocytosis, acidosis or elevated C-reactive protein. Blood cultures (days 1 to 2) and baseline serum BDG (days 1 to 3) were performed. Patients with candidemia were treated for ≥14 days, those without candidemia but ≥1 positive BDG (≥80 pg/ml) received AFT for ≥10 days, and patients with negative blood cultures and negative BDG discontinued anidulafungin.i 1. Tabah A, Koulenti D, Laupland K, et al. Characteristics and determinants of outcome of hospital-acquired bloodstream infections in intensive care units: the EUROBACT International Cohort Study. Intensive Care Med. 2012;38:1930-45. 2. Prowle JR, Echeverri JE, Ligabo EV, et al. Acquired bloodstream infection in the intensive care unit: incidence and attributable mortality. Crit Care. 2011;15:R100. Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA N 1 AL C l b 2 P E t 2 T G i 3 F S i ll 4 Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA N 1 AL C l b 2 P E 2 T G i 3 F S i ll 4 SA Nouer , AL Colombo , P Esteves , T Guimaraes , F Scapinello , B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Results We analyzed 246 consecutive episodes of severe sepsis (38%) or septic shock (62%). The 28-day mortality was 36.2%. The profi le of dead patients had a signifi cantly higher average age (65 (IQR: 75.5 to 57.5) vs. 63 (47 to 72); P <0.06), APACHE II (27 (22 to 30) vs. 23 (18 to 27); P <0.001) and SOFA (11 (9 to 12.75) vs. 9 (7 to 10); P <0.001). CRP (168.4 (106 to 285) vs. 165.4 (87.8 to 275) mg/dl; P = NS), PCT (6.5 (0.94 to 23.8) vs. 5.8 (0.97 to 19.59) ng/ml; P = NS) and WBC 14.7 (9.5 to 21.4) vs. 12.9 (5.5 to 17.5); P = NS) were increased in those who died, but CHOL (102 (75 to 134) vs. 108 (86 to 141) mg/dl; P = NS) had lower values. These Introduction Early initiation of antifungal therapy (AFT) improves the outcome in candidemic patients, but empiric AFT is not considered the standard of care. Methods We used a scoring system based on the presence of a central venous catheter and receipt of antibiotics, plus at least two of the S25 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P70 P70 Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database S Fernandes1, D Bruno2, J Morgado3, C Calle4, C Ferreira5 1Centro Hospitalar Lisboa Norte CHLN, Lisbon, Portugal; 2Hospital Fernando Fonseca, Lisbon, Portugal; 3Centro Hospitalar de Lisboa Central, Lisbon, Portugal; 4Instituto Português de Oncologia, Lisbon, Portugal; 5National Institute of Legal Medicine and Forensic Sciences, Coimbra, Portugal Critical Care 2015, 19(Suppl 1):P70 (doi: 10.1186/cc14150) Estimating the duration of a central venous catheter at time of insertion Pearson’s correlation coeffi cient was used to assess the correlation between estimated CVC time and actual time. Duration of CVC use was dichotomized into long (≥7 days) or short (<7 days), based on previous literature, and sensitivity and specifi city for predicting long duration was calculated. We performed a logistic regression analysis to identify variables associated with long CVC duration and calculated the area under the ROC curve (AUC). P72 Preventing catheter-related infections in ICUs: comparing catheter care techniques Preventing catheter-related infections in ICUs: comparing catheter care techniques S Ozden, R Iscimen, H Akalin, N Kelebek Girgin, F Kahveci, M Sinirtas Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P72 (doi: 10.1186/cc14152) Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database We aim to determine whether iron defi ciency is a risk or protective factor for bacteremia in the ICU. Conclusion Our results suggest a low correlation between clinician prediction at time of insertion and actual duration of CVC. We did not fi nd any good predictors of long duration of CVC. Given our relatively low sample size, we may have been underpowered. It may not be feasible to identify patients at the time of insertion who may benefi t from antimicrobial-coated catheters. Methods We performed a retrospective analysis of patients included in the MIMIC-II database, an ICU database that collected data from patients admitted to the medical, surgical, coronary and cardiac surgery ICU of Boston’s Beth Israel Deaconess Medical Center during a period of 7 years. We performed logistic regression models to assess the association between iron and bloodstream infection. Results We included 3,980 patients, 2,988 with low serum iron (<60 ng/ ml) and 992 with normal/high serum iron (≥60 ng/ml). During their fi rst stay in the ICU, 351 (8.82%) patients developed bloodstream infections. Low serum iron was associated with increased odds of bloodstream infection (OR: 1.37; 95% CI: 1.04 to 1.80). After adjusting for age, gender, Simplifi ed Acute Physiology Score, presence of central venous catheter, ICU type, transfusions performed before iron measured, neoplastic disease, diabetes mellitus, hepatic disease, congestive heart failure and ferritin levels, low levels of iron were still associated with an increased odds of bacteremia (OR: 1.41; 95% CI: 1.03 to 1.9). In contrast, low serum iron was associated with a decreased risk of death in the hospital (adj OR: 0.73, CI: 0.57 to 0.95). Catheter-associated bloodstream infections in an ICU of a university hospital in Wroclaw, Poland: an international nosocomial infection control consortium’s fi ndings i g W Duszynska1, VD Rosenthal2, A Litwin1, E Woznica1, A Kubler1 1University Hospital, Wroclaw, Poland; 2International Nosocomial Infection Control Consortium, Buenos Aires, Argentina Critical Care 2015, 19(Suppl 1):P74 (doi: 10.1186/cc14154) i g W Duszynska1, VD Rosenthal2, A Litwin1, E Woznica1, A Kubler1 1University Hospital, Wroclaw, Poland; 2International Nosocomial Infection Control Consortium, Buenos Aires, Argentina Critical Care 2015, 19(Suppl 1):P74 (doi: 10.1186/cc14154) Introduction Catheter-associated bloodstream infections are serious but potentially possible to reduce complication of treatment in the ICU. The aim of the study was to evaluate the frequency and etiology of central line-associated bloodstream infections (CLA-BSI) in ICU patients according to the International Nosocomial Infection Control Consortium (INICC) project. y Conclusion Continued education is important in preventing CRBSIs. Maximum precautions must be taken. Usage of antiseptic solutions with clorhexidine and chlorhexidine gluconate impregnated dressing decreased insertion side infections and usage of silver-coated needleless connectors reduced microorganism entry through the catheter lumen and provided a severe decrease in infection ratio. Reference p j Methods A prospective, observational study was conducted in the 20-bed ICU of the University Hospital in Wroclaw from January 2011 to November 2014. CLA-BSI were diagnosed and evaluated according to protocols standardized by the INICC. The density of CLA-BSI/1,000 central line-days, the incidence index/100 admissions to the hospital, the central line utilization ratio (CL-UR) as well as the microbiological profi le of CLA-BSI were evaluated. The results were compared with our earlier published data and with the fi ndings of international reports. Results Among 1,746 ICU patients, CLA-BSI were diagnosed in 69 cases. The incidence index was 3.88/100 admissions to the ICU. CLA-BSI were diagnosed in 18% of the overall number (381) of device-associated healthcare-associated infections. Central line was used in 91.41 ± 4.4% patients during 19,819 patient-days and 18,155 central line-days. The median density of CLA-BSI/1,000 central line-days was 3.88/3.77/3.36 and 0.0 accordingly in years 2011/2012/2013 and 2014 (from January to November). The main pathogens of CLA-BSI were CN staphylococci (22%), Staphylococcus aureus (21%), and Enterobacteriaceae (29%). In this study, the density of CLA-BSI was about 50% lower (2.75 (2.0 to 6.06)) than in our previous study and in the INICC’s report (2014), but higher than in the CDC’s NHSN (2012) report. j Methods A prospective, observational study was conducted in the 20-bed ICU of the University Hospital in Wroclaw from January 2011 to November 2014. P73 Results Among 1,746 ICU patients, CLA-BSI were diagnosed in 69 cases. The incidence index was 3.88/100 admissions to the ICU. CLA-BSI were diagnosed in 18% of the overall number (381) of device-associated healthcare-associated infections. Central line was used in 91.41 ± 4.4% patients during 19,819 patient-days and 18,155 central line-days. The median density of CLA-BSI/1,000 central line-days was 3.88/3.77/3.36 and 0.0 accordingly in years 2011/2012/2013 and 2014 (from January to November). The main pathogens of CLA-BSI were CN staphylococci (22%), Staphylococcus aureus (21%), and Enterobacteriaceae (29%). In this study, the density of CLA-BSI was about 50% lower (2.75 (2.0 to 6.06)) than in our previous study and in the INICC’s report (2014), but higher than in the CDC’s NHSN (2012) report. Comparison of three cutaneous antiseptic solutions for the prevention of catheter colonization in an ICU for adult patients: a multicenter prospective randomized controlled trial H Yasuda1, M Sanui2, T Komuro2, J Hatakeyama3, S Matsukubo4, S Kawano5, H Yamamoto6, K Andoh7, R Seo8, N Shime9, E Noda10, N Saito11 1Japanese Red Cross Musashino Hospital, Tokyo, Japan; 2Saitama Medical Center Jichi Medical University, Saitama, Japan; 3YokohamaCity Minato Red Cross Hospital, Kanagawa, Japan; 4Kurashiki Central Hospital, Okayama, Japan; 5The Jikei University School of Medicine, Tokyo, Japan; 6Toyonaka Municipal Hospital, Osaka, Japan; 7Sendai City Hospital, Miyagi, Japan; 8Kobe City Medical Center General Hospital, Hyogo, Japan; 9National Hospital Organization Kyoto Medical Center, Kyoto, Japan; 10Kyushu University Hospital, Fukuoka, Japan; 11Nippon Medical School Chiba Hokusoh Hospital, Tiba, Japan Conclusion The implementation of the infection control program and preventive interventions for patients with central venous catheters improved the safety and quality of healthcare in the ICU by reducing CLA-BSI rate. Critical Care 2015, 19(Suppl 1):P73 (doi: 10.1186/cc14153) Introduction The current CDC guideline for the prevention of intravascular catheter-related infections recommends skin preparation with a greater than 0.5% chlorhexidine with alcohol solution before central venous catheter (CVC) or peripheral arterial catheter (AC) placement. However, few studies investigated the superiority of 1% alcoholic chlorhexidine gluconate (1% CHG) over either 0.5% alcoholic chlorhexidine gluconate (0.5% CHG) or 10% aqueous povidone iodine (10% PVI) for the prevention of catheter colonization. The aim of this study is to compare the eff ectiveness of three skin antiseptic solutions for the prevention of intravascular catheter colonization. Reference 1. Kübler A, Duszyñska W, Rosenthal VD, et al. Device-associated infection rates and extra length of stay in an intensive care unit of a university hospital in Wroclaw, Poland: International Nosocomial Infection Control Consortium’s (INICC) fi ndings. J Crit Care. 2012;27:105.e5-10. Catheter-associated bloodstream infections in an ICU of a university hospital in Wroclaw, Poland: an international nosocomial infection control consortium’s fi ndings CLA-BSI were diagnosed and evaluated according to protocols standardized by the INICC. The density of CLA-BSI/1,000 central line-days, the incidence index/100 admissions to the hospital, the central line utilization ratio (CL-UR) as well as the microbiological profi le of CLA-BSI were evaluated. The results were compared with our earlier published data and with the fi ndings of international reports. 1. Safdar N, et al. Chlorhexidine-impregnated dressing for prevention of catheter-related bloodstream infection: a meta-analysis. Crit Care Med. 2014;42:1703-13. Preventing catheter-related infections in ICUs: comparing catheter care techniques S Ozden, R Iscimen, H Akalin, N Kelebek Girgin, F Kahveci, M Sinirtas Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P72 (doi: 10.1186/cc14152) Introduction Catheter-related bloodstream infections (CRBSI) are common and an important cause of morbidity and mortality in critical patients. Optimum approaches for preventing infections are presented in guidelines. This study aims to evaluate effi cacy of diff erent care techniques and education, to defi ne risk factors for decreasing ratio of CRBSIs and to analyze eff ects on morbidity and mortality. Introduction Catheter-related bloodstream infections (CRBSI) are common and an important cause of morbidity and mortality in critical patients. Optimum approaches for preventing infections are presented in guidelines. This study aims to evaluate effi cacy of diff erent care techniques and education, to defi ne risk factors for decreasing ratio of CRBSIs and to analyze eff ects on morbidity and mortality. Conclusion Low serum iron increases the risk of bloodstream infection in the ICU, and should be considered as a risk factor to stratify patients’ risk of bacteremia during ICU stay. S26 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods After ethics committee approval, patients admitted to the ICU, older than 18 years, who were thought to have a central venous catheter (CVC) for more than 48 hours, and whose fi rst catheter was inserted in the ICU were included in the study. Staff were educated before the study and periodically during the study. Catheter care and insertion were applied according to the guidelines. The study was planned as three sequences. In the fi rst group, catheter care was made with a sterile gauze pad. In the second and third groups, catheter care was made with chlorhexidine gluconate impregnated dressing. Also in the third group, a silver-coated needleless connector was inserted into the tip of venous catheters. median duration of catheter indwelling in the entire population was 3.7 days with an interquartile range of 2.0 to 6.7 days, with no signifi cant diff erence between the groups (P = 0.36). Thirteen catheters (5.1%) in the 10% PVI group were positive for catheter-tip colonization, whereas six catheters (2.2%) in the 1% CHG group and fi ve catheters (1.9%) in the 0.5% CHG group were positive (P = 0.07). Preventing catheter-related infections in ICUs: comparing catheter care techniques The probability of catheter colonization was signifi cantly higher in the 10% PVI group than each CHG groups (P  =  0.028, log-rank test). The incidence of catheter colonization and CRBSI is compared between the three groups. Conclusion In this randomized controlled trial comparing the eff ective- ness of three cutaneous antiseptic solutions for the prevention of catheter colonization, either 0.5% or 1.0% CHG was superior to 10% PVI. median duration of catheter indwelling in the entire population was 3.7 days with an interquartile range of 2.0 to 6.7 days, with no signifi cant diff erence between the groups (P = 0.36). Thirteen catheters (5.1%) in the 10% PVI group were positive for catheter-tip colonization, whereas six catheters (2.2%) in the 1% CHG group and fi ve catheters (1.9%) in the 0.5% CHG group were positive (P = 0.07). The probability of catheter colonization was signifi cantly higher in the 10% PVI group than each CHG groups (P  =  0.028, log-rank test). The incidence of catheter colonization and CRBSI is compared between the three groups. Conclusion In this randomized controlled trial comparing the eff ective- ness of three cutaneous antiseptic solutions for the prevention of catheter colonization, either 0.5% or 1.0% CHG was superior to 10% PVI. p Results Totally 105 patients were included in the study and every group included 35 patients. There was no diff erence between groups when evaluating reasons for catheter insertion. There was no statistically signifi cant diff erence according to emergent or elective catheterization, trying times, or catheter insertion side (P >0.05). CRBSI was determined in two patients in group 1, in one patient in group 2, and in no patient in group 3. In group 1 it was observed on the 4th and 11th days. In group 2 it was observed on the 18th day after catheterization. Before the study, a statistically signifi cant decrease was determined in CRBSI ratios before and after education (16.4/1,000, 12.9/1,000 catheter-days (P <0.001)). According to Group 1 a statistically meaningful decrease was assigned in CRBSI ratios in Groups 2 and 3 (4.84/1,000, 2.22/1,000, 0/1,000 catheter-days) (P <0.001, P <0.001, P <0.001). P75 Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source B Civantos Martin, I Pozuelo Echegaray, C Guallar Espallargas, A Robles Caballero, C Briones, P Extremera Navas, P Millan Estañ, A Garcia de Lorenzo y Mateos Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P75 (doi: 10.1186/cc14155) Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source B C P l h C G ll ll p p y B Civantos Martin, I Pozuelo Echegaray, C Guallar Espallargas, A Robles Caballero, C Briones, P Extremera Navas, P Millan Estañ, A Garcia de Lorenzo y Mateos Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P75 (doi: 10.1186/cc14155) Methods This multicenter prospective randomized controlled trial was conducted in 15 Japanese ICUs from December 2012 to March 2014. Patients over 18 years of age undergoing CVC and AC placement in the ICU are randomized to have one of three skin antiseptic preparations before catheter insertion. After removal of the catheter, the distal tip is cultured using semiquantitative or quantitative techniques. The incidence of catheter colonization and catheter-related bloodstream infection (CRBSI) is compared between the three groups. Introduction Long experience in the treatment of neutropenic patients admitted to the ICU has taught us the importance of removing the permanent central venous catheter when infection is suspected, because of the great mortality associated. The problem usually comes when the origin of sepsis is not clear and we assume that mortality is Results A total of 997 catheters were placed, including 339 catheters using 1% CHG, 329 using 0.5% CHG, and 329 using 10% PVI. The S27 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 immune systems to suppress dissemination of infection by the netted chromatin decorated with antibacterial molecules. It was reported that NETs play an important role in various kinds of infections such as pneumonia. However, there is no report on NETs in patients of soft tissue infections. In this study, we evaluated NET production in pus and clarifi ed the role of NETs as a host defense in patients of soft tissue infections. not easy to avoid. It is important to know what happens to neutropenic patients admitted to the ICU because of sepsis from any source, in whom catheter infection cannot be excluded. Methods A retrospective, cohort, descriptive study was carried out between January 2013 and November 2014. Epidemiology data were collected from all neutropenic patients admitted to the ICU who came from hemato-oncology services and had an implanted central venous catheter. Microbiology results and data related to the catheter removal were described. Methods This study was conducted in the ICU of the Trauma and Acute Critical Care Center at Osaka University Hospital. Use of nanotechnology-based surface antiseptic solutions in the ICU Y Kuplay, N Akgun, C Agalar, H Aydýn, O Alýcý, G Turan FSM Teaching and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P78 (doi: 10.1186/cc14158) Y Kuplay, N Akgun, C Agalar, H Aydýn, O Alýcý, G Turan FSM Teaching and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P78 (doi: 10.1186/cc14158) Introduction In our study, we aimed to compare the application of benzalkonium chloride (BC) – a nanotechnology-based product – for 24-hour periods and didecyl dimethyl ammonium chloride (DDAC) for 12-hour periods regarding effi ciency in application of surface antiseptics in the ICU.f Introduction In our study, we aimed to compare the application of benzalkonium chloride (BC) – a nanotechnology-based product – for 24-hour periods and didecyl dimethyl ammonium chloride (DDAC) for 12-hour periods regarding effi ciency in application of surface antiseptics in the ICU.f y Methods A retrospective observational study in a medical and surgical ICU in a tertiary care hospital on adult patients admitted from January 2010 to December 2013. The study enrolled 845 patients divided into 283 internal jugular CVC (IJC), 270 subclavian CVC (SCC) and 287 femoral CVC (FC) in which the catheters were inserted in the ICU by experienced physicians with at least 50 previously successful trials of central line insertion, using CVC bundle checklist. ICU length of stay, incidence of complications, APACHE II score adjusted severity and mortality were calculated for each group. Methods Two diff erent areas with eight beds at both sides of a common corridor in the ICU were named as areas A and B. BC was applied in area A with 24-hour periods and DDCA was applied in area B with 12- hour periods for surface cleaning. Samples were taken from a total of 20 diff erent surfaces including nurse-station desks, phones, keyboards, beds, bedside monitors and ventilators by the same infection control nurse every 24 hours from area A and every 12 hours from area B for 7  days. Swab samples were cultured on 5% sheep bloody agar and McConkey agar in the laboratory. Then the cultured mediums were incubated at 35 to 37°C in an aerobic environment for 18 to 24 hours. NCSS (Number Cruncher Statistical System) 2007 and PASS 2008 Statistical Software (UT, USA) programs were used for the statistical analysis.f Results Patient and catheter characteristics including the duration of catheterization were similar in all groups. Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source B C P l h C G ll ll We collected pus from the patients of soft tissue infections at the time when drainage or debridement was performed and when clinical improvement was observed. The smears of pus specimens were examined by immunohistochemistry to visualize the major NET components: DNA, neutrophil elastase, and histone H1. Concurrently, the patients’ clinical data and laboratory data of blood were recorded to analyze the relationship with NET production.i Results A total of 15 patients were included, mean age was 53 years old and 66% were male. The implanted catheter was removed in 80% of patients. Platelet transfusion was needed in 100% of patients before catheter removal and no complication was observed during catheter removal or in the insertion of a new one. In 53% of patients, catheter infection was confi rmed a posteriori. i p Conclusion Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source can be benefi cial for this kind of patient as it was found that in more than 50% of patients catheter infection was confi rmed a posteriori. Results A total of fi ve patients were included in this study and drainage of abscess or debridement of infection site was performed in all of the cases. Four patients of them were diagnosed as necrotizing soft tissue infections by Clostridium spp. (n = 1) and Bacteroides spp. (n = 3) and the other was diagnosed as cervical abscess by Streptococcus spp. In all cases, no NETs but neutrophils were identifi ed in the fi rst pus: however, NETs appeared in the later smears as the patients’ condition was getting better. Eff ect of insertion route on risk of central line-associated bloodstream infection in critically ill patients R Alhubail, N Hassan g g Conclusion These results suggested that NETs also worked as an immune system against soft tissue infections. Drainage or debridement of infection focus might promote NET production. KFSH-D, Dammam, Saudi Arabia Introduction Femoral, jugular or subclavian central venous catheterization (CVC) is routinely performed during the care of the critically ill. These invasive procedures contribute to additional morbidity, mortality, and costs derived from the interactions between traumatic, infectious and other complications. The aim of this study is to determine whether the subclavian, jugular or femoral central venous access (CVA) routes have an eff ect on the incidence of CLABSI in critically ill patients and to compare between these routes regarding major complications and ICU mortality. P78 Use of nanotechnology-based surface antiseptic solutions in the ICU The rate of CLABSI in the IJC, SCC and FC groups was 5.8 versus 7.2 versus 3.45 per 1,000 catheter- days respectively with P = 0.35. ICU mortality was 134 (47%) cases of the IJC group, 108 (39%) cases of the SCC group and 113 (39%) cases of the FC group. There was no signifi cant diff erence between the three groups of CVC in the incidence of CLABSI rate in the critically ill patients, and a slight increase in ICU mortality in the IJC group compared with the other two groups. Pneumothorax occurred in six (2.2%) cases of SCC and 11 (3.8%) cases of IJC with no signifi cant diff erence between the two groups as the P value was 0.3.f Results There were no statistical diff erences between two groups (Table 1). Conclusion Site of insertion of CVC does not appear to aff ect the rate of CLABSI among critically ill patients. Pneumothorax was recorded in SCC and IJC groups with no statistical preference to either group. Table 1 (abstract P78). Isolated pathogen ratio percentage A (BC) B (DDCA) P value First day 25 20 1.000 Second day 5 15 0.605 Third day 30 20 0.715 Fourth day 65 50 0.527 Fifth day 45 60 0.527 Sixth day 25 25 1.000 Seventh day 60 45 0.527 Table 1 (abstract P78). Isolated pathogen ratio percentage Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials h 1 k2 Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials E Choi1, J Park2 1Yeungnam University College of Medicine, Daegu, South Korea; 2Uijeongbu St. Mary’s Hospital, Uijeongbu, South Korea Critical Care 2015, 19(Suppl 1):P81 (doi: 10.1186/cc14161) Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials Results We observed a signifi cant reduction in DSSI rates in cardiac surgery following implementation of a multimodal ICP from 3.1% in 2010 down to 0.23% in November 2014 (Figure 1). Introduction Whole-body skin decolonization with chlorhexidine in critically ill patients reduces multidrug-resistant bacterial colonization, and catheter-related bloodstream infection (BSI). We performed a meta-analysis of randomized controlled trials to determine whether daily bathing with chlorhexidine decreased hospital-acquired BSIs in critically ill patients. Introduction Whole-body skin decolonization with chlorhexidine in critically ill patients reduces multidrug-resistant bacterial colonization, and catheter-related bloodstream infection (BSI). We performed a meta-analysis of randomized controlled trials to determine whether daily bathing with chlorhexidine decreased hospital-acquired BSIs in critically ill patients. Conclusion Implementing a multimodal ICP signifi cantly reduced the incidence of DSSI in our hospital but it remains diffi cult to identify which interventions were most eff ective. Reference 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. Figure 1 (abstract P79). Incidence of DSSI. Figure 1 (abstract P79). Incidence of DSSI. y Methods We searched the MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials databases to identify randomized controlled trials that compared daily bathing with chlorhexidine and a control (daily bathing with soap and water or nonantimicrobial washcloths, or implementation of MRSA screening and isolation) in critically ill patients. The primary outcome was hospital-acquired BSIs. Secondary outcomes were adverse eff ects of chlorhexidine and the incidence of identifi ed pathogens. i Results This meta-analysis included four studies. The overall incidence of hospital-acquired BSIs was signifi cantly lower in the chlorhexidine group compared with the control 0.80 (95% CI, 0.71 to 0.90; P <0.001; I2 = 29.4%). Gram-positive (RR = 0.59, 95% CI, 0.44 to 0.79, P = 0.000; I2 = 46.0%) and MRSA-induced (pooled RR = 0.64; 95% CI, 0.47 to 0.88, P  =  0.006; I2  = 0.0%) bacteremias were signifi cantly less common in the chlorhexidine group. Chlorhexidine did not aff ect Gram-negative bacteremia or fungemia. Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU y GM Plantier1, CE Bosso1, BN Azevedo2, AC Correa3, AL Silva3, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil; 3Santa Casa de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P80 (doi: 10.1186/cc14160) Introduction Urinary catheter insertion is a common procedure in ICUs and can be an important cause of infection in the hospital environment [1,2]. We aimed to analyze the eff ect of chlorhexidine on long-term urinary catheter insertion and urinary tract infection (UTI) during a 5-year period in patients admitted to a coronary ICU. y p p y Methods Analysis of patients admitted to a coronary ICU of a medium- sized hospital in Brazil from January 2010 to May 2014. The institutional protocol of periprocedural antisepsis was changed from iodine-based antiseptic to chlorhexidine in 2012. The UTI diagnosis was based on urine culture (>105 colony-forming units per ml of urine) associated with at least one clinical/laboratory abnormality (fever >38°C, urination urgency, increased urinary frequency, dysuria, or suprapubic or lumbar pain). The UTI rate represents the urinary tract infections associated with long-term urinary catheter (patient with UTI associated with long-term urinary catheter divided by patients with long-term urinary catheter × 1,000). Results The urinary tract infection rates were 4.8 (year 2010: patients·day–1 (n: 2,511), long-term urinary catheter·day–1 (n: 1,455), device usage rate (958%)), 4.4 (year 2011: patients·day–1 (n: 2,529), long- term urinary catheter·day–1 (n: 1,140), device usage rate (45%)), 0.0 (year 2012: patients·day–1 (n: 2,660), long-term urinary catheter·day–1 (n: 783), device usage rate (29%)), 0.0 (year 2013: patients·day–1 (n: 2,573), long- term urinary catheter·day–1 (n: 960), device usage rate (37%)), and 0.0 (year 2014: patients·day–1 (n: 1,070), long-term urinary catheter·day–1 (n: 444), device usage rate (42%)). y y Methods Analysis of patients admitted to a coronary ICU of a medium- sized hospital in Brazil from January 2010 to May 2014. The institutional protocol of periprocedural antisepsis was changed from iodine-based antiseptic to chlorhexidine in 2012. The UTI diagnosis was based on urine culture (>105 colony-forming units per ml of urine) associated with at least one clinical/laboratory abnormality (fever >38°C, urination urgency, increased urinary frequency, dysuria, or suprapubic or lumbar pain). P79 P79 Reduction of deep surgical site infections in cardiac surgery by introducing a multimodal infection control program A Rutten, JP Ory, L Jamaer, A Van Assche, J Dubois Jessa Ziekenhuis, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P79 (doi: 10.1186/cc14159) Introduction Deep surgical site infections (DSSI) are a major compli- cation after cardiac surgery with a high mortality rate and reported incidences between 0.5 and 5%. Implementing a comprehensive infection control program (ICP) reduces this incidence [1]. The incidence in our hospital varied from 3.1 to 3.8%, which was considered too high. We evaluated the impact of introducing a multimodal ICP on the incidence of DSSI. Methods We noticed a too high incidence of DSSI after cardiac surgery during an observational 3-year period (Figure 1). In February 2013 we introduced a bundle of interdisciplinary infection control measures. Medical and nursing staff of all involved departments took part in developing and implementing these guidelines. Besides emphasizing the importance of existing guidelines (antiseptic shower, hair removal by clipper, strict hand hygiene, prophylactic antibiotics, limiting OR traffi c, tight glycemic control (80 to 110 mg/ dl), and so on), new strategies were introduced. The most important new strategies were nasal decolonization with mupirocin twice daily 48  hours perioperatively, preoperative antiseptic skin preparation twice (chlorhexidine gluconate 0.5%), applying topical skin adhesive to the sternal wound postoperatively and in the case of CABG procedures maintaining a strict barrier between the vein harvesting procedure and the chest procedure. Conclusion The use of chlorhexidine in the periprocedural antisepsis of urinary catheterization contributed to the decrease of urinary tract infections associated with long-term urinary catheter in patients admitted to the coronary ICU. P80 Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU GM Plantier1, CE Bosso1, BN Azevedo2, AC Correa3, AL Silva3, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil; 3Santa Casa de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P80 (doi: 10.1186/cc14160) References 1. Silva E, et al. Crit Care, 2004;8:R251-60. 2. Vincent JL, et al. JAMA. 2009;302:2323-9. Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU The UTI rate represents the urinary tract infections associated with long-term urinary catheter (patient with UTI associated with long-term urinary catheter divided by patients with long-term urinary catheter × 1,000). 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. P77 P77 Role of neutrophil extracellular traps against soft tissue infections N Yamamoto1, M Ojima2, S Hamaguchi1, T Hirose2, R Takegawa2, N Matsumoto2, T Irisawa2, M Seki1, O Tasaki3, T Shimazu2, K Tomono1 1Division of Infection Control and Prevention, Osaka University Graduate School of Medicine, Suita, Japan; 2Osaka University Graduate School of Medicine, Suita, Japan; 3Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Critical Care 2015, 19(Suppl 1):P77 (doi: 10.1186/cc14157) Critical Care 2015, 19(Suppl 1):P77 (doi: 10.1186/cc14157) Introduction Neutrophils work as the frontline of defense against infections and neutrophil extracellular traps (NETs) are one of the S28 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The eff ect of a good surface disinfectant should begin immediately and it should have a long-lasting disinfecting eff ect on the surface. DDAC is an effi cient disinfectant used in medicine and the food industry to protect the surfaces. However, it may cause severe skin itching. BC, which is a nanotechnology-based product, leaves its active metabolites on the surface; it is applied by constituting a spongy layer. Since the effi ciency of BC lasts for 24 hours and it is applied to perform cleaning with 24-hour intervals, we think that it is preferable with regards to workforce gain and cost. Clinical validation of an electronic hand hygiene surveillance system y Methods A quasi-experimental study was done in three level III ICUs of a tertiary care hospital in Kolkata (January to April 2014). Data were collected on existing hand hygiene compliance rate, ventilator- associated pneumonia (VAP) rate, catheter-related bloodstream infection (CRBSI) rate, catheter-related urinary tract infection (CAUTI) rate, standardized mortality ratio (SMR) and average ICU length of stay in the abovementioned units. Root cause analysis was done and interventions were developed to improve hand hygiene compliance and was implemented (July to October 2014). Comparison was done between preintervention and postintervention periods. y P Levin, R Razon, MJ Cohen, CL Sprung, S Benenson Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2015, 19(Suppl 1):P84 (doi: 10.1186/cc14164) P Levin, R Razon, MJ Cohen, CL Sprung, S Benenson Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2015, 19(Suppl 1):P84 (doi: 10.1186/cc14164) Introduction Good hand hygiene (HH) is critical to infection control in the ICU. Electronic HH surveillance systems are purported to improve HH practices. Such a system was recently trialed in our ICU. The system is based on radiofrequency transponders in three locations: bracelets worn by ICU personnel; on all HH product dispensers; and above each patient’s bed. By correlating input from these three sources the system detects whether HH was performed before and after each patient contact. In the event that HH is not performed, the bracelet alerts the user (by vibration) in real time. This study represents a clinical validation of the system. Results In the preintervention period (January to April 2014) the hand hygiene compliance among the caregivers was found to be 40%, VAP rate (8.77), CRBSI rate (3.42), CAUTI rate (5.27), SMR (1.14) and average ICU length of stay was 6 days ± 5.85 SD (median 4.5). Interventions were developed and implemented as follows: education and awareness – road shows; positive reinforcement; secret watch nurse; e-ICU  – electronic surveillance; ring the bell once every hour – baseline hand hygiene; visual reminders; availability of alcohol-based hand rub, soap and water and sinks; random hand swabs; and compliance audits. In the postintervention period (July to October 2014) data showed a signifi cant improvement in the hand hygiene compliance (75%). Further analysis showed an association with decrease in the incidence of VAP rate (4.71), CAUTI rate (3.51), CRBSI rate (2.65), SMR (1.05) and average ICU LOS 5.05 days ± 4.03 SD (median 4). Improving hand hygiene compliance leads to improved health outcome: an analysis y V Rao, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India p Conclusion Introduction of infection control bundle in the ICU reduced the incidence of nosocomial MDRO transmission and infection, which resulted in the reduction of anti-MRSA antibiotics and carbapenems use in critically ill patients. p p y p Critical Care 2015, 19(Suppl 1):P82 (doi: 10.1186/cc14162) Introduction Hand hygiene is the single most eff ective but least practiced action in breaking the chain of transmission of microbes. Studies have shown a correlation between the compliance of hand hygiene and its impact on the health outcome. Eff ects of infection control bundle to prevent nosocomial infection in the ICU in the ICU A Matsushima, M Kawanami, S Fujimi, N Inadome, N Kubo, T Yoshioka Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P83 (doi: 10.1186/cc14163) Introduction Multidrug-resistant organism (MDRO) infections in critically ill patients are often life-threatening. To prevent nosocomial infections of MDRO, we made an infection control bundle in our ICU in 2013. In this study we evaluated the eff ect of our infection control bundle to prevent nosocomial MDRO transmission and infection. Conclusion The electronic HH system consistently underestimated both HH opportunities and HH performance. The main reason for dissatisfaction with the system was inaccuracy of bracelet alerts. These data suggest that for an electronic system to be accepted by ICU staff , it has to be highly accurate and comfortable for the user. Methods Our infection control bundle consists of preemptive contact precaution to all care, active surveillance culture and isolation of patients with MDRO. This bundle was applied to all patients admitted to our ICU since 2013. The study period to evaluate the eff ects of the bundle was from April 2012 to March 2014, and we divided it into two periods; fi rst period (before introduction of the bundle) and second period (after introduction of the bundle). We compared the incidence of nosocomial transmission and infection of MDRO between the two periods. MDRO was defi ned as bacteria that were resistant to more than three kinds of antibiotics. Nosocomial transmission was defi ned when MDRO was detected later than 48  hours after admission. Nosocomial infection was diagnosed according to the National Nosocomial Infection Surveillance Manual. P83f Eff ects of infection control bundle to prevent nosocomial infection in the ICU A Matsushima, M Kawanami, S Fujimi, N Inadome, N Kubo, T Yoshioka Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P83 (doi: 10.1186/cc14163) Clinical validation of an electronic hand hygiene surveillance system y Methods ICU staff (nurses and physicians) were followed by a trained observer over 60-minute periods. Each movement and contact during the period was documented. HH opportunities were determined according to WHO criteria and actual HH performance recorded. Observer and electronic data were compared for number of opportunities, HH performance and compliance. A satisfaction questionnaire was distributed to all users. Paired Student’s t test was used for comparison of the observer and electronic data. Results Observations were made over 56 time periods that included 836 HH opportunities and 485 occasions when HH was performed. The observer recorded 10.9  ±  7.6 HH opportunities/hour compared with 6.8 ± 6.9 for the electronic system (P <0.001). HH performance occurred on 8.7 ± 3.9 occasions/hour versus 6.0 ± 3.1 occasions/hour as recorded by the electronic system (P <0.001). Overall HH compliance was 62.5  ±  17.7% versus 57.5  ±  21.0% respectively (P  =  0.523). On comparison of specifi c observation periods, there was poor correlation between compliance as recorded by the observer and electronic system (r = 0.03, P = 0.915). Satisfaction questionnaires were completed by 41 personnel. Satisfaction with the system was low or very low for 21/41 (61%). System inaccuracy (either bracelet alerts without cause, or lack of bracelet alerts when HH was required) was the most common reason for dissatisfaction (31/41, 76%), followed by physical discomfort from the bracelet (18/41, 44%). Conclusion Improved hand hygiene compliance can be attributed to decreased incidence of VAP, CRBSI, CAUTI, SMR and average ICU LOS. This does defi nitely impact the overall clinical outcome. However, continued surveillance of hand hygiene compliance and regular audits is of utmost importance to make the change sustainable. Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials h 1 k2 The overall incidence of adverse events, such as skin rashes, was similar in both groups. Conclusion Implementing a multimodal ICP signifi cantly reduced the incidence of DSSI in our hospital but it remains diffi cult to identify which interventions were most eff ective. Reference 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. S29 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Daily bathing with chlorhexidine was associated with a reduction in the rates of hospital-acquired BSI without signifi cant complications in critically ill patients. It also decreased the incidence of Gram-positive hospital-acquired BSIs, especially MRSA. Results Admission to the ICU comprised 363 patients in the fi rst period and 380 patients in the second period. The incidence of transmission was decreased from 48 (13.2%) to 21 (5.5%) in methicillin-resistant Staphylococcus aureus (MRSA), from 16 (4.4%) to zero (0%) in multidrug- resistant Acinetobacter baumannii. The incidence of nosocomial infection by MDRO was also decreased from 23 (6.3%) to 17 (4.5%) in pneumonia, from fi ve (1.4%) to two (0.3%) in urinary tract infection, and from 12 (3.3%) to one (0.3%) in surgical site infection. The incidence of antibiotic use for MDRO infection was decreased from 41 (11.3%) to 24 (6.3%) in anti-MRSA antibiotics, and from 19 (5.2%) to eight (2.1%) in carbapenems. P86 A survey of UK acute clinicians’ knowledge of personal protective requirements for infectious diseases and chemical, biological, and radiological warfare agents AR Bond1, A Buckingham2, J Schumacher1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P86 (doi: 10.1186/cc14166) Evaluation of the microbial tightness of closed system transfer devices by simulating airborne and touch contamination University of Bonn, Germany Introduction The use of intravascular catheter devices is often associated with serious bloodstream infections due to microbial contaminations. To minimize risk of such infections NIOSH recommends S30 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 the use of closed system transfer devices (CSTDs). To evaluate the microbial tightness of CSTDs we developed two methods which simulate the bioburden in ambient air of operating rooms and ICUs. the use of closed system transfer devices (CSTDs). To evaluate the microbial tightness of CSTDs we developed two methods which simulate the bioburden in ambient air of operating rooms and ICUs. for clinicians treating patients exposed to infectious diseases and CBRN agents, ideally in a simulation setting. Further research into whether the required levels of PPE are readily available to clinicians would be pertinent. Methods The methods simulate airborne and touch contamination. We tested the microbial tightness of the integrated Safefl ow® valve of a Mini-Spike® which is used for drug admixture. The airborne contamination was done in an exposure chamber in which a nebulizer distributed defi ned B. subtilis spore aerosols [1]. A Mini-Spike® was inserted into a vial of 0.9% sodium chloride solution (NaCl). A nebulizer with a suspension of 4.8 × 105 CFU spores of B. subtilis per ml was used to generate an aerosol for 1 minute. The volume of B. subtilis suspension nebulized per minute was 0.278  ml. This corresponds to 1.34  ×  103 aerosolized spores in the exposure chamber, which has a volume of 0.24 m3 (5.6 × 103 CFU per m3 air). The used concentration was 100 times higher than the microbial burden found in hospitals [2]. After nebulization the valve was disinfected and NaCl was withdrawn into a syringe at certain time intervals. The NaCl was incubated on tryptic soy agar at 37°C for 48 hours. Results were documented as CFU. For touch contamination, a Mini-Spike® was attached to a vial of NaCl. The valve of the Mini-Spike® was contaminated with 105 CFU Staphylococcus aureus. The subsequent procedure was done as described above. g References 1. Dunkelberg H, Fleitmann-Glende F. Measurement of the microbial barrier eff ectiveness of sterilization containers in terms of the log reduction value for prevention of nosocomial infections. Am J Infect Control. 2006;34:285-9. 1. Dunkelberg H, Fleitmann-Glende F. Measurement of the microbial barrier eff ectiveness of sterilization containers in terms of the log reduction value for prevention of nosocomial infections. Am J Infect Control. 2006;34:285-9. 2. Qudiesat K. Assessment of airborne pathogens in healthcare settings. AJMR. 2009;3:66-76. Results During the study period, 40 patients with TB were admitted to the ICU; 75% male and median age of 52 years (IQR 37.5 to 62.8). Overall, 22 (55%) patients died in the hospital, of whom 16 (40%) died in the ICU. Comorbid illness was identifi ed in 32 (80%) patients, with HIV infection being the most common, present in 15 (37.5%) patients. The main reason for ICU admission was respiratory failure (70%), followed by sepsis/septic shock (22.5%). Twenty-eight (70%) patients had isolated pulmonary disease, four (10%) had isolated extrapulmonary disease and eight (20%) had association of pulmonary and extrapulmonary disease. Mycobacterial cultures were positive in 31 (77.5%) patients; three patients presented monoresistant strains. Twenty-nine (72.5%) patients required mechanical ventilation and 21 (52.5%) required vasopressor infusion in the ICU; two patients were treated with ECMO. Thirty-four (85%) patients received antituberculosis therapy. The median length of stay was 11.5 (IQR 3.25 to 28.5) days in the ICU and 40.5 (IQR 21.0 to 62.8) days in the hospital. The presence of at least one comorbidity, smoking, age, sepsis/septic shock on admission, high SAPS II and APACHE II score, positive direct examination and PCR in respiratory samples, the need for mechanical ventilation or vasopressor infusion were signifi cantly associated with mortality (P <0.05). There was no association between mortality and HIV status, site of TB disease, concomitant acute disease or development of hospital infections. 2. Qudiesat K. Assessment of airborne pathogens in healthcare settings. AJMR. 2009;3:66-76. R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento Introduction To describe the characteristics of the patients with tuberculosis (TB) requiring intensive care and to identify the factors associated with in-hospital mortality in an ICU in Portugal. Results Out of nine tested valves, none showed transmission of B. subtilis spores after airborne contamination. Three out of nine tested valves were contaminated with S. aureus after touch contamination. Conclusion Our study shows that both methods are suitable for evaluating the microbial tightness of CSTDs. References Methods A retrospective cohort study between January 2007 and July 2014 of all patients with TB admitted to the ICU of the Infectious Diseases Department of Centro Hospitalar de São João. Comorbid diagnoses, clinical features, radiological and laboratory investigations and outcomes were reviewed. The primary outcome was the in- hospital mortality. A univariate analysis was performed to identify risk factors for death. References References 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West Africa. http://www.cdc.gov/vhf/ebola/outbreaks/guinea/. 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West Africa http://wwwcdc gov/vhf/ebola/outbreaks/guinea/ 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West p g g 2. Brinker A, et al. Personal protection during resuscitation of CBW victims. A survey among medical fi rst receivers in the UK. Prehosp Disaster Med. 2009;24:525-8. A survey of UK acute clinicians’ knowledge of personal protective requirements for infectious diseases and chemical, biological, and radiological warfare agents AR Bond1, A Buckingham2, J Schumacher1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P86 (doi: 10.1186/cc14166) Introduction We conducted a survey to assess clinicians’ knowledge of personal protective equipment (PPE) requirements for infectious diseases and biochemical warfare agents. A safe level of PPE is essential when treating patients with highly infectious diseases or those contaminated with hazardous substances. The recent Ebola virus disease (EVD) outbreak in West Africa has highlighted that, although uncommon, contagious diseases with high mortality rates can be a threat to healthcare systems at local, national, and international levels [1]. Chemical, biological, radiological or nuclear (CBRN) contamination presents similar risks. Conclusion In this cohort we found a high mortality rate in the TB patients requiring intensive care. The risk factors for mortality due to severe TB are mainly related to the severity of organ failure, patient characteristics and burden of disease and not to HIV status or site of TB disease. Methods A validated, hand-delivered, multiple-choice questionnaire [2] was used to assess intensive care, emergency medicine, and anesthetics specialist registrars’ knowledge of respiratory and skin protection needed during a resuscitation scenario with advanced life support. Participants selected the PPE required for the biological hazards: EVD, severe acute respiratory syndrome (SARS), inhalational anthrax, plague and smallpox; and the biochemical hazards: sarin, hydrogen cyanide, phosgene and mustard gas (dichlordiethyl sulfi de). Responses were compared with UK national recommendations and a previous survey in 2009 [2]. Tuberculosis in the ICU: a retrospective cohort study R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento Centro Hospitalar de São João/Faculty of Medicine University of Porto, Portugal Critical Care 2015, 19(Suppl 1):P87 (doi: 10.1186/cc14167) Tuberculosis in the ICU: a retrospective cohort study R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 Introduction Changes in infection agents and their sensitivity to antibiotics are the main cause of severity of surgical infections. In spite of development and introduction of new drugs and methods of treatment, the number of patients with sepsis increases, so the problem in diagnosing and treatment is still far from resolution. Introduction Changes in infection agents and their sensitivity to antibiotics are the main cause of severity of surgical infections. In spite of development and introduction of new drugs and methods of treatment, the number of patients with sepsis increases, so the problem in diagnosing and treatment is still far from resolution. Methods A comparative retrospective analysis of 52 histories of patients with sepsis, which were treated in the Department of Surgical Infections in 1999 and 2013. g g Methods A comparative retrospective analysis of 52 histories of patients with sepsis, which were treated in the Department of Surgical Infections in 1999 and 2013. Results The number of patients with sepsis in 2013 was raised 2.7 times, in comparison with 1999. Mortality decreased from 79% in 1999 to 55% in 2013. In most cases sepsis was accompanied with immunosuppressive disorders, such as diabetes, oncology, alcohol and drug addiction, and HIV infection. We analyzed crops of discharge from the wound and blood cultures in 52 patients with sepsis. Crops of wound were taken during the initial surgical intervention, then every 3 to 7 days, as well as the surgical interventions being repeated. Blood cultures were performed in the presence or suspected diagnosis of sepsis, in accordance with the classifi cation Bone criteria. In comparison of spectrum of infection agents, Staphylococcus aureus is still leading (1999 – 36.6% of isolates, 2013 – 25%), and the percentage of MRSA was 0% in 1999 and 37.5% in 2013. The frequency of Gram-negative fl ora has increased: E. coli (8.5%/20%), P. aeruginosa (8.5%/12%), Klebsiella pneumoniae (0%/16%) and Acinetobacter spp. (0%/16%). Speaking about the resistance of microorganisms, there is still a high percentage of sensitivity to aminoglycoside antibiotics (79.4%/75%), glycopeptides (77.2%/71%), carbapenems (88.4%/78%) and also to the combination therapy (71.8%/62.4%), but also a reduction in sensitivity to the group of beta-lactam antibiotics (58.2%/32.5%) and fl uoroquinolones (64.6%/36.4%). Conclusion Cutaneous mucormycosis is less common than other clinical forms, most frequently seen in inmunocompetent patients but potentially lethal if treatment is not rapid. Patients at risk are those with disruption of the normal protective cutaneous barrier. Low-pathogenicity mycoplasma species induce immunoparesis and are highly prevalent amongst patients with ventilator-associated pneumonia TJ Nolan1, N Gadsby2, TP Hellyer3, K Templeton2, R McMullan4, J McKenna5, J Rennie1, CT Robb1, TS Walsh1, AG Rossi1, AJ Simpson3, A Conway Morris6 1University of Edinburgh, UK; 2NHS Lothian, Edinburgh, UK; 3Newcastle University, Newcastle, UK; 4Queen’s University, Belfast, UK; 5Belfast Health and Social Care Trust, Belfast, UK; 6University of Cambridge, UK Critical Care 2015, 19(Suppl 1):P89 (doi: 10.1186/cc14169) Introduction Ventilator-associated pneumonia (VAP) remains a signifi cant problem within ICUs. There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms of this immunoparesis remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent amongst patients with VAP than previously recognised, and that these organisms potentially impair immune cell function. Conclusion The number of patients with sepsis has increased; the mortality of sepsis has decreased. The frequency of S. aureus isolation is still high, MRSA is the same. The frequency of Gram- negative fl ora isolation has increased, especially K. pneumoniae and Acinetobacter spp. The resistance of microorganisms to beta-lactams and fl uoroquinolones is rising but the sensitivity to aminoglycosides, glycopeptides, and carbapenems is still maintained. y Methods Two cohorts [1,2], totalling 159 patients, were recruited from 12 UK ICUs; all patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage. VAP was defi ned as growth of organisms at >104 CFU/ml on conventional culture. Thirty- six healthy donors underwent lavage for comparison. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. Additionally, healthy donor monocytes and macrophages (MDM) were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed. P91 2. Hellyer T, et al. Thorax. 2014 [Epub ahead of print]. 1. Conway Morris A, et al. Thorax. 2010;65:201-7. Infectious events and prescription of antimicrobials in the coronary ICU CE Bosso1, SV Ferreira2, GE Valerio2, JV Moraes2, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P91 (doi: 10.1186/cc14171) Introduction The eff ectiveness of initially used antimicrobials represents an important factor in infectious events in coronary intensive care units (CICU) [1]. This study aimed to analyze the prevalence of infectious events and the prescribed antimicrobial in CICU. Results Mycoplasmataceae were found in 48% of patients with VAP, compared with 14% of patients without VAP (P <0.0001). Patients with sterile lavage had a similar prevalence to healthy donor lavage (10 vs. 8%, P = 0.54). The commonest organism identifi ed was M. salivarium. Human blood monocytes and MDM incubated with M. salivarium displayed impaired cytokine responses to lipopolysaccharide and MDM demonstrated impaired phagocytosis. Methods We analyzed the data of 2,005 patients admitted to the CICU for 3 years. The infectious events were based on general characteristics, main sites and outbreaks of infectious events in addition to the main microorganisms and pathogens. The prescription of antimicrobials was analyzed based on the isolated or associated use of antimicrobials. We also analyzed the adequacy of initial empirical antimicrobial according to the microbiological evidence. The general characteristics of events – that is, time, evidence of infection, infections by multidrug-resistant pathogens – are also presented. y Conclusion This study demonstrates a high prevalence of Mycoplasmataceae amongst patients with VAP, with a markedly lower prevalence amongst patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The commonest organism found, M. salivarium, is able to profoundly impair the functions of key immune cells and thus suggests that Mycoplasmataceae may contribute to VAP pathogenesis. Results The prevalence of infection was 4% (n  = 81). Ventilator- associated pneumonia was 35% (n = 28), whereas urinary and primary bloodstream associated with catheters was 14% (n = 11) and 9% (n = 7), respectively. There was 82% (n = 66) evidence of microbiological infection. The main pathogens and microorganisms found were Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 In these patients, if signs are of sepsis it is very important to suspect the possibility of infection by Mucor and initiate empiric antifungal treatment with surgery to avoid high mortality. Surprisingly, in our series, determination of procalcitonin showed high levels in spite of not having value in fungal infection. P88 Cutaneous mucormycosis in the ICU Cutaneous mucormycosis in the ICU EH Herrero, M Sánchez, A Agrifoglio, L Cachafeiro, MJ Asensio, B Galván, A García de Lorenzo Hospital La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P88 (doi: 10.1186/cc14168) Results Ninety-eight clinicians (anesthetics n  = 51, emergency medicine n = 21, intensive care medicine n = 26) completed surveys. The best knowledge (76% correct) was for SARS, with less knowledge for anthrax, plague, EVD, and smallpox (60%). We found limited knowledge for chemical warfare agents (20 to 30%). Sixty to 80% of all incorrect responses were over-rated. There was no diff erence in knowledge compared with previous published results [2]. Introduction Mucormycosis is a devastating disease most commonly seen in immunosuppressed individuals. It has the propensity to disseminate in humans and cause rhinocerebral, pulmonary, gastrointestinal, and cutaneous infections. This study focuses on cutaneous mucormycosis, incidence, epidemiologic characteristics and mortality in intensive care medicine. Conclusion Despite national and regional training since previous surveys [2], the results indicate that further training on PPE is required S31 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We present a descriptive study in an ICU between the years 2001 and 2013 on the incidence of patients with cutaneous mucormycosis. Sociodemographics, comorbidities and laboratory data were recorded. Clinical data were collected to calculate the APACHE score. The main outcome was to analyze the epidemiological characteristics of patients with cutaneous mucormycosis and mortality. Results Seven patients were identifi ed with cutaneous mucormycosis between the years 2001 and 2013. The mean age of patients was 52 ± 4, with an APACHE score of 19 ± 9, and 57% died. All patients were admitted for trauma-related injury suff ering blast, abrasive injuries or burns. Mortality among patients with signs of sepsis was 100%, and only in one of them was empirically antifungal therapy started; in the others antibiotic treatment was directed. Among patients without signs of sepsis, the survivor was treated with amputation where mucoral infection was isolated. Procalcitonin rose in all patients with signs of sepsis. P90 Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 IV Avdoshin, LG Akinchits, ES Konstantinova, MA Shatil, ON Dobrydin, NA Bubnova Saint-Petersburg City Hospital of St. George, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P90 (doi: 10.1186/cc14170) Analysis of Gram-negative rod bacteremia in the surgical and medical ICU Analysis of Gram-negative rod bacteremia in the surgical and medical ICU D Adukauskiene, D Valanciene D Adukauskiene, D Valanciene Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P92 (doi: 10.1186/cc14172) Introduction The aim was to analyze the Gram-negative bacteremia profi le and the predisposing factors for length of stay in the surgical and medical ICU and outcome. Methods Retrospective data analysis of patients during 4 years treated in a surgical and medical ICU with positive blood culture for Gram- negative rod. g Results Gram-negative rod monobacteremia (n = 160) cultures revealed: Escherichia coli (n  = 52, 32.5%), Acinetobacter spp. (n  = 47, 29.4%), Klebsiella spp. (n = 22, 13.7%), Enterobacter spp. (n = 20, 12.5%), Proteus spp. (n = 11, 6.9%), anaerobes (n = 3, 1.9%) and other Gram-negative rods, including Stenotrophomonas maltophilia, Haemophilus infl uenzae, Neisseria meningitidis, Achromobacter spp. and Actinobacillus limirensi (n = 5, 3.1%). Both E. coli and Acinetobacter spp. were responsible for the vast majority of Gram-negative rod monobacteremia (n  = 99, 61.8%, P = 0.0128). Also most often (n = 50, 72.5%, P = 0.049) primary bacteremia was caused by E. coli and Acinetobacter spp. Separate group’s multidrug resistance was found: E. coli in 12 (23.1%) cases, Acinetobacter spp. in 45 (95.7%, P = 0.02), Klebsiella spp. in nine (40.9%), Enterobacter spp. in 11 (55.0%), Proteus spp. in six (54.6%) cases. The vast majority of patients with multidrug-resistant bacteremia were aged over 65 years (n = 64, 77.1%, P = 0.042), stayed in the ICU less than 14 days (n = 70, 84.3%, P = 0.039), and had lethal outcome (n = 74, 89.2%, P = 0.03). Patients who stayed in the ICU less than 14 days presented with primary Gram-negative rod bacteremia (n = 67, 57.7%, P = 0.03), need for mechanical ventilation (n = 90, 77.6%, P = 0.043) and lethal outcome (n  = 112, 96.6%, P  =  0.01). Lethal outcome was confi rmed in patients with primary Gram-negative rod bacteremia (n = 55, 79.7%, P = 0.03), MDR strain (n = 74, 89.2%, P = 0.03), presence of shock (n = 120, 75.0%, P <0.001), mechanical ventilation (n = 133, 74.3%, P <0.001), cancer chemotherapy (n = 18, 90.0%, P = 0.03), and chronic obstructive pulmonary disease (n = 13, 100%, P = 0.03). Conclusion The carriage of MDRO in ICU-admitted patients is important, especially for ESBL-GN. Is carriage of multidrug-resistant organisms a risk factor for nosocomial infections in an infectious diseases ICU? Introduction The objective was to evaluate whether asymptomatic carriage of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and extended-spectrum beta- lactamase producing Gram-negative bacilli (ESBL-GN) on admittance to the ICU of the University Hospital of Infectious Diseases Cluj-Napoca, Romania is a risk factor for infection due to these multidrug-resistant organisms (MDRO) during hospitalization. fi Conclusion We conclude that infection is prevalent even in CICU, and that the microbiological profi le is quite diverse, as well as the antibiotics. This allows us to better understand the profi le of this kind of unit. Reference g g p Methods A prospective study during a 6-month period (June to November 2014), including all adult patients admitted to our ICU. All patients were screened on admittance for nasal MRSA, intestinal VRE and ESBL-GN carriage. Patients admitted with any localization of infections due to these organisms were excluded. Patients were monitored for developing nosocomial infections due to MDRO during hospitalization. We evaluated previous colonization as a risk factor for future infections. We used bioMerieux selective chromogenic media for MDRO for screening and Vitek2Compact for identifi cation. Statistical analysis was performed with chi-square test and univariate analysis. 1. Silva E, et al. Crit Care. 2004;8:R251-60. y p q y Results From 119 screened adult patients, 65 women (54.6%), average age 67  years, we had at screening on admittance into the ICU: 14 positive MRSA (11.8%), 63 positive ESBL-GN (52.9%  – 41 strains of Escherichia coli, 26 strains of Klebsiella sp., 11 strains of Proteus mirabilis and one strain of Enterobacter cloacae) and 35 positive VRE (29.4% – 33 strains of Enterococcus faecium and two strains of Enterococcus faecalis) without concomitant infection with these MDRO. The average duration of ICU stay was 7.32 days. During hospitalization, 14 patients (11.8%) developed nosocomial infections due to MDRO. Colonization with MDRO preceded nosocomial infections in: one of four patients with MRSA-positive blood cultures (P = 0.96), seven of eight patients with ESBL-GN infections (P = 0.10) and three of four patients with VRE urinary tract infections (P = 0.14). Although not statistically signifi cant, owing to the low number, most patients who developed infections with ESBL-GN had previous intestinal colonization. 1. Silva E, et al. Crit Care. 2004;8:R251-60. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P93 Gram-positive bacteria (n = 24, 30%; Staphylococcus aureus (n = 16, 20%), Enterococcus faecalis (n = 4, 5%), Staphylococcus epidermidis (n = 3, 4%)), Gram-negative (n = 43, 53%; klebsiella sp. (n = 13, 16%), Pseudomonas aeruginosa (n = 7, 9%), Escherichia coli (n = 7, 9%)) and fungi (n = 5, 6%; candida sp. (n  = 2, 3%), Candida albicans (n  = 1, 1%), Candida dubliniensis (n  = 1, 1%)). The commonly prescribed antimicrobials were piperacillin/tazobactam (n = 32, 40%), vancomycin (n = 30, 37%), polymyxin B (n = 23, 28%), cefepime (n = 16, 20%), meropenem (n = 12, 15%), cefuroxime (n = 8, 10%), ciprofl oxacin (n = 6, 7%), tigecycline (n = 6, 7%), ampicillin (n = 5, 6%), clindamycin (n = 4, 5%), chloramphenicol (n = 4, 5%), oxacillin (n = 4, 5%) and others (n = 32, 28%). There was 75% (n = 46) infection during hospitalization in the unit. Approximately 32% of infections were caused by multidrug-resistant pathogens, although there was effi ciency of 81% in the proper use of initial antimicrobials. Conclusion We conclude that infection is prevalent even in CICU, and that the microbiological profi le is quite diverse, as well as the antibiotics. This allows us to better understand the profi le of this kind of unit. Reference Is carriage of multidrug-resistant organisms a risk factor for nosocomial infections in an infectious diseases ICU? M Lupse1, M Flonta2, L Herbel2, A Petrovan2, A Binder2, N Todor3, A Cioara1 1University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Teaching Hospital of Infectious Diseases, Cluj-Napoca, Romania; 3‘Ion Chiricuta’ Institute of Oncology, Cluj-Napoca, Romania Critical Care 2015, 19(Suppl 1):P93 (doi: 10.1186/cc14173) p g References S32 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P96 admission; 4.8% required the extrarenal depuration technique. In total, 497 patients (44.1%) were coronary, 49.5% male, mean age 66.18 (SD ±12.6), CI (64.88 to 67.48); mean APACHE II 9.74 (SD ± 6.1), CI (9.1 to 10.3); and a mean time stay of 3.62 days (SD ±4.7), CI (3.1 to 4.1). Mortality in this group was 3.7%. In 61.9% of cases the diagnosis of admission was AMI, 16% arrhythmia and 11.6% unstable angina. Of patients, 629 were polyvalent (55.8%), 53.85% male, mean age 58.05 (SD ±17.2), CI (56.7 to 59.4); mean APACHE II 14.6 (SD ±9.1), CI (13.8 to 15.3); and a mean time stay of 4.64 days (SD ±7.7), CI (4 to 5.25). Mortality was 11.6%. In 33.2% the cause of income was digestive, 23.2% acute or chronic exacerbated respiratory failure, 12.4% severe sepsis/septic shock and 10.1% postoperative cardiovascular surgery. The incidence density (ID) of catheter-related bacteremia was 5.5, 92.8% from the fourth day of ICU admission; ID of ventilator-associated pneumonia (VAP) was 5.94, 88.9% since the fourth day; and ID of urinary tract infections (UTI) related to urinary catheter was 2.88, 80% of them since the fourth day. From all patients who developed intra-ICU infections, mean APACHE II in admission was 21.3 (SD ±9.6) with a mean time stay of 23.4 days (SD ±12.9) and a mortality percentage of 19.6%. 1. Rit K, et al. IJCP. 2013;24:451-5. P95 Emergence of isolates that are intrinsically resistant to colistin in critically ill patients: are we selecting them out? MN Sivakumar, M Hisham, V Nandakumar, T Gopinathan Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P95 (doi: 10.1186/cc14175) Introduction Poor infection control practices along with irrational usage of antibiotics lead to emergence of multidrug-resistant (MDR) organisms. Increasing use of colistin for treating MDR infections leads to selection of organisms that are intrinsically resistant to colistin. There are limited Indian literatures which evaluated the incidence of intrinsically resistant isolates to colistin in critically ill patients. Our study aimed to investigate the incidence of true pathogen or colonizer with the prior antibiotic exposure and patient’s clinical outcome. Conclusion Being a broad-spectrum bactericidal agent usable both orally and parenterally with low toxicity profi les and lesser prevalence of cross-resistance with other antimicrobials, fosfomycin can be an alternative to other broad-spectrum agents to treat uncomplicated infections as well as in the case of infections with MDR organisms where treatment options are very few. This study possibly reveals a much- needed solution for the rising carbapenem resistance and also for the treatment of infections with such MDR pathogens, thereby bringing down the length of stay in hospital, cost of therapy and suff ering on the part of the patients. Methods The prospective, cross-sectional study was carried out from March 2013 to April 2014. Clinical samples included culture positivity for isolates intrinsically resistant to colistin from patients who were admitted to the ICU or had a prior ICU stay in the same admission. Methods The prospective, cross-sectional study was carried out from March 2013 to April 2014. Clinical samples included culture positivity for isolates intrinsically resistant to colistin from patients who were admitted to the ICU or had a prior ICU stay in the same admission. Results A total of 93 unusual Gram-negative rods were isolated from 76 patients. This included 19.4% (n = 18) Serratia marcescens, 12.9% (n = 12) Stenotrophomonas maltophilia, 14% (n = 13) Burkholderia cepacia, 24.7% (n = 23) Proteus mirabilis, 17.2% (n = 16) Morganella morganii, 9.7% (n = 9) Elizebethkingia meningoseptica and 2.1% (n = 2) Providencia species. A total of 68.4% (n = 52) patients had prior exposure to either colistin or carbapenems or both. In total, 71% (n  = 66) of the total isolates from patients had previous antibiotic exposure. 2. Garbati MA, et al. J Infect Dev Ctries. 2013;7:213-6. Analysis of Gram-negative rod bacteremia in the surgical and medical ICU The incidence of nosocomial infections with MDRO in the ICU is high. ESBL-GN intestinal colonization could be a risk factor for nosocomial infections but further studies are needed to confi rm this. Patient epidemiology in a level II hospital ICU and how main nosocomial infections aff ect morbidity and mortality M Muñoz, E Yuste, O Moreno, R Fernandez, R Ramirez Hospital Universitario San Cecilio, Granada, Spain Critical Care 2015, 19(Suppl 1):P94 (doi: 10.1186/cc14174) Introduction We describe the type of patient and the main nosocomial infections in a level II hospital ICU unit, 18 beds (12 polyvalent-general, six coronary). Introduction We describe the type of patient and the main nosocomial infections in a level II hospital ICU unit, 18 beds (12 polyvalent-general, six coronary). y Methods We used the ENVIN-HELICS database and made statistical calculations for all patients admitted to the ICU between 1 October 2012 and 30 September 2013 using SPSS v.15. g Results Patients admitted (1,126): 65.1% were male; mean age 61.72 (SD ±15.8), CI (60.7 to 62.7); mean APACHE II 12.6 (SD ±8.42), CI (12.12 to 13.11); and a mean time stay of 4.84 days (SD ±6.26). In total, 68.9% were provided from the community. A total of 44.1% were coronary, 2.84% trauma and 53.02% medical–surgical patients. A total of 29.8% had antibiotic therapy in the ICU, 20% had it before incoming. In total, 18.38% were treated with artifi cial airway (MV, tracheostomy). In total, 54.09% used a urinary catheter and 38.8% needed a central venous catheter. Fifteen percent of patients had some kind of surgery before Conclusion E. coli and Acinetobacter spp. – the most often pathogens of Gram-negative rod bacteremia – were mostly multidrug resistant. Multidrug-resistant bacteremia was related to age, length of stay less than 14  days, and lethal outcome. Predisposing factors for shorter length of stay: primary bacteremia, mechanical ventilation, lethal outcome, and for lethal outcome: primary bacteremia, multidrug resistance, presence of shock, mechanical ventilation, cancer chemotherapy, chronic obstructive pulmonary disease. S33 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P95 Among the total 93 intrinsically resistant isolates to colistin, 37.6% (n  = 35) of isolates from all clinical sources (endotracheal, pus, urine and blood samples) were true pathogens and the remaining 62.3% (n = 58) were colonizers. There was a statistically signifi cant increase in length of ICU stay and duration of hospitalization in the presence of true pathogen. Conclusion Selection pressure due to extensive use of higher antibiotics may lead to emergence of intrinsically resistant isolates, which narrows the therapeutic options in the ICU. Our study emphasizes the paramount importance of establishing clinical relevance of these organisms before treating them as true pathogens. This calls for judicious use of higher antibiotics, implementation of an antibiotic stewardship program and strict infection control practices. f Kolkata A Chakraborty, S Roy, S Chakraborty, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P96 (doi: 10.1186/cc14176) Introduction In the era of rising prevalence of serious infections caused by multidrug-resistant (MDR) organisms and the paucity of in-fl ow of newer antimicrobial agents, the relatively older antibiotics that had been left out of clinical practice for various reasons are now being increasingly considered as the potential agents to combat such infections. Fosfomycin, known for almost four decades, has a broad spectrum of activity against several Gram-negative and Gram-positive bacteria. Methods This study, conducted in the Microbiology Department of Medica Superspecialty Hospital between July and November 2014, was aimed at testing the in vitro sensitivity of fosfomycin against isolates identifi ed from various clinical specimens from diff erent parts of Kolkata. After confi rming the identity and antibiogram by Microscan Autoscan 4, the isolates were tested for fosfomycin sensitivity by the Epsilometer test. MIC values were interpreted in accordance with the currently recommended Clinical and Laboratory Standards Institute (CLSI) criteria for urinary tract isolates of Escherichia coli and Enterococcus faecalis and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria for Enterobacteriaceae and Staphylococcus aureus. y Conclusion In our ICU the main cause of admission was the polyvalent patient, who is younger and has more severity with not much diff erence in mean time of stay compared with the coronary patient. The intra-ICU infections provide an increase of morbi-mortality risk and consumption of resources. p y Results Out of the 1,895 isolates tested, fosfomycin displayed an overall in vitro susceptibility against 90%, but only 64% against MDR strains. Among the MDR organisms nearly 78% of E. coli and 70% of Klebsiella spp. and 40% of MRSA isolates showed provisional MICs in the sensitive range while among the sensitive strains fosfomycin showed around 92% susceptibility. Our study results were comparable with the results obtained from an Indian study published from CMC Vellore in 2013 showing a fosfomycin susceptibility of around 75% among MDR uropathogenic E. coli. P97 Results A total of 93 unusual Gram-negative rods were isolated from 76 patients. This included 19.4% (n = 18) Serratia marcescens, 12.9% (n = 12) Stenotrophomonas maltophilia, 14% (n = 13) Burkholderia cepacia, 24.7% (n = 23) Proteus mirabilis, 17.2% (n = 16) Morganella morganii, 9.7% (n = 9) Elizebethkingia meningoseptica and 2.1% (n = 2) Providencia species. A total of 68.4% (n = 52) patients had prior exposure to either colistin or carbapenems or both. In total, 71% (n  = 66) of the total isolates from patients had previous antibiotic exposure. Among the total 93 intrinsically resistant isolates to colistin, 37.6% (n  = 35) of isolates from all clinical sources (endotracheal, pus, urine and blood samples) were true pathogens and the remaining 62.3% (n = 58) were colonizers. There was a statistically signifi cant increase in length of ICU stay and duration of hospitalization in the presence of true pathogen. Antibiotic synergy testing for multidrug-resistant Gram-negative pathogens in a Greek ICU E Douka, E Perivoliot, E Kraniotaki, M Nepka, C Routsi, K Fountoulis, A Skoutelis, S Zakynthinos Evangelismos General Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P97 (doi: 10.1186/cc14177) Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma p A Chakraborty, S Roy, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P99 (doi: 10.1186/cc14179) A Chakraborty, S Roy, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P99 (doi: 10.1186/cc14179) Introduction Urinary tract infection (UTI) is one of the most common bacterial infections in humans. Gram-negative organisms being the most common causative agent, the rising prevalence of resistance to a number of antibiotics and more importantly the production of extended spectrum beta-lactamase (ESBL) by these organisms is a growing concern worldwide. As the scenario is no better in community isolates, the choice of empirical antimicrobials for such infections becomes a great challenge for the clinicians. fi Results Against 59 MDR A. baumannii strains, the synergy eff ect of CRB/ COL was 55.9%, RIF/COL 38.9%, CRB/GEN 22%, CRB/AMK 20.3% and TG/COL 16.9%, respectively. Against 41 K. pneumoniae strains, synergy rates were: CRB/COL 43.9%, CRB/GEN 31.7%, PIP/TAZ/GEN 29.2% and TG/COL 24.4% respectively. Against 64 P. aeruginosa strains, synergy rates were: AMK/PIP/TAZ 64.6%, AMK/AZT 64.6%, AMK/CEF 58.3%, CRB/ COL 52%, AMK/CRB 25%. Conclusion The most eff ective combination for both the A. baumannii and K. pneumoniae strains tested was CRB/COL. The next most eff ective combination was RIF/COL and CRB/GEN respectively. No competitive eff ect was observed for RIF/COL combination in all cases tested. The most eff ective combinations for P. aeruginosa strains were AMK plus PIP/TAZ or AZT or CEF. The next most eff ective combination was CRB/ COL. We recommend implementation of an antibiotic synergy test for MDR pathogens as a routine antimicrobial test in the hospitals’ microbiology laboratories, especially for critically ill patients, since some combinations seem to excel. Further studies are needed for the correlation of these combinations with clinical effi cacy. Methods In this retrospective observational study we aimed at knowing the prevalence of ESBL production by organisms causing UTI in the community and to study the antibiogram of such isolates. Urine samples from patients with suspected UTI in the community were cultured for uropathogen by routine microbiological methods and susceptibility testing was done on Microscan Autoscan 4 (Siemens). y Results Out of 527 isolates of Enterobactereaceae, 314 (59.58%) were ESBL producers from the community samples compared with 315 (67.30%) from hospital samples, with Escherichia coli being the most commonly isolated pathogen. Enterobacter spp. P100 Is it possible to predict multidrug-resistant organism colonization and/or infection at ICU admission? F Callejo-Torre1, JM Eiros2, S Ossa-Echeverri1, P Olaechea3, F Alvarez-Lerma4, M Palomar5, Envin-Helics Study Group1 1Hospital Universitario de Burgos, Spain; 2Hospital Clínico Universitario de Valladolid, Spain; 3Hospital de Galdakao-Usansolo, Galdakao, Spain; 4Hospital del Mar, Barcelona, Spain; 5Hospital Arnau de Villanova, Lleida, Spain Critical Care 2015, 19(Suppl 1):P100 (doi: 10.1186/cc14180) y Methods We retrospectively assessed two hypothetical empirical antibiotic treatment algorithms for VAP on an 18-bed ICU. Data on consecutive episodes of microbiologically confi rmed VAP were collected over a period of 22  months and divided into a derivation (1 February 2013 to 30 November 2013) and validation (1 December 2013 until 15 November 2014) cohort. We constructed two algorithms in the derivation cohort. One is a local ecology-based algorithm (LEBA), according to clinical risk factors for MDR and susceptibility results in our hospital. The other is a Gram stain-based algorithm (GSBA). The selection of antibiotics on GSBA was directed against pathogens predicted from the results of bedside Gram staining of tracheal aspirates collected just before antibiotic therapy. Subsequently, LEBA and GSBA were retrospectively reviewed and compared with actually prescribed antibiotics in the validation cohort.i p Critical Care 2015, 19(Suppl 1):P100 (doi: 10.1186/cc14180) Introduction We tried to develop a predictive model for patients colonized/infected by any multidrug-resistant organism (MDRO-C/I) at ICU admission based on risk factors easy to obtain (not depending on complex clinical records), being aware that foreseeing MDRO-C/I at ICU admission is key for appropriate empirical treatment and infection control. Results The fi rst 50 VAP episodes made up the derivation cohort and the subsequent 50 VAP episodes the validation cohort. Antibiotic coverage rates by applying LEBA and GSBA were identical (96% vs. 96%). GSBA proposed more narrow spectrum therapy as compared with LEBA (P <0.001). GSBA recommended carbapenems in signifi cantly less episodes than LEBA (P <0.001) and the same episodes as actually prescribed initial therapy (P  =  1). However, there was signifi cant increase of antibiotic coverage rates in GSBA compared with the actually prescribed initial therapy (96% vs. 78%, P = 0.015). Conclusion Antibiotic coverage rates on GSBA were comparable with LEBA. The use of GSBA would result in a signifi cant reduction of the administration of broad-spectrum antibiotics. Bedside Gram staining may be useful to guide appropriate initial antibiotic therapy for VAP. Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma showed highest prevalence (80%) of ESBL production from the community samples. Among the ESBL producing strains from the community, the sensitivity to ciprofl oxacin, levofl oxacin and nitrofurantoin was 18%, 21% and 44% respectively while in the non-ESBL producers the sensitivity rates were 52%, 51% and 73% respectively. P98 Development of antibiotic treatment algorithms based on Gram stain to restrict use of broad-spectrum antibiotics in the treatment of ventilator-associated pneumonia: a retrospective analysis J Yoshimura, T Kiguchi, A Matsushima, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P98 (doi: 10.1186/cc14178) P98 Development of antibiotic treatment algorithms based on Gram stain to restrict use of broad-spectrum antibiotics in the treatment of ventilator-associated pneumonia: a retrospective analysis J Yoshimura, T Kiguchi, A Matsushima, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P98 (doi: 10.1186/cc14178) p y Conclusion Organisms producing the ESBL phenotype present with an added possibility of being resistant to other broad-spectrum antimicrobial agents which are commonly prescribed in the community to empirically treat such infections. This makes the choice of empirical antibiotic much more challenging in the community, drawing errors in judgment. A possibility of frequent overcorrection lies on the other side of the coin. This study also shows the possible need for empirical institution of class I carbapenems as one of the treatment options and outpatient parenteral antimicrobial therapy. Introduction Ventilator-associated pneumonia (VAP) is a common and serious problem in ICUs. Several studies have been conducted to determine the eff ectiveness of Gram stain of tracheal aspirates for diagnosing VAP. However, the eff ectiveness for predicting causative microorganisms and guiding appropriate initial antibiotic therapy has not been evaluated. The purpose of this study is to determine whether Gram stain of tracheal aspirates can guide appropriate initial antibiotic therapy for VAP. Antibiotic synergy testing for multidrug-resistant Gram-negative pathogens in a Greek ICU Introduction The emergence of multidrug-resistant (MDR) pathogens is a major cause of infection-related mortality among critically ill patients. The synergistic eff ect between commonly used antibiotics against diffi cult to treat nosocomial MDR Gram-negative strains, if present, could provide a viable option as an alternative therapy. The aim of this study was to investigate the potential of antibiotic synergy against MDR A. baumannii, K. pneumonia and P. aeruginosa strains, isolated from critically ill patients in a Greek ICU. y Methods We tested 59 A. baumannii, 41 K. pneumoniae and 64 P. aeruginosa strains, isolated during the period 2010 to 2013. All strains were resistant to carbapenems and showed reduced susceptibility or resistance to tigecycline or colistin (MIC >2), in accordance with CLSI guidelines. We evaluated double-drug combinations of carbapenem (CRB)/colistin (COL), tigecycline (TG)/COL, rifampicin (RIF)/COL, CRB/ gentamicin (GEN), CRB/amikacin (AMK) for A. baumannii, TG/COL, S34 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 CRB/COL, piperacillin–tazobactam (PIP/TAZ)/GEN, CRB/GEN for K. pneumoniae and AMK/(PIP/TAZ), AMK/aztreonam (AZT), AMK/cefepime (CEF), AMK/CRB and CRB/COL for P. aeruginosa strains. In order to perform synergy tests, the E-test methodology (BioMerieux, Marcy l’E’toile, France) was used. Synergy was defi ned as a fraction inhibitory concentration (FIC) index ≤0.5, additive eff ect 0.5 to 1, indiff erent or antagonistic eff ect >2 (Lorian defi nition). CRB/COL, piperacillin–tazobactam (PIP/TAZ)/GEN, CRB/GEN for K. pneumoniae and AMK/(PIP/TAZ), AMK/aztreonam (AZT), AMK/cefepime (CEF), AMK/CRB and CRB/COL for P. aeruginosa strains. In order to perform synergy tests, the E-test methodology (BioMerieux, Marcy l’E’toile, France) was used. Synergy was defi ned as a fraction inhibitory concentration (FIC) index ≤0.5, additive eff ect 0.5 to 1, indiff erent or antagonistic eff ect >2 (Lorian defi nition).f P103 Novel infl uenza A antibodies reduce severity of secondary pneumococcal pneumonia after infl uenza infection in mice KF Van der Sluijs, F Van Someren Greve, MD De Jong, MJ Schultz, NP Juff ermans Novel infl uenza A antibodies reduce severity of secondary pneumococcal pneumonia after infl uenza infection in mice KF Van der Sluijs, F Van Someren Greve, MD De Jong, MJ Schultz, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P103 (doi: 10.1186/cc14183) y Methods Data were collected prospectively from 69,894 patients admitted consecutively (stay >24  hours) to 147 Spanish ICUs participating in the National Surveillance Study of Nosocomial Infections in ICU registry (ENVIN) during April to June 2006 to 2010. Univariable and multivariable analysis was performed for both objectives but we used only easy-to-obtain variables for the predictive model exclusively from those admitted in 2010 (n  = 16,950, 2/3 for analysis and 1/3 for subsequent validation). Introduction Secondary bacterial pneumonia after infl uenza infection can cause severe disease with a high mortality. Recently, a new group 2 infl uenza A antibody (AT10_002) has been developed, which binds to multiple H3 and H7 subtypes. In a mouse model of primary infl uenza infection, treatment with AT10_002 as a fusion antibody protects against lethal infection, and reduces loss of bodyweight [1]. We hypothesized that treatment with AT10_002 reduces weight loss, lung injury and bacterial outgrowth, in a mouse model of viral infection followed by secondary pneumococcal infection. y q Results In the 2006 to 2010 period, 1,046 were C/I by MRSA (note that relative risks are not included due to space limitations). First objective: previous antibiotic, APACHE II score >18, skin-soft tissue or postsurgical superfi cial skin infections, trauma or medical patient, age >65 (especially >75), urinary catheter and admitted from a long- term care facility were independent risk factors for MRSA-C/I in ICU. Multicolonization increased signifi cantly the risk of MRSA-C/I, and immunodefi ciency and gender male emerged as protective factors. Second objective: independent risk factors on ICU admission were male gender, trauma critical patient, urgent surgery, admitted from other ICU, community or long-term facility, being immunosuppressed and skin-soft tissue infection. All confi gured the risk model for which, although showing good discrimination (AUC-ROC, 0.77; 95% CI, 0.72 to 0.82), sensitivity (67%) and specifi city (76.5%) were insuffi cient for the ICU setting. P101 P101 Methicillin-resistant Staphylococcus aureus in the ICU: risk factors and a predictive model to detect it at ICU admission F Callejo-Torre1, JM Eiros2, S Ossa-Echeverri1, P Olaechea3, M Palomar4, F Alvarez-Lerma5, Envin-Helics Study Group1 1Hospital Universitario de Burgos, Spain; 2Hospital Clínico Universitario de Valladolid, Spain; 3Hospital de Galdakao-Usansolo, Galdakao, Spain; 4Hospital Arnau de Villanova, Lleida, Spain; 5Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P101 (doi: 10.1186/cc14181) g y p y Results Among 41 patients, 15 (37%) underwent FMS. There was no diff erence in age, sex, underlying disease, target temperature management, and prophylactic antibiotic use between two groups. Mean duration of ECMO was 4 days in both groups. The incidence of bacteremia was none in the group with FMS and fi ve (19%) in the group without FMS. Within fi ve cases of bacteremia, three were caused by enterobacterium. Introduction Being capable of predicting MRSA on ICU admission is crucial to enhance infection control and to avoid inappropriate empirical treatment. Two objectives were studied: to describe risk factors for MRSA colonization/infection (MRSA-C/I) once admitted to the ICU; and to develop a predictive model at ICU admission, based on easy-to-obtain admission factors. Conclusion FMS may be protective against bacteremia for OHCA patients undergoing ECMO. Protective eff ect of a fecal incontinence management system against bacteremia for out-of-hospital cardiac arrest patients undergoing extracorporeal membrane oxygenation S Kikuta, R Miki, S Ishihara, S Nakayama Hyogo Emergency Medical Center, Chuo, Kobe, Hyogo, Japan Critical Care 2015, 19(Suppl 1):P102 (doi: 10.1186/cc14182) Introduction Recently, extracorporeal cardiopulmonary resuscitation (ECPR) has become a common measure against cardiopulmonary arrest. In cases with ECPR, we usually insert cannulae for extracorporeal membrane oxygenation (ECMO) via the femoral artery and vein. However, the cannulation site is often contaminated by feces due to incontinence. Moreover, patients tend to be compromised by hypothermia due to the target temperature management, so we often experience central line-associated bloodstream infection of patients undergoing ECMO. We investigated the protective eff ect of a fecal incontinence management system (FMS) against bacteremia in patients undergoing ECMO. Conclusion MDRO prediction at ICU admission could not be based merely on clinical–demographic risk factors. Taking into account local particularities and combining risk factors with a rapid laboratory test might be the most eff ective way forward. g g Methods We studied 41 consecutive patients undergoing ECMO for out-of-hospital cardiac arrest (OHCA) between April 2010 and May 2014. Patients were divided into two groups according to the use or no use of FMS (Flexi-Seal™). Patients who died within 48 hours or from whom cannulae for ECMO were removed within 48  hours were excluded. Patients’ characteristics, underlying disease, target temperature management, prophylactic antibiotic use and incidence of bacteremia during admission were recorded and analyzed retrospectively. Results Among 41 patients, 15 (37%) underwent FMS. There was no diff erence in age, sex, underlying disease, target temperature management, and prophylactic antibiotic use between two groups. Mean duration of ECMO was 4 days in both groups. The incidence of bacteremia was none in the group with FMS and fi ve (19%) in the group without FMS. Within fi ve cases of bacteremia, three were caused by enterobacterium. g g Methods We studied 41 consecutive patients undergoing ECMO for out-of-hospital cardiac arrest (OHCA) between April 2010 and May 2014. Patients were divided into two groups according to the use or no use of FMS (Flexi-Seal™). Patients who died within 48 hours or from whom cannulae for ECMO were removed within 48  hours were excluded. Patients’ characteristics, underlying disease, target temperature management, prophylactic antibiotic use and incidence of bacteremia during admission were recorded and analyzed retrospectively. P102 (relative risk not shown due to space limitation): age 65 to 74, medical or surgical critical patient (especially urgent surgery), admitted from other ICU or long-term facility, immunosuppression and deep postsurgical skin or skin-soft tissue infections. Admitted from the community and female gender emerged as protective factors. Although the predictive model showed good discrimination (AUC-ROC = 0.775 (95% CI, 0.744 to 0.807)), sensitivity was only 67.4%. Validation with the remaining 4,952 patients (1/3) showed an AUC-ROC = 0.712 (95% CI, 0.665 to 0.759) and a P value on the Hosmer–Lemeshow goodness of fi t test of 0.855. Even creating a new model, including variables obtained after ICU admission (severity by APACHE score, mechanical ventilation, central venous, arterial or urinary catheter, immunodefi ciency, parenteral nutrition, ventricular derivation, extrarenal depuration, non-invasive ventilation, tracheotomy, enteral nutrition and nasogastric tube), prediction capability did not improve (AUC-ROC = 0.801 (95% CI, 0.774 to 0.828), sensitivity 71.4%). Protective eff ect of a fecal incontinence management system against bacteremia for out-of-hospital cardiac arrest patients undergoing extracorporeal membrane oxygenation S Kikuta, R Miki, S Ishihara, S Nakayama Hyogo Emergency Medical Center, Chuo, Kobe, Hyogo, Japan Critical Care 2015, 19(Suppl 1):P102 (doi: 10.1186/cc14182) P100 Results The fi rst 50 VAP episodes made up the derivation cohort and the subsequent 50 VAP episodes the validation cohort. Antibiotic coverage rates by applying LEBA and GSBA were identical (96% vs. 96%). GSBA proposed more narrow spectrum therapy as compared with LEBA (P <0.001). GSBA recommended carbapenems in signifi cantly less episodes than LEBA (P <0.001) and the same episodes as actually prescribed initial therapy (P  =  1). However, there was signifi cant increase of antibiotic coverage rates in GSBA compared with the actually prescribed initial therapy (96% vs. 78%, P = 0.015). Methods Data were collected prospectively from admission to discharge of 16,950 patients admitted consecutively (at least >24  hours) to 147 Spanish ICUs of the ENVIN (National Surveillance Study of Nosocomial Infections in ICUs) registry, from April to June 2010. To create the predictive model, 11,998 (2/3) patients were used for univariable and multivariable logistic regression model and 4,952 (1/3) for subsequent validation. Results With a MDRO prevalence of 2.12% (359 MDROs at ICU admission were detected in 314 patients), 87.58% patients had only one MDRO, meanwhile 12.42% were MDRO-C/I by two or more simultaneously. Risk factors used in the development of the predictive model and independently associated with MDRO-C/I at ICU admission were Conclusion Antibiotic coverage rates on GSBA were comparable with LEBA. The use of GSBA would result in a signifi cant reduction of the administration of broad-spectrum antibiotics. Bedside Gram staining may be useful to guide appropriate initial antibiotic therapy for VAP. S35 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Source of MDR infections in an ICU: busting the myth R Agrawal Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Results In total, 1,499 patients were included, of whom 265 patients (18%) had a viral respiratory tract infection with at least one virus. In 17 patients, two viruses were found; two patients had an infection with three viruses. The most prevalent was parainfl uenzavirus-3 (5.7%); 17 patients (1.1%) had an infection with infl uenza. The lowest prevalence of viral infections occurred in September (12%), the highest in October and February (both 26%). Of the patients tested positive in TA, only 46% also tested positive in NP. The median cp values were not signifi cantly diff erent between TA and NP swabs (31.1 vs. 31.6, P = 0.75). Introduction MDR infections in the ICU are not nosocomial all the time, as perceived commonly. We performed a 2-year retrospective study to analyze the source of culture positivity in a medical ICU and to identify which types of infections are more prevalent. Methods The data of a 35-bed medical ICU were analyzed from November 2012 to October 2014. The source of culture positivity was divided into three groups: patients admitted from the ER to the ICU who were referred from other hospitals or direct admissions, the second group was patients admitted within the hospital but outside the ICU for the fi rst 48 hours, and the third group was ICU-acquired infections. We also analyzed the data for type of infections, whether Gram-negative, Gram-positive or fungal. Conclusion The prevalence of viral respiratory tract infections is high in unselected ICU patients. Testing tracheal aspirate in combination with nasopharynx greatly increased detection of viruses, and yields similar cp values. Whether these viral infections are associated with prolonged mechanical ventilation and worse outcomes remains to be determined. g p g Results There were 1,051 cultures positive in a 2-year period. In total, 46.8% (n = 492) of cultures were already positive on admission, which denotes community-acquired and referred patients from other hospitals. A total of 31.1% (n  = 327) of cultures were positive from patients admitted to general wards for more than 48 hours and then transferred to the ICU. Twenty-two percent (n = 232) of cultures were ICU-acquired infections. The data show community-acquired and hospital-acquired infections are the bulk of the culture load in an ICU. P103 Afterwards validation with the remaining 4,952 (1/3) showed AUC-ROC  = 0.72 (95% CI, 0.65 to 0.79) and P value on the Hosmer–Lemeshow goodness of fi t test = 0.539. The model did not improve even after including more complex variables (AUC-ROC = 0.82; 95% CI, 0.77 to 0.86, sensitivity 63.64%, specifi city 78.48%). y y p Methods Male C57Bl/6 mice were intranasally inoculated with 400 TCID50 Infl uenza A (H3N2). Two days after infection, mice were injected with either AT10_002 i.v. (n = 8) or a control antibody (n = 7). After 7 days, both groups were intranasally inoculated with 5 × 103 S. pneumoniae type 3 and were sacrifi ced 18  hours later. Outcome measures were weight loss, wet lung weight, cell count in bronchoalveolar lavage fl uid (BALF), and colony-forming units (CFUs) in lung homogenate. Data are represented as medians, and treatment groups are compared using nonparametric tests.i Results Mice receiving AT10_002 showed signifi cantly lower weight loss at the time of sacrifi ce compared with the control group (+1% vs. –12% change in weight; P = 0.0003). Also wet lung weight was lower (68 vs. 96 mg; P = 0.0003), cell counts in BALF were lower (4.9 × 105 vs. 7.0 × 105 cells/ml; P = 0.0037) and CFUs in lung homogenate were lower (33 vs. 25 × 104 CFUs/mg; P = 0.0003) compared with controls. Conclusion Early treatment with infl uenza antibody AT10_002 signifi cantly reduces weight loss, lung injury and bacterial outgrowth, i Conclusion Independent risk factors for MRSA-C/I in the ICU and at ICU admission are described. To predict MRSA-C/I at ICU admission we should not rely on clinical–demographic risk factors alone. Its combination with a rapid laboratory test could be the way to proceed in future studies. Conclusion Early treatment with infl uenza antibody AT10_002 signifi cantly reduces weight loss, lung injury and bacterial outgrowth, S36 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 hospitals – 17 high-volume care units) in the Czech Republic from 1 January 2011 to 5 November 2013. Patients were divided into two groups: survivors (n = 274) and nonsurvivors (n = 261). in a mouse model of infl uenza infection followed by secondary pneumococcal pneumonia. Reference in a mouse model of infl uenza infection followed by secondary pneumococcal pneumonia. P103 Reference g Results Survivors versus nonsurvivors were similar in: age 65.8 (64.2; 67.5) versus 66.5 (64.7; 68.3) P = 0.583, men 159 (58.0%) versus 160 (62.0%) P = 0.376, APACHE II score 27 (15 to 40) versus 28 (15 to 40) P  =  0.737. Statistically signifi cant diff erences between survivors versus nonsurvivors were found in the parameter ‘Consensus initial antimicrobial therapy with microbial cultures’ 178 (79.5%) versus 128 (58.4%) P <0.001 and in the parameter ‘Administration antimicrobials within the fi rst hour’ 163 (59.9%) versus 171 (70.7%) P  =  0.001. Administration of 30  ml/kg crystalloid for hypotension or lactate 4 mmol/l (3 hours) and application of vasopressors (6 hours) were in both groups without statistically signifi cant diff erences. 1. Wagner K, et al. Proc Natl Acad Sci U S A. 2014;111:16820-5. Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock 1 l 1 Š 2 l 1 Š k1 Introduction The early identifi cation of severe sepsis and septic shock and early implementation of the SSC bundles were associated with reduced mortality [1]. The failure to initiate appropriate antimicrobial therapy increased mortality of septic shock patients [2]. We hypothesized that the parameter ‘Consensus initial antimicrobial therapy with microbial cultures’ correlates with outcome of septic shock patients. Conclusion Antibiotic stewardship and strict adherence to infection control protocols in hospitals and guidelines for general practitioners can signifi cantly reduce the load of resistant organisms in the ICU. This may eventually improve patient outcomes and help in preserving the antibiotics for future generations. Methods We analyzed 535 consecutive patients with septic shock (sepsis-induced hypotension persisting despite adequate fl uid resuscitation) from the EPOSS database (Data-based Evaluation and Prediction of Outcome in Severe Sepsis), which was developed to monitor and assess treatment effi cacy in patient with severe sepsis and septic shock. Patients were admitted to participating ICUs (12 Source of MDR infections in an ICU: busting the myth R Agrawal This could be attributed to increased surveillance and adherence to infection control practices in the ICU which may not be followed stringently in other parts of the hospital. Overuse of broad-spectrum antibiotics in community and primary care hospitals has resulted in a spurt in growth of resistant infections. This has reached an alarming level in developing countries. Out of total cultures positive 78.3% (n = 822) were Gram-negative infections which included community-based and non-ICU infections. P105 Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock P Szturz1, P Folwarczny1, J Švancara2, R Kula1, P Ševèík1 1University Hospital and Faculty of Medicine Ostrava University, Ostrava, Czech Republic; 2Institute of Biostatistic and Analyses, Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P105 (doi: 10.1186/cc14185) P105 Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock P Szturz1, P Folwarczny1, J Švancara2, R Kula1, P Ševèík1 1University Hospital and Faculty of Medicine Ostrava University, Ostrava, Czech Republic; 2Institute of Biostatistic and Analyses, Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P105 (doi: 10.1186/cc14185) Prevalence of viral respiratory tract infections in acutely admitted and ventilated ICU patients: a prospective multicenter observational study F Van Someren Greve1, KF Van der Sluijs1, R Molenkamp1, AM Spoelstra-de Man2, OL Cremer3, RB De Wilde4, PE Spronk5, MD De Jong1, MJ Schultz1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU Medical Center, Amsterdam, the Netherlands; 3University Medical Center Utrecht, the Netherlands; 4Leiden University Medical Center, Leiden, the Netherlands; 5Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P104 (doi: 10.1186/cc14184) g p y gif Conclusion We found that correct choice of antibiotics improves outcome of septic shock patients. The choice of empirical antimicrobial therapy depends on complex factors related to the underlying disease, susceptibility of pathogens, patient’s history and clinical syndrome. Adequate initial antimicrobial therapy as an important factor of survival along with suitable initial fl uid resuscitation and application of vasopressors should be a priority for healthcare in human septic shock. References p , p , Critical Care 2015, 19(Suppl 1):P104 (doi: 10.1186/cc14184) Introduction The prevalence of viral respiratory tract infections in critically ill patients is uncertain, as well as the optimal diagnostic method to detect these. The aim of this study was to assess the prevalence of viral respiratory tract infections in mechanically ventilated patients, in both the upper and lower respiratory tract.i Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) References 1. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 2. Kumar A, et al. Crit Care Med. 2006;34:1589-96. 1. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 2. Kumar A, et al. Crit Care Med. 2006;34:1589-96. Methods A prospective observational study was performed in fi ve ICUs in the Netherlands. From September 2013 to April 2014, consecutive acutely admitted, mechanically ventilated patients were included, regardless of diagnosis at admission. Nasopharyngeal (NP) swabs and tracheal aspirates (TA) were collected at intubation, and were tested via multiplex RT-PCR for the following viruses: infl uenza A and B, parainfl uenzaviruses, RSV, human metapneumoviruses, bocaviruses, coronaviruses, rhinoviruses, enteroviruses, parechoviruses and adenoviruses. Viral DNA/RNA copies were expressed by crossing-point (cp) values. References Concordance between qPC in VAP patients Positive Agreement culture qPCR (%) S. aureus 28/20 31/25 96.7/89.7 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 (BAL/ETA) Conclusion Sensitivity and specifi city of the ne for these main bacteria found in VAP could en antibiotic therapy. In the future, the developme aim at obtaining a bedside diagnostic in only a P108 Use of Cepheid Xpert Carba-R® for rapid detec carbapenemase-producing bacteria in critica surgical patients: fi rst report of an observatio A Cortegiani, V Russotto, P Capuano, G Tricoli, DM L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/ Introduction Xpert Carba-R® (Cepheid®, USA) i rapid (<1 hour) detection of bacteria carrying genes (KPC, NDM, VIM, OXA-48, IMP-1). The compare PCR with microbiological cultures in surgical patients. Methods We performed an observational study P107 P107 Concordance between a new molecular real-time approach and traditional culture in suspected VAP patients M Clavel1, O Barraud2, V Moucadel3, MC Ploy2, E Karam4, F Meynier3, B François for Valibi Study Group5 1Hopital Dupuytren, Limoges, France; 2UMRS-1092, Hopital Dupuytren, Limoges, France; 3bioMérieux SA, Grenoble, France; 4Service de Réanimation, Brive, France; 5Inserm, Limoges, France Critical Care 2015, 19(Suppl 1):P107 (doi: 10.1186/cc14187) the ICU stay. We obtained two rectal swab specimens and two drainage samples to perform PCR assay and classic culture tests. We used Cohen’s K to test concordance of results. We considered concordant those results of positive detection of carbapenemase-producing bacteria by both methods (even if a polymicrobial growth was observed by cultures) or negative results by both methods. Concordance was studied for rectal swab and drainage specimens. Antibiotic susceptibility testing was performed through a semiquantitative method. the ICU stay. We obtained two rectal swab specimens and two drainage samples to perform PCR assay and classic culture tests. We used Cohen’s K to test concordance of results. We considered concordant those results of positive detection of carbapenemase-producing bacteria by both methods (even if a polymicrobial growth was observed by cultures) or negative results by both methods. Concordance was studied for rectal swab and drainage specimens. Antibiotic susceptibility testing was performed through a semiquantitative method. Results Eight complete samples sets were collected from seven patients. Seven rectal swab specimens were negative for both PCR and cultures. In one patient a positive culture from carbapenem-resistant P. aeruginosa was detected from the rectal swab resulting negative to PCR. References In one patient a positive culture from carbapenem-resistant A. baumanii was detected by drainage culture resulting negative to PCR. In two cases a positive result was observed from both PCR and cultures of rectal swab and drainage specimens. Vim and KPC genes were detected in one case and A. baumanii and K. pneumoniae with carbapenem resistance were isolated from cultures. A KPC gene was detected by PCR in the other case, and K. pneumoniae with carbapenem resistance was isolated from cultures. In all other cases a negative result was observed by both PCR and cultures. Cohen’s K of 0.71 (95% CI = 0.21 to 1) was observed for rectal swab and drainage specimens. Introduction Early microbiological documentation may reduce attributable mortality and excessive use of broad-spectrum antibiotics in ventilator-associated pneumonia (VAP). Using bronchoalveolar lavage (BAL) and endotracheal aspirates (ETA), we studied a new molecular biology-based approach to detect and quantify bacteria in less than 3 hours. This prospective multicenter trial aimed at comparing the microbiological results obtained using this molecular protocol (easyMAG® system) and semiquantitative culture in suspected VAP. y y q p Methods ETA and BAL samples were consecutively collected during 10 months in adult patients in four ICUs of France. The molecular method includes a preprocessing liquefaction for ETA before DNA extraction. DNAs were extracted using the easyMAG® system. Real- time PCR (qPCR) was run using the ABI7500FastDx PCR instrument. The results presented here concern: Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacteriaceae. Quantifi cation was performed using qPCR standard curves, by converting the cycle threshold to CFU/ ml. Conclusion We need more data to evaluate the performance of PCR for rapid detection of carbapenemase-producing bacteria from rectal swabs and drainage of critically ill surgical patients even though its concordance with cultures seems to be good. Results A total of 125 suspected VAP were included from 122 patients. In total, 125 BAL and 107 ETA were collected. Sex ratio (M/F) was 76%, and CPIS ≥6 was calculated in 74.6% of the suspected VAP patients. Mean ventilation duration before sampling was 6 days. Seventy-eight percent and 65% of the BAL and ETA culture were positive respectively. Correlations between molecular method and culture on BAL and ETA are reported in Table 1. 09 Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Positive Agreement Sensitivity Specifi city culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/93.8 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/92.4 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/88.4 (BAL/ETA) Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Introduction Antimicrobial resistance constitutes a growing global threat, driven in part by inappropriate antimicrobial prescribing [1]. Most hospitals implement antibiotic policies to promote antimicrobial stewardship. This audit examined the Royal Cornwall Hospital Trust (RCHT) Critical Care Department’s compliance with the current standard defi ned in our local antimicrobial policy. This states that all antimicrobial prescriptions are to have an indication and review date recorded [2]. Sequential strategies to improve compliance were introduced prior to re-auditing the eff ects. Methods The RCHT Critical Care Department utilizes the Phillips Care Vue electronic patient record. Data from this system were interrogated at three stages to assess our compliance with the trust’s antimicrobial policy. The fi rst data interrogation was performed prior to any intervention, and refl ected baseline antimicrobial prescribing habits. The second data interrogation was performed during a period of active antibiotic stewardship promotion. The third data interrogation was performed following the addition of a care bundle to the prescribing module of Care Vue. This daily tick-box prompt reminded clinicians to check that all antimicrobial prescriptions had an indication and review date recorded. The records of all of the patients admitted to the critical care department during the periods of data interrogations were assessed for antimicrobial indication and review date transcription.i References . Klevens, et al. Estimating healthcare associated infections. Public Health Rep. 2007;122:160-6. . Klevens, et al. Estimating healthcare associated infections. Public Health Rep. 2007;122:160-6. . Hecker, et al. Unnecessary use of antimicrobials. Arch Intern Med. 2003;163:972-8. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S37 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P107 Concordance between a new molecular real-time approach and traditional culture in suspected VAP patients M Clavel1, O Barraud2, V Moucadel3, MC Ploy2, E Karam4, F Meynier3, B François for Valibi Study Group5 1Hopital Dupuytren, Limoges, France; 2UMRS-1092, Hopital Dupuytren, Limoges, France; 3bioMérieux SA, Grenoble, France; 4Service de Réanimatio Brive, France; 5Inserm, Limoges, France Critical Care 2015, 19(Suppl 1):P107 (doi: 10.1186/cc14187) Introduction Early microbiological documentation may red attributable mortality and excessive use of broad-spectrum antibio in ventilator-associated pneumonia (VAP). Using bronchoalve lavage (BAL) and endotracheal aspirates (ETA), we studied a molecular biology-based approach to detect and quantify bacter less than 3 hours. This prospective multicenter trial aimed at compa the microbiological results obtained using this molecular prot (easyMAG® system) and semiquantitative culture in suspected VAP. Methods ETA and BAL samples were consecutively collected du 10 months in adult patients in four ICUs of France. The molec method includes a preprocessing liquefaction for ETA before D extraction. DNAs were extracted using the easyMAG® system. R time PCR (qPCR) was run using the ABI7500FastDx PCR instrument. results presented here concern: Staphylococcus aureus, Pseudomo aeruginosa and Enterobacteriaceae. Quantifi cation was perform using qPCR standard curves, by converting the cycle threshold to C ml. Results A total of 125 suspected VAP were included from 122 patie In total, 125 BAL and 107 ETA were collected. Sex ratio (M/F) was 7 and CPIS ≥6 was calculated in 74.6% of the suspected VAP patie Mean ventilation duration before sampling was 6 days. Seventy-e percent and 65% of the BAL and ETA culture were positive respectiv Correlations between molecular method and culture on BAL and are reported in Table 1. Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ in VAP patients Positive Agreement Sensitivity Specif culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/9 (BAL/ETA) P. References aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/9 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/8 (BAL/ETA) Conclusion Sensitivity and specifi city of the new molecular appro for these main bacteria found in VAP could enable targeted fi rst- antibiotic therapy. In the future, the development of this approach aim at obtaining a bedside diagnostic in only a few hours. P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay rapid (<1 hour) detection of bacteria carrying carbapenem-resista genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study i compare PCR with microbiological cultures in critically ill, abdom surgical patients. Methods We performed an observational study at University Hospit Giaccone’ Palermo. We enrolled abdominal surgical patients admi to the ICU with suspected abdominal sepsis or developing sepsis du P107 Concordance between a new molecular real-t traditional culture in suspected VAP patients M Clavel1, O Barraud2, V Moucadel3, MC Ploy2, E Ka B François for Valibi Study Group5 1Hopital Dupuytren, Limoges, France; 2UMRS-1092, H Limoges, France; 3bioMérieux SA, Grenoble, France; 4 Brive, France; 5Inserm, Limoges, France Critical Care 2015, 19(Suppl 1):P107 (doi: 10.1186/ Introduction Early microbiological docum attributable mortality and excessive use of bro in ventilator-associated pneumonia (VAP). lavage (BAL) and endotracheal aspirates (ET molecular biology-based approach to detect a less than 3 hours. This prospective multicenter t the microbiological results obtained using t (easyMAG® system) and semiquantitative cultu Methods ETA and BAL samples were consecu 10 months in adult patients in four ICUs of method includes a preprocessing liquefactio extraction. DNAs were extracted using the e time PCR (qPCR) was run using the ABI7500Fast results presented here concern: Staphylococcu aeruginosa and Enterobacteriaceae. Quantif using qPCR standard curves, by converting the ml. Results A total of 125 suspected VAP were inclu In total, 125 BAL and 107 ETA were collected. S and CPIS ≥6 was calculated in 74.6% of the s Mean ventilation duration before sampling wa percent and 65% of the BAL and ETA culture we Correlations between molecular method and c are reported in Table 1. Table 1 (abstract P107). Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries gi q Results A total of 102 mechanically ventilated patients, 75 men and 27 women, were included in the study. All patients showed VAP caused by MDR bacteria. They were stratifi ed by outcome into survivors and nonsurvivors. ICU mortality was 55%. Gender, cause of admission, the causative microbe, colonization of bronchial secretions and secondary bacteremia had no correlation with outcome. Age and APACHE II score were higher in nonsurvivors (P <0.01 and P <0.05 respectively). The time-onset of pneumonia after admission was longer in patients with VAP caused by Klebsiella or Pseudomonas than those with VAP caused by acinetobacter (P <0.01). Patients with Klebsiella or Pseudomonas pneumoniae needed more time on mechanical ventilation than those with pneumonia from acinetobacter (P <0.01). Introduction Micafungin (MCF) is an echinocandin agent with broad activity against Candida spp., which are frequently isolated in blood and eschar cultures of burned patients, who present diff erent pharmacokinetics (PK) characteristics. Due to the limited information about its PK, we investigate MCF levels in plasma and burn eschar tissues in this population. Methods A PK study of MCF at standard dosage (100 mg/day). Cmax (end of the infusion) and Cmin (before next dose) plasma levels of MCF were obtained after fi rst dose and at steady state (days 4 and 5 of therapy); and on day 5 in eschars (1 to 3 hours after infusion). They were measured by HPLC. Spearman’s rho test was used for bivariate correlations between MCF exposure and patient’s clinical factors. Conclusion VAP caused by MDR bacteria is a leading cause of ICU death. Age and APACHE II score are signifi cant risk factors of death. References 1. Golia S, et al. J Clin Diagn Res. 2013;7:2462-6. 1. Golia S, et al. J Clin Diagn Res. 2013;7:2462-6. 1. Golia S, et al. J Clin Diagn Res. 2013;7:2462-6. 2. Charles MP, et al. Australas Med J. 2013;6:430-4. 2. Charles MP, et al. Australas Med J. 2013;6:430-4. Results There were 10 patients (eight men; age: 18 to 77 years). Patients’ characteristics and PK are shown in Table  1. A high interindividual variability was observed in the concentrations of MCF. Peak plasma concentrations after the fi rst and repeated doses of MCF were inversely correlated with % burned TBSA (Spearman’s ρ  = –0.695 and –0.750 (P <0.05), respectively), but not with the time from burn injury. P108 P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Results From the fi rst data interrogation, antimicrobial prescriptions had an indication and review date transcription in 57% and 60% of cases respectively. Following the awareness campaign, the indication and review date transcription rate increased to 78% and 85% respectively. A daily electronic prompt was then added to our care bundle list. The fi nal data interrogation, performed after this intervention, demonstrated that the transcription rates for both the indication and the review date had increased to 96%. Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay for rapid (<1 hour) detection of bacteria carrying carbapenem-resistance genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study is to compare PCR with microbiological cultures in critically ill, abdominal surgical patients. Conclusion We have demonstrated that the use of a daily prompt within an electronic patient record can greatly improve compliance in recording the indication and review date for all antimicrobials. These data support the widespread implementation of an electronic prescribing system where daily reminders are integrated in an eff ort to improve compliance with antimicrobial stewardship. Methods We performed an observational study at University Hospital ‘P. Giaccone’ Palermo. We enrolled abdominal surgical patients admitted to the ICU with suspected abdominal sepsis or developing sepsis during Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S38 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Eventually, the IC was confi rmed for 403/835 patients (peritonitis: 177; candidaemia: 141; deep candidiasis: 61; mixed infection sites: 24). Candida albicans was the main pathogen (67%), then C. glabrata (16%). At inclusion, CIC were treated with caspofungin (Cas): 55%, and fl uconazole (Flu): 34%, whereas these antifungals were administered to 46% and 45% of SIC, respectively. Patients with SIC were more severe than those with CIC. The two main criteria for initiating empirically an AFT were a central venous catheter (79%) and severe septic shock (70%). The rate of change of the initial AFT was higher in the CIC group (49%) than in the SIC group (33%, P <0.0001). P111 Epidemiological cohort study of systemic antifungal therapy for suspected or confi rmed invasive candidiasis in the ICU: the Amarcand2 study J Constantin1, JF Timsit2, JP Gangneux3, JP Mira4, P Montravers2, H Dupont5, P Perrigault6, O Lortholary7, E Azoulay8, O Leroy9 1CHU Estaing, Clermont-Ferrand, France; 2Paris Diderot University/Bichat Hospital, Paris, France; 3Rennes University Hospital, Rennes, France; 4Cochin University Hospital, Paris, France; 5Amiens University Hospital, Amiens, France; 6Montpellier University Hospital, Montpellier, France; 7Necker University Hospital, Paris, France; 8Saint-Louis University Hospital, Paris, France; 9Tourcoing University Hospital, Tourcoing, France Critical Care 2015, 19(Suppl 1):P111 (doi: 10.1186/cc14191) P108 In the CIC group, it was mostly for changing the antifungal agent (de-escalation Cas  Flu in half of the patients) based on mycological tests results. In the SIC group, the AFT was modifi ed almost as often for changing the drugs (including 22% de-escalation Cas  Flu) as for stopping the AFT. The 28-day mortality of candidaemia was 42% in cases of C. glabrata, 40% in cases of C. albicans, and 20% in cases of C. parapsilosis. Among survivors, the median duration of treatment was 17 to 21 days according to the infection site in cases of CIC, and 10 days in cases of SIC. Epidemiological cohort study of systemic antifungal therapy for suspected or confi rmed invasive candidiasis in the ICU: the Amarcand2 study J Constantin1, JF Timsit2, JP Gangneux3, JP Mira4, P Montravers2, H Dupont5, P Perrigault6, O Lortholary7, E Azoulay8, O Leroy9 1CHU Estaing, Clermont-Ferrand, France; 2Paris Diderot University/Bichat Hospital, Paris, France; 3Rennes University Hospital, Rennes, France; 4Cochin University Hospital, Paris, France; 5Amiens University Hospital, Amiens, France; 6Montpellier University Hospital, Montpellier, France; 7Necker University Hospital, Paris, France; 8Saint-Louis University Hospital, Paris, France; 9Tourcoing University Hospital, Tourcoing, France l l d y Conclusion This is the largest PK study of 100  mg daily of MCF in severely burned critically ill patients. The inverse correlation between MCF exposure and % burned TBSA suggests that patients with large burned TBSA may need higher doses of MCF. Nevertheless, MCF levels in plasma and burn eschar tissues after the fi rst and multiple doses were above the MIC90 against most clinically important Candida species. g y p g Critical Care 2015, 19(Suppl 1):P111 (doi: 10.1186/cc14191) Introduction Prescription of antifungal treatments (AFT) in ICUs in case of suspected or confi rmed invasive candidiasis (SIC or CIC) has been challenged by diff erent guidelines. The study aimed to describe the epidemiology of the invasive candidiasis (IC), analyze the criteria for the AFT initiation, the AFT type, and its changes during patient follow-up. Methods A prospective observational multicenter cohort study. Consecutive adult patients with SIC or CIC and treated with systemic AFT were included between October 2012 and September 2013 in 104 French ICUs. References 1. World Health Organisation. Antimicrobial resistance: global report on surveillance. 2014. http://www.who.int/drugresistance/documents/ surveillancereport/en/. 1. World Health Organisation. Antimicrobial resistance: global report on surveillance. 2014. http://www.who.int/drugresistance/documents/ surveillancereport/en/. 2. Royal Cornwall Hospital Trust. Automatic stop/review date policy for antimicrobials. 2012. Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries MCF concentrations in burn eschars were not correlated with % burned TBSA. MCF was well tolerated. One patient had candidemia. The crude mortality was 40%. P112 Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries A Agrifoglio1, MJ Asensio1, M Sánchez1, B Galván1, E Herrero1, L Cachafeiro1, E Perales1, S Luque2, A García de Lorenzo1 1La Paz/IdiPAZ University Hospital, Madrid, Spain; 2Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P112 (doi: 10.1186/cc14192) Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries A Agrifoglio1, MJ Asensio1, M Sánchez1, B Galván1, E Herrero1, L Cachafeiro1, E Perales1, S Luque2, A García de Lorenzo1 1La Paz/IdiPAZ University Hospital, Madrid, Spain; 2Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P112 (doi: 10.1186/cc14192) P113 P113 Tedizolid clearance by in vitro continuous renal replacement therapy model SJ Lewis, L Switaj, BA Mueller University of Michigan, Ann Arbor, MI, USA Critical Care 2015, 19(Suppl 1):P113 (doi: 10.1186/cc14193) Factors associated with survival of ICU patients with pneumonia caused by multidrug-resistant Gram-negative bacteria M Georgiadou, E Pappa, E Papandreou, H Pavlou, M Eforakopoulou KAT-EKA General Hospital Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P110 (doi: 10.1186/cc14190) Introduction Multidrug-resistant (MDR) bacterial pneumonia is associated with signifi cant morbidity and mortality in severely ill ICU patients. The assessment of factors associated with the onset and clinical course of MDR pneumonia may improve treatment eff ectiveness. The purpose of this study is to identify factors associated with outcome in mechanically ventilated patients with ventilator- associated pneumonia (VAP) caused by MDR bacteria. y Conclusion French ICU patients are treated with antifungal agents selected according to the candidiasis severity, contrary to ESCMID guidelines which recommend initiating with echinocandins regardless of severity. As recommended, the therapy was secondarily adapted to microbiological results. p y Methods We studied retrospectively all mechanically ventilated patients treated in the A’ ICU of KAT General Hospital in Athens from 1 January 2011 to 31 December 2013 and showed ventilator-associated pneumonia from MDR Gram-negative bacteria. Standard demographic and clinical data, the causative organisms and outcome were recorded. For statistical signifi cance, chi-square and Student t tests were used. P112 P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration G Kennedy1, J Osborn1, S Flanagan2, N Alsayed3, S Bertolami1 1Cubist Pharmaceuticals, Lexington, MA, USA; 2Cubist Pharmaceuticals, San Diego, CA, USA; 3Cubist Pharmaceuticals, Zurich, Switzerland Critical Care 2015, 19(Suppl 1):P114 (doi: 10.1186/cc14194) Introduction Tedizolid phosphate, a novel oxazolidinone antibacterial prodrug recently approved by the US Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections, is available as oral (that is, tablets) and intravenous formulations. The clinical pharmacokinetics of tedizolid, the active moiety of tedizolid phosphate, are similar when orally administered tedizolid phosphate is given as powder in a capsule or as tablets. This suggests that crushing tablets prior to administration is unlikely to alter tedizolid pharmacokinetics, provided no drug is lost during administration. To determine whether the expected dose of tedizolid phosphate can be delivered via nasogastric (NG) tube in critically ill patients who have diffi culty swallowing, this study evaluated the stability and recovery of tedizolid phosphate 200 mg tablets after crushing, dispersion in water, and passage through an NG tube. Introduction Tedizolid phosphate, a novel oxazolidinone antibacterial prodrug recently approved by the US Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections, is available as oral (that is, tablets) and intravenous formulations. The clinical pharmacokinetics of tedizolid, the active moiety of tedizolid phosphate, are similar when orally administered tedizolid phosphate is given as powder in a capsule or as tablets. This suggests that crushing tablets prior to administration is unlikely to alter tedizolid pharmacokinetics, provided no drug is lost during administration. To determine whether the expected dose of tedizolid phosphate can be delivered via nasogastric (NG) tube in critically ill patients who have diffi culty swallowing, this study evaluated the stability and recovery of tedizolid phosphate 200 mg tablets after crushing, dispersion in water, and passage through an NG tube. for treatment of nosocomial pneumonia, common in critically ill patients with acute kidney injury. There are limited data on tedizolid disposition in continuous renal replacement therapy (CRRT). This study’s purpose was to assess continuous hemofi ltration (CHF) and continuous hemodialysis (CHD) infl uence on tedizolid clearance. Methods Validated, bovine blood-based, in vitro CHF and CHD models were used with six new HF 1400 (polysulfone) and six new Multifl ow 150 (AN 69) hemodiafi lters. P113 Tedizolid clearance by in vitro continuous renal replacement therapy model P113 Tedizolid clearance by in vitro continuous renal replacement therapy model SJ Lewis, L Switaj, BA Mueller University of Michigan, Ann Arbor, MI, USA Critical Care 2015, 19(Suppl 1):P113 (doi: 10.1186/cc14193) Introduction Tedizolid is an oxazolidinone antibiotic approved to treat acute bacterial skin and soft tissue infection and is under investigation Results In total, 870 patients were included and 835 evaluable, the IC was confi rmed at study inclusion for 291 and suspected for 544 patients. S39 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P112). Clinical and pharmacokinetic characteristics of patients Table 1 (abstract P112). Clinical and pharmacokinetic characteristics of patients Plasma Plasma Days from SOFA MCF dose Cmax/Cmin Cmax/Cmin Burn eschar admission at the % burned (mg/kg after fi rst dose at steady state tissue on day 5 to the start beginning LOS in BICU Patient TBSA % FT ABSI body weight) (μg/ml) (μg/ml) (μg/g) of MCF of MCF (days) 1 35 20 9 1.3 8.6/0.8 7.4/1.0 2.3 38 1 75 2 40 35 8 2.0 8.5/1.1 9.4/1.8 <LQ 15 6 23 3 23 16 8 1.3 6.4/0.8 10.3/1.2 <LQ 12 2 17 4 70 40 12 1.1 3.9/0.5 4.5/0.8 0.4 8 6 43 5 23 12 7 1.3 7.5/1.8 8.0/1.4 0.6 10 5 19 6 70 60 11 1.2 3.4/0.5 5.0/0.9 1.5 12 5 70 7 80 70 12 1.1 3.8/0.4 4.0/0.4 0.2 34 2 61 8 60 50 10 1.4 4.8/0.5 4.3/1.0 0.2 34 2 90 9 44 34 10 1.3 4.5/1.1 9.1/2.3 0.2 8 6 34 10 34 28 9 1.3 4.1/0.7 5.4/1.0 0.7 10 5 35 Median 42 34.5 9.5 1.3 4.7/0.7 6.4/1.0 0.5 12 5.0 39 IQR 31.3 to 70.0 19.0 to 52.5 8 to 11.3 1.1 to 1.4 3.9 to 7.5/0.5 to 1.1 4.5 to 9.1/0.9 to 1.4 0.3 to 1.1 9.5 to 19.8 3.5 to 5.6 22.7 to 71.3 ABSI, Abbreviated Burn Severity Index; BICU, burn intensive care unit; FT, full thickness; IQR, interquartile range; LOS, length of hospital stay; LQ, limit of quantifi cation (<0.1 μg/ml); SOFA, Sequential Organ Failure Assessment; TBSA, total body surface area. CLTM appears modest relative to total body clearance and is unlikely to require dose adjustments. CRRT adsorption in the clinical setting is likely less than what we observed in this in vitro, continuously recirculating blood model. CLTM appears modest relative to total body clearance and is unlikely to require dose adjustments. P113 Tedizolid clearance by in vitro continuous renal replacement therapy model CRRT adsorption in the clinical setting is likely less than what we observed in this in vitro, continuously recirculating blood model. Figure 1 (abstract P113). P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration G Kennedy1, J Osborn1, S Flanagan2, N Alsayed3, S Bertolami1 1Cubist Pharmaceuticals, Lexington, MA, USA; 2Cubist Pharmaceuticals, San Diego, CA, USA; 3Cubist Pharmaceuticals, Zurich, Switzerland Critical Care 2015, 19(Suppl 1):P114 (doi: 10.1186/cc14194) P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration P115 P115 Antiviral prophylaxis inhibits cytomegalovirus reactivation in critical illness NJ Cowley1, A Owen1, J Millar1, SC Shiels1, RL Woolley2, NJ Ives2, H Osman1, P Moss2, JF Bion1 1University Hospital Birmingham, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P115 (doi: 10.1186/cc14195) Introduction Reactivation of latent cytomegalovirus (CMV) can lead to viraemia or CMV disease and has been detected in up to 30% of critically ill patients without prior history of immune suppression. However, the clinical importance of this observation remains unclear. We report a proof-of-concept randomised controlled trial of two antiviral drugs in intensive care patients to determine their impact on CMV reactivation. Methods We conducted a single-centre randomised controlled study of high-dose valaciclovir or low-dose valganciclovir prophylaxis, as compared with standard care, in CMV seropositive patients in the ICU at Queen Elizabeth Hospital Birmingham, UK. Patients were excluded if CMV seronegative. Study participants randomised to a study drug received either 450  mg valganciclovir daily enterally (or ganciclovir intravenously) or 2 g valaciclovir four times daily enterally (or aciclovir intravenously) for a period of up to 28 days. Blood was collected for CMV viral load during the 28-day study period. The primary outcome measure was reactivation of CMV in blood above 20 copies.ml–1 (assay detection limit) by day 28. Methods DAP (6  mg/kg) was administered intravenously every 48 hours to CVVHDF patients in the ICU. Blood and fi ltrate samples were collected at 0, 1, 1.5, 2, 5, 12, 24, and 48 hours after infusion. All collected samples were analyzed using HPLC according to the method of Tobin and colleagues [3]. Maximum concentration (Cmax), elimination half- life (t1/2), area AUC, Cmin, volume of distribution (Vd), clearance (CL), fraction unbound, and sieving coeffi cient (Sc) were evaluated. Patient characteristics and CVVHDF parameters including blood, dialysate, and fi ltration fl ow rates were recorded. il Results Three patients were included in the study. Mean blood, dialysate, and fi ltration fl ow rates were 86.7  ±  11.5  ml/minute, 417  ±  29  ml/hour, and 417  ±  29  ml/hour, respectively, confi rming that CVVHDF was performed under low-fl ow setting. P115 Cmax was 50.1 ± 12.7 mg/l (31.9, 70.5, 49.7 mg/l); t1/2, 35.1 ± 34.8 hours (18.6, 11.5, 70.5 hours); AUC, 889 ± 399 mg hour/l (471, 967, 1,260 mg hour/l); Cmin, 16.0  ±  10.3  mg/l (2.3, 24.7, 14.0  mg/l); Vd, 26.0  ±  20.9 l (23.8, 6.34, 47.9 l); CL, 9.47  ±  4.56  ml/minute (14.7, 6.35, 7.37  ml/minute); and fraction unbound, 5.8% (5.7, 4.1, 7.6%). Sc and CL of dialyzer were 0.08  ±  0.03 (0.11, 0.04, 0.07) and 1.20  ±  0.39  ml/minute (1.70, 0.88, 0.96 ml/minute), respectively. Results A total of 124 patients were randomised; 44 control, 34 valaciclovir, and 46 valganciclovir. Recruitment to the valaciclovir arm was halted early because of an imbalance in mortality (44% mortality vs. 19% in other arms). Independent blinded review of all deaths did not reveal any deaths attributable to unexpected causes. Fourteen patients were excluded from the primary analysis because of baseline CMV reactivation. CMV reactivation occurred in 30% (12/40) of the control arm but only 3% (1/39) in the valganciclovir arm (RR: 0.09 (95% CI: 0.01, 0.6)). When the two treatment arms were considered together, reactivation was observed in only 4% (3/70) (RR: 0.1 (95% CI: 0.04, 0.5)). See Figure 1. Conclusion DAP (6  mg/kg daptomycin every 48  hours) in patients receiving low-fl ow CVVHDF resulted in showing variability of AUC and avoiding accumulation. Owing to small case numbers, it needs further study. g Conclusion This is the fi rst study in critical care to assess the feasibility of antiviral prophylaxis to prevent CMV reactivation in a mixed population of critically ill patients. Low-dose valganciclovir was shown to suppress CMV reactivation as eff ectively as higher-dose valaciclovir. P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration Tedizolid’s transmembrane clearances (CLTM) during CHF and CHD were assessed by measuring sieving (SC) and saturation (SA) coeffi cients at various ultrafi ltrate (Quf) (1, 2, 3 l/ hour) and dialysate fl ow rates (Qd) (1, 2, 3 and 6 l/hour), using a blood fl ow rate (Qb) of 200 ml/minute. Tedizolid adsorption was tested in a 1 l recirculating CHF model at Quf of 2 l/hour and Qb of 200 ml/minute over 4  hours. Adsorption (%) was calculated after correcting for the dilution by CHF priming volume. Urea was added as a control in all experiments. Methods For each assay, run in triplicate, one 200 mg tablet of tedizolid phosphate was crushed, dispersed in water, drained under gravity through one of two types of NG tubes (type 1, Kangaroo Nasogastric Feeding Tube, 10 Fr 43" (109 cm); type 2, Salem Sump Dual Lumen Stomach Tube, 18 Fr/CH (6.0 m) 48" (122 cm)), and collected for recovery analysis by high-performance liquid chromatography with UV detection. To analyze the chemical stability of the crushed tablet dispersed in water, the aqueous preparation was assayed initially after dispersion and again after 4 hours at room temperature, without NG tube passage. The prespecifi ed limit for tedizolid phosphate in recovery samples was 90 to 110% of the dose. Limits were also specifi ed for levels of certain impurities. Results Urea SC and SA were ~1 in all experiments. In CHF, mean tedizolid SC ranged from 0.52 to 0.57 for HF1400 and from 0.50 to 0.54 for M150. CLTM did not diff er between fi lter types for Quf of 1, 2, and 3 l/hour. In CHD, mean tedizolid SA ranged from 0.46 to 0.56 for HF1400 and from 0.38 to 0.44 for M 150. Tedizolid CLTM with the HF1400 was higher than M150 values at Qd of 6 l/hours (P <0.02). Tedizolid exhibited irreversible adsorption within 10 minutes. See Figure 1. Results The average and individual recovery values of tedizolid phosphate were within 90 to 110% of the 200 mg dose when crushed tablets, dispersed in water at room temperature, were transferred through the 2 NG tubes (type 1: 95.8%; type 2: 93.6%). There was Conclusion Tedizolid’s CLTM is dependent on hemodiafi lter type and Qd for CHD and Quf in CHF. P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration At conventional CRRT rates, tedizolid S40 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Acknowledgements Research funded by the NIHR under the RfPB Programme (PG 1010 23225). Views expressed are of the authors and not necessarily the NHS, NIHR, or DOH. no signifi cant change in recovery values after 4  hours of storage at room temperature (93.9% initially and 94.7% after 4  hours). Results for degradation products and impurities were also within specifi ed limits in NG recovery samples and in the 0-hour and 4-hour aqueous preparations. P116 p p Conclusion The stability and recovery of tedizolid phosphate were not infl uenced by crushing the tablets and passing through an NG tube. Therefore, administration of crushed tedizolid phosphate tablets to patients is unlikely to alter the pharmacokinetics of tedizolid compared with whole tablets. Pharmacokinetics of daptomycin in patients undergoing low-fl ow continuous venovenous hemodiafi ltration TI Ide1, Y Takesue1, K Ikawa2, S Nishi1 1Hyogo College of Medicine, Nishinomiya City, Japan; 2Hiroshima University, Hiroshima, Japan Critical Care 2015, 19(Suppl 1):P116 (doi: 10.1186/cc14196) Introduction In daptomycin (DAP), 1,061  mg hour/l of the area under the concentration–time curve (AUC)/MIC was required to obtain clinical success [1], and a trough serum concentration (Cmin) cutoff point of 24.3 g/ml was most signifi cantly associated with CPK elevation [2]. Reportedly, DAP at a recommended dosage of 8 mg/kg is removed in patients undergoing high-fl ow continuous venovenous hemodiafi ltration (CVVHDF) (blood fl ow and fi ltration rates were 150 ± 48 and 2 l/hour). In Japan, CVVHDF is preferentially performed with lower fl ow rates. Investigating eff ects of fl ow rate on DAP removal during continuous renal replacement therapy is essential to adjust therapeutic dosages. We aimed to investigate the pharmacokinetics of DAP in CVVHDF patients in this setting. PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? S De Winter1, J Wauters1, E Van Wijngaerden1, W Peetermans1, P Annaert2, J Verhaegen1, JB Gillet1, D Knockaert1, I Spriet1 1University Hospitals Leuven, Belgium; 2Catholic University Leuven, Belgium Critical Care 2015, 19(Suppl 1):P118 (doi: 10.1186/cc14198) Methods A total of four colistin-resistant (MIC ≥4) GNB were isolated from ICU patients with nosocomial MDR infections. All four isolates were Klebsiella pneumonia. Among these isolates three were from blood and one from endotracheal aspirate and all four isolates were sensitive to fosfomycin in vitro. All of these patients had multiple comorbidities with recent history of colistin exposure. Intravenous fosfomycin sodium (inj Fosmicin; Meiji, Japan) was started as a combination therapy with carbapenem. Introduction Studies in the ICU showed that a single amikacin dose of ≥25 mg/kg should be used in conditions of increased distribution volume (Vd) such as severe sepsis/shock [1]. However, no data are available for emergency department (ED) patients in the early phase of sepsis/septic shock. The purpose of this study was to determine whether a single amikacin dose of 25 versus 15 mg/kg results in PK/PD target attainment for ED patients. Results Among the three bacteremic patients, two recovered completely from sepsis as well as the patient with ventilator-associated pneumonia. There was clinical as well as microbiological cure with normalization of sepsis markers. The only one bacteremic patient who died during the course of therapy was later diagnosed to have azole- resistant fungemia as a superinfection. g p Methods ED patients with severe sepsis/shock were randomly treated with a single amikacin dose of 25 versus 15  mg/kg. Blood samples were collected at  +1 (peak),  +6  hours and  +24  hours (trough) after the start of infusion. Primary outcome was PK/PD target attainment defi ned as a peak/MIC >8, corresponding with both actual MIC values documented from isolated pathogens, as well as EUCAST susceptibility breakpoints for Enterobacteriaceae and P. aeruginosa; that is, 8 mg/l. Noncompartmental analysis was used to calculate PK parameters. g p Conclusion Based on the evidence of clinical experience and available studies, intravenous fosfomycin therapy may be considered as the last option for the treatment of MDR GNB infection where there is documented colistin resistance and where there is literally no other choice of antibiotic therapy. PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? Results During a study duration of 20 months, 50 patients were enrolled in each dosing regimen resulting in 100 peak concentrations, 92 and 88 +6 hours and +24 hours concentrations respectively. Target attainment using local MIC values (median 2  mg/l, documented in 56 isolated Gram-negative pathogens) was achieved in 95% in both groups (P = 0.98). Using EUCAST susceptibility breakpoints, the target Table 1 (abstract P118) Introduction In vitro studies suggest that there is signifi cant adsorption of amikacin, netilmicin, gentamicin and tobramycin to polyacrylonitrile haemofi lters. This occurs rapidly and has the potential to substantially reduce the peak aminoglycoside concentration, which will reduce effi cacy [1]. However, whether signifi cant adsorption occurs in vivo is unknown. We therefore carried out a controlled in vivo study of the eff ect of amikacin adsorption by polyacrylonitrile fi lters during haemofi ltration, using a porcine model of acute renal failure. l, clearance. aMann–Whitney U <0.05, 15 versus 25 mg/kg ED patients Conclusion The EUCAST-based PK/PD target was only attained in 76% of patients treated with 25 mg/kg. However, in contrast to ICU patients, the majority of ED patients are treated for community-acquired infections, so MIC values are signifi cantly lower than the EUCAST susceptibility breakpoints, warranting PK/PD target attainment in both 25 and 15 mg/kg dosing regimens when local epidemiology is taken into account. i g p Methods A porcine model of acute renal failure was created by bilateral ligation of the renal arteries and veins. Eight pigs underwent haemofi ltration using a 0.6 m2 polyacrylonitrile fi lter, blood fl ow 200 ml/ minute, ultrafi ltration rate 1,000 ml/hour. All ultrafi ltrate was returned to the pigs via a separate venous catheter so that any elimination of amikacin by haemofi ltration could only be due to adsorption. Another eight pigs underwent sham haemofi ltration in which blood was pumped around a haemofi ltration circuit without a haemofi lter and without ultrafi ltration. Both groups of pigs were given intravenous amikacin, 15 mg/kg body weight over 30 minutes, and blood samples were taken from the arterial limb of the haemofi lter circuit at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120, 150, and 180 minutes after the start of the amikacin administration to assay amikacin concentrations. Results Post-distribution peak concentration of amikacin was slightly, but signifi cantly, lower in the CRRT group than that in sham group (55.0 ± 4.5 vs. 61.1 ± 5.9 mg/l, P <0.05).f Reference 1. Taccone et al. Crit Care. 2013;14:R53. Intravenous fosfomycin therapy in critically ill patients infected with colistin-resistant enterobacteriacae Conclusion This study shows that the eff ect of adsorption by polyacrylonitrile haemofi lters on in vivo amikacin peak concentrations is small, and less than would be expected from in vitro data. Introduction Carbapenem-resistant enterobacteriacae emerged in recent years as one of the most challenging groups of antibiotic- resistant pathogens. Polymyxins are considered as the last resort for the treatment of infections with carbapenem-resistant Gram-negative bacilli (GNB). Inadequate or extensive use of colistin leads to emergence of colistin resistance in GNB, jeopardizing treatment options in ICUs, potentially increasing mortality and morbidity and necessitating prudent use of alternative antibiotics. Fosfomycin, a phosponic acid derivative which acts primarily by disrupting bacterial cell wall synthesis, is a broad-spectrum antibiotic. Fosfomycin tromethamine is an oral formulation approved for the treatment of uncomplicated urinary tract infection caused by multidrug-resistant (MDR) bacteria. Recently fosfomycin is also available as a sodium/disodium formulation for intravenous use, which is showing promising result against MDR/ potentially drug-resistant pathogens. Acknowledgement This work was supported by a grant from the Hong Kong Research Grants Committee, CUHK 4644/08M. Reference 1. Tian Q, Gomersall CD, Ip M, Tan PE, Joynt GM, Choi GY. Adsorption of amikacin, a signifi cant mechanism of elimination by hemofi ltration. Antimicrob Agents Chemother. 2008;52:1009-13. References 1. Safdar N, et al. Antimicrob Agents Chemother. 2004;48:63-8. 2. Bhavnani SM, et al. Clin Infect Dis. 2010;50:1568-74. . Safdar N, et al. Antimicrob Agents Chemother. 2004;48:63-8. 3. Tobin CM, et al. J Antimicrob Chemother. 2008;62:1462-3. Figure 1 (abstract P115). CMV reactivation over time. Each line represents a single patient. Figure 1 (abstract P115). CMV reactivation over time. Each line represents a single patient. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S41 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 was attained in 76% versus 40% in the 25 versus 15  mg/kg group, respectively (P < 0.0001). Single-dose PK parameters are displayed in Table 1 and compared with the ones reported in the ICU [1]. P117 P117 Adsorption of amikacin during continuous venovenous haemofi ltration in a swine model of acute renal failure CD Gomersall, Q Tian, D Reynolds, M Ip, G Choi, G Joynt The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P117 (doi: 10.1186/cc14197) Table 1 (abstract P118) PK parameter 15 mg/kg ED 25 mg/kg ED 25 mg/kg ICU Peak (mg/l) 58 (47 to 70)a 91 (72 to 105)a 73 (62 to 90) Trough (mg/l) 6 (3 to 12) 5 (3 to 15) 7 (2 to 15) Vd (l/kg) 0.3 (0.3 to 0.5) 0.4 (0.2 to 0.6) 0.4 (0.3 to 0.5) Cl (ml/minute/kg) 1.6 (1 to 2.3)a 2.2 (1.4 to 3)a 1.9 (1.3 to 3.5) Cl, clearance. aMann–Whitney U <0.05, 15 versus 25 mg/kg ED patients. 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation Introduction Amikacin Inhale is an integrated drug–device combi- nation in development by Bayer HealthCare through a collaboration with Nektar Therapeutics, to improve clinical outcome in intubated and mechanically ventilated patients with Gram-negative pneumonia. It is available in two confi gurations: on-vent for intubated patients and hand-held for extubated patients to complete aerosolized antibiotic therapy. Amikacin Inhale is a smart system that consists of the pulmonary drug delivery system with a vibrating mesh nebulizer and the specially formulated Amikacin Inhalation Solution (400 mg every 12  hours for 10  days). The objectives of this study were to evaluate the performance of the Amikacin Inhale hand-held confi guration with supplemental O2 concentration supplied at diff erent fl ow rates and in diff erent orientations. We hypothesize that the delivered dose of amikacin will not signifi cantly change with increased O2 fl ow rate or varying orientation.i q Results Preventive administration of IT as an adjunct to systemic antibiotics was associated with a lower incidence of NP in group 1 (group 1 33.3%, group 2 66.7%, χ2 = 6,000; P = 0.014) and a shorter duration of ICU stay (group 1 8.0  ±  4.6  days vs. 17.1  ±  18.4  days, P = 0.03). The mortality did not diff er between groups: 11.1% in group 1 and 22.2% in group 2 (P ≥0.99). On day 3 Acinetobacter spp. (30.5%), K. pneumoniae (22.0%), B. cepacia (13.2%) and P. aeruginosa (34.3%) were detected in BAL, there were no diff erences between groups. In group 1 CPIS remained stable and APACHE II decreased. CPIS and APACHE II were lower in group 1 on day 5 (P = 0.0004). y Conclusion Early administration of IT as an adjunct to systemic antibiotics is eff ective in prevention of NP in multiple trauma patients: it promotes decrease of NP incidence and decrease of ICU stay. Methods In the hand-held confi guration of Amikacin Inhale, amikacin is aerosolized into a holding chamber. Amikacin aerosol is inhaled with ambient air entering the bottom of the chamber through the inhalation valve. Supplemental O2 may be supplied through the O2 port and mixes with ambient air entering through the inhalation valve. O2 concentration and delivered dose at the mouthpiece exit were characterized in vitro at various O2 fl ow rates (2 to 10  l/minute). O2 concentrations were measured every minute until the end of dosing. Early preventive administration of inhaled tobramycin in severe polytrauma Early preventive administration of inhaled tobramycin in severe polytrauma A Kuzovlev1, A Shabanov2, T Chernenkaya2, V Moroz1, A Goloubev1 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.V. Sklifosofsky Research Institute, Moscow, Russia Critical Care 2015, 19(Suppl 1):P121 (doi: 10.1186/cc14201) y Results Overall, nine cases with CNS infection were recorded aged from 22 to 74, all males. LP was performed between the second and 17th day (average 8.3 days) and the CSF analysis showed 40 to 6,000 cells – mainly PMNs, protein 161 mg% to 287 mg% and glucose from 3 to 58 mg/dl. They were all colonized with Acinetobacter baumannii sensitive only to colistin. CSF cultures were negative for all patients besides one, who grew A. baumannii. Of those, seven (77%) were receiving i.v. colistin, eight (88%) carbapenems, and eight (88%) glycopeptides, all in combination with other antibiotics. Median i.t. administration time was 9.1 days. All patients responded to i.v. and i.t. antibiotics but there was one case in which fever relapsed and increased number of cells in subsequent LP was observed which was attributed to colistin, which was withdrawn. All these patients survived, and were discharged to the ward. p y A Kuzovlev1, A Shabanov2, T Chernenkaya2, V Moroz1, A Goloubev1 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.V. Sklifosofsky Research Institute, Moscow, Russia Critical Care 2015, 19(Suppl 1):P121 (doi: 10.1186/cc14201) Introduction Nosocomial pneumonia (NP) occurs in 30 to 50% of multiple trauma patients. It is mostly caused by multiresistant Gram- negative bacteria. Use of inhaled antibiotics as adjuncts to systemic antibiotics presents a great outlook for the prevention of NP in multiple trauma patients. The aim of the study was to evaluate the effi cacy of early administration of inhaled tobtamycin (IT) as an adjunct to systemic antibiotics for the prevention of NP in polytrauma. Methods Fifty-four ICU mechanically ventilated patients with multiple trauma (ISS >30; car accident 55.6%; fall 29.6%; train accident 11.1%; domestic 3.7%) were enrolled in the single-center randomized trial. Groups were comparable in ISS, age, sex, type of trauma, and blood loss. Patients were randomized into two groups: Group 1 (n  = 27), addition of IT to systemic antibiotics (ciprofl oxacin 800  mg/day; metronidazol 1,500 mg/day); Group 2 (n = 27), only systemic antibiotics (same regimen). Early preventive administration of inhaled tobramycin in severe polytrauma Inhaled tobramycin (300 mg twice daily via nebulizer) and systemic antibiotics were administered within the fi rst 24 hours Conclusion Patients treated with the abovementioned regime showed clinical and biochemical improvement. The above drug combination turned out to be successful in neurosurgical ICU patients with CNS infection. Intrathecal administration of colistin, vancomycin and amikacin for central nervous system infections in ICU neurosurgical patients P Alexandropoulos, S Georgiou, V Chantziara, A Tsimogianni, E Chinou, V Karagiannisa, G Michaloudis Introduction Central nervous system (CNS) infections in ICU patients after neurosurgery are a diffi cult and life-threatening complication demanding immediate action. In many cases intravenous (i.v.) administration of antibiotics is not suffi cient; thus, intrathecal (i.t.) administration is required. g pi pp 2 Results The mean O2 concentration ranged from 36 to 70% over 2 to 10 l/minute and was ≥40% at ≥3 l/minute. The delivered dose did not change substantially with increasing enriched O2 fl ow rate (72 to 82% of nominal dose). At 0° and 45° orientations, the delivered dose of amikacin was 74 to 80% and 73 to 76% of the nominal dose (400 mg), respectively. Methods From January 2013 to November 2014 all cases with CNS infections were recorded. Inclusion criteria were the presence of fever ≥38.5°C, increased infl ammatory markers, compatible lumbar puncture (LP) fi ndings (increased number of polymorphonuclear leukocytes, increased protein and low glucose compared with serum levels) and no evidence of other site of infection. All subjects were receiving appropriate i.v. antibiotic treatment based on cultures. Intrathecal administration of 300,000 iu colistin, 25 mg vancomycin and 25 mg amikacin was performed taking under consideration that neurosurgical patients in the ICU have CNS infection attributed to Gram-negative bacteria or/and to Staphylococcus species. Conclusion Amikacin Inhale was shown in vitro to be suitable for extubated patients who require supplemental O2. The delivered dose was independent of supplemental O2 and device orientation. P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation A ventilator was connected to the set up to simulate patient breathing. The delivered dose was measured at the exit of the mouthpiece. Drug distribution within the test setup compartments was analyzed using HPLC. The in vitro delivered amikacin dose was also measured at nebulizer orientations of 0° and 45° (n = 3 per orientation) using a simulated breathing profi le with no supplemental O2. P122 Intrathecal administration of colistin, vancomycin and amikacin for central nervous system infections in ICU neurosurgical patients P Alexandropoulos, S Georgiou, V Chantziara, A Tsimogianni, E Chinou, V Karagiannisa, G Michaloudis Saint Savvas Oncology Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P122 (doi: 10.1186/cc14202) Reference 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. S42 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P120 P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation N Kadrichu1, K Corkery1, T Dang1, P Challoner2 1Novartis Pharmaceuticals, San Carlos, CA, USA; 2Nektar Therapeutics, San Francisco, CA, USA Critical Care 2015, 19(Suppl 1):P120 (doi: 10.1186/cc14200) after ICU admission. After obtaining the results of bronchoalveolar lavage microbiology, the antibiotic regimen was switched according to the sensitivity. The primary outcome measure was new onset of NP and duration of ICU stay. Microbiological, X-ray, CPIS, signs of sepsis and oxygenation index were used as objective indicators of the clinical progress. The secondary outcome measure was 30-day mortality. Diagnosis of NP was made according to the standard clinical and CPIS criteria. The data were statistically analyzed by SPSS 11.5 (M, σ, Newman–Keuls test; chi-square-test P <0.05). after ICU admission. After obtaining the results of bronchoalveolar lavage microbiology, the antibiotic regimen was switched according to the sensitivity. The primary outcome measure was new onset of NP and duration of ICU stay. Microbiological, X-ray, CPIS, signs of sepsis and oxygenation index were used as objective indicators of the clinical progress. The secondary outcome measure was 30-day mortality. Diagnosis of NP was made according to the standard clinical and CPIS criteria. The data were statistically analyzed by SPSS 11.5 (M, σ, Newman–Keuls test; chi-square-test P <0.05). Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Bl 1 N Ni 1 M B i l1 P S h 2 E Ull 3 I S Wid 1 Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Blum1, N Nigro1, M Briel1, P Schuetz2, E Ullmer3, I Suter-Widmer1, B Winzeler1, R Bingisser1, H Elsaesser3, D Drozdov2, B Arici2, SA Urwyler1, J Refardt1, P Tarr4, S Wirz4, R Thomann5, C Baumgartner6, H Duplain7, D Burki8, W Zimmerli3, N Rodondi6, B Mueller2, M Christ-Crain1 1University Hospital Basel, Switzerland; 2Medical University Clinic, Kantonsspital Aarau, Switzerland; 3Kantonsspital Baselland/Liestal, Liestal, Switzerland; 4Kantonsspital Baselland/Bruderholz, Bruderholz, Switzerland; 5Bürgerspital, Solothurn, Switzerland; 6Inselspital, Bern University Hospital, Bern, Switzerland; 7Hôpital du Jura, Site de Delémont, Delémont, Switzerland; 8Viollier SA, Basel, Switzerland Results We included nine trials enrolling 2,637 patients. Eight trials were of unclear risk of bias and one was classifi ed as having low risk of bias. In trials comparing heparin with placebo or usual care, the risk ratio for death associated with heparin was 0.88 (95% CI = 0.77 to 1.00, I2 = 0%, 2477 patients, six trials). In trials comparing heparin with other anticoagulants, the risk ratio for death was 1.30 (95% CI = 0.78 to 2.18, I2  = 0%, 160 patients, three trials). In trials comparing heparin with placebo or usual care, major hemorrhage was not statistically signifi cantly increased (risk ratio 0.79, 95% CI  = 0.53 to 1.17, I2 = 0%, 2,392 patients, three trials). In one small trial of heparin compared with other anticoagulants, the risk of major hemorrhage was signifi cantly increased (2.14, 95% CI = 1.07 to 4.30, 48 patients). Important secondary and safety outcomes, including minor bleeding, were sparsely reported. Critical Care 2015, 19(Suppl 1):P125 (doi: 10.1186/cc14205) Critical Care 2015, 19(Suppl 1):P125 (doi: 10.1186/cc14205) Introduction Clinical trials yielded confl icting data about the benefi t of adding systemic corticosteroids for community-acquired pneumonia (CAP). We evaluated whether short-term corticosteroid treatment reduces time to clinical stability in patients hospitalized for CAP. Methods This randomized, placebo-controlled multicenter trial compared prednisone 50  mg for 7  days with placebo in patients hospitalized with CAP. The primary endpoint was time to clinical stability. Results Overall, 802 patients were randomized in seven Swiss hospitals from December 2009 to May 2014. Time to clinical stability was shorter in the prednisone group compared with placebo (3.0 vs. 4.4  days, HR = 1.33, 95% CI = 1.15 to 1.50, P <0.001). The prednisone group as compared with the placebo group had a shorter time to hospital discharge (6 vs. i References 1. Alejandria MM, Lansang MD, Dans LF, Mantaring III JBlas. Intravenous immunoglobulin for treating sepsis, severe sepsis and septic shock. Cochrane Database Syst Rev. 2013;9:CD001090. doi:10.1002/14651858.CD001090.pub2. 2. Department of Health. Clinical guidelines for immunoglobulin use. London: Department of Health; 2011. y p 2. Department of Health. Clinical guidelines for immunoglobulin use. London: Department of Health; 2011. Methods We included randomized controlled trials from MEDLINE, EMBASE, CENTRAL, Global Health, Scopus, Web of Science, the International Clinical Trials Registry Platform (inception to April 2014), conference proceedings, and reference lists of relevant articles. Two reviewers independently identifi ed and extracted trial-level data from randomized trials investigating unfractionated or low molecular heparin administered to patients with sepsis, severe sepsis, septic shock or DIC associated with infection. Internal validity was assessed in duplicate using the Risk of Bias tool. Our primary outcome was mortality. Safety outcomes included hemorrhage, transfusion and thrombocytopenia. Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis g Conclusion The use of IVIg does not appear to aff ect mortality in sepsis. There was also no statistical benefi t or harm demonstrated by using IVIg. This also holds true whether IVIg is given either according to the guidelines or not; however, stricter adherence to the guidelines does have fi nancial implications. Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) Introduction Septic shock is characterized by systemic infl ammation coupled with upregulation of coagulation. Heparin is an inexpensive and widely available anticoagulant with anti-infl ammatory properties. The objectives our study were to evaluate the effi cacy and safety of heparin in patients with sepsis, septic shock or disseminated intravascular coagulation (DIC) associated with infection. P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis R Zarychanski1, AM Abou-Setta1, S Kanji2, AF Turgeon3, A Kumar1, DS Houston1, E Rimmer1, BL Houston4, L McIntyre2, AE Fox-Robichaud5, PC Hebert6, DJ Cook5, DA Fergusson2 1University of Manitoba, Winnipeg, MB, Canada; 2Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3Université Laval, Québec City, QC, Canada; 4University of Toronto, ON, Canada; 5McMaster University, Hamilton, ON, Canada; 6Centre hospitalier de l’Université de Montreal (CHUM) – Hopital Notre-Dame, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Bl 1 N Ni 1 M B i l1 P S h 2 E Ull 3 I S Wid 1 7 days (HR = 1.19, 1.04 to 1.38), P = 0.012) and a shorter duration of intravenous antibiotic treatment (4 vs. 5 days (diff erence, –0.89 days, –0.20 to –1.57 days), P = 0.011). All-cause mortality, ICU stay, recurrent pneumonia and rehospitalization rate were similar in both groups. Incidence of pneumonia-associated complications until day 30 tended to be lower in the prednisone group (2.8% vs. 5.6%, OR = 0.49, 0.23 to 1.02, P = 0.06). The prednisone group had a higher rate of in-hospital hyperglycemia needing insulin treatment (19.4% vs. 10.9%, OR = 1.96, 1.31 to 2.93, P = 0.001). Other adverse events compatible with corticosteroid use were rare and similar in both groups. Conclusion Heparin in patients with sepsis, septic shock, and DIC associated with infection may be associated with decreased mortality; however, the overall impact remains uncertain. Safety outcomes have been under-reported and require further study. Large randomized trials are needed to evaluate the effi cacy and safety of heparin in patients with sepsis, severe sepsis, and septic shock. Reference 1. Karaiskos I, Galani L, Baziaka F, Giamarellou H. Intraventricular and intrathecal colistin as the last therapeutic resort for the treatment of multidrug-resistant and extensively drug-resistant Acinetobacter baumannii ventriculitis and meningitis: a literature review. Int J Antimicrob Agents. 2013;41:499-508. S43 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis R Zarychanski1, AM Abou-Setta1, S Kanji2, AF Turgeon3, A Kumar1, DS Houston1, E Rimmer1, BL Houston4, L McIntyre2, AE Fox-Robichaud5, PC Hebert6, DJ Cook5, DA Fergusson2 1University of Manitoba, Winnipeg, MB, Canada; 2Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3Université Laval, Québec City, QC, Canada; 4University of Toronto, ON, Canada; 5McMaster University, Hamilton, ON, Canada; 6Centre hospitalier de l’Université de Montreal (CHUM) – Hopital Notre-Dame, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) guidelines for prescription, with a mortality rate of 25%. Nine patients did not meet the national guidelines for prescription, with a mortality rate of 44%. The diff erence in mortality rates between the two groups did not reach statistical signifi cance (P = 0.6). There was no signifi cant diff erence in APACHE II scores between the two groups. There was also no diff erence in mortality between those receiving IVIg and those who did not. We also found no diff erence in those receiving single or double doses of IVIg. P126 Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) P124 P124 Use of intravenous immunoglobulin to treat sepsis in a general ICU Y Drakeford, J Kelly, P Morgan, J Melville, A Holland Surrey and Sussex Healthcare NHS Trust, Redhill, UK Critical Care 2015, 19(Suppl 1):P124 (doi: 10.1186/cc14204) Use of intravenous immunoglobulin to treat sepsis in a general ICU Y Drakeford, J Kelly, P Morgan, J Melville, A Holland Surrey and Sussex Healthcare NHS Trust, Redhill, UK Critical Care 2015, 19(Suppl 1):P124 (doi: 10.1186/cc14204) Introduction Sepsis is a major cause of admission to the ICU, and a leading cause of death for ICU patients. Intravenous immunoglobulin (IVIg) is indicated in the treatment of some patients with sepsis, although the evidence for this remains controversial. The use of IVIg is regulated due to its high cost, and prescription guidelines have been revised by the NHS, coordinated by the National Demand Management Programme for Immunoglobulin. Conclusion Prednisone treatment for 7 days in hospitalized patients with CAP shortens time to clinical stability, time to hospital discharge and duration of intravenous antibiotic treatment without an increase in complications. Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model T Masuda1, C Mitaka2, M Khin Hnin Si2, K Kido2, Y Qi2, T Uchida2, S Abe2, T Miyasho3, M Tomita4 1Tokyo Medical and Dental University, Tokyo, Japan; 2Tokyo Medical and Dental University Graduate School, Tokyo, Japan; 3Rakuno Gakuen University, Hokkaido, Japan; 4Tokyo Medical and Dental University Hospital of Medicine, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P127 (doi: 10.1186/cc14207) g gi Results The mortality rate of 28 days in the L-group was 32.8%, and was 18.8% in the H-group. Mean arterial pressure increased signifi cantly (P <0.01) in the H-group compared with the L-group. WBC counts in the L-group increased and in the H-group decreased (P <0.01) during PMX- DHP treatment. Platelet counts in both groups decreased signifi cantly (P <0.01).There was no signifi cant diff erence between before and after PMX-DHP in IL-6 levels. On the other hand, IL-1ra decreased signifi cantly before and after PMX-DHP. Also, IL-6 and IL-1ra in the L-group were signifi cantly higher than those in the H-group at the start of PMX- DHP. PCT values in the L-group were increased compared with the H-group at the start of PMX-DHP (P <0.01). PCT in the L-group increased signifi cantly (P <0.01), but no signifi cant changes in the H-group. PAI- 1 showed no signifi cant changes before and after PMX-DHP and no changes in both groups at the start of PMX-DHP. Introduction Direct hemoperfusion with a polymyxin B immobilized column (PMX-DHP) adsorbs endotoxin and has been used for the treatment of septic shock [1]. However, the mechanisms of action behind PMX-DHP are not fully understood. Therefore, the purpose of this study was to elucidate mechanisms of action behind PMX-DHP in a rat model of cecal ligation and puncture. Introduction Direct hemoperfusion with a polymyxin B immobilized column (PMX-DHP) adsorbs endotoxin and has been used for the treatment of septic shock [1]. However, the mechanisms of action behind PMX-DHP are not fully understood. Therefore, the purpose of this study was to elucidate mechanisms of action behind PMX-DHP in a rat model of cecal ligation and puncture. Methods Sprague–Dawley rats were anesthetized and were mechanical ventilated after tracheostomy. The right internal carotid artery was cannulated with a catheter for continuous measurement of the arterial pressure and heart rate. P128 y p Results RecAP administration resulted in a terminal elimination half- life and plasma clearance of 49 to 58 hours and 2.8 to 3.4 l/hour after single ascending doses, respectively, and 63 to 66 hours and 3.1 to 3.8 l/hour after multiple ascending doses. Peak recAP concentrations and AP activity levels were reached at the end of the 1-hour infusion and showed a rapid decline with about 10% of the maximum concentration remaining at 4 hours and less than 5% at 24 hours post start. Although the maximal concentration and total systemic exposure of recAP and AP activity increased slightly more than dose proportionally this is of no signifi cance in the estimated therapeutic dose range. RecAP treatment was generally well tolerated and anti-drug antibodies could not be detected in serum. P129 Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network White blood cell counts have an impact on septic patient outcome followed by polymyxin-B immobilized fi ber with direct hemoperfusion p H Tanaka, T Ikeda, S Ono, S Suda, T Ueno Tokyo Medical University, Hachioji Medical Center, Hachioji, Japan Critical Care 2015, 19(Suppl 1):P128 (doi: 10.1186/cc14208) Introduction The mortality rate of severe sepsis and septic shock is varied and high (25 to 70%). In our institute, the indication for polymyxin-B immobilized fi ber with direct hemoperfusion (PMX-DHP) has been that circulatory failure (systolic blood pressure <90 mmHg or required catecholamines and high lactacidemia) continued despite following early goal-directed therapy by the Surviving Sepsis Campaign guidelines 2012. Conclusion RecAP is characterized by a long serum terminal half-life, by stable serum AP levels and did not exert any safety concerns when administered to healthy volunteers. These results pave the way to investigate the potential of recAP as a new treatment option for sepsis- associated AKI in a phase II clinical trial, which will start at the end of 2014 [Clinical Trial Register:NCT02182440]. g Methods This study included 80 patients with severe sepsis or septic shock due to abdominal infection retrospectively. These subjects were divided into two groups: those with WBC counts <4,000 (L-group: 64 patients) and those with WBC counts >12,000 (H-group: 16 patients). Mean arterial pressure, WBC counts, platelet counts, interleukin-6 (IL-6), and plasminogen activator inhibitor-1 (PAI-1) were measured immediately before the initiation and after the completion of PMX- DHP. Statistical analysis was performed using the chi-squared test for background factors, with Wilcoxon’s rank-sum test for comparison within a group, and Mann–Whitney’s U test for comparison between groups. The signifi cance level was set at P <0.05. Pharmacokinetics, safety and tolerability of h alkaline phosphatase in healthy volunteers 1 d2 3 l 2 Methods We conducted a retrospective audit of pharmacy records of IVIg prescriptions issued to ICU patients with severe sepsis and septic shock from 2009 to 2014 against national prescription guidelines. Microbiology results were examined to support prescriptions, and admission APACHE II scores and unit outcomes were examined. Results From 2009 to 2014, 644 patients were admitted to the ICU with severe sepsis and septic shock, with a mortality rate of 41%. Seventeen patients received IVIg. Of these, eight patients met the national Methods We conducted a retrospective audit of pharmacy records of IVIg prescriptions issued to ICU patients with severe sepsis and septic shock from 2009 to 2014 against national prescription guidelines. Microbiology results were examined to support prescriptions, and admission APACHE II scores and unit outcomes were examined. p p y E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) Results From 2009 to 2014, 644 patients were admitted to the ICU with severe sepsis and septic shock, with a mortality rate of 41%. Seventeen patients received IVIg. Of these, eight patients met the national Introduction Clinical trials showed renal protective eff ects of bovine intestinal alkaline phosphatase in critically ill patients with S44 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion PMX-DHP improved hemodynamics, acid–base balance, and creatinine levels through reducing cytokines and renal tubular cell death in a rat model of cecal ligation and puncture. These fi ndings suggest the preventive role of PMX-DHP in the development of sepsis- related acute kidney injury. sepsis-associated acute kidney injury (AKI) [1,2]. Recently, human recombinant AP (recAP) was developed as a pharmacological attractive replacement. We conducted a phase I clinical trial to evaluate tolerability, safety and pharmacokinetics of recAP in healthy volunteers. Methods In a randomized, double-blind, placebo-controlled phase I trial, healthy volunteers received via a 1-hour i.v. infusion a single dose of recAP (200, 500, 1,000 or 2,000 U/kg; n = 33) or multiple doses of recAP (500 or 1,000 U/kg; n = 18) on three consecutive days (n = 18). Serum recAP concentrations, AP activity levels and anti-drug antibodies were measured, and safety parameters were monitored. References 1. Pickkers P, et al. Crit Care. 2012;16:R14. , ; 2. Heemskerk S, et al. Crit Care Med. 2009;37:417-23.e1. Reference 1. Ronco C, et al. Polymyxin B hemoperfusion: a mechanistic perspective. Crit. Care. 2014;18:309. 1. Ronco C, et al. Polymyxin B hemoperfusion: a mechanistic perspective. Crit. Care. 2014;18:309. Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model The right femoral vein was cannulated with a catheter for infusion of saline (10 ml/kg/hour) during the study period. The rats were randomized into three experimental groups: cecal ligation and puncture (CLP) + dummy column (Dummy- DHP) group (n = 10), CLP + PMX-DHP group (n = 10), and sham group (n = 4). Four hours after CLP, Dummy-DHP or PMX-DHP was performed for 1  hour. Blood was drawn from the right internal carotid artery, perfused through PMX column or dummy column, and returned to the right femoral vein. The heart rate, mean arterial pressure, arterial blood gases, and plasma concentrations of creatinine, lactate, potassium, and cytokines (IL-6 and IL-10) were measured at baseline and at 4, 5, and 8 hours after CLP. At the completion of the experiment, the rats were killed overdose of pentobarbital. The kidney, liver, and lung were harvested, and histopathologic examinations of these organs were performed. g g p Conclusion The mortality rate of the L-group tended to be higher than that of the H-group. Infl ammatory and anti-infl ammatory cytokines in the L-group were higher than those of the H-group. These results indicate that leukopenia (WBC <4,000) in severe sepsis patients leads to more severe outcome and hypercytokinemia than leukocytosis (WBC >12,000) in severe sepsis patients. P129 Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network E Sevketoglu1, D Yildizdas2, O Horoz2, H Kihtir1, T Kendirli3, S Bayraktar4, J Carcillo5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Istanbul, Turkey; 2Cukurova University Medical Faculty, Adana, Turkey; 3Ankara University Medical Faculty, Ankara, Turkey; 4Haseki Research and Training Hospital, Istanbul, Turkey; 5University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Results Hypotension and metabolic acidosis occurred in the CLP  + Dummy-DHP group, whereas hemodynamics and acid–base balance were better maintained in the CLP  + PMX-DHP group. Plasma concentrations of lactate, creatinine, potassium, and cytokines were signifi cantly higher in the CLP + Dummy-DHP group than in the CLP + PMX-DHP group at 8 hours. Renal tubular cell death was observed in the CLP + Dummy-DHP group, but not in the CLP + PMX-DHP group. Critical Care 2015, 19(Suppl 1):P129 (doi: 10.1186/cc14209) Introduction Thrombocytopenia-associated multiple organ failure (TAMOF) can lead to high mortality in critically ill children, possibly related to consequences of thrombotic microangiopathy. Plasma Introduction Thrombocytopenia-associated multiple organ failure (TAMOF) can lead to high mortality in critically ill children, possibly related to consequences of thrombotic microangiopathy. Plasma S45 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 APACHE scores before and after ECAC therapy were available for eight patients who died; APACHE score decreased >5 points in fi ve patients after single application of ECAC. exchange therapy may improve thrombotic microangiopathy [1]. The purpose of this observational cohort study is to describe whether there is an association between use of plasma exchange therapy and outcome in the Turkish TAMOF network. Conclusion ECAC can be used as adjuvant therapy in treatment of severe sepsis/septic shock/MOF. Our patients had high PM and four could be saved with use of ECAC. We could expect a better outcome if ECAC was used early (<24 hours) during treatment. However, future well-designed studies are needed to clarify the role of ECAC in patients with MOF/septic shock. Methods We performed a retrospective cohort analysis in patients with TAMOF at three diff erent pediatric ICUs comparing those who received plasma exchange (+) plus standard therapies with those who did not receive plasma exchange (–) and only received standard therapies. Results Among the 42 TAMOF patients enrolled, all had a primary or secondary sepsis diagnosis. P130 P130 Clinical experience of using a novel extracorporeal cytokine adsorption column for treatment of septic shock with multiorgan failure Methods We retrospectively evaluated 387 patients who received a broad-spectrum antimicrobial treatment for septic shock due to GNB between January 2009 and December 2012 in the ICU of 10 Japanese tertiary hospitals. After alignment of the treatment time phase for each patient, we divided the patients into two groups according to whether PMX treatment was performed within 24  hours after ICU admission (PMX group: n  = 129 and non-PMX group: n  = 258). The primary endpoint was 28-day mortality. Ruby Hall Clinic, Pune, India Introduction Severe sepsis and multiorgan failure (MOF) are major causes of death in the ICU. The extracorporeal cytokine adsorption column (ECAC; Cytosorb®, CytoSorbents Corporation, USA), a critical care focused therapeutic device, results in rapid in vitro and in vivo elimination of several key cytokines and prevents organ failure. Use of ECAC in patients with sepsis is a new area of research with insuffi cient data to promote large prospective RCTs. Studies published to date have shown promising results. We report our clinical experience with ECAC for severe sepsis/septic shock/MOF patients. Results The mean (SD) age and SOFA scores on ICU admission were 72.5 (12.5) years and 10.0 (3.4), respectively. The infection site was intra-abdominal (47.0%), pulmonary (17.6%), and urinary tract (27.8%). Two-thirds of all patients had bacteremia due to GNB. No diff erence in 28-day mortality was observed between the two groups (PMX: 33.9% vs. non-PMX: 33.1%, P = 0.87). In the Cox regression analysis adjusted for age, sex and facilities, the PMX treatment (hazard ratio = 0.87; 95% confi dence interval, 0.53 to 1.43) did not improve the outcome.f Methods A retrospective evaluation of ECAC in patients admitted to a tertiary ICU from November 13 to October 14 to analyze: clinical safety; selection of a subgroup of patients where it could be used; selection of timing for initiation; number of device fi lters required per patient; and selective markers to identify above initiation. Patients were managed with standard of care (SOC; antibiotics, vasopressors, i.v. fl uids, sepsis dosed steroids) and ECAC as adjuvant therapy. Vitals, APACHE II and SOFA scores were measured. i Conclusion No diff erence in mortality rate was observed after adjustment for the endotoxin adsorption therapy with PMX in the patients with septic shock due to GNB. Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network Fifteen received plasma exchange therapy (PE(+) group) and 27 received standard medical treatment without plasma exchange (PE(–) group). The mean age was 17.69 months (8.24 to 54.22) in the PE(+) group, and 13.46 months (6.47 to 20.55) in the PE(–) group. Age (P = 0.232), gender (P = 0.206), thrombocyte count (P = 0.09), OFI score (P = 0.111) and Pelod score (P = 0.177) on admission were not statistically diff erent between groups. The overall 28-day mortality was higher in the PE(–) group 70.37% compared with 26.67% in the PE(+) group (univariate P =0.006; multivariate controlling for Pelod, OFI, PRISM scores and neurological failure P = 0.048). Length of stay was increased in the PE(+) group (P = 0.004). Reference Introduction Mortality from septic shock in the ICU remains high, ranging from 30 to 50%. In particular, Gram-negative bacilli (GNB) account for 40% of the causative bacteria of severe sepsis, which progresses to multiorgan failure due to signifi cant infl ammation. Hemoperfusion with polymyxin B-immobilized fi ber (PMX) adsorbs endotoxin and can reduce the infl ammatory cascade of sepsis due to GNB. However, the clinical effi cacy of this treatment has not been demonstrated. We aimed to verify the effi cacy of endotoxin adsorption therapy by using PMX. Reference 1. Nguyen TC, Carcillo JA. Bench-to-bedside review: Thrombocytopenia- associated multiple organ failure – a newly appreciated syndrome in the critically ill. Crit Care. 2006;10: 235. Reference 1. Nguyen TC, Carcillo JA. Bench-to-bedside review: Thrombocytopenia- associated multiple organ failure – a newly appreciated syndrome in the critically ill. Crit Care. 2006;10: 235. Eff ectiveness of polymyxin B immobilized fi ber hemoperfusion in patients with septic shock due to Gram-negative bacillus infection: the PMXHP study N Saito1, K Sugiyama2, T Ohnuma3, T Kanemura4, M Nasu5, Y Yoshidomi6, H Adachi7, H Koami8, Y Tsujimoto9, A Tochiki10, Y Wagatsuma11, T Myumi12 1Chiba Hokusou Hospital, Nippon Medical School, Chiba, Japan; 2Tokyo Metropolitan Bokutoh Hospital, Tokyo, Japan; 3Saitma Medical Center, Jichi Medical University, Saitama, Japan; 4National Hospital Organization Disaster Medical Center, Tokyo, Japan; 5Urasoe General Hospita, Okinawa, Japan; 6Saga-ken Medical Center, Koseikan, Saga, Japan; 7Iizuka Hospital, Fukuoka, Japan; 8Saga University Hospital, Saga, Japan; 9Yamagata Prefectural Central Hospital, Yamagata, Japan; 10Tsukuba Medical Center Hospital, Ibaraki, Japan; 11University of Tsukuba, Ibaraki, Japan; 12University of Occupational and Environment Health, Fukuoka, Japan Conclusion The positive association found between use of plasma exchange therapy and improved survival supports the potential of this therapy in Turkish children with TAMOF. The positive, although less so, associated treatment eff ect observed after controlling for illness severity provides further rationale for performing a randomized controlled trial in the pediatric Turkish TAMOF network. Sample size calculations call for a 100-patient trial with a pre hoc interim analysis after enrollment of 50 TAMOF patients. p Critical Care 2015, 19(Suppl 1):P131 (doi: 10.1186/cc14211) Introduction Mortality from septic shock in the ICU remains high, ranging from 30 to 50%. In particular, Gram-negative bacilli (GNB) account for 40% of the causative bacteria of severe sepsis, which progresses to multiorgan failure due to signifi cant infl ammation. Hemoperfusion with polymyxin B-immobilized fi ber (PMX) adsorbs endotoxin and can reduce the infl ammatory cascade of sepsis due to GNB. However, the clinical effi cacy of this treatment has not been demonstrated. We aimed to verify the effi cacy of endotoxin adsorption therapy by using PMX. Impact of evolving cardiac catheterisation services on admissions to a regional ICU Results Nineteen ICU patients (14 men, fi ve women; 24 to 72 years; average ICU stay 10 days; average ventilator days 9) with APACHE II >17 (except one with dengue shock syndrome), SOFA score ≥11 (n = 16) and the majority having infection largely in the lung (n = 8; alone or with UTI and blood infection) followed by the abdomen (n = 4), UTI (n = 3) and others (n = 4) were given ECAC (total ECAC = 31). Predicted mortality (PM) was >40% in 16, >30% in two and <30% in two (tropical infections) patients. Duration of therapy was 6 hours (no. of ECAC = 18) and 8 hours (n = 4; no. of ECAC = 5) for the majority of patients. Overall, four patients (two with tropical infections and two with PM >40%) survived; three of them had were ECAC early (<24 hours of admission). The majority of patients (n = 11) who died could be given ECAC only once. Of patients who died, seven were given ECAC late (>24 hours). g EA Gorman, D Trainor Royal Victoria Hospital, Belfast, UK Critical Care 2015, 19(Suppl 1):P132 (doi: 10.1186/cc14212) EA Gorman, D Trainor Introduction National UK audit data demonstrate cardiac catheterisation services, including percutaneous coronary intervention and noncoronary interventions, are increasing [1-3]. National mortality rates post cardiac catheterisation are also increasing, refl ecting an increasing proportion of sicker patients undergoing interventional procedures [3]. National audit procedures do not evaluate patients admitted to intensive care post cardiac catheterisation. We aimed to Introduction National UK audit data demonstrate cardiac catheterisation services, including percutaneous coronary intervention and noncoronary interventions, are increasing [1-3]. National mortality rates post cardiac catheterisation are also increasing, refl ecting an increasing proportion of sicker patients undergoing interventional procedures [3]. National audit procedures do not evaluate patients admitted to intensive care post cardiac catheterisation. We aimed to S46 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P133). evaluate the impact of an evolving regional cardiac catheterisation service on a regional intensive care unit (RICU) serving a population of 1.8 million. Methods A retrospective review was carried out. Patients admitted from the regional cardiac catheterisation laboratory to the regional ICU, between September 2009 and September 2014, were identifi ed using validated RICU admission records. Clinical data were extracted from computerised patient records. Impact of the introduction of e-learning prior to a basic transthoracic echo course P Madhivathanan1, S Jain2, D Walker3 Introduction Deep venous thrombosis (DVT) is an increasing major cause of mortality and morbidity. There is a need for quick, easy, cheap, convenient and reliable diagnostic tools. The objectives were to ascertain the diagnostic concordance of emergency doctor- performed ultrasound (EDUS) of the lower extremities with specialist doctor-performed (radiologist or vascular surgeon) echo-Doppler (SDED), in the diagnosis of DVT, and to identify possible causes of nonconcordance. P Madhivathanan1, S Jain2, D Walker3 1Barts Health NHS Trust, London, UK; 2Homerton University Hospitals NHS Foundation Trust, London, UK; 3University College London Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P133 (doi: 10.1186/cc14213) P Madhivathanan , S Jain , D Walker 1Barts Health NHS Trust, London, UK; 2Homerton University Hospitals NHS Foundation Trust, London, UK; 3University College London Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P133 (doi: 10.1186/cc14213) Introduction Focused Intensive Care Echo accreditation is a nationally approved pathway for training and accreditation in basic transthoracic echocardiography (TTE) in the UK. Recently, an e-learning module, the Intensive Care Echo and Basic Lung Ultrasound (ICE-BLU), has been introduced to facilitate TTE learning [1]. Previous work from our group has shown that incorporating simulation-based teaching elements into a basic TTE course improves candidates’ satisfaction [2]. We assessed the impact of introducing the ICE-BLU e-learning programme prior to our simulation-based basic TTE course. Methods A prospective, multicentre study. Adult patients (>18 years old) with clinical suspicion of DVT, with high or moderate risk (on Wells scoring), or low risk with increased D-dimer levels, were eligible for the study. Emergency doctors performed two EDUS in femoral and popliteal areas (these results were blinded). After this, echo-Doppler was performed by specialist doctors. Both procedures were done within 24 hours of each other. The fi nal result was considered nonconcordant if one or both of the EDUS did not match with the SDED. These SDED were used as reference (as standard clinical practice). Methods Prior to the August 2014 course, all candidates were required to complete the ICE-BLU e-learning module. On the morning of the course, the candidates completed a questionnaire to assess the impact of the e-learning module. The survey included questions on the quality of content, user friendliness, whether the content was pitched at the right level and any problems faced whilst accessing the e-learning module. P133 p Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) References 1. http://www.e-lfh.org.uk/programmes/icu-echoultrasound. Accessed 1 Dec 2014. Accessed 1 Dec 2014. 2. Madhivathanan PR, et al. Simulation-based transthoracic echo teaching: a tertiary centre experience. Intensive Care Med. 2014;40 Suppl 1:S158-9. 2. Madhivathanan PR, et al. Simulation-based transthoracic echo teaching: a tertiary centre experience. Intensive Care Med. 2014;40 Suppl 1:S158-9. 1. British Cardiovascular Interventional Society. Audit returns for adult interventional procedures. January 2013–December 2013. http://www.bcis. org.uk/documents/BCIS_Audit_2013_for_web_Version_23-11-2014.pdf. Diagnostic concordance of emergency doctor-performed bedside ultrasonography versus specialist-performed echo-Doppler ultrasonography in the diagnosis of deep venous thrombosis of lower limbs 2. National Institute for Cardiovascular Outcomes. Myocardial ischaemia national audit project. Public report April 2012–March 2013. http://www.ucl.ac.uk/nicor/audits/minap/reports. 3. British Cardiovascular interventional society. National coronary interventional procedures. Public report. January 2012–December 2013. http://www.bcis.org.uk/resources/PCI_Audit_Report_2012_fi nal.pdf. DL Ly-Pen1, JP Penedo Alonso2, MS Sánchez Perez2, FR Roldán Moll2, MZ Zamorano Serrano2, LD Díaz Vidal2, SJ Justo3 1Southend University Hospital, Westcliff -on-Sea, UK; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) DL Ly-Pen1, JP Penedo Alonso2, MS Sánchez Perez2, FR Roldán Moll2, MZ Zamorano Serrano2, LD Díaz Vidal2, SJ Justo3 1Southend University Hospital, Westcliff -on-Sea, UK; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) Impact of evolving cardiac catheterisation services on admissions to a regional ICU p p Results A total of 170 patients were identifi ed (representing 2.9% of critical care admissions during this time). Baseline characteristics: 71.7% male, median age 66 (IQR 55 to 74), median APACHE score 18 (IQR 15 to 23). Seventy-one patients (41.7%) had an APACHE score >20. Fifteen patients (8.8%) were aged >80  years. Admissions increased yearly – 20 in 2010, 26 in 2011, 35 in 2012, 47 in 2013, 37 at the end of the third quarter of 2014 (projected 59 admissions by year end 2014). Median length of stay was 3.5 days (IQR 1.8 to 7.2). Average length of stay reduced yearly (9.14 days in 2010 to 5.01 days in 2014). ICU bed- days per year remained static over the 5-year period. Critical care and hospital mortality rates were 33% and 39% respectively. There was a trend towards increasing mortality yearly, and with increasing age and APACHE score. Conclusion Our survey has shown that the e-learning initiative was welcome by the candidates. We conclude that introduction of e-learning prior to a simulation-based basic TTE course enhances candidates’ satisfaction and feedback. Conclusion An evolving cardiac catheterisation service is having a signifi cant impact on intensive care services within a regional centre. Increasing mortality trends in this critical care population refl ects post- cardiac catheterisation mortality trends nationally. We suggest intensive care admissions post cardiac catheterisation should be included in the national audit, to allow forward planning of intensive care services and to promote quality improvement within this population. References Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) y Results From January 2014 to December 2014, 32 patients with septic shock (study group) and 20 patients with sepsis but no septic shock (control group) were recruited. The baseline characteristics were similar. Conventional echocardiographic measurements, including LVEF (59.53% vs. 60.67% in the control group, P  =  0.450) and fractional shortening (31.98% vs. 32.98%, P = 0.323), did not diff er between the two groups. The study group had a greater degree of myocardial dysfunction measured by left ventricular global longitudinal strain (–14.6 vs. –17.6, P = 0.005, with a less negative value implying worse myocardial contractility). The hemodynamic profi les (cardiac index 3.49 l/minute/m2 vs. 3.41 l/minute/ m2 respectively, P = 0.764) were not statistically diff erent.i Introduction Myocardial depression is common among septic patients [1]. The aim of this study was to assess whether the values of cardiac index (CI) and velocity–time integral (VTI) calculated by echocardiography diff er between survivors and nonsurvivors of sepsis. Methods This was a prospective observational study. We included adult newly admitted septic patients, regardless of disease severity. Exclusion criteria were concomitant pregnancy or obstetric/gyne- co logical sepsis and co-existing or terminal diseases that may limit life expectancy. At the moment of recruitment, additional exclusion criteria included: concomitant pulmonary embolism, trauma or acute ischemic coronary disease; pericardial tamponade; aortic valve disease; tachyarrhythmias and absence of adequate echocardiographic windows. Echocardiographic evaluations were made within the fi rst 10 minutes of initiation of fl uid therapy in the emergency room. All measurements and images were obtained with a 1.5 to 3.5 MHz phased array transducer using a standard cardiac preset. CI is the quotient of the cardiac output (CO) divided by the body surface area. The CO is the product of the stroke volume by the heart rate. Stroke volume is calculated as the product between aortic VTI (measured using pulsed- wave Doppler) and aortic cross-sectional area. The latter is calculated in the long axis parasternal window using the left ventricular outfl ow tract diameter measurement. Impact of the introduction of e-learning prior to a basic transthoracic echo course We also analysed candidates’ feedback from our January and August 2014 courses (Figure 1). Results From September 2013 to September 2014, a total of 328 patients were enrolled. Fifty-one investigators from seven hospitals performed the EDUS. Each patient had the EDUS (femoral and popliteal areas) and SDED (also in femoral and popliteal areas). Of 328 pairs of US studies, 37 were nonconcordant between EDUS and SDED. Two EDUS were incomplete, so the concordance analysis was performed with 326 US studies, with 35 discordant. The percentage of agreement between EDUS and SDED was 89.26%. The kappa index was 0.76 (95% CI = 0.69 to 0.84), and this means a substantial agreement. Results The response rate of the survey was 100%. Eighty per cent of candidates completed the e-learning module. The e-learning module was rated high by most candidates (80%). However, nearly one-half of the candidates faced problems accessing the module, online. Analysis of candidates’ feedback (from the January and August 2014 courses) revealed that candidates’ overall impression was better with the introduction of e-learning prior to the course. Conclusion There is substantial agreement between EDUS and SDED in the diagnosis of DVT, in routine clinical practice. This confi rms the results of previous papers. The largest nondiagnostic concordance in thrombus occurs in the early performances of emergency doctors, S47 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Comparing group I versus group II, the mortality rate was 45%, and there was a statistically signifi cant diff erence for temperature (P = 0.001), HR (P = 0.001) and WBC count (P = 0.01) on admission. Upon comparing survivors versus nonsurvivors in group I there was a statistical diff erence in HR on day 7 (P = 0.02), successful vasopressor withdrawal (P = 0.02), P/F ratio (P = 0.02) and ScVO2 on day 7 (P = 0.03). Regarding IL-1α, IL-1β, TNFα and troponin I there was no statistical signifi cant diff erence between groups I and II but IL-6, IL-10 and CRP showed statistically signifi cant diff erence on admission PV and CS. Pro- BNP shows statistically signifi cant diff erence in all CS samples between septic and nonseptic groups. Regarding echo upon comparing the survivors versus nonsurvivors, E’d/t on day 0 shows a statistically signifi cant diff erence between both groups. Reference 1. Angus DC. Epidemiology of severe sepsis in USA. Crit Care Med. 2001;29:1303-10. Impact of the introduction of e-learning prior to a basic transthoracic echo course SAPS II and seventh-day SOFA are good predictive scores for mortality in sepsis. especially until the fi fth performance. After the sixth one, the incidence of mismatches falls dramatically. It seems advisable to mentor the training programmes with at least fi ve shadowed performances in order to lower the incidence of mistakes. P135 P135 Cardiac abnormalities in patients with septic shock detected by speckle tracking echocardiography PY Ng1, WC Sin1, AK Ng2, WM Chan1 1Queen Mary Hospital, Hong Kong; 2Grantham Hospital, Hong Kong Critical Care 2015, 19(Suppl 1):P135 (doi: 10.1186/cc14215) P135 Cardiac abnormalities in patients with septic shock detected by speckle tracking echocardiography PY Ng1, WC Sin1, AK Ng2, WM Chan1 1Queen Mary Hospital, Hong Kong; 2Grantham Hospital, Hong Kong Critical Care 2015, 19(Suppl 1):P135 (doi: 10.1186/cc14215) Introduction Sepsis-induced myocardial dysfunction is a well- recognized condition and confers worse outcomes in septic patients. However, the diagnostic criteria remain poorly described. Echocardiographic assessment by conventional parameters such as left ventricular ejection fraction (LVEF) is often aff ected by ongoing changes in preload and afterload conditions. Novel echocardiographic technologies such as speckle tracking imaging have evolved for direct assessment of the myocardial function. In this study, we investigate the measurement of myocardial strain by speckle tracking imaging for the diagnosis of sepsis-induced myocardial dysfunction. g p y p Conclusion Diastolic dysfunction was seen in 90% of patients. Fever, HR, and WBC counts are still good early indicators for diagnosis of sepsis. Vasopressor withdrawal on the seventh day was a good predictor for survival. Admission serum IL-6, IL-10 and CRP from PV were better indicators for sepsis than IL-1, pro-BNP and troponin I. Admission TNFα and seventh-day IL-6 levels were highly prognostic for mortality. CS samples proved that NT pro-BNP is a good indicator for sepsis diagnosis and a good predictor for survival. TNFα from CS samples was also a good predictor of mortality. SAPS II and a slower E’d/t on admission was a good predictor of mortality. R f Methods This is a prospective, case–control study at a university- affi liated tertiary care adult medical ICU. Consecutive patients admitted with a diagnosis of septic shock meeting the international consensus criteria were included. Patients with other causes of myocardial dysfunction were excluded. They are compared with age-matched, gender-matched, and cardiovascular risk factor-matched controls, who were admitted to hospital for sepsis but did not develop septic shock. Conventional echocardiographic parameters, as well as speckle tracking imaging of myocardial function, were obtained within 24  hours of diagnosis. Offl ine analyses of endocardial tracings were performed by two independent operators. Reference Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) Conclusion This is a fi rst study in the adult population to show that the use of speckle tracking imaging can diagnose signifi cant sepsis- induced myocardial dysfunction, which was not otherwise detectable by conventional echocardiography. P138 Speckle tracking imaging for evaluation of eff ects of positive end-expiratory pressure level on right ventricular function M Türker, A Pirat, A Camkiran Firat, B Pirat, A Sezgin, G Arslan Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P138 (doi: 10.1186/cc14218) Introduction Positive end-expiratory pressure (PEEP) is commonly used to correct hypoxemia in the ICU. However, PEEP may impair right ventricular functions by increasing its afterload. Speckle tracking imaging (STI) is a new echocardiography strain analysis technique that provides direct assessment of myocardial contractility during systole and diastole. The aim of this study was to evaluate the eff ects of diff erent PEEP levels on right ventricular functions by using STI in patients undergoing coronary artery bypass grafting surgery. Methods After ethics committee approval and patients’ written consent, we prospectively analyzed 20 CABG surgery patients. After initiation of mechanical ventilation and before sternotomy, 5, 10, and 20 cmH2O PEEP were applied in 5-minute intervals consequently. After stabilization at each PEEP level, four-chamber and two-chamber images of the right ventricle were recorded using TEE. The right ventricle diameter, velocity, longitudinal strain, SR, and fractional area change (RVFAC) were calculated and evaluated from the recorded images. PEEP at which LS disappeared or reappeared were compared using the Wilcoxon signed-rank test to assess the infl uences of anatomical side and PEEP increase or decrease. The values of PEEP at disappearance of LS for the right lung were not statistically signifi cantly diff erent from the left lung (P  =  0.844 for increases, P  =  0.938 for decreases). The values of PEEP at which LS disappeared obtained during increases were not statistically signifi cantly diff erent from values obtained during decreases (P = 1.000 for left lung, P = 0.875 for right lung; Figure 1). From data pooled from both sides and protocols, the median value of PEEP at which LS disappeared as a false positive sign of pneumothorax was 25 cmH2O (interquartile range = 20 to 30 cmH2O). At PEEP = 10 cmH2O, no patient showed absence of LS, whereas at PEEP = 35 cmH2O, all patients showed absence of LS (Figure 2). g Results The mean age of study patients (85% male) was 59.7 ± 10.5 years. Intraoperative mean, systolic, and diastolic arterial blood pressures and heart rate were similar at the three PEEP levels. P136 Evaluating the eff ect of sepsis and septic shock on myocardial functions by echocardiography and serum biomarker level in peripheral veins and coronary sinus M Soliman1, A Alazab1, R El Hossainy1, M Nirmalan2, H Nagy1 1Cairo University, Cairo, Egypt; 2University of Manchester, UK Critical Care 2015, 19(Suppl 1):P136 (doi: 10.1186/cc14216) Introduction Sepsis is a leading cause of death in critically ill patients despite the use of modern antibiotics and resuscitation therapies. Biomarkers and cardiovascular changes have an important place in this process. Myocardial depression occurs in 40% of septic patients. Methods Twenty patients (group I) with sepsis or septic shock were included and 10 patients (group II) served as the nonseptic group. Group I morbidity and mortality at day 28 in the ICU were targeted as the endpoint. Laboratory investigations, APACHE IV, SAPS II and SOFA scores were calculated. Biomarkers IL-1α, IL-1β, IL-6, IL-10, TNFα, CRP, NT-proBNP and troponin level were estimated on admission and day 7 in the peripheral vein (PV) and coronary sinus (CS). Transthoracic echocardiography and tissue Doppler imaging was done on admission and on day 7. Results In 3 months, 58 patients were included. The average age was 46.6 years, and 36 were male. Overall mortality was 14%. We included 16 patients with sepsis syndrome, 27 patients with severe sepsis and 15 patients with septic shock. Severe sepsis patients presented with higher values of CI, when compared with sepsis syndrome and septic shock patients (3.46, 3.08 and 2.92 l/minute/m2, respectively, P = NS). The same occurred with VTI (19.27, 18.81 and 16.74 cm for severe sepsis, sepsis syndrome and septic shock, respectively; P = NS). Mean values of CI were lower in nonsurvivors of sepsis (2.51 vs. 3.35 l/minute/ m2, P  =  0.018). Mean values of VTI were also lower in nonsurvivors (14.83 vs. 19.01 cm, P = 0.022). Conclusion In our study, nonsurvivors of sepsis presented with lower values of both CI and VTI in the emergency department. Therefore, CI S48 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P139). Absence of LS during increasing and decreasing PEEP. Figure 2 (abstract P139). Values of PEEP above which LS was not present. and VTI may be good markers of sepsis severity and mortality in newly admitted patients. In addition, further studies are warranted to assess the role of CI and VTI as therapeutic targets. P139 Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP J Golub1, A Markota1, A Stožer2, G Prosen3, A Bergauer1, F Svenšek1, A Sinkovič1 1University Medical Centre Maribor, Slovenia; 2University of Maribor, Slovenia; 3Community Health Centre, Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P139 (doi: 10.1186/cc14219) P140 Lung ultrasound in quantifying lung water in septic shock patients L De Geer, A Oscarsson, M Gustafsson Linkoping University Hospital, Linkoping, Sweden Critical Care 2015, 19(Suppl 1):P140 (doi: 10.1186/cc14220) P136 Reference 1. Silva E, Pedro Mde A, Sogayar AC, et al. Brazilian Sepsis Epidemiological Study (BASES study). Crit Care. 2004;8:R251-60. P138 P138 Compared with 5 and 10 cmH2O PEEP, mean RVFAC signifi cantly decreased at 20 cmH2O PEEP (P  =  0.001). Right ventricle velocity reduced with incremental PEEP increases (P <0.05). Mean SR values decreased at 20 cmH2O PEEP when compared with 5 cmH2O PEEP (P = 0.03). Mean right ventricle diameter measurements decreased with incremental PEEP increases; however, this decrease was signifi cantly diff erent between 20 cmH2O PEEP and other two PEEP levels (P = 0.01). The mean right ventricle strain value signifi cantly decreased at 20 cmH2O PEEP when compared with other two PEEP levels (P <0.001 for both). p g Conclusion According to this study, higher PEEP levels correlate with disappearance of LS without pneumothorax. Absence of LS in patients with high PEEP should be interpreted with caution and other signs of pneumothorax should be sought before therapeutic interventions are attempted. Conclusion Compared with 5 and 10 cmH2O PEEP levels, right ventricle functions in terms of strain, SR, right ventricle diameter, and RVFAC were signifi cantly impaired at 20 cmH2O PEEP level. P140 Variations in near-infrared spectroscopy-derived oxygen downslope during a vascular occlusion test in critically ill patients: relationship with outcome p A Donati, E Damiani, A Carsetti, V Monaldi, E Montesi, P Pelaia A Donati, E Damiani, A Carsetti, V Monaldi, E Montesi, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P141 (doi: 10.1186/cc14221) Università Politecnica delle Marche, Ancona, Italy y Critical Care 2015, 19(Suppl 1):P141 (doi: 10.1186/cc14221) Results No changes in mean arterial pressure were observed. The perfused small vessel density tended to decrease at t1 and normalize at t2 (Figure 1) in the hyperoxia group. These variations appeared early after 2 minutes of FiO2 changes. A signifi cant increase in lactate levels over time (from 1.1 (0.9 to 1.7) at t0 to 1.4 (1.1 to 1.9) mmol/l at t2, P = 0.01) was seen in the hyperoxia group. Introduction Near-infrared spectroscopy (NIRS) with a vascular occlusion test (VOT) can be used to extrapolate information regarding the tissue oxygen extraction rate. We explored the meaning of variations in tissue oxygen saturation downslope (StO2down) during a VOT in critically ill patients. Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Figure 1 (abstract P142). y p Methods In this prospective observational study, NIRS (thenar eminence) was applied every day in 93 patients admitted to the ICU. A VOT was performed using a 40% StO2 target. The slope of the desaturation curve was assessed separately for the fi rst part (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between Down2 – Down1 was calculated.if Results No signifi cant diff erences were seen in StO2 Down1 or Down2 between ICU survivors (n  = 76) and ICU nonsurvivors (n  = 17) over Figure 1 (abstract P141). Figure 1 (abstract P142). Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Normobaric hyperoxia and the microcirculation in critically ill patients: a prospective observational study Normobaric hyperoxia and the microcirculation in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, E Montesi, S Ciucani, M Rogani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P142 (doi: 10.1186/cc14222) Conclusion LUS non-invasively and user-independently quantifi es lung water in concordance with, but does not replace, invasive measurements. Further studies are needed establish the role of LUS as a monitoring and diagnostic tool in septic shock patients. References Introduction It is well known that oxygen acts as a vasoconstrictor. We evaluated the impact of normobaric hyperoxia on the sublingual microcirculation in critically ill patients. Introduction It is well known that oxygen acts as a vasoconstrictor. We evaluated the impact of normobaric hyperoxia on the sublingual microcirculation in critically ill patients. 1. Frassi F, et al. J Card Fail. 2007;13:830-5. 1. Frassi F, et al. J Card Fail. 2007;13:830-5. 2. Prosen G, et al. Crit Care. 2011;15:R114. Methods Forty mechanically ventilated (FiO2 ≤50%) patients with hemodynamic stability were enrolled in a prospective observational study. The fi rst 20 patients underwent a 2-hour period of hyperoxia (FiO2  = 100%), and 20 patients were studied as controls (no FiO2 variations). The sublingual microcirculation (three sites) was evaluated with sidestream dark-fi eld imaging at baseline (t0), after 2  hours of hyperoxia (t1), and 2  hours after return to baseline (t2). Continuous video recording was also performed during FiO2 variations on one and the same area (2-minute video). 3. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP Introduction Quantifi cation of lung ultrasound (LUS) artifacts (B-lines) is used to assess pulmonary congestion in emergency medicine and cardiology [1,2]. We investigated B-lines in relation to extravascular lung-water index (EVLWI) from invasive transpulmonary thermodilution in septic shock patients. Our aim was to evaluate the role of LUS in an intensive care setting. Methods Twenty-one patients admitted with septic shock to a general ICU underwent LUS of eight zones, four per hemithorax, within 24  hours after ICU admission. EVLWI was calculated simultaneously by transpulmonary thermodilution using a pulse-contour continuous cardiac output system, and NT-proBNP and clinical data were collected. Two physicians blinded to other data independently quantifi ed the number of B-lines. Spearman’s rho was used to test the correlation of B-lines to EVLWI and clinical data, and linear regression and Bland– Altman analysis were used to assess the agreement between B-lines and EVLWI. Interobserver variability was tested using Bland–Altman analysis and intraclass correlation coeffi cient (ICC). Introduction The objective of our study was to estimate correlation between PEEP and disappearance of lung sliding (LS) due to lung overdistension in the absence of pneumothorax. Methods We performed a prospective study from September 2013 to May 2014 in adult patients with respiratory failure who required mechanical ventilation, lung CT and recruitment manoeuvre. Lung CT was used as the gold standard to exclude pneumothorax. A staircase recruitment manoeuvre was used with 5 cmH2O increases of PEEP from baseline to 35 cmH2O and decreases in reverse order. The duration of each step was 1 minute. Lung ultrasound was performed to evaluate LS at each step in one intercostal window in the highest point of left and right hemithoraces by physicians trained in lung ultrasound and blinded to changes in PEEP.i Results Fourteen patients (67%) were male, the median age was 62 years (IQR 55 to 68) and eight (38%) patients had cardiac comorbidities. In median, SAPS 3 was 64 (IQR 60 to 74), ICU length of stay was 3 days (IQR 2 to 8) and seven patients (33%) died within 30 days of ICU admission. All patients were mechanically ventilated and treated according to Results In all, eight patients were included; fi ve (62.5%) males, mean age 70.1 ± 7.4 years. Mean auto-PEEP was 0.7 ± 0.4 cmH2O. Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP The values of S49 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 guidelines [3]. The median number of B-lines was 15 (IQR 10 to 30) and the median (IQR) NT-proBNP, EVLWI and oxygenation index (OI) were 7,800 ng/l (3,690 to 15,050), 11 ml/kg (IQR 8 to 18) and 9.2 (5.7 to 15.7), respectively. None of the characteristics diff ered signifi cantly between survivors and nonsurvivors. The number of B-lines correlated to EVLWI (ρ = 0.45, P = 0.04; r2 = 0.20, P = 0.04), but not to NT-proBNP (ρ = –0.42, P = 0.06), OI (ρ = 0.25, P = 0.31) or ICU length of stay (ρ = 0.14, P = 0.57). On Bland–Altman analysis, mean diff erences and 95% limits of agreements between B-lines and EVLWI was 4.9 (–14.5 to 24.5), and 5.9 (–3.5 to 15.3) when assessing observer agreement. The ICC between methods was 0.52 (95% CI = –0.17 to 0.81) and 0.90 (95% CI = 0.73 to 0.92) between observers. the fi rst 10 days in the ICU, while Down2 – Down1 was higher in ICU nonsurvivors (Figure 1). Patients in the upper quartile of mean Down2 – Down1 showed the highest 90-day mortality (P = 0.014).l Conclusion ICU nonsurvivors tended to show a fl attening in the last part of the desaturation curve during a VOT, suggesting a reduced tissue oxygen extraction. This may depend on microvascular dysfunction and/or cellular hypometabolic status. P142 P143 P143 Near-infrared spectroscopy for assessing the tissue oxygen extraction rate during sepsis: relationship with outcome A Donati, E Damiani, C Scorcella, S Tondi, S Ciucani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P143 (doi: 10.1186/cc14223) Prospective nonrandomized observational study of the use of an impedance threshold device in patients with spontaneous respiration and hemodynamic instability C Pantazopoulos1, I Floros1, N Archontoulis1, D Xanthis1, D Barouxis2, N Iacovidou2, T Xanthos2 1Laiko General Hospital of Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P144 (doi: 10.1186/cc14224) Conclusion The length of time to lactate normalization in the severe burn patient is a marker of survival and a simple parameter to guide the endpoint of resuscitation; however, the percentage of lactate clarifi ed in the fi rst 24 hours is not valid. Introduction The use of an impedance threshold device (ITD) in cardiac arrest victims has been shown to increase the systolic arterial pressure (SAP) by increasing venous return [1]. There are limited studies concerning the use of ITD in patients with spontaneous respiration and hemodynamic instability. The purpose of this study is to evaluate changes of hemodynamic parameters with the use of ITD in patients with spontaneous respiration and hemodynamic instability. P144 P144 Prospective nonrandomized observational study of the use of an impedance threshold device in patients with spontaneous respiration and hemodynamic instability C Pantazopoulos1, I Floros1, N Archontoulis1, D Xanthis1, D Barouxis2, N Iacovidou2, T Xanthos2 1Laiko General Hospital of Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P144 (doi: 10.1186/cc14224) Lactate in the burn patient This may refl ect a tissue hypometabolic status, which may be better elicited in the fi nal part of the ischemic challenge. Healthy volunteers (n = 27) were studied as controls. The slope of the desaturation curve was assessed separately for the fi rst (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between, Down2 – Down1, was calculated. Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P  <0.05); no diff erence was seen between ICU survivors (n  = 7) and nonsurvivors (n  = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P  <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P  <0.05); no diff erence was seen between ICU survivors (n  = 7) and nonsurvivors (n  = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P  <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Conclusion Tissue oxygen extraction was reduced in septic patients. Nonsurvivors showed a fl attening in the last part of the desaturation curve during a VOT, while the fi rst part of the StO2 downslope did not show any diff erence between survivors and nonsurvivors. This may refl ect a tissue hypometabolic status, which may be better elicited in the fi nal part of the ischemic challenge. y Results During a period of 2  years we studied 143 patients; their mean age was 46.98  ±  19.38 years, mean TBSA burn injury of the study population was 22.82 ± 20.25. The fl ame was the most frequent mechanism. A total of 83 patients were in mechanical ventilation and 13.6% of them developed ARDS. The mortality range in the study group was 17%. Serum lactate at admission ≥2 mmol/l was associated with 31.3% mortality versus 6% in patients with a serum lactate at admission <2 mmol/l (P <0.05). Length of time to lactate normalization variable is associated with mortality (P <0.02). The average lactate normalization time was 4.6 days in nonsurvivors while in survivors it was 2.02 days. A relation does not exist between the lactate clearance and mortality in all patients. P143 Near-infrared spectroscopy for assessing the tissue oxygen extraction rate during sepsis: relationship with outcome C S ll S d S C l Introduction Microcirculatory dysfunction impairs tissue oxygenation during sepsis. We applied near-infrared spectroscopy (NIRS) to evaluate the tissue oxygen extraction rate in sepsis and its relationship with outcome. Methods A prospective observational study; 14 septic patients underwent NIRS monitoring (thenar eminence) with a vascular occlusion test (using a 40% StO2 target) at admission to the ICU. Figure 1 (abstract P141). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S50 Figure 1 (abstract P143). 10 minutes after the intervention. Endpoint of the study was a change of patient’s SAP after application of ITD. Results The SAP of patients that were included in the study increased 15 to 22  mmHg (P  <0.05). Heart rate remained unchanged. Eighty percent of patients declared good to very good tolerance from ITD application. Conclusion In this observational study of patients with spontaneous respiration and hypotension, ITD increased the SAP, while it seems that it was well tolerated by patients. Additional and larger studies will be needed in the future in order to investigate the benefi ts of ITD when used to patients with spontaneous respiration and hemodynamic instability. Reference 1. Pirrallo RG, et al. Eff ect of an inspiratory impedance threshold device on hemodynamics during conventional manual cardiopulmonary resuscitation. Resuscitation. 2005;66:13-20. 1. Pirrallo RG, et al. Eff ect of an inspiratory impedance threshold device on hemodynamics during conventional manual cardiopulmonary resuscitation. Resuscitation. 2005;66:13-20. Lactate in the burn patient p EH Herrero, M Sánchez, L Cachafeiro, A Agrifoglio, B Galván, MJ Asensio, A García de Lorenzo Hospital La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P145 (doi: 10.1186/cc14225) Introduction Severe burns result in rapid loss of intravascular volume due to development of a severe capillary leak and hypovolemic shock. It is widely accepted that traditional markers, such as blood pressure and urinary output, are useful but do not suffi ciently refl ect global perfusion, regional microcirculation or reversal shock. Blood lactate concentration is widely used in ICUs as a reliable prognostic marker of global tissue hypoxia. Our aim is to determine whether the percentage of lactate clarifi ed in the fi rst 24 hours is valid as a guide for resuscitation. Methods We prospectively studied 143 consecutive burn patients admitted to our Burn Unit. Sociodemographics and comorbidities data were recorded. Clinical data were collected to calculate the Acute Burn Severity Index. Resuscitation according to the Parkland formula was guided by a urinary output of 0.5 to 1 ml/hour and the results of monitoring the blood pressure. Crystalloid solution (Ringer´s acetate) was given exclusively during the fi rst 24 hours. Early surgical excision of burn eschar and early coverage of excised burn wounds with autografts was performed. Initial and subsequent serum lactate levels were measured to calculate lactate clearance according to the formula: lactate basal – lactate at 24 hours / lactate basal × 100. The primary outcome was mortality. Figure 1 (abstract P143). Healthy volunteers (n = 27) were studied as controls. The slope of the desaturation curve was assessed separately for the fi rst (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between, Down2 – Down1, was calculated. Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P  <0.05); no diff erence was seen between ICU survivors (n  = 7) and nonsurvivors (n  = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P  <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Conclusion Tissue oxygen extraction was reduced in septic patients. Nonsurvivors showed a fl attening in the last part of the desaturation curve during a VOT, while the fi rst part of the StO2 downslope did not show any diff erence between survivors and nonsurvivors. Is the shock index a universal predictor in the emergency department? A cohort study Results Twenty-three patients underwent VA ECMO for refractory CS in the study period. Etiologies of CS were: 11 acute myocarditis, fi ve acute myocardial infarction and seven acute decompensation of chronic cardiomyopathy. The median time of ECMO was 10 days (4 to 15). Thirteen patients died during hospital stay and 10 survived. Three patients were bridged to LVAD and two to heart transplant; eight were bridged to recovery. The main cause of ICU death was multiple organ dysfunction (12/13). Nonsurvivors showed signifi cantly higher LAC0 (5 (2 to 6) vs. 8 (5 to 11), P = 0.021). Lactate clearance at 48 hours was not signifi cantly diff erent between survivors and nonsurvivors (79%, 95% CI  = 67 to 86 vs. 60%, 95% CI  = 32 to 72, P  =  0.08). However, LAC48 was predictive for ICU mortality (AUC 0.82; 95% CI = 0.64 to 1.0; P = 0.011). ROC curve analysis identifi ed the accuracy was highest by setting the lactate <2 mmol/l. Patients that did not normalize lactate (LAC <2 mmol/l) after 48 hours despite hemodynamic restoration had poorer outcome at 30 days, as is shown in the Kaplan–Meier curve (log- rank P = 0.006) (Figure 1). Introduction The shock index (SI; heart rate/systolic blood pressure) is a widely reported tool to identify acutely ill patients at risk for circulatory collapse in the emergency department (ED). Because old age, diabetes, essential hypertension, and β-/Ca2+ channel-blockers might reduce the compensatory increase in heart rate and mask blood pressure reductions in shock or pre-shock states, we hypothesized that these factors weaken the association between SI and mortality, reducing the utility of SI to identify patients at risk. Methods This was a cohort study from Odense University Hospital of all fi rst-time visits to the ED between 1995 and 2011 (n = 111,019). The outcome was 30-day mortality. We examined whether age ≥65 years, diabetes, essential hypertension, and use of β-/Ca2+ channel-blockers modifi ed the association between SI and mortality. The prognostic value of SI ≥1 was evaluated with diagnostic likelihood ratios. Conclusion Failing to normalize patient’s LAC in the fi rst 48 hours of VA ECMO assistance for CS is a predictor of ICU mortality. Targeting LAC level <2  mmol/l at 48  hours post ECMO institution might be a reasonable goal for these patients. Results We observed a 30-day mortality of 3%. Blood lactate normalization following venoarterial ECMO institution for refractory cardiogenic shockfi Methods A 5-month prospective nonrandomized observational study that included 50 adult patients with spontaneous respiration and hypotension in the emergency room and the wards of multiple causes except trauma. After measurement of the SAP and verifi cation of hypotension (SAP ≤90  mmHg), a mask-style ITD was added. Hemodynamic parameters were evaluated every 1  minute and for M Bottiroli, D Decaria, T Maraffi , S Nonini, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P146 (doi: 10.1186/cc14226) Introduction Venoarterial (VA) ECMO is used to support patients with refractory cardiogenic shock (CS). Elevated lactate level (>2  mmol/l) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S51 Figure 1 (abstract P146). an inotropic score as a predictor of mortality and morbidity among children who diagnosed septic shock. Figure 1 (abstract P146) Methods A multicenter retrospective chart review was performed in two pediatric ICUs. A total of 93 children with septic shock were recruited. Hourly doses of following inotropes were recorded for the fi rst 48 hours after admission: dopamine, dobutamine, adrenaline and noradrenaline. The inotrope score for every hour, minimum, maximum and mean values for the fi rst 24 hours, and subsequent 24 hours were calculated. In our analysis, the inotrope score was calculated as described by Wernovsky. We expanded this formula to include norephinephrine as follows: Wernovsky Inotrope Score = dopamine dose (μg/kg/minute) + dobutamine dose (μg/kg/minute) + 100 × epinephrine dose (μg/kg/ minute). Our adjusted inotrope score = Wernovsky Inotrope Score + 100 × norepinephrine dose (μg/kg/minute).f p p μg g Results Forty-two of 93 patients died. Age and sex were not diff erent between survivors and nonsurvivors. Signifi cantly higher mean and maximum inotropic score for the fi rst 24 hours and fi rst 48 hours were found in nonsurvivors than those of survivors (P <0.05). Using 15 as a cutoff point for predicting mortality, the sensitivity and specifi city were 69.76% and 50.98% respectively. The association between Prism scores and minimum, mean and maximum inotrope scores were statistically signifi cant for 0 to 24 hours, 25 to 48 hours and 0 to 48 hours. Mean 0 to 24 hours and maximum 0 to 48 hours inotrope scores were weakly associated with prolonged ICU stay (P = 0.047, P = 0.042 respectively). There were no signifi cant relationships between inotrope scores and receiving mechanical ventilation.i Figure 1 (abstract P146). Is the shock index a universal predictor in the emergency department? A cohort study With SI <0.7 as reference, a SI of 0.7 to 1 was associated with an adjusted OR of 2.9 (CI 2.7 to 3.2) for 30-day mortality while the adjusted OR for SI ≥1 was 10.3 (CI 9.2 to 11.5). ORs for SI ≥1 were reduced (but still signifi cant) in patients who were older, hypertensive, or on β-/Ca2+ channel-blockers, whereas diabetes had no eff ect. The OR for SI ≥1 in patients ≥65 years was 8.2 (CI 7.2 to 9.4) compared with 18.9 (CI 15.6 to 23.0) in younger patients. β-/Ca2+ channel-blocked patients had an OR of 6.4 (CI 4.9 to 8.3) versus 12.3 (CI 11.0 to 13.8) in nonusers, and the OR for hypertensive patients was 8.0 (CI 6.6 to 9.4) versus 12.9 (CI 11.1 to 14.9) in nonhypertensive patients. The OR for SI ≥1 of 9.3 (CI 6.7 to 12.9) in diabetics did not diff er from the OR of 10.8 (CI 9.6 to 12.0) in nondiabetic patients. A SI of 0.7 to 1 was associated with ORs signifi cantly greater than 1 (range: 2.2 to 3.1) with no evident diff erences within the subgroups. A SI measurement ≥1 was associated with lower positive likelihood ratios in patients ≥65 years, with hypertension, diabetes or using β-/Ca2+ channel-blockers (range 4.9 to 6.5) compared with patients not exposed to these factors (range 7.6 to 11.6). Blood lactate normalization following venoarterial ECMO institution for refractory cardiogenic shockfi is an indicator of end-organ hypoperfusion. We hypothesize that the lactate (LAC) normalization had prognostic value in this cohort of patients. Methods We performed a retrospective observational study on patients admitted to the ICU for refractory CS from January 2010 to November 2014. Patients with postcardiotomy and/or post-transplant CS were excluded. Demographics, clinical, hemodynamic and biochemical values were collected. LAC was measured on arterial blood before ECMO institution (LAC0) and after 48 hours (LAC48). Lactate clearance was calculated as follows: (LAC0  – LAC48) / LAC0  ×  100. Data were analyzed by comparative statistic; sensibility and specifi city were tested with ROC. Conclusion The mean and maximum inotropic scores in the fi rst 24 hours and 0 to 48 hours are an independent predictor of mortality in critically ill children with septic shock. P148 Is the shock index a universal predictor in the emergency department? A cohort study AK Kristensen1, JG Holler1, J Hallas2, A Lassen1, N Shapiro3 1Odense University Hospital, Odense, Denmark; 2University of Southern Denmark, Odense, Denmark; 3Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P148 (doi: 10.1186/cc14228) P149 Risk factors for severe vasodilatory shock after cardiac surgery J Almeida, F Galas, J Fukushima, E Almeida, A Gerent, E Osawa, C Park, R Nakamura, A Leme, M Sundin, R Kalil Filho, F Jatene, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P149 (doi: 10.1186/cc14229) Introduction Vasodilatory shock is a well-known complication in patients who undergo cardiac surgery with cardiopulmonary bypass (CPB) and its occurrence is associated with higher morbidity and mortality. Despite that, clinical characteristics of vasoplegic shock and its spectrum of severity are poorly described. The aim of this study was to compare patients who developed mild to moderate vasoplegic shock with patients who developed a severe form and to identify predictive factors for the severe form of vasoplegic shock. Results Heterogeneity of LS eff ects was registered in a number of values. The most signifi cant positive eff ect which allowed one to evaluate myocardial reserve was demonstrated by decrease of PPA (93.5% of patients) and increase of EF (77.6% of patients). The most signifi cant changes were also noted in decrease of TrI, PH and MI (in 53.2%, 36.6% and 36% of patients, respectively). In 69.2% of patients with noncoronarogenic dilated cardiomyopathy the eff ect of LS exposure was marked. In the majority of patients with ischemic cardiomyopathy the eff ect was moderate. In case of the absence of LS-positive eff ect, perioperative use of mechanical circulatory support was considered. Conclusion Preoperative use of LS allows one to evaluate myocardial reserves and prepare high-risk patients with CHF for surgery. Our fi ndings may serve as one of the additional criteria to choose the type of surgical treatment: reconstructive surgery (with or without perioperative mechanical circulatory support) or heart transplantation. Methods We performed an observational study in 300 patients who underwent cardiac surgery with CPB and presented within the fi rst 24  hours after surgery with refractory hypotension and used a vasopressor agent. Severe vasoplegic shock was defi ned as a requirement of norepinephrine higher than 1  μg/kg/minute or the use of two or more vasopressors. Baseline characteristics, laboratorial, clinical and intraoperative data, such as amount of fl uids, bleeding, blood transfusion, inotropes and length of CPB were collected at ICU admission. Logistic regression was performed using severe vasodilatory shock as the outcome. Results There were 46 (15%) patients who develop the severe form of vasodilatory shock within 24  hours after cardiac surgery. Reference Reference 1. Jochberger S, et al. Intensive Care Med. 2009;35:489-97. 1. Jochberger S, et al. Intensive Care Med. 2009;35:489-97. P149 In a univariate analysis, patients with the severe form were more likely to be older, to receive more blood transfusion and inotropic agents, to have higher levels of serum lactate, lower hemoglobin concentration and lower SvO2 at the end of the procedure, lower cardiac output index, higher heart rate and higher levels of reactive C protein at ICU admission. These patients also experienced more postoperative organ dysfunction, had a longer length of ICU stay and higher mortality. There were no diff erences between patients regarding amount of fl uids and length of CPB. In a multivariate analysis we identify age (OR = 1.04, 95% CI = 1.01 to 1.08, P = 0.016), intraoperative use of epinephrine (OR = 5.49, 95% CI = 2.42 to 12.43, P <0.001), higher serum lactate at the end of the procedure (OR = 1.04, 95% CI = 1.01 to 1.06, P = 0.001) and intraoperative blood transfusion (OR = 5.06, 95% CI = 2.19 to 11.69, P <0.001). P151 Levosimendan versus dobutamine in cardiac surgery MD Delgado-Amaya, EC Curiel-Balsera, CJ Joya-Montosa Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P151 (doi: 10.1186/cc14231) Introduction Early studies suggested a signifi cant increase in survival in patients treated with levosimendan compared with dobutamine or placebo (LIDO, RUSSLAN and CASINO trials). However, two subsequent studies (SURVIVE and REVIVE II) have not confi rmed these fi ndings. Methods A prospective observational study of all patients undergoing cardiac surgery at Malaga’s Regional Hospital from March 2009 to March 2013. We analyzed patients who used levosimendan compared with those that used dobutamine in the fi rst  hours after cardiac surgery, discarding patients in which neither of these two drugs were used or surgical cases that arrived at the ICU with both inotropics. We analyzed demographic variables as well as clinical complications in the ICU and overall perioperative mortality of patients. We performed a second analysis using the propensity score, obtaining the probability of patients being treated with either drug, pairing each patient who received levosimendan with its nearest neighbor receiving dobutamine. Results We collected 875 patients: 331 received one of the two drugs, 50 received both drugs and 494 did not receive any drug. ICU mortality was 7.2% (levosimendan group) and 12.5% (dobutamine group), P = 0.1. After adjustment for severity and type of surgery, the use of levosimendan in the postoperative period was not a protective factor for ICU mortality (P = 0.18, OR = 0.5, 95% CI = 0.18 to 1.3). In the matched sample, mortality was 7.4% (levosimendan group) and 5.9% (dobutamine), P = 0.73. After logistic regression adjusted for severity, measured with EuroSCORE and type of surgery, levosimendan was not a protective factor for ICU mortality (P = 0.8, OR = 1.2, 95% CI = 0.26 to 5.45).f Conclusion This study demonstrated that older patients, intraoperative blood transfusion and utilization of epinephrine were independently associated with a more severe form of vasodilatory shock after cardiac surgery with CPB. Also, we identifi ed that a higher lactate at the end of the procedure was an independent predictive factor for this severe form of shock. Is an inotrope score a predictor of mortality and morbidity in children with septic shock? E Sevketoglu1, A Anil2, S Kazanci1, O Yesilbas1, M Akyol1, S Bayraktar3, N Aksu4, S Hatipoglu1, M Karabocuoglu5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Bakýrkoy, Turkey; 2Izmir Katip Celebi University Medicine Faculty, Izmir, Turkey; 3Haseki Research and Training Hospital, Istanbul, Turkey; 4Tepecik Research and Training Hospital, Izmir, Turkey; 5Sisli Memorial Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P147 (doi: 10.1186/cc14227) E Sevketoglu1, A Anil2, S Kazanci1, O Yesilbas1, M Akyol1, S Bayraktar3, N Aksu4, S Hatipoglu1, M Karabocuoglu5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Bakýrkoy, Turkey; 2Izmir Katip Celebi University Medicine Faculty, Izmir, Turkey; 3Haseki Research and Training Hospital, Istanbul, Turkey; 4Tepecik Research and Training Hospital, Izmir, Turkey; 5Sisli Memorial Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P147 (doi: 10.1186/cc14227) Introduction Inotropes and vasoactive drugs in septic shock are commonly used to maintain cardiac output, tissue perfusion and oxygenation. We undertook this study with the purpose of evaluating S52 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion SI is independently associated with 30-day mortality in a broad population of ED patients. Old age, hypertension and β-/Ca2+ channel-blockers weaken this association, but the association remains prognostic. SI ≥1 suggests substantial risk of 30-day mortality in all ED patients. for the surgical intervention (2 to 4  days before surgery). In total, 44.9% of the patients had CHF caused by noncoronarogenic dilated cardiomyopathy and 55.1% by ischemic cardiomyopathy. Indication for LS therapy was left ventricular ejection fraction (EF) <35% (28 ± 6%). Seventy percent of patients had mitral insuffi ciency (MI), grades II to IV, 63% tricuspid insuffi ciency (TrI), grades II to III, 84% pulmonary hypertension (PH), 47% arterial hypertension, grades II to III, and 27% of the patients had left ventricular aneurysm. Mean level of BNP was 1,803 ± 124 pg/ml. LS was administered as i.v. infusion in doses of 0.025 to 0.1 μg/kg/minute without previous bolus injection. Mean duration of infusion was 27.5 ± 5.3 hours. After infusion all patients underwent control assessment of values. All patients were operated: 25 (23.3%) underwent reverse cardiac remodeling, 63 (58.9%) myocardium revascularization (MR) with mitral or aortic valve replacement, 17 (15.9%) MR and/or resection of left ventricular aneurism and two (1.9%) heart transplantation. Levosimendan in the emergency department: a useful tool to improve cellular perfusion Introduction Levosimendan was originally developed for the treat ment of decompensated heart failure in situations for which conventional therapy is not suffi cient. It is an eff ective calcium- sensitising drug with vasodilatory and inotropic eff ects and improves cardiac contractility. Trials have shown positive outcome benefi t with the use of levosimendan [1]. We reviewed the usage levosimendan at our institution and outcome of these patients. Introduction Levosimendan is an inotropic seldom used in emergency departments (EDs). It has shown improved mortality in patients with shock [1] with few adverse eff ects [2]. This work denotes the experience of levosimendan use in the ED of Hospital Universitario Mayor between 2012 and 2014. p Methods We reviewed the use of levosimendan at Harefi eld from January 2013 through December 2013. Patient demographics, logistic EuroSCORE (Figure  1), diagnosis, surgical or intervention details, inotropic support, dosage and duration of levosimendan use, length of stay in the ICU, cost (Table 1) and patient outcome were collected. Results Levosimendan was used in 30 patients, 23 (77%) male and seven (23%) female. Median age was 69 (59 to 72.8). Levosimendan was used post cardiac surgery, post angioplasty and in patients with ventricular assist devices (VAD) and extracorporeal membrane oxygenator (ECMO). Most of the patients received a standard regimen of 12.5 mg administered at a dose of 0.1 μg/kg/minute for 24 hours. Concurrent noradrenaline was used in most of the patients ranging from 0.02 to 0.2 μg/kg/minute. The median length of stay in the ICU was nine (6 to 14.5) days for survivors and 23.5 (7.5 to 36) days in nonsurvivors. Sixteen patients (55%) survived and were discharged from the hospital. Methods We reviewed the use of levosimendan at Harefi eld from January 2013 through December 2013. Patient demographics, logistic EuroSCORE (Figure  1), diagnosis, surgical or intervention details, inotropic support, dosage and duration of levosimendan use, length of stay in the ICU, cost (Table 1) and patient outcome were collected. Methods We present a retrospective study which analyzes the eff ect, after 24  hours, of levosimendan administration on perfusion parameters of 166 ED patients. Patients had to have shock diagnosis of any cause. Diff erences between the initial and fi nal mean value of the following parameters were evaluated: lactate, central venous oxygen saturation (ScvO2) and venoarterial diff erence of CO2 (DvaCO2). Data were stratifi ed according to levosimendan categories (initial or rescue). Reference Reference Preoperative treatment with levosimendan helps to evaluate myocardial reserves in cardiosurgical patients with chronic heart failure Conclusion In our environment, we have observed diff erences in the use of levosimendan compared with dobutamine (higher rate of men undergoing CABG, diabetes and worse EF). After homogenizing the sample of patients by propensity score, an eff ect on mortality is discarded and we observed a signifi cant need for use of norepinephrine and a nonsignifi cant trend for prolonged mechanical ventilation and renal failure requiring renal replacement therapy, both probably related with the greatest need for vasopressors observed. Introduction The aim of the study was to assess the possibility of preoperative levosimendan (LS) administration in myocardial reserve evaluation and choice of the method of surgical treatment in patients with chronic heart failure (CHF). Introduction The aim of the study was to assess the possibility of preoperative levosimendan (LS) administration in myocardial reserve evaluation and choice of the method of surgical treatment in patients with chronic heart failure (CHF). Methods LS was used in 107 (female and male) patients (mean age 53  ±  3  years) as a component of CHF therapy to prepare them S53 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P152 Reference 1. Follath F, et al. Lancet. 2002;360:196-202. Levosimendan in the emergency department: a useful tool to improve cellular perfusion In addition, association between diff erent variables with mortality was sought. Diff erences were considered statistically signifi cant at probability levels below 0.05.f y , ( ) p Results Levosimendan was used in 30 patients, 23 (77%) male and seven (23%) female. Median age was 69 (59 to 72.8). Levosimendan was used post cardiac surgery, post angioplasty and in patients with ventricular assist devices (VAD) and extracorporeal membrane oxygenator (ECMO). Most of the patients received a standard regimen of 12.5 mg administered at a dose of 0.1 μg/kg/minute for 24 hours. Concurrent noradrenaline was used in most of the patients ranging from 0.02 to 0.2 μg/kg/minute. The median length of stay in the ICU was nine (6 to 14.5) days for survivors and 23.5 (7.5 to 36) days in nonsurvivors. Sixteen patients (55%) survived and were discharged from the hospital. p y Results There were no diff erences in APACHE II values between patients who received levosimendan as initial therapy from those who received it as a rescue measure. A total of 41 patients fulfi lled lactate normalization requirements (lactate <2.0 or clearance >50%) (Table 1). Forty-four patients reached normal values of SvcO2 and 37 patients of DavCO2 after levosimendan initiation. There were no associations between the normalization of lactate, SvO2 and DvaCO2 and diff erent types of shock. Twenty-nine patients who received initial therapy with levosimendan normalized their lactate values and 12 who received it as a rescue therapy (P <0.05). Sixty-three patients developed hypotension, and none had adverse eff ects requiring discontinuation of the drug. Hospital mortality was 47.7%. Variables associated with mortality in the study group were lactate value at admission (OR = 1.3, 95% CI = 1.0 to 1.7), the use of vasopressin after start levosimendan (OR = 7.5, 95% CI = 1.9 to 28.6) and the use of norepinephrine before starting (OR = 10.8, 95% CI = 1.9 to 60.7). Figure 1 (abstract P152). Logistic EuroSCORE of all patients. Table 1 (abstract P153). Perfusion variables before and after levosimendan Variable Initial Final Lactate (mmol/l) 3.3 (0.5 to 18) 2.38 (0.4 to 17)* SvcO2 (%) 56.3 (23 to 85) 65.2 (37 to 91)* DvaCO2 (mmHg) 8.2 (–16 to 26) 7.3 (–2 to 21) *P <0.01. McNemar test. Table 1 (abstract P153). Perfusion variables before and after levosimendan Variable Initial Final Figure 1 (abstract P152). Logistic EuroSCORE of all patients. Figure 1 (abstract P152). Levosimendan in the emergency department: a useful tool to improve cellular perfusion Logistic EuroSCORE of all patients. Conclusion Levosimendan use in the ED, as initial or rescue therapy, normalizes lactate values and improves the SvcO2 after 24  hours, without an increase in adverse eff ects. f Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Levosimendan Adrenaline Milrinone Maximum dose (μg/kg/minute) 0.2 1 0.7 Cost per vial (£) 894 2.30 16 Cost per 24 hours (£) 894 7 112 Conclusion We have successfully used this drug in high-risk patients during the perioperative period with good results without major complications. Levosimendan seems to reduce catecholamine requirement, the need for mechanical circulatory support, and the duration of critical care, which can justify the cost of this drug. It can be also useful in weaning patients from short-term VAD and ECMO. Larger studies are required in this area. Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Levosimendan Adrenaline Milrinone P153 Levosimendan in the emergency department: a useful tool to improve cellular perfusion P153 Levosimendan in the emergency department: a useful tool to improve cellular perfusion G Devia, J Torres, S Lopez Hospital Universitario Mayor, Bogotá, Colombia Critical Care 2015, 19(Suppl 1):P153 (doi: 10.1186/cc14233) Levosimendan: use, cost-eff ectiveness and outcome in a tertiary cardiothoracic centre Levosimendan: use, cost-eff ectiveness and outcome in a tertiary cardiothoracic centre A Ranjan, N Bhudia, I McGovern, C Walker, L Kuppurao Royal Brompton & Harefi eld NHS Foundation Trust, Harefi eld, London, UK Critical Care 2015, 19(Suppl 1):P152 (doi: 10.1186/cc14232) A Ranjan, N Bhudia, I McGovern, C Walker, L Kuppurao Royal Brompton & Harefi eld NHS Foundation Trust, Harefi eld, London, UK Critical Care 2015, 19(Suppl 1):P152 (doi: 10.1186/cc14232) P153 References 1. Landoni G, et al. Minerva Anestesiol. 2010.76:276-86. 2. Mebazaa A, et al. JAMA. 2007.297:1883–91. Conclusion We have successfully used this drug in high-risk patients during the perioperative period with good results without major complications. Levosimendan seems to reduce catecholamine requirement, the need for mechanical circulatory support, and the duration of critical care, which can justify the cost of this drug. It can be also useful in weaning patients from short-term VAD and ECMO. Larger studies are required in this area. Introduction The inotropic agents used in the ICU are dobutamine and milrinone; unfortunately, they have shown signifi cant side eff ects when used for myocardial depression during septic shock. Our objective is to describe Mn behavior in septic shock. S54 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 adaptive threshold for low amplitude QRS detection; QRS fi lter with an extended frequency range; management of VT alarm priorities. Conclusion A majority of the FPs was linked to user settings and patient conditions. The algorithm changes resulted in a clear reduction of ECG algorithm-related FP alarms, while the magnitude of the reduction depends strongly on the settings at the bedside. References 1. ECRI Institute. 2013;42:354-9. 2 Drew BJ et al PLoS ONE 2014 9: e110274 Methods We reviewed 72 clinical records of patients with diagnosis of septic and mixed shock who received Mn through January to December 2013. Demographic, hemodynamic, metabolic and gasometric data were recorded before and after Mn infusion. The PiCCO monitoring system was used. Data were expressed as mean and standard deviation. The statistical analysis used Student’s t test. P <0.05 was considered signifi cant. adaptive threshold for low amplitude QRS detection; QRS fi lter with an extended frequency range; management of VT alarm priorities. Conclusion A majority of the FPs was linked to user settings and patient conditions. The algorithm changes resulted in a clear reduction of ECG algorithm-related FP alarms, while the magnitude of the reduction depends strongly on the settings at the bedside. References 1. ECRI Institute. 2013;42:354-9. 2. Drew BJ, et al. PLoS ONE. 2014.9: e110274. 1. ECRI Institute. 2013;42:354-9. P155 Admission to the ICU with cerebrovascular event (CVE) and trauma was related to arrhythmia (P = 0.021, P = 0.032, respectively). There was a signifi cant relationship between VIP and sepsis presence (P  <0.001, P <0.001). Atrial fi brillation was the most frequent type of arrhythmia (53%), and the most frequently used medication was diltiazem (28.5%). The patients who had arrhythmias had a longer ICU stay (P = 0.021). The mortality rate for all patients was 48.1%. There was no statistically signifi cant relationship between arrhythmia and mortality (P >0.05). False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 y j 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA , , ; , , , Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) Introduction The ECRI Institute has identifi ed alarm fatigue as the number one health technology hazard [1]. A recent study on 461 ICU patients investigated 2,558,760 alarms [2]. In total, 88.8% of the annotated 12,671 arrhythmia alarms were false positives (FPs). It was concluded that the excessive number of alarms is ‘a complex interplay of inappropriate user settings, patient conditions, and algorithm defi ciencies’. Nine conditions causing alarms, four of which were ECG algorithm related, were reported [2]. In this study, we investigated a new algorithm in which improvements targeting three of the reported four ECG-related conditions were implemented: low amplitude QRS; wide QRS; nonactionable ventricular tachycardia (VT). gi p y y Conclusion Older age, higher APACHE II scores, trauma, CVE, VIP and sepsis increases arrhythmia risk in critically ill patients. Atrial fi brillation is most common and the most preferred treatment for all arrhythmias is diltiazem. References Results Seventy-two patients were studied: 36.5% were women, mean and SD of age, APACHE II, mechanical ventilation days and long ICU stay were: 67 ± 16 years, 18.5 ± 5.9 points, 14.9 ± 12.9 and 24.5 ± 21.9 days, respectively. A total 20.3% of the patients received dobutamine. Thirty- nine percent presented mixed shock. Global mortality was 23%. After Mn infusion: cardiac index (CI) increased: 3.1 ± 1 to 3.3 ± 1.1, cardiac rate increased: 82.4 ± 14.4 to 88.3 ± 18 and ScvO2 increased: 71.1 ± 10.3 to 76.1 ± 7.3 (P <0.05). PaCO2 arteriovenous diff erence and lactate were reduced: 7.36 ± 3.3 to 6.04 ± 3.6 and 18.7 ± 14.9 to 13.1 ± 9.1 (P <0.05). CVP, MAP, RVSI, VSTI, EVLWI and base excess showed no signifi cant diff erence. Mn initial average dose was 0.35  ±  0.13. NE before and after Mn infusion showed no signifi cant diff erence: 0.11 ± 0.20 versus 0.12 ± 0.22. References 1. Kanji S, et al. Crit Care Med. 2008;36:1620-4 1. Kanji S, et al. Crit Care Med. 2008;36:1620-4. j , ; 2. Annane D, et al. Am J Respir Crit Care Med. 2008;178:20-5. 2. Annane D, et al. Am J Respir Crit Care Med. 2008;178:20-5. P156 Arrhythmia incidence and risk factors in critically ill patients G Tasdemir, N Kelebek Girgin, A Aydin Kaderli, E Cizmeci, R Iscimen, F Kahveci, A Aydinlar Uludag University Medical Faculty, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P156 (doi: 10.1186/cc14236) Introduction Cardiac arrhythmias may be observed at any time during the ICU stay. The prognosis may suff er due to these arrhythmias. In our study, we aimed to evaluate incidence and risk factors of arrhythmias occurring in patients in the ICU. Introduction Cardiac arrhythmias may be observed at any time during the ICU stay. The prognosis may suff er due to these arrhythmias. In our study, we aimed to evaluate incidence and risk factors of arrhythmias occurring in patients in the ICU. Conclusion Mn optimizes cardiovascular performance in septic shock and mixed shock, without aff ecting hemodynamic variables and global tissue perfusion. In addition, we observed that the IC, ScvO2, PaCO2 arteriovenous diff erence and lactate are related variables. References Methods Patients treated in the ICU were included in the study if they fulfi lled the following: age >18, no cardiac valvular disease, no cardiac surgery in the recent 6-month period, no history of myocardial infarction (MI), need for mechanical ventilation, and one or more organ failure. Demographic, hemodynamic and laboratory parameters, APACHE II score, presence of sepsis, acute renal failure, MI, and VIP during the ICU stay were recorded. Therapies used for arrhythmia and response to therapies were also recorded. 1. Holmes CL, et al. Vasoactive drugs for vasodilatatory shcok in ICU. Curr Opin Crit Care. 2009;15:398-402. 2. Jentzer JC, et al. Pharmacotherapy update on the use of vasopressors and inotropes in the intensive care unit. J Cardiovasc Pharmacol Ther [Epub ahead of print].l p p Results Two hundred and fourteen patients were included in the study. Twenty-six percent (n = 56) of patients had arrhythmias. Incidence was higher in females (P = 0.045). Average age of arrhythmic patients was 69 (19 to 86), and they were older than nonarrhythmic patients (P <0.001). APACHE II scores were higher in arrhythmic patients (P = 0.001). 3. Deep A, et al. Evolution of haemodynamics and outcome of fl uid-refractory septic shock in children. Intensive Care Med. 2013;39:1602-9. 3. Deep A, et al. Evolution of haemodynamics and outcome of fl uid-refractory septic shock in children. Intensive Care Med. 2013;39:1602-9. High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery N De Mey, I Brandt, C Van Mieghem, K De Decker, G Cammu, L Foubert OLV AALST, Aalst, Belgium Critical Care 2015, 19(Suppl 1):P158 (doi: 10.1186/cc14238) N De Mey, I Brandt, C Van Mieghem, K De Decker, G Cammu, L Foubert OLV AALST, Aalst, Belgium Introduction Hs-cTn is the new standard cardiac biomarker for the diagnosis of myocardial necrosis. We conducted a prospective study to compare the course and values of Hs-cTn and CK-MB after CABG and OPCAB. We also evaluated the relationship between values >10 × 99th percentile URL of CK-MB and Hs-cTn as a possible marker for perioperative myocardial infarction. g g Results The cTnI concentrations measured in the control group ranged from 0.4 to 17.2, mean 2.1 (95% CI: 1.6 to 2.6) ng/l, without age dependency. Men revealed higher levels than women, means (IQR) were 2.8 (1.2 to 2.6) and 1.1 (0.7 to 1.3) ng/l. The CLSI nonparametric method revealed a 99th percentile value of 16 ng/l. The quantifi cation of cTnI above the LoD (1.0 ng/l) and below the 99th percentile was possible in 79 of 120 individuals. The imprecision profi le according to NCCLS revealed 20%, 10% and 5% CVs at concentrations of 2, 3 and at 20 ng/l, respectively. The discharge diagnosis was NSTEMI in 71 patients. The cTnI median values at 0, 3 and 6 hours were 46, 166 and 399 ng/l, respectively. AUC values at 0, 3 and 6  hours were 0.923, 0.964 and 0.969 for hs-cTnT and 0.919, 0.962 and 0.958 for cTnI, respectively. cTnI revealed AUC values of absolute changes from admission to 3 hours and from admission to 6 hours were 0.920 and 0.931, respectively.i Methods All adult patients undergoing cardiac surgery between February and August 2014 were included. Exclusion criteria were urgent surgery, GFR <60  ml/minute/1.73 m2, CK-MB >4  μg/l and/or Hs-cTn >14 ng/l at baseline (BL). Hs-cTn and CK-MB were measured before induction (BL), upon arrival in the ICU and at fi xed times after arrival. Patients with perioperative AMI as defi ned by the third universal defi nition of AMI were excluded for post hoc analysis [1]. Results Of the 93 patients admissible for inclusion, 40 in the CABG and 14 in the OPCAB group met all inclusion criteria in this preliminary dataset. High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery CK-MB values are higher from ICU arrival up to T24 versus baseline in both CABG and OPCAB (P <0.0001) with a peak at T3. For Hs-cTn, ICU up to T48 values are higher (P <0.01) in CABG with a peak Conclusion The PATHFAST cTnI demonstrated complete fulfi llment of the analytical criteria for high-sensitive cTn assays: The imprecision (CV) at the manufacturer-recommended 99th percentile value was 5%. The quantifi cation of cTnI in was possible in 65.8% of healthy individuals. The examination of the diagnostic characteristics revealed complete concordance with the hscTnT assay for detection of NSTEMI and for assessment of absolute changes of cTn values (rise and/or fall) in NSTEMI patients. PATHFAST cTnI showed highly sensitive detection of NSTEMI with increasing sensitivity at admission and after 3 to 6 hours, not going along with decreased specifi city. The PATHFAST cTnI might be useful at the point-of-care setting for early rule-in and rule-out diagnosis of NSTEMI. Figure 1 (abstract P158). Figure 1 (abstract P158). Analytical and diagnostic characteristics of the high-sensitivity PATHFAST troponin I assay p y E Spanuth1, R Thomae2, E Giannitsis3 E Spanuth1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P159 (doi: 10.1186/cc14239) E Spanuth1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P159 (doi: 10.1186/cc14239) Conclusion Among ADHF patients, the coupling of pulmonary hypertension and RVD carries an incremental risk, having a portentous impact on the survival rate. Introduction The POC PATHFAST cTnI assay (Mitsubishi Medience, Japan) has shown promising analytical validity. We thought to evaluate whether the assay could be classifi ed ‘highly-sensitive’. P158 yi g y Methods cTnI was determined using PATHFAST in 120 healthy individuals (60 men and 59 women, 21 to 69  years old, median 42  years). Cardiac disorders were excluded by cardiac magnetic resonance imaging. The diagnostic characteristics were investigated by determination of cTnI and cTnT (Roche hs-cTnT) in 181 patients admitted to the chest pain unit at presentation, and 3 and 6 hours later. The results were related to the discharge diagnoses. Pulmonary hypertension, right ventricular dysfunction and acute heart failure: a portentous consortium y Methods The false alarm rate of the new algorithm (GE Carescape, 2012) was compared with that of the algorithm evaluated in the study (GE Solar, 2003) [2] on the collected ECG waveform data. User settings such as QRS detection sensitivity (high/normal) were not available. Therefore, normal sensitivity was assumed for both versions. With the old algorithm, 10 patients with low QRS amplitudes gave a signifi cantly higher number of FPs than were reported [2]. For those patients, both sensitivity modes were tested with the old algorithm. Sixty-six percent of patients with a pacemaker did not have the pacemaker mode selected [2]. Outlier patients in which false alarms were due to user settings (20 patients with a pacemaker) or patient condition (four patients with a bundle branch block) rather than algorithm defi ciency were excluded. HD Michalopoulou, HM Michalopoulou, P Stamatis, E Kostetsos, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P157 (doi: 10.1186/cc14237) Introduction Pulmonary hypertension and right ventricular dysfunc- tion (RVD) are frequently encountered in patients with acute heart failure. We sought a better understanding of the coupling between RVD and pulmonary hypertension in the setting of acute decompensated heart failure (ADHF) as it might improve the prognostic stratifi cation and infl uence the survival rates. l Methods Echocardiography was performed in 329 patients with ADHF and right ventricular function was assessed by measuring the right ventricular fractional area, and a right ventricular ejection fraction (RVEF) <35% was taken as the cutoff value for RV systolic dysfunction. The systolic pulmonary pressure (PASP) was calculated from the tricuspid regurgitation signal applying the modifi ed Bernoulli equation, Results Improved algorithm resulted in 66% reduction of FP alarms. When using the high-sensitivity mode for the 10 patients with low QRS, FP reduction was 18%. No compromises regarding detection of true events were found. The 24 outlier patients contributed to 81.3% of FP alarms. The algorithm changes responsible for the reduced FPs were: S55 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 at T6, and from T3 to T48 in OPCAB (P <0.05) versus baseline (Figure 1). In CABG patients CK-MB levels are higher versus OPCAB from ICU up to T12 (P <0.03), and from ICU to T48 for Hs-cTn levels (P <0.02). Pulmonary hypertension, right ventricular dysfunction and acute heart failure: a portentous consortium In 39 CABG patients (97.5%) and 10 OPCAB patients (71.4%) all individual Hs-cTn values are above 140 ng/l (= 10 × 99th percentile of URL). at T6, and from T3 to T48 in OPCAB (P <0.05) versus baseline (Figure 1). In CABG patients CK-MB levels are higher versus OPCAB from ICU up to T12 (P <0.03), and from ICU to T48 for Hs-cTn levels (P <0.02). In 39 CABG patients (97.5%) and 10 OPCAB patients (71.4%) all individual Hs-cTn values are above 140 ng/l (= 10 × 99th percentile of URL).i and pulmonary hypertension was considered as PASP >35  mmHg. Based on the values of PASP and RVEF the study group was classifi ed into four subgroups: group 1, normal PASP/preserved RVEF; group 2, high PASP/preserved RVEF; group 3, normal PASP/ low RVEF; group 4, high PASP/low RVEF. The primary endpoint was all-cause mortality. The median follow-up was 18 months. Survival analysis was performed according to the Cox regression method, adjusted for age, gender, LV function, estimated glomerular fi ltration rate, troponin I, hemoglobin, serum sodium and BNP levels. Conclusion Both CK-MB and Hs-cTn levels increase signifi cantly after cardiac surgery. Postoperative Hs-cTn levels exceed the 10  ×  99th percentile of URL in nearly all CABG patients. Our data show an important discrepancy between the 10  ×  99th percentile for both biomarkers, and suggest that a diff erent defi nition for postoperative AMI may be needed when Hs-cTn is used. Results Pulmonary hypertension was found in 78% of the patients (median PASP: 53 mmHg). As compared with the patients with normal PASP the patients with pulmonary hypertension were more likely to be in New York Heart Association functional class (NYHA) III or IV (86% vs. 49%, P <0.001), had a lower RVEF (23 ± 9% vs. 32 ± 8%, P <0.001), and had signifi cantly higher BNP levels (280 ± 107 pg/ml vs. 540 ± 320 pg/ ml, P <0.001). In a Cox model, compared with patients with normal right ventricular function and without pulmonary hypertension (group 1), the adjusted hazard ratio for mortality was 3.1 (95% CI: 1.6 to 4.2, P <0.01) in group 2, 0.3 (95% CI: 0.2 to 1.9, P = 0.3) in group 3 and 4.2 (95% CI: 1.9 to 6.1, P <0.001) in group 4. y Reference y Reference 1. Thygesen et al. J Am Coll Cardiol. 2012;60:1581-98. P160 P160 Combining therapeutic hypothermia and primary coronary intervention in comatose survivors of ventricular fi brillation due to ST-elevation myocardial infarction R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) Combining therapeutic hypothermia and primary coronary intervention in comatose survivors of ventricular fi brillation due to ST-elevation myocardial infarction R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) y R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) Introduction Primary percutaneous coronary intervention (PPCI) is the preferred reperfusion strategy for ST-elevation acute myocardial infarction (STEMI). In comatose survivors of cardiac arrest, mild induced hypothermia (MIH) improves neurological recovery. Figure 1 (abstract P158). S56 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Of the 3,835 nontraumatic patients treated in the ED, 3,196 were adults. In this adult population, 500 ECGs were performed in patients whose symptoms suggest ACS. The median age was 62.3 years and the sex ratio was 1.16. Clinical presentation was chest pain (31%), dyspnea (14%), palpitations (5%), disturbance of consciousness (3%) or others (47%). Fifty-six (11.2%) were diagnosed as ACS, including 20 ST-elevation myocardial infarction (STEMI), 28 non-STEMI and eight unstable angina. Of the 20 STEMI patients, eight (40%) and fi ve (25%) were diagnosed as STEMI complicated by right ventricular and posterior wall ischemia respectively, which means that these complications could have been missed by standard 12-lead ECG. Methods A total of 112 patients undergoing PPCI and MIH were compared with 32 comparable consecutive patients who underwent PPCI but no MIH. We hypothesized that combining both methods lead to better survival rate. MIH was induced (propofol, fentanyl, saline 4 ml/kg BW, 2°C) and maintained for 24 hours, targeting 32 to 34°C. Spontaneous rewarming was allowed (0.5°C).if g Results There were no signifi cant diff erences between the MIH and Control group in general characteristics, cardiac arrest circumstances and angiographic features. Except for decreases in heart rate during MIH, there was no diff erence between MIH and no MIH groups in arterial pressure, peak lactate (7.7 vs. 6.2 mmol/l; P = 0.36), need for vasopressors (57% vs. 41%; P = 0.09), aortic balloon counterpulsation (13% vs. 22%; P  =  0.19), repeat cardioversion/defi brillation (17% vs. 25%; P = 0.30). P160 There was lower incidence of inotropic use (36% vs. 59%; P = 0.01) and use of antiarrhythmics (11% vs. 53%; P = 0.002). There was no diff erence in FiO2 during mechanical ventilation and in renal function. See Table 1. y Conclusion Eighteen-lead ECG with synthesized right-sided and posterior precordial leads was an effi cient method to diagnose ACS in a Caucasian population within 10 minutes of ED arrival. It is particularly performant to detect right ventricular ischemia early, which can modify acute therapeutic strategy. References 1. Thygesen K, et al. Circulation. 2007;116:2634-53. 2. Katoh T, et al. J Nippon Med Sch. 2011;78:22-9. 3. Tamura A, et al. Am J Cardiol. 2014;114:1187-91. References 1. Thygesen K, et al. Circulation. 2007;116:2634-53. 2. Katoh T, et al. J Nippon Med Sch. 2011;78:22-9. 3. Tamura A, et al. Am J Cardiol. 2014;114:1187-91. Table 1 (abstract P160). Survival after 12 months MIH Control P CPC 1/2 50 (45%) 5 (15%) 0.002 CPC 3/4 0 0 NS Cardio 20 (18%) 6 (19%) 0.95 CNS 18 (16%) 17 (53%) 0.00001 Table 1 (abstract P160). Survival after 12 months Conclusion Hospital survival with CPC 1/2 was signifi cantly better in the MIH group (45% vs. 15%; P = 0.01). Our study clearly demonstrates that PPCI and MIH are feasible and may be combined safely in comatose survivors of ventricular fi brillation in STEMI setting. Such strategy improves survival with good neurological recovery. References Introduction After an acute myocardial infarction with ST-segment elevation (STEMI) treated with percutaneous coronary intervention (PCI), the left ventricle (LV) can undergo negative remodeling (R–). We aimed to investigate whether global longitudinal strain (SGL) of the left ventricle (LV) predicts remodeling. e e e ces 1. HACA Study Group. Mild therapeutic hypothermia to improve the neurological outcome after cardiac arrest. N Engl J Med. 2002;346:549-56. 2. Knafelj R, Radsel P, Ploj T, Noc M. Primary percutaneous coronary intervention and mild induced hypothermia in comatose survivors of ventricular fi brillation with ST-elevation acute myocardial infarction. Resuscitation. 2007;74:227-34. g g ; 2. Knafelj R, Radsel P, Ploj T, Noc M. Primary percutaneous coronary intervention and mild induced hypothermia in comatose survivors of ventricular fi brillation with ST-elevation acute myocardial infarction. Resuscitation. 2007;74:227-34. Methods Transthoracic echocardiography with speckle tracking imaging (TTE-STI) was performed 2 to 3 days after primary PCI and 6 months later in patients with diagnosis of STEMI. P160 LV R– criteria were: LVEF increase ≤5% and end-diastolic volume increase ≥15%. Logistic regression and ROC curve analysis was used for the statistical analysis. Results Eighty-three patients (56  ±  11  years) with STEMI at any LV localization and subjected to primary PCI were studied during 2012: LV P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) y y Methods A consecutive registry of patients seen in 2013 (January to October) in the ICU of a hospital without a HU. The total transfer time is considered from the call to the Emergency Coordination Center until arrival at the HU. In turn, this time is divided into activation time, arrival time of the relocation team, patient preparation time and transfer time. Methods A consecutive registry of patients seen in 2013 (January to October) in the ICU of a hospital without a HU. The total transfer time is considered from the call to the Emergency Coordination Center until arrival at the HU. In turn, this time is divided into activation time, arrival time of the relocation team, patient preparation time and transfer time. In the case of primary PCI, the door-to-balloon time was estimated by adding to the total transfer time the initial assessment and completion time of catheterization and balloon infl ation. The times are expressed in minutes, as the median and interquartile range. In the case of primary PCI, the door-to-balloon time was estimated by adding to the total transfer time the initial assessment and completion time of catheterization and balloon infl ation. The times are expressed in minutes, as the median and interquartile range. Introduction Diastolic dysfunction as evaluated by tissue Doppler imaging (TDI), particularly by E/E’ ratio (peak early diastolic transmitral velocity/peak early diastolic mitral annular velocity) and mitral annular E’-wave, is common and crucial in critical illness. Our prospective observational study assessed the prognostic signifi cance of TDI variables versus cardiac biomarkers, B-type natriuretic peptide (BNP), troponin-T (TnT) and investigated determinants of plasma BNP rise, in septic shock. g Results During 10 months of 2013, we treated 162 STEMI. Of these, 104 had evidence of reperfusion (64%). Primary PCI was performed in 24 patients (23%), of which 10 were transferred from the hospital to the HU. Fibrinolytic therapy was used in 62 patients (59%), of these 20 (32.2%) required rescue PCI. The transfer time for primary PCI was 0:39:44 (0:31:41 to 0:44:32) minutes. The transfer time for rescue PCI was 0:38:56 (0:37:25 to 0:51:29) minutes. Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) p y gi Results Mean  ±  SD APACHE II score was 21.22  ±  7.28, mean  ±  SD admission SOFA score was 10.25 ± 2.76. Hospital mortality was 55%. Nonsurvivors had increased E/E’ (15.56  ±  1.48; P  <<0.00001) and reduced E’ 6.32  ±  0.68 cm/second (P  <<0.00001) compared with survivors, who exhibited inverse correlations with an E/E’ signifi cantly lower (9.30 ± 2.88) and higher E’ (9.01 ± 0.85 cm/second). In contrast, BNP and TnT levels displayed remarkably lower statistical signifi cance in nonsurvivors (P  =  0.005, P  =  0.007 respectively). The ROC curves had an area under the curve of 0.98 for the E’, and 0.92 for the E/E’. Vasopressor management (noradrenaline dose) (P  =  0.0001), fl uid balance (P <0.001) and E/E’ (P = 0.00004) were independent predictors of plasma BNP concentration. Introduction The goal of the study was to analyse cerebral blood fl ow in pregnancy complicated by preeclampsia. Introduction The goal of the study was to analyse cerebral blood fl ow in pregnancy complicated by preeclampsia. Methods This was a prospective study. I group: 45 patients, 17 to 38 years (mean age 27.5 ± 5.3 years) with verifi ed diagnosis of severe preeclampsia; control group: 72 healthy women with normal pregnancy, third trimester, 19 to 34 years (mean age 24.5± 4.3 years). Exclusion criteria: potentially haemodynamically signifi cant stenosis; congestive heart disease; arrhythmia; large changes in haemorheology; diabetes mellitus; and craniospinal trauma and syncope. Study of cerebral fl ow was improved by the method of transcranial dopplerography (TCD). All patients underwent duplex scan of extracranial portions of brachiocephalic arteries and transcranial duplex scan in the area of middle cerebral artery (MCA) (segment M1). During duplex scan of brachiocephalic arteries lumen, the presence of extravasal causes for basic blood fl ow disturbances was estimated. We determined lumen of large basilar arteries and quantitative features of blood fl ow in MCA. Synthesized 18-lead electrocardiogram as routine myocardial ischemia detection in an emergency department: a preliminary evaluation in Europe Figure 1 (abstract P162). Clinique Notre-Dame de Grâce, Gosselies, Belgium q g Critical Care 2015, 19(Suppl 1):P161 (doi: 10.1186/cc14241) Introduction Standard 12-lead electrocardiogram (ECG) is, with biomarkers, the most accurate method in the diagnosis of acute coronary syndrome (ACS). However, posterior (V7-V8-V9) and right (V3R-V4R-V5R) derivations are not systematically performed due to the time-consuming procedure involved, despite major therapeutic implications (fl uid loading instead of nitrates use in right ventricular involvement) and published guidelines [1]. Recently, an 18-lead ECG system, standard 12-lead ECG and six additional synthesized leads (assessing posterior and right ventricular areas) in only one recording procedure has been developed. The reliability of this material (ECG 2550; Nihon Kohden Co. Ltd, Japan) was already validated in this indication in an Asian population [2,3]. Methods We conducted a prospective, observational study with patients admitted to our emergency department (ED), during a 6-month period. Requirement for ECG was guided by physician’s discretion according to patient’s history. All patients with chest pain, dyspnea, palpitations, disturbance of consciousness, malaise or abdominal complaint underwent synthesized 18-lead ECG within 10 minutes of ED arrival. The aim of the study was to evaluate the eff ectiveness of the synthesized 18-lead ECG as an ischemia triage tool in the ED, and particularly the ability to early detect a right ventricular involvement. Figure 1 (abstract P162). S57 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 R– patients (n = 35, 42%) and no LV R– patients (n = 48, 58%). Diabetes mellitus (41% vs. 19%; P <0.001) and TnI levels (1.2 ± 2.1 μg/l vs. 0.4 ±0.3 μg/l; P = 0.005) showed higher incidence in LV R– patients. SGL was –12.5 ± 5.6% in no LV R– patients and –6.5 ± 3.4 in LV R– patients. In the regression analysis just LV SGL and SL in left anterior descending territory remained signifi cant, OR: 1.85 (1.24 to 2.76) (P <0.001) and OR: 1.63 (1.15 to 2.31) (P <0.001), respectively. P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) The door-to-balloon time estimated for primary PCI was 80 minutes. Methods Twenty-seven mechanically ventilated adult patients admitted to our ICU were evaluated within 72 hours of evolving septic shock. Patients underwent two transthoracic echocardiographies within 72 hours of the onset of septic shock: shortly after diagnosis and 24 hours later (confi rmatory), alongside relevant measurements of cardiac biomarkers. Peak mitral infl ow E and A velocity waves were recorded using pulsed-wave Doppler at the mitral valve tips from the apical four-chamber view, peak early (E’) and late (A’) diastolic myocardial velocities were obtained by TDI at the septal mitral annulus in the apical four-chamber view. E/E’ was calculated. P ≤0.01 was reported as statistically signifi cant. Conclusion Times for interhospital transfer of patients with STEMI who had undergone urgent catheterization are within the range considered optimal. In the case of primary PCI, times are lower than the 90 to 120 minutes recommended practice guidelines. Synthesized 18-lead electrocardiogram as routine myocardial ischemia detection in an emergency department: a preliminary evaluation in Europe The analysis of ROC curves revealed that at the cutoff level of –12.46%, SGL identifi es LV R– with a sensibility of 81% and a specifi city of 86% (AUC = 0.88: 95% CI: 0.79 to 0.96; P <0.001) (Figure 1).i fl ow velocity (Vps), maximal end-diastolic velocity (Ved), time-adjusted maximal velocity (TAMX), resistance index (RI), pulsative index (PI), and systolic/diastolic ratio (S/D) were determined. Signifi cance of mean value diff erences were calculated using the STATISTICA 6.0 program with determination of Student’s t criteria with normal spread in the group. g p Results All haemodynamic values in the M1 segment of MCA in preeclamptic patients were decreased in comparison with the same values in healthy pregnant women with diff erent signifi cance: PI (mean 0.77 vs. 0.84, P <0.01); RI (mean 0.52 vs. 0.54, P <0.05); Vps (mean 90.22 vs. 104.74 cm/second, P <0.001); Ved (mean 43.25 vs. 48.53 cm/second, P <0.001); TAMX (mean 61.48 vs. 67.30 cm/second, P <0.01); and S/D (mean 2.02 vs. 2.06, P  <0.05). Found pathophysiological changes of cerebral haemodynamics were consistent with a dopplerographic pattern of diminished perfusion and are typical for vascular segments, which are located proximally to the zone of abnormally high haemodynamic resistance: prestenotic arterial segments, episodes of arterial hypertension and distal vasoconstriction. Conclusion SGL assessment in the fi rst days after primary PCI is useful in the prediction of LV R– independently of the myocardial infarction localization. P164 Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) Analysis of the interhospital transfer times in patients with ST-elevation acute coronary syndrome for undergoing urgent coronariography Analysis of the interhospital transfer times in patients with ST-elevation acute coronary syndrome for undergoing urgent coronariography FM Acosta Diaz, O Moreno, M Muñoz, R Fernandez, J Soto, M Colmenero San Cecilio Universitary Hospital, Granada, Spain Critical Care 2015, 19(Suppl 1):P163 (doi: 10.1186/cc14243) y Conclusion With TCD we obtained a possibility to determine and estimate changes in cerebrovascular fl ow in pregnant patients with severe preeclampsia. This enhances diagnostic possibilities of some serious pregnancy complications, and gives us deep understanding of some components of pathogenesis and increased treatment effi cacy. y p p Critical Care 2015, 19(Suppl 1):P163 (doi: 10.1186/cc14243) Introduction The aim was to analyze the related assistance times to transfer patients with ST-elevation acute coronary syndrome (STEMI) referred to another hospital with a hemodynamics unit (HU) for performing emergency catheterization (primary or rescue PCI). P165 P168 Results A total of 207 male and 144 female patients, APACHE II score 21  ±  7, 62  ±  14  years old, one to 126 TPTDs per patient. Diagnosis: cirrhosis/liver failure n = 112 patients (31.9%), sepsis 55 (15.7%), ARDS 46 (13.1%), GI aff ection 21 (6.0%), cardiogenic shock 19 (5.4%), various 98 (27.9%). Patients with liver failure were slightly younger than the other patients (58 ± 11 vs. 64 ± 15 years; P <0.001). All other baseline characteristics were comparable including APACHE II (20 ± 7 vs. 21 ± 8; NS), SAPS (39 ± 12 vs. 41 ± 14; NS), height (172 ± 9 vs. 170 ± 9 cm; NS) and weight (76 ± 20 vs. 73 ± 17 kg; NS). Among haemodynamic parameters, preload markers GEDVI (753  ±  168 vs. 790  ±  226  ml/m2; P  =  0.182) and CVP (14.4 ± 8.8 vs. 14.9 ± 7.1 mmHg; P = 0.250) were comparable. Despite comparable preload parameters, the following parameters were signifi cantly diff erent: patients with acute or chronic liver failure had signifi cantly higher cardiac index (4.3 ± 1.3 vs. 3.3 ± 1.3 l/minute/ m2; P <0.001), stroke volume index (50 ± 15 vs. 37 ± 15; P <0.001), pulse pressure (75 ± 19 vs. 65 ± 21 mmHg; P = 0.021) and cardiac power index (0.7 ± 0.24 vs. 0.60 ± 0.28 W/m2; P <0.001). By contrast, MAP (77 ± 15 vs. 80 ± 15 mmHg; P = 0.045), SVRI (1,305 ± 638 vs. 1,877 ± 898 dyn*s/ cm5*m2; P <0.001) and heart rate (84 ± 19 vs. 92 ± 22/minute; P <0.001) were signifi cantly lower in patients with liver failure. P168 Room temperature transpulmonary thermodilution (TPTD) with increased indicator 20 ml TPTD bolus compared with standard TPTD with 15 ml iced saline: a prospective observational study W Huber1, E Maendl1, A Beitz1, M Messer1, T Lahmer1, B Henschel1, S Rasch1, C Schnappauf1, RM Schmid1, ML Malbrain2 1Klinikum rechts der Isar, Technical University of Munich, Germany; 2Ziekenhuis Netwerk, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P168 (doi: 10.1186/cc14248) Introduction Use of ice-cold saline is assumed to provide best accuracy of TPTD to obtain the cardiac index (CI), global end-diastolic volume (GEDVI) and extravascular lung-water (EVLWI). However, room- temperature injectate might facilitate TPTD outside the ICU. Accidental intra-arterial injection: an under-reported preventable never event femoral TPTD (Pulsion Medical Systems, Germany). Results Fifteen female and 16 male patients, APACHE II score 21 ± 7. Mean values of CI (4.02 ± 0.98 vs. 3.96 ± 0.91 l/minute*m2; P <0.001), GEDVI (800 ± 166 vs. 796 ± 163 ml/m2; P = 0.011) and EVLWI (10.3 ± 3.7 vs. 9.7 ± 3.6 ml/kg; P <0.001) were slightly higher when measured at room temperature compared with cold saline. Mean bias and PE values were 0.06  ±  0.37 l/minute*m2 and 18.6% for CI, 4  ±  81  ml/m2 and 20.2% for GEDVI and 0.5 ± 1.1 ml/kg and 22.7% for EVLWI. Bias values in case of femoral compared with jugular indicator injection were not signifi cantly diff erent for CI (0.04 ± 0.41 vs. 0.11 ± 0.30 l/minute*m2; P = 0.161) and EVLWI (0.56 ± 1.19 vs. 0.42 ± 1.07 ml/kg; P = 0.492). Bias for GEDVI-room was signifi cantly lower for femoral CVC compared with jugular indicator injection (–6.0 ± 81.1 vs. 18.9 ± 78.3 ml/m2; P = 0.008). Conclusion Compared with previous data using 15  ml room- temperature injectate, our data with 20 ml room-temperature injectate in general provide acceptable bias and percentage error when compared with standard TPTD with 15 ml iced saline. This also applies for femoral CVC room-temperature TPTD which might also be related to a new PiCCO-2 algorithm correcting for femoral CVC site. Reference M Mariyaselvam, A Hutton, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P166 (doi: 10.1186/cc14246) Introduction Depending upon the medication administered, accidental administration of medication into the arterial line can cause devastating complications. This wrong-route injection is a never event in the UK but may be under-reported especially when occurring in the unconscious patient who may not notice associated pain temporally. Under-reporting may occur because resultant complications may be delayed a number of hours and the accountable healthcare worker may not recognise or choose not to report the error. In 2008 the UK National Patient Safety Agency (NPSA) reported only 76 incidents related to poor sampling technique but few wrong route arterial injections. Of these 21% suff ered moderate to severe harm [1]. The NPSA suggests that training and the use of clear labelling alongside red arterial tubing and standard red lock caps be used to prevent arterial sampling errors. Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka By the transtemporal approach in the MCA M1 segment, peak systolic Conclusion Diastolic dysfunction as evaluated by E/E’ and E’ constitutes a major independent predictor of outcome in septic shock, compared with cardiac biomarkers, suggesting that echocardiographic techniques assessing diastolic dysfunction in sepsis may replace cardiac biomarkers for mortality prediction. Fluid balance, vasopressor management and diastolic dysfunction are independent predictors of BNP elevation in septic shock. Our fi ndings should be confi rmed by an extended prospective study. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S58 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P166 the PiCCO-2 device with the latest algorithm correcting GEDVI for femoral TPTD (Pulsion Medical Systems, Germany). Accidental intra-arterial injection: an under-reported preventable never event Methods In 2014, we conducted a national postal survey of ICUs in the UK to attempt to determine the rate of accidental intra-arterial injections. The survey was sent to the clinical director of every ICU and they were asked whether they were aware of any unintentional arterial line injection having occurred in their hospital in the last 5 years. 1. Huber W, et al. J Crit Care. 2014;29:1133. 1. Huber W, et al. J Crit Care. 2014;29:1133. y Results Of the 56 ICUs that responded, 16 (28.5%) reported that they had personally seen an accidental injection into the arterial line. P169 Transpulmonary thermodilution-derived haemodynamics in patients with liver failure: a prospective study in 351 patients W Huber, A Breitling, B Henschel, S Mair, S Goetz, J Tschirdewahn, J Frank, A Beitz, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2015, 19(Suppl 1):P169 (doi: 10.1186/cc14249) Conclusion Despite the arterial line safety recommendations made by the NPSA in 2008, we demonstrate that intra-arterial injection is still a problem and that it remains under-reported. Our incidence is likely to be an underestimate as it relies on the recollections of a single individual in each institution. Medical errors can be mitigated by consideration of human factors and system engineering to improve patient safety. A focus on clinical awareness, colour coding and training may lead to improvements; however, institutions and clinical directors also bear a responsibility to prevent never events and a number of engineered solutions are now available such as needle-free non-injectable arterial sampling devices to protect the healthcare environment and make this error impossible [2,3]. Introduction Patients with acute or chronic liver failure are considered to have an altered pattern of haemodynamics. Nevertheless, there is a lack of studies systematically investigating haemodynamics in patients with liver failure. Therefore, it was the aim of this study to compare transpulmonary thermodilution (TPTD)-derived haemodynamics of 112 patients with acute or chronic liver failure with 239 patients without liver failure. Acknowledgement Funding from Eastern Academic Health Science Network, UK. References Acknowledgement Funding from Eastern Academic Health Science Network, UK. Methods We analyzed a prospectively maintained database including 6,016 TPTD measurements in 351 patients. To account for diff erent numbers of TPTDs in diff erent patients, comparison of fi rst measurements of patients with and without liver failure was the primary endpoint. Statistics: Wilcoxon test for unpaired samples; IBM SPSS Statistics 22. 1. Rapid response alert 06. NPSA; 2008. 1. Rapid response alert 06. NPSA; 2008. 2. Mariyaselvam et al. Anaesthesia. 2014;70:51-5. 3. Mukhopadhyay et al. Crit Care. 2010;14:R7. Reference 1. Romano M, et al. Care Med. 2002;30:1834-41. Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report Measures were performed by a single trained operator, who was blind to CVP values. Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. Measures were performed by a single trained operator, who was blind to CVP values. Results Forty-eight patients were included. A poor correlation was found between CVP and IJV and EJV circumference and area in mechanically ventilated patients. A strong correlation was found between CVP and EJV circumference (r: 0.74; P = 0.0004; 95% CI: 0.421 to 0.897) and area (r: 0.702; P = 0.0012; 95% CI: 0.35 to 0.88) in spontaneously breathing patients. Conventional receiver-operating characteristic curves were generated to assess the utility of EJV circumference and area to predict low (≤8  mmHg) versus high (>8  mmHg) CVP values. AUC for EJV circumference and area was 0.935 (P <0.0001; 95% CI: 0.714 to 0.997) and 0.87 (P <0.0001; 95% CI: 0.63 to 0.98) respectively (Figure 1). Conclusion These results highlight a potentially evolving role of Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report Introduction There are few methods of cardiac output (CO) estimation validated in children. The aim of this study is to investigate the reliability of an uncalibrated pulse contour method of CO estimation, the pressure recording analytical method (PRAM), in pediatric patients scheduled for diagnostic right and left heart catheterization, compared with the oxygen-direct Fick method. yg Methods Cardiac index (CI) was simultaneously estimated by Fick, and PRAM applied to pressure signals recorded invasively from a femoral catheter. All measurements were performed in steady-state condition. PRAM CI measurements were obtained for 10 consecutive beats simultaneously during the Fick CI estimation. Agreement between Fick and PRAM was assessed using the Bland–Altman method. Correlation coeffi cient, bias, and percentage of error were calculated. fi Results Forty-three CI measurements were performed in 43 patients. The data showed good agreement between CIFick and CIPRAM: r2 = 0.98; bias –0.0074 l/minute/m2; limits of agreement from –0.22 to 0.22  l/minute/m2. The percentage error was 8%. Figure  1 shows the Bland–Altman plot. Figure 1 (abstract P171). Figure 1 (abstract P170). Bland–Altman plot of the cardiac index measured with Fick versus PRAM. Figure 1 (abstract P170). Bland–Altman plot of the cardiac index measured with Fick versus PRAM. Figure 1 (abstract P171). Nonetheless, the external jugular vein (EJV) circumference and area have not been evaluated. Considering the role of EJV visual assessment in the clinical estimation of CVP, we hypothesized that EJV ultrasound evaluation could be used to reliably estimate CVP. Conclusion PRAM provides reliable estimates of cardiac output in hemodynamically stable pediatric cardiac patients compared with the Fick method. Reference Conclusion PRAM provides reliable estimates of cardiac output in hemodynamically stable pediatric cardiac patients compared with the Fick method. f Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. Measures were performed by a single trained operator, who was blind to CVP values. Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. P168 A recent study [1] showed acceptable bias and percentage error (PE) for CI-room derived from TPTD with 15  ml room temperature saline compared with CI-cold using 15 ml iced saline for TPTD. However, GEDVI-room and EVLWI-room had borderline PE values close to 30%, and the bias of GEDVI-room markedly increased with higher values of GEDVI and in case of femoral CVC. Since imprecision of TPTD-room might be reduced by a larger volume of injectate, it was the aim of our study to compare CI, GEDVI and EVLWI derived from TPDT using 20 ml room temperature injectate with standard TPTD with 15 ml iced saline. Conclusion Our data derived from a large TPTD database demonstrate markedly diff erent haemodynamics in patients with cirrhosis or acute liver failure with the only exception of static preload markers GEDVI and CVP. These fi ndings should be considered in instable patients with liver failure. Methods In 31 patients 236 sets with two 20 ml TPTDs with 21°C and subsequently two standard TPTDs with 4°C saline were obtained using Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S59 Figure 1 (abstract P171). P170 P170 Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report J Alonso Iñigo1, F Escribá1, J Carrasco1, J Encarnación1, M Fas2, M Barberá1 1Hospital Universitario y Politécnico La Fe, Valencia, Spain; 2Hospital Universitario de la Ribera, Alzira, Spain Critical Care 2015, 19(Suppl 1):P170 (doi: 10.1186/cc14250) P172 Do intravascular hypovolaemia and hypervolaemia result in changes in pulmonary blood volume? g p y JJ Vos1, TW Scheeren1, SA Loer2, A Hoeft3, JK Wietasch1 1University of Groningen, University Medical Center Groningen, the Netherlands; 2Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, the Netherlands; 3University of Bonn, Germany Critical Care 2015, 19(Suppl 1):P172 (doi: 10.1186/cc14252) Introduction Hypovolaemia is generally believed to induce centrali sa- tion of blood volume. Therefore, we evaluated whether hypovolaemia and hypervolaemia result in a change in central blood volume (that is, pulmonary blood volume (PBV)) and we explored the eff ects on the distribution between PBV and circulating blood volume (Vd circ). Introduction Hypovolaemia is generally believed to induce centrali sa- tion of blood volume. Therefore, we evaluated whether hypovolaemia and hypervolaemia result in a change in central blood volume (that is, pulmonary blood volume (PBV)) and we explored the eff ects on the distribution between PBV and circulating blood volume (Vd circ). Methods Twenty anesthetized Landrace/Large-White pigs (19 ± 2 kg, 10 to 15 weeks) were subjected to a fi xed hemorrhage (50% over 30 minutes). The pigs were randomly allocated into two groups (n = 10 per group). In group A, ITD was the only treatment for hypotension, while in group B, an intravenous administration of 1 l Ringer lactate was applied for treatment of hypotension. Hemodynamic parameters were continuously assessed for the fi rst 30 minutes after blood loss. Methods After local District Governmental Animal Investigation Committee approval, blood volume was altered in both directions randomly in steps of 150  ml (mild) to 450  ml (moderate) either by haemorrhage, retransfusion of blood, or infusion of colloids in six Foxhound dogs. The anaesthetised dogs were allowed to breathe spontaneously. Blood volumes were measured using the dye dilution technique: PBV was measured as the volume of blood between the pulmonary and aortic valve, and Vd circ by two-compartmental curve fi tting [1,2]. The PBV/Vd circ ratio was used as a measure of blood volume distribution. A linear mixed model was used for analysing the infl uence of blood volume alterations on the measured haemodynamic variables and blood volumes. yi Results Mean systolic arterial pressures (SAPs) 30  minutes after the intervention in each group were as follows: group A 80 ± 5 mmHg and group B 90 ± 4 mmHg. Maximum SAPs during the assessment period were: group A 89 ± 2 mmHg and group B 128 ± 5 mmHg. p P Balsorano, S Romagnoli, A De Gaudio P Balsorano, S Romagnoli, A De Gaudio AOUC Careggi, Florence, Italy AOUC Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P171 (doi: 10.1186/cc14251) AOUC Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P171 (doi: 10.1186/cc14251) Introduction Although recognized as a questionable indicator of the intravascular volume, central venous pressure (CVP) is integrated in many therapeutic algorithms for hemodynamic resuscitation of critically ill patients [1]. In an attempt to simplify CVP estimation, several clinical and ultrasonographic approaches have been suggested [2-5]. p y g Conclusion These results highlight a potentially evolving role of EJV circumference and area in the hemodynamic management S60 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of spontaneously breathing patients. An important aspect of the suggested approach is its simplicity, requiring basic technical skills and making it suitable in any scenario where an ultrasound machine is available. P175 Relation between global end-diastolic volume and left ventricular end-diastolic volume A Pironet1, P Morimont1, S Kamoi2, N Janssen1, PC Dauby1, JG Chase2, B Lambermont1, T Desaive1 1University of Liège, Belgium; 2University of Canterbury, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P175 (doi: 10.1186/cc14255) (Figure 1). Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. References 1. Anesthesiology. 1994;81:76-86. 2. Intensive Care Med. 2001;27:767-74. Figure 1 (abstract P172). Figure 1 (abstract P172). 1University of Liège, Belgium; 2University of Canterbury, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P175 (doi: 10.1186/cc14255) Introduction Measurement of global end-diastolic volume (GEDV) is provided by cardiovascular monitoring devices using thermodilution procedures. The aim of this study was to assess the relation between this clinically available index and left ventricular end-diastolic volume (LVEDV), which is typically not available at the patient bedside. Methods Measurements were performed on six anaesthetised and mechanically ventilated pigs. Volume loading via successive infusions of saline solution was fi rst performed and was followed by dobutamine infusion. These two procedures provided a wide range of LVEDV values. During these experiments, GEDV was intermittently measured using the PiCCO monitor (Pulsion AG, Germany) during thermodilutions and LVEDV was continuously measured using an admittance catheter (Transonic, NY, USA) inserted in the left ventricle. Results Table  1 presents the linear correlations obtained between LVEDV and GEDV. These correlations are good to excellent, with r2 values from 0.59 to 0.85. However, the coeffi cients of the linear regressions present a large intersubject variability, which prevents the precise estimation of LVEDV using GEDV. Nevertheless, variations in LVEDV are well reproduced by the GEDV index. The variations in LVEDV actually equal 21 to 48% of those in GEDV. The coeffi cient b is always nonzero, indicating that some proportion of the GEDV index is actually not linked to LVEDV. Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. R f Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. Comparative study between fl uidless resuscitation with permissive hypotension using the impedance threshold device versus aggressive fl uid resuscitation with Ringer lactate in a swine model of hemorrhagic shock C Pantazopoulos1, I Floros1, N Archontoulis1, D Xanthis1, D Barouxis2, N Iacovidou2, T Xanthos2 1Laiko General Hospital of Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P174 (doi: 10.1186/cc14254) References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. Introduction Permissive hypotension, which results in avoidance of intravascular overpressure and thereby avoidance of platelet plug dislodgement early in the clotting mechanism, improves the results after trauma and hemorrhage. The research hypothesis is that augmentation of negative intrathoracic pressure with the use of an impedance threshold device (ITD) will improve hemodynamic parameters, without aff ecting permissive hypotension or causing hemodilution. On the other hand, aggressive resuscitation with Ringer lactate will cause hemodilution and intravascular pressures that are very high for permissive hypotension, capable of platelet plug dislodgement. 2. Intensive Care Med. 2001;27:767-74. P172 Do intravascular hypovolaemia and hypervolaemia result in changes in pulmonary blood volume? Mean pulse pressure was higher in the ITD group versus the fl uid resuscitation group (P  <0.05). After the assessment period, mean hematocrit in group A was 24 ± 2%, while in group B it was 18 ± 1% (P <0.001). Conclusion In our study, the ITD increased SAP and pulse pressure without overcompensation. On the other hand, aggressive fl uid resuscitation led to a signifi cant increase of SAP >100 mmHg capable of clot dislodgement and in addition led to hemodilution. Results A total of 68 alterations in blood volume resulted in changes in Vd circ ranging from –33 to +31% (Figure 1). PBV decreased during mild and moderate haemorrhage, while during retransfusion PBV increased during moderate hypervolaemia only. The PBV/Vd circ ratio remained constant during all stages of hypovolaemia and hypervolaemia (Figure 1). P175 R l i 1. Anesthesiology. 1994;81:76-86. P175 However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) p S Hutchings1, P Hopkins1, A Campanile2 pp Methods This prospective, single-center study included 80 patients with sepsis from the Department of Critical Care Medicine of Zhejiang Hospital. Patients were randomly assigned to either Group A or Group B, with patients of in Group A fi rst taking the passive leg raising test and then taking the fl uid infusion test, while patients in Group B followed the opposite protocol. NICOM was used to continuously record hemodynamic parameters such as cardiac output (CO), heart rate (HR) and central venous pressure (CVP), at baseline1, PLR, baseline2, and volume expansion (VE). Fluid responsiveness was defi ned as the change in CO (ΔCO) ≥10% after VE. Introduction We performed an evaluation of three devices used for assessment of volume status in critically ill patients in our institution: transthoracic echocardiography (TTE) (CX50; Philips Ultrasound), bio reactance (NICOM; Cheetah Medical) and pulse contour-based thermodilution (PiCCO; Pulsion Medical). Methods Ten mechanically ventilated critically ill patients with PiCCO monitoring in situ and a good quality of images on transthoracic view were included. All study measurements were made in triplicate. A single trained cardiologist, blinded to the results from the other monitors, performed the TTE study. Diff erences among the three methods were assessed for signifi cance using one-way ANOVA, Spearman’s coeffi cient and Bland–Altman analysis. All statistical analyses were performed using Graph-pad Prism 5 and P <0.05 was taken as signifi cant. g ( ) Results CO increased during PLR (from 5.21 ± 2.34 to 6.03 ± 2.73 l/ minute, P <0.05); and after VE (from 5.09 ± 1.99 to 5.60 ± 2.11 l/minute, P <0.05). The PLR-induced change in CO (ΔCOPLR) and the VE-induced change in CO (ΔCOVE) were highly correlated (r = 0.80 (0.64 to 0.90)), while the CVP and ΔCOVE were uncorrelated (r = 0.12 (–0.16 to 0.32)). The areas under the ROC curves of ΔCOPLR and ΔCVP for predicting fl uid responsiveness were 0.868 and 0.514 respectively. ΔCOPLR ≥10% was found to predict fl uid responsiveness with a sensitivity of 86% and a specifi city of 79%.l g p p gi Results Ninety measurements were obtained. NICOM and TTE-derived stroke volume appeared well matched but PICCO-derived values showed signifi cant variation (F = 2.4, P = 0.09). References Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S61 Table 1 (abstract P175). Linear regressions between LVEDV and GEDV Subject a b (ml) r2 1 0.26 7.64 0.82 2 0.43 –47.10 0.66 3 0.21 –12.99 0.75 4 0.25 –11.42 0.59 5 0.41 –65.42 0.85 6 0.48 –65.75 0.68 LVEDV = a × GEDV + b. regards fl uid administration between PiCCO and echocardiography. NICOM appeared unreliable in this setting. regards fl uid administration between PiCCO and echocardiography. NICOM appeared unreliable in this setting. Conclusion The results show that GEDV and LVEDV are generally well correlated, but the correlation coeffi cients are subject specifi c. A preliminary calibration step (for instance using echocardiography) is thus necessary to infer LVEDV from GEDV. Introduction Fluid administration is always important and diffi cult during the therapy of patients with sepsis. Accurately predicting fl uid responsiveness and thus estimating whether the patient will benefi t from fl uid therapy seems particularly important. The present study intended to predict fl uid responsiveness in patients with sepsis using a bioreactance-based passive leg raising test, and to compare this approach with the commonly used central venous pressure (CVP) approach. P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) There was no correlation between TTE velocity time integer (VTI) and NICOM stroke volume variation (SVV) (r = 0.24, P = 0.20; Figure 1A) but a good correlation and small bias between TTE-VTI and PiCCO-SVV (r = 0.76, P <0.0001; Figure 1B). Applying the following indications for volume expansion (PiCCO and NICOM SVV >15% and TTE VTI variability >15%) we found an agreement in 71% of cases between TTE and PiCCO and in 42% of cases between echocardiography and NICOM. i Conclusion Bioreactance-based PLR could predict fl uid responsiveness in patients with sepsis, while CVP could not. P176 P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment H Barrasa, J Maynar, S Castaño, Y Poveda, P Garcia Domelo, A Tejero, G Baziskueta, A Quintano, B Fernández Miret, M Iturbe, S Cabañes, F Fonseca Alava University Hospital-Santiago, Vitoria, Spain Critical Care 2015, 19(Suppl 1):P180 (doi: 10.1186/cc14260) Introduction Dynamic parameters are becoming standard for fl uid responsiveness assessment. Cutoff values are diff erent in the literature. The aim was to assess the accuracy of diff erent preload parameters to predict fl uid responsiveness using pulse power analysis and to compare diff erent levels of hemodynamic response due to passive leg raising (PLR) against the eff ect of a fl uid challenge (FC). y Results We evaluated 27 patients, age 68 (95% CI: 61 to 74) and APACHE II score 22 (95% CI: 18 to 26). Seven patients were high responders, eight patients were moderate responders and 12 were nonresponders. DO2 was signifi cantly increased in high responders (37  ±  35%, P <0.01) as compared with moderate responders or nonresponders. Furthermore, nonresponders had a decrease in their DO2 (–10 ± 7%, P <0.01), while moderate responders showed no change in their DO2 (1.6% ± 10, P = 0.73) after fl uid challenge. We found no diff erences in changes in lactate levels and central venous oxygen saturation (ScvO2) between high responders, moderate responders and nonresponders. No diff erences in the changes of VCO2 or VO2/VCO2 ratio were found between high responders, mild responders and nonresponders too. Changes in DO2/VCO2 ratio were found to be signifi cantly increased only in high responders (47 ± 73% vs. –14 ± 31%, P = 0.02) and not in mild responders (15 ± 54% vs. –14 ± 31%, P = 0.15) as compared with nonresponders. g gfl g Methods A prospective study in a 17-bed mixed ICU. Patients were fully ventilated and CO monitored with LiDCOplus® and underwent a FC due to hypotension and/or hypoperfusion and preload dependence (SVV and/or PPV >10%). PLR was performed before FC. Hemodynamic data were recorded prePLR, postPLR and postFC with 0.5 l crystalloids. We compared diff erent cutoff values of increase in CO and SV (10 to 15%) to assess the ability of PLR, SVV, PPV and CVP to predict the response to FC. Statistical analysis: continuous variables expressed as mean ± SD. P180 P180 Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment H Barrasa, J Maynar, S Castaño, Y Poveda, P Garcia Domelo, A Tejero, G Baziskueta, A Quintano, B Fernández Miret, M Iturbe, S Cabañes, F Fonseca Alava University Hospital-Santiago, Vitoria, Spain Critical Care 2015, 19(Suppl 1):P180 (doi: 10.1186/cc14260) Global end-diastolic volume: a better indicator of cardiac preload in patients with septic shock L Mirea, R Ungureanu, D Pavelescu, I Grintescu g Clinical Emergency Hospital of Bucharest, Romania g y p , Critical Care 2015, 19(Suppl 1):P179 (doi: 10.1186/cc14259) g y p , Critical Care 2015, 19(Suppl 1):P179 (doi: 10.1186/cc14259) Introduction The aim of the study was to assess the value of the global end-diastolic volume (GEDV) evaluated by transpulmonary thermodilution as an indicator of cardiac preload comparing with stroke volume variation (SVV) in patients with septic shock. g p p Conclusion Our results support the idea that a reversible FC (PLR; CO cutoff 12.6%) is best at identifying responder patients to a FC. Dynamic parameters (SVV/PPV) are also eff ective when appropriate. Beat-to- beat SV and CO using pulse power analysis is a valid tool for these tests. Methods A prospective, observational study performed in an interdisciplinary ICU including 91 patients with septic shock. Hemodynamic monitoring was performed with a new calibrated pulse wave analysis method (VolumeView/EV1000; Edwards Lifesciences) in 37 patients (group EV1000) or with an uncalibrated method (FloTrac/ Vigileo; Edwards Lifesciences) in 54 patients (group Vigileo) during the fi rst 72 hours. All patients were receiving mechanical ventilation and vasopressors. Measurements were performed before and immediately after volume loading using 500 ml Ringer solution over a short period (<30 minutes).l p References p p Referencesl 1. De Backer D. Can passive leg raising be used to guide fl uid administration? Crit Care. 2006;10:170. 2. Pinsky MR. Functional haemodynamic monitoring. Curr Opin Crit Care. 2014;20:288-93. 3. Teboul JL, Monnet X. Pulse pressure variation and ARDS. Minerva Anestesiol. 2013;79:398-407. Conclusion Stroke volume produced by bioreactance appeared to be comparable with that measured by echocardiography but not with PiCCO. There was a good agreement between decision-making as 4. Monnet X, Teboul JL. Volume responsiveness. Curr Opin Crit Care. 2007;13:549-53. Figure 1 (abstract P176). Figure 1 (abstract P176). t P176) S62 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P178 Conclusion The transpulmonary thermodilution GEDV is a better indicator of cardiac preload than SVV in patients with septic shock. Acknowledgements This paper was cofi nanced from the European Social Fund, through the Sectorial Operational Programme Human Resources Development 2007–2013, project number POSDRU/159/1.5/S/138907 ‘Excellence in scientifi c interdisciplinary research, doctoral and postdoctoral, in the economic, social and medical fi elds – EXCELIS’; coordinator, The Bucharest University of Economic Studies. References P178 Acute changes of metabolic parameters after fl uid challenge T Nguyen, D De Bels, M Pustetto, P Cottignies, J Devriendt, C Pierrakos Brugmann Hospital, Brussels, Belgium Critical Care 2015, 19(Suppl 1):P178 (doi: 10.1186/cc14258) Introduction The detection of heart response to fl uid administration is still a challenge in clinical practice. Changes in metabolic parameters may be useful to detect changes in cardiac output (CO) after fl uid expansion. 1. Michard F, et al. Chest. 2003;124:1900-8. 2. Bendjelid K, et al. Br J Anaesth. 2013;111:573-9. Methods This is a prospective observational study in adult critically ill patients. CO was measured either by echocardiography or by a thermodilution method (PiCCO, Swan–Ganz catheter). Hemodynamic measurements and blood gas analysis were obtained before and after a fl uid challenge with either 1,000 ml crystalloid or 500 ml colloid. Arterial and central venous blood gas samples were taken simultaneously. Oxygen delivery (DO2), oxygen consumption (VO2) and carbon dioxide production (VCO2) were calculated according to well-known formulas. Patients were divided into three groups (high responders, mild responders and nonresponders) according to their change in CO (>20%, 10 to 20%, <10%, respectively). P181 P181 Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment Comparison before and after was done using a paired Student’s t test, and receiver operating characteristic (ROC) curves were generated by varying the discriminating threshold of each variable. p Conclusion Only signifi cant increases of CO (>20%), after fl uid administration, lead to improved oxygen delivery; DO2 may be decreased in nonresponders. The changes of ScvO2 and lactate levels do not track the changes of CO after fl uid challenge. The DO2/VCO2 ratio may be a useful index to identify signifi cant increases of CO after fl uid challenge in cases where CO measurement is not feasible. Results Thirty-one patients were included. Baseline parameters: MAP 70.5 mmHg (SD 13.3) 87% under catecholamine, SV 55.32 ml (SD 20.2), CO 5.2 l (SD 2), SVV 16.8% (SD 12), PPV 19.1% (SD 14), HR 96 bpm (SD 18) and CVP 9.2 mmHg (SD 2.5). In total, 41.9% of patients increased 15% CO after FC (selected as responders), 38.7% after the PLR. Diff erences in responders versus nonresponder patients were: baseline SVV (23.9 vs. 11.6; P = 0.02) and PPV (28.4 vs. 12.4; P = 0.01). Diff erences in SV and CO were not statistically signifi cant. The best parameter to predict positive response to FC was PLR with cutoff 12.6% for CO increase: sensitivity 84.6% (95% CI = 65 to 104), specifi city 94.4% (95% CI = 84 to 105) and AU ROC 0.94 (95% CI = 0.86 to 1.0). ROC was also good for SVV 0.835 (95% CI = 0.66 to 1.0; P = 0.002) and PPV 0.833 (95% CI = 0.681 to 0.985; P = 0.002) in this cutoff value. In SV increase, PLR, SVV and PPV had P <0.05, but with worse ROC. In addition, SVV <13% identifi ed patients who will not increase MAP with FC: sensitivity 91.7% (95% CI = 76 to 107.3%), negative predictive value 93.5 (95% CI  = 80.7 to 106). CVP failed to distinguish responders from nonresponders. References volume status. In this way, dynamic echocardiographic parameters have been proposed in mechanically ventilated children [1,2], using the heart–lung interactions. This study aimed to investigate whether respiratory variations of aortic blood fl ow velocity (DELTA Vpeak ao) and inferior vena cava diameter (DELTA IVC) by transthoracic echocardiography (TTE) could accurately predict fl uid responsiveness in ventilated children. volume status. In this way, dynamic echocardiographic parameters have been proposed in mechanically ventilated children [1,2], using the heart–lung interactions. This study aimed to investigate whether respiratory variations of aortic blood fl ow velocity (DELTA Vpeak ao) and inferior vena cava diameter (DELTA IVC) by transthoracic echocardiography (TTE) could accurately predict fl uid responsiveness in ventilated children. 1. Monnet X, et al. Esophageal Doppler monitoring predicts fl uid responsiveness in critically ill ventilated patients. Intensive Care Med. 2005;31:1195-201. 2. Tibby SM, et al. Are transoesophageal Doppler parameters a reliable guide to paediatric haemodynamic status and fl uid management? Intensive Care Med. 2001;27:201-5. 2. Tibby SM, et al. Are transoesophageal Doppler parameters a reliable guide to paediatric haemodynamic status and fl uid management? Intensive Care Med. 2001;27:201-5. Methods A prospective observational and interventional study conducted in a pediatric ICU investigated 40 mechanically ventilated children with preserved left ventricular (LV) function using TTE. Each patient had tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs. Intervention: standardized volume expansion (VE). P183 Fluid management in mechanically ventilated children with acute circulatory failure based on the pleth variability index in a pediatric ICU H Bouguetof, M Negadi, K El Halimi, L Zemour, D Boumendil, Z Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P183 (doi: 10.1186/cc14263) Results The VE-induced increase in LV stroke volume was ≥10% in 28 patients (responders) and <10% in 12 patients (nonresponders). Before VE, the DELTA Vpeak ao and DELTA IVC in responders was respectively higher than that in nonresponders (18.75% (12 to 32) vs. 13.5% (6 to 16) and 31% (18 to 57) vs. 17.5% (14 to 25)). The prediction of fl uid responsiveness was higher with DELTA Vpeak ao (ROC curve area 0.894 (95% CI = 0.756 to 0.969), P = 0.0001) and DELTA IVC (ROC curve area 0.869 (95% CI = 0.717 to 0.957), P = 0.0001). P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Introduction Prediction of fl uid responsiveness is defi ned by an increase in stroke volume (SV) of at least 10% after volume expansion. Dynamic [1] and static [2] esophageal Doppler (OD) parameters have been proposed in mechanically ventilated children to guide fl uid therapy. This study aimed to compare dynamic parameters using the respiratory variation in aortic blood fl ow with static parameters using Doppler corrected fl ow times (FTc) obtained by OD. Introduction The accuracy of predicting fl uid responsiveness (FR) using IVC collapsibility is high in patients on controlled mechanical ventilation, but remains unknown in spontaneously breathing patients with mechanical ventilation. Also, adequate ultrasound images of IVC are diffi cult to obtain in a substantial number of patients. The aim of the current study is to evaluate utility of collapsibility of jugular veins (IJV) and subclavian veins (SCV) in comparison with collapsibility of IVC in patients on pressure support ventilation. ppl y Methods A prospective, observational and interventional study was conducted in our pediatric ICU from March 2012 to September 2014. We investigated 18 mechanically ventilated children with acute circulatory failure (ACF)  – tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs – using OD for each patient. Intervention: standardized volume expansion (VE). Results The VE-induced increase in stroke volume was ≥10% in 14 patients (responders) and <10% in four patients (nonresponders). Before VE, the DELTA Vpeak ao in responders was higher than in nonresponders (19.5% (12 to 29) vs. 11.5% (7 to 13)), whereas FTc was lower in responders than in nonresponders (262.5 milliseconds (180 to 340) vs. 285 milliseconds (205 to 300)). The prediction of fl uid responsiveness was higher with DELTA Vpeak ao (ROC curve area 0.964 (95% CI = 0.756 to 1.000); P = 0.0001) than with FTc (ROC curve area 0.562 (95% CI = 0.314 to 0.790); P = 0.7203). pediatric PICU pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Results A total of 211 fl uid challenges were studied in 91 patients. We observed a signifi cant relationship between the GEDV index before volume loading and the percentage increase in GEDV index in the EV1000 group and changes in GEDV index were signifi cantly correlated with changes in stroke volume index (r = 0.75, P <0.001), but an insignifi cant relationship between SVV variation and cardiac index variation (P >0.05) in the Vigileo group. Introduction Volume expansion remains the fi rst treatment step for most children with acute circulatory failure in order to assess blood Introduction Volume expansion remains the fi rst treatment step for most children with acute circulatory failure in order to assess blood S63 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Prediction of fl uid responsiveness in mechanically ventilated children using dynamic and static parameters by esophageal Doppler in a pediatric ICU Prediction of fl uid responsiveness in mechanically ventilated children using dynamic and static parameters by esophageal Doppler in a pediatric ICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P182 (doi: 10.1186/cc14262) Conclusion In this study, PVI seems to predict fl uid responsiveness in ventilated children with ACF. pp p K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P182 (doi: 10.1186/cc14262) References The best cutoff value for DELTA Vpeak ao was 16% with sensitivity and specifi city predictive values of 71.6% and 83.3%, respectively, and DELTA IVC was 20% with sensitivity and specifi city predictive values of 88.5% and 90.9%, respectively. Introduction The pleth variability index (PVI) is a new dynamic index obtained by automatic estimation of respiratory variations in the pulse oximeter waveform amplitude. This noninvasive and continuous hemodynamic monitoring has been recently proposed in mechanically ventilated patients to guide fl uid therapy. We recently acquired a PVI monitor in 2014. PVI is calculated by measuring changes in perfusion index (PI) during the respiratory cycle as follows: PVI = ((PImax – Pimin) / PImax) × 100. This study aimed to investigate whether PVI at baseline can predict fl uid responsiveness. pl p Methods In our pediatric ICU we started a prospective and observational study. Between January and November 2014, nine mechanically ventilated children were investigated using PVI and transthoracic echocardiography for each patient with acute circulatory failure (ACF): tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs. Intervention: standardized volume expansion. Conclusion In this study, DELTA Vpeak and DELTA IVC were appropriate variables to predict fl uid responsiveness by TTE in ventilated children. References 1. Durand P, et al. Respiratory variations in aortic blood fl ow predict fl uid responsiveness in ventilated children. Intensive Care Med. 2008;34:888-94. 1. Durand P, et al. Respiratory variations in aortic blood fl ow predict fl uid responsiveness in ventilated children. Intensive Care Med. 2008;34:888-94.l 2. Choi DY, et al. Respiratory variation in aortic blood fl ow velocity as a predictor of fl uid responsiveness in children after repair of ventricular septal defect. Pediatr Cardiol. 2010;31:1166-70. 2. Choi DY, et al. Respiratory variation in aortic blood fl ow velocity as a predictor of fl uid responsiveness in children after repair of ventricular septal defect. Pediatr Cardiol. 2010;31:1166-70. Results Signifi cant changes in stroke volume were observed after volume loading (VL) ≥10% in eight patients (responders (R)) and <10% in one patient (nonresponder (NR)). Before VL, PVI was signifi cantly higher in R than NR at baseline ((19.75  ±  3.15%) vs. (9%  ±  0.00%), P <0.0001), and decreased signifi cantly in R from baseline to after VL ((19.75% ± 3.15) vs. (12.5% ± 2.828), P <0.0001). P187 Assessing fl uid status with the vascular pedicle width: relationship to IVC diameter, IVC variability and lung comets N Salahuddin, I Hussain, Q Shaikh, M Joseph, H Alsaidi, K Maghrabi King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P187 (doi: 10.1186/cc14267) Assessing fl uid status with the vascular pedicle width: relationship to IVC diameter, IVC variability and lung comets Methods Mechanically ventilated patients having cardiac arrhythmia who have been considered for volume expansion were recruited in this prospective study. Each patient was sedated, paralyzed and monitored with a central venous catheter and a thermistor-tipped femoral arterial VolumeView catheter connected to the EV1000 monitor. We assessed hemodynamic changes after PLRT via a pulse wave contour analysis method. Then we compared it with hemodynamic changes after volume expansion (NSS 500 ml in 15 minutes) via the transpulmonary thermodilution (TPTD) method. N Salahuddin, I Hussain, Q Shaikh, M Joseph, H Alsaidi, K Maghrabi King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P187 (doi: 10.1186/cc14267) Introduction This study attempts to determine a vascular pedicle width (VPW) cutoff value that identifi es a fl uid replete state defi ned as an IVC diameter ≥2 cm and ≤15% respiratory variation. p y Methods In a cross-sectional design, consecutive, critically ill patients underwent simultaneous chest radiographs and ultrasounds. The Research Ethics Committee approved the study. Results A total of 17 patients were included in this study. Six patients were volume responders (TPTD cardiac index change ≥15%). A PLRT change cardiac index ≥10% from the pulse wave contour analysis method had predicted VR with a sensitivity of 50%, a specifi city of 72.7% and an area under the ROC curve of 0.591 (P = 0.546). Results Eighty-four data points on 43 patients were collected. VPW correlated with IVC diameter (r  = 0.64, P ≤0.001) and IVC variation (r = –0.55, P ≤0.001). No correlation was observed between VPW and number of lung comets (r = 0.12, P = 0.26) or positive fl uid balance (r  = 0.3, P  =  0.058). On multivariate linear regression, standardized coeffi cients demonstrated that a 1 mm increase in IVC diameter corresponded to a 0.28 mm (Beta) increase in VPW. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Results A total of 204,680 patients met the search criteria, and 76,807 patients developed one or more postsurgical complications (morbidity rate 37.5%). In patients with and without complications, hospital costs (including 30  days readmission costs) were $27,607  ±  32.788 and $15.783  ±  12,282 (P  <0.0001), median (interquartile range) hospital lengths of stay (fi rst stay) were 7 (4 to 10) days and 4 (3 to 5) days (P  <0.0001), and 30-day readmission rates were 17.2% and 11.9% (P <0.0001), respectively. With PGDT, the morbidity rate was anticipated to decrease from 26.6 to 31.1%, yielding gross cost savings of $153 million to 263 million for the study period, $61 million to 105 million per year, or $754 to 1,286 per patient. used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean difference in total costs volume was 9.8 ml/predicted body weight. The area under the ROC curve of IVC collapsibility was 0.576 (95% confi dence interval (CI): 0.38 to 0.77), while the area under the ROC curves of right IJV, left IJV, right SCV and left SCV collapsibility were 0.870 (95% CI: 074 to 1.0), 0.54 (95% CI: 0.34 to 0.74), 0.62 (95% CI: 0.43 to 0.81) and 0.54 (95% CI: 0.34 to 0.74), respectively. Greater than 11% of right jugular vein collapsibility predicted fl uid responsiveness with a sensitivity of 79% and a specifi city of 94%. volume was 9.8 ml/predicted body weight. The area under the ROC curve of IVC collapsibility was 0.576 (95% confi dence interval (CI): 0.38 to 0.77), while the area under the ROC curves of right IJV, left IJV, right SCV and left SCV collapsibility were 0.870 (95% CI: 074 to 1.0), 0.54 (95% CI: 0.34 to 0.74), 0.62 (95% CI: 0.43 to 0.81) and 0.54 (95% CI: 0.34 to 0.74), respectively. Greater than 11% of right jugular vein collapsibility predicted fl uid responsiveness with a sensitivity of 79% and a specifi city of 94%. pi y Conclusion Our results suggest collapsibility of the right jugular vein can be a useful predictor of fl uid responsiveness in patients on pressure support ventilation, compared with other central large veins. Collapsibility of IVC does not predict FR in those patients. P185 Passive leg raising cannot predict volume responsiveness in septic shock patients having cardiac arrhythmia P Ratanawatkul1, A Wattanathum2 1Srinagarind Hospital, Khon Kaen, Thailand; 2Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P185 (doi: 10.1186/cc14265) Reference 1. Pearse et al. Eff ect of a perioperative, cardiac output-guided hemodynamic therapy algorithm on outcomes following major gastrointestinal surgery: a randomized clinical trial and systematic review. JAMA. 2014;311:2181-90. Introduction The passive leg raising test (PLRT) is a self-volume challenge used in order to predict volume responsiveness (VR) in both spontaneous and mechanically ventilated critically ill patients. However, there were small numbers of arrhythmic patients included in previous studies. Therefore, the accuracy of the PLRT for prediction of VR in arrhythmic patient is still inconclusive. We hypothesized that the PLRT can predict VR in mechanically ventilated patients having cardiac arrhythmia. P187 P187 ROC curve analysis yielded an AUC of 0.843 (95% CI = 0.75 to 0.93), P ≤0.001 and provided the best accuracy with a cutoff VPW value of 64 mm (sensitivity 81%, specifi city 78%, PPV = 88.5%, NPV = 66%, correct classifi cation rate = 79.6%). See Figure 1. Conclusion The PLRT may not be used for prediction of VR in mechanically ventilated patients having cardiac arrhythmia; however, further and larger studies are needed to confi rm this fi nding. References 1. Monnet X, et al. Crit Care Med. 2006;34:1402-7. 1. Monnet X, et al. Crit Care Med. 2006;34:1402-7. 2. Cavallaro F, et al. Intensive Care Med. 2010;36:1475-83. 2. Cavallaro F, et al. Intensive Care Med. 2010;36:1475-83. Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) p y p p Conclusion Postsurgical complications occurred in more than one- third of our study population and had a dramatic impact on hospital costs, length of stay, and readmission rates. Potential cost savings with PGDT were $754 to 1,286 per patient. These projections should help hospitals estimate the return on investment for implementation of PGDT. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) The best cutoff value for DELTA Vpeak ao was 13% with sensitivity and specifi city predictive values of 85.7% and 100%, respectively; and the best cutoff value for FTc was 265 milliseconds with sensitivity and specifi city predictive values of 57.1% and 75%, respectively. Methods Patients on pressure support ventilation were prospectively included when fl uid challenges were clinically indicated. Bilateral IJV were examined at the level of cricoid cartilage. Bilateral SCV were measured where the veins crossed the clavicle. Anteroposterior diameter, cross-sectional area (CSA) of IJV and SCV were measured using frame by frame analysis. IVC was measured 2 cm from the right atrial border in a long axis view. Fluid responsiveness was defi ned as 8% increase of stroke volume calculated by the Vigileo monitor (Vigileo, FloTrac; Edwards Lifesciences) after passive leg raising (started from supine position). Receiver operating characteristic (ROC) curves were generated using EZR. Results Twenty-nine patients (35 measurements) were included. Nineteen measurements had fl uid responsiveness. The mean tidal Conclusion In our study, DELTA Vpeak was the most appropriate variable to predict fl uid responsiveness by OD in ventilated children with ACF. S64 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean diff erence in total costs. Results A total of 204,680 patients met the search criteria, and 76,807 patients developed one or more postsurgical complications (morbidity rate 37.5%). In patients with and without complications, hospital costs (including 30  days readmission costs) were $27,607  ±  32.788 and $15.783  ±  12,282 (P  <0.0001), median (interquartile range) hospital lengths of stay (fi rst stay) were 7 (4 to 10) days and 4 (3 to 5) days (P  <0.0001), and 30-day readmission rates were 17.2% and 11.9% (P <0.0001), respectively. With PGDT, the morbidity rate was anticipated to decrease from 26.6 to 31.1%, yielding gross cost savings of $153 million to 263 million for the study period, $61 million to 105 million per year, or $754 to 1,286 per patient. used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean diff erence in total costs. Bioimpedance as a measure of fl uid overload in patients recently admitted to intensive care Bioimpedance as a measure of fl uid overload in patients recentl admitted to intensive care M O’Connor, E Galtrey, CJ Kirwan, JR Prowle Barts Health NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) y Barts Health NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) Introduction Fluid overload is associated with adverse outcomes in critical illness; however, better methodology is required for its quantifi cation. Bioelectrical impedance analysis (BIA) represents a non- invasive method for quantifi cation of fl uid overload [1], but has not been widely taken up in the ICU. Results Fifty patients were included, 40.8% of them were responders. The proportion of responders increases with the increase of dose of fl uids (Table 1). The regression equation was: change of Pmsf (%) = 4.4 (dose of fl uids ml/kg, 95% CI 2.3 to 6.5) – 1.6 (95% CI 7.4 to 4.3, R2 = 0.28, F(1.47) = 17.8, P <0.001). The predicted dose of fl uids required to achieve a change in Pmsf of 15% is 3.7 ml/kg crystalloids. y p Methods We assessed changes in fl uid balance and performed daily BIA (using a Maltron BioScan 920-II; Maltron International Ltd, UK) over 3 days in consecutive ICU admissions with LOS >72 hours. Table 1 (abstract P189). Change of Pmsf-arm and CO Table 1 (abstract P189). Change of Pmsf-arm and CO 1 ml/kg 2 ml/kg 3 ml/kg 4 ml/kg (n = 12) (n = 12) (n = 13) (n = 13) P ΔPmsf-arm (%) 0.0 6.5 9.0 18 0.05 (–4 to 9) (3 to 21) (8 to 16) (9 to 21) ΔCO (%) 3.9 6 9.9 12.9 0.1 (0.4 to 10) (2.1 to 9.1) (–1.6 to 14.3) (2.6 to 23.5) Responders (%) 25.0 18.2 46.2 69.2 0.04 Values are median (interquartile range). Results Of 24 patients 71% were male, median age was 65 years and APACHE II score was 15. Eleven patients had a medical diagnosis and 13 a surgical or trauma reason for admission. Seventy-one percent were mechanically ventilated and 67% were on vasopressors or inotropes. Median BIA-estimated extracellular water was 25.2 l (IQR 22 to 28) on day 1, equating to excess fl uid of 7.2 l (IQR 5 to 13.9). Median right body resistance normalized to height at 50 kHz (R50/h) on day 1 was 214 Ω/m (IQR 187 to 256). Return on investment for implementation of perioperative goal-directed therapy in major surgery: a nationwide database study Introduction Preventable postsurgical complications are increasingly recognized as a major healthcare burden. A recent meta-analysis showed a 17 to 29% decrease in complications after major surgery with perioperative goal-directed therapy (PGDT) [1]. We assessed the fi nancial consequences of postsurgical complications in a large population from 541 US hospitals in order to predict potential savings with PGDT. Introduction Preventable postsurgical complications are increasingly recognized as a major healthcare burden. A recent meta-analysis showed a 17 to 29% decrease in complications after major surgery with perioperative goal-directed therapy (PGDT) [1]. We assessed the fi nancial consequences of postsurgical complications in a large population from 541 US hospitals in order to predict potential savings with PGDT. Methods Data from adults who had any one of 10 major noncardiac surgical procedures between January 2011 and June 2013 were selected from the Premier research database. Twenty-six postsurgical complications were tabulated. Hospital costs, length of stay, and readmission rates were compared in patients with and without complications. Risk ratios reported by Pearse’s meta-analysis were Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. S65 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 The objective of this study was to determine the minimum volume of intravenous fl uid required to signifi cantly increase the Pmsf. Conclusion A VPW value of 64 mm accurately identifi es a fl uid replete state. Increased extravascular lung water, however, was not relatable to the VPW measurements. The VPW can be confi dently used to discriminate fl uid repletion from fl uid responsiveness. li Methods Patients following cardiac surgery were randomly allocated to receive 1, 2, 3 or 4 ml/kg (body weight) of crystalloid over 5 minutes using a 60 ml syringe. Pmsf was measured using the arterial pressure after stopping blood fl ow in the arm with a pneumatic tourniquet infl ated for 1 minute. Cardiac output (CO) was also recorded at baseline and immediately after the fl uid infusion. CO was measured with LiDCO or pulmonary artery catheter, and a positive response was considered an increase of 10% from baseline. From previous data, the least signifi cant change for Pmsf was 15%. Return on investment for implementation of perioperative goal-directed therapy in major surgery: a nationwide database study Medians were compared using the independent samples media test, and proportions were compared using a chi-square test. Statistical signifi cance was considered when P <0.05. P188 Bioimpedance as a measure of fl uid overload in patients recently admitted to intensive care Daily change in ECW and R50/h correlated with daily fl uid balance between BIA measurements (R2 = 0.48 and 0.37 respectively) (Figure 1).i y Conclusion BIA suggests many patients already have signifi cant fl uid overload on the fi rst day of ICU admission. Overall, changes in device-specifi c algorithms for ECW estimation and measured resistances correlated with recorded fl uid balance; however, there were inconsistencies in the number of individual patients. Prospective assessment is required to establish whether BIA measurements can be used to assist fl uid management in the ICU. Conclusion The minimum volume required to perform an eff ective fl uid challenge is 4 ml/kg infused in 5 minutes. However, only 30% of the variation of change in Pmsf can be explained by the dose of i.v. fl uid given. The proportion of responders increases with the volume of fl uids. 1. Earthman C, et al. Bioimpedance spectroscopy for clinical assessment of fl uid distribution and body cell mass. Nutr Clin Pract. 2007;22:389. P189 P189 Minimal volume for a fl uid challenge in postoperative patients H Aya, A Rhodes, RM Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P189 (doi: 10.1186/cc14269) Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery 1ICESP, São Paulo, Brazil; 2Heart Institute, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P191 (doi: 10.1186/cc14271) Introduction After cardiac surgery, about 15% of patients develop vasoplegic shock, characterized by systemic vasodilation, increased capillary permeability and edema. We hypothesized that large-volume resuscitation, resulting in positive fl uid balances in the fi rst 24 hours of ICU admission, would be associated with mortality and would not be protective against AKI in this subset of patients. Conclusion Postsurgical complications have a signifi cant impact on hospital margins. Enhanced Recovery Programs have the potential not only to improve quality of care but also to improve hospital margins. References g Methods This is a retrospective analysis of 362 patients submitted to cardiac surgery at the Heart Institute of University of São Paulo in a period of 2 years. Of a total of 2,383 patients, we enrolled 362 patients. Vasoplegic shock was diagnosed if in the 24 hours of ICU admission patients had hypotension, need of vasoactive drugs after fl uid replacement and cardiac index ≥2.2 l/minute/m2. Data were analyzed in logistic regression models for 30-day mortality and acute kidney injury through Acute Kidney Injury Network (AKIN) score as outcomes. Results The mean age of patients was 57 years. Of 362 patients, 53 died at 30 days (14.6%). Nonsurvivors as compared with survivors were slightly older (59 ± 12 vs. 55 ± 13, P = 0.063), had a higher prevalence of AKI through AKIN score ((0) 6.9%, (1) 11.1%, (2) 28.9%, (3) 31.9%, P  <0.001), a higher 24-hour fl uid balance (421  ml (–55 to 695) vs. 2,686 ml (1,321 to 2,856), P <0.001), and higher lactate levels at the intraoperative and at 48 hours (5 mmol/l (4.0 to 7.6) vs. 4.4 (3.33 to 6.55), P <0.001; and 8.11 (5.49 to 12.3) vs. 1.5 (1.33 to 1.88), P <0.001). In the multivariate analysis, positive fl uid balance in the fi rst 24 hours (OR = 1.006, 95% CI = 1.003 to 1.008, P <0.001) and higher lactate after 48 hours (OR = 1.204, 95% CI = 1.072 to 1.353, P = 0.002) were predictors of 30-day mortality. Forty-three percent of patients developed AKI during 30 days. P192 Impact of postsurgical complications on hospital costs and margins R Lavender1, M Mythen2, J Bao3, RH Chapman3, F Michard1 1Edwards Lifesciences, Irvine, CA, USA; 2UCLH National Institute of Health Research, London, UK; 3Avalere Health, Washington, DC, USA Critical Care 2015, 19(Suppl 1):P192 (doi: 10.1186/cc14272) y Results Three hundred and thirty-nine patients were included; mean age was 51 ± 20.4 years, 167 (49%) patients were male. Mean APACHE II score was 22 ± 12.8 and SAPS II score was 35.4 ± 18.9. Severe sepsis/ septic shock was the admitting diagnosis in 129 (38%) patients, 108 (32%) patients were postoperative. AKI developed in 148 (44%) patients; Risk 29 (9%); Injury 26 (8%); Failure 89 (26%) by the RIFLE criteria. On univariate regression analysis; positive fl uid balance >2 l on the fi rst ICU admission day, OR 2 (95% CI = 1.3, 3.3, P = 0.002); age, OR 2.7 (95% CI = 1.7, 4.2, P = 0.000); CHF, OR 3.1 (95% CI = 1.2, 7.9, P = 0.013); APACHE II score, OR 1.02 (95% CI = 1.0, 1.04, P = 0.006); SAPS II score, OR 1.04 (95% CI = 1.02, 1.05, P = 0.000); mean MAP on admission, OR 0.98 (95% CI = 0.96, 0.99, P = 0.033); need for vasopressors on admission, OR 2.7 (95% CI = 1.7, 4.2, P <0.001) and for >24 hours, OR 2.7 (95% CI = 1.7, 2.5, P <0.001); and vancomycin use, OR 1.5 (95% CI = 1.02, 2.53, P = 0.04) signifi cantly predicted the development of AKI. On multivariate regression, CHF, OR 3.8 (95% CI = 1.4, 10, P = 0.007); age, OR 1.02 (95% CI = 1.01, 1.03, P = 0.001); vasopressors for >24 hours, OR 2.6 (95% CI = 1.6, 4.2, P <0.001) and a >2 l positive fl uid balance on the fi rst ICU day, OR 1.6 (95% CI = 1.02, 2.7, P = 0.04) remained signifi cant predictors. Introduction The impact of postsurgical complications (PSC) on hospital cost has been studied but the impact on margins remains controversial [1]. We assessed economic consequences of PSC in US Medicare patients, and benefi ts expected from reducing PSC by 14% to 40% with Enhanced Recovery Programs [2]. y Methods Data from patients with ≥1 comorbidity and major cardiac, vascular, gastrointestinal and orthopedic surgeries in 2011 were extracted from Medicare Standard Analytic Files. Hospital margin was calculated as payment minus cost. Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery In the multivariate analysis, positive fl uid balance in the fi rst 24 hours (OR = 1.001, 95% CI = 1.000 to1.001, P <0.001) and higher lactate at 48 hours (OR = 1.011, 95% CI = 1.000 to 1.021, P = 0.0043) were predictors of 30-day AKI. 1. Eappen S, et al. JAMA. 2013;309:1599-606. P192 Patients with and without PSC were compared, and the economic impact of a 14 to 40% relative reduction in PSC was calculated. Results Of 303,432 patients, 37% had ≥1 PSC. Median length of stay was 10 days for patients with ≥1 PSC and 6 days without (P <0.0001 with vs. without PSC), with readmissions for 21% and 16%, respectively (P <0.0001 with vs. without PSC). Average margins for cases with PSC converted into without PSC would be $1,870 higher. A 14 to 40% reduction in patients with PSC (from 37% to 32% to 22%) would result in saving $153 million to $438 million, and increase hospital margins overall by $28 million to $79 million. See Table 1. i Conclusion Fluid overload, defi ned as a >2 l positive fl uid balance on the fi rst day of ICU admission, is an independent risk factor for the development of AKI in the general ICU population. Table 1 (abstract P192) With PSC Without PSC Mean 2011 US$/patient Cost ($) Margin ($) Cost ($) Margin ($) Cardiac 46,535 –2,286 32,887* –778* Gastrointestinal 33,280 –3,088 18,942* –752* Orthopedic 20,798 –3,567 15,194* –1,872* Vascular 31,042 –4,782 17,667* –2,267* *P <0.0001 with versus without PSC. Table 1 (abstract P192) P191 Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery A Rezende1, L Camara2, A Leme2, J Ribeiro2, I Bispo2, S Zeferino2, J Jardim2, C Park1, E Osawa2, J Almeida2, A Gerent1, F Galas2, D Fonseca2, J Fukushima1, L Hajjar2 1ICESP, São Paulo, Brazil; 2Heart Institute, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P191 (doi: 10.1186/cc14271) Minimal volume for a fl uid challenge in postoperative patients H A A Rh d RM G d M C i H Aya, A Rhodes, RM Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK g Critical Care 2015, 19(Suppl 1):P189 (doi: 10.1186/cc14269) Introduction In critical illness, fl uid overload may predispose to acute renal dysfunction by a number of mechanisms. Once acute kidney Introduction An eff ective fl uid challenge should increase the mean systemic fi lling pressure (Pmsf) in order to increase the venous return. Figure 1 (abstract P188). Change in BIA-measured ECW (a) or R/h at 50 kHz (b) versus daily fl uid balance. Hz (b) versus daily fl uid balance. Figure 1 (abstract P188). Change in BIA-measured ECW (a) or R/h at 50 kHz (b) versus daily fl uid balance. S66 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Positive fl uid balance after cardiac surgery is an independent risk factor for mortality and for acute kidney injury in patients presenting vasoplegic shock. injury (AKI) develops, positive fl uid balance has been described as a risk factor for overall mortality and delayed renal recovery. We hypothesized that fl uid overload may be an independent risk factor for AKI in the critically ill. injury (AKI) develops, positive fl uid balance has been described as a risk factor for overall mortality and delayed renal recovery. We hypothesized that fl uid overload may be an independent risk factor for AKI in the critically ill. Methods In a cross-sectional design, we collected data on consecutive, critically ill, adult patients admitted over a 5-month period to the medical and surgical ICUs of a single center. AKI was defi ned according to the RIFLE Classifi cation. Logistic regression analysis was performed to determine the predictive ability of variables for AKI. The institutional Research Ethics Committee approved the study. P195 Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Results We included a total of 1,267 patients. The median age was 68 (quartiles: 59, 76), 32% were female, 68% underwent coronary artery bypass grafting and 59% underwent valve surgery. Median length of hospital stay was 6 days (quartiles: 5, 9). Median length of stay in the normal, elevated and high lactate groups were 5 days (quartiles: 4, 7), 6 days (quartiles: 5, 9) and 9 days (quartiles: 6, 17), P <0.001 for comparison. In multivariable analysis, patients with an elevated lactate had a 1.12 times (95% CI: 1.02 to 1.23, P = 0.02) longer length of stay compared with those with normal lactate. Patients with a high lactate had a 1.30 times (95% CI: 1.10 to 1.53, P = 0.002) longer length of stay compared with those with normal lactate. Introduction National University Hospital, Singapore, recently formed a Division of Critical Care – Respiratory Therapy. This service rapidly expanded to provide 24/7 Respiratory Therapy Services in the cardiothoracic intensive care unit (CTICU). One goal of service expansion was a reduction in duration of mechanical ventilation after cardiac surgery. We hypothesized that introduction of a team- based extubation protocol would reduce the duration of mechanical ventilation and ultimately aff ect ICU length of stay. Conclusion Postoperative lactate levels are associated with increased length of hospital stay in patients undergoing major cardiac surgery. Interventions aimed at decreasing postoperative lactate levels may decrease hospital length of stay. f Methods A multidisciplinary group created a team-based extubation protocol. The protocol was applied to all elective postoperative cardiac surgery patients. To assess the protocol’s impact, data were collected in a registry 3 months before and 3 months after protocol initiation. Data collection included cardiopulmonary bypass time, McCormack airway assessment, ICU admission time, initial pH, lactate, inotropes upon arrival at the CTICU, blood gas analysis prior to extubation, time of extubation and length of stay. Patients were excluded from data analysis if they experienced events which contraindicated application of the protocol, such as signifi cant intraoperative or postoperative complications. These events were explicitly stated in the extubation protocol. Lactate levels after major cardiac surgery are associated with hospital length of stay Introduction The objective of the study was to evaluate whether postoperative lactate values are associated with hospital length of stay in patients undergoing major cardiac surgery. Previous studies have shown an association between postoperative lactate levels and increased morbidity and mortality after major cardiac surgery. However, the association between lactate and hospital length of stay has not been adequately characterized. Methods We performed a retrospective analysis of all patients presenting for coronary artery bypass grafting and/or valve surgery between 2002 and 2014 at a tertiary care center in Boston, who had a lactate level measured within 3 hours of skin closure. Lactate values S67 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 were categorized into clinical meaningful categories: 0 to 2  mmol/l (normal), 2 to 4 mmol/l (elevated) and ≥4 mmol/l (high) to allow for nonlinear eff ects. The unadjusted association between lactate group and length of stay was assessed with the Kruskal–Wallis test and post hoc Wilcoxon rank-sum tests. To assess the association between postoperative lactate levels and hospital length of stay we performed multivariable Poisson regression with robust variance estimates. We adjusted for more than 30 variables including patient demographics, comorbidities, cardiac characteristics (for example, New York Heart Association class and ejection fraction), and surgical characteristics (for example, year, status (elective, urgent, emergent), type of procedure, perfusion time, and cross clamp time). severe hypotension (MAP <65 mmHg) and the study was not stopped in any case. The length of the hospital stay was shorter among patients in the intensive group (10.9 (9.9 to 11.9) vs. 12.4 days (11.3 to 13.6); P = 0.045). severe hypotension (MAP <65 mmHg) and the study was not stopped in any case. The length of the hospital stay was shorter among patients in the intensive group (10.9 (9.9 to 11.9) vs. 12.4 days (11.3 to 13.6); P = 0.045). Conclusion An intensive alveolar recruitment protocol did not result in hemodynamic instability in hypoxemic patients after cardiac surgery (NCT01502332). P195 P195 Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Singapore’s Domain-specifi c review board granted waiver of patient consent to analyze and present these data. Hemodynamic behavior in a randomized trial of intensive alveolar recruitment after cardiac surgery recruitment after cardiac surgery A Leme, M Amato, E Osawa, J Fukushima, M Feltrim, E Nozawa, J Almeida L Hajjar, F Galas Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P194 (doi: 10.1186/cc14274) A Leme, M Amato, E Osawa, J Fukushima, M Feltrim, E Nozawa, J Almeida, L Hajjar, F Galas jj Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P194 (doi: 10.1186/cc14274) Introduction The potential benefi ts of a protocol of intensive alveolar recruitment may be outweighed by its detrimental eff ects in hemodynamic stability after cardiac surgery. The aim of this study was to analyze the hemodynamic behavior of patients included in a trial of intensive alveolar recruitment after cardiac surgery. p y p Results A total of 201 patients undergoing elective open cardiac surgery were included; 99 patients before protocol implementation (pre-protocol) and 102 patients after implementation (post-protocol). There was no signifi cant diff erence in mean age (60 vs. 61 P = 0.823), gender (79.8% vs. 79.4% P  =  1.00), EuroSCORE (26 vs. 32 P  =  0.576) and proportion receiving bypass surgery (72% vs. 80% P = 0.206) or valve surgery (21% vs. 19% P = 0.722) between the two groups. Median extubation time was reduced by 3.5 hours (620 minutes vs. 408 minutes P <0.001). ICU length of stay was also reduced following introduction of the pre-protocol 48 hours versus 24 hours post protocol (P <0.05).i Methods In this randomized trial, we assigned adult patients with PaO2/ FIO2 <250 at a PEEP of 5 cmH2O to either intensive alveolar recruitment or a standard protocol, both using low-tidal volume ventilation (6 ml/ kg/ibw) after adequate volemia status. Our hypothesis was that an intensive alveolar recruitment protocol with controlled pressure of 15 cmH2O and PEEP of 30 cmH2O during 1  minute, repeated three times at 1-minute intervals between each maneuver, would not cause hemodynamic instability. Conclusion A team-based extubation protocol signifi cantly reduced the duration of mechanical ventilation and this translated to reduced ICU length of stay in patients undergoing elective open-heart surgery. Results In total, 163 patients were included in the standard and 157 in the intensive group. Patients of the intensive group had a signifi cant reduction of the MAP at T1, T2 and T3 (1 hour, 2 hours and 3 hours of the protocol), returning to baseline after T4 (Figure 1). No patients had P196 Impact of patient frailty on outcome in cardiothoracic surgery J Brohan, P Delaney, B O’Brien Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 JL Iribarren, J Jiménez, N Perez, M Brouard, R Perez, E Hurtado, S Diosdado, M Buitrago, A Arbesu, R Martinez, M Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P199 (doi: 10.1186/cc14279) Introduction Cardiopulmonary bypass can lead to postoperative hemodynamic disorders. Several genetic polymorphisms have been studied in this setting. We investigated the possible existence of a synergism between polymorphisms of plasminogen activator inhibitor type 1 (PAI-1) and tumoral necrosis factor beta (TNF-B) on hemodynamic response after cardiac surgery. Introduction Cardiopulmonary bypass can lead to postoperative hemodynamic disorders. Several genetic polymorphisms have been studied in this setting. We investigated the possible existence of a synergism between polymorphisms of plasminogen activator inhibitor type 1 (PAI-1) and tumoral necrosis factor beta (TNF-B) on hemodynamic response after cardiac surgery. g Results A total of 8,026 were recorded, in 77 of them an IABP was inserted before the surgery. We performed a propensity score analysis by pairing 72 patients with and without BCIAO based on epidemiological factors and type of surgery. In the analysis of all-cause 30-day mortality, 27% of patients in whom IABP was inserted prior surgery died versus 13.1% of patients without IABP preoperative implantation (P = 0.043). A combined endpoint that included need for prolonged mechanical ventilation over 24 hours or reoperation or mediastinitis or stroke after surgery or 30-day mortality was performed and occurred in 58.3% of patients with preoperative IABP versus 41.7% without it (P = 0.046). When stratifi ed by preoperative risk (analyzed with EuroSCORE), no diff erence between groups was observed (P = 0.62, OR 0.75 (0.23 to 2.35)) for mortality rate and (P = 0.11, OR 0.47 (0.19 to 1.18)) for the combined endpoint. The patients with preoperative IABP implantation had a higher ICU length of stay (10.6  ±  7.7 vs. 4.6  ±  6.7, P  =  0.046) with no diff erences in terms of overall hospital stay (21.8  ±  18.7 vs. 18.9 ± 22.08, NS). y y Methods We prospectively studied the association between hemodynamic response and polymorphisms of TNF-B and PAI-1 in 563 patients undergoing elective cardiac surgery during the years 2008 to 2011. We tested the Hardy–Weinberg equilibrium in the sample. V18 SPSS was used.if Results We studied 563 patients. We found signifi cant diff erences in TNF-B polymorphisms regarding norepinephrine requirements at 4 hours (F: 15.9; P <0.001), post hoc Scheff é (GG vs. P198 Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery J Muñoz-Bono, MD Delgado-Amaya, E Curiel-Balsera, C Joya-Montosa, G Quesada-García Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P198 (doi: 10.1186/cc14278) Results A total of 120 patients were included in this study, including 100 patients who underwent cardiac surgery and 20 patients who underwent thoracic surgery. Eighty-fi ve patients (70.8%) were male. The mean age was 65.4 years (range 25 to 89 years). The mean baseline frailty score also varied widely within our cohort. Four patients died in the ICU following their surgery (3% ICU mortality rate). Mean length of ICU stay was 2.7 days (range 0 to 20 days), with a mean duration of ventilation of 20 hours (range 0 to 264 hours). Follow-up of these patients at 6 months following their surgery is currently underway. Introduction The aim of the study is to analyze IABP use in patients undergoing cardiac surgery included in the ARIAM Registry of Cardiac Surgery. Introduction The aim of the study is to analyze IABP use in patients undergoing cardiac surgery included in the ARIAM Registry of Cardiac Surgery. g y Methods An observational, retrospective, multicenter study of all patients undergoing cardiac surgery included in the ARIAM- ANDALUCIA database of Cardiac Surgery from March 2008 to July 2012. We used the chi-square test and Student t test as needed, establishing the level of statistical signifi cance at 95%. y y y Conclusion Owing to advances in life expectancy, health and perioperative medicine, it has become more diffi cult to determine fi tness for major surgery. Our data suggest that frailty may be a useful prognostic measure to help inform such decisions. R f gi Results Of the 8,026 patients who underwent cardiac surgery during the study period, BCIAO was implemented in 358 (4.5%) of them. In total, 65.4% were male. Surgical times in those patients where IABP was implanted were 146  ±  81  minutes and 90  ±  66  minutes (cardiopulmonary and aortic clamping times, respectively). The in- surgery room mortality was 4.7%, 30-day mortality in these patients was 40.2%. Patients in whom IABP was implanted had a mortality rate eight times higher than those who did not require it during surgery or postoperatively (40.2% vs. 8.4%, P = 0.0001. OR 8.1, 95% CI (6.4 to 10.3)). Impact of patient frailty on outcome in cardiothoracic surgery J Brohan, P Delaney, B O’Brien Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) Figure 1 (abstract P194). J Brohan, P Delaney, B O’Brien , y, Cork University Hospital, Cork, Ireland Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) y p , , Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc142 Introduction Frailty is defi ned as a multidimensional syndrome involving loss of physical and cognitive reserve leading to greater vulnerability to adverse events [1]. Such events include susceptibility to unplanned hospital admissions, and death [1-3]. Frailty is associated with increased ICU and 6-month mortality, and reduced quality of life [4]. The aim of this study is to investigate the impact of baseline frailty on postoperative quality of life indicators and postoperative frailty following cardiothoracic surgery. Methods Adult patients undergoing cardiac surgery or thoracic surgery (involving thoracotomy) were included in this study. Baseline measures of frailty [4] and performance status were prospectively recorded using validated tools. Informed consent was obtained prior to inclusion. Outcome measures of APACHE II scores, duration of ventilation, length S68 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P198 Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery Besides mortality was higher, the later IABP was implanted the higher the mortality rate was (29.6% of the preoperative, 44.2% of surgical and 54.4% of those starting in ICU, P = 0.015). The ICU length of stay was 9 ± 22 days while the hospital length of stay was 21 ± 28 days. In patients who needed IABP, the ICU stay was higher than for those who did not need it (9 ± 22 vs. 5 ± 10 days, P = 0.002) whereas there was no diff erence in hospital stay (21 ± 28 vs. 20 ± 24 days, P = 0.054). 1. Rockwood K, et al. A global clinical measure of fi tness and frailty in elderly people. CMAJ. 2005;173:489-95. 2. Rockwood K, et al. Changes in relative fi tness and frailty across the adult lifespan: evidence from the Canadian National Population Health Survey. CMAJ. 2011;183:E487-94. 3. Makary MA, et al. Frailty as a predictor of surgical outcomes in older patients. J Am Coll Surg. 2010;210:901-8. 4. Le Maguet P, et al. Prevalence and impact of frailty on mortality in elderly ICU patients: a prospective, multicentre observational study. Intensive Care Med. 2014;40:674-82. P198 of ICU stay and mortality were recorded. Follow-up at 6 months was conducted by telephone to assess recovery patterns. of ICU stay and mortality were recorded. Follow-up at 6 months was conducted by telephone to assess recovery patterns. Registry of Cardiac Surgery MD Delgado-Amaya, C Joya-Montosa, J Muñoz-Bono, E Curiel-Balsera Hospital Regional de Málaga, Spain MD Delgado-Amaya, C Joya-Montosa, J Muñoz-Bono, E Curiel-Balsera Hospital Regional de Málaga, Spain f p y y Conclusion The intra-aortic balloon pump was used by 4.5% of surgeries performed during the study period and in patients with an increased risk of perioperative complications, estimated by EuroSCORE. ICU length of stay was higher in patients requiring IABP, with no diff erences in overall hospital stay. Mortality rate was 40% higher, and increases with the delay in the implantation. p g g p Critical Care 2015, 19(Suppl 1):P197 (doi: 10.1186/cc14277) g g Critical Care 2015, 19(Suppl 1):P197 (doi: 10.1186/cc14277) Introduction The aim of our study was to assess whether the preoperative use of IABP is benefi cial in patients undergoing cardiac surgery of any kind. Introduction The aim of our study was to assess whether the preoperative use of IABP is benefi cial in patients undergoing cardiac surgery of any kind. Methods An observational, retrospective, multicenter study of all patients undergoing cardiac surgery and included in the ARIAM- ANDALUCIA Registry of Cardiac Surgery from March 2008 to July 2012. The probability of placing IABP in the preoperative period has been calculated, making a propensity analysis to obtain two homogeneous groups treated with or without the IABP, based on personal history, functional status and type of surgery. Seventy-seven patients with preoperative IABP were matched with 77 patients without BCIAO with the nearest propensity score. We used the chi-square test or Student t test as needed and binary logistic regression for multivariate analysis so we can rule out possible confounding variables. We used the statistical package R v2.12 for MAC. P199 Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 JL Iribarren, J Jiménez, N Perez, M Brouard, R Perez, E Hurtado, S Diosdado, M Buitrago, A Arbesu, R Martinez, M Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P199 (doi: 10.1186/cc14279) P200 Pharyngeal oxygenation during apnoea following conventional pre-oxygenation and high-fl ow nasal oxygenation D Stolady, M Mariyaselvam, H Young, E Fawzy, M Blunt, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P200 (doi: 10.1186/cc14280) y Methods We performed a prospective randomized cross-over study on hypoxemic non-intubated patients (PaO2/FiO2 ≤300 mmHg) admitted to the ICU of the San Gerardo Hospital and prescribed to receive oxygen by facial mask. We delivered the same air/oxygen mix by HFNC (Optifl ow; Fisher & Paykel Healthcare, Auckland, New Zealand) and facial mask (20 minutes per step). Continuous recordings of regional lung volumes by EIT (Pulmovista 500; Drager Medical GmbH, Lubeck, Germany) and of inspiratory eff ort by esophageal pressure (Pes) were obtained and analyzed offl ine by dedicated software. Introduction We hypothesised that pharyngeal oxygen concentrations would be maintained higher and for longer with transnasal humidifi ed rapid insuffl ation ventilatory exchange (THRIVE) than conventional bag-mask pre-oxygenation (CPO). CPO requires the mask to be removed during laryngoscopy; this means that air may enter the mouth so subsequent apnoeic oxygenation will be less eff ective. Oral suctioning could exacerbate this process. However, if high pharyngeal oxygen concentrations and an open airway are maintained, apnoeic oxygenation could be substantially improved. Methods used have included NO-DESAT [1] and recently THRIVE [2], which has been shown to extend apnoea times for up to 1 hour. yfl y Results We enrolled 15 patients (10 male), age 57 ± 16 years. Compared with standard facial mask, HFNC signifi cantly improved PaO2/FiO2 (199 ± 60 vs. 150 ± 46, P <0.001) and end-expiratory lung impedance (corresponding to aeration) (866  ± 568 au vs. baseline, P  <0.001). Moreover, HFNC decreased the respiratory rate (22 ± 5 bpm vs. 20 ± 5 bpm, P <0.001), as well as negative Pes swings (ΔPes 8.3 ± 5 mmHg vs. 6.6 ± 1 mmHg, P <0.01) and corrected minute ventilation (that is, actual MV × actual PaCO2 / 40 mmHg) (49,887 ± 16,176 au vs. 41,811 ± 14,042 au, P <0.001). Finally, central venous pressure increased (6 ± 5 mmHg vs. 4 ± 5 mmHg, P <0.01), possibly indicating positive end-expiratory pressure eff ect. Methods A volunteer with a nasopharyngeal sampling catheter underwent simulated emergency airway management (EAM), using both CPO and THRIVE, with and without suction. Reference 1. Sotello D, Rivas M, Mulkey Z, Nugent K. High-fl ow nasal cannula oxygen in adult patients: a narrative review. Am J Med Sci. 2015;349:179-85. Results Pharyngeal oxygen concentrations (mean and SEM) are shown in Figure 1 (all points are signifi cant P <0.05). Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 AA, 0.32 (0.11 to 0.65) vs. 0.06 (0.04 to 0.09) μg/kg/minute, P <0.001; GG vs. AG, 0.32 (0.11 to 0.65) vs. 0.06 (0.03 to 0.08), P <0.001)) and at 24 hours (F: 8; P = 0.005), post hoc Scheff é (GG vs. AA, 0.27 (0.01 to 0.52) vs. 0.10 (0.06 to 0.14), P = 0.019; GG vs. AG, 0.27 (0.01 to 0.52) vs. 0.07 (0.04 to 0.09), P = 0.003)). Unfavorable TNF-B (G homozygous vs. allele A) and PAI-1 unfavorable (4G homozygous vs. allele 5G) were grouped, after adjusting for perioperative signifi cant variables. The homozygous GG and 4G alleles were signifi cant for NA 4 hours (F: 5.5; P = 0.02 and F: 4.1; P = 0.04, respectively) and GG–4G allele interaction (F: 6; P = 0.01) (Figure 1), Conclusion The use of IABP prior to cardiac surgery in patients at high risk does not reduce the mortality rate nor the combined endpoint described above. ICU length of stay was greater in those patients in whom IABP was implanted prior to surgery; there were no diff erences in overall hospital stay. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S69 THRIVE. Assessment of NO-DESAT (15 l/minute) was abandoned due to discomfort. R f Figure 1 (abstract P199). THRIVE. Assessment of NO-DESAT (15 l/minute) was abandoned due to discomfort. Figure 1 (abstract P199). P200 Following 3 minutes of pre-oxygenation with CPO (FiO2 = 1, FEO2 >0.8) or THRIVE (60 l/minute; Optifl ow, Fisher and Paykel), EAM was simulated by voluntary apnoea and pharyngoscopy with the laryngoscope blade tip placed 2 cm from the posterior pharyngeal wall. Capnography at the laryngoscope tip confi rmed apnoea. Pharyngeal gas samples (20  ml) were collected during apnoea, and after 5 seconds of oropharyngeal suctioning. Pre- oxygenation was repeated between sampling. Samples (n = 100) were analysed using calibrated fuel cells. f Conclusion In non-intubated hypoxemic critically ill patients, HFNC improves oxygenation and end-expiratory aeration; moreover, HFNC reduces the inspiratory eff ort and the minute ventilation needed to maintain normal arterial CO2 tension. 0 Eff ects of high-fl ow nasal cannula therapy on oxygenation, lung volumes and CO2 removal in critically ill hypoxemic patients: preliminary results p y T Mauri1, N Eronia2, G Bellani2, G Grasselli2, R Marcolin2, S Marocco Arrigoni2, A Pesenti2 1IRCC Ca’ Granda Maggiore Policlinico Hospital, Milan, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P201 (doi: 10.1186/cc14281) Figure 1 (abstract P199). Figure 1 (abstract P199). while for NA at 24 hours statistics showed GG (F: 3.2; P = 0.07), 4G allele (F: 2; P = 0.15) and interaction (F: 3.6, P = 0.05). Conclusion GG homozygous polymorphism TNF-B is associated with an increased dependence on norepinephrine after cardiopulmonary bypass, showing a synergistic action with the 4G allele of PAI-1. while for NA at 24 hours statistics showed GG (F: 3.2; P = 0.07), 4G allele (F: 2; P = 0.15) and interaction (F: 3.6, P = 0.05). Introduction High-fl ow nasal cannula (HFNC) is increasingly proposed as respiratory support for hypoxemic non-intubated acute respiratory failure patients. Clinically, HFNC therapy decreases dyspnea, improves patient’s comfort, improves oxygenation and enhances clearance of upper airway secretions [1]. We present preliminary results from a clinical study aimed at measuring the eff ects of HFNC on gas exchange, lung volumes and inspiratory eff ort in hypoxemic non-intubated critically ill patients. Conclusion GG homozygous polymorphism TNF-B is associated with an increased dependence on norepinephrine after cardiopulmonary bypass, showing a synergistic action with the 4G allele of PAI-1. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness A similar pattern was seen was seen in the vast majority of ICUs: a single institution reported no capnography available. However, in 141 (66.8%) of the hospitals surveyed, no facility to measure ETCO2 was present on the general wards. Where available, 86.7% used capnography to confi rm ETT placement. Less than 50% used ETCO2 to determine CPR eff ectiveness and 8% to prognosticate.i is located in the same position as the light source on the standard Macintosh blade thus providing a view angle of up to 290° and the USB camera is connected to a laptop. A total of the fi rst 50 patients who presented to the emergency department over a period of 6 months in need of intubation were included in the study and every alternate patient participated in the evaluation of the assembled video laryngoscope (VAL). Information about patient demographics and airway characteristics, Cormack-Lehane (C/L) views and the ease of intubation using the VAL was collected. Failure was defi ned as more than one attempt at intubation. given the option to decline participation. All data were anonymised. Results A total of 211 hospitals met the inclusion criteria. The response rate was 100%. Arrest calls were mainly attended by anaesthesia (47.8%) and ICU doctors (38.3%) with around 2% physicians only. Most were a registrar grade (56.3%). The ability to measure ETCO2 was available in all but four EDs; most used waveform capnography. A similar pattern was seen was seen in the vast majority of ICUs: a single institution reported no capnography available. However, in 141 (66.8%) of the hospitals surveyed, no facility to measure ETCO2 was present on the general wards. Where available, 86.7% used capnography to confi rm ETT placement. Less than 50% used ETCO2 to determine CPR eff ectiveness and 8% to prognosticate. Results Excellent (C/L1) or good (C/L2) laryngeal exposure was obtained in 92% and 8% of patients respectively. In 25 patients in whom VAL was performed, there was a comparable or superior view. Intubation with direct laryngoscopy was successful in 95.2% of patients and VAL was successful in 95.4% of patients. Three patients from the VAL group and four patients from the direct laryngoscopy group were excluded. See Figure 1. Conclusion We believe this is the fi rst study of its kind to follow NAP4 and investigate the availability of capnography throughout for use during cardiac arrest. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness Whilst equipment levels appear adequate (albeit not perfect) in resuscitation areas, there appears a lack of availability of suitable devices on general wards. Figure 1 (abstract P202). New video laryngoscope connected to laptop. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness i Conclusion Pharyngeal oxygen concentration rapidly falls following CPO. This may be detrimental for apnoeic oxygenation during con ven- tional laryngoscopy. Conversely, THRIVE maintains high pharyngeal oxygen concentrations over time. Suction has an immediate negative eff ect on pharyngeal oxygen concentration that is attenuated by f SM Ayyan, Z Ali Figure 1 (abstract P200). Pharyngeal oxygen concentration. Introduction Video laryngoscopes have been introduced in recent years as an alternative choice to facilitate tracheal intubation. Diffi culties with tracheal intubation are mostly caused by diffi cult direct laryngoscopy with impaired view to the vocal cords. Many endoscopic intubation laryngoscopes have been designed to visualize the vocal cords around the corner looking through a proximal viewfi nder. Although they are useful devices, they have limitations for doing direct laryngoscopy and are very expensive, hence they are not used for routine tracheal intubation.i Methods A Macintosh intubating laryngoscope has been modifi ed by attaching a waterproof USB camera with a inbuilt light source, which S70 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 is located in the same position as the light source on the standard Macintosh blade thus providing a view angle of up to 290° and the USB camera is connected to a laptop. A total of the fi rst 50 patients who presented to the emergency department over a period of 6 months in need of intubation were included in the study and every alternate patient participated in the evaluation of the assembled video laryngoscope (VAL). Information about patient demographics and airway characteristics, Cormack-Lehane (C/L) views and the ease of intubation using the VAL was collected. Failure was defi ned as more than one attempt at intubation. examining practice regarding intubation for cardiac arrest and the availability and utilisation of capnography within the ED, ICU and general wards. Questions were directed at the anaesthetist or intensive care doctor ‘responding to cardiac arrest calls’. The respondent was given the option to decline participation. All data were anonymised. Results A total of 211 hospitals met the inclusion criteria. The response rate was 100%. Arrest calls were mainly attended by anaesthesia (47.8%) and ICU doctors (38.3%) with around 2% physicians only. Most were a registrar grade (56.3%). The ability to measure ETCO2 was available in all but four EDs; most used waveform capnography. P203 P203 Availability of appropriate airway monitoring at UK in-hospital cardiac arrest S Turle1, PB Sherren1, T Callaghan1, S Nicholson2, SJ Shepherd1 1Pan-Thames Intensive Care Training Scheme, London, UK; 2KSS Intensive Care Training Scheme, London, UK Critical Care 2015, 19(Suppl 1):P203 (doi: 10.1186/cc14283) Using a laryngoscope and endotracheal tube succeeds in a diffi cult case of nasogastric tube insertion J P k Y L Using a laryngoscope and endotracheal tube succeeds in a diffi cult case of nasogastric tube insertion J Park, Y Lee Ewha Womans Unversity Hospital, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P204 (doi: 10.1186/cc14284) , Ewha Womans Unversity Hospital, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P204 (doi: 10.1186/cc14284) Introduction Nasogastric (NG) tube insertion is necessary in a variety of critically ill patients for intra-abdominal decompression, prevention of aspiration, route of medication administration and nutrition. However, it often fails in patients who showed sedated or comatose mentality with poor cooperation during the procedure. Although there are many reports inserting a NG tube in diffi cult cases, most methods need a special guide wire, tube or nasoendoscope. We report a case of NG tube insertion in a comatose patient using a laryngoscope and endotracheal tube which are easily available. Figure 1 (abstract P202). New video laryngoscope connected to laptop. Conclusion This new assembled VAL is the cheapest video-assisted laryngoscope available costing around $60, which can even be introduced into primary healthcare setup in developing countries. VAL consistently yielded a comparable or superior glottic view compared with direct laryngoscopy despite the limited or lack of prior experience with the device. Because the device can be used for both routine as well diffi cult tracheal intubation, it may be a helpful tool to intubate trauma cases where C-spine immobilization is unavoidable. The presented video-assisted laryngoscope is a useful tool for documentation, teaching and monitoring tracheal intubation. Figure 1 (abstract P204). Insertion of a nasogastric tube through an endotracheal tube in the esophagus. References 1. Grmec S. Intensive Care Med. 2002;28:701-4. 2. Deakin CD, et al. Resuscitation. 2010;81:1305-52. 3. Cook TM, et al. Br J Anaesth. 2011;106:617-31. 1. Grmec S. Intensive Care Med. 2002;28:701-4. Admissions with airway emergencies in the ICU at a tertiary referral centre Results Forty-fi ve patients (31 male) were included with a mean age of 67  ± 15  years. The commonest admission diagnoses were sepsis (10), cardiogenic shock (10), primary respiratory failure (nine) and intracranial haemorrhage (eight). The median transfer delay time was 47 minutes. Only 27 (60%) patients were actually transferred and they were signifi cantly younger than nontransferred patients (62 vs. 73 years, P = 0.02). In-transit mortality was zero. Mean length of stay in the critical care centre was 14.8 ± 16.8 days. Survival to discharge was signifi cantly higher in transferred (14/27) compared with nontransferred (3/18) patients (52% vs. 17%, P = 0.017). Overall mortality rates were 62% and 69% at 1 and 6 months respectively and were signifi cantly lower in the transferred group (P = 0.02). M Gresoiu, M Singer M Gresoiu, M Singer University College London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P205 (doi: 10.1186/cc14285) Introduction As a tertiary referral centre for ENT and maxillo-facial surgery, our ICU receives complex elective and emergency cases. The frequency, aetiology and outcomes of airway emergencies are poorly described. Understanding these factors is key to improving management. g Methods We conducted a retrospective review of the ICU electronic patient database examining unplanned admissions with airway emergencies between December 13 and November 14. Data on demographics, aetiology of airway obstruction (including postprocedural), APACHE II score, therapeutic intervention(s) administered, and outcomes were collected. Conclusion Overall mortality rates of medical patients intubated urgently at BGH were high. Forty per cent of intubated patients were not transferred, indicating signifi cant modifi cation of the protocol over time. Patients transferred to the critical care centre were younger and had signifi cantly better outcomes than patients remaining in BGH, probably due to decisions not to transfer patients with poor prognoses. Most patients who survived to discharge were still alive 6 months later. Reference Results Of 1,516 unplanned admissions, airway emergencies represented 6.3% (96 patients) of whom 40 (41.7%) had malignancy (26 maxillo-facial/trachea, three pulmonary, four haematological, seven other) and 24 infection (abscesses, epiglottitis, Ludwig’s angina). Referring specialties were maxillo-facial surgery (n  = 34), internal medicine (n = 25), ENT (n = 21) and other surgical specialties (n = 16). Thirteen patients had complications post bronchoscopy (vocal cord palsy, need for NIV or intubation), one post microlaryngoscopy, and 20 were admitted after diffi cult intubation. Outcomes of medical patients requiring emergency intubation in a rural Irish hospital Outcomes of medical patients requiring emergency intubation in a rural Irish hospital Results A 15-year-old male patient admitted due to abrupt mental change and brain imaging showed severe subdural hemorrhage. NG tube insertion was done for enteral feeding but failed several times though changing position. As we had no guide wire and no nasoendoscope, an endotracheal tube was used as guidance for the NG tube. After making a longitudinal midline cut on the endotracheal tube, it was inserted into the esophagus under a laryngoscope. The NG tube was pushed into the endotracheal tube, and then the endotracheal tube was removed through the cut, reserving the NG tube. We checked the position of the NG tube by air sound and X-ray, and started enteral feeding without complication, such as nasal bleeding, emphysema, and gastric perforation. See Figure 1. p A O’ Connor, MP D’Alton, B Carey Bantry General Hospital, Co. Cork, Ireland Critical Care 2015, 19(Suppl 1):P206 (doi: 10.1186/cc14286) Introduction Bantry General Hospital (BGH) is a small rural hospital serving a large, geographically isolated part of southwest Ireland. Following an infl uential national review of adult critical care services [1], a protocol was introduced in late 2010 mandating the immediate transfer of all medical patients intubated on an emergency basis to a large critical care centre 100 km away. Similar mandatory transfer protocols were introduced at the same time throughout the island of Ireland but few data are available regarding patient outcomes. We designed a study to look at the outcomes of all patients encompassed by the protocol at BGH. Conclusion We report a new method of NG tube insertion using a laryngoscope and endotracheal tube. y Methods We retrospectively reviewed the charts and electronic data of medical patients requiring emergency intubation at BGH from November 2010 to December 2013. We recorded the following data: age, sex, admission diagnosis, comorbidities, time delay to transfer, in- transit mortality, length of stay, survival to discharge and 1-month and 6-month mortality.i p Reference 1. Towards Excellence in Critical Care. Review of adult critical care services in the Republic of Ireland fi nal report. Submitted to the Health Service Executive; September 2009. Admissions with airway emergencies in the ICU at a tertiary referral centre Eighteen were admitted post drainage of abscess (dental, retropharyngeal) and seven for observation for epiglottitis. Thirteen patients had stridor (three tracheal stenosis, one vocal cord cyst, one post CVA, four post vocal cord palsy, one post oesophagoscopy, one post thyroidectomy, two post decannulation). Seven were admitted after emergency tracheostomy, one after blocked tracheostomy, one after emergency laryngectomy, six post bleeding (epistaxis, haemoptysis, bleed form laryngectomy site), and four post evacuation haematoma. Three were admitted following anaphylaxis/ angioedema and one after laryngospasm. Twenty-nine patients required medical management only (for example, steroids, nebulisers, and so forth), 25 were extubated post diffi cult intubation and six needed haemostasis control. Ten (nine surgical) tracheostomies were performed during their ICU stay. Sixteen patients died in hospital, of whom fi ve were in the ICU at the time; 14 of these had an underlying malignancy. Twenty-three patients deteriorated during their ICU stay including HAP (n = 3), bleeding from airway (n = 3), PEA arrest (n = 1), airway swelling (n = 2), blocked laryngectomy (n = 1), and tracheostomy dislodgement (n = 1). 03 Availability of appropriate airway monitoring at UK in-hospital cardiac arrest S Turle1, PB Sherren1, T Callaghan1, S Nicholson2, SJ Shepherd1 1Pan-Thames Intensive Care Training Scheme, London, UK; 2KSS Intensive Care Training Scheme, London, UK Critical Care 2015, 19(Suppl 1):P203 (doi: 10.1186/cc14283) Introduction Airway complications are more common outside the operating theatre and in emergency situations. Capnography remains the gold standard of confi rming correct endotracheal tube (ETT) placement, retaining high sensitivity and specifi city in cardiac arrest [1]. The 2010 European Resuscitation Council guidelines for adult advanced life support recommended waveform capnography in this setting [2]. Failure to use capnography was also identifi ed as a major contributor to airway-related morbidity and mortality in a national UK audit [3]. We sought to investigate current practice relating to the availability and use of capnography equipment cardiac arrest within UK hospitals. Methods Between June and November 2014, a telephone survey was conducted of all UK acute hospitals with adult level 3 ICUs and an emergency department (ED). Hospitals were identifi ed using nationally available data. A standardised telephone questionnaire was developed Figure 1 (abstract P204). Insertion of a nasogastric tube through an endotracheal tube in the esophagus. S71 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 http://ccforum.com/supplements/19/S1 Methods A NG tube was inserted using a laryngoscope and endotracheal tube. Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes Centro Hospitalar Tâmega e Sousa, Penafi el, Portugal Critical Care 2015, 19(Suppl 1):P209 (doi: 10.1186/cc14289) Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes g Centro Hospitalar Tâmega e Sousa, Penafi el, Portugal Critical Care 2015, 19(Suppl 1):P209 (doi: 10.1186/cc14289) g g j p Results A total of 348 patients were included. VL attained better glottic visualization than DL (92.3% vs. 82.6%, respectively: P <0.001). In total, 299 patients with good glottic visualization were included in the analysis. Of these patients, 185 (61.9%) were male, median age and body mass index were 69 (interquartile range (IQR), 51 to 77) and 22 (IQR, 20 to 24) respectively. In univariate analysis, VL group had less respiratory failure (18.3% vs. 46.8%: P <0.001) and included more trauma patients (21.1% vs. 7.9%: P <0.001). The fi rst-attempt success rates were similar between two groups (82.6% vs. 77.4%: P = 0.286). Multivariable logistic regression analysis adjusted for potential confounders showed that the success rate of VL was similar to that of DL (odds ratio, 1.17; 95% confi dence interval, 0.57 to 2.39).i Introduction β-Adrenergic agonists increase the ciliary beat frequency in experimental models, raising the possibility that they may be useful for airway hygiene [1]. Salbutamol increases large airway mucociliary clearance [2], although this may not be true for smaller airways [3]. There are no data from ICU patients, so we decided to test the eff ectiveness of transtracheal instillation of a mixture of ipratropium and salbutamol, assessing the reduction of the number of aspirations and hours of ventilation. Methods An open randomized prospective study was held during 2014. All admitted patients were alternately selected as the study or control group and included if submitted to invasive ventilation for at least for 24 hours. Four patients who had secondary cardiac rhythm eff ects attributable to β-adrenergic agonists were excluded. In the study group, 3 ml of a dilution of 2.5 ml ipratropium (0.52 mg) plus salbutamol (2.5 mg) with 2.5 ml distilled water was instilled after the aspiration of secretions. During the ventilation period we noted for both groups, in addition to the demographic data, the number of tracheal aspirations by day and hours of ventilation. The statistical analysis was done with XLSTAT 2014 and we used the Kolmogorov– Smirnov test to compare the normal distribution of the groups. P210 Simultaneous use of a heat and moisture exchanger and a heated humidifi er causes critical airway occlusion in less than 24 hours M Mariyaselvam, A Doyle, G Wijewardena, N English, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P210 (doi: 10.1186/cc14290) p , g y , Critical Care 2015, 19(Suppl 1):P210 (doi: 10.1186/cc14290) q Results There were 62 RSI intubations using THRIVE (ICU and ED = 30; OR = 33). Diffi cult airway equipment used in 36 cases (videolaryngoscopy in 23). Mean apnoea time was 118 seconds (30 to 480 seconds), with a median SpO2 fall of 1% (0 to 33%). There was no correlation between arterial desaturation and apnoeic time. OR cases had a mean apnoea of 113 seconds with a median SpO2 fall of 0% (0 to 13%). ICU and ED cases had a mean apnoea time of 119 seconds and median SpO2 fall of 1% (0 to 33%). THRIVE was universally readily accepted. Reasons cited included: simplifi cation of pre-oxygenation (hands free) and increased confi dence. Six outlying arterial desaturation events suggested poor airway maintenance at induction or use in particularly high-risk patients. Many anaesthetists reinstituted THRIVE following extubation in selected patients (for example, obesity). No complications occurred during implementation. Introduction We hypothesised that the simultaneous use of a heat and moisture exchanger (HME) and a heated humidifi er (HH) would increase the incidence of airway occlusion over a 24-hour period in comparison with each device in isolation. This bench study compares the incidence of airway occlusion when using (group 1) no airway humidifi cation, (group 2) a HME alone, (group 3) a HH alone and (group 4) both a HME and a HH in combination. Tracheal intubation requires the use of artifi cial humidifi cation systems. HMEs are less effi cient but convenient especially for a short period of intubation and HHs are commonly more expensive. Both devices are often used in close proximity on the ICU depending on the particular clinical scenario and/ or clinical practitioner. Following a critical incident of HME obstruction due to waterlogging on our ICU we realised that HH and HME may be used together inadvertently. This airway obstruction was only resolved by the removal of the HME from the patient’s breathing circuit. Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction M Mariyaselvam, D Stolady, G Wijewardena, M Blunt, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P208 (doi: 10.1186/cc14288) Results These preliminary results included 107 patients. The only normal distribution, according to the Kolmogorov–Smirnov test, was for the number of hours of ventilation: 76.3 ± 100 (24; 478) in the study group versus 111 ± 167 (24; 780) in the control group.i Conclusion The preliminary analysis, referring to the fi rst 6 months of study, showed a tendency in the reduction of both ventilation hours and length of stay in the study group. No signifi cant diff erence was found in the number of aspirations, which may be explained by ICU nursing routines. Further studies will try to fi nd whether signifi cant diff erences in the incidence of VAP exist, allowing this procedure to be implemented in ICU routines. Introduction Rapid sequence induction (RSI) in the ICU, emergency department (ED) and operating room (OR) carries the risk of hypoxemia if laryngoscopy is prolonged especially in high-risk patients. Bag and mask pre-oxygenation is normally used to extend the apnoea time; however, arterial desaturation may still rapidly occur. Transnasal humidifi ed rapid insuffl ation ventilatory exchange (THRIVE) is a new technique that provides modest CPAP during pre-oxygenation and crucially also continuous oxygenation of the pharyngeal space throughout the apnoeic period. In elective surgery, THRIVE provides apnoea times as long as 60 minutes due to apnoeic oxygenation [1]. We report the fi rst implementation of THRIVE with emergency patients into the ICU, ED and OR. References 1. Frohock JI, Wijkstrom-Frei C, Salathe M. Eff ects of albuterolenantiomers on ciliary beat frequency in ovine trachealepithelial cells. J Appl Physiol. 2002;92:2396-402. 1. Frohock JI, Wijkstrom-Frei C, Salathe M. Eff ects of albuterolenantiomers on ciliary beat frequency in ovine trachealepithelial cells. J Appl Physiol. 2002;92:2396-402. 2. Lafortuna CL, Fazio F. Acute eff ect of inhaled salbutamol onmucociliary clearance in health and chronic bronchitis. Respiration. 1984;45:111-23. 2. Lafortuna CL, Fazio F. Acute eff ect of inhaled salbutamol onmucociliary clearance in health and chronic bronchitis. Respiration. 1984;45:111-23. 3. Svartengren K, Philipson K, Svartengren M, Camner P. Eff ect ofadrenergic stimulation on clearance from small ciliatedairways in healthy subjects. Exp Lung Res. 1998;24:149-58. Methods Following training a THRIVE system was installed in each location either as a fi xed system on the anaesthetic machine (OR) or a mobile solution on a wheeled stand (ICU, ER). This was a simplifi ed Optifl ow system (Fisher and Paykel, New Zealand) consisting of a high-fl ow rotameter, a reusable humidifi er, a reusable circuit and a disposable nasal interface. Anaesthetists of all grades were encouraged to use THRIVE (60 l/minute) prior to and during all high-risk intubations. Prospective data of pre and post intubation SpO2 and time to intubate were collected. Anaesthetists were interviewed on acceptability of the technique. 1. Patel A, et al. Anaesthesia. 2014 [Epub ahead of print]. Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes i Conclusion Despite the possible poor alignment of airway, the fi rst- attempt success rate of VL is similar to that of DL among patients with good glottic visualization. P210 Methods A lung simulator underwent pressure controlled ventilation Conclusion We conclude that THRIVE provides a convenient, safe and easy to implement technique for pre-oxygenation and apnoeic oxygenation during laryngoscopy. y p g Methods A lung simulator underwent pressure-controlled ventilation (Pinsp = 25 cmH2O; PEEP = 5 cmH2O; Vt = 500 ml) for 24 hours for seven P209 The primary exposure was use of VL. Potential confounders of success rate examined were age, sex, primary indication of intubation, methods of intubation, and operator level of training and specialty. Among patients with good glottic visualization, we conducted a multivariable logistic regression adjusted for potential confounders. Comparison of video laryngoscopy with direct laryngoscopy in patients with good glottic visualization: an observational study of 348 emergency intubations g y Y Kato, H Okamoto, H Uchino, T Fukuoka Kurashiki Central Hospital, Kurashiki Okayama, Japan Critical Care 2015, 19(Suppl 1):P207 (doi: 10.1186/cc14287) g y Y Kato, H Okamoto, H Uchino, T Fukuoka Introduction Video laryngoscopy (VL) is known to improve glottic visualization and the fi rst-attempt success rate compared with direct laryngoscopy (DL) in emergency tracheal intubations (ETIs). Since VL does not align the oral, pharyngeal, and laryngeal axes of the upper airway, it sometimes leads to failed intubation despite good glottic visualization. We tested the hypothesis that VL has a lower fi rst- attempt success rate of ETI than DL among patients with good glottic visualization. Methods We performed a prospective observational study examining ETIs at our tertiary care institution from July 2012 to June 2014. All consecutive patients who underwent ETIs in the emergency department and ICU were included. Patients under 18  years of age, intubated with VL not using C-MAC, were excluded. After each ETI eff ort, the operator completed a standardized data collection form. We classifi ed glottic visualization as good (C-L grade 1 or 2), and poor (C-L grade 3 or 4). The primary outcome was the fi rst-attempt success rate. Conclusion Marked variation was seen in the type and aetiology of airway emergencies admitted to the ICU. A broad training programme is thus required to off er wide-ranging awareness of potential problems, communication (including an emergency airway plan), and acute management. S72 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P211 P211 Endobronchial streptokinase for airway thrombus: a case series D Lloyd, J Bomford, M Barry, W Berry, N Barrett, L Camporota, N Ioannou, B Lams, C Langrish, C Meadows, A Retter, D Wyncoll, G Glover Guys and St Thomas’ NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P211 (doi: 10.1186/cc14291) Introduction Pulmonary haemorrhage (PH) is common in patients receiving mechanical ventilation and especially during ECMO, due to severe lung pathology and systemic anticoagulation. Whilst PH manifests as worsening ventilation and gas exchange, in ECMO patients who already have low tidal volume and who do not rely on pulmonary gas exchange, deterioration may not be evident until extensive airway thrombus (AT) has developed. Management of AT is challenging, with lavage, suctioning, mechanical disruption and extraction of limited effi cacy in severe cases. Limited reports suggest that topical thrombolytics may have a role in the management of AT [1]. We report the safety and effi cacy of endobronchial streptokinase (EBSK) in patients with extensive AT. Results Control group: 100 patients who received standard intubation and treatment in 2009 to 2010. Of these, four dropped out due screening failure of pneumonia on the day of enrollment. Study group: in 2011 to 2014, 192 patients were screened. Of these, 49 were found eligible and were enrolled. Of these, 14 dropped out (four screening failures with pneumonia on day of enrollment and 10 withdrawals with MV of less than 24 hours). Mean age was 51 (control) and 49 (study). Males were 75% of both groups and mean weight was 81 kg in both. VAP was diagnosed in 26 (27%) of controls and only three (8.5%) of the study group (P = 0.03). Mean time from admission to VAP diagnosis was 4.7 days in controls versus 5.12 in the study group (NS). No serious adverse events occurred. Methods A retrospective case series in a UK ECMO centre. Patients who received EBSK between 2010 and 2014 were identifi ed from pharmacy records. Conclusion Patients connected to the AnapnoGuard system demonstrated a statistically signifi cant lower VAP rate compared with the control group (8.5% vs. 27% respectively, P = 0.03). The estimated relative risk of VAP occurring in the control group was more than three times higher than the study group. Rinsing and aspiration of subglottic secretions combined with cuff pressure and seal management may be an eff ective method to prevent VAP. P211 Results Five patients were identifi ed, 80% were male. Median age was 40 years, APACHE II score 36.5 and Murray score 3.75. Four were on ECMO with systemic heparin. All had ARDS secondary to lung infections (community-acquired pneumonia (two), lung abscess (one), TB (one) and PJP (one)). All had extensive AT, diagnosed on bronchoscopy, causing occlusion of the trachea or major bronchi, refractory to physiotherapy, lavage, suctioning ± rigid bronchoscopy. Patients received up to three administrations of EBSK, 1,000 u/ml in saline 0.9% under bronchoscopic guidance. Dose per administration was 30,000 to 80,000 u and total dose was 30,000 to 150,000 u (375 to 1,500  u/kg), with interval bronchoscopy after several hours for lavage and suctioning of lysed clot. In all cases EBSK was well tolerated with no immediate complications and no clinically signifi cant change in systemic laboratory coagulation parameters at 12 or 24  hours compared with pretreatment baseline. In all cases, signifi cant clearance of airway thrombus was achieved. Median tidal volume increased from 60 ml pre treatment to 170 ml at 24 hours. Median PaO2 during the ‘FiO2 1.0 test’ improved from 9.0 to 17.6 kPa at 24 hours. No major bleeding, intracerebral haemorrhage or ECMO cannulae bleeding was seen up to 7 days post treatment.i P213 Risk factors for bleeding complications after percutaneous dilatational tracheostomy: a 10-year institutional analysis K Pilarczyk1, G Marggraf1, M Dudasova1, E Demircioglu1, D Wendt1, B Huschens2, H Jakob1, F Dusse1 1West German Heart Center Essen, University Hospital Essen, Germany; 2University Hospital Essen, Germany Critical Care 2015, 19(Suppl 1):P213 (doi: 10.1186/cc14293) Introduction Percutaneous dilatational tracheotomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. Bleeding complications after PDT are infrequently observed but have a tremendous impact on further clinical course CFA risk stratifi cation for patients scheduled for PDT. Introduction Percutaneous dilatational tracheotomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. Bleeding complications after PDT are infrequently observed but have a tremendous impact on further clinical course CFA risk stratifi cation for patients scheduled for PDT. Conclusion In this series, the largest reported to date, and the fi rst on ECMO, EBSK was highly eff ective in achieving clearance of AT with subsequent improvements in pulmonary mechanics and gas exchange. No major disturbance of systemic coagulation parameters or major haemorrhagic complications occurred. P212 test periods for each group (n = 24). A HME (Filta-Therm; Intersurgical, Berkshire, UK) was placed between the breathing circuit and catheter mount or the HH (MR850; Fisher & Paykel, Auckland, New Zealand) was used, or both in combination. Circuit manipulation was performed 4-hourly to simulate patient movement. Hourly Vt was recorded to determine airway occlusion. Critical airway occlusion (defi ned as a drop on the TV to <50 ml) was assessed using a Fisher’s exact test. 1. Keane et al. Chest. 1999;115:293-300. P211 The use of EBSK may be considered for refractory AT. Methods We retrospectively reviewed the records of all patients who underwent PDT (using the Ciaglia technique with bronchoscopic guidance) on our cardiothoracic ICU between 2003 and 2013. Patients were stratifi ed into two groups: patients suff ering from acute moderate, severe or major bleeding (Group A) and patients who presented none or only mild bleeding (Group B). y Reference S73 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Reduction of ventilator-associated pneumonia using the AnapnoGuard system Reduction of ventilator-associated pneumonia using the AnapnoGuard system p y Y Bar-Lavie Rambam Medical Center, Haifa, Israel Critical Care 2015, 19(Suppl 1):P212 (doi: 10.1186/cc14292) Introduction Ventilator-associated pneumonia (VAP) is a common complication in mechanically ventilated patients. Frequently the pathogens responsible derive from aspirated secretions of the upper respiratory tract or the stomach. In order to prevent aspiration, two missions should be attained: a good tracheal cuff seal with a well- tolerated pressure, together with continuous evacuation of secretions from the subglottic space. These two goals can be achieved using the AnapnoGuard system and its related endotracheal tube (ETT). Results In all seven of the breathing circuits in group 4 (both a HME and a HH in combination), critical airway occlusion occurred suddenly between 19 and23  hours. No episodes occurred in the other three groups (P <0.0001). g p Conclusion The combination of the use of HME and HH within a single ICU risks inadvertent dual use in a single patient and if uncorrected this is likely to result in a ventilator circuit obstruction. Medical errors can be mitigated by consideration of human factors and system engineering to improve patient safety. A focus on clinical awareness and training may lead to improvements; however, the numbers, experience and turnover of critical care staffi ng would indicate that a systems approach is appropriate and either HME or HH should be used exclusively in an ICU. y Methods A single-center, open-label study in a general ICU. Control group: (retrospective data) mechanically ventilated patients on standard of care regular ETT, manual suction of the trachea and oral– pharyngeal space by nursing staff . Study group: (prospective data) connected at all times to the AnapnoGuard system: an ETT with two above-the-cuff suction ports and a third port and lumen for rinsing and CO2 measurement. A triple lumen harness is connected to a control system designed to measure CO2 levels above the cuff (to identify leaks), infl ate the cuff accordingly, rinse and suction secretions above the cuff . To be included in the study patients had to have no pneumonia on admission and at least 3 days of mechanical ventilation. VAP was diagnosed for a new chest X-ray infi ltrate accompanied by fever, leucocytosis and positive sputum culture. The study was approved by the hospital IRB. Reference Study groups showed signifi cant diff erences in Simplifi ed Acute Physiology Score (SAPS) on the day of PDT (Group A: 47.0 ± 13.1, Group B: 32.9 ± 11.2, P = 0.042), renal replacement therapy on the day of PDT (Group A: 53 (60.2%), Group B: 439 (48.1%), P  = 0.026), presence of coagulopathy (Group A: 48 (54.5%), Group B: 393 (43.0%), P = 0.043), platelet count (Group A: 91.6 ± 59.2, Group B: 111.5 ± 79.8 × 1,000/μl, P = 0.037), fi brinogen levels (Group A: 373.6 ± 159.1, Group B: 450.6 ± 259.0 mg/dl, P = 0.012), proportion of PDTs performed by residents (Group A: 72 (81.8%), Group B: 632 (69.2%), P = 0.034) and moderately to very diffi cult PDT (Group A: 31 (35.2%), Group B: 141 (15.4%), P = 0.001). Using logistic regression analyses, diffi cult PDT, low-experienced operator, SAPS >40 and low fi brinogen were independent predictors of relevant bleedings after PDT. Methods The study was conducted in a Danish eight-bed, non- university ICU. Since 2007, all patients admitted to the ICU have been registered on an electronic patient record system, in which daily vital values, diagnoses, procedures and healthcare providers’ notes are entered. When searching for ‘percutaneous dilatation tracheostomy’ in the electronic system, we found all patients who had undergone this specifi c procedure. Afterwards we analyzed each of these patients’ hospital records, looking for any periprocedure or postprocedure complications noted within 7 days. In addition we registered patients’ age, sex, BMI, SOFA score, methods used in procedures and experience of operators. p Results A total of 136 patients admitted to the UCI had undergone a PDT between 2007 and 2014. Of these, two were excluded due to the PDT being performed in another hospital before admission to our ICU. All 134 PDTs were performed with the Ciaglia Blue Rhino Method. No PDTs were performed with bronchoscopic guidance. In 12 cases some kind of complication due to the PDT was registered: six cases with need of surgical hemostasis, three cases of bleeding with need of transfusion of blood products, one case of PDT displacement, one case of ventilation-related problems during procedure and, fi nally, one case of tracheal cartilage fracture. There were no incidents of pneumothorax. No PDTs had a lethal outcome due to the procedure itself. The total complication rate was 9.0%. Reference Results A total of 1,001 patients (46% male, mean age 68.1  years) that underwent PDT were analyzed. In the majority of patients, no or S74 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of PDT are described in the literature, each with disadvantages and benefi ts. The aim of this study was to analyze complications due to PDTs performed without the use of bronchoscopic assistance. only mild bleeding during PDT occurred (none: 425 (42.5%), mild: 488 (48.8%)). In 84 patients (8.4%), bleeding was classifi ed as moderate. Three patients suff ered from severe bleeding, only one major bleeding with need for emergency surgery was observed. Study groups showed signifi cant diff erences in Simplifi ed Acute Physiology Score (SAPS) on the day of PDT (Group A: 47.0 ± 13.1, Group B: 32.9 ± 11.2, P = 0.042), renal replacement therapy on the day of PDT (Group A: 53 (60.2%), Group B: 439 (48.1%), P  = 0.026), presence of coagulopathy (Group A: 48 (54.5%), Group B: 393 (43.0%), P = 0.043), platelet count (Group A: 91.6 ± 59.2, Group B: 111.5 ± 79.8 × 1,000/μl, P = 0.037), fi brinogen levels (Group A: 373.6 ± 159.1, Group B: 450.6 ± 259.0 mg/dl, P = 0.012), proportion of PDTs performed by residents (Group A: 72 (81.8%), Group B: 632 (69.2%), P = 0.034) and moderately to very diffi cult PDT (Group A: 31 (35.2%), Group B: 141 (15.4%), P = 0.001). Using logistic regression analyses, diffi cult PDT, low-experienced operator, SAPS >40 and low fi brinogen were independent predictors of relevant bleedings after PDT. only mild bleeding during PDT occurred (none: 425 (42.5%), mild: 488 (48.8%)). In 84 patients (8.4%), bleeding was classifi ed as moderate. Three patients suff ered from severe bleeding, only one major bleeding with need for emergency surgery was observed. Percutaneous dilatational tracheostomy: complications and safety without the use of bronchoscopic guidance Sygehus Lillebælt, Vejle, Denmark Sygehus Lillebælt, Vejle, Denmark yg j Critical Care 2015, 19(Suppl 1):P214 (doi: 10.1186/cc14294) Reference Of the 12 cases, four (33%) complications occurred during the procedure, the rest (66%) occurred after the procedure. The overall periprocedure complication rate was 3%. Conclusion In this study, PDTs without the use of bronchoscopic guidance were performed safely with a low rate of complications. Conclusion Periprocedural bleeding complications during PDT are rare. However, low fi brinogen levels as well as diffi cult PDT, low-experienced operator and SAPS >40 are associated with an increased risk for bleeding complications. Therefore, preprocedural risk evaluation for bleeding complications should include these factors and further studies are necessary to prove whether modifi cation of risk factors – for example, substitution of fi brinogen prior to PDT – is able to reduce incidence of bleeding complications. P215 Introduction Since the introduction and development of percu- taneous dilatational tracheostomy (PDT), this procedure is accepted and incorporated in ICUs worldwide. In spite of obvious benefi ts for the patients, who obtain more comfort and mobility and less use of sedatives, the procedure also implies the risk of several complications, some of which may be lethal. Severe complications include hemorrhage, displacement and pneumothorax. Diff erent methods P216 Impact of antibiotic therapy during a bedside percutaneous tracheotomy procedure in an ICU E Brotfain1, A Borer1, L Koyfman1, A Frenkel1, S Gruenbaum2, A Smolikov1, A Zlotnik1, M Klein1 1Ben Gurion University of The Negev, Soroka Medical Center, Beer Sheva, Israel; 2Yale University School of Medicine, New Haven, CT, USA Critical Care 2015, 19(Suppl 1):P216 (doi: 10.1186/cc14296) Results In total 12,839 records of patients discharged from the ICU were analyzed. Tracheostomy was present in 133 patients. Two groups were defi ned: (1) TCU (n = 56) and (2) GW (n = 77). Patients of the TCU group were older (60.1 ± 13.1 vs. 54.9 ± 15.8 years; P <0.05) with higher APACHE II score (23 (CI: 21.5 to 25.6) vs. 18.5 (CI: 17.1 to 19.9); P <0.001), and had longer stay in the ICU (45.8 (CI: 38.2 to 53.3) vs. 28.4 (CI: 24.2 to 32.6) days; P <0.001) and on the ward (71.1 (CI: 57.4 to 84.8) vs. 46.1 (CI: 33.7 to 58.2) days; P <0.001) than those of the GW group. The GW group had category 1 of Sabadell score more frequently than the TCU group (25.9% vs. 8.9%; P = 0.019). Rates of nosocomial infections were similar in both groups. No signifi cant diff erences on vasoactive use (50% vs. 40.2%), renal failure (23.2% vs. 20.7%), blood transfusions (25% vs. 23.2%), parenteral nutrition (10.7% vs. 12.9%), in-hospital deaths (14.3% vs. 24.6%), decannulation (55% vs. 50%), or discharge to home (53.6% vs. 37.7%) were found between groups 1 and 2, respectively. Introduction Percutaneous bedside tracheostomy (PBT) is a frequently done procedure in the ICU. PBT is a clean-contaminated procedure, and the duration of the procedure is 15 to 20 minutes depending on the physician’s procedural skills. The rate of infectious complications and effi cacy of perioperative therapy in reducing infections after PBT is currently unknown. Currently there have been no defi nitive recommendations for prophylactic antibiotic therapy before PBT in the ICU. Methods All clinical and microbiological data were retrospectively collected and analyzed during the ICU stay before PBT performance and 72 hours after the PBT procedure from 110 patients in our ICU. Controls were defi ned as patients in whom the PBT procedure was performed in the ICU, with antibiotics administered 72 hours prior to and during the procedure (Group 1, n  = 82). P216 Cases were defi ned as patients in whom the PBT procedure was performed in the ICU without antibiotics administered 72 hours prior to and during the procedure (Group 2, n = 28). Secondary bacteremia, line sepsis and VAP during the 72 hours after PBT were considered infectious complications. Two- tailed P <0.05 was considered to be signifi cant.f Conclusion In our setting the TCU helps to care more safely for severe tracheostomized patients after ICU discharge, and furthermore facilitates discharge home. References 1. Martinez GH, et al: Respir Care. 2009;54:1644-52. 1. Martinez GH, et al: Respir Care. 2009;54:1644-52. 2. Fernandez R, et al: Crit Care Med. 2011;39:2240-5. 2. Fernandez R, et al: Crit Care Med. 2011;39:2240-5. i Results No diff erences were found in age, gender, admission diagnoses, length of ICU stay and in-hospital mortality rate between the two study groups. Overall Gram-negative, Gram-positive and fungal fl ora were Reference Reference Methods We retrospectively reviewed medical records of ICU adults patients who had TQ when discharged to a new TCU and to a GW. The study was carried out in two tertiary care university hospitals from January 2007 to November 2014. Study variables were age, sex, APACHE II score, principal diagnosis, associated major procedures, length of stay in ICU and out in hospital, TCU and GW, Sabadell score, in-hospital mortality, types of tracheotomy procedure, decision to decannulate and discharge to home or long-care facilities. 1. Kollig E, et al. Injury. 2000;31:663-8. 1. Kollig E, et al. Injury. 2000;31:663-8. 1. Kollig E, et al. Injury. 2000;31:663-8. Decision-making algorithm for TS in the ICU Second University of Naples, Italy Critical Care 2015, 19(Suppl 1):P215 (doi: 10.1186/cc14295) Introduction Nowadays more percutaneous dilatational tracheostomy (PDT) methods are in use, but there is no ideal risk-free technique. We Figure 1 (abstract P215). Figure 1 (abstract P215). Figure 1 (abstract P215). S75 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 similar in both groups before and after PBT. Patients who received antibiotic therapy had a lower incidence of new ventilator-associated pneumonia (VAP) episodes (15/82 (18.2%) in Group 1 vs. 14/28 (50%) in Group 2, P  <0.001 (0.23, 0.87 to 0.13)) (Table  1). There were no diff erences in the incidence of bacteremia or line sepsis (Table 1). Conclusion Our fi ndings highlight the importance of conducting a prospective randomized control trial to better understand the role of antibiotic prophylaxis in PBT. have outlined a decisional algorithm to choose the most appropriate technique in each case to reduce the incidence of complications. have outlined a decisional algorithm to choose the most appropriate technique in each case to reduce the incidence of complications. Methods A retrospective review was performed using data from the last 14 years. Two hundred patients were selected. Patients were divided into two groups: one including the fi rst 100 PDTs treated without the algorithm (nA-group) and the other including the last 100 patients treated with the algorithm (A-group). Valuation of clinical and anatomical features of the patients, neck ultrasound and fi brobronchoscopy came before the procedure [1]. The algorithm was formulated by our experience with PDT techniques, comparing the specifi c characteristics of each one with the physiopathological characteristics of each patient. Conclusion Our fi ndings highlight the importance of conducting a prospective randomized control trial to better understand the role of antibiotic prophylaxis in PBT. P217 P217 Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards J Rubio1, JA Rubio2, E Palma2, R Sierra1, F Carmona1, F Fuentes2 1Hospital Universitario Puerta del Mar, Cadiz, Spain; 2Hospital Infanta Cristina, Badajoz, Spain Critical Care 2015, 19(Suppl 1):P217 (doi: 10.1186/cc14297) Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards Results We recorded complications (bleeding, tracheoesophageal fi stula, subglottic stenosis, tracheal rings’ fracture, diffi culty of placement, change of procedure) related to PDTs performed with and without applying the algorithm. We considered complications that occurred in our experience and we changed our modality in technique choice (Figure 1). Compared with the complications reported in the nA-group, use of the algorithm as a guide to choose the kind of PDT technique seems to reduce the incidence of complications (37% vs. 19%; P = 0.001 chi-square test). Introduction Patients with a tracheostomy (TQ) tube in place discharged from the ICU to a general ward (GW) are a fragile group and the TQ may be a risk factor for morbidity and mortality [1,2]. For this reason they need closer monitoring and more airway care. The Transitional Care Unit (TCU) assists patients with serious medical conditions and bridges the gap between the ICU and home or long-term care facilities providing the necessary medical, nursing, psychological and rehabilitative care. The purpose of this study was to evaluate the impact of the TCU on outcomes of tracheostomized patients discharged from the ICU. Conclusion In our experience the application of the proposed algorithm may reduce the incidence of complications related to PDT in the ICU. However, a randomized controlled multicenter study would be necessary in order to confi rm the effi ciency and validity of the proposed algorithm. P218 Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT procedure Infection Group 1 Group 2 P value OR Bacteremia 21/82(25.6%) 11/28(39.3%) 0.149 Colonization 67/82(82.7%) 23/28(82.1%) 0.718 Line sepsis 10/82 (12%) 4/28 (14.3%) 0.39 New VAP (%) 15/82(18.2%) 14/28 (50%) 0.001 Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT procedure Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT d Introduction Incidence of obesity in developed countries is rising. Currently, Europe has a prevalence of 9 to 30% with signifi cant impact on public health systems. Obese patients in the ICU require special management and treatment. Altered anatomy in obese patients complicates procedures such as mechanical ventilation. Obesity aff ects cardiopulmonary physiology and requires elevated ventilation pressures. In our retrospective study, we determined the eff ect of early S76 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion There is a clear reduction in the need for unplanned IPPV in both patient groups. An audit in 2013 showed incomplete protocol adherence in the ITU, therefore benefi ts may be underestimated. Conclusion There is a clear reduction in the need for unplanned IPPV in both patient groups. An audit in 2013 showed incomplete protocol adherence in the ITU, therefore benefi ts may be underestimated. percutaneous dilatational tracheotomy (PDT) and cessation of sedation on respiratory parameters in severely obese patients. percutaneous dilatational tracheotomy (PDT) and cessation of sedation on respiratory parameters in severely obese patients. p y p y p Methods From June 2010 to July 2014, we included all patients with a body weight of >130  kg, respiratory failure and PDT who were admitted to the ICU of the University Hospital of Muenster. We compared respirator parameters and blood gas analysis before and after PDT. Parameters were recorded on days –1, 0, 1, 3, 5, and 10, with day 0 describing the day of PDT. P220 P220 Is the gastric tube a burden for noninvasive ventilation? I Minev, C Stefanov University Hospital ‘St. Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H K d J N i J S id l Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H Kennedy, J Navein, J Seidel Doncaster Royal Infi rmary, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P219 (doi: 10.1186/cc14299) H Kennedy, J Navein, J Seidelifi y Doncaster Royal Infi rmary, Sheffi eld, UK Methods In this study, six of the COPD patients admitted to our ICU, who required NGT placement, were ventilated with the Draeger Evita 2 dura through a modifi ed reusable silicone face mask (UMDNS code: 12-453 with 22 mm ID connection; sizes 4 and 5) with silicone headgear and a hook ring. All of them had a NGT during their stay in the ICU. We evaluated the effi cacy of our modifi cation comparing the achieved Vt with modifi ed and unmodifi ed face mask, during two periods of 10 minutes. The mode and parameters of ventilation were not changed. We assessed patient comfort with a visual analogue scale. Introduction Respiratory failure is a well-known complication of aortic aneurysm surgery. We describe the impact of a protocol, using CPAP after elective surgery to reduce the need for unplanned invasive ventilation. Methods In 2012 we introduced a CPAP protocol for patients undergoing elective aortic aneurysm surgery, either open (AAA) or as an endovascular repair (EVAR). According to pre-existing risk factors (see Table 1) and arterial blood gas analysis in the anaesthetic room, they were assigned to two alternative options on the ITU: prophylactic CPAP for 9 hours in each of the fi rst two postoperative nights or oxygen via face mask. CPAP was applied at any time in the patients stay, if their P/F ratio dropped below 40. Criteria to stop CPAP were also predefi ned. Previously, CPAP was initiated at the discretion of nursing staff , P/F ratios were not utilised. p g Results The average duration of NIV was 3.5  days (SD  = 1.6). We examined two sets of 10 consequent breathing cycles for every patient. The mean Vt was 472 ml (SD = 76 ml) with standard face mask and 460 ml (SD = 86 ml) with the modifi ed one. There was statistically signifi cant correlation between the two datasets (P  <0.05). No additional leaks were detected. According to the VAS evaluation, fi ve of the patients (83%) had comfort improvement with the modifi ed mask. Table 1 (abstract P219). Criteria for the use of prophylactic CPAP FEV1: <1.5 l/minute Poor exercise tolerance (<100 yards) due to chest problems Poor exercise tolerance (<100 yards) due to chest problems SpO2 <92% on FiO2 >0.4 in theatre SpO2 <92% on FiO2 >0.4 in theatre Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H K d J N i J S id l Conclusion With this modifi cation of the face mask we achieved adequate drainage of the stomach and/or the enteral nutrition of the patients and improvement in their comfort during NIV, compared with the ventilation with a standard mask, without additional air leaks and at a low cost. Table 1 (abstract P219). Criteria for the use of prophylactic CPAP P218 Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) George’, Plovdiv, Bulgaria Critical Care 2015, 19(Suppl 1):P220 (doi: 10.1186/cc14300) y g y Results Twenty-one patients were included in the study. Mean age was 56 ± 10.3 years and 14 (66.7%) of the patients were male. Body weight was 164.5 ± 39.4 kg, body height accounted for 176.8 ± 8.7 cm (n = 20) and body mass index was 49.7 ± 16.9 kg/m2. Patients stayed in the ICU for 18.4 ± 13.8 days. Mean time of mechanical ventilation by endotracheal tube was 2.4 ± 1.5 days (n = 20) and via tracheostomy 9.8  ± 7.0  days. After PDT, peak inspiratory pressure (P  <0.0001), positive end-expiratory pressure (P <0.0001) and insuffl ated oxygen concentration (P <0.0001) could signifi cantly and rapidly be reduced. Respiratory minute volume increased signifi cantly (P = 0.004). PDT was not associated with relevant complications. Introduction The application of noninvasive ventilation (NIV) in ICUs has spread widely during the years. It is used in the treatment of diff erent forms of acute respiratory failure and COPD exacerbations. Although NIV is thought to be more comfortable for patients than invasive mechanical ventilation, its failure rates in the ICUs range between 10 and 40%. Except for the interface-related problems, there are some specifi c considerations for the patient–ventilator interaction and the applied mechanical forces. During NIV there is a predisposition for the stomach to be infl ated with gas, which could cause severe respiratory complications, especially in COPD patients, and thus prolong the mechanical ventilation and the weaning process. This remains one of the major causes for NIV failure. Although a lot of face masks with diff erent interfaces are available on the market, just a few have additional ports for a NGT. They are characterized by higher price and a complex setup. In order to perform NIV in patients, requiring NGT placement, without additional air leaks and to be able to ensure their enteral nutrition and/or stomach drainage, we installed a port for a NGT on a standard face mask. Conclusion Early PDT rapidly improves respiratory distress in severely obese patients due enabling of spontaneous breathing and reduction of dead space ventilation. Lung ultrasound aeration assessment: comparison of two techniques S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) p p Methods The authors prospectively enrolled 17 patients with septic shock who were admitted to the medical ICU, Phramongkutklao Hospital between September 2013 and June 2014. EVLWI was measured by TPTD (VolumeView Set, EV1000; Edwards Lifesciences) method. According to international evidence-based recommendations for point-of-care lung ultrasound 2012, three methods of LUS (LOGIQ e ultrasound; GE Healthcare) were compared to assess EVLW daily in each patient until no indication for invasive blood pressure monitoring [1]. Firstly, B-lines were measured in 28 lung zones. The total numbers of B-lines seen in each patient were counted as total B-line scores (TBS). Secondly, upper and lower BLUE points were anterior two-region scans each side marked by physician hands. Pulmonary edema was diagnosed if three or more B-lines were presented in all regions. Lastly, scanning eight regions, two anterior and two laterals per side, was considered abnormal if more than one scan per side had three or more B-lines.i Introduction Lung ultrasound (LUS) allows semiquantifi cation of lung aeration in PEEP trials [1], pneumonia [2] and weaning [3]. LUS score is based on number/coalescence of vertical artifacts (B-lines) in longitudinal scan (LONG) [4]: the pleura is identifi ed between two ribs and its visualization limited by intercostal space (ICS) width. We hypothesized that a transversal scan (TRANSV) aligned with ICS would visualize longer pleura and a higher number of artifacts, with better assessment of loss of aeration (LoA). Methods LONG and TRANSV were performed in six areas per lung (anterior, lateral and posterior, each divided into superior and inferior). Once LONG was performed, TRANSV was obtained by a probe rotation until the ribs disappeared. We considered pleural length, B-line number/coalescence, and subpleural/lobar consolidations. P221 P221 Lung ultrasonography as a marker of pulmonary edema in cardiac surgery patients: visual versus quantitative evaluation F Corradi1, C Brusasco2, T Manca3, F Nicolini3, F Nicosia1, T Gherli3, V Brusasco2, A Vezzani3 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria di Parma, Italy Critical Care 2015, 19(Suppl 1):P221 (doi: 10.1186/cc14301) Results We compare patient cohorts in the years 2010 and 2011 (pre protocol) with 2013 and 2014 (post protocol). Results are reported as the split between open surgery and endovascular repair. Table  2 presents requirements for invasive ventilation (IPPV) and length of stay (LOS) for both patient groups. Table 2 (abstract P219). IPPV requirements and length of stay data Table 2 (abstract P219). IPPV requirements and length of stay data 2010 to 2011 2013 to 2014 LOS LOS LOS LOS ITU hospital ITU hospital IPPV (days) (days) IPPV (days) (days) EVAR 2/67 (3%) 2 6 1/77 (1.3%) 2 5 AAA 10/46 (21.7%) 5 9 6/37 (16.2%) 5 12 Introduction Lung ultrasonography (LUS) has been used for non- invasive detection of pulmonary edema. LUS visual scores (V-LUS) based on B-lines are poorly correlated with either pulmonary capillary wedge pressure (PCWP) or extravascular lung water (EVLW). A new quantitative LUS analysis (Q-LUS) has been recently proposed [1,2]. The aim of the study was to investigate whether Q-LUS is better correlated with PCWP and EVLW than V-LUS, and to what extent positive end- expiratory pressure (PEEP) aff ects the assessment of pulmonary edema by Q-LUS and V-LUS. S77 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Thirty-nine patients mechanically ventilated with PEEP of 5 cmH2O (n = 47) or 10 cmH2O (n = 30) and PCWP (n = 77) or EVLW (n = 38) monitored were studied.i Methods Thirty-nine patients mechanically ventilated with PEEP of 5 cmH2O (n = 47) or 10 cmH2O (n = 30) and PCWP (n = 77) or EVLW (n = 38) monitored were studied.i versus 3.9 ± 0.1 cm (range 3.0 to 4.3; variance 0.1) (P <0.0001), B-lines per scan were 1.1 ± 1.6 versus 1.8 ± 2.5 (P <0.0001), coalescent B-lines were detected in 24 versus 30% (P <0.05) and subpleural consolidations in 16 versus 22% (P <0.05), respectively. LUS scores’ prevalence signifi cantly diff ered in LONG versus TRANSV (Figure 1). P221 Results PCWP was signifi cantly and strongly correlated with Q-LUS Grey Unit value (r2 = 0.64) but weakly with V-LUS B-line score (r2 = 0.19). EVLW was signifi cantly and strongly correlated with QLUS Grey Unit mean value (r2 = 0.65) more than with V-LUS B-line score (r2 = 0.42). Q-LUS showed a better diagnostic accuracy than V-LUS for the detection of PCWP >15 mmHg or EVLW >10 ml/kg. With a PEEP of 5 cmH2O, the correlations with PCWP or EVLW were stronger with Q-LUS than V-LUS. With a PEEP of 10 cmH2O, the correlations with PCWP or EVLW were still signifi cant for Q-LUS but insignifi cant for V-LUS. Intraobserver repeatability and interobserver reproducibility were much better for Q-LUS than V-LUS. f Conclusion TRANSV visualizes signifi cantly longer pleura and greater number of artifacts useful for lung disease assessment. References f Conclusion TRANSV visualizes signifi cantly longer pleura and greater number of artifacts useful for lung disease assessment. References 1. Bouhemad B, et al. Am J Resp Crit Care Med. 2011;183:341-7. 2. Bouhemad B, et al. Crit Care Med. 2010;38:84-92. 3. Soummer A. et al. Crit Care Med. 2012;40:264-72. 4. Volpicelli G. et al. Int Care Med. 2012;38:577-91. Lung ultrasound aeration assessment: comparison of two techniques LUS score was assigned: 0 normal lung, 1 moderate LoA (≥3 well-spaced B-lines), 2 severe LoA (coalescent B-lines), 3 complete LoA (tissue-like pattern). Results We enrolled 38 patients (21 males, age 60 ± 16 years, BMI 24.7 ± 4.7 kg/m2) corresponding to 456 ICSs. In 63 ICSs, a tissue-like pattern was visualized in both techniques. In the other 393, LONG versus TRANSV pleural length was 2.0 ± 0.6 cm (range 0.8 to 3.8; variance 0.31) Results A total of 40 comparisons were obtained. Signifi cant positive linear correlations were found between TBS and EVLWI determined by TPTD (r  = 0.637, P <0.001). The TBS ≥39 has sensitivity of 91.7% and specifi city of 75.0% to defi ne EVLWI >10  ml/kg. There was low sensitivity (33.3% and 50.0% respectively) but high specifi city (100% and 96.0% respectively) of the positive BLUE points and eight regions to defi ne EVLWI >10 ml/kg. Figure 1 (abstract P222). LUS scores. *P <0.01 TRANSV versus LONG. i Conclusion TBS is the best method for assessing EVLW compared with BLUE points and eight regions. These data support the benefi t of LUS with summation of B-line scores of 28 rib interspaces for assessment of the increment of EVLW in septic shock patients. f Reference Reference 1. Volpicelli G, et al. Intensive Care Med. 2012;38:577-91. 1. Volpicelli G, et al. Intensive Care Med. 2012;38:577-91. Ultrasound assessment for extravascular lung water in patients with septic shock Conclusion Both V-LUS and Q-LUS are acceptable indicators of pulmonary edema in patients mechanically ventilated with low PEEP but at high PEEP only Q-LUS provides data that are signifi cantly correlated. Computer-aided Q-LUS has the advantages of being not only independent of operator perception but also of PEEP. References P Pirompanich1, A Wattanathum2 1Thammasat University, Pathumthani, Thailand; 2Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P223 (doi: 10.1186/cc14303) 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. Introduction Extravascular lung water (EVLW) refers to fl uid within the lung but outside the vascular compartment. Increment of EVLW was associated with mortality in critically ill patients. Extravascular lung water index (EVLWI) >10 ml/kg was found in patients with cardiogenic pulmonary edema and correlated with pulmonary capillary wedge pressure >20 mmHg. Measurement of EVLW needs sophisticated tools and an invasive method by transpulmonary thermodilution (TPTD) technique. In contrast, multiple B-lines by lung ultrasound (LUS) have been recently proposed to correlate with increased EVLW in patients with pulmonary edema. This study aims to compare three methods of LUS and EVLWI measured by TPTD to assess pulmonary edema in patients with septic shock. bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) Introduction One of the aims of lung recruitment is to improve oxygenation [1], but it has not yet been investigated in spontaneously breathing patients. Our objective was to evaluate the eff ects of recruitment maneuvers on oxygenation in patients ventilated in CPAP/ pressure support (CPAP/PS) mode. p pp Methods In a prospective, observational study, 30 patients with a Lung Injury Score ≥2 were recruited. Following baseline measurements (t0) PEEP was increased by 5 cmH2O (t1). Recruitment maneuver was applied for 40 seconds with 40 cmH2O PS. Measurements were taken immediately after recruitment (t2) then 15 minutes (t3) and 30 minutes later (t4).f 2 3 4 Results According to the diff erence of PaO2/FiO2 between t2 and t0, three groups were defi ned: nonresponders (NR: diff erence of PaO2/FiO2 ≤0%, n = 8), low responders (LR: diff erence of PaO2/FiO2 = 0 to 50%, n = 11) and high responders (HR: diff erence of PaO2/FiO2 >50%, n = 11). In the NR-group, PaO2/FiO2 decreased signifi cantly: median (interquartile), PaO2/FiO2 = 178 (159 to 240) versus 165 (118 to 210) mmHg; in the LR- group and in the HR-group there was signifi cant improvement: 119 (98 to 164) versus 161 (123 to 182) mmHg and 141 (130 to 183) versus 239 (224 to 369) mmHg, P <0.05, respectively. Dynamic compliance (Cdyn) signifi cantly dropped at t2 as compared with t0 in the NR-group, Cdyn = 62 (48 to 87) versus 53 (43 to 78) ml/cmH2O, while there was no signifi cant change in the LR- and HR-groups, P <0.05. At the same time points the dead space to tidal volume ratio (Vds/Vte) signifi cantly increased in the NR-group, Vds/Vte = 30 (23 to 37) versus 37 (26 to 42)%, but not in the LR- and HR-groups, P <0.05. Figure 1 (abstract P224). Sample ultrasound recording. Lines 1 and 2 are diaphragm thickness measurements. Results We were successfully able to record the diaphragm thickness in all included patients. Median time on the ventilator was 9 days (IQR 4 to 15 days). Mean baseline thickness was 1.9 mm (SD ±0.4 mm), and mean nadir was 1.3 mm (SD ±0.4 mm), corresponding with a mean change in thickness of 32% (SD ±18%). As early as after only 72 hours of MV, we already noted an average drop of diaphragm thickness of 20%, illustrating the rapid progression of the atrophy in VIDD. P226f Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) References 1. Grosu H, et al. Chest. 2012;142:1455-60. Conclusion Recruitment maneuvers improved PaO2/FiO2 in the majority of patients (73%) without aff ecting Cdyn or Vds/Vte; therefore it may be a safe approach to improve oxygenation in patients ventilated in CPAP/PS mode. 1. Grosu H, et al. Chest. 2012;142:1455-60. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) Conclusion On average, diaphragm thickness decreased 32% in our cohort. The decrease occurred rapidly, with two-thirds of the maximal thinning already present after 72 hours of MV. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Thirty-two spontaneously breathing patients with suspected community-acquired pneumonia undergoing computed tomography examination were consecutively enrolled. Each hemithorax was evaluated for the presence or absence of abnormalities by chest X-ray and quantitative or visual ultrasonography. Methods Thirty-two spontaneously breathing patients with suspected community-acquired pneumonia undergoing computed tomography examination were consecutively enrolled. Each hemithorax was evaluated for the presence or absence of abnormalities by chest X-ray and quantitative or visual ultrasonography. diaphragmatic atrophy can be assessed using ultrasound, the biggest trial in humans published so far included seven patients and only measuring the thickness at two moments during the disease process [1]. We aimed to assess the time course of diaphragm atrophy in a larger cohort of MV patients using ultrasound. y Results Quantitative ultrasonography showed higher sensitivity (93%), specifi city (95%), and diagnostic accuracy (94%) than chest X-ray (64%, 80%, and 69%, respectively), or visual ultrasonography (68%, 95%, and 77%, respectively), or their combination (77%, 75%, and 77%, respectively). Methods A total of 54 patients from an adult ICU were included in this prospective single-centre cohort trial. Patients who needed <72 hours of MV or had been recently admitted to an ICU were excluded. Patients were ventilated in a controlled, assisted, and/or hybrid ventilation mode. The thickness of the diaphragm was assessed daily; the fi rst recording was within 24 hours after the start of mechanical ventilation and we continued the measurements until the patients were extubated or tracheotomised. We measured the diaphragm at the zone of apposition, as described by McCool and colleagues [2] using a linear 13 MHz ultrasound probe. Figure 1 shows a sample measurement. y Conclusion Quantitative lung ultrasonography was considerably more accurate than either chest X-ray or visual ultrasonography in the diagnosis of community-acquired pneumonia and it may represent a useful fi rst-line approach for confi rmation of clinical diagnosis in emergency settings. Quantitative ultrasonography for pneumonia F C di1 C B 2 T M 3 F Ni i 1 A V 1. Lachman B. Intensive Care Med. 1992;18:319-21. y F Corradi1, C Brusasco2, T Manca3, F Nicosia1, A Vezzani3, V Brusasco2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria, Parma, Italy Critical Care 2015, 19(Suppl 1):P225 (doi: 10.1186/cc14305) F Corradi1, C Brusasco2, T Manca3, F Nicosia1, A Vezzani3, V Brusasco2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria, Parma, Italy Critical Care 2015, 19(Suppl 1):P225 (doi: 10.1186/cc14305) References Figure 1 (abstract P224). Sample ultrasound recording. Lines 1 and 2 are diaphragm thickness measurements. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. g p y 2. Quanta Imaging Technology. www.quanta.camelotbio.com. A better way to determine sample size to detect changes in length of mechanical ventilation? A better way to determine sample size to detect changes in length of mechanical ventilation? YS Chiew1, C Pretty1, D Redmond1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P227 (doi: 10.1186/cc14307) Atrophy of diaphragm muscle visualized with ultrasound in mechanically ventilated patients T Schepens, M Mergeay, W Verbrugghe, P Parizel, M Vercauteren, PG Jorens Antwerp University Hospital, Edegem, Belgium p y p , g , g Critical Care 2015, 19(Suppl 1):P224 (doi: 10.1186/cc14304) Introduction Mechanical ventilation (MV) induces diaphragmatic muscle atrophy and contractile fi bre dysfunction, the so-called ventilator-induced diaphragm dysfunction (VIDD). Although Figure 1 (abstract P222). LUS scores. *P <0.01 TRANSV versus LONG. S78 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 A better way to determine s of mechanical ventilation? Introduction Chest-X-ray is recommended for routine use in patients with suspected pneumonia, but its use in emergency settings is limited. In this study, the diagnostic performance of a new method for quantitative analysis of lung ultrasonography was compared with bedside chest X-ray and visual lung ultrasonography for detection of community-acquired pneumonia, using thoracic computed tomography as a gold standard. YS Chiew1, C Pretty1, D Redmond1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P227 (doi: 10.1186/cc14307) Introduction Estimation of eff ective sample size (N/arm) is important to ensure power to detect signifi cant treatment eff ects. However, S79 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 monitoring of the eff ect of benzodiazepines on respiratory status in non-intubated patients has been diffi cult, putting patient safety at risk. A non-invasive respiratory volume monitor (RVM) that provides continuous measurement of minute ventilation (MV), tidal volume (TV) and respiratory rate (RR) was used to quantify the eff ects of midazolam on respiratory status in spontaneously breathing patients. traditional parametric sample size estimations depend upon restrictive assumptions that often do not hold in real data. This study estimates N to detect changes in length of mechanical ventilation (LoMV) using Monte-Carlo simulation (MCS) and mechanical ventilation (MV) data to better simulate the cohort. Methods Data from 2,534 MV patients admitted to Christchurch Hospital ICU from 2011 to 2013 were used. N was estimated using MCS to determine a sample size with power of 80%, and compared with the Altman’s nomogram for two patients groups, (1)  all patients and (2)  targeted patients with 1 <LoMV ≤15 days. MCS allows any range of intervention eff ect to be simulated, where this study tested a 10 and 25% diff erence in LoMV (0.5 to 1.25 days for mean LoMV of 5 days). The simulated LoMV for the intervention group is compared with the LoMV in a control group using the one-sided Wilcoxon rank-sum test, Student t test, and Kolmogorov– Smirnov test to assess central tendency and variation. Methods An impedance-based RVM (ExSpiron; Respiratory Motion Inc., Waltham, MA, USA) was used in 30 patients who received 2 mg midazolam prior to induction of anesthesia and were sedated but spontaneously breathing. A better way to determine s of mechanical ventilation? Further studies are ongoing to quantify hypoventilation after administration of other anesthetic medications. Conclusion Continuous monitoring with RVM provides a valuable depiction of hypoventilation from benzodiazepines, not demonstrated by other methodologies such as pulse oximetry and RR alone. RVM monitoring can help uncover potentially life-threatening hypoventilation in older patients. Further studies are ongoing to quantify hypoventilation after administration of other anesthetic medications. Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. A better way to determine s of mechanical ventilation? Eleven of these patients (58  ± 19  years, average BMI 27.7) received midazolam at least 20  minutes prior to induction. Digital RVM data were collected and MV, TV and RR calculated and evaluated from 30-second segments 10 minutes before and after the fi rst dose of midazolam. Ten patients were analyzed as a group and one patient was analyzed separately (due to idiosyncratic reaction). y Results The distribution of LoMV is heavily skewed. Altman’s nomogram assumes a normal distribution and found N >1,000 to detect a 25% LoMV change. Figure 1 panels (1) and (2) show N for 80% power if all patients were included, and panels (3) and (4) for the targeted patient group. Panels (1) and (3) show that it is impossible to achieve 80% power for a 10% intervention eff ect. For 25% eff ect, MSC found N = 400/arm (all patients) and N = 150/arm (targeted cohort). Results Following administration of midazolam, the group MV and TV decreased an average of 19 ± 7% and 16 ± 5%, respectively (mean ± SEM, P <0.01, both) while RR remained essentially unchanged (decrease of 3 ± 8%, P >0.3). In the younger half of the cohort (45 ± 16 years), the decreases in MV and TV were not signifi cant, only 6 ± 3% and 8 ± 5%, respectively. The older half of the cohort (72 ± 8 years) displayed fourfold greater MV and TV decreases (32 ± 11%, P <0.05 and 25 ± 6%, P <0.05), when compared with the younger cohort, P <0.01, Figure 1). Figure 1 (abstract P227). Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. P228 Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G M ll 1 D L dd2 Figure 1 (abstract P227). Figure 1 (abstract P228). Figure 1 (abstract P228). Figure 1 (abstract P227). Conclusion Continuous monitoring with RVM provides a valuable depiction of hypoventilation from benzodiazepines, not demonstrated by other methodologies such as pulse oximetry and RR alone. RVM monitoring can help uncover potentially life-threatening hypoventilation in older patients. P229 P229 Nebulized heparin for patients under mechanical ventilation: a conventional data meta-analysis GJ Glas1, A Serpa Neto2, J Horn1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Israelita Albert Einstein, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P229 (doi: 10.1186/cc14309) Nebulized heparin for patients under mechanical ventilation: a conventional data meta-analysis y GJ Glas1, A Serpa Neto2, J Horn1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Israelita Albert Einstein, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P229 (doi: 10.1186/cc14309) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 1Vidant Medical Center, Greenville, NC, USA; 2Respiratory Motion, Inc., Waltham, MA, USA Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 1Vidant Medical Center, Greenville, NC, USA; 2Respiratory Motion, Inc., Waltham, MA, USA Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1 D Ladd2 Introduction Mechanical ventilation has the potential to induce pulmonary coagulopathy. Local treatment by nebulization of heparin could be benefi cial in ventilated patients. The aim of this data meta- analysis is to determine the association between nebulization of heparin and outcome of mechanically ventilated critically ill patients. Methods PubMed, Scopus, EMBASE, and Web of Science were searched for relevant articles. Articles were selected if they compared Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Introduction Benzodiazepines are used in many of settings to induce sedation, but can cause a reduction in respiratory drive. Objective Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S80 Figure 1 (abstract P229). Eff ect of heparin nebulization on mortality. Figure 1 (abstract P229). Eff ect of heparin nebulization on mortality. nebulization of heparin with standard care. The primary endpoint was overall mortality. Secondary endpoints included occurrence of pneumonia and number of ventilator-free days and alive at day 28.i pulse oximetry with derived heart rate; snoring and nasal airfl ow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic diffi culties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI). pulse oximetry with derived heart rate; snoring and nasal airfl ow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic diffi culties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI). Results Thirty-one patients with the mean age of 67 ± 9 years were included in the study. Their mean APACHE II score was 16 ± 5 and BMI was 33 ± 9 kg/m2. Admission arterial blood gases were as follows (mean ± SD); pH: 7.33 ± 0.07, PaO2: 74 ± 12 mmHg, PaCO2: 69 ± 11 mmHg, HCO3 –: 31 ± 5, O2Sat%: 92 ± 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 ± 6 in patients with OAI >5. Diagnosis of obstructive sleep apnea with respiratory polygraph in hypercapnic ICU patients P231 G Gursel, A Zerman, M Aydogdu, B Basarik Aydogan, K Gonderen, S Memmedova, N Sevimli, I Koroglu, Z Isikdogan, M Badoglu, O Ozdedeoglu Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P230 (doi: 10.1186/cc14310) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1 D Ladd2 Diagnosis of obstructive sleep apnea with respiratory polygraph in hypercapnic ICU patients G Gursel, A Zerman, M Aydogdu, B Basarik Aydogan, K Gonderen, S Memmedova, N Sevimli, I Koroglu, Z Isikdogan, M Badoglu, O Ozdedeoglu Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P230 (doi: 10.1186/cc14310) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1 D Ladd2 Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identifi ed to have OSA. Conclusion Although it underestimates AHI, RPLG is important and technically feasible in ICU patients in suggesting the presence of OSA and in providing information for appropriate NIV management. Results Six articles were found: fi ve retrospective cohorts with historical controls, one randomized controlled trial, covering 423 patients. Dosages of nebulized heparin varied from 30,000 to 150,000 IU/day. Fifty out of 222 patients (22.5%) receiving nebulized heparin and 48 out of 201 patients (23.9%) receiving standard care died (risk ratio (RR) 0.79 (95% CI 0.47 to 1.35)) (see Figure 1). Occurrence of pneumonia (RR 1.36 (95% CI 0.54 to 3.45); I2 = 59%), and number of ventilator-free days and alive at day 28 (standardized mean diff erence 0.11 (95% CI –0.14 to 35); I2 = 0%), were not diff erent between the two groups. Results Thirty-one patients with the mean age of 67 ± 9 years were included in the study. Their mean APACHE II score was 16 ± 5 and BMI was 33 ± 9 kg/m2. Admission arterial blood gases were as follows (mean ± SD); pH: 7.33 ± 0.07, PaO2: 74 ± 12 mmHg, PaCO2: 69 ± 11 mmHg, HCO3 –: 31 ± 5, O2Sat%: 92 ± 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 ± 6 in patients with OAI >5. Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identifi ed to have OSA. Conclusion Nebulization of heparin is not associated with improved outcome in mechanically ventilated critically ill patients. This meta- analysis is limited by methodological problems in most included studies. Only one randomized controlled trial could be included. Also, most patients in the meta-analyzed studies suff ered from inhalation trauma, and heparin dosages diff ered widely. Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU G Besch1, J Revelly2, P Jolliet2, L Piquilloud-Imboden2 1CHRU Besançon, France; 2CHUV, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P232 (doi: 10.1186/cc14312) G Besch1, J Revelly2, P Jolliet2, L Piquilloud-Imboden2 1CHRU Besançon, France; 2CHUV, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P232 (doi: 10.1186/cc14312) y 2 2 Results The study group was comprised of four men and three women. Median age was 69 (IQR: 61.0 to 72.5) years old. The reason for admission was ARDS (n  = 5) and acute exacerbation of idiopathic pulmonary fi brosis (n = 2). The reasons for ARDS are bacterial pneumonia (n = 3), necrotizing fasciitis (n = 1) and extensive burn (n = 1). We identifi ed NETs in the bronchial aspirates of all the patients. NET formation had persisted in four cases during the study period, their P/F ratio did not improve and all patients were dead due to respiratory failure. On the other hand, NETs decreased and vanished in three cases, their P/F ratio improved and all patients recovered from ARDS. Introduction As the objective clinical criteria [1] are imperfect to assess patients before extubation, simple physiological parameters are used to try to improve extubation failure (EF) prediction. The rapid shallow breathing index (RSBI) (respiratory rate (RR) over tidal volume (VT) ratio) recorded during a T-piece spontaneous breathing trial (SBT) is known as the most reliable physiologic predictor. However, RSBI is nowadays usually computed during a pressure support (PS) SBT using the values displayed on the ventilator screen and not based on spirometry measurements without any assist as initially published. The aim of the present study was to prospectively assess the ability of currently measured RSBI to predict EF. Conclusion NET formation was observed in bronchial aspirates of all the patients diagnosed with ARDS or acute exacerbation of idiopathic pulmonary fi brosis. It may be one of the prognostic factors of ARDS or acute exacerbation of idiopathic pulmonary fi brosis. Methods Retrospective analysis of prospectively collected data from patients intubated for more than 48 hours admitted in the medico- surgical ICU of Lausanne, Switzerland, from January 2007 to December 2008. Respiratory muscle training during mechanical ventilation: a systematic reviewfi a systematic review D Brace, M Parrotto, C Urrea, A Goffi , A Murray, E Fan, L Brochard, N Ferguson, E Goligher UHN, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P231 (doi: 10.1186/cc14311) Introduction The most frequent reasons for hypercapnic respiratory failure (HRF) in ICUs are COPD and in recent years obesity hypoventilation syndrome (OHS) and obstructive sleep apnea (OSA). Even 15 to 30% of COPD patients also have accompanying OSA. Due to increased upper airway resistance, those patients require higher expiratory pressures (EPAP) during noninvasive ventilation (NIV). In order to prescribe optimal mode and pressures during the ICU stay and at discharge, the intensivist should diagnose the underlying OSA. Portable recording devices have been developed and they were approved at least for the diagnosis in high pretest probability patients with results equal to in-laboratory polysomnography. The aim of this study is to assess whether respiratory polygraph (RPLG) can be used for obtaining diagnostic information of OSA in hypercapnic ICU patients. Methods Patients, with HRF requiring NIV, were included in the study. RPLG studies were conducted under nasal oxygen before NIV, using the Philips Respironics Alice PDx® device, which provides the records of Introduction Respiratory muscle weakness is common in mechanically ventilated patients and impairs liberation from ventilation. Inspiratory muscle training (IMT) might accelerate liberation from mechanical ventilation. We undertook to summarize previously published IMT protocols and the impact of IMT on respiratory muscle function and clinical outcomes. Methods We searched multiple databases using a sensitive search strategy combining MeSH headings and keywords for studies of IMT during MV. Studies were adjudicated for inclusion and data were abstracted independently and in duplicate. Methodological quality was assessed using the GRADE system. Results Eleven studies met the inclusion criteria; of these, six were randomized controlled trials and fi ve were observational studies. Figure 1 (abstract P231). Eff ect of IMT on the rate of successful weaning from mechanical ventilation. S81 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 A variety of IMT techniques were employed including inspiratory threshold loading (eight studies), biofeedback to increase inspiratory eff ort (one study), chair-sitting (one study) and diaphragmatic breathing pattern training (one study). Threshold loading was achieved by application of an external device (six studies) or increases in the inspiratory pressure trigger setting (two studies). University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) Introduction Acute lung injury (ALI) and its hypoxemic form, acute respiratory distress syndrome (ARDS), has no approved pharma co- logical treatment and is a condition with high mortality. Vascular leakage is one of the early events of ALI/ARDS. CD73 activity (ecto- 5’-nucleotidase) maintains the endothelial barrier function of lung capillaries via its enzymatic endproduct adenosine. Interferon-beta (IFNβ) increases synthesis of CD73 and has been eff ective in a mouse model of ALI [1]. Results A total of 478 extubated patients were included, 25 of whom (5.2%) were reintubated. ICU mortality (ICU-m) and in-hospital mortality (H-m) were higher in reintubated patients: ICU-m = 6 (24) versus 22 (5), P = 0.002 and H-m = 9 (36) versus 63 (15), P = 0.009. The reasons for EF were: acute lung failure (n = 15), congestive heart failure (n = 4) and aspiration/bronchial congestion (n = 6). Demographic data were similar between patients successfully and nonsuccessfully extubated: age: 58 ± 17 versus 58 ± 19 years, P = 0.85; male gender: 15 (60) versus 263 (61), P = 0.99. SAPS II score was higher in the EF group: 30 ± 22 versus 42 ± 27, P = 0.04. RSBI were signifi cantly higher in patients who experienced EF: RSBI=59 ± 44 versus 43 ± 26, P = 0.04. The area under the ROC curve for currently measured RSBI was: 0.617 (95% CI = 0.571 to 0.662), P = 0.035. Methods Therefore we conducted a phase I/II trial [2], in which intravenously administered human recombinant IFNβ1a (FP-1201) was used in the study, which consisted of dose escalation (phase I) and expansion (phase II) parts to test the FP-1201 safety, tolerability and effi cacy in ALI/ARDS patients. CD73, MxA (a marker for IFN β response) and other biomarkers were measured to follow pharmacokinetics/ dynamics of the intravenously administered FP-1201 and therapeutic effi cacy. Conclusion In a cohort of 458 medico-surgical ICU patients, RSBI measured during a pressure support SBT was higher in patients experiencing EF but very imperfect to predict EF. Reference fi y Results The optimal tolerated dose of FP-1201 (10 μg/day for 6 days) resulted in maximal MxA stimulation. Also soluble CD73 values increased, while IL-6 decreased in sera of the FP-1201-treated ALI/ARDS patients. Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU EF was defi ned as the need for reintubation within 48 hours after extubation. Reintubations for a procedure requiring general anesthesia were not considered as EFs. RR and VT during the PS SBT were recorded from the ventilator and RSBI was computed accordingly. Baseline characteristics and currently measured RSBI were compared between patients who experienced EF versus success (t test or chi-square test as appropriate). The ability of currently measured RSBI to predict EF was assessed using ROC curve analysis. P234 1. MacIntyre NR, et al. Chest. 2001;120:375S. Respiratory muscle training during mechanical ventilation: a systematic reviewfi Most studies implemented IMT in the weaning phase (n = 5) or after diffi cult weaning (n = 5); one study implemented IMT within 24 hours of intubation. IMT was associated with greater increases in maximal inspiratory pressure compared with control (six studies, mean diff erence 7.6 cmH2O (95% CI 5.8, 9.3), I2 = 0%). There were no signifi cant diff erences in the duration of MV (six studies, mean diff erence –1.1 days (95% CI –2.5, 0.3), I2 = 71%) or the rate of successful weaning (Figure 1; fi ve studies, risk ratio 1.13 (95% CI 0.92, 1.40), I2 = 58%). The GRADE quality of evidence was low for all these outcomes; risk of bias was high for most studies and summary eff ects were imprecise and inconsistent. No serious adverse events related to IMT were reported. P233 Presence of neutrophil extracellular traps in bronchial aspirate of patients diagnosed with acute respiratory distress syndrome or acute exacerbation of idiopathic pulmonary fi brosis M Ojima1, N Yamamoto1, T Hirose1, S Hamaguchi1, N Matsumoto1, R Takegawa1, M Seki1, O Tasaki2, K Tomono1, T Shimazu1 1Osaka University Graduate School of Medicine, Suita, Japan; 2Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Critical Care 2015, 19(Suppl 1):P233 (doi: 10.1186/cc14313) Introduction Neutrophil extracellular traps (NETs) are fi brous structures that are produced extracellularly from activated neutrophil. They can trap and kill various pathogens, and their release is one of the fi rst lines of immune system. Meanwhile, recently it was reported that NETs also exert adverse eff ects in infl ammatory diseases. Acute respiratory distress syndrome (ARDS) and acute exacerbation of idiopathic pulmonary fi brosis is acute-onset respiratory failure. It is characterized by excessive neutrophil infi ltration into the alveolus, and great amounts of neutrophil elastase are released. The purpose of this study is to evaluate whether NETs are produced in bronchial aspirate of patients with ARDS or acute exacerbation of idiopathic pulmonary fi brosis, and to identify correlations with the respiratory function. p Conclusion IMT in mechanically ventilated patients appears safe and well tolerated and improves respiratory muscle function. IMT was not associated with accelerated liberation from mechanical ventilation. However, because the included studies had important methodological limitations and employed varying methods of IMT, we cannot draw fi rm conclusions about the eff ect of IMT on clinical outcomes. i y y Methods This was a prospective observational study of seven patients admitted to the ICU of a large urban tertiary referral hospital. All patients were mechanically ventilated at the time of admission. The bronchial aspirates were collected serially from the patients by suction through a tracheal tube. To identify NETs, extracellular components including DNA, histone H3, and citrullinated histone H3 were simultaneously detected using immunohistochemistry. The respiratory function was evaluated by PaO2/FiO2 ratio (P/F ratio). New molecules controlling endothelial barrier S Jalkanen New molecules controlling endothelial barrier S Jalkanen New molecules controlling endothelial barrier S Jalkanen University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314 References 1. Crit Care Med. 2006;34:532-7. 2. J Crit Care. 2012;27:522.e11-7. 3. Crit Care. 2013;17:R132. Methods Forty-one CD-1 mice were divided into two groups: an HCl group underwent orotracheal instillation of hydrochloric acid on day 0, and a group control. Mice were evaluated on days 0, 1, 2 and 4 after a 30-minute period of mechanical ventilation. Blood and lung edema fl uid (EF) were sampled. Before initiation of MV, all mice received a tracheal instillation of bovine serum albumin (5%) to detect changes in alveolar protein levels over 30 minutes. Plasma levels of sRAGE and total protein levels were measured. AFC rate values were corrected after measurement of mouse serum albumin in EF. P235 P235 Endocan can be a predictive marker of severity of sepsis but cannot be a marker of acute respiratory distress syndrome in ICU patients M Mizunuma, H Ishikura, Y Nakamura, K Muranishi, S Morimoto, H Kaneyama, Y Izutani, T Nishida, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P235 (doi: 10.1186/cc14315) f References 1. Kiss et al. Eur J Immunol. 2007;37:3334-8. 2. Bellingan G, et al. Lancet Respir Med. 2014; 2:98-107. 1. Kiss et al. Eur J Immunol. 2007;37:3334-8. 2. Bellingan G, et al. Lancet Respir Med. 2014; 2:98-107. P237 Elevated levels of soluble RAGE predict impaired alveolar fl uid clearance in a translational mouse model of acute respiratory distress syndrome R Blondonnet1, M Jabaudon1, G Clairefond2, J Audard1, D Bouvier2, G Marceau1, P Blanc2, P Dechelotte1, V Sapin2, JM Constantin1 1CHU Clermont-Ferrand, France; 2Laroratoire R2D2-EA7281, Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P237 (doi: 10.1186/cc14317) Results We enrolled 70 ARDS patients during the investigation periods. We met six patients with nonsepsis, 27 with severe sepsis and 37 with septic shock. The serum Endocan levels were signifi cantly higher in patients with septic shock (3.7 to 3.9 ng/ml) than in patients with severe sepsis (1.7 to 2.3 ng/ml, P <0.05), nonsepsis (0.6 to 0.3 ng/ml, P <0.05) and healthy donors (0.4 to 0.1 ng/ml, P <0.05). However, there was no signifi cant correlation between the Endocan level and the severity of ARDS. In addition, signifi cant correlation between the Endocan level and EVLWi and PVPI was not observed. Introduction Receptor for advanced glycation endproducts (RAGE) is a transmembrane receptor expressed in the lung and primarily located on alveolar type I cells. RAGE is implicated in acute respiratory distress syndrome to alveolar infl ammation and, when its soluble form sRAGE is assayed in plasma or pulmonary edema fl uid, as a marker of AT I cell injury. Functional activity of AT 1 cells can be assessed by the measurement of the alveolar fl uid clearance (AFC) rate [1], but the relationship between sRAGE plasma levels of sRAGE and AFC rates has never been investigated. Our objectives were to report plasma levels of sRAGE in a mouse model of direct acid-induced epithelial injury, and to test their correlation with AFC rates. Conclusion These results suggested that there was good relationship between the Endocan level and the severity of sepsis. But unlike the trauma patients, correlation between the severity of ARDS and Endocan value was not recognized. P236 Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Q Chen, A Liu, H Qiu, Y Yang Southeast University, Nanjing, China Critical Care 2015, 19(Suppl 1):P236 (doi: 10.1186/cc14316) Endocan can be a predictive marker of severity of sepsis but cannot be a marker of acute respiratory distress syndrome in ICU patients M Mizunuma, H Ishikura, Y Nakamura, K Muranishi, S Morimoto, H Kaneyama, Y Izutani, T Nishida, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P235 (doi: 10.1186/cc14315) l Results The permeability signifi cantly increased after LPS stimulation in a dose-dependent and time-dependent manner (P <0.01). Mean- while, MSC-EC interaction more signifi cantly decreased endothelial permeability induced by LPS (P  <0.05 or P  <0.01). Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group (P  <0.01). However, neutralizing HGF with anti- HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability (P  <0.05). Compared with the MSC group, MSC-EC interaction increased VE-cadherin protein expression (P <0.01), and restored remodeling of F-actin and junctional localization of VE- cadherin. However, the MSC eff ect was signifi cantly blocked by anti- HGF antibody (P <0.05 or P <0.01). Introduction Endocan (endothelial cell specifi c molecule-1), a 50 kDa dermatan sulfate proteoglycan, is expressed by endothelial cells in the lung and kidney. It was reported that the serum Endocan level is related to the severity of sepsis and positive correlation with the mortality rate. On the other hand, it was also reported that lower levels of serum Endocan are associated with subsequent development of chronic kidney disease, and chronic liver disease acute lung injury (ALI) in trauma patients. The aim of this study is confi rmation of the relationship between serum Endocan level and the severity of sepsis, and also the severity of acute respiratory distress syndrome (ARDS) in septic patients. y Conclusion These data suggest that MSC-EC interaction decreased endothelial permeability induced by LPS, which was mainly attributed to HGF secreted by hMSCs. The main mechanisms of HGF restoring the integrity of EC monolayers are remodeling of endothelial intercellular AJs and decreasing caveolin-1 protein expression. p p Methods This study was conducted as a single-center, retrospective, observational study in the emergency department of Fukuoka University Hospital from April 2010 to August 2013. Blood samples were collected within 2 hours when the patients were diagnosed with ARDS. In this time we adopted the Berlin defi nition as the categorized of ARDS severity. Furthermore, we evaluated the extravascular lung water index (EVLWi) and pulmonary vascular permeability index (PVPI) as a condition of ARDS using the transpulmonary thermodilution method. Additionally, 10 healthy donors were entered as a control. The patients were divided into nonsepsis, severe sepsis and septic shock using the ACCP/SCCM guidelines. University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) The overall mortality of the 37 patients treated with FP-1201 S82 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secreted more factors by MSC–endothelial cell (MSC-EC) interaction. We hypothesized that MSC-EC interaction restored endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF. was only 8.1%, fourfold to fi vefold less than the expected rate based on APACHE II score values of 21.9. The control group (n = 59), which was eligible for the trial but not possible to recruit, had mortality of 32.2% (P = 0.01) and APACHE II score 23.0. Conclusion Restriction of vascular leakage with FP-1201 seems to signifi cantly benefi t ALI/ARDS patients. Our results suggest that FP- 1201 could be the fi rst eff ective, mechanistically targeted, disease- specifi c pharmacotherapy for patients with ARDS. However, these fi ndings warrant conduction of a large multicenter study to establish safe and eff ective FP-1201 treatment of ARDS. f p p y p Methods We investigated the endothelial permeability induced by LPS under two co-culture conditions in transwells. HPMECs were added into the upper chambers of cell-culture inserts, while there two diff erent co-culture conditions in the lower side of transwells as follows: MSC-EC interaction group: MSCs and HPMEC contact co- culture in the lower chambers; and MSC groups: MSCs only in the lower chambers. The endothelial permeability in the upper side of transwells was detected. Then the concentration of HGF was measured in the culture medium using an enzyme-linked immunosorbent assay kit, following by neutralizing HGF with anti-HGF antibody in the co-culture medium. In addition, VE-cadherin protein expression were measured under the co-culture conditions by western blot, adherens junctions (AJs) protein including F-actin and VE-cadherin were detected by immunofl uorescence technique as well.i P240 y Results Of those 4,652 patients, there were 2,947 nonsmokers, 1,148 exsmokers, and 557 active smokers. There was no diff erence of APACHE II score between three groups of patients. The active smokers exhibited the highest incidence of ARDS (active smokers 5.4%, exsmokers 4.8%, and nonsmokers 3%, P  = 0.003). There was no diff erence of 28-day mortality between the three groups of patients. Active smokers had the highest incidence of SIRS (active smokers 41%, exsmokers 37%, and nonsmokers 34%, P  = 0.006). Compared with nonsmokers and exsmokers, active smokers had a longer SICU LOS (P <0.01) and higher total SICU cost (P = 0.02). Patients who smoked more than 15 pack- years were 2.5 times more likely to develop ARDS than patients who smoked ≤15 pack-years (95% CI: 1.65 to 3.66, P <0.001). In multivariate analysis we found that every 1 pack-year of cigarette smoking before References 1. Hoshino Y, et al. Am J Physiol Lung Cell Mol Physiol. 2001;281:L509-16. 2. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 (Suppl 1):S45-54. P238 Smoking increased risk of ARDS in surgical critically ill patients: results from the multicenter THAI-SICU study P Wacharasint1, P Fuengfoo1, N Sukcharoen1, R Rangsin2, THAI-SICU Trial Group3 1Phramongkutklao Hospital, Bangkok, Thailand; 2Phramongkutklao College of Medicine, Bangkok, Thailand; 3THAI-SICU Collaborating Centre, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P238 (doi: 10.1186/cc14318) 2 Results A total of 101 ARDS patients was enrolled; 44 (43.6%) patients were normal weight, 36 (35.6%) overweight and 21 (20.8%) obese. Lung and chest wall elastance were not diff erent between groups, both at PEEP levels of 5  cmH2O and 15  cmH2O (P  = 0.580 and P  = 0.113, respectively), and the end-inspiratory transpulmonary pressure was also similar. We did not observe any diff erence between groups regarding PaO2/FiO2 ratio (P = 0.178 at PEEP 5 cmH2O; P = 0.073 at PEEP 15 cmH2O) and PaCO2 (P = 0.491 at PEEP 5 cmH2O; P = 0.237 at PEEP 15 cmH2O). Introduction Cigarette smoking slowly and progressively damages the respiratory system [1]. In surgical critically ill patients, whether active cigarette smoking until admission to the surgical intensive care unit (SICU) is associated with increased risk of acute respiratory distress syndrome (ARDS) is not clearly identifi ed. 2 Conclusion In ARDS obese patients the chest wall elastance and the end-inspiratory transpulmonary pressure are not aff ected by the body weight, suggesting that normal weight and obese patients present similar risks for lung stress and VILI onset. The severity of hypoxemia was not diff erent between groups. y yi Methods We conducted a cohort study using the THAI-Surgical Intensive Care Unit (THAI-SICU) study databases [2], which recruited 4,652 Thai patients admitted to the SICUs from nine university-based hospitals in Thailand (April 2011 to November 2012). The enrolled patients were divided into three groups (active smokers, exsmokers, and nonsmokers). Primary outcome was the incidence of patients diagnosed with ARDS and the secondary outcomes included 28-day mortality, incidence of systemic infl ammatory response syndrome (SIRS), SICU length of stay (LOS), and total SICU cost. f References 1. Pelosi et al. Chest. 1996;109:144-51. 1. Pelosi et al. Chest. 1996;109:144-51. . Suratt et al. J Appl Physiol Respir Environ Exerc Physiol. 1984;57:403 2. Suratt et al. J Appl Physiol Respir Environ Exerc Physiol. 1984;57:403-7. P239l P239 Infl uence of body weight on lung mechanics and respiratory function in ARDS patients I Algieri1, D Chiumello2, C Mietto1, E Carlesso1, A Colombo1, G Babini1, M Cressoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS, ‘Ospedale Maggiore Policlinico Mangiagalli Regina Elena’ di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P239 (doi: 10.1186/cc14319) Figure 1 (abstract P237). Relation between AFC rates and sRAGE plasma levels. (P = 0.03) and day 2 (P = 0.02). The rate of AFC was inversely correlated with sRAGE levels in the plasma (Spearman’s ρ = –0.73, P <0.001). See Figure 1. Introduction Traditionally, ARDS obese patients are ventilated with higher tidal volumes and higher PEEP due to expected increased in pleural pressure. However, data from the literature regarding the infl uence of body mass on lung mechanics and, particularly, on chest wall elastance are not univocal [1,2]. Failure to account for how the increase in body weight could aff ect the respiratory function can result in injurious mechanical ventilation and in the onset of VILI. The aim of this study was to evaluate the role of the body weight on respiratory mechanics in ARDS patients. g Conclusion The highest impairment in AFC is reported on day 1. sRAGE levels are also higher in injured mice and may be a good surrogate marker of AT I cell injury. This is a newly described relationship between AFC rates and sRAGE plasma level in a mouse model of direct epithelial injury. Our results support further translational investigation on the role of RAGE in alveolar injury and recovery. 1. Eur Respir J. 2012;39:1162-70. Methods A group of deeply sedated and paralyzed ARDS patients was divided into three classes according to the body mass index: normal weight (between 18.5 and 24.9 kg/m2), overweight (between 25.0 and 29.9 kg/m2) and obese (between 30.0 and 39.9 kg/m2). They were mechanically ventilated in volume-controlled mode with a tidal volume of 6 to 8 ml/kg according to predicted body weight. Respiratory mechanics and gas exchange were assessed at PEEP levels of 5 and 15 cmH2O. Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Q Chen, A Liu, H Qiu, Y Yang Results Basal AFC rate was 35% over 30 minutes in HCl-injured mice, but it was signifi cantly depressed on day 1 (16% over 30 minutes; P = 0.02). Over time, AFC reached basal levels again. Plasma levels of sRAGE were higher in HCl-treated animals than in control animals on day 1 Introduction Mesenchymal stem cells (MSCs) have potent stabilizing eff ects on the vascular endothelium injury, inhibiting endothelial Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S83 Figure 1 (abstract P237). Relation between AFC rates and sRAGE plasma levels. admission to the SICU is associated with increased risk of new ARDS with a hazard ratio of 1.02 (95% CI: 1.01 to 1.02, P  = 0.001) after adjustment for APACHE II score, age, gender, and chronic obstructive pulmonary disease. Conclusion In surgical critically ill patients, active smokers are associated with increased risk of new ARDS, longer SICU LOS, and higher total ICU cost, compared with exsmokers and nonsmokers. Our fi ndings emphasize the essential need for a smoking cessation program. f Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Introduction Acute respiratory distress syndrome (ARDS) develops on the basis of nosocomial pneumonia (NP) in 30 to 35% of cases. A complex clinical approach is required for diagnosing it. The aim of this study was to investigate into the role of the PaO2/FiO2 ratio (P/F), extravascular lung water index (EVLWI) and surfactant protein D (SPD) as a complex diagnostic tool for ARDS in NP. S84 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The observational study in ICU ventilated septic patients with peritonitis (70%), pancreonecrosis (25%) and mediastinitis (5%) was done in 2010 and 2014. ARDS was diagnosed and staged according to the V.A. Negovsky Research Institute criteria and the Berlin defi nition. Plasma SPD was measured on ARDS diagnosis (day 0) and days 3 and 5 by the immunoenzyme essay (BioVendor, USA). Patients were treated according to the international guidelines. Data were statistically analyzed by STATISTICA 7.0, ANOVA and presented as median and 25 to 75th percentiles (ng/ml); P  <0.05 was considered statistically signifi cant. Areas under the receiver operating curves were calculated. Results Sixty-fi ve patients (out of 450 screened) were enrolled in the study according to the inclusion/exclusion criteria. Patients were assigned into groups: NP + ARDS (n = 43, 43 ± 4.9 years old, M/F 39/4, mortality 23%) and NP (n = 22, 40 ± 5.1 years old, M/F 20/2, mortality 18%). Groups were comparable in APACHE II and SOFA scores on the baseline. In the NP + ARDS group SPD was higher at all points than in the NP group. Plasma SPD on day 0 >111.2 ng/ml yielded a sensitivity of 68.2% and specifi city of 92.3% (AUC 0.85; 95% CI 0.684 to 0.945; P <0.0001) for diagnosing ARDS in NP. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Results Forty-fi ve oesophageal balloon pressure–volume curves were obtained in 31 patients undergoing controlled mechanical ventilation (PEEP 12 ± 5 cmH2O, FiO2 0.7 ± 0.2, tidal volume/ideal body weight 8.0 ± 1.6 ml/kg). According to the graphically detected minimum slope section of the curve, the minimum and maximum appropriate balloon volumes were 1.5 ± 0.6 ml and 5.3 ± 0.9 ml, respectively. Between these two volumes, the slope of the curve was 1.1 ± 0.5 cmH2O/ml, ranging from 0.3 to 3.1 cmH2O/ml. Eff ects of a recruitment maneuver on plasma soluble rage in patients with diff use ARDS: a prospective randomized crossover study M Jabaudon, N Hamroun, L Roszyk, R Blondonnet, R Guerin, JE Bazin, V Sapin, B Pereira, JM Constantin CHU Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P241 (doi: 10.1186/cc14321) y M Jabaudon, N Hamroun, L Roszyk, R Blondonnet, R Guerin, JE Bazin, V Sapin, B Pereira, JM Constantin CHU Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P241 (doi: 10.1186/cc14321) Introduction The soluble form of the receptor for advanced glycation endproducts (sRAGE) is a promising marker for epithelial dysfunction, but it has not been fully characterized as a biomarker during ARDS. Whether sRAGE could inform on the response to ventilator settings has been poorly investigated, and whether recruitment maneuver (RM) may infl uence plasma sRAGE remains unknown. p p gl Results Forty-fi ve oesophageal balloon pressure–volume curves were obtained in 31 patients undergoing controlled mechanical ventilation (PEEP 12 ± 5 cmH2O, FiO2 0.7 ± 0.2, tidal volume/ideal body weight 8.0 ± 1.6 ml/kg). According to the graphically detected minimum slope section of the curve, the minimum and maximum appropriate balloon volumes were 1.5 ± 0.6 ml and 5.3 ± 0.9 ml, respectively. Between these two volumes, the slope of the curve was 1.1 ± 0.5 cmH2O/ml, ranging from 0.3 to 3.1 cmH2O/ml. l Methods Twenty-four patients with moderate/severe, nonfocal ARDS were enrolled in this prospective monocentric crossover study and randomized into a ‘RM-SHAM’ group when a 6-hour-long RM sequence preceded a 6-hour-long sham evaluation period, or a ‘SHAM-RM’ Figure 1 (abstract P241). Evolution of plasma levels of sRAGE (pg/ml) in both randomization sequences. 2 Conclusion The oesophageal artefact  – that is, the reaction of the oesophageal wall to balloon infl ation – may be clinically signifi cant, being on average 1  cmH2O for each millilitre of volume injected in the catheter, but reaching values as high as 3 cmH2O/ml. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Our fi ndings could help to design future studies of sRAGE as a marker of response to therapeutic interventions during ARDS. P242 Oesophageal artefact may signifi cantly aff ect oesophageal pressure measurement in mechanically ventilated patients F Mojoli, F Torriglia, M Pozzi, S Bianzina, G Tavazzi, A Orlando, A Braschi Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy Critical Care 2015, 19(Suppl 1):P242 (doi: 10.1186/cc14322) Introduction Oesophageal pressure is increasingly used to monitor and manage mechanically ventilated patients. Even if the oesophageal balloon catheter is correctly positioned, the measurement can be aff ected by inappropriate balloon fi lling and/or oesophageal reaction to balloon infl ation. We aimed to assess the oesophageal reaction to oesophageal balloon fi lling in mechanically ventilated patients. i Methods An oesophageal balloon catheter (NutriVent; Sidam, Mirandola, Italy) was introduced in mid/distal thoracic position in 31 patients under invasive mechanical ventilation for acute respiratory failure. At ambient pressure, the balloon of the NutriVent catheter can be infl ated up to 6 ml without generation of recoil pressure. The balloon was progressively infl ated in 0.5  ml steps up to 9  ml and end-expiratory values of balloon pressure were used to assemble the balloon pressure–volume curve. The minimum slope section of the curve was graphically detected and infl ation volumes corresponding to this part of the curve were considered appropriate. Overdistension of the balloon being excluded by defi nition in this section of the curve, its slope was attributed to the oesophageal reaction to balloon infl ation.i Methods An oesophageal balloon catheter (NutriVent; Sidam, Mirandola, Italy) was introduced in mid/distal thoracic position in 31 patients under invasive mechanical ventilation for acute respiratory failure. At ambient pressure, the balloon of the NutriVent catheter can be infl ated up to 6 ml without generation of recoil pressure. The balloon was progressively infl ated in 0.5  ml steps up to 9  ml and end-expiratory values of balloon pressure were used to assemble the balloon pressure–volume curve. The minimum slope section of the curve was graphically detected and infl ation volumes corresponding to this part of the curve were considered appropriate. Overdistension of the balloon being excluded by defi nition in this section of the curve, its slope was attributed to the oesophageal reaction to balloon infl ation. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) The pressure generated by the oesophageal reaction leads to overestimation of pleural pressure. Therefore, the oesophageal artefact may signifi cantly aff ect clinical decision-making based on absolute values of oesophageal pressure. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) P/F ratio on day 0 <280 yielded a sensitivity of 94.1% and specifi city of 76.9% (AUC 0.89; 95% CI 0.744 to 0.952; P <0.0001) and EVLWI on day 0 >8.3 ml/kg yielded a sensitivity of 94.1% and specifi city of 92.3% (AUC 0.92; 95% CI 0.810 to 0.982; P <0.0001) for the diagnosis of ARDS in NP. A complex ROC analysis (for SPD in the group of patients with P/F <280 and EVLWI >8.3) yielded a much better diagnostic accuracy of SPD: cutoff >93.7 ng/ml, sensitivity 81.0%, specifi city 100.0% (AUC 0.96; 95% CI 0.817 to 0.998; P <0.0001). Conclusion A complex approach (P/F <280, EVLWI >8.3, SPD >93.7) presents as a sensitive and highly specifi c method for diagnosing ARDS in NP patients. group (inverted sequences). Protective ventilation was applied, and RM consisted of the application of 40  cmH2O airway pressure for 40 seconds. Arterial blood was sampled for gas analyses and sRAGE measurements, 5  minutes pre RM (or 40-second-long sham period), 5 minutes, 30 minutes, 1 hour, 4 hours and 6 hours after the RM (or 40-second-long sham period). g p ) Results Mean PaO2/FiO2, tidal volume, PEEP and plateau pressure were 125  mmHg, 6.8  ml/kg (ideal body weight), 13 and 26  cmH2O, respectively. Median baseline plasma sRAGE levels were 3,232 pg/ml. RM induced a signifi cant decrease in sRAGE (–1,598 ± 859 pg/ml) in 1 hour (P = 0.043). At 4 and 6 hours post RM, sRAGE levels increased back toward baseline values. Pre-RM sRAGE was associated with RM- induced oxygenation improvement (AUC 0.87). See Figure 1. Conclusion We report the fi rst kinetics study of plasma sRAGE after RM in ARDS. Our fi ndings could help to design future studies of sRAGE as a marker of response to therapeutic interventions during ARDS. Results Mean PaO2/FiO2, tidal volume, PEEP and plateau pressure were 125  mmHg, 6.8  ml/kg (ideal body weight), 13 and 26  cmH2O, respectively. Median baseline plasma sRAGE levels were 3,232 pg/ml. RM induced a signifi cant decrease in sRAGE (–1,598 ± 859 pg/ml) in 1 hour (P = 0.043). At 4 and 6 hours post RM, sRAGE levels increased back toward baseline values. Pre-RM sRAGE was associated with RM- induced oxygenation improvement (AUC 0.87). See Figure 1. Conclusion We report the fi rst kinetics study of plasma sRAGE after RM in ARDS. P244 P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation Regional distribution of excess tissue mass in ARDS lung I Algieri1, D Massari1, A Colombo1, G Babini1, F Crimella1, M Brioni1, A Cammaroto1, K Nikolla1, C Montaruli1, M Guanziroli1, M Gotti1, C Chiurazzi1, M Amini1, M Chiodi1, M Cressoni1, D Chiumello2, L Gattinoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS, ‘Ospedale Maggiore Policlinico Mangiagalli Regina Elena’ di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P244 (doi: 10.1186/cc14324) Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation y p g y T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) Introduction ARDS has a wide variability of lung morphological charac- teristics. Both alveolar collapse and airway narrowing or closing are present, often heterogeneously. Despite advances in ARDS imaging, we have thus far been unable to distinguish regional airway opening from airway dilatation in PEEP-induced lung recruitment. We demonstrate the technique of functional respiratory imaging (FRI) to diff erentiate these two entities. Introduction ARDS is characterized by edema diff use to all lung fi elds. Distribution of excess tissue mass had been studied with CT scan in a few patients on a single slice, comparing with data obtained in healthy controls. Methods ARDS patients underwent CT scan imaging during their ICU stay at 45  cmH2O end-inspiratory pressure. After hospital discharge, patients underwent a follow-up CT scan performed at end inspiration. Each lung was divided into three sections along the apex–base axis and into three sections along the sternum–vertebral axis (nine regions per lung). Excess tissue mass in each lung region was defi ned as the diff erence in lung tissue (grams) between the CT scan performed during ARDS course and the follow-up CT scan. Results are presented as mean ± SD. Methods Six patients with early-stage ARDS were included in this prospective single-centre cohort trial. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) Figure 1 (abstract P244). Figure 1 (abstract P244). demonstrated recently its eff ectiveness on prognosis. Extrapulmonary etiologies of ARDS include abdominal emergencies. In cases of severe hypoxemia in the early postoperative period, intensivists discuss prone positioning based on the risk/benefi t ratio. demonstrated recently its eff ectiveness on prognosis. Extrapulmonary etiologies of ARDS include abdominal emergencies. In cases of severe hypoxemia in the early postoperative period, intensivists discuss prone positioning based on the risk/benefi t ratio. p gi Methods We conducted a retrospective two-center study of 5 years. The aim was to compare the prevalence of surgical complication potentially related to prone positioning between patients who had at least one prone positioning session and patients that remained in a supine position after abdominal surgery. Patients with ARDS in a context of recent (<7  days) abdominal surgery (except laparoscopy) were included. The primary outcome was the number of patients who had at least one surgical complication potentially related to prone positioning. We defi ned a priori these complications: scar dehiscence, abdominal compartment syndrome, stoma leakage, stoma necrosis, scar necrosis, wound infection, displacing of a drainage system, removal of gastro- or jejunostomy feeding, digestive fi stula, evisceration.i g j j y g gi Results We identifi ed 43 patients with postoperative ARDS (62  ± 8 years, SAPS II 50 ± 13), among whom 34 (79%) had emergent surgery. Fifteen patients had at least one stoma after surgery. Nineteen patients (44%) had at least one prone positioning session (number of sessions: 2 (1 to 3)). At baseline, prone group patients had minimum PaO2/FiO2 ratio lower than the supine group (77 ± 23 vs. 110 ± 46 mmHg, P = 0.005). Plateau pressure was higher in the prone group (28 ± 4 vs. 23 ± 5 cmH2O, P = 0.002). The fi rst prone positioning session signifi cantly increased the PaO2/FiO2 ratio: 106  ± 52 vs. 192  ± 90  mmHg (P  = 0.001). Mean duration of the fi rst prone positioning session was 20 ± 10 hours. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 In the prone group, 11 patients (58%) had at least one surgical complication, in comparison with nine (38%) in the supine group (P = 0.2). These complications resulted in revision surgery for two (10%) patients in the prone group and two (8%) in the supine group (P = 0.8). Mortality in the ICU was respectively 42% and 38% in prone group and supine group (P = 0.8).if Conclusion These preliminary results confi rm the eff ectiveness of prone positioning in terms of oxygenation in ARDS after abdominal surgery without signifi cant increase in surgical complications and no eff ect on the need for surgical revisions. Hence, if necessary, clinicians should not refrain from proning patients with postabdominal surgery ARDS. P243 P243 Prone positioning in acute respiratory distress syndrome after abdominal surgery S Gaudry1, S Tuff et1, AC Anne-Claire2, N Zucman1, S Msika1, M Pocard2, D Payen2, D Dreyfuss1, JD Ricard1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Lariboisière, Paris, France Critical Care 2015, 19(Suppl 1):P243 (doi: 10.1186/cc14323) Introduction Prone positioning has been used for many years as an alveolar recruitment strategy in acute respiratory distress syndrome (ARDS). Prone positioning in ARDS improves oxygenation and Introduction Prone positioning has been used for many years as an alveolar recruitment strategy in acute respiratory distress syndrome (ARDS). Prone positioning in ARDS improves oxygenation and Figure 1 (abstract P241). Evolution of plasma levels of sRAGE (pg/ml) in both randomization sequences. S85 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Effi cacy and safety of open lung ventilation in patients with impaired peripheral chemorefl ex sensitivity N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Kuban State Medical University, Krasnodar, Russia Critical Care 2015, 19(Suppl 1):P246 (doi: 10.1186/cc14326) g Results Piglets ventilated with higher energy loads (RR 15 and 12) developed ventilator-induced lung injury (VILI), while piglets ventilated with lower energy loads (RR 9, 6 and 3) did not. The energy dissipated in the lung parenchyma at each breath was 0.56  J (0.52 to 0.62). Dissipated energy increased with time in piglets that developed VILI, while it remained near-constant in all the other piglets. Recruitability was 6% (3 to 8) of lung parenchyma, strain was 3.1 (2.6 to 3.5) and lung inhomogeneity extent (that is, the percentage of lung parenchyma that is inhomogeneous) was 10% (9 to 11). The energy dissipated in the lung parenchyma was well related to lung recruitability (r2 = 0.50, P <0.0001), strain (r2 = 0.57, P <0.0001) and lung inhomogeneity extent (r2 = 0.42, P <0.0001). Multiple linear regression showed that dissipated energy was independently related to all of the three determinants of energy dissipation: lung opening and closing (P = 0.025), strain (P <0.0001) and lung inhomogeneities (P <0.01). Introduction Mechanical ventilation during anesthesia leads to the development of atelectasis, poor oxygenation and postoperative pulmonary complications. Application of PEEP and recruitment maneuver (RM) can signifi cantly reduce the severity of atelectasis and improve lung function. But the application of this strategy often leads to hemodynamic instability, which may be associated with impaired reactivity of the cardiovascular system. The purpose of this study was to evaluate the effi cacy and safety of RM in patients with increased sensitivity of peripheral chemoreceptors (SPCR), which refl ects the decreasing reactivity of the cardiovascular system. g y y Methods We conducted a prospective study in 116 patients with high SPCR, evaluated using the breath-holding test. The test was performed by measuring of voluntary breath-holding duration (BHD) after two- thirds of maximal inspiration. The end of breath-hold was determined by a palpation of contraction of the diaphragm. BHD <38 seconds was the marker of high SPCR [1]. All patients received a major abdominal surgery and were randomized into an open lung ventilation group or a PEEP group. P244 The lower infl iction point on a pressure–volume curve was considered as the clinically acceptable minimal PEEP value. Subsequently, four distinct PEEP levels were chosen to perform CT scans: at 20 cmH2O; median value between 1 and 3; clinically acceptable minimal; and 0 cmH2O. FRI methods as described by De Backer and colleagues [1] were used to evaluate airway opening and airway dilatation. Results We studied eight ARDS patients (55  ± 18  years) with a BMI of 27 ± 6 kg/m2. At ICU admission, patients had the following clinical parameters: PaO2/FiO2 106 ± 33 with PEEP 15 ± 5 cmH2O; PaCO2 43 ± 10 mmHg; pH 7.35 ± 0.05. The average increase in lung weight during ARDS compared with follow-up CT scan was 68 ± 40% (680 ± 320 g). Figure 1 presents the tissue volume during ARDS (white bars) and after ARDS resolution (black bars) and compares the ratio between the two (*P <0.01 vs. dependent region). Results Airway stretching (that is, bronchodilatation) could be quantifi ed and distinguished from airway recruitment with this technique. Higher PEEP pressures not only recruit, but also expand the bronchi. The ratio of dilation/recruitment of bronchi was higher in the upper lobes than in the lower lobes, as illustrated in Figure 1. We were able to phenotype each patient, allowing a prediction on when an increase in PEEP further recruits atelectasis/bronchi or distends certain airway regions. Conclusion The excess tissue mass was not diff erent between apex, hilum and base, but was increased in the dependent lung regions at apex and hilum, being uniformly distributed at the lung base. y g Conclusion The novel technique of FRI can be used to visualise the airway structures in ARDS and distinguish airway stretching from Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S86 more pronounced. So CI on average decreased by 34% (from 3.7 to 2.5 l/minute/m2) compared with 10% with no RM (P <0.05), and SVR decreased by 19% (from 1,310 to 1,150 dyn × sec–1 × cm–5, P <0.05), while in the PEEP group it did not change. No signifi cant diff erences between groups in the incidence of complications, length of stay in the ICU and in the hospital were noted. Figure 1 (abstract P245). Bronchodilation and bronchial recruitment split per lobe for one patient. Effi cacy and safety of open lung ventilation in patients with impaired peripheral chemorefl ex sensitivity The concept of open lung ventilation was performed as follows: PEEP was increased from 4 to 10 cmH2O for three breaths, from 10 to 15 cmH2O for three breaths, and from 15 to 20 cmH2O for 10 breaths [2]. Then PEEP was reduced to 12 cmH2O. This RM was repeated every hour. In the PEEP group PEEP was maintained at 12 cmH2O during the whole anesthesia. Hemodynamics, blood gases and dynamic compliance were evaluated. Conclusion Energy dissipation was largely dependent on strain, obtained with very high tidal volumes, but also lung inhomogeneities and lung opening and closing played a signifi cant role. Reference 1. Cressoni M. et al. Am J Respir Crit Care Med. 2014;189:149-58. P244 Conclusion RM patients with high SPCR and with reduced reactivity of the cardiovascular system improve lung function, but this is associated with the risk of hemodynamic instability. References 1. Zabolotskikh I, et al. Eur J Anesth. 2014;31:62. 2. Weingarten TN, et al. Br J Anesth. 2010;104:16-22. Determinants of energy dissipation in the respiratory system during mechanical ventilation Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P247 (doi: 10.1186/cc14327) Introduction Mechanical ventilation performs at each breath mechanical work on the lung parenchyma. Part of this energy is recovered and part is dissipated into the respiratory system. The purpose of this study is to assess the respective role of three known determinants of energy dissipation: lung opening and closing, strain and lung inhomogeneities [1]. Figure 1 (abstract P245). Bronchodilation and bronchial recruitment split per lobe for one patient. g g Methods Thirteen female piglets (21 ± 2 kg) were ventilated with a strain (VT/FRC) greater than 2.5 (VT ~ 38 ± 3 ml/kg) for 54 hours or until massive lung edema developed. Piglets were divided into fi ve groups characterized by diff erent energy loads obtained varying the respiratory rate: 15 breaths/minute (n = 3), 12 (n = 3), 9 (n = 3), 6 (n = 2) and 3 (n = 2). Every 6 hours two CT scans were performed (end-expiration and end- inspiration) and a static pressure–volume curve was obtained. A total of 51 CT scan couples and 51 corresponding pressure–volume curves was analyzed. The energy dissipated in the lung parenchyma at each breath was determined as the hysteresis area of the pressure–volume curve. CT scans were quantitatively analyzed with dedicated software. Data are presented as median (interquartile range). airway recruitment. This pilot study shows that, in ARDS, the upper lung regions are subject to airway dilation, whereas the lower (atelectatic) lung lobes have more airway opening with higher PEEP levels. Reference 1. De Backer et al. Radiology. 2010;257:854-62. P246fi P248 P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Results RM improved oxygenation compared with the PEEP group. The mean increase in the oxygenation index at the end of surgery was 31% (from 340 to 445 mmHg, P <0.05), in the PEEP group the increase was less signifi cant and amounted to 12% (from 330 to 370 mmHg, P <0.05). Dynamic compliance increased by 35% in the RM group and did not change in the PEEP group. Hemodynamic changes at RM were Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) Introduction Mechanical ventilation and hyperoxia independently promote pulmonary injury and infl ammation. However, the time course of the immune response following concurrent exposure is unclear. The Introduction Mechanical ventilation and hyperoxia independently promote pulmonary injury and infl ammation. However, the time course of the immune response following concurrent exposure is unclear. The Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S87 lungs. We used EIT with increased signal quality and spatial resolution to describe and quantify the regional changes in aeration caused by body position, both during spontaneous breathing and mechanical ventilation in pulmonary healthy patients undergoing laparoscopic prostatectomy. lungs. We used EIT with increased signal quality and spatial resolution to describe and quantify the regional changes in aeration caused by body position, both during spontaneous breathing and mechanical ventilation in pulmonary healthy patients undergoing laparoscopic prostatectomy. Figure 1 (abstract P248). Infl ammatory mediators independent of tidal volumes after 12 hours of MV. Methods In 40 patients we performed EIT measurements at fi ve points of time (Table 1) with the Swisstom BB2 prototype. Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) p g g Methods Healthy male C57Bl/6J mice, aged 9 to 10  weeks, were randomly assigned to experimental groups (n = 8), in which the applied fractions of oxygen (FiO2) were 30%, 50% or 90% and tidal volumes were either 7.5 or 15 ml/kg. Anesthetized mice were tracheotomized and ventilated for 8 or 12 hours. Infl ammatory cells and mediators were measured in bronchoalveolar lavage fl uid (BALf).i Introduction The still unclear mechanisms causing ventilator- induced diaphragm dysfunction (VIDD) are considered intrinsic to the diaphragm muscle fi bers. VIDD delays and complicates weaning from mechanical ventilation (MV) and accordingly contributes to prolonged ICU stay by 50%, with older patients being more aff ected than the young. The main aim of this study was to measure the eff ects of aging and 5 days of MV on rat diaphragm muscle fi ber structure and function. We also aimed to investigate the biological age of the old rats to obtain data useful to design future experimental studies focusing on the eff ects of age in an ICU setting. Results Mice exposed to higher FiO2 had signifi cantly higher PaO2 levels at the end of the experiment. The total number of infl ammatory cell in the BALf was not signifi cantly diff erent between the experimental groups (P = 0.28), yet an increasing trend in the percentage of neutrophils was observed with increasing FiO2 (P = 0.03). Cytokine and chemokine levels did not diff er between FiO2 groups at 8  hours of ventilation. In mice ventilated for 12 hours, a signifi cantly increasing trend in IFNγ, IL-1β, IL- 10, MCP-1 and TNFα (Fig. 1, P <0.01) was measured with increasing FiO2, whereas IL-6, KC, MIP-2, GM-CSF and VEGF remained virtually unchanged. Diff erences between the tidal volume groups were small and did not markedly infl uence the eff ects of hyperoxia. See Figure 1.f Methods We used a unique ICU rat model, which allows us to maintain the vital parameters stable under deep sedation and MV for long durations (several weeks). Diaphragm fi ber cross-sectional area (CSA) and force-generating capacity (specifi c force = absolute force / CSA) were measured in young (6 months) and old (28 to 32 months) F344/ BN hybrid rats in response to 5 days of deep sedation and volume- controlled MV. P250 Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) To investigate the biological age of the old rats, we performed a second set of experiments, comparing muscle fi ber CSA and specifi c force in fast and slow-twitch distal hind limb muscles in three diff erent age groups: young adults (6  months), middle aged (18 months) and old rats (28 months). ylf yp g Conclusion Hyperoxia induced a time-dependent and diff erentiated immune response that was independent of tidal volumes in a model of mechanically ventilated mice. The presence of cytokines and chemokines in the pulmonary compartment was more pronounced with prolonged and severe hyperoxia. P248 Thirty-two electrodes were used to apply weak alternating currents to the thorax and to measure the resulting voltages, from which tomographic images of the changes in regional impedance caused by ventilation were created. We describe the ventilation distribution using a novel EIT lung function parameter called Silent Spaces that provides information about areas that do not receive much or any air during tidal breathing and are divided into nondependent (NSS) and dependent Silent Spaces (DSS) using a reference line that runs perpendicular to the gravity vector right through the centre of ventilation. NSS and DSS are expressed as a percentage of the total lung area. Results Perioperative changes of NSS and DSS are shown in Table 1 as mean ± SD. Statistically signifi cant diff erences marked by § when compared with 1 or by * when compared with 3 (P <0.05). Conclusion We describe for the fi rst time the mapping of Silent Spaces during spontaneous breathing and changing ventilation conditions and body positions in patients with healthy lungs using EIT. This mapping of Silent Spaces might prove useful for developing perioperative protective ventilation strategies. Figure 1 (abstract P248). Infl ammatory mediators independent of tidal volumes after 12 hours of MV. aim of this preclinical study was to study both time-dependent and dose-dependent eff ects of supplemental oxygen during prolonged ventilatory support on pulmonary infl ammation in a well-established murine model of ventilation comparing low and high tidal volumes. aim of this preclinical study was to study both time-dependent and dose-dependent eff ects of supplemental oxygen during prolonged ventilatory support on pulmonary infl ammation in a well-established murine model of ventilation comparing low and high tidal volumes. Methods Healthy male C57Bl/6J mice, aged 9 to 10  weeks, were randomly assigned to experimental groups (n = 8), in which the applied fractions of oxygen (FiO2) were 30%, 50% or 90% and tidal volumes were either 7.5 or 15 ml/kg. Anesthetized mice were tracheotomized and ventilated for 8 or 12 hours. Infl ammatory cells and mediators were measured in bronchoalveolar lavage fl uid (BALf).i Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods All 89 inpatients on the CCU who received mechanical ventilation continuously for 7  days or more between October 2012 and December 2013 were initially included. Forty patients who were intubated prior to arrival at RCHT, had incomplete notes, or were extubated during end-of-life care were excluded. Patients were divided into groups by fi rst airway intervention; 31 TOE, 18 TR. fi bers from young, middle aged and old animals confi rmed the 28 to 32 month rats to be senescent from a skeletal muscle point of view. p Conclusion These results demonstrate intrinsic changes in diaphragm muscle fi bers of signifi cant importance for the prolonged and complicated weaning from MV. Moreover, the increased number of frail diaphragm muscle fi bers observed after MV in old age, both controls and mechanically ventilated, off ers a further age-related possible mechanism which may be of signifi cant clinical importance. These results also provided useful information to design future experimental studies focused on the eff ect of age in an ICU setting, pharmacological intervention strategies as well as mechanisms underlying rat strain diff erences. g p yi y ; , Results A total 52% (16/31) of patients had TOE, required no other airway intervention and survived to discharge from hospital, compared with 72% (13/18) of TR patients. Four patients from each group failed the fi rst intervention and died prior to a second intervention. In total, 8/11 patients who had a second intervention after failed TOE survived to discharge from hospital. One patient had a second TR but died before discharge. This gave an in-hospital mortality rate of 19% for the TOE group and 28% for the TR group. TOE was performed earlier, all 31 on days 7 to 15. TR was performed later; 14/18 on days 7 to 15, and 4/18 on days 17 to 23. Early TR was more successful; 11/11 survived to discharge without a second intervention who had TR on days 7 to 12, compared with 29% (2/7) after day 12. TOE was more successful when performed later; 64% (7/11) survived to discharge without a second airway intervention when TOE was after day 10, 45% (9/20) between days 7 and 10. Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Results Twenty-eight patients were included in G1 and 22 patients in G2. There was no signifi cant diff erence between the patients’ admission parameters and daily NIV usage times. PaCO2 decreased >5 mmHg in 93% of G1 patients and in 60% of G2 patients in the fi rst 6 hours (P = 0.044). A 10 mmHg reduction in PaCO2 occurred in more patients (93% vs. 60%, P = 0.004) and in a shorter time (1.8 ± 1.2 vs. 3 ± 3 days, P = 0.044) in G1. At the time of discharge, PaCO2 levels were <50 mmHg in 79% of G1 and 41% of G2 patients (P = 0.006). Both groups showed similar and signifi cant improvements in PaO2, PaCO2 and HCO3 – levels within the fi rst 4 days but only in G1 patients were HCO3 – levels decreased more rapidly than G2 patients (P = 0.007). Duration of NIV (6 ± 2 vs. 8 ± 3 days, P = 0.002) and the number of mode and pressures changes (0.3 ± 1.8 vs. 2 ± 2 times, P >0.0001) were signifi cantly less in G1. While mean IPAP was similar in both groups, maximum and minimum EPAP titrated automatically in G1 were signifi cantly diff erent from G2. Mean tidal volume and amount of leakage were also signifi cantly higher in G1. fi g pfi Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) fi g pfi Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Introduction Bilevel non-invasive ventilation (NIV) is an established therapy in chronic obstructive pulmonary disease (COPD) but confl icting evidence exists for its use in patients with pneumonia. Initial arterial pH <7.25 is used as a marker of severity and need for admission to critical care (CC) [1]. We examined the impact of pH and condition on outcome in patients with acute respiratory failure (ARF) of mixed aetiology treated with NIV. Methods Data were collected retrospectively for a 5-year period from 2008 to 2013 using the Metavision electronic patient record. We identifi ed all patients admitted with ARF treated with bilevel NIV. Patients who received continuous positive airway pressure or had a primary surgical problem were excluded. Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) We recorded primary cause of respiratory failure, arterial blood gas values and mortality.i g gi y g Conclusion These results suggest that the AVAPS-AE mode may provide some advantages in hypercapnic ICU patients such as rapid PaCO2 reduction, less NIV duration and workload. Results A total of 145 patients were identifi ed. Mean age was 64 and 51% were male. The primary diagnosis was pneumonia in 69 patients and exacerbation of COPD in 57. The overall mortality was 19% on CC and 39% at 1 year. In patients with COPD, infective exacerbations had a higher CC mortality (17%) compared with non-infective (0%). However, by 1 year the mortality was 28% in infective and 29% in non-infective. Patients with pneumonia had a higher mortality on CC (25%) and at 1 year (48%). Patients with an initial pH <7.25 were less likely to survive. The mortality at discharge from CC was 16% (pH ≥7.25) and 26% (pH <7.25) but narrowed to 38% and 39% by 1 year. When subdivided, it was found that patients with infective COPD and pH <7.25 had the lowest 1-year mortality (17%) while those with pneumonia and pH <7.25 had the highest mortality (67%). P251 P251 Does it make a diff erence to add automatic EPAP titration to the volume-targeted pressure support mode in noninvasive ventilation of hypercapnic ICU patients? G Gursel, A Zerman, B Basarik, K Gonderen, M Aydogdu, S Memmedova Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P251 (doi: 10.1186/cc14331) Introduction Obese patients are increasing in number in ICUs and more than 90% of them also have sleep apnea syndrome. Variability in upper airway resistance during sleep and awakening periods makes it diffi cult to set EPAP in these patients. A new mode that automatically titrates EPAP according to upper airway resistance and IPAP according to target tidal volume may be more eff ective. The aim of this study is to evaluate whether adding automatic EPAP titration to the volume-targeted pressure support mode will provide any therapeutic advantages in hypercapnic ICU patients. Conclusion TOE is more common and is associated with shorter time spent ventilated, in the CCU and in hospital than TR. It is also associated with a lower in-hospital mortality rate. TOE is more successful when performed after day 10; TR is more successful when performed before day 13. After failed TOE, a second TOE is associated with longer time in hospital but a better mortality rate than secondary tracheostomy. y Methods The hypercapnic patients treated with average volume- assured pressure support-automatic EPAP (AVAPS-AE) mode (Group1 (G1)) were compared with those treated with AVAPS mode (Group 2 (G2)). G2 was recruited retrospectively and matched with G1 according to diagnoses, demographic characteristics, arterial blood gas values and daily noninvasive ventilation (NIV) usage times. Trilogy 100® devices and their software Directview® (Philips Respironics) were used to reveal the respiratory data such as pressures, volumes, and daily usage times. For statistical analyses, t test, chi-square test and repeated measures of ANOVA were used. P253 Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) P249 Perioperative assessment of regional ventilation during changing body positions and ventilation conditions by electrical impedance tomography with increased spatial resolution and signal quality A März1, A Ukere1, K Wodack1, C Trepte1, A Waldmann2, S Böhm2, D Reuter1 1University Medical Center Hamburg-Eppendor, University Hamburg, Germany; 2Swisstom AG, Landquart, Switzerland Critical Care 2015, 19(Suppl 1):P249 (doi: 10.1186/cc14329) Perioperative assessment of regional ventilation during changing body positions and ventilation conditions by electrical impedance tomography with increased spatial resolution and signal quality A März1, A Ukere1, K Wodack1, C Trepte1, A Waldmann2, S Böhm2, D Reuter1 1University Medical Center Hamburg-Eppendor, University Hamburg, Germany; 2Swisstom AG, Landquart, Switzerland Critical Care 2015, 19(Suppl 1):P249 (doi: 10.1186/cc14329) Results This study demonstrated an unexpected increase in CSA (P  <0.001) of the diaphragm fi bers in response to 5  days of MV in both young and old animals. Maximum force decreased 39.8 to 45.2% (P  <0.001) in both young and old animals compared with controls, resulting in a dramatic loss of specifi c force. This increase in CSA and the concomitant decrease in specifi c force observed in both young and old diaphragm fi bers are compatible with an ineff ective compensatory hypertrophy in response to the MV. The comparison of the limb muscles Introduction Electrical impedance tomography (EIT) is a functional imaging technology allowing one to regionally monitor aeration of the Table 1 (abstract P249) 1 2 3 4 5 (sitting, (supine, (ventilated, (ventilated, (ventilated, spontaneous breathing) spontaneous breathing) supine position) 30° head down position) supine position) NSS (%) 5.25 ± 2.9 4.12 ± 1.89§ 3.05 ± 1.9§ 2.8 ± 2.7§ 2.67 ± 1.9§ DSS (%) 0.07 ± 0.3 2.29 ± 2.3§ 9.23 ± 6.3§ 11.5 ± 8.9§* 8.5 ± 5.8§ Table 1 (abstract P249) 1 2 3 4 5 (sitting, (supine, (ventilated, (ventilated, (ventilated, spontaneous breathing) spontaneous breathing) supine position) 30° head down position) supine position) NSS (%) 5.25 ± 2.9 4.12 ± 1.89§ 3.05 ± 1.9§ 2.8 ± 2.7§ 2.67 ± 1.9§ DSS (%) 0.07 ± 0.3 2.29 ± 2.3§ 9.23 ± 6.3§ 11.5 ± 8.9§* 8.5 ± 5.8§ S88 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 After fi rst failed TOE, four patients had a successful second TOE; all four survived to discharge resulting in a median CCU stay of 29 days and median hospital stay of 39 days (excluding prior to CCU admission). Seven patients had TR after the fi rst failed TOE, fi ve survived to discharge from the CCU and four to discharge from the hospital. This group had shorter median stays in both the CCU (27 days) and hospital (32 days). Overall, the median duration of time ventilated, in the CCU, and in hospital was shorter for the TOE group; 13, 17 and 24 days respectively, compared with 22, 27.5 and 34 days for the TR group. P252 Retrospective study of patients receiving long-term mechanical ventilation I D d B H d R Si l i I Dunwoody, B Hopwood, R Sinclair Royal Cornwall Hospital, Truro, UK Critical Care 2015, 19(Suppl 1):P252 (doi: 10.1186/cc14332) Introduction This study analysed the practice of clinicians managing patients requiring long-term mechanical ventilation in the critical care unit (CCU) of the Royal Cornwall Hospital, Truro (RCHT), comparing outcomes of primary tracheostomy (TR) with trial of extubation (TOE). Conclusion NIV is used in our unit with comparable success rates to published series [2,3]. COPD patients responded well to NIV, while Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S89 patients with pneumonia treated with NIV have the highest mortality. A low presenting pH is associated with a higher mortality in patients with pneumonia treated with NIV. However, in COPD patients, pH <7.25 is not associated with higher mortality in CC or at 1 year. Further work defi ning the precise role of pH as a prognostic indicator is warranted. References patients with pneumonia treated with NIV have the highest mortality. A low presenting pH is associated with a higher mortality in patients with pneumonia treated with NIV. However, in COPD patients, pH <7.25 is not associated with higher mortality in CC or at 1 year. Further work defi ning the precise role of pH as a prognostic indicator is warranted. References care units within the NoECCN. Patient data collected included patient demographics, admission diagnosis and speciality, hospital length of stay (LOS) pre and post critical care admission, severity of illness scores, critical care LOS and status at hospital discharge. p g Results During the study period 134 patients met the criteria for PMV representing 1% of annual admissions and 6.9% NoECCN bed- days. The majority of patients receiving PMV were medical (50.7%), followed by emergency surgery (20.1%), elective surgery (16.4%) and specialist services such as spinal cord injury (8.2%) and cardiothoracic transplant (4.5%). The commonest admission diagnosis in the medical population was pulmonary infection followed by acute neurological disorders, while 89.4% of surgical patients were admitted to critical care during the perioperative period. At the end of the study period the highest hospital mortality was observed in the nonspecialist surgical population (26.5%). In contrast, the medical population had one of the lowest hospital mortality rates (11.8%), lower than predicted using the intensive care national audit research network illness severity score. P254 Lung protective ventilation with lower tidal volumes and development of pulmonary complications in critically ill patients without ARDS FD Simonis1, A Serpa Neto2, M Gama de Abreu3, P Pelosi4, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Isrealita Albert Einstein, São Paulo, Brazil; 3University Hospital Carl Gustav Carus, Dresden, Germany; 4IRCCS San Martino IST, Genoa, Italy Critical Care 2015, 19(Suppl 1):P254 (doi: 10.1186/cc14334) Reference 1. Pilcher DV, et al. Outcomes, cost and long term survival of patients referred to a regional weaning unit. Thorax. 2005;60:187-92. Methods An individual patient data meta-analysis of studies of ventilation in ICU patients without ARDS. Corresponding authors of retrieved studies provided individual patient data. The primary outcome, pulmonary complications, was a composite of development of ARDS or pneumonia during hospital stay. Secondary outcomes included ICU and hospital length of stay, and in-hospital mortality. Patients were assigned to three groups based on tidal volume size (≤7 ml/kg predicted body weight (PBW), 7 to 10 ml/kg PBW, or ≥10 ml/ kg PBW). P252 Retrospective study of patients receiving long-term mechanical ventilation I D d B H d R Si l i Comparable rates of hospital discharge were found in both medical (85%) and nonspecialist surgical patients (88.9%). 1. 2013 BTS NIV audit. https://www.brit-thoracic.org.uk/document-library/ audit-and-quality-improvement/audit-reports/ bts-adult-niv-audit-report-2013/ 2. Carillo et al. ICM. 2012;38:458-66. 3. Lightowler et al. BMJ. 2003;326:185-9. Lung protective ventilation with lower tidal volumes and development of pulmonary complications in critically ill patients without ARDS FD Simonis1, A Serpa Neto2, M Gama de Abreu3, P Pelosi4, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Isrealita Albert Einstein, São Paulo, Brazil; 3University Hospital Carl Gustav Carus, Dresden, Germany; 4IRCCS San Martino IST, Genoa, Italy Critical Care 2015, 19(Suppl 1):P254 (doi: 10.1186/cc14334) g Conclusion The results of this study highlight an expanding proportion of NoECCN critical care bed-days occupied by stable patients undergoing PMV. In keeping with published UK data, elevated hospital mortality was observed in the nonspecialist surgical subpopulation. Although the literature suggests the medical cohort of patients has poorer prognosis, within our region all were liberated from mechanical ventilation and over 80% were discharged from hospital. R f Introduction A large meta-analysis suggests that use of low tidal volumes benefi ts patients without ARDS [1] but most studies in this meta-analysis included patients receiving ventilation during general anesthesia for surgery. The aim of the present meta-analysis is to determine the association between tidal volume size and development of pulmonary complications in ICU patients. Eff ects of positive end-expiratory pressure on lung ventilation/ perfusion matching: a clinical study N Eronia1, T Mauri2, G Bellani1, R Marcolin1, S Marocco Arrigoni1, G Grasselli1, A Pesenti1 , 1San Gerardo Hospital, Monza, Italy; 2IRCC Ca’ Granda Maggiore Policlinico Hospital, Milan, Italy Critical Care 2015, 19(Suppl 1):P256 (doi: 10.1186/cc14336) Results Seven investigations (2,184 patients) were meta-analyzed. Pulmonary complications occurred in 23%, 28% and 31% respectively in the ≤7 ml/kg PBW, 7 to 10 ml/kg PBW and ≥10 ml/kg PBW group (adjusted RR, 0.72; 95% CI, 0.52 to 0.98; P  = 0.042). Occurrence of pulmonary complications was associated with a lower number of ICU- free days and alive at day 28, a lower number of hospital-free days and alive at day 28 and increased in-hospital mortality. Introduction Positive end-expiratory pressure (PEEP) exerts multiple protective eff ects in hypoxemic critically ill patients: PEEP can increase end-expiratory lung volume (EELV) and induce recruitment, thus reducing lung strain and opening and closing of alveoli and potentially improving the ventilation/perfusion matching. In particular, estimation of PEEP-induced ventilation/perfusion matching might help identify the optimal PEEP setting, but bedside non-invasive methods are few and complex to be applied in daily clinical practice. Electrical impedance tomography (EIT) is a non-invasive bedside technique that claims to track global and regional changes in perfusion and ventilation over time. In the present study we aimed at verifying the eff ects of PEEP on ventilation/perfusion matching, as assessed by EIT, in acute respiratory failure patients. Conclusion Ventilation with low tidal volumes is associated with a lower risk of development of pulmonary complications. Occurrence of pulmonary complications is associated with an increased ICU and hospital length of stay and in-hospital mortality in ICU patients without ARDS. Reference Reference 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. Reference 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. p y p Methods We enrolled 20 intubated critically ill patients undergoing controlled mechanical ventilation, sedated, paralyzed and with PaO2/FiO2 ≤300 at PEEP ≥5  cmH2O. We started EIT monitoring (Pulmovista500®; Dräger Medical GmbH, Lübeck, Germany) and applied two PEEP levels (clinical and clinical + 5 cmH2O) for 20 minutes each. We collected ventilatory and EIT parameters and, by offl ine analysis, we calculated the increase of EELV at higher PEEP and the EIT- based indexes of ventilation heterogeneity (VtHetend-insp) and of the regional homogeneity of ventilation/perfusion matching (HA/P). P255 Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 1Sunderland Royal Hospital, Sunderland, UK; 2James Cook University Hospital, Middleborough, UK Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 1Sunderland Royal Hospital, Sunderland, UK; 2James Cook University Hospital, Middleborough, UK Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) Pressure reconstruction method for spontaneous breathing eff ort monitoring Pressure reconstruction method for spontaneous breathing eff ort monitoring N Damanhuri1, YS Chiew1, NA Othman1, PD Docherty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P259 (doi: 10.1186/cc14339) N Damanhuri1, YS Chiew1, NA Othman1, PD Docherty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P259 (doi: 10.1186/cc14339) Introduction Estimating respiratory mechanics of mechanically venti lated patients is unreliable when patients exhibit spontaneous breathing (SB) eff orts on top of ventilator support. This reverse triggering eff ect [1] results in an M-wave shaped pressure wave. A model-based method to reconstruct the aff ected airway pressure curve is presented to enable estimation of the true underlying respiratory mechanics of these patients. Table 1 (abstract P257) Ki <27.8 ml/ Ki ≥27.8 ml/ minute/ml minute/ml × 104 (n = 7) × 104 (n = 8) P value PaO2/FiO2 280 (261 to 346) 239 (212 to 271) 0.31 pH 7.46 (7.43 to 7.48) 7.41 (7.39 to 7.44) 0.15 PaCO2 (mmHg) 37 (34 to 44) 50 (46 to 53) 0.01 WBCs (103 cell/mm3) 8 (8 to 9) 9 (9 to 16) 0.34 Total lung volume (ml) 1,298 (1,092 to 1,494) 1,516 (1,408 to 1,665) 0.18 Total lung weight (g) 592 (488 to 741) 732 (597 to 774) 0.39 Total lung gas (ml) 706 (551 to 843) 804 (755 to 1,080) 0.15 Not-infl ated lung tissue (%) 25 (12 to 30) 23 (17 to 30) 0.95 Poorly infl ated lung tissue (%) 34 (42 to 42) 31 (29 to 34) 0.39 Well-infl ated lung tissue (%) 36 (27 to 47) 47 (35 to 52) 0.53 Conclusion Patients clinically defi ned as having primary graft dysfunction did not have an increased rate of 18-FDG uptake. 18-FDG uptake was not diff erent in single-lung versus bilateral transplantation and, in single-lung procedures, the native lung showed elevated infl ammatory activity. Methods Airway pressure and fl ow data from 72 breaths of a pneu- monia patient were used for proof of concept. A pressure wave reconstruction method fi lls parts of the missing area caused by SB eff orts and reverse triggering by connecting the peak pressure and end-inspiration slope (Figure  1). A time-varying elastance model [2] was then used to identify underlying respiratory elastance (AUCEdrs). P258 4.8) vs. 1.9 (1.5 to 2.1), P <0.01) and lower compliance of dependent lung regions (Crsdep, 13 ± 3 ml/cmH2O vs. 18 ± 6 ml/cmH2O, P <0.05), as compared with patients with smaller improvement. 4.8) vs. 1.9 (1.5 to 2.1), P <0.01) and lower compliance of dependent lung regions (Crsdep, 13 ± 3 ml/cmH2O vs. 18 ± 6 ml/cmH2O, P <0.05), as compared with patients with smaller improvement. P257 18-FDG PET in lung transplantation I Al 1 F V l 1 M G l 1 B S f P257 18-FDG PET in lung transplantation I Al 1 F V l 1 M G l 1 B S f 18-FDG PET in lung transplantation I Algieri1, F Valenza1, M Guanziroli1, B Safaee Fakhr1, M Cressoni1, M Brioni1, A Colombo1, G Babini1, F Crimella1, D Massari1, K Nikolla1, G Crisafulli1, S Paladini1, L Rosso2, A Palleschi2, F Zito2, D Chiumello1, L Gattinoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda- Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2015, 19(Suppl 1):P257 (doi: 10.1186/cc14337) p g p g p g p g p Results The median value of LU comets was signifi cantly lower in Group A compared with Group B. There was a signifi cant increase in hypoxic index in Group A compared with Group B. There was no signifi cant diff erence between both groups as regards PIP, while Pplat showed a signifi cant increase in Group B compared with Group A and Cs showed a signifi cant decrease in Group B. Introduction Lung transplantation is associated with an infl ammatory reaction known as primary graft dysfunction. This clinical syndrome occurs within the fi rst 72  hours after transplantation and is characterized by hypoxemia (PaO2/FiO2 <300) and bilateral infi ltrates not secondary to cardiac dysfunction, viral or bacterial pneumonia and venous anastomotic obstruction. gi p Conclusion LU is a useful to quantify EVLW; it is a good predictor of weaning. References Methods 18-FDG PET scan was used to study 15 lung transplantation patients. The rate of 18-FDG uptake (Ki) was computed voxel by voxel with the Patlak method. Patients were divided according to the median Ki (27.8 (20.3 to 34.6) ml/minute/ml × 104). Data are reported as median and interquartile range. 1. Nektaria X, Eumorfi a K, George P, et al. Impact of lung ultrasound on clinical decision making in critically ill patients. Intensive Care Med. 2014;40:57-65. 2. Jambrik Z, Monti S, Coppola V, et al. Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water. Am J Cardiol. 2004;93:1265-70. 2. Jambrik Z, Monti S, Coppola V, et al. Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water. Am J Cardiol. 2004;93:1265-70. Results Five patients developed primary graft dysfunction; median Ki in these patients was not diff erent from patients who did not (24.5 (18.2 to 33.6)  ml/minute/ml  × 104 vs. 29.1 (23 to 35.4)  ml/minute/ ml × 104 respectively, P = 0.64). Bilateral lung transplantation patients were characterized by a median Ki of 30.5 (22.9 to 34.5) ml/minute/ ml  × 104, while patients undergoing single-lung transplantation presented a median Ki of 24.4 (21 to 34.1) ml/minute/ml × 104 (P = 0.61). Considering single-lung transplantation, graft and native lung had similar Ki: 24.4 (21 to 34.1) ml/minute/ml × 104 versus 24.2 (17.7 to 30.1) ml/minute/ml × 104 respectively (P = 0.64). When patients were divided according to the median Ki value, higher Ki was associated with higher PaCO2 values (50 (46 to 53) mmHg vs. 37 (34 to 44) mmHg, P = 0.01). See Table 1. Usefulness of extravascular lung water assessment as a predictor of weaning from mechanical ventilation Usefulness of extravascular lung water assessment as a predictor of weaning from mechanical ventilation Conclusion EIT might represent a feasible, bedside method to estimate PEEP-induced improvement in ventilation/perfusion matching. Assessing regional ventilation and mechanical lung properties might help identify patients who would benefi t more from higher PEEP. g Alexandria University Hospital, Alexandria, Egypt Critical Care 2015, 19(Suppl 1):P258 (doi: 10.1186/cc14338) Introduction Quantitative measurement of extravascular lung water (EVLW) would be extremely useful for clinical management, both as an index of severity and to guide treatment lung ultrasonography (LU) as a tool to quantify EVLW. q y Methods Forty mechanically ventilated patients were examined for 5  successive days after being eligible for weaning; counting B-lines (comets) in both lung fi elds by LU, peak airway pressure, plateau airway pressure, static compliance and ABG analysis every 6 hours. Patients were divided into two groups according to success of the weaning process: group A (weaned group), and group B (nonweaned group). Results The median value of LU comets was signifi cantly lower in Group A compared with Group B. There was a signifi cant increase in hypoxic index in Group A compared with Group B. There was no signifi cant diff erence between both groups as regards PIP, while Pplat showed a signifi cant increase in Group B compared with Group A and Cs showed a signifi cant decrease in Group B. Pressure reconstruction method for spontaneous breathing eff ort monitoring The area of the unreconstructed M-wave has less pressure, resulting in a lower overall AUCEdrs without reconstruction. The missing area of the airway pressure or AUCEdrs is hypothesized to be a surrogate of patient- specifi c inspiratory to assess the strength of SB eff orts. AUCEdrs and missing area A2 are compared with/without reconstruction.if 2 Results Median AUCEdrs and breath-specifi c eff ort using reconstruction were 24.99 (IQR: 22.90 to 25.98) cmH2O/l and 3.64 (IQR: 0.00 to 3.87)% versus AUCEdrs of 20.87 (IQR: 15.24 to 27.48) cmH2O/l for unreconstructed M-wave data, indicating signifi cant patient and breath-specifi c SB eff ort, and the expected higher elastance (P <0.05). Conclusion A simple reconstruction method enables the real-time measurement of respiratory system properties of a SB patient and measures the surrogate of the SB eff ort, that latter of which has clinical use in deciding whether to extubate or re-sedate the patient. Conclusion Patients clinically defi ned as having primary graft dysfunction did not have an increased rate of 18-FDG uptake. 18-FDG uptake was not diff erent in single-lung versus bilateral transplantation and, in single-lung procedures, the native lung showed elevated infl ammatory activity. Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) g g y p g Results Patients were 62 ± 12 years old, mean PaO2/FiO2 was 197 ± 52, lower PEEP level was 7 (7 to 9) cmH2O, while higher PEEP was 12 (12 to 14) cmH2O (P <0.001). At higher PEEP, EELV increased (391 (334 to 555) ml vs. PEEP low, considered as baseline, P <0.001); VtHetend- insp was reduced (1.8 (1.5 to 2.4) vs. 2.2 (1.8 to 2.6), P <0.001) and HA/P increased (0.29 ± 0.19 vs. 0.2 ± 0.15, P <0.05). Interestingly, the increase of HA/P was signifi cantly correlated with the decrease of VtHetend- insp (r = –0.48, P <0.05). Moreover, patients with higher potential for improvement of ventilation/perfusion matching (that is, patients with increase of HA/P >16%) had higher baseline VtHetend-insp (2.6 (2.3 to Introduction The combination of a global demographic shift and increased survival following critical illness has led to an increasing number of patients requiring prolonged mechanical ventilation (PMV) and longer critical care stay. This is a prospective observational study evaluating the characteristics and speciality-based outcome of critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network (NoECCN). Methods A weekly survey was conducted over a 1-year period screening patients older than 16 years of age requiring PMV in all 18 adult critical S90 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P257 q y Methods Forty mechanically ventilated patients were examined for 5  successive days after being eligible for weaning; counting B-lines (comets) in both lung fi elds by LU, peak airway pressure, plateau airway pressure, static compliance and ABG analysis every 6 hours. Patients were divided into two groups according to success of the weaning process: group A (weaned group), and group B (nonweaned group).i Breath-to-breath respiratory mechanics variation: how much variation should we expect? Each patient was sedated to prevent spontaneous breathing eff ort, and ventilated using the volume control mode with a square fl ow profi le. Varied PEEP levels were maintained for 30 minutes before 1 minute of data were collected for analysis. This dataset provides a wide range of respiratory mechanics values, and the clinical protocol detail is in [1]. A clinically proven, single compartment model respiratory system elastance (Ers) is identifi ed from data for every breathing cycle at each PEEP level using a linear regression method. The dynamic elastance (Edynamic = (peak airway pressure – positive end-expiratory pressure) / tidal volume) of the corresponding breathing cycle is calculated for comparison.fii p g g y p Results The coeffi cient of variation (CV) of identifi ed Ers across all patients was low (<0.005), as expected, as the 30-minute period allows time-dependent alveolar recruitment to fully occur and stabilise. However, even with substantial stabilisation periods, there remains a diff erence between the minimum and maximum estimated Ers within each 1-minute period of analysed data. The diff erences were median 2.8% (interquartile range (IQR): 1.5 to 4.6%, 90% range (90R): 0.8 to 13.4) for Ers and 3.6% (IQR: 2.3 to 5.2, 90R: 0.9 to 10.8) for Edynamic, and in extreme cases, can be up to 16%. Results Over 275 paired samples were analysed over the following concentration ranges: pH 7.249 to 7.545; pCO2 3.32 to 11.01 kPa; pO2 5.59 to 21.80 kPa; Hct 23.6 to 41.4%; K+ 3.3 to 4.79 mM. The imprecision for each sensor was calculated to be: pH –0.00 ± 0.03; pCO2 –0.48 ± 0.34 kPa; pO2 –0.85 ± 0.96 kPa; Hct –4.49 ± 3.42%; K+ 0.08 ± 0.15 mM, respectively. The data collected on the new analyser for Hct showed relative imprecision of 3.42%. The pooled SD was calculated to be 1.21% and the mean SD of each of the novel devices used was 1.14%. This indicates that scatter observed on the Hct sensor was largely due to inter-device rather than intra-device factors. Conclusion This study quantifi ed the variability (over short periods) of identifi ed and estimated respiratory mechanics properties used to (potentially) guide ventilation care in sedated patients. It is also important to note that this minimum level of variability occurs even when stabilisation is achieved. Breath-to-breath respiratory mechanics variation: how much variation should we expect? 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) p KT Kim1, YS Chiew1, C Pretty1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P260 (doi: 10.1186/cc14340) KT Kim1, YS Chiew1, C Pretty1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P260 (doi: 10.1186/cc14340) Introduction Patient-dedicated arterial blood analysers off er the potential to allow close monitoring of critically ill patients without leaving the patient’s bedside or net loss of blood for the patient. A new patient-attached blood gas analyser was evaluated in a clinical environment to compare the results against the reference bench-top analyser. Introduction Model-based respiratory mechanics can be used to guide mechanical ventilation therapy. However, identifi ed mechanical properties vary breath to breath, leading to potential treatment errors when using model-based care that requires accuracy. This study investigates and quantifi es this variability to improve its application in guiding clinical interventions. Methods Twenty ICU patients with a range of clinical conditions, including trauma, head injury, post-surgical recovery and sepsis, were included in the study. The new analyser was operated by clinical staff at the Queen Elizabeth Hospital, Birmingham, who each underwent a 90-minute training programme. Patients were monitored using the patient-dedicated analyser (Proxima; Sphere Medical) for up to 3 days. Each time a sample was tested on the patient-dedicated analyser, a concurrent sample was drawn and tested on the hospital’s bench- top analyser (Cobas b221; Roche Diagnostics). Samples were assessed using methods described by Bland and Altman for repeated measures. Performance of the novel device was analysed by calculating bias as the mean diff erence between the new analyser and the comparator device, imprecision as ±1 standard deviation (SD) from the mean and limits of agreement as ±1.96 SD from the mean. Percentage values for bias and precision were calculated from analysis of the percentage diff erence between the two methods of analysis. Intra-device imprecision for the haematocrit (Hct) sensor was calculated using a pooled variance calculation. Methods Retrospective data from 12 acute respiratory distress syndrome (ARDS) patients were used [1]. 1. Bersten. Eur Respir J. 1998;12:526-32. References 1. Akoumianaki E, et al. Chest. 2013;143:927-38. 2. Chiew YS, et al. Biomed Eng Online. 2011;10:111. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 S91 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P259). Edrs for M-wave and reconstructed airway pressure at PEEP = 15 cmH2O. t PEEP = 15 cmH O Figure 1 (abstract P259). Edrs for M-wave and reconstructed airway pressure at PEEP = 15 cmH2O. P261 P261 Evaluation of a patient-dedicated blood gas analyser JJ Fox1, TH Clutton-Brock2 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) Breath-to-breath respiratory mechanics variation: how much variation should we expect? Thus, clinically, if this information was to be used to guide ventilation in real time, such as titrating PEEP to minimal elastance, larger errors, at least up to 15% variation in Ers, could be expected, which could well aff ect care. Such levels thus also begin to defi ne the minimum levels of change necessary to be larger than natural variation. Conclusion The patient-dedicated blood gas analysers demonstrated excellent agreement with the bench-top analyser for pH, pCO2, pO2 and K+, while Hct provides a reasonable trend monitor with variable bias. Proxima is well suited to enable staff to more closely manage unstable and critically ill patients. This device may be of signifi cant benefi t to patients at risk of iatrogenic anaemia or those being treated in side rooms/isolation rooms. Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) Introduction Maintaining the appropriate tidal volume (VT) is important for success of lung protective ventilation. However, in neonates, the presence of airway leaks may increase the errors in the delivery of tiny VT, which raises a concern of ventilator-induced lung injury. This study is to investigate the accuracy of VT delivery during non-invasive ventilation (NIV) with modern neonatal ventilators. Results There was a signifi cant correlation between the indexed EE and indexed VE in both groups (R = 0.51, P <0.0001 in the control group; R = 0.63, P <0.0001 in the MV group). The VR was signifi cantly suppressed in the MV group compared with the control group (0.041 ± 0.003/minute/ BEE vs. 0.069 ± 0.003/minute/BEE, P = 0.012; respectively). Although the indexed VE was comparable in the MV and control groups (0.19 ± 0.07 l/ kg vs. 0.17 ± 0.04 l/kg, P = 0.23; respectively), the indexed EE was shifted to a higher range in the MV group than in the control group (maximal indexed EE; 2.26 ± 0.68 vs. 1.74 ± 0.20, P = 0.008; respectively). The TV was smaller (maximal TV; 985 ± 592 ml vs. 1,410 ± 299 ml, P = 0.018; respectively) and the RR was more frequent (maximal RR; 30 ± 8/minute vs. 16 ± 4/minute, P <0.0001; respectively) in the MV group than in the control group. Methods Using a lung simulator for a patient body weight of 3  kg, we measured the actual delivered VT in the lung and compared it with the value displayed on the ventilator in six ventilators. We tested 18 conditions with various combinations of respiratory mechanics (normal, restrictive, obstructive), leak levels (0, 1.0, 1.5 l/minute), and PEEP settings (5, 10 cmH2O). All conditions were tested in NIV mode. The pressure level was set to achieve VT to the lung at 6 to 7 ml/kg. All other settings were: FIO2 0.21, Itime 0.6 seconds, f 25/minute, and default rise time. We calculated the mean errors of the ventilator-displayed VT at various levels of airway leak. Eff ective capnography training in the ICU using the ‘hats and caps’ training tool Eff ective capnography training in the ICU using the ‘hats and caps training tool CA Lobo, FE Kelly, S Steynberg, G Thomas, C Pope, M Eveleigh, M Charlton, TM Cook Royal United Hospital, Bath, UK Critical Care 2015, 19(Suppl 1):P264 (doi: 10.1186/cc14344) Reference Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S92 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P262 The VE was indexed by body weight and the EE was also indexed by the basal energy expenditure (BEE) estimated by the Harris–Benedict formula. VR was defi ned as the slope in the indexed VE–indexed EE plot with an assumption of those relationship in the linear manner. We examined the correlation between indexed EE and indexed VE in both groups, and the diff erences of the maximal indexed EE, the maximal indexed MV, and the others between both groups were investigated using the unpaired t test. For all the data, signifi cance was accepted at values of P <0.05. P262 Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) Table 1 (abstract P262) Leak amount Hamilton Hamilton (l/minute) C3 G5 Servo i PB840 PB980 VN500 0 26 (15) 4 (6) 3 (2) 2 (7) –4 (2) 2 (2) 1.0 –9 (5) 9 (5) –2 (2) –5 (4) –2 (4) 20 (5) 1.5 16 (8) –14 (8) –2 (2) –6 (4) –2 (3) 33 (6)a Data presented as mean (SD), %. aUnable to measure in one case. Introduction Failure to use or correctly interpret capnography in patients dependent on an artifi cial airway in ICUs was thought to have contributed to 74% of ICU airway-related deaths in the NAP4 study [1]. However, capnography is only of value if those using it can interpret it correctly, with recommendations for training all ICU staff in capnography [1,2]. A recent UK survey identifi ed that only 48% of ICUs have trained all staff in capnography interpretation (TM Cook, personal communication). In this study, we used a capnography teaching aid (‘hats and caps’) to educate all ICU staff during a 1-month period, and evaluated its eff ectiveness. Conclusion Tidal volume accuracy during neonatal NIV varies greatly among diff erent ventilators and leak conditions. This must be considered in neonatal ventilation management to avoid over- ventilation or underventilation. Figure 1 (abstract P264). ‘Hats and caps’ capnography training guide. Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators Conclusion The VR to external stress with mechanical ventilation is more suppressed than in healthy volunteers. The VE in the mechanical ventilation was earned by a higher RR rather than by increased TV. Careful monitoring of VE or RR would be benefi cial in early rehabilitation with mechanical ventilation. y Results The VT mean error values are presented in Table 1. When no leak existed, the mean error was less than 5% in all ventilators except one (C3) which showed a mean error of 26%. As the leak level increased, three ventilators (C3, G5, and VN500) showed marked diff erences between the delivered and displayed VT. In particular, the VN500 could not operate in the large leak condition. The other three ventilators (PB840, PB980, Servo i) showed acceptable VT accuracy across all conditions tested. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods ‘Hats and caps’ was devised on our ICU [3] and used for the training: this teaches that capnography traces on the left signify the airway is functional, in contrast to the traces on the right which indicate immediate attention is required (Figure 1). This was presented to staff working on the ICU in individual bedside teaching sessions with feedback obtained and evaluated. percentage variation –1.4040 ± 4.73 (–2.15 to –0.67); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.82 ± 3.85 (3.04 to 4.69); fl ow percentage variation 9.76  ± 8.08 (8.11 to 11.41); r  = 0.969 and r2 = 0.939 (P <0.000). (3) FiO2 variation –0.005 ± 0.26 (–0001 to 0009); FiO2 percentage variation –0.72 ± 5.2 (–1.5 to 0.1); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.91 ± 1.26 (3.69 to 4.13); fl ow percentage variation 12.77 ± 5.33 (11.84 to 13.7); r = 0.996 and r2 = 0.992 (P <0.000). See Figure 1. percentage variation –1.4040 ± 4.73 (–2.15 to –0.67); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.82 ± 3.85 (3.04 to 4.69); fl ow percentage variation 9.76  ± 8.08 (8.11 to 11.41); r  = 0.969 and r2 = 0.939 (P <0.000). (3) FiO2 variation –0.005 ± 0.26 (–0001 to 0009); FiO2 percentage variation –0.72 ± 5.2 (–1.5 to 0.1); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.91 ± 1.26 (3.69 to 4.13); fl ow percentage variation 12.77 ± 5.33 (11.84 to 13.7); r = 0.996 and r2 = 0.992 (P <0.000). See Figure 1. Results We delivered teaching sessions to 100% (9/9) of junior doctors, 100% (71/71) of nursing staff and other health professionals. We obtained feedback from 90% (76/84), showing an improvement in understanding of capnography from 73% of respondents to 100%, with 87% reporting that the teaching aid made capnography interpretation much easier. All felt the training would improve patient safety, and 97% felt it would be worthwhile training in other ICUs. g Conclusion The FiO2 percentage variation in the Airvo® is higher than the other two devices, with no clinical relevance. The fl ow percentage variation of Evita XL® is superior to the other two devices; this may have some clinical relevance. Comparison of three methods of applying high fl ow nasal oxygen: in vitro study yl y in vitro study M Muñoz Garach1, O Olga1, ME Yuste Osorio1, R Fernandez Fernandez2, R Ramirez Puerta1, F Acosta Díaz1, AM Perez Bailón1, S Narbona Galdó1, L Peñas Maldonado1 1Hospital Universitario San Cecilio, Granada, Spain; 2Hospital Nuestra Señora del Prado, Talavera de la Reina, Spain Critical Care 2015, 19(Suppl 1):P265 (doi: 10.1186/cc14345) Methods Twenty-one adult and clinically stable patients undergoing assisted mechanical ventilation for more than 48  hours were investigated during pressure support (baseline) and during a 2-hour CPAP trial. sEMG of diaphragm (costmar), intercostal and sternocleidal (accessory muscles) was recorded with a dedicated device (Dipha16; Inbiolab, Groningen, the Netherlands) simultaneously with airway waveforms and expressed as the ratio of the signal during baseline. Diaphragmatic electrical activity from a nasogastric tube (EAdi) of 14 of those patients was also measured. 1Hospital Universitario San Cecilio, Granada, Spain; 2Hospital Nuestra Señora del Prado, Talavera de la Reina, Spain Critical Care 2015, 19(Suppl 1):P265 (doi: 10.1186/cc14345) Introduction High fl ow nasal (HNF) requires precise control of the fraction of inspired oxygen (FiO2) and fl ow contributed as well as an adequate adjustment of temperature and humidity of the gas provided. There are several equipments for HNF. We evaluated the FiO2 and fl ow supplied with three diff erent systems Results The rapid shallow breathing index was lower than 105 in all patients and only one patient failed the trial. We observed that the mean inspiratory value of costmar increased immediately after switch to CPAP but did not signifi cantly vary during the CPAP trial (ANOVA, P = 0.7). On the other hand, the activation of accessory muscles increased signifi cantly during the same period (P  = 0.01) and was strongly correlated with respiratory rate (r = 0.41, P <0.001) and inversely with f y Methods There have been analyzed: (1)  ‘Oxygen Therapy’ from Dräger Evita-XL®; (2)  Fisher & Paykel Airvo® option; and (3)  pack of fl owmeters Debson®. Measurements were made in the distal part of the circuit that is used in clinical practice. Variables: programmed and measured FiO2, programmed and measured fl ow. We used the Oxygen Monitor Ohmeda 5120® and Flow Meter® Fisher-Porter. Before each measurement we checked and/or calibrated each of them. All measurements were performed at room temperature in the ICU of our hospital (23 to 26º).The data were processed using SPSS v.15.0.1, accepting a signifi cance level of 95%. Figure 1 (abstract P266). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Use of ‘hats and caps’ enabled delivery of short bedside teaching sessions to clinical staff in ICU during everyday work. Feedback shows a marked improvement in confi dence around capnography interpretation. It may have value in other ICUs to improve staff understanding of capnography and improve patient safety. References P266 Surface electromyography of respiratory muscles during a CPAP trial for weaning g S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) 1. Cook TM, et al. BJA. 2011;106:617-31. 2. McGrath BA. BJA. 2014;113:521. 3. Cook TM, et al. Anaesthesia. 2013;68:421. Introduction Weaning from mechanical ventilation is an important concern in ICU clinical practice. Surface electromyography (sEMG) [1] is a non-invasive tool to assess activity of diff erent muscles. We describe sEMG patterns of respiratory muscles during a CPAP trial [2] in patients undergoing pressure support ventilation. Surface electromyography of respiratory muscles during a CPAP trial for weaning S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) Surface electromyography of respiratory muscles during a CPAP trial for weaning S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) Ventilatory response during intentional early rehabilitation in patients with mechanical ventilation K Obata1, N Shiba2, T Takahashi3, S Ichiba2, Y Ujike2 1 d C Ok l Ok , , , , j 1Japanese Red Cross Okayama Hospital, Okayama, Japan; 2Okayama University Graduate School of Medicine, Okayama, Japan; 3Tokyo University of Technology, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P263 (doi: 10.1186/cc14343) Introduction Intentional early rehabilitation with mechanical ventilation in ICUs is performed in clinical settings. However, strict indexes for safe rehabilitation have not been fully elucidated. The purpose of this study is to analyze ventilator response (VR) between healthy volunteers and patients with mechanical ventilation. Methods Sixteen healthy volunteers (Control group) and 13 patients on mechanical ventilation (MV group) were enrolled in this study. Both groups were positioned in a variety of postures (baseline in a supine position, settled back 30°, settled back 60°, and sitting position) and measured with an indirect calorimeter. The instantaneous energy expenditure (EE), tidal volume (TV), respiratory rate (RR) and minute expiratory volume (VE) were non-invasively measured in each posture. Figure 1 (abstract P264). ‘Hats and caps’ capnography training guide. S93 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Systematic procedures including non-invasive ventilation improve morbidity in sleeve gastrectomy I Auriant, N Devos, S Rossi I Auriant, N Devos, S Rossi Critical Care 2015, 19(Suppl 1):P269 (doi: 10.1186/cc14349) Table 1 (abstract P267). Mean ventilator-days/reintubation rates Table 1 (abstract P267). Mean ventilator-days/reintubation rates 2009 2010 2011 2012 2013 2014 MICU mean ventilator-days 7.71 6.81 5.90 5.88 5.50 5.50* MICU reintubation rates 14.5% 9.8% 9.7% 9.8% 13.9% 8.3% SICU mean ventilator-days 5.91 5.93 5.68 5.93 4.93** 5.20* SICU reintubation rates 5.2% 3.0% 4.1% 4.3% 4.7% 7.5% *, **Signifi cance as described in Results. Introduction Postoperative morbidity after sleeve gastrectomy is decreasing, but remains signifi cant. Bleeding and surgical fi stules remain the leading causes of morbidity and mortality. In several studies in postoperative care of obese patients, non-invasive positive pressure ventilation (NPPV) reduced the risk of lower respiratory tract infection and pneumonia [1], thereby reducing in-hospital morbidity. The aim of study was to describe whether systematic use of NPPV improves morbidity in the postoperative care of sleeve gastrectomy. Methods A 4-year before–after study was conducted in a 19-bed intermediate care unit of a private hospital. Before period: standard treatment – all patients received oxygen supplementation to achieve SaO2 above 90%. After period: standard treatment plus NPPV  – all patients were submitted to a systematic postoperative protocol: NPPV was provided using an oxygen CIPAP system with 5 cmH2O. Statistical analysis: complication rates were compared using the chi-square test. P <0.05 was considered statistically signifi cant. Conclusion Initiatives to reduce ventilator-days per patient realized signifi cant reductions from 2009 to 2014 while reintubation rates were unaff ected. One component of the bundle, early mobilization, introduced in the SICU in 2013 was associated with an additional reduction in mean ventilator-days. y gi Results A total of 857 patients were included. Inclusion characteristics were similar in the two groups: Before group – noNPPV: 352 patients, 2010 to 2011. Age: 40.58 ± 10.94, BMI: 42.79 ± 5.51, sex ratio F/M: 0.81. After group – NPPV: 504 patients, 2012 to 2013. Age: 40.81 ± 11.24, BMI: 42.92 ± 5.09, sex ratio F/M: 0.77. There is a signifi cant between- group diff erence in the complication rate: Before group – noNPPV: 10 surgical fi stula (2.84%) and six postoperative bleeding (1.70%); After group – NPPV: seven surgical fi stula (1.39%) and three postoperative bleeding (0.6%). The overall complication rate fell from 4.54% to 1.98%. The chi-square statistic = 4.58. The number of degrees of freedom is 1. References 1. Chittawatanarat K, Thongchai C. Spontaneous breathing trial with low pressure support protocol for weaning respirator in surgical ICU. J Med Assoc Thai. 2009;92:1306-12. 1. Chittawatanarat K, Thongchai C. Spontaneous breathing trial with low pressure support protocol for weaning respirator in surgical ICU. J Med Assoc Thai. 2009;92:1306-12. Methods Ventilator-day data were compiled from 2009 to 2014 for MICU and SICU in our facility. Reintubation rates were calculated when intubations were required >1 day after an extubation. 2. Esteban A, Alia I, Gordo F, et al. Extubation outcome after spontaneous breathing trials with T-tube or pressure support ventilation. The Spanish Lung Failure Collaborative Group. Am J Respir Crit Care Med. 1997;156:459-65. 2. Esteban A, Alia I, Gordo F, et al. Extubation outcome after spontaneous breathing trials with T-tube or pressure support ventilation. The Spanish Lung Failure Collaborative Group. Am J Respir Crit Care Med. 1997;156:459-65. Results Ventilator volume ranged from 639 to 766 distinct patients/ year in the MICU and from 555 to 687 for the SICU. Ventilator-day reduction was signifi cant (P <0.01) for the MICU* (7.7 to 5.5, –29%) and the SICU* (5.91 to 5.20,  –12%). Reduction patterns diff ered between the units as the SICU had a distinct reduction (**P = 0.007) between 2012 and 2013 coinciding with implementation of an early mobilization program. Reintubation rates diff ered between the units and rates did not increase with decreasing mean patient ventilator- days. See Table 1. Comparison of three methods of applying high fl ow nasal oxygen: in vitro study p g gi Results (1) FiO2 variation –0.001 ± 0.09 (–0.01 to 0.002); FiO2 percentage variation –0.012  ± 1.88 (–0.27 to 0.25); r2  = 0.999 and r  = 0.998 (P  <0.000). Flow variation (l/minute) 5.45  ± 3.23 (4.94 to 5.96); fl ow percentage variation 19.59 ± 11.63 (17.75 to 21.43); r = 0. 997 and r2 = 0.994 (P <0.000). (2) FiO2 variation –0.007 ± 0.26 (–0.011/–0.003); FiO2 Figure 1 (abstract P265). Programmed and measured fl ow. Figure 1 (abstract P265) Programmed and measured flow Figure 1 (abstract P266). Figure 1 (abstract P265). Programmed and measured fl ow. S94 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 before discontinue mechanical ventilation and extubation in the surgical intensive care unit (SICU). tidal volume (r = –0.16, P = 0.02). In patients with EAdi we confi rmed a tight correlation between costmar and EAdi (r = 0.62, P <0.001). See Figure 1. tidal volume (r = –0.16, P = 0.02). In patients with EAdi we confi rmed a tight correlation between costmar and EAdi (r = 0.62, P <0.001). See Figure 1. Methods We performed a prospective open-label randomized control study (non-inferiority trial) in SICU patients who were intubated and used mechanical ventilation, and appropriated discontinuation of the ventilator between June 2011 and November 2013. All patients underwent the same weaning protocol. The appropriated patients for discontinuation of MV were randomized into low pressure support up to 7  cmH2O and T-piece method. Reintubation within 72  hours, pneumonia after extubation, and hospital mortality were recorded. The statistical signifi cant diff erence was considered when P <0.05. Conclusion sEMG indicated that while diaphragm activation remains constant during the CPAP period, accessory muscles were progressively recruited and particularly in the conditions of increased respiratory rate and lower tidal volumes. Ventilator-day reductions not associated with reintubations and further reduced by an early mobilization program Ventilator-day reductions not associated with reintubations and further reduced by an early mobilization program M Palmquist-Tess, A Adams, J Mangan, D Owen, M LeClaire HCMC, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P267 (doi: 10.1186/cc14347) y y p g M Palmquist-Tess, A Adams, J Mangan, D Owen, M LeClaire HCMC, Minneapolis, MN, USA Introduction Mechanical ventilation is associated with increased risk of pneumonia, barotrauma, VILI, VAP, ARDS and mortality. From 2009 to 2014 in the MICU/SICU of our facility, eff orts to reduce ventilator-days included: noninvasive ventilation, sedation reduction, daily sedation vacations and weaning protocols. In 2013, an early mobilization of ventilated patients in the SICU was initiated. Aggressive ventilator-day reduction eff orts may be expected to lead to premature extubations and reintubations. Conclusion The outcomes after discontinuation of the mechanical ventilator between low pressure support and T-piece was not diff erence in term of reintubation, pneumonia after extubation and hospital mortality. Systematic procedures including non-invasive ventilation improve morbidity in sleeve gastrectomy The value returned from the chi-square statistic is P <5%.i References 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. gif Results A total of 520 patients were randomized into two groups: low pressure support group (260 patients) and T-piece group (260 patients). There was no diff erence in age, gender, body mass index, comorbidity, site of surgery, Charlson Comorbidity Index and Acute Physiologic and Chronic Health II score between groups (P >0.05). Regarding the intention to treat analysis, there were no diff erences between groups in reintubation rate (PSV 10% vs. T 14.6%; P = 0.109), pneumonia after extubation (PSV 14.2% vs. T 11.9%; P = 0.435) and hospital mortality rate (PSV 3.1% vs. T 3.5%; P = 0.805). Chiumello D, et al. Non-invasive ventilation in postoperative patients: a sytematic review. Intensive Care Med. 2011;37:918-29. P271 Systematic alveolar recruitment after cardiac surgery AL Lafarge, CK Kerneis, F Scalbert, LL Larnier, AB Brusset, PE Estagnasie, PS Squara Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P271 (doi: 10.1186/cc14351) Introduction The aim of our study was to investigate the value of C-reactive protein (CRP) and procalcitonin (PCT) in identifi cation of the systemic infl ammatory response syndrome (SIRS) and other complications in the early postoperative period after cardiac surgery with cardiopulmonary bypass (CPB) [1]. Methods In 93 patients undergoing coronary artery bypass graft surgery with CPB, after Ethical Committee approval in a prospective study, serum PCT and CRP values were collected before operation and daily until postoperative day 5. All patients were divided post hoc into patients with SIRS (n = 42) and patients without SIRS (n = 51). Student’s t test, the Mann–Whitney U test and receiver operating characteristic (ROC) curves were used. Introduction We designed a pilot study to evaluate the interest of an early systematic acute recruitment maneuver (ARM) in postcardiac surgery hypoxemic patients in order to properly design a larger trial. Methods This randomized controlled trial included consecutive patients operated on in our institution. Three hours after surgery, hypoxemic patients (PaO2 <300  mmHg, FIO2  = 1) were randomly assigned to ARM or control (H0). ARM was performed by applying once a positive end-expiratory pressure of 35  cmH2O during 45  seconds. Blood gases and hemodynamic variables were collected at H1, H8, H24 and H48. The primary endpoint was the duration of mechanical ventilation (MV). Secondary endpoints were survival rate, ICU length of stay and the occurrence of pneumonia. Results The comparison of serum CRP values in patients with or without SIRS on postoperative day 1 until postoperative day 5 demonstrated an increase in both groups without signifi cant diff erences (P >0.05). The PCT levels increased more signifi cantly in SIRS patients (5.78  ± 3.21 ng/ml vs. 1.23 ± 0.31 ng/ml) compared with patients without SIRS (P  = 0.0001) on postoperative day 1. In patients with postoperative complications (21/93, 22%) (circulatory failure  = 10, pneumonia  = 2, respiratory insuffi ciency  = 9, sepsis  = 0), PCT levels remained elevated until postoperative day 5 (6.11 ± 2.87 ng/ml) but diminished in patients with SIRS (0.96 ± 0.23 ng/ml) (P <0.0001). Early postoperative pulmonary complications following heart transplantation A Camkiran Firat, Ö Kömürcü, P Zeyneloglu, M Türker, A Sezgin, A Pirat Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P270 (doi: 10.1186/cc14350) Introduction The aim of this study was to determine the types, incidence, and risk factors for early postoperative pulmonary complications in heart transplantation recipients. Methods We retrospectively collected data from the records of consecutive heart transplantations from January 2003 to December 2013. A total of 83 patients underwent heart transplantation. Those patients younger than 10  years (n  = 9) and the patients who died intraoperatively (n = 1) or during the fi rst postoperative day (n = 1) were not included in the analyses. The data collected for each case were demographic features, duration of mechanical ventilation, respiratory problems that developed during the ICU stay, and early postoperative mortality (<30  days). The respiratory complications that we sought were pleural eff usion, pneumonia, pulmonary atelectasis, pulmonary edema, pneumothorax, and acute respiratory failure. Figure 1 (abstract P271). p p y Results Of the 72 patients considered, 52 (72.2%) were male. The mean age at the time of transplantation was 32.1  ± 16.6  years. The mean duration of postoperative mechanical ventilation was 71.8  ± 126.6 hours. The mean length of ICU stay was 13.5 ± 18.0 days. Two patients (2.8%) and one patient (1.4%) required extracorporeal membrane oxygenation support and intra-aortic balloon pump support, respectively, due to low cardiac output or primary graft failure postoperatively. Twenty-fi ve patients (34.7%) developed early postoperative respiratory complications. The most frequent problem was pleural eff usion (n = 19, 26.4%) followed by atelectasis (n = 6, 8.3%), acute respiratory distress syndrome (n = 5, 6.9%), pulmonary edema (n = 4, 5.6%), and pneumonia (n = 3, 4.2%). Postoperative duration of mechanical ventilation (44.2 ± 59.2 hours vs. 123.8 ± 190.8 hours, P = 0.005) and the length of ICU stay postoperatively (10.1  ± 5.8  hours vs. 19.8 ± 28.9 hours, P = 0.03) were longer among patients who had respiratory problems. Postoperative length of stay in the hospital (22.3 ± 12.5 days vs. 30.3 ± 38.3 days, P = 0.75) was similar in the two groups. The overall mortality rate was 12.5% (n  = 9 patients). The patients who had respiratory problems did not show higher mortality than those who did not have respiratory problems (16.0% vs. 10.6%, P = 0.71). the two groups. Early postoperative pulmonary complications following heart transplantation At H1, PaO2/FIO2 was 367 ± 15 in the recruited group versus 299 ± 15 mmHg in the control group, P = 0.002. At H8 and 24 the diff erence was not signifi cant. At H48, the PaO2/FIO2 was lower in the recruited group (296 ± 10 vs. 343 ± 11 mmHg, P = 0.003) (Figure 1). The duration of mechanical ventilation (invasive + non-invasive) was lower in the recruited group (total 6.4 ± 1.4 vs. 8.4 ± 1.4 hours, P = 0.02). The survival rate, the length of stay in the ICU and the occurrence of pneumonia were similar in the two groups (P >0.2). Conclusion We can speculate that the inverse evolution of the blood oxygenation between the ARM group versus control may be due to: barotraumatism of normal alveoli during the ARM and/or a higher de- recruitment rate after ARM due to the shorter mechanical ventilation support. This pilot study shows that a unique ARM decreased the duration of MV in cardiac surgery patients but this may have subsequent detrimental eff ects on blood oxygenation. P272 Open-label randomized control trial between low pressure support and T-piece method for discontinuation from mechanical ventilator and extubation in general surgical ICUs g g K Chittawatanarat, S Orrapin, S Orrapin Chiang Mai University, Chiang Mai, Thailand Chiang Mai University, Chiang Mai, Thailand g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) Introduction In routine practice for most surgical patients in Thailand, all appropriated patients with planned discontinuation of mechanical ventilator (MV) are routinely changed to T-piece before extubation. However, this method needs to alter the instrument for testing tolerability of the patient. The objective of this study was to compare continuous low pressure support (PSV) and the T-piece method (T) Conclusion Systematic use of NPPV signifi cantly improves morbidity in the postoperative care of sleeve gastrectomy. Reference Conclusion Systematic use of NPPV signifi cantly improves morbidity in the postoperative care of sleeve gastrectomy. Chiumello D, et al. Non-invasive ventilation in postoperative patients: a sytematic review. Intensive Care Med. 2011;37:918-29. S95 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P271). P270 Figure 1 (abstract P271). P270 Early postoperative pulmonary complications following heart transplantation A Camkiran Firat, Ö Kömürcü, P Zeyneloglu, M Türker, A Sezgin, A Pirat Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P270 (doi: 10.1186/cc14350) Is procalcitonin a valuable marker for identifi cation of postoperative complications after coronary artery bypass graft surgery with cardiopulmonary bypass? y y A Baysal1, M Dogukan2, H Toman3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Adiyaman University Research and Training Hospital, Adiyaman, Turkey; 3Canakkale 18 Mart University Hospital, Canakkale, Turkey Critical Care 2015, 19(Suppl 1):P272 (doi: 10.1186/cc14352) A Baysal1, M Dogukan2, H Toman3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Adiyaman University Research and Training Hospital, Adiyaman, Turkey; 3Canakkale 18 Mart University Hospital, Canakkale, Turkey Critical Care 2015, 19(Suppl 1):P272 (doi: 10.1186/cc14352) Conclusion Respiratory complications were relatively common in our cohort of heart transplant recipients. However, these complications were mostly self-limiting and did not result in increased mortality. Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery A Baysal1, I Ozkaynak2, M Dogukan3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Kahramanmaraþ Sütçü Ýmam University Hospital, Kahramanmaraþ, Turkey; 3Adiyaman University Research and Training Hospital, Adiyaman, Turkey Critical Care 2015, 19(Suppl 1):P273 (doi: 10.1186/cc14353) A Baysal1, I Ozkaynak2, M Dogukan3 A Baysal , I Ozkaynak , M Dogukan 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Kahramanmaraþ Sütçü Ýmam University Hospital, Kahramanmaraþ, Turkey; 3Adiyaman University Research and Training Hospital, Adiyaman, Turkey Critical Care 2015, 19(Suppl 1):P273 (doi: 10.1186/cc14353) Conclusion Increasing sweep gas fl ow results in eff ective CO2 removal which can be further reinforced by raising blood fl ow. The clinically relevant oxygenation eff ect even in this setting of low invasivity could broaden the range of indications towards hypercapnic lung failure with mild to moderate hypoxia. Figure 1 (abstract P274). PaO2 with increasing blood fl ow (*P <0.0001). Introduction Our goal was to investigate the incidence of postoperative jaundice in open heart surgery patients and to determine the risk factors associated with hepatic dysfunction. Methods A total of 292 patients were included in a prospective study design. Patients undergoing on-pump coronary artery bypass graft surgery (CABG) (n = 154) and valve repair surgery (mitral, mitral and aortic valve and/or tricuspid valve) (n  = 138) were included. Postoperative hyperbilirubinemia was defi ned as occurrence of a plasma total bilirubin concentration of more than 34  μmol/l (2  mg/ dl) in any measurement during the postoperative period. All patients were divided into groups with or without hyperbilirubinemia. Liver enzymes were collected on postoperative days 1, 7, 14 and 30. The risk factors including age, cardiopulmonary bypass time, number of blood transfusions, inotropic support, use of intra-aortic balloon pump and ICU stay were evaluated with logistic regression. Figure 1 (abstract P274). PaO2 with increasing blood fl ow (*P <0.0001). y g g Results Postoperative hyperbilirubinemia was observed in 27 of 292 patients (9.3%). The numbers of valves replaced, preoperative total bilirubin concentration, increased cardiopulmonary bypass time, higher number of inotropic support agents, and use of intra-aortic balloon pump correlate with hyperbilirubinemia on postoperative day  7 (P  <0.05). P271 A PCT threshold value of 2.79 ng/ml was able to discriminate between postoperative complications in patients with or without SIRS with a sensitivity of y Results We included 124 patients, age 67.5 ± 10.6 years, M/F sex ratio 95/29, left ventricle ejection fraction 58.8 ± 10.6%, forced expiratory volume 94 ± 23% of the predicted value, bypass/valve ratio 82/53. The preoperative and postoperative PaO2/FIO2 were 401 ± 66 and 204 ± 66 mmHg, respectively (P <0.0001). The hemodynamic and ventilation status as well as the fl uid and inotrope supports were comparable in S96 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 developed miniaturized device consisting of a centrifugal pump and a membrane ventilator (iLA Activve®; Novalung, Germany) allows eff ective decarboxylation via a jugular double lumen cannula. So far no data on gas exchange in this setting exist to date. 82.5% and a specifi city of 70% (area under the curve: 0.76  ± 0.05; P <0.01) on postoperative day 1. Conclusion After cardiac surgery with CPB, PCT values increased signifi cantly in the SIRS group of patients compared with patients without SIRS on postoperative day 1 and remained elevated until postoperative day 5. In the early postoperative period, early rise of PCT values may help to discriminate the development of postoperative complications in patients with or without SIRS. Methods We included 10 patients receiving iLA Activve® due to hypercapnic respiratory failure as bridge-to-transplant or obstructive lung disease. Sweep gas fl ow was increased in steps from 1 to 14 l/ minute at constant blood fl ow (phase 1). Similarly, blood fl ow was gradually increased at constant sweep gas fl ow (phase 2). At each step, gas transfer via the membrane as well as arterial blood gas samples were obtained. 1. Delannoy B, Guye ML, Slaiman DH, Lehot JJ, Cannesson M. Eff ect of cardiopulmonary bypass on activated partial thromboplastin time waveform analysis, serum procalcitonin and C-reactive protein concentrations. Crit Care. 2009;13:R180. 1. Delannoy B, Guye ML, Slaiman DH, Lehot JJ, Cannesson M. Eff ect of cardiopulmonary bypass on activated partial thromboplastin time waveform analysis, serum procalcitonin and C-reactive protein concentrations. Crit Care. 2009;13:R180. P275 P275 Safety and effi cacy of extracorporeal CO2 removal combined with continuous renal replacement therapy in patients presenting both acute respiratory distress syndrome and acute kidney injury J Allardet-Servent, M Castanier, T Signouret, A Lepidi, R Soundaravelou, J Seghboyan Hopital Européen Marseille, France Critical Care 2015, 19(Suppl 1):P275 (doi: 10.1186/cc14355) P271 Results During phase 1, we observed a signifi cant increase in CO2 transfer together with a decrease in PaCO2 levels from a median of 66 mmHg (range 46 to 85) to 49 (31 to 65) mmHg from 1 to 14 l/ minute sweep gas fl ow, while arterial oxygenation deteriorated with high sweep gas fl ow rates. During phase 2, oxygen transfer signifi cantly increased leading to an increase in PaO2 from 67 (49 to 87) at 0.5 l/ minute to 117 (66 to 305) mmHg at 2.0 l/minute. Higher blood fl ow rates also signifi cantly enhanced decarboxylation. Increasing blood fl ow to 2.0 l/minute led to negative suction pressures of more than –100 mmHg and signs of hemolysis. See Figure 1. Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery Independent risk factors of early postoperative jaundice are; multiple valve replacement surgery, ejection fraction and use of intraaortic balloon pump (R = 0.58, R2 = 0.33, F = 26.44, P <0.001). The ICU stay was signifi cantly longer in group 2 (11.52 ± 3.76 days) as compared with group 1 (2.79 ± 1.36 days) (P <0.001). g y Conclusion Patients undergoing multiple valve replacement procedures are at greater risk for the development of postoperative hyperbilirubinemia and an association with prolonged ICU stay was observed. Other risk factors including ejection fraction, increased cardiopulmonary bypass time and use of intra-aortic balloon pump are also important as they have been reported to increase postoperative complications [1]. Conclusion Increasing sweep gas fl ow results in eff ective CO2 removal which can be further reinforced by raising blood fl ow. The clinically relevant oxygenation eff ect even in this setting of low invasivity could broaden the range of indications towards hypercapnic lung failure with mild to moderate hypoxia. p Reference 1. Nishi H, Sakaguchi T, Miyagawa S, et al. Frequency, risk factors and prognosis of postoperative hyperbilirubinemia after heart valve surgery. Cardiology. 2012;122:12-9. Interhospital transfer of patients in extracorporeal membrane oxygenation F Socci, S Di Valvasone, M Ciapetti, A Franci, M Bonizzoli, G Cianchi, S Batacchi, A Peris Careggi Hospital, Firenze, Italy Critical Care 2015 19(Suppl 1):P276 (doi: 10 1186/cc14356) gg y Critical Care 2015, 19(Suppl 1):P276 (doi: 10.1186/cc14356) gg y Critical Care 2015, 19(Suppl 1):P276 (doi: 10.1186/cc14356) Introduction The transfer of patients in extracorporeal membrane oxygenation (ECMO) from a peripheral hospital to a tertiary center represents a high-risk situation of adverse events [1]. The aim of this retrospective study is to determine the feasibility and safety of interhospital transfer for critically ill patients with ECMO support. Methods We collected data for the ECMO Regional Reference Centre Careggi Hospital activity from September 2009 to June 2014. In this study, 57 transfers were examined. The ECMO service is activated by a telephone call from a peripheral hospital. The team is represented by an intensivist, a heart surgeon, a cardiologist, a perfusionist and an intensive care nurse, all previously trained in the management of patients with ECMO. Medical personnel and the necessary equipment are transported by an ambulance and a van, specially designed and equipped for the transfer of patients with ECMO. Methods Retrospectively, we examined 20 patients with cerebral MRI (including SWI) who had suff ered from severe ARDS and received ECMO therapy. The MRI slides were anonymized and analyzed by two experienced neuroradiologists. Based on the distribution pattern and characteristic, a modifi ed HACE score (mHCS) was surveyed [2]. i y Results Six of 20 patients (30%) showed multiple MH with emphasis in the splenium of the corpus callosum. Eight patients had sporadic MH in the parenchyma of the brain but not in the corpus callosum. The remaining six patients had no intracerebral alterations. The distribution of MH with involvement of the splenium resembled that seen in HACE survivors. Results In this study, 57 patients transferred from the peripheral hospital to the ECMO center were examined; in all cases the ECMO system was implanted in the peripheral hospital (54 venovenous ECMO and three venoarterious ECMO). On average, trails were 271  km  ± 304 (round trip) (minimum 14 km to maximum 939 km). The activation time from the call to the ambulance departure from our hospital was an average of 2 hours 27 minutes 13 seconds ± 1 hour 25 minutes 35 seconds. Determinants of gas exchange during extracorporeal CO2 removal using a novel pump-driven venovenous gas exchange system in a minimally invasive setting y g A Hermann, K Riss, P Schellongowski, A Bojic, P Wohlfarth, O Robak, W Sperr, T Staudinger Medical University of Vienna, Austria Critical Care 2015, 19(Suppl 1):P274 (doi: 10.1186/cc14354) A Hermann, K Riss, P Schellongowski, A Bojic, P Wohlfarth, O Robak, W Sperr, T Staudinger Medical University of Vienna, Austria Critical Care 2015, 19(Suppl 1):P274 (doi: 10.1186/cc14354) Introduction Pump-driven venovenous extracorporeal CO2 removal (ECCO2-R) increasingly takes root in hypercapnic lung failure to minimize ventilation invasiveness or to avoid intubation. A recently Introduction Pulmonary overdistension has been observed in 33% of patients with acute respiratory distress syndrome (ARDS) despite low tidal volume (6 ml/kg ideal body weight) ventilation [1]. Tidal volume S97 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (VT) reduction from 6 to 4  ml/kg attenuates overdistension but is associated with hypercarbia [2]. We thought to combine extracorporeal CO2 removal (ECCO2R) with continuous renal replacement therapy (CRRT) through the insertion of an oxygenator membrane within the hemofi ltration circuit in patients presenting both ARDS and acute kidney injury (AKI).i the devices during transport were not recorded; in some cases it was necessary to manage minor complications (circuit cavitation, minor vascula accesses bleeding). Conclusion Some studies have found several complications during transfer of patients in ECMO [2]. In our experience, there were no complications during the transfer of ECMO patients, even for longer trips. A wide and thorough clinical evaluation and multidisciplinary ECMO team allowed the optimization of clinical parameters before transport and a safely transfer. The start of ECMO treatment at peripheral hospitals and the transfer of patients in ECMO may be a viable option compared with conventional ventilation. Our data suggest that ECMO can be set up safely in peripheral hospitals by a multidisciplinary highly specialized ECMO team [3]. y j y Methods A fi rst set of measurement was performed at 6 ml/kg before and after ECCO2R. Twenty minutes later, VT was reduced to 4 ml/kg for the remainder of the study period (72 hours). Ventilator settings were those of the ARMA trial. The CRRT mode was hemofi ltration with 33% of predilution. Ultrafi ltration was adjusted to achieve a fi ltration fraction of 15%. Sweep gas fl ow was constant at 8 l/minute. The primary endpoint was a 20% reduction of PaCO2 at 20 minutes after initiation of ECCO2R. P276 h Introduction Cerebral microhemorrhages (MH) are diminutive focal bleedings which can be detected best by MRI using susceptibility- weighted imaging sequences (SWI). They can be found in a variety of neurologic diseases. The pattern of distribution can lead to the underlying pathomechanism [1]. Survivors of high-altitude cerebral edema (HACE) showed multiple MH, predominantly in the splenium of the corpus callosum. Mountaineers with a lack of acclimatization to high altitudes tend to suff er from HACE. Hypoxemia in great heights is discussed to be the main trigger of HACE [2]. Acute respiratory distress syndrome (ADRS) is characterized by oxygenation failure in mechanically ventilated patients. The severity is classifi ed by the ratio of arterial oxygen tension to fraction of inspired oxygen [3]. In some patients suff ering from severe ARDS, refractory to conventional therapy, venovenous extracorporeal membrane oxygenation therapy is the therapeutic option to ensure oxygenation. References 2 2 Results Eight patients were studied. Age was 69 ± 11 years, SAPS II was 68 ± 9 and SOFA score was 13 ± 4 at inclusion. Blood fl ow, at the inlet of the oxygenator membrane, was 400 ± 4 ml/minute. CO2 removal rate was 84 ± 4 ml/minute. Initiating ECCO2R, at 6 ml/kg, induced a mean PaCO2 reduction of 17% (41 ± 5.5 to 33.9 ± 5.6 mmHg, P <0.001). Then, lowering the VT to 4  ml/kg induced a mean PaCO2 increase of 25% (33.9 ± 5.6 to 42.6 ± 8 mmHg) and a mean PaO2/FIO2 ratio increase of 8% (176 ± 63 to 190 ± 61). Minute ventilation decrease from 7.4 ± 1.6 to 5 ± 1.2 l/minute. Respiratory system compliance did not vary. No major complications were observed. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 2. Haneya A, Philipp A, Foltan M, Mueller T, Camboni D, Rupprecht L, et al. Extracorporeal circulatory systems in the interhospital transfer of critically ill patients: experience of a single institution. Ann Saudi Med. 2009;29:110-4. 3. Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 2. Haneya A, Philipp A, Foltan M, Mueller T, Camboni D, Rupprecht L, et al. Extracorporeal circulatory systems in the interhospital transfer of critically ill patients: experience of a single institution. Ann Saudi Med. 2009;29:110-4. p p g 3. Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. p p g 3. Determinants of gas exchange during extracorporeal CO2 removal using a novel pump-driven venovenous gas exchange system in a minimally invasive setting Results Eight patients were studied. Age was 69 ± 11 years, SAPS II was 68 ± 9 and SOFA score was 13 ± 4 at inclusion. Blood fl ow, at the inlet of the oxygenator membrane, was 400 ± 4 ml/minute. CO2 removal rate was 84 ± 4 ml/minute. Initiating ECCO2R, at 6 ml/kg, induced a mean PaCO2 reduction of 17% (41 ± 5.5 to 33.9 ± 5.6 mmHg, P <0.001). Then, lowering the VT to 4  ml/kg induced a mean PaCO2 increase of 25% (33.9 ± 5.6 to 42.6 ± 8 mmHg) and a mean PaO2/FIO2 ratio increase of 8% (176 ± 63 to 190 ± 61). Minute ventilation decrease from 7.4 ± 1.6 to 5 ± 1.2 l/minute. Respiratory system compliance did not vary. No major complications were observed. y j y Methods A fi rst set of measurement was performed at 6 ml/kg before and after ECCO2R. Twenty minutes later, VT was reduced to 4 ml/kg for the remainder of the study period (72 hours). Ventilator settings were those of the ARMA trial. The CRRT mode was hemofi ltration with 33% of predilution. Ultrafi ltration was adjusted to achieve a fi ltration fraction of 15%. Sweep gas fl ow was constant at 8 l/minute. The primary endpoint was a 20% reduction of PaCO2 at 20 minutes after initiation of ECCO2R. Interhospital transfer of patients in extracorporeal membrane oxygenation Transfer duration (from activation to return to the ECMO center) was an average of 8  hours 25  minutes 6  seconds  ± 3  hours 27  minutes 58 seconds (minimum 3 hours to maximum 16 hours 55 minutes). The stop time (necessary for evaluation of the patient and for placement of the ECMO system) was an average of 3 hours 53 minutes 40 seconds ± 1 hour 6 minutes 35 seconds (minimum 2 hours 5 minutes to maximum 7 hours 30 minutes). Major complications related to malfunctions of Conclusion Based on these results, we postulate that hypoxemia is one of the main players in the development of splenium-associated MH, not only in HACE but also in severe ARDS and other diseases accompanied with severe hypoxemia. Further investigations have to examine potential triggers and special circumstances concerning ARDS treatment which lead to MH in this distinctive pattern. 1. Renard D, et al. J Neurol Neurosurg Psychiatry. 2014;85:1041-8. 2. Schommer K, et al. Neurology. 2013;81:1776-9. 3. Force ADT, et al. JAMA. 2012;307:2526-33. References Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. Conclusion Combining ECCO2R and CRRT in patients with ARDS and AKI is safe and feasible through the insertion of an oxygenator membrane within a RRT circuit. References 1. Terragni PP, Rosboch G, Tealdi A, et al. Tidal hyperinfl ation during low tidal volume ventilation in acute respiratory distress syndrome. Am J Respir Crit Care Med. 2007;175:160-6. P277 Microhemorrhages in the corpus callosum after treatment with extracorporeal membrane oxygenation S Riech, P Hellen, O Moerer, K Kallenberg, M Müller, M Quintel, M Knauth University Medical Center Goettingen, Germany Critical Care 2015, 19(Suppl 1):P277 (doi: 10.1186/cc14357) 2. Terragni PP, Del Sorbo L, Mascia L, et al. Tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal CO2 removal. Anesthesiology. 2009;111:826-35. 2. Terragni PP, Del Sorbo L, Mascia L, et al. Tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal CO2 removal. Anesthesiology. 2009;111:826-35. P279 Introduction Diff erential hypoxia is a pivotal problem in cardio- pulmonary failure patients with femoral venoarterial extracorporeal membrane oxygenation (VA ECMO) support. Although there was some attempt to deliver more oxygenated blood to the upper body, the mechanism of diff erential hypoxia has not been well investigated. Successful approach for emergent consent for ECMO research J Board1, S Vallance1, C Aubron2, D Pilcher1, V Pellegrino1, DJ Cooper1 1The Alfred Hospital, Melbourne, Australia; 2Monash University, Melbourne, Australia Critical Care 2015, 19(Suppl 1):P279 (doi: 10.1186/cc14359) f y Methods We used a sheep model of acute respiratory failure that was supported with femoral VA ECMO (from inferior vena cava to femoral artery (IVC-FA)), ECMO from superior vena cava to FA (SVC-FA), ECMO from IVC to carotid artery (IVC-CA) and ECMO adding an additional return cannula to internal jugular vein based on femoral VA ECMO (FA- IJV). Angiography and blood gas analysis were performed. Introduction The HELP-ECMO pilot study (Heparin low dose protocol versus standard care in critically ill patients undergoing ECMO; ACTRN12613001324707) is a randomised controlled phase II study evaluating two levels of heparin anticoagulation in patients with no requirement for full anticoagulation. This work is a substudy of the HELP-ECMO trial and describes the consent process of the parent study. At our site, consent for research is often obtained by the research coordinator with little involvement from investigators. However, the nature of the ECMO population required a modifi ed consent approach to be implemented given that ECMO is often inserted emergently. It required a model that would be successful out of hours and could be delivered by any member of the treating team. g g p y g y p Results Blood oxygen saturation (SO2) of IVC (83.6 ± 0.8%) was higher than that of SVC (40.3  ± 1.0%) in sheep with IVC-FA. Oxygen-rich blood was drained back to the ECMO circuit and poorly oxygenated blood in the SVC entered the right atrium (RA). SVC-FA achieved the oxygen-rich blood return from IVC to RA without shifting the arterial cannulation. SO2 of SVC and pulmonary artery increased (70.4 ± 1.0% and 73.4 ± 1.1%, respectively) subsequently. Compared with IVC-FA, Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. e 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. References 1. Renard D, et al. J Neurol Neurosurg Psychiatry. 2014;85:1041-8. 2. Schommer K, et al. Neurology. 2013;81:1776-9. 3. Force ADT, et al. JAMA. 2012;307:2526-33. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S98 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P278f less diff erences of venous oxygen return and attenuated diff erential hypoxia were also observed in IVC-CA and FA-IJV. See Figure 1.f P278 Diff erential venous oxygen return: a key factor of diff erential hypoxia in venoarterial extracorporeal membrane oxygenation X Hou1, X Yang1, Z Du1, J Xing1, C Jiang1, J Wang1, Z Xing1, H Wang1, H Zeng2 1Beijing Anzhen Hospital, Beijing, China; 2Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China Critical Care 2015, 19(Suppl 1):P278 (doi: 10.1186/cc14358) Conclusion Diff erential venous oxygen return is a key factor of diff erential hypoxia in VA ECMO. We can take advantage of the notion of diff erential venous oxygen return to choose better cannula confi guration in clinical practice. In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock M Bottiroli, T Maraffi , D Decaria, S Nonini, E Montrasio, K Gervasio, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P281 (doi: 10.1186/cc14361) y g M Bottiroli, T Maraffi , D Decaria, S Nonini, E Montrasio, K Gervasio, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P281 (doi: 10.1186/cc14361) Introduction Venoarterial (VA) ECMO is used for mechanical support in patients with cardiogenic shock (CS) unresponsive to medical therapy. Long-term survival and quality of life after hospital discharge have not yet been well analyzed. Methods We performed a retrospective observational study of patients admitted to the ICU for refractory CS from January 2010 to November 2014. Patients with postcardiotomy and/or post-transplant CS were excluded. Demographic, clinical and biochemical variables were collected. Continuous variables are presented as mean (standard deviation) and categorical variables as percentage. Long-term outcome and quality of life were assessed during scheduled follow-up evaluations or telephonic interviews. g p Results From April to December 2014, 30 patients were screened. Fourteen met the eligibility criteria and were approached for consent. Twelve patients were enrolled and randomised to receive either standard anticoagulation or low-dose heparin as per the study protocol. Consent was provided by the person responsible for 10 patients. One patient was competent to consent for themselves and one was enrolled under legislation allowing enrolment into research in the absence of a person responsible. There were two refusals. Seventy- one per cent of participants were approached out of hours. Eighty-six per cent were consented by clinicians. Twenty-one per cent of patients were consented by a non-investigator. Results We analyzed 23 consecutive patients undergoing VA ECMO for refractory CS. Etiologies of cardiac collapse were: 11 acute myocarditis, fi ve acute myocardial infarctions and seven acute decompensation of chronic cardiomyopathies (CCM). Thirteen patients died during the hospital stay and 10 survived. The main cause of ICU death was progressive multiple organ dysfunction (12/13). Baseline variables are presented in Table 1. All patients discharged from the hospital are still alive at follow-up (median 27 months, range 4 to 56) with a median NYHA class of 1 (range 1 to 2). All patients except one returned to an active style of life. Extracorporeal membrane oxygenator and ventricular assist device activity of a tertiary cardiothoracic centre: survival rates and length of ITU stay y D Sarridou1, CP Walker1, N Rashid1, D Heaton1, I McGovern1, N Marczin2, J Mitchell1 1The Royal Brompton & Harefi eld NHS Foundation Trust, London, UK; 2Imperial College, London, UK Critical Care 2015, 19(Suppl 1):P280 (doi: 10.1186/cc14360) Introduction Harefi eld Hospital accepts patients as a tertiary centre for end-stage heart and respiratory failure for the south of England. Interventions include VA-ECMO, VV-ECMO as a bridge to lung transplantation, left ventricular assist devices (LVAD) as a bridge for heart transplantation, right ventricular assist devices (RVAD) and BIVADs. The aim of this review was to identify the total number of such patients, analyse the individual length of ITU stay and calculate survival and mortality rates for each intervention. Conclusion VA-ECMO is an eff ective treatment tool for refractory CS in patients with acute life-threatening heart failure. Patients aff ected by acute decompensation of CCM had poorer outcomes characterized by multiple organ dysfunction, as already known in the literature. y Methods Patients consisted of six groups: Group 1: VA ECMO, Group 2: VV-ECMO, Group 3: LVAD, Group 4: RVAD, Group 5: BIVAD, Group 6: combination of devices. Data were extracted from the Perfusion Department records and the intensive care dataset from 2011 to 2013. Data included length of ITU stay, outcome, indication for device insertion and device-related major complications.i P280 P280 Extracorporeal membrane oxygenator and ventricular assist device activity of a tertiary cardiothoracic centre: survival rates and length of ITU stay D Sarridou1, CP Walker1, N Rashid1, D Heaton1, I McGovern1, N Marczin2, J Mitchell1 1The Royal Brompton & Harefi eld NHS Foundation Trust, London, UK; 2Imperial College, London, UK Critical Care 2015, 19(Suppl 1):P280 (doi: 10.1186/cc14360) P282 h P282 Characteristics of trauma patients with creatine kinase elevation N Assanangkornchai, O Akaraborworn, C Kongkamol, K Kaewsaengrueang Prince of Songkla University, Hatyai, Thailand Critical Care 2015, 19(Suppl 1):P282 (doi: 10.1186/cc14362) j p Results Forty patients were identifi ed. Twenty-nine were male and 11 female. Group 1 included 22 patients, Group 2: two patients, Group 3: four patients, Group 4: four patients, Group 5: zero, Group 6: eight patients treated with various combinations of ECMO or ventricular assist devices. ITU stay varied from 1 day to a maximum of 6 months intermittently for one patient. Duration of ITU stay for all 40 patients was 1,052  days with an average of 26.3  days per patient. Sixteen patients survived and were discharged to the Transplant Unit. Twenty- four patients died, putting the survival rate at 40% for this group. Conclusion This review demonstrates that the majority of these patients occupied intensive care beds for a prolonged period of time and despite the use of advanced support devices survival rates were signifi cantly lower than mortality rates. Results Forty patients were identifi ed. Twenty-nine were male and 11 female. Group 1 included 22 patients, Group 2: two patients, Group 3: four patients, Group 4: four patients, Group 5: zero, Group 6: eight patients treated with various combinations of ECMO or ventricular assist devices. ITU stay varied from 1 day to a maximum of 6 months intermittently for one patient. Duration of ITU stay for all 40 patients was 1,052  days with an average of 26.3  days per patient. Sixteen patients survived and were discharged to the Transplant Unit. Twenty- four patients died, putting the survival rate at 40% for this group. Introduction Rhabdomyolysis is a condition that results in the release of mainly creatine kinase (CK) and myoglobin from the breakdown of myocytes. Myoglobin has been known to cause renal failure (RF) and the CK level is routinely used as an indicator. A CK level >5,000  U/l was found to be associated with the risk of RF [1]. However, data are lacking on the level of CK to predict RF, especially in general trauma patients. The purpose of this study was to determine the initial CK level that predicts markedly elevated CK and the characteristics of trauma patients with elevated CK. Introduction Rhabdomyolysis is a condition that results in the release of mainly creatine kinase (CK) and myoglobin from the breakdown of myocytes. P279 Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. S99 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The HELP-ECMO pilot study is enrolling patients admitted to a large metropolitan ICU who require ECMO. Education on eligibility criteria and study processes was given to all ICU senior medical staff . Consent must be performed prior to the commencement of anticoagulation, often only a short time after ECMO cannulation. To facilitate recruitment at all hours, a HELP-ECMO study box is located in the ICU. Within the box is an instruction page outlining the screening, consent and randomisation process, as well as administrative tasks. Plain language statements, randomisation envelopes and consent documentation stickers are provided. A consent script is provided to ensure consistency across consenting personnel. The process is reviewed by the research coordinator the following day to confi rm local governance compliance. In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock Multivariate analysis (Cox) revealed pre-ECMO SOFA score (HR = 2.18, 95% CI = 1.016 to 4.6) and history of CCM (HR = 19, 95% CI = 2 to 178) and pre-ECMO lactate (HR = 1.2, 95% CI = 1.02 to 1.4) as independent risk factors for hospital mortality. Conclusion The model of consent described has proven to be successful in this challenging patient population. The ability of all staff to perform consent for the study has been a signifi cant factor in the success of the pilot. The review of study processes by research coordinators has supported this model. Table 1 (abstract P281) Alive Dead P value Age (years) 30 ± 18 43 ± 14 0.012 CCM 0 (0%) 7 (100%) 0.014 SOFA 8 ± 1.4 10 ± 1.9 0.002 Creatinine (mg/dl) 1.1 ± 0.3 2.2 ± 0.7 0.001 Bilirubin (mg/dl) 1 ± 0.4 2 ± 1.8 0.09 Lactate (mmol/l) 5 ± 2.1 8 ± 5.3 0.021 Platelets (103/μl) 257 ± 79 162 ± 99 0.03 Conclusion VA-ECMO is an eff ective treatment tool for refractory CS in patients with acute life-threatening heart failure. Patients aff ected by acute decompensation of CCM had poorer outcomes characterized by multiple organ dysfunction as already known in the literature References 1. Cartin Ceba R, et al. Risk factors for development of acute kidney injury in critically ill patients: a systematic review and meta-analysis of observational studies. Crit Care Res Pract. 2012:691013. Methods A retrospective observational study of adults with confi rmed rhabdomyolysis admitted to the Neurosciences Critical Care Unit between 2002 and 2012. Data collection included APACHE score, daily CK (with PEAK CK defi ned as the maximum CK recorded throughout ICU stay), creatinine, calcium, phosphate and bicarbonate levels, AKI, RRT, ICU length of stay and mortality. 1. Cartin Ceba R, et al. Risk factors for development of acute kidney injury in critically ill patients: a systematic review and meta-analysis of observational studies. Crit Care Res Pract. 2012:691013. 2. Suh SH, et al. Acute kidney injury in patients with sepsis and septic shock: risk factors and clinical outcomes. Yonsei Med J. 2013;54:965-72. 2. Suh SH, et al. Acute kidney injury in patients with sepsis and septic shock: risk factors and clinical outcomes. Yonsei Med J. 2013;54:965-72. g y y Results A total of 232 patients met the inclusion criteria. Rhabdo- myolysis was associated with trauma (76%), medical (15%) and surgical (9%) admission diagnoses. Forty-fi ve (19%) patients developed AKI, with 29 (12.5%) requiring RRT. Mortality was signifi cantly higher in patients who developed AKI (62% vs. 18%, P <0.001). Average CK on admission was 5,009 IU/l (SD 12,403 IU/l). CK values remained greater than 2,000 IU/l for an average of 3.3 days (range 1 to 10 days). Although PEAK CK was greater in patients requiring RRT compared with those that did not (PEAK CK: 32,354 IU/l vs. 7,353 IU/l, P = 0.001), receiver operator characteristic curves revealed that a threshold for PEAK CK >5,000 IU/l is only 55% specifi c and 83% sensitive for the prediction of need for RRT. CK peaks on the day of admission in 46% of patients, on day 2 in 37%, and on day 3 or later in 17% of cases. A McMahon Score >6 calculated on admission is 68% specifi c and 86% sensitive for RRT. P284 Methods Data from the Songklanagarind Hospital trauma registry were reviewed over 1 year (January 2013 to December 2013). Patients with at least two records of CK and creatinine (Cr) levels were included. Creatine kinase levels were analyzed during the fi rst 3 days of hospital admission. RF was defi ned as a Cr increment >0.3 mg/dl within 48 hours. Results Of the 1,491 patients admitted to the trauma service, 47 patients had CK levels drawn twice. These patients had a mean age of 32  years and a median Injury Severity Score (ISS) of 14. The predominant mechanism of injury was motorcycle crash. Only three patients developed RF. The median CK during the fi rst 3  days after admission was 3,088 (IQR 1,327, 6,072) U/l. The CK peaked at 11 hours after admission at a mean value of 16,114.167 (SD 34,010.80) U/l. There were no signifi cant diff erences in demographic data, ISS scores and fl uid balance between the groups of CK level over or below 5,000 U/l. A mean positive fl uid balance observed; however, initial CK was signifi cantly diff erent between the two groups. None of the patients with initial CK of <900 U/l had a peak of CK >5,000 U/l. Risk factors for acute kidney injury in patients with complicated intra-abdominal infection A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) P283 Rhabdomyolysis: early prognostication of renal failure and other adverse outcomes J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) Rhabdomyolysis: early prognostication of renal failure and other adverse outcomes J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) Introduction The clinical diagnosis of rhabdomyolysis is confi rmed by creatine kinase (CK) levels >1,000 IU/l [1]. A local therapeutic protocol triggers aggressive renoprotective treatment in all patients with CK >2,000 IU/l. To evaluate local practice and refi ne CK thresholds for the instigation of renoprotective treatment, we studied the correlation between CK time trends and adverse outcomes such as acute kidney injury (AKI), the need for emergency renal replacement therapy (RRT) and mortality. We also evaluated the McMahon Score, a risk prediction model based on demographic, clinical, and laboratory variables available on admission [2].i p y Conclusion Severe AKI with RRT need is highly associated with previous HTN. The number of previous medications is related to severe AKI too. HTN has been described as a risk factor for developing AKI in critically ill patients [1]. ACEI and ARB use has been associated with AKI development in septic patients [2]. To our knowledge, this is the fi rst study that investigates risk factors associated with AKI in surgical septic patients with CIAI. 2. McMahon GM, et al. JAMA Intern Med. 2013;73:1821-8. 1. Khan FY. Neth J Med. 2009;67:272-83. Risk factors for acute kidney injury in patients with complicated intra-abdominal infection Risk factors for acute kidney injury in patients with complicated intra-abdominal infection A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) Introduction AKI has been poorly studied in surgical septic patients. The aim of our study was to determine the factors related to AKI in surgical septic patients with complicated intra-abdominal infection (CIAI) and mortality associated with AKI in this setting. Methods An observational study was performed of all adult patients with CIAI requiring surgery and ICU admission from June 2011 to June 2013. We recorded demographic data, SAPS II, SOFA score at admission, presence of septic shock, history of pre-existing comorbidities, angiotensin-converting enzyme inhibitor (ACEI), angiotensin receptor blocker (ARB), NSAIDs, statins or diuretics consumption, baseline creatinine and at admission, and standard biomarkers. Factors associated with developing AKI and renal replacement therapy (RRT) were studied using a multivariate analysis. Association between mortality and AKI and RRT need was also analyzed. Conclusion Trauma patients had varying levels of elevated CK. Initial CK shows a promising result as a predictor of high peak CK levels. A larger sample size is needed to demonstrate the predictors of RF in trauma patients with elevated CK levels. Reference 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. Results A total of 114 patients were included, with a mean SAPS II of 42.14. Sixty-seven patients (58.8%) developed AKI and 33 (28.9%) required RRT. Development of AKI (R2 = 0.498; P <0.0001; AUC = 0.926) was independently associated with SOFA (OR = 1.57; 95% CI = 1.29, 2.02) and creatinine at admission (OR for 0.1 units = 1.56; 95% CI = 1.30, 1.99). RRT need (R2 = 0.382; P <0.0001; AUC = 0.892) was independently associated with arterial hypertension (HTN) (OR  = 4.90; 95% CI  = 1.50, 15.97) and SOFA score (OR = 1.71). In another model with more predictive capacity (R2 = 0.433; P <0.0001; AUC = 0.918) the number of previous medications (OR = 3.73; 95% CI = 1.92, 8.38) and SOFA score (OR = 1.86; 95% CI = 1.47, 2.54) were related to RRT need. Both AKI and RRT need were related to ICU (RR = 8.41, 95% CI = 1.14, 62.5; and RR = 8, 95% CI = 2.40, 27.85 respectively) and 28-day mortality (RR = 2.8, 95% CI = 1.00, 7.86; and RR = 4.65, 95% CI =1.99, 10.40 respectively). P282 h Myoglobin has been known to cause renal failure (RF) and the CK level is routinely used as an indicator. A CK level >5,000  U/l was found to be associated with the risk of RF [1]. However, data are lacking on the level of CK to predict RF, especially in general trauma patients. The purpose of this study was to determine the initial CK level that predicts markedly elevated CK and the characteristics of trauma patients with elevated CK. Conclusion This review demonstrates that the majority of these patients occupied intensive care beds for a prolonged period of time and despite the use of advanced support devices survival rates were signifi cantly lower than mortality rates. S100 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Introduction Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients [1]. Early detection and treatment may improve outcome. Introduction Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients [1]. Early detection and treatment may improve outcome. y Methods A retrospective analysis of prospectively collected data from 2,158 patients without end-stage renal disease from the EPaNIC trial [2]. For early detection of AKI, defi ned according to the creatinine-based KDIGO guidelines [3], three multivariate logistic regression models (LR) were developed using data available at baseline (LR_B), upon ICU admission (LR_BA), and at the end of the fi rst day in the ICU (LR_BAD1). In a subpopulation (n  = 580) where plasma neutrophil gelatinase- associated lipocalin (pNGAL), an early biomarker of AKI, was measured at ICU admission, the value of adding pNGAL to LR_BA and LR_BAD1 was assessed. The models were evaluated via bootstrapping, by comparing receiver operator characteristic (ROC) and decision curves. pi Conclusion Although higher CK levels are associated with adverse outcomes, instigation of renoprotective treatment should not be based solely on CK levels. A McMahon Score >6 on admission allows for a more sensitive, specifi c and timely identifi cation of patients at risk of renal failure requiring RRT. S101 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P285). Area under ROC curves for the diff erent models LR_BA + pNGAL on LR_BA on pNGAL on subpopulation subpopulation subpopulation LR_B LR_BA LR_BAD1 with pNGAL with pNGAL with pNGAL (n = 2,158) (n = 2,067) (n = 1,808) (n = 528) (n = 528) (n = 528) 0.73 0.76 0.82 0.64 0.70 0.76 with the AMPK activator AICAR (100 mg/kg intraperitoneal, 24 hours before CLP), and sacrifi ced 24 hours after CLP. Blood and tissue samples were collected for all animals. AMPK activation (pThr172), B-ENaC, and mitophagy (LC3 II/I) were examined by western blot of kidney lysates. Plasma creatinine (Scr) was assessed using ELISA. Results Table 1 presents the performance of the models and admission pNGAL. References 1. De Geus H, et al. Am J Respir Crit Care Med. 2011;183:907-14. 2. Casaer M, et al. N Engl J Med. 2011;365:506-17. 3. Acute Kidney Injury Work Group. Kidney Int. 2012;2:1-138. 1. De Geus H, et al. Am J Respir Crit Care Med. 2011;183:907-14. 2. Casaer M, et al. N Engl J Med. 2011;365:506-17. 3. Acute Kidney Injury Work Group. Kidney Int. 2012;2:1-138. P286 Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? K Jin, H Li, J Volpe, D Emlet, N Pastor-Soler, MR Pinsky, BS Zuckerbraun, K Hallows, JA Kellum, H Gomez University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P286 (doi: 10.1186/cc14366) Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Performance improved when predictions were made at a later time point, and was highest for LR_BAD1. Similar results were obtained in subgroups of septic and cardiac surgery patients. As an independent predictor, pNGAL alone did not perform better than a model using routine clinical data available upon admission. However, when combining pNGAL with LR_BA, predictive performance improved. The performance of LR_BAD1 was not improved by including pNGAL. with the AMPK activator AICAR (100 mg/kg intraperitoneal, 24 hours before CLP), and sacrifi ced 24 hours after CLP. Blood and tissue samples were collected for all animals. AMPK activation (pThr172), B-ENaC, and mitophagy (LC3 II/I) were examined by western blot of kidney lysates. Plasma creatinine (Scr) was assessed using ELISA. g Results The acute response to sepsis was characterized by early activation of AMPK which increased from 6 to 18  hours, peaked at 24 hours, and decreased by 48 hours (Figure 1A). This activation was associated with a consistent decrease in B-ENaC expression. In AICAR pretreated animals, AMPK was only activated in WT mice, which was associated with a decrease in the expression of B-ENaC as compared with AMPK KO mice (Figure 1B). p p y g p Conclusion This study shows the potential of data-driven models based on routinely collected patient information for early detection of AKI during the fi rst week of ICU stay. Although adding admission pNGAL to admission data improved early detection of AKI, this added value is lost upon inclusion of data from the fi rst day of ICU. R f Conclusion AMPK was activated early after induction of sepsis, and was associated with a consistent decrease in Beta-ENaC expression in the apical membrane of tubular epithelial cells. In addition, absence of AMPK activation in KO animals was associated with increased expression of Beta-ENaC at 24 hours after CLP. These data support the hypothesis that early activation of AMPK decreases energy consumption through ion channel downregulation. Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? S102 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 AKI as per the AKIN KDIGO defi nition, as well as their worst level of TIMP2*IGFBP7 during their fi rst 2-day stay. Values of mean and 25th to 75th percentile for the worst value of TIMP2*IGFBP7 were 0.24 (0.11 to 0.46), 0.50 (0.28 to 1.24), 0.94 (0.34 to 3.28) and 3.34 (1.47 to 6.22) for no AKI, AKIN I, II and II respectively (P <0.0001). The worst values for no AKI/no sepsis, no AKI/sepsis, AKI/no sepsis and AKI/sepsis were 0.21 (0.10 to 0.4), 0.32 (0.15 to 0.63), 1.05 (0.41 to 2.31) and 0.98 (0.36 to 3.94) respectively, with P <0.05 for AKI and P = NS for sepsis. The AUC ROC curve for prediction of AKI of the worst value was 0.80 with sensitivity of 73.5% and specifi city of 71.4% (P <0.0001). In contrast to the Sapphire study, in our population cutoff values of 0.4 and 0.8 (ng/ml)2/1,000 predict AKI and AKIN ≥II respectively, regardless of the presence of sepsis. See Figure 1. Results There were 101 patients without AKI, 95 patients with KDIGO 1 AKI, and 42 patients with KDIGO 2 to 3 AKI within 48 hours of the start of surgery. In patients without AKI, median TIMP-2·IGFBP7 values were less than 0.3 (ng/ml)2/1,000 (dashed line in Figure 1) for all time points. In patients with KDIGO 1 AKI, median [TIMP-2·IGFBP7] signifi cantly exceeded this cutoff at 24 and 36 hours following the start of surgery (*one-sided P  <0.025). Median [TIMP-2·IGFBP7] increased earlier in KDIGO 2 to 3 AKI patients, remaining signifi cantly elevated relative to the cutoff from 12 to 60 hours after the start of surgery. The highest median [TIMP-2·IGFBP7] was observed at 24 hours for KDIGO 2 to 3 AKI patients and was nearly fi ve times the 0.3 (ng/ml)2/1,000 cutoff .i p yi gf Conclusion Urinary [TIMP-2·IGFBP7] was signifi cantly elevated as early as 12 to 24 hours from the start of surgery in patients who developed AKI within 48 hours. Monitoring of these biomarkers in the immediate postsurgical period might enable improved management of patients at risk for AKI. Conclusion TIMP-2 and IGFBP-7 can predict AKI in both septic and nonseptic critically ill patients. Further pragmatic randomised controlled trials are needed to prove their role on clinical basis. Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? P287 Urinary TIMP-2 and IGFBP7 elevate early in critically ill postoperative patients that develop AKI P Honore1, LS Chauwla2, A Bihorac3, AD Shaw4, J Shi5, JA Kellum6 1VUB, Brussels, Belgium; 2VAMC, Washington, DC, USA; 3UF, Gainesville, FL, USA; 4VUSM, Nashville, TN, USA; 5WB, Carlsbad, CA, USA; 6PiU, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P287 (doi: 10.1186/cc14367) Introduction This study tested the hypothesis that the cellular response in the kidney to sepsis is characterized by early activation of AMP activated protein kinase (AMPK), and that such activation is temporally associated with downregulation of the epithelial sodium channel (B-ENaC). Introduction Little is known about temporal changes in [TIMP- 2·IGFBP7] relative to injury in patients who develop AKI. In this analysis, we examined [TIMP-2·IGFBP7] in serial urine collections from the subset of Sapphire patients who were admitted to the ICU after major surgery. Methods We stratifi ed 238 Sapphire patients into three groups by their maximum AKI stage within 48 hours of the start of surgery using KDIGO criteria (No AKI, KDIGO 1, and KDIGO 2 to 3). Median TIMP-2·IGFBP7 values were calculated from all samples collected at 12 (±6)-hour intervals for 4 days following the start of surgery. Methods Fifteen C57BL/6 wildtype (WT) mice were subjected to cecal ligation and puncture (CLP), and sacrifi ced at 2, 6, 18, 24, or 48 hours. In addition, we pretreated three WT and three AMPK Beta 1 knockout mice Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. intervals for 4 days following the start of surgery. Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Figure 1 (abstract P287). Figure 1 (abstract P287). Figure 1 (abstract P287). Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? Reference 1. Kashani et al. Crit Care. 2013;17:R25. 1. Kashani et al. Crit Care. 2013;17:R25. Single point measurement of cystatin C has similar performance as serum creatinine for assessment of kidney function in critically ill patients J Houthoofd1, M Carlier2, J De Waele2, R Vanholder2, J Delanghe2, J Decruyenaere2, E Hoste2 Introduction Sepsis and acute kidney injury (AKI) have a high prevalence in the ICU population. The aim of this study is to describe the composite of tissue inhibitor of metalloproteinases-2 (TIMP2) and insulin-like growth factor-binding protein-7 (IGFBP7) as novel urinary renal biomarkers in both septic and nonseptic patients. Introduction The gold standard for routine evaluation of kidney function is measurement of serum creatinine concentration (Scr). In ICU patients, muscle loss and dilution leads to decreased Scr. Scr is distributed in total body water, resulting in delay of Scr changes when the glomerular fi ltration rate (GFR) changes (lag time). Cystatin C (CysC) is a protein produced by all cells with a nucleus and therefore less aff ected by muscle mass. Also, the lag time may be shorter as the volume of distribution is only extracellular fl uid. The objective of this study was to evaluate whether single point measurement of CysC is more adequate than Scr for monitoring of kidney function in ICU patients. Methods We conducted a prospective, observational study in two university hospitals. Patients were admitted in ICU either from the emergency department or after undergoing an acute surgery at hospital admission. Two months prior to the admission, recruited patients had not been admitted to hospital. We collected epidemiological, clinical and laboratory data at admission, 24 and 48 hours. TIMP2*IGFBP7 was analysed in urine samples by a point-of-care device (Nephrocheck®; Astute Medical). Results The sample included 98 patients (65 men) with mean age 55 ± 17.3 years, length of ICU stay 11.1 ± 14.6 days. In total, 41.4% had sepsis at ICU admission; 59.2% were diagnosed of sepsis within the fi rst 48 hours of stay. We stratifi ed patients based on the presence of Methods Data were collected in two prospective single-center studies on a convenience sample of ICU patients. During the 24-hour study period, we measured CysC, Scr, and urinary inulin clearance (Cinu) as a Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2]*[IGFBP7] value within the fi rst 48 hours of ICU admission to predict (a) AKI and (b) AKIN KDIGO ≥2. Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2]*[IGFBP7] value within the fi rst 48 h predict (a) AKI and (b) AKIN KDIGO ≥2. P288 Urinary TIMP2 and IGFBP7 as early biomarkers of acute kidney injury in septic and nonseptic critically ill patients M Cuartero 1, A Betbesé 1, J Sabater2, J Ballús2, J Ordóñez1 1Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Spain; 2Hospital Universitari Bellvitge, Hospitalet Llobregat, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P288 (doi: 10.1186/cc14368) P291 gold standard for assessment of GFR. We compared Scr and CysC, with Cinu. Also, we assessed Cinu in patients who had CysC and Scr within the normal sex and age corrected limits. Finally, we determined the ability of CysC and Scr to detect normal range and decreased GFR (80 to 120 ml/minute/1.73m2 resp. <60). Association between urinary TIMP-2 and IGFBP7 as early biomarkers of AKI and oliguria during liver surgery: a prospective pilot study F Desmet1, M D’Hondt1, H Pottel2, S Carlier1, E Hoste3, J Kellum4, W De Corte1 Association between urinary TIMP-2 and IGFBP7 as early biomarkers of AKI and oliguria during liver surgery: a prospective pilot study F Desmet1, M D’Hondt1, H Pottel2, S Carlier1, E Hoste3, J Kellum4, W De Corte1 1AZ Groeninge, Kortrijk, Belgium; 2Catholic University Leuven, Kortrijk, Belgium; 3Ghent University Hospital, Ghent University, Ghent, Belgium; 4University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P291 (doi: 10.1186/cc14371) 1AZ Groeninge, Kortrijk, Belgium; 2Catholic University Leuven, Kortrijk, Belgium; 3Ghent University Hospital, Ghent University, Ghent, Belgium; 4University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P291 (doi: 10.1186/cc14371) p Results We included 68 patients, with median age 58  years (IQR 29 years), length of stay in the hospital before study 11 days (IQR 16), and APACHE II score 19 (IQR 9). Scr was 1.12 (IQR 1.55), CysC 0.64 (IQR 0.73), and Cinu 80  ml/minute/1.73 m2 (IQR 82). Cinu was markedly decreased in patients with Scr in normal range (n  = 12) compared with patients with CysC in normal range (n = 22) (55 ml/minute/1.73 m2 (IQR 57.4) vs. 100 (IQR 42.2), P <0.001). Patients with normal range Scr had similar proportion of patients with Cinu in the normal range compared with normal range CysC patients (33.3% vs. 45.5%, P = 0.23). ROC analysis showed that Scr and CysC had similar performance for detection of normal range Cinu (AUC: 0.66; 95% CI = 0.53 to 0.77 vs. 0.77; 95% CI = 0.65 to 0.87; P = 0.118), and decreased Cinu (AUC: 0.86; 95% CI = 0.76 to 0.97 vs. 0.94; 95% CI = 0.88 to 1; P = 0.113). Introduction Patients undergoing elective liver surgery have an increased risk for developing AKI [1]. This study was intended to assess [TIMP-2]*[IGFBP7] and its possible association with urine output (UO) in this population. References 1. Slankamenac et al. Development and validation of a prediction score for postoperative ARF following liver resection. Ann Surg. 2009;250:720-8. 2. Hoste et al. Derivation and validation cutoff s for clinical use of cell cycle arrest biomarkers. Nephrol Dial Transplant. 2014;29:2054-61. 2. Hoste et al. Derivation and validation cutoff s for clinical use of cell cycle arrest biomarkers. Nephrol Dial Transplant. 2014;29:2054-61. g Results Mean age was 81  ± 5.6  years (16 male, 40.0%). Thirty-fi ve patients underwent TA-TAVI and fi ve patients TAo-TAVI. AKI developed in 17 patients (42.5%); seven patients (17.5%) suff ered from AKI 3 and required renal replacement therapy (RRT). Mean maximum value of UTI within 24 hours after TAVI was signifi cantly higher in patients with AKI compared with patients without renal impairment (2.19 ± 3.11 vs. 0.67 ± 0.816, P = 0.037) and higher in patients with AKI 3 compared with patients with AKI 2 (4.73 ± 3.58 vs. 0.59 ± 0.71, P = 0.022). In contrast, preoperative creatinine (AKI (mg/dl) 1.22 ± 0.41 vs. no AKI 1.30 ± 0.59; AKI 3 1.32 ± 0.49 vs. AKI 2 1.26 ± 0.53, P = NS) and early postoperative serum creatinine levels (maximum within 24 hours after TAVI: AKI 1.41 ± 0.50 vs. no AKI 1.34 ± 0.60; AKI 3 1.69 ± 0.56 vs. AKI 2 1.32 ± 0.54, P = NS) did not show any association with the development of AKI. ROC analyses revealed a very good predictive value of early UTI levels for the development of AKI 3 within the next 72 hours after TAVI with a sensitivity of 100% and a specifi city of 80% for a cutoff value of 0.815 (AUC = 0.919, 95% CI = 0.824 to 1.0, SE 0.048, P = 0.001). Single point measurement of cystatin C has similar performance as serum creatinine for assessment of kidney function in critically ill patients Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2]*[IGFBP7] value within the fi rst 48 hours of ICU admission to predict (a) AKI and (b) AKIN KDIGO ≥2. S103 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P290 Early prediction of acute kidney injury after transcatheter aortic valve implantation with urinary G1 cell cycle arrest biomarkers F Dusse, M Dudásová, D Wendt, M Thielmann, E Demircioglu, HG Jakob, K Pilarczyk University Hospital Essen, Germany Critical Care 2015, 19(Suppl 1):P290 (doi: 10.1186/cc14370) Introduction Acute kidney injury (AKI) is a common complication following transcatheter aortic valve implantation (TAVI) and has been shown to increase mortality. The concentration of the G1 cell cycle arrest proteins TIMP-2 and IGFBP7 in the urine have recently been suggested as sensitive biomarkers for early detection of AKI in critically ill patients. Whether postoperatively elevated levels of urinary [TIMP-2][IGFBP7] (UTI) predict the development of an AKI in patients undergoing TAVI is currently unknown. Conclusion This pilot study demonstrated the association between [TIMP-2]*[IGFBP7] increase and oliguria and may therefore indicate kidney damage during liver surgery. As Scr could not diff erentiate for these changes, patients did not meet the classical biomarker criteria for AKI. y Methods In a prospective cohort study, 40 patients undergoing TAVI, either trans-apical (TA) or trans-aortic (TAo), were enrolled. Serial measurements of UTI were performed every 12  hours in the postoperative course. Results were calculated for their multiplication and presented as arbitrary values. Urinary output and serum creatinine were recorded simultaneously. The primary clinical endpoint was the occurrence of AKI according to the AKI Network. P291 Secondly we sought to compare [TIMP-2]*[IGFBP7] with serum creatinine concentration (Scr). Methods A prospective single-center pilot study performed on 12 patients undergoing elective liver surgery. Serial urine samples were analyzed for [TIMP-2]*[IGFBP7] measured with the Nephrocheck device (Astute Medical, San Diego, CA, USA). Serial Scr was analyzed, UO, blood losses, and mean arterial pressure (MAP) were recorded. Fluid management was standardized, oliguria defi ned as a UO <0.5 ml/kg/ hour. [TIMP-2]*[IGFBP7] values of >0.3 identify patients at high risk and >2 at highest risk for AKI [2]. Conclusion Single point measurement of Scr and CysC has similar performance for detection of normal and decreased GFR in this cohort of ICU patients. Performance was weak for detection of normal GFR, but both biomarkers had moderate good performance for detection of decreased GFR. g [ ] Results Males comprised 66.7%, median age was 72  years. Median surgical time was 195  minutes. Peroperative median MAP was 71  mmHg (IQR 69; 77). Baseline median GFR was normal in eight patients and decreased in four patients (eGFR >90 and 66.5  ml/ minute/1.73 m2 respectively). Median baseline Scr was 0.75 mg/dl (IQR 0.61; 1.10), 0.74 mg/dl (IQR 0.64; 1.04) at ICU admission and 0.74 mg/ dl (0.64; 1.04) on day 1 postoperatively. No diff erence in Scr and eGFR was seen between these time points (P = 0.457 and P = 0.517 respectively; repeated-measures ANOVA). Median peroperative and postoperative UO was 0.18 ml/kg/hour (IQR 0.13; 0.23) and 0.93 ml/kg/ hour (IQR 0.79; 1.49) respectively. Median baseline [TIMP-2]*[IGFBP7] was 0.10 (IQR 0.04; 0.34), 2.02 (1.44; 6.23) during surgery, 0.61 (IQR 0.27; 1.22) at ICU admission and 0.74 (0.67; 0.97) on day 1 postoperatively. [TIMP-2]*[IGFBP7] diff ered at these time points (P <0.0001; repeated- measures ANOVA). Peroperative oliguria was associated with increased [TIMP-2]*[IGFBP7] (P = 0.018, chi-squared test). Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data y J Dixon1, K Lane1, R Dalton2, I MacPhee1, B Philips1 J Dixon1, K Lane1, R Dalton2, I MacPhee1, B Philips1 p 1St George’s Hospital and University of London, UK; 2King’s College, London, UK Critical Care 2015, 19(Suppl 1):P292 (doi: 10.1186/cc14372) 1St George’s Hospital and University of London, UK; 2King’s College, London, UK Critical Care 2015, 19(Suppl 1):P292 (doi: 10.1186/cc14372) Introduction There is currently no accurate method of measuring the glomerular fi ltration rate (GFR) during acute kidney injury (AKI). Four- hour creatinine clearance (4-CrCl) is often used. We have previously validated a method of measuring the GFR using a continuous infusion of low-dose iohexol (CILDI) in patients with stable renal function (GFR from normal to <30  ml/minute/1.73  m2). Steady state was achieved in <10 hours in all subjects and we calculate that variations >10.3% suggest an AKI. In this study we compare GFR measured by CILDI with 4-CrCl and 1-hour creatinine clearance (1-CrCl). Conclusion Early elevation of urinary [TIMP-2][IGFBP7] after TAVI is associated with the development of postoperative AKI. These biomarkers have an excellent diagnostic accuracy in the prediction of severe AKI requiring RRT that is superior to that of serum creatinine. Further studies are necessary to prove whether UTI-guided therapy of patients with AKI can reduce morbidity and mortality. Methods Critically ill patients with evolving AKI and patients following nephrectomy were recruited. Demographics were compared using the t test. CIDLI was connected for up to 72 hours. Plasma and renal iohexol and creatinine concentrations were measured by tandem mass spectrometry four times daily. Iohexol renal clearance (IRC) and S104 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 1-CrCl and 4-CrCl were calculated and compared using Bland–Altman analysis. P294 Impact of kidney injury on fl uid overload and impaired oxygenation A Akcan Arikan, LL Loftis, MA Arnold, CE Kennedy Baylor College of Medicine, Houston, TX, USA Critical Care 2015, 19(Suppl 1):P294 (doi: 10.1186/cc14374) y Results Baseline estimated GFR was similar in the postnephrectomy (88 ± 28) to the evolving AKI group (92 ± 23), P = 0.70. The evolving AKI group had a higher APACHE score (17.8 ± 5.1 vs. 10.6 ± 3.9; P <0.001). Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data When 1-CrCl was compared with IRC, a bias of 0.8% (SD 26%, limits of agreement –52 to 50%; Pearson’s r = 0.90) was observed in the evolving AKI group, whereas bias was –3.3% (SD 16, limits of agreement –35 to 29%; Pearson’s r = 0.95) in the postnephrectomy group. When 4-CrCl was compared with IRC, bias was 5.1% (SD 54, limits of agreement –102 to 112%, Pearson’s r = 0.45) in the established AKI group and bias was –4.5% (SD 38, limits of agreement –79 to 70%; Pearson’s r = 0.78) in the postnephrectomy group. Introduction Severity of acute kidney injury (AKI) and fl uid overload (FO) are not incorporated into current severity of illness measures and are invisible to the practitioner. The causal relationship and timing between AKI and FO and oxygenation is not clear. The Fluid Overload Kidney Injury Score (FOKIS) is a daily score incorporating subscores for AKI (pRIFLE (creatinine (Cr) and urine output (UOP))), FO (total fl uid (in – out) / ICU admission weight) >15% in fi ve percentile increments, and exposure to nephrotoxic medications. We previously reported that FOKIS outperforms PRISM in mortality prediction in our pediatric intensive care unit (PICU). We hypothesized that patients with AKI on admission to the PICU developed worse fl uid overload and in turn worse oxygenation. y g Conclusion Our data suggest that 4-CrCl is not as accurate and precise as 1-CrCl in patients with AKI and following nephrectomy. IRC appears to be more accurate and precise in patients with a predicted AKI risk and outcome (post nephrectomy) than in patients with evolving AKI. We hypothesise that IRC will be useful alternative to creatinine-based measures of AKI. Methods We prospectively calculated daily FOKIS scores and subscores (Cr, UOP, FO) in PICU patients. We excluded patients with <7 day stays in order to properly explore the association between timing of AKI and FO and oxygenation by oxygenation index (OI). y y y Results Over 18 months, there were 2,830 patients, 436 patients with PICU stay >7  days, 361 patients had complete data for all 7  days. Mortality was 4.5% overall and 11% cohort. A total of 246 patients (68%) had AKI (by FOKISCr or FOKISuop); 205 patients (57%) on day 1, 85 patients (24%) on day 3. Admission or day 3 AKI by either FOKIS subscore (FOKISCr or FOKISuop) did not predict maxFO or mortality. P295 P295 Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs M Ostermann1, L Forni2, K Kashani3, M Joannidis4, A Shaw5, M Chawla6, JA Kellum7, on Behalf of Sapphire Investigators7 1Guy’s & St Thomas Foundation Hospital, London, UK; 2Royal Surrey County Hospital, Guilford, UK; 3Mayo Clinic, Rochester, MN, USA; 4University Hospital Innsbruck, Austria; 5Vanderbilt University Hospital, Nashville, TN, USA; 6George Washington University Medical Center, Washington, DC, USA; 7University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P295 (doi: 10.1186/cc14375) FOKIS f Methods Retrospective data from all nondialysed patients undergoing isolated CABG at our institution were collected for the year 2013. Propensity scoring using the software platform MatchIt® was used to match subjects from ONCAB and OPCAB groups with regard to preoperative variables: logistic EuroSCORE, creatinine clearance (CrCl), gender and operative urgency. Postoperative AKI was defi ned as a rise of 50% or more in baseline serum creatinine [2]. Chi-square analysis was used to determine statistically signifi cant diff erences. Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. yif Results From 500 cases (369 OPCAB, 131 ONCAB), 262 subjects were included in the fi nal analysis, with 131 in each group. There was a higher incidence of AKI and renal replacement therapy (RRT) in the ONCAB group, although this was not signifi cantly greater than in the OPCAB group (Table 1). The mortality rate was identical with three deaths in each group. The average length of ICU stay for the OPCAB group was 1.96 days versus 2.49 days for the ONCAB group. 2. Acute Kidney Injury Network criterion. http://www.akinet.org. 1. Lamy A, et al. N Engl J Med. 2012;366:1489-97. Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data Increasing total FOKIS score was associated with increasing mortality and increasing OI (Table  1). FOKIS, controlled for PRISM, was an independent predictor of OI (P = 0.03). Does cardiopulmonary bypass increase the risk of postoperative acute kidney injury after coronary artery bypass grafting? A Karmali, C Walker, L Kuppurao Harefi eld Hospital, London, UK Critical Care 2015, 19(Suppl 1):P293 (doi: 10.1186/cc14373) Does cardiopulmonary bypass increase the risk of postoperative acute kidney injury after coronary artery bypass grafting? A Karmali, C Walker, L Kuppurao Harefi eld Hospital, London, UK Critical Care 2015, 19(Suppl 1):P293 (doi: 10.1186/cc14373) Introduction Acute kidney injury (AKI) following coronary artery bypass grafting (CABG) results in signifi cant morbidity, mortality and prolonged stay on the cardiothoracic ICU. We sought to determine the incidence of AKI in our cardiothoracic centre, hypothesizing a higher occurrence in patients undergoing CABG using cardiopulmonary bypass (ONCAB) compared with off -pump (OPCAB) surgery [1]. Table 1 (abstract P294) P FOKIS 0 <4 4 to 7 >7 value maxOI, median (IQR) 7.4 11.1 16.4 14.2 0.03 (5.9 to 16.4) (6.2 to 23.6) (7.3 to 29.6) (10 to 38.7) Mortality, % 3.6 7.7 13 38 <0.001 Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. Table 1 (abstract P294) P FOKIS 0 <4 4 to 7 >7 value maxOI, median (IQR) 7.4 11.1 16.4 14.2 0.03 (5.9 to 16.4) (6.2 to 23.6) (7.3 to 29.6) (10 to 38.7) Mortality, % 3.6 7.7 13 38 <0.001 Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. Table 1 (abstract P294) Table 1 (abstract P294) Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK g Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Introduction This study investigated renal outcomes following radio- contrast (RC) administration in patients from two intensive care units (ITUs), where one gave RC-induced nephropathy (RCIN) prophylaxis, while the other did not. Acute kidney injury (AKI) during critical illness increases morbidity and mortality. ITU patients, who already suff er a variety of renal insults, often require RC, increasing their risk of developing AKI, and requiring renal replacement therapy (RRT). Evidence suggests that hydration alone is inadequate for the prevention of RCIN in ITU patients, and is contraindicated in some disease states [1]. The European Society of Intensive Care Medicine (ESICM) provides recommendations for prophylaxis [2]. The current study aimed to establish the effi cacy of the ESICM recommended prophylaxis. p p y Conclusion Both SOFA score and BD may be used to predict AKI in surgical oncology patients at ICU admission. These variables allow physicians to recognize early patients who might be under risk, and anticipate measures to avoid further renal impairment. References 1. Rocktaeschel J, Morimatsu H, Uchino S, Goldsmith D, Poustie S, Story D, et al. Acid–base status of critically ill patients with acute renal failure: analysis based on Stewart–Figge methodology. Crit Care. 2003;7:R60. 1. Rocktaeschel J, Morimatsu H, Uchino S, Goldsmith D, Poustie S, Story D, et al. Acid–base status of critically ill patients with acute renal failure: analysis based on Stewart–Figge methodology. Crit Care. 2003;7:R60. gg gy 2. Smith I, Kumar P, Molloy S, Rhodes A, Newman PJ, Grounds RM, et al. Base excess and lactate as prognostic indicators for patients admitted to intensive care. Intensive Care Med. 2001;27:74-83. fi Methods Retrospective data from 140 Maidstone (M) ITU patients (men 101, women 39, mean age 63.5, mean APACHE 15.3) and 73 Tunbridge Wells (T) ITU patients (men 41, women 32, mean age 60.2, mean APACHE 20.2) admitted between 22 September 2011 and 22 September 2013, who underwent RC-enhanced CT, were collected. Patients on MITU received ESICM-recommended RCIN prophylaxis: 200 mg aminophylline i.v. over 30 minutes prior to RC, 1.26% sodium bicarbonate 3 ml/kg/hour for 1 hour prior to RC and 1 ml/kg/hour for 6  hours post RC. Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs 1 2 h 3 d 4 h 5 h l 6 Table 1 (abstract P293) OPCAB ONCAB P value AKI 14 (10.69%) 19 (14.50%) 0.35 RRT 6 (4.58%) 9 (6.87%) 0.43 Table 1 (abstract P293) g Critical Care 2015, 19(Suppl 1):P295 (doi: 10.1186/cc14375) Conclusion Our study is limited by its size and the use of logistic EuroSCORE as a composite measure of risk factors. However, it has demonstrated that in our predominantly off -pump cardiac unit, OPCAB conferred no statistically signifi cant advantage over ONCAB with regard to postoperative AKI. Studies to date have standardised patient-specifi c variables, but not the conduct of the procedure itself; for example, minimising renal hypoperfusion during OPCAB. This may be a reason for the lack of clear superiority in the continuing debate over choice of CABG procedure and reducing the risk of AKI. References Introduction Tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) are specifi c urinary biomarkers which can predict acute kidney injury (AKI) in critically ill patients within 12 hours [1]. A [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 is associated with seven times the risk of AKI compared with a test result ≤0.3 [2]. The aim of our study was to explore the use of potentially nephrotoxic medications within the window between a positive biomarker test and the diagnosis of AKI stage 2 or 3.i Introduction Tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) are specifi c urinary biomarkers which can predict acute kidney injury (AKI) in critically ill patients within 12 hours [1]. A [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 is associated with seven times the risk of AKI compared with a test result ≤0.3 [2]. The aim of our study was to explore the use of potentially nephrotoxic medications within the window between a positive biomarker test and the diagnosis of AKI stage 2 or 3. Methods We identifi ed all patients enrolled into the Sapphire study [1] who received at least one potentially nephrotoxic drug on the day of AKI (defi ned by stage 2 or 3 as per KDIGO classifi cation). We subsequently Methods We identifi ed all patients enrolled into the Sapphire study [1] who received at least one potentially nephrotoxic drug on the day of AKI (defi ned by stage 2 or 3 as per KDIGO classifi cation). We subsequently 2. Acute Kidney Injury Network criterion. http://www.akinet.org. pp References 1. Kashani K, et al. Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury. Crit Care. 2013;17:R25. 2. Bihorac A, et al. Validation of cell-cycle arrest biomarkers for acute kidney injury: using clinical adjudication. Am J Resp Crit Care Med. 2014;189:932-9. 2. Bihorac A, et al. Validation of cell-cycle arrest biomarkers for acute kidney injury: using clinical adjudication. Am J Resp Crit Care Med. 2014;189:932-9. Results There were 76 (26.7%) patients who developed AKI within the fi rst 48 hours after ICU admission. In a univariate analysis, patients with AKI were more likely to be male, had higher Sequential Organ Failure Assessment (SOFA) score, higher baseline serum creatinine and urea levels, higher serum lactate levels and had more metabolic academia at admission. These patients also had a higher 24-hour Simplifi ed Acute Physiology III score and higher length of mechanical ventilation as compared with non-AKI patients. There were no diff erences between patients regarding intraoperative vasopressors, type and amount of fl uids, diuresis and blood transfusion. In a multivariate analysis we identifi ed admission base defi cit (BD) (OR = 1.13, 95% CI = 1.02 to 1.24, P = 0.017) and SOFA score (OR = 1.35, 95% CI = 1.2 to 1.51, P <0.001) as independent predictive factors of early AKI. 2. Joannidis M, et al. Intensive Care Med. 2010;36:392-411. 1. Huber W, et al. Radiology. 2006;239:793-804. Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs 1 2 h 3 d 4 h 5 h l 6 S105 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 determined the proportion of patients who had a [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 before meeting the criteria for AKI. determined the proportion of patients who had a [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 before meeting the criteria for AKI. Predictors of renal recovery in critically ill patients with AKI: observations from the ongoing FBI clinical trial f S Robinson, U Larsen, A Zincuk, S Zwisler, P Toft , , , , Odense University Hospital, Odense, Denmark , , , , Odense University Hospital, Odense, Denmark y p Critical Care 2015, 19(Suppl 1):P298 (doi: 10.1186/cc14378) y Critical Care 2015, 19(Suppl 1):P298 (doi: 10.1186/cc14378) Introduction The predictive value of NGAL for renal recovery is not established. Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients Results Of 184 patients who developed AKI, 58% received one or more potentially nephrotoxic drug on the day of AKI. Eighty-nine percent of these patients had a positive biomarker test ≥12 hours earlier. In 41% of patients receiving one or more nephrotoxic drug on the day of AKI, at least one nephrotoxic medication was stopped within 1 day of AKI, and in 24% of patients all nephrotoxic drugs were stopped within 1 day of AKI, which implies that these medications were not absolutely necessary. Introduction Patients who undergo major oncology surgery are under high risk to develop postoperative acute kidney injury (AKI), mainly due to infl ammatory and ischemic insults. This complication results in worse outcomes. The aim of this study is to identify predictive factors of AKI in this population. Conclusion Nephrotoxic medications are commonly used in patients who develop moderate or severe AKI. The [TIMP-2]·[IGFBP7] test could have identifi ed many of these patients earlier and would have off ered an opportunity to reduce exposure to non-essential nephrotoxic drugs. References Methods We performed an observational study in 285 consecutive patients admitted to a surgical ICU after major abdominal oncology surgery. Baseline characteristics, laboratorial, clinical and intraoperative data, such as type of fl uids, blood transfusion, bleeding and use of vasopressor, were collected at ICU admission. Early acute kidney injury was defi ned according to the Acute Kidney Injury Network classifi cation at 48 hours of ICU admission. Logistic regression model was performed using AKI as the outcome. P297 Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients A Gerent, J Almeida, E Almeida, A Lousada, C Park, J Ribeiro, J Fukushima, A Leme, E Osawa, A Rezende, I Bispo, F Galas, L Hajjar Instituto do Cancer do Estado de São Paulo – ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P297 (doi: 10.1186/cc14377) Introduction The predictive value of NGAL for renal recovery is not established. Methods Data from the fi rst 19 patients were assessed during a multicentre low molecular weight heparin trial (FBI, EudraCT Number: 2012-004368-23). Critically ill patients with AKI are randomly assigned into either a treatment arm (1  mg/kg enoxaparin) or a control arm (40  mg enoxaparin) upon commencement of CRRT. The primary outcome is the occurrence of venous thromboembolism. NGAL was measured at baseline and during CRRT-free intervals. Results The total number of patients developing renal injury falling into any RIFLE category for MITU at 24, 28 and 72 hours was eight (0.06%), 12 (0.09%) and 14 (0.1%), while for TITU it was fi ve (0.07%), six (0.08%), and four (0.05%) respectively. A repeated-measures ANOVA revealed no signifi cant diff erences in outcomes between the two groups overall (F = 2.35, P = 0.127) or at each time point (F = 1.93, P = 0.123). Results Patients were comparable at baseline with respects to demographics, APACHE II, creatinine, NGAL, start of dialysis, and the duration of dialysis. The main cause of AKI was sepsis (42%). In 63% of the patients, the reason for starting dialysis was a combination of anuria and electrolyte disturbances. Twenty-six percent of patients were dialysis dependent after the fi rst CRRT-free interval. Plasma NGAL levels were higher in nonrenal recovery patients (1,074 (±694) ng/ml) compared with renal recovery patients (296 (±197) ng/ml; P = 0.01). Urine NGAL levels were higher in nonrenal recovery patients (3,885 Conclusion While RCIN is a recognised problem within the critical care population, there is little clear evidence for any prophylactic strategy to reduce this risk. This study suggests that a RCIN prophylaxis protocol based on the ESICM recommendations has no eff ect on the incidence of RCIN. However, further studies are needed. Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) TITU patients received standard critical care alone. Exclusion criteria were: those undergoing RRT prior to CT, >1 CT in 48 hours, no creatinine (Cr) data available post scan. The Cr prior to CT (baseline), at 24, 48 and 72 hours post CT scan were identifi ed. The RIFLE criteria was used to classify the changes of Cr from baseline into low risk (% change >1.25), risk (% change >1.5), injury (% change >2) or failure (% change >3, or Cr>355 and increase of >44). P299 Comparison of two strategies for initiating renal replacement therapy in the ICU: study protocol plan for a multicenter, randomized, controlled trial from the AKIKI research group S Gaudry1, D Hajage2, F Schortgen3, L Martin-Lefevre4, J Ricard1, D Dreyfuss1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Bichat, Paris, France; 3Hôpital Henri Mondor, Créteil, France; 4CHD La Roche sur Yon, France Critical Care 2015, 19(Suppl 1):P299 (doi: 10.1186/cc14379) Results The total baseline plasma concentration of all standard amino acids was similar between IHD versus SLEDf groups (1,812  ± 517 vs. 2,675 ± 527 μmol/l, respectively) but were higher in the CVVH group (3,194  ± 564  μmol/l). RRT reduced the plasma concentration of amino acids in the SLEDf group (to 1,732 ± 529 μmol/l; P = 0.02), but had no eff ect in the IHD or CVVH groups (IHD; 1,853 ± 523, CVVH; 2,845 ± 512 μmol/l). The average, unadjusted loss of amino acids was signifi cantly infl uenced by mode of RRT (IHD, 5.13  ± 3.1 vs. SLEDf, 8.21 ± 4.07 vs. CVVH, 18.69 ± 3.04 g; P <0.01). The total baseline plasma concentration of trace elements was similar in the IHD, SLEDf and CVVH groups (3,797 ± 827, 3,667 ± 791, 3,642 ± 481 μg/l, respectively). By the end of the RRT session, the plasma concentration of trace elements had reduced (IHD, to 3,103 ± 827; SLEDf, to 2,805 ± 797; CVVH, to 3,433 ± 481 μg/l; P = 0.01). By the end of each RRT session, total losses of trace elements were estimated at IHD, 5,051 ± 2,312; SLEDf, 8,751 ± 2,421; CVVH, 11,258 ± 2,547 μg/l; P = 0.02 for treatment. Introduction There is currently no validated strategy for the timing of renal replacement therapy (RRT) for acute kidney injury (AKI) in the ICU when short-term life-threatening metabolic abnormalities are absent. No adequately powered prospective randomized study has to date addressed this issue. As a result, signifi cant practice heterogeneity exists and may expose patients either to unnecessary hazardous procedures or to undue delay in RRT. Methods This is a multicenter, prospective, randomized, open- label parallel-group clinical trial that compares the eff ect of two RRT initiation strategies on overall survival of critically ill patients receiving intravenous catecholamines and/or invasive mechanical ventilation and presenting with RIFLE F stage of AKI. In the early strategy, RRT is initiated immediately. References S106 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (±2,722) ng/ml) compared with renal recovery patients (597 (±565) ng/ml; P = 0.006). See Figures 1 and 2. Infl ammatory parameters (WBCs, CRP, and procalcitonin) did not diff er signifi cantly between the groups. Conclusion NGAL may be able to predict renal recovery, and allow proper utilization of resources. Figure 2 (abstract P298). Urine neutrophil gelatinase-associated lipocalin concentration by recovery. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. more than 72  hours after randomization, serum urea concentration >40  mmol/l, serum potassium concentration >6  mmol/l, serum potassium concentration >5.5  mmol/l persisting despite medical treatment, arterial blood pH <7.15 in a context of pure metabolic acidosis (PaCO2 <35 mmHg) or in a context of mixed acidosis with a PaCO2 >50 mmHg without possibility of increasing alveolar ventilation, acute pulmonary edema due to fl uid overload despite diuretic therapy leading to severe hypoxemia requiring oxygen fl ow rate >5 l/minute to maintain SpO2 >95% or FiO2 >50% under invasive or non-invasive mechanical ventilation. The primary endpoint is overall survival, measured from randomization (D0) until death, regardless of the cause. The minimum follow-up duration for each patient will be 60 days. To demonstrate a 14% decrease in mortality, a total of 546 subjects (273 per group) should be randomized.i p g p Results Enrollment is ongoing. After the fi rst interim analysis, the DSMB recommended to continue the study. On 5 December 2014, 318 patients were included in the trial. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. Conclusion The AKIKI study will be one of the very few large randomized controlled trials evaluating mortality according to the timing of RRT in critically ill patients with RIFLE F stage of AKI. Results should help clinicians better decide when to initiate RRT. Figure 2 (abstract P298). Urine neutrophil gelatinase-associated lipocalin concentration by recovery. P300 Micronutrient loss in renal replacement therapy for acute kidney injury W Oh1, M Devonald1, D Gardner2, R Mahajan3, D Harvey3, A Sharman3, B Mafrici1, M Rigby1, S Welham2 1Nottingham University Hospitals NHS Trust, Nottingham, UK; 2University of Nottingham, Sutton Bonington, UK; 3University of Nottingham, UK Critical Care 2015, 19(Suppl 1):P300 (doi: 10.1186/cc14380) Micronutrient loss in renal replacement therapy for acute kidney injury j y W Oh1, M Devonald1, D Gardner2, R Mahajan3, D Harvey3, A Sharman3, B Mafrici1, M Rigby1, S Welham2 1Nottingham University Hospitals NHS Trust, Nottingham, UK; 2University of Nottingham, Sutton Bonington, UK; 3University of Nottingham, UK Critical Care 2015, 19(Suppl 1):P300 (doi: 10.1186/cc14380) Introduction The prevalence of malnutrition in acute kidney injury (AKI) is high. Patients with AKI may require renal replacement therapy (RRT), which could result in loss of water-soluble micronutrients. Little is known about these losses in RRT and whether they diff er between types of RRT. This study aims to quantify micronutrient losses during RRT in patients with AKI and to compare them in three diff erent RRT modalities: continuous venovenous haemofi ltration (CVVH), intermittent haemodialysis (IHD) and sustained low-effi ciency diafi ltration (SLEDf). (±2,722) ng/ml) compared with renal recovery patients (597 (±565) ng/ml; P = 0.006). See Figures 1 and 2. Infl ammatory parameters (WBCs, CRP, and procalcitonin) did not diff er signifi cantly between the groups. Conclusion NGAL may be able to predict renal recovery, and allow proper utilization of resources. Methods A prospective observational study is being conducted at NUH. Thirty-three adult patients with AKI requiring RRT (13 IHD, 10 SLEDf, 10 CVVH) have been recruited. Samples of blood and RRT effl uent were obtained at baseline, mid and end-session from each participant during their fi rst RRT treatment. Samples were processed and stored at –80°C for subsequent analysis of amino acids by high- performance liquid chromatography and trace elements by inductively coupled mass spectrometry after derivatization from physiological fl uids. Micronutrient losses were calculated by multiplying mass- corrected concentrations by total volume of RRT effl uent, adjusted for baseline plasma concentrations and RRT dose. Data were analysed by restricted maximum likelihood estimating equations. P299 In the delayed strategy, clinical and metabolic conditions are closely monitored and RRT is initiated only when one or more events (severity criteria) occur, including: oliguria or anuria for Conclusion Micronutrients are lost during RRT in AKI. The degree of micronutrient loss is infl uenced by the type of RRT used. S107 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P301 Methods A comparison of patients receiving CVVHDF on the 11-bed critical care unit at Conquest Hospital, Hastings was undertaken, before and after the implementation of new CVVHDF protocols. All patients receiving CVVHDF were identifi ed from the electronic patient record system between March 2012 to 2013 and September 2013 to 2014. Patient demographics, the duration of CVVHDF and sodium bicarbonate supplementation were analysed between the groups to assess the impact of the new protocols. P301 Super high-fl ux CVVHD using regional citrate anticoagulation: long-term stability of middle molecule clearance M Siebeck, D Kindgen-Milles University Hospital, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P301 (doi: 10.1186/cc14381) Introduction Conventional membranes used for CRRT have a limited middle molecule clearance. New membranes called super high-fl ux (SHF) or high cutoff membranes have been investigated. The loss of albumin with hemofi ltration is a major drawback, but these membranes can be used in CVVHD with regional citrate anticoagulation (Ci-Ca® CVVHD), which may limit albumin loss, and contribute to a prolonged fi lter patency and an improved and stable middle molecule clearance. We evaluated saturation coeffi cients (SC), plasma clearances (PCL) and serum levels of eight small and middle molecules during 72 hours of Ci-Ca® CVVHD with a SHF membrane (Ultrafl ux®EMiC®2). p p Results Sixty-four patients received CVVHDF in 2012 to 2013, 61 receiving citrate and three receiving unfractionated heparin due to fulminant liver failure. Forty-seven patients received CVVHDF in 2013 to 2014, two receiving no anticoagulation due to severe coagulopathy and one receiving unfractionated heparin. The two patient cohorts assessed were similar in age (median 65.5 for March 2012 to 2013 cohort vs. 66 for September 2013 to 2014 cohort), gender mix (64% male vs. 57% male) and severity of illness as assessed by APACHE II score (23 vs. 24). Mean duration of CVVHDF was also similar (71.5 hours vs. 75 hours). P299 A total 30/64 of 2012 to 2013 patients did not require a fi lter change prior to completion of RRT, compared with 23/47 of 2013 to 2014 patients. Sodium bicarbonate was added to the dialysate fl uid in 29/64 2012 to 2013 patients, compared with just 2/47 2013 to 2014 patients.i l Methods After approval of the local committee of medical ethics and written informed consent we enrolled patients on a surgical ICU with AKI RIFLE-F who were treated with a Ci-Ca® CVVHD with a SHF membrane for 72  hours. We measured urea (0.006  kDa), creatinine (0.113  kDa), osteocalcin (5.8  kDa), B2MG (12  kDa), myoglobin (17.2 kDa), FreeLightChains (FLC) kappa (25 kDa) and lambda (50 kDa) and albumin (66 kDa) at 0 hours, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours. PCL, SC and serum levels during 72 hours were compared, using the Wilcoxon signed-rank test with P <0.05. Conclusion Changing protocols resulted in a signifi cant reduction in off -license addition of sodium bicarbonate to dialysate bags without impacting on fi lter life, thus reducing nursing workload and removing a potential source of adverse events in this high-risk group of patients. Results Four females and 10 males (mean age 68.1 ±15.1 years; mean APACHE II score 13.7 ± 14.7; mean SAPS II 38.7 ± 12.7) were included. The SC and the PCL (ml/minute) of small solutes like creatinine at 1 hour (1.0 ± 0.0/23.72 ± 1.04) and 72 hours (0.95 ± 0.16/22.19 ± 3.99) were not statistically signifi cantly diff erent (P = 0.5/P = 0.42), the PCL was slightly reduced by 6%. The creatinine serum level was reduced by 42%. The SC and PCL of B2MG from 1 hour (0.61 ± 0.09/14.49 ± 2.5) to 72 hours (0.48 ± 0.13/11.6 ± 2.96) were signifi cantly decreased (P = 0.0024/P = 0.0061). The reduction was 23% only; the overall clearance still was high. There was almost no reduction in SC or PCL for FLC kappa from 1 hour (0.176 ± 0.047/4.14 ± 1.08) to 72 hours (0.164 ± 0.078/3.854 ± 1.87), not reaching statistical signifi cance (P = 0.94/P = 0.81). The serum levels of B2MG and FLC kappa were decreased by 39% and 23%. The SC of albumin was low (1 hour: 0.0009 ± 0.0004) and clearance decreased rapidly within the fi rst 6  hours from 0.021  ± 0.01 to 0.011  ± 0.009. Descriptive study of the haematological management of adult patients with severe respiratory failure receiving venovenous extracorporeal membrane oxygenation p yg O Tavabie, R Pocock, N Barrett, A Retter Introduction Venovenous extracorporeal membrane oxygenation (VV-ECMO) is a novel therapy for severe respiratory failure (SRF). Its introduction has reduced mortality; however, patients require substantial blood product support and between 10 and 20% of cases develop a life-threatening haemorrhage. p g g Methods We contacted 336 practitioners at 135 centres, examining their haematological management. Conclusion This study shows high middle molecular clearances using a SHF membrane with Ci-Ca® CVVHD for 72 hours with no loss of albumin. This set-up may improve blood purifi cation in critically ill patients with acute kidney injury. Results In total 25% of practitioners contacted responded; 85% were attending physicians, predominantly based in North America and Europe, 41 and 32% respectively. Ninety-six per cent of units used a polymethylpentene membrane oxygenator and all used a centrifugal pump. Thirty-four per cent of responders managed <10 cases a year and 60% worked in units handling <20 annually, 6% saw >50 patients. One centre did not use unfractionated heparin. Monitoring of anticoagulation varied; 52% used the APTT, 43% the ACT and 5% the APTTr. Sixty per cent did not routinely measure antithrombin. Results In total 25% of practitioners contacted responded; 85% were attending physicians, predominantly based in North America and Europe, 41 and 32% respectively. Ninety-six per cent of units used a polymethylpentene membrane oxygenator and all used a centrifugal pump. Thirty-four per cent of responders managed <10 cases a year and 60% worked in units handling <20 annually, 6% saw >50 patients. One centre did not use unfractionated heparin. Monitoring of anticoagulation varied; 52% used the APTT, 43% the ACT and 5% the APTTr. Sixty per cent did not routinely measure antithrombin. Scenario 1 was based on a patient with H1N1. Practitioners targeted a haemoglobin (Hb) of 80 to 100 g/l; however, 20% targeted a Hb outside this range; 38% favouring a transfusion threshold of <80  g/l when the patient was improving compared with 32% when the patient had just started on ECMO. Seventeen per cent of practitioners transfused platelets when the count was <30 × 109/l whilst 21% maintained the platelet count >100 × 109/l. Scenario 2 described a patient with SRF secondary to a hospital-acquired pneumonia. The patient developed a haemothorax, with persistent blood loss of 200 ml/hour. Descriptive study of the haematological management of adult patients with severe respiratory failure receiving venovenous extracorporeal membrane oxygenation Practitioners targeted a higher haemoglobin concentration of 100 g/l and targeted a higher platelet count of >100 × 109/l when compared with the patient in scenario 1, neither of these diff erences was statistically signifi cant. Seventy-one per cent stated they would manage the patient off anticoagulation. There was no agreement as to the length of time off anticoagulation; 26% restarted anticoagulation in <12  hours, compared with 22% who advised no anticoagulation for >5  days. Scenario 3 examined the management of an incidental intracranial haemorrhage. There was a lack of consensus regarding the duration off anticoagulation; 14% of responders held anticoagulation for less than P299 Serum levels of albumin did not decrease (1 hour: 2.64 ± 0.51; 72 hours: 2.63 ± 0.25). P302 P302 Citrate anticoagulation for continuous venovenous haemodiafi ltration: the impact of a novel protocol on patients receiving therapy in one regional hospital J Highgate1, G Escott2, A Lowe2, F Stedman2, N McNeillis2 1Worthing Hospital, Worthing, UK; 2Conquest Hospital, Hastings, UK Critical Care 2015, 19(Suppl 1):P302 (doi: 10.1186/cc14382) Introduction Citrate has been used to anticoagulate extracorporeal haemofi ltration circuits since the 1960s, and has been used as the fi rst-line anticoagulant for continuous venovenous haemodiafi ltration (CVVHDF) at Conquest Hospital since 2009. Benefi ts of citrate demonstrated in clinical trials include increased fi lter life and increased bicarbonate formation from metabolism of the citrate complex; citrate also lacks the increased bleeding risk associated with unfractionated heparin use. One of the main issues with new renal replacement therapies is the development of ideal dialysate fl uids. During the initial period of citrate use at Conquest, hyponatraemia was identifi ed as an issue, with off -license supplementation of dialysate fl uid with sodium bicarbonate often necessary to prevent this. New protocols were therefore developed, designed to maximise the fi ltration dose and maintain normal electrolyte balance. S108 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Data were collected from our computerised information system on all AKI patients receiving RRT on our ICU, between October 2008 and October 2013. This included demographics, APACHE II and SOFA scores, modality and dose of RRT and ICU length of stay (LOS). Renal and patient survival at ICU discharge was collected, in addition to outcome data at 28 and 90 days and 12 months. Data were examined 12 hours whilst 37% held anticoagulation for >5 days and tranexamic was considered useful by 25%. y Conclusion There was wide variation in the use of blood products and the intensity of anticoagulation. This is not surprising given the current lack of evidence. Further work is required to provide a standardised approach. using Cox proportional hazard multivariate analysis, with Stata 10.1. Results A total of 620 patients with AKI received RRT on our ICU between October 2008 and October 2013. Sixty-one per cent were males. Median age was 65 years (IQR 54 to 74). Median APACHE II score was 23 (IQR 18 to 27). Median SOFA score was 11 (IQR 8 to 13). Fifty-fi ve per cent were mechanically ventilated. Comparison between nafamostat mesilate and unfractionated heparin as anticoagulant during continuous renal replacement therapy Comparison between nafamostat mesilate and unfractionated heparin as anticoagulant during continuous renal replacement therapy S Makino, H Kita, Y Miyatake, T Yokoyama, K Kubota, N Obata, M Egi, T Misumi, S Izuta, S Mizobuchi Kobe University Hospital, Kobe, Japan Critical Care 2015, 19(Suppl 1):P304 (doi: 10.1186/cc14384) Introduction For continuous renal replacement therapy (CRRT), continuous administration of anticoagulant would be necessary to prevent the circuit clotting. Nafamostat mesilate (NM) is commonly used as its anticoagulant in Japan, although unfractionated heparin (UFH) is the most frequently used anticoagulant internationally. There is little study to compare the risk and benefi t of NM with UFH as an anticoagulant during CRRT. Methods We conducted a single-center retrospective observational study to compare NM with UFH as anticoagulant during CRRT. We screened subsequent critically ill patients requiring CRRT in our ICU from January 2011 to December 2013. We excluded patients who required any other extracorporeal circuit including extracorporeal membrane oxygenation, who used both NM and UFH simultaneously, or who were administered any other anticoagulant including gabexate mesilate or urokinase. The primary outcome of this analysis was fi lter life, and the secondary outcome was the incidence of bleeding complications during CRRT. As an initial dose, NM and UFH were given pre fi lter at 15 to 25 mg/hour and 1,500 to 3,000 IU/hour, respectively. The dose of both drugs was adjusted to maintain activated clotting time at post fi lter between 150 and 200 seconds. Filter life was assessed using the Kaplan–Meier method and the incident of bleeding complications was compared using the chi-square test. P <0.05 was considered to be statically signifi cant. Conclusion Results from our cohort suggest that, in patients with AKI presenting to ICU for RRT, long-term patient survival is signifi cantly impaired. Renal outcomes are poor with 35% being either dialysis dependent or having severe chronic kidney disease (eGFR <15  ml/ minute), at 1 year from ICU discharge. Our data do not suggest a benefi t of using HVHF in AKI patients presenting to ICU for RRT. P306 Plasma antioxidant capacity in critical traumatized patients: severity and anatomical location y G Papakitsos1, A Kapsali1, T Papakitsou2, A Roimba3 1GHA, Arta, Greece; 2GHM, Messologi, Greece; 3General Practitioner, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P306 (doi: 10.1186/cc14386) i Results We included 101 patients in this study. Among them, 76 patients were with NM and 25 patients were with UFH. They used 239 fi lters in total; 173 with NM, 66 with UFH. There were signifi cantly more post- surgical patients in the NM group (P = 0.001). There was no diff erence in age, APACHE II score, days from ICU admission to commencement of CRRT, length of ICU stay and mortality between two groups. There was no diff erence in median number of fi lters used by one patient (NM vs. heparin; median of 1.5 (IQR) vs. 2 (IQR), P = 0.27). Filter life in the UFH group was signifi cantly longer than those in the NM group (NM vs. UFH; median of 24 hours vs. 36 hours; P = 0.01). The incidence of bleeding complications was not signifi cantly diff erent between two groups (P = 0.15). Introduction Oxidative stress (OS) has been invoked as a relevant factor in the evolution and outcome of critical care patients. Indeed, antioxidant therapies have been used in critical care patients, but with controversial results. This may be explained by assuming OS as a homeostatically regulated parameter and both its excess and its defi cit infl uencing severity progression. Nonetheless, antioxidant agents could mask an OS signaling role, blocking otherwise physiological responses aimed at recovery of homeostasis. We have evaluated plasma total antioxidant capacity (TAC) in traumatized patients in the emergency department (ED) and we determined its potential relationship with severity and trauma location. Conclusion In our retrospective analysis with 101 patients, fi lter life with UFH was signifi cantly longer than those with NM. The incidence of bleeding complications was not signifi cantly diff ered between patients with NM and UFH. y Methods In a prospective observational study of ED polytraumatized patients (n = 23, mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score of 11 ± 6) we measured (in the fi rst 24 hours) plasma TAC by the ferric reducing activity/antioxidant power (FRAP). For control subjects, we used age-matched and gender-matched volunteers (n = 32). We also evaluated the contribution of antioxidant molecules (uric acid, bilirubin, and albumin) to these values. P302 A total of 96.7% received CVVH as the principal RRT modality. Twenty-one per cent received a period of high-volume haemofi ltration (HVHF) (80 ml/kg/hour), median LOS was 6 days (IQR 3 to 14). In total, 331 (53.4%) patients recovered their renal function at ICU discharge, whilst 237 (38.2%), 220 (35.4%), and 220 (35.4%) patients did not at 28 and 90 days and 12 months respectively. A total of 414 (66.7%) patients survived to ICU discharge, with 368 (59.3%), 341 (55%) and 308 (49.6%) patients being alive at 28 and 90 days and 12 months respectively. Overall patient survival at the end of follow-up was 43%. Adjusting for age and sex; APACHE II score, SOFA score and use of HVHF were associated with worse patient survival at ICU discharge (HR: 1.07, 95% CI: 1.03 to 1.11, P <0.001, HR: 1.11, 95% CI: 1.03 to 1.19, P = 0.006 and HR: 2.27, 95% CI: 1.4 to 3.66, P = 0.001, respectively). Adjusting for age and sex; APACHE II score and use of HVHF were associated with worse renal recovery at ICU discharge (HR: 1.06, 95% CI: 1.03 to 1.09, P <0.001 and HR: 1.55, 95% CI: 1.03 to 2.3, P = 0.032 respectively). SOFA score did not appear to signifi cantly impact renal recovery (HR: 0.99, 95% CI: 0.94 to 1.04, P = 0.81). Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu Suzhou Municipal Hospital, Nanjing Medical University, Suzhou, China Critical Care 2015, 19(Suppl 1):P309 (doi: 10.1186/cc14389) Introduction The aim was to analyze the injury features and treatment strategies of surviving casualties in the explosion accident on 2 August 2014 in Kunshan city (Kunshan ‘August 2’ Explosion Accident). Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. Introduction The aim was to analyze the injury features and treatment strategies of surviving casualties in the explosion accident on 2 August 2014 in Kunshan city (Kunshan ‘August 2’ Explosion Accident). Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. p p p p Results In total, 722 trauma patients were included, of which 300  patients were hypothermic. The mortality in the hypothermia group was signifi cantly higher than in normotherm patients (OR = 3.73, 95% CI = 2.02 to 7.13, P <0.001). A cutoff point of 36°C was observed as the best threshold for hypothermia (sensitivity 74%, specifi city 56%). Besides hypothermia, other predictors found for 28-day mortality were APACHE II score corrected for temperature, minimum thrombocytes in fi rst 24 hours and urea and included in the fi nal model with an AUC of 0.89 (95% CI = 0.85 to 0.92). External validation of the model was associated with a predicted probability of an AUC of 0.64 (95% CI = 0.51 to 0.77).i Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. Results A total of 40  cases were admitted to our hospital within 6.3  ± 0.8  hours after the explosion accident on 2 August 2014 in Kunshan city, including 28 males and 12 females. The major injury types included burn injury, inhalation injury, blast injury (lung, eye, eardrum, and so forth), traumatic brain injury and bone fractures. All cases suff ered from burn injury caused by the explosion. The mean burned area in the surviving casualties accounted for 92  ± 14% total body surface area (TBSA) and those with third-degree burns for 77 ± 19% TBSA. Additionally, incidence rate of inhalation injuries was 97.5%. There were 34 cases complicated by multiple organ dysfunction syndrome, which accounted for 85.0%. Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu The common organ dysfunction of surviving casualties included the lungs, circulation, liver, gastrointestinal tract, kidney, brain, and coagulation. During hospitalization, the most common infectious site in surviving casualties was a burn wound, followed by blood and lung. The most common infectious strain of bacteria was Gram- negative bacteria, which accounted for 91.3%. Further analysis showed that Proteus mirabilis ranked fi rst in occurrence, followed by Acinetobacter baumannii and Pesudomonas aeruginosa, and Klebsiella pneumonia and Enterobacter cloacae ranked fourth and fi fth. After intensive treatment, the mean 28-day mortality was 20.0% and 90- day mortality was 62.5%, mainly due to septic shock and multiple organ dysfunction syndrome. Conclusion Hypothermia, defi ned as <36°C, is associated with an increased 28-day mortality. The discriminative ability of the developed model for predicting mortality in a new patient population is moderate. P309 Methods A retrospective cohort study was performed in adult trauma patients admitted to a level 1 trauma center and who were transferred to the ICU between 2007 and 2012. Diff erent cutoff points for hypothermia were compared to fi nd the best defi nition for hypothermia. Logistic regression analysis was performed to quantify the net eff ect of hypothermia on admission to the ICU on 28-day mortality and to develop a model with predictors. The developed model was externally validated in data from another level 1 trauma center with a comparable patient population. Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu Suzhou Municipal Hospital, Nanjing Medical University, Suzhou, China Critical Care 2015, 19(Suppl 1):P309 (doi: 10.1186/cc14389) P305 Long-term renal and survival outcomes in acute kidney injury patients receiving renal replacement therapy in intensive care I Elsayed1, N Pawley1, J Rosser1, MJ Heap1, GH Mills1, A Tridente2, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P305 (doi: 10.1186/cc14385) Results Polytraumatized patients show diff erences in TAC with reference to control subjects. ED polytraumatized patients show high FRAP values. We found that FRAP values were inversely correlated with APACHE II score (r = –0.266, P <0.01) suggesting that, in trauma patients, increased antioxidant response, as measured by FRAP assay, could be a pathophysiological response to stress. Albumin and uric acid concentrations reproduced the FRAP trend with severity. Introduction Acute kidney injury (AKI) aff ects 40% of critically ill patients, with UK data reporting 5% needing renal replacement therapy (RRT). Hospital mortality is reported as being up to 60%. We sought to evaluate renal and long-term patient survival outcomes in AKI patients receiving RRT on our ICU. Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, S109 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion An increase in StO2 and lower tissue oxygen extraction rates were associated with H-survival in polytrauma patients. Figure 1 (abstract P308). Figure 1 (abstract P308). P  <0.002) and head trauma (β  = –0.219, P  <0.045). These results accentuate the infl uence of trauma location and severity in TAC changes. TAC response in ED patients reinforces the need for an adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. P  <0.002) and head trauma (β  = –0.219, P  <0.045). These results accentuate the infl uence of trauma location and severity in TAC changes. TAC response in ED patients reinforces the need for an adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. P307 Hypothermia as a predictor for mortality in trauma patients K Balvers1, JM Binnekade1, C Boer2, JC Goslings1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU University Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P307 (doi: 10.1186/cc14387) Introduction Previous studies reported hypothermia as an independent predictor for mortality. However, diff erent cutoff points were used in these studies and external validation has never been applied. The aim of this study was to quantify the net eff ect of hypothermia on admission to the ICU on the 28-day mortality and to test the predictors from the developed model in another level 1 trauma center with a comparable patient population to validate the model. Figure 1 (abstract P308). Conclusion An increase in StO2 and lower tissue oxygen extraction rates were associated with H-survival in polytrauma patients. Near-infrared spectroscopy to assess tissue oxygenation in patients with polytrauma: relationship with outcome A Donati, E Damiani, R Domizi, S Pierantozzi, S Calcinaro, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) Università Politecnica delle Marche, Ancona, Italy , , y Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) y Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) Introduction We evaluated tissue oxygenation by means of near- infrared spectroscopy (NIRS) and explored its relationship with outcome in polytrauma patients. Methods A prospective observational study; 37 polytrauma patients underwent NIRS monitoring (thenar eminence) every day during their stay in the ICU. A VOT was performed with a 40% tissue oxygen saturation (StO2) target. Healthy volunteers (n = 27) were studied as controls. y y Conclusion During the Kunshan ‘August 2’ Explosion Accident, burn injury was the leading cause of injuries. Most surviving casualties are accompanied by multiple organ dysfunction syndrome and infection. Results StO2 increased over the fi rst 7 days only in hospital survivors (n = 29), who showed higher values as compared with healthy volunteers at days 5 and 7 (Figure 1). StO2 downslope and upslope tended to be lower in H-nonsurvivors (n = 8) (P <0.05 at days 2 and 4) as compared with H-survivors. Tissue hemoglobin index was lower in H-no survivors over the fi rst 7  days and tended to normalize only in H-survivors (P >0.05 vs. healthy at day 7). Five patients were discharged from the ICU but did not survive until H-discharge. At discharge from the ICU, these patients were similar to H-survivors in SOFA score, heart rate, mean arterial pressure and lactate, but showed lower StO2 downslope (–13 (–16.5, –11.7)%/minute vs. –8.6 (–11.7, –6.5)%/minute, P = 0.01). P310 In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users A Franci1, S Piantini2, M Pierini2, A Peris1, M Mangini1 1A.O.U. Careggi, Firenze, Italy; 2University of Florence, Italy Critical Care 2015, 19(Suppl 1):P311 (doi: 10.1186/cc14391) Introduction The purpose of this study is to analyze anatomic distributions, diagnostic methods, and prognosis of missed fractures in patients with severe injury. Introduction The purpose of this study is to analyze anatomic distributions, diagnostic methods, and prognosis of missed fractures in patients with severe injury. gg y y y Critical Care 2015, 19(Suppl 1):P311 (doi: 10.1186/cc14391) Methods A review of single-institutional medical records between January 2001 and May 2012 identifi ed 58 patients with 62 delayed diagnoses of fractures among 4,643 severely injured patients older than 20 years with Injury Severity Scores higher than 16. We evaluated combined injuries, location of fractures, diagnostic methods, and reasons for missed diagnosis at initial examination. Introduction Road accidents are the leading cause of death for young people, 50% being represented by vulnerable road users (VRU) (pedestrians, cyclists). In-depth accident studies assess the consequences of lack of use of safety devices and the need to develop new ones. Since 2009 a permanent team (physicians and engineers) has performed in-depth studies on road trauma admitted to our ICU [1]. Results Among 62 missed fractures, there were eight cases of spine fracture, 10 cases of peri-shoulder joint fracture, eight cases of upper extremity fracture, 10 cases of pelvis of acetabulum fracture, and 26 cases of lower extremity fracture. Head injury was the most common concomitant injury (23 cases). Initially missed fractures were most commonly discovered by offi cial reading by radiologists. The most common reasons for misdiagnosis were the use of improper radiologic study and missed-reading of proper radiologic studies. Methods The team studied 52 VRU crashes that occurred in an urban area. The clinical data included an injury assessment using total body CT scan, Injury Severity Score (ISS), Abbreviated Injury Score (AIS), ICU and hospital length of stay and outcome score. Engineers collect data onsite with the partnership of the police, and assess the dynamics of the vehicles with the most advanced reconstruction techniques. Medical and engineering data were cross-matched during the correlation process. Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) I Turriziani, A Cecchi, A Giugni, L Copertino Introduction In the literature, duration of mechanical ventilation (DMV) is often considered an important risk factor (RF) [1] for VAP [2] in critical patients; generally the whole duration of MV is taken into S110 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P311). Frequency of lesions. account, including days before and after infection onset. We tried to assess whether, counting only MV days prior to VAP development (MVp), something would change. Methods We considered, in a 10-year period, data prospectively collected in our database (4D solution, V11) on trauma patients admitted to the ICU directly from the emergency department. Inclusion criteria were: age ≥16 years, ICU length of stay (ICUlos) ≥4 days, DMV ≥48 hours; we excluded patients who received antibiotics before VAP (or during the whole stay, for patients without VAP) and with incomplete data. Data were: age, sex, prehospital GCS <9, prehospital intubation (preHTI), admission base excess (BE), Injury Severity Score (ISS), surgery, massive transfusion, feeding, antacids, nursing, DMV, ICUlos and MVp. MVp was calculated as the diff erence between the fi rst day of VAP and the fi rst day of MV in patients who developed VAP (vapY) and whole DMV in patients that did not (vapN). We only considered the fi rst infectious episode. The outcome was VAP onset. Group comparison was made with Fisher’s exact test and Student’s t test. Signifi cant variables were evaluated in a logistic regression (LR) model; the Hosmer–Lemeshow test (HL) was used as the post-estimation test. Odds ratio (OR) and 95% confi dence interval (95% CI) were calculated. Statistical signifi cance for P <0.05. We used Stata/IC 10.1 for analysis. Figure 1 (abstract P311). Frequency of lesions. Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Dynamic N injured Speed (SD) Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Dynamic N injured Speed (SD) Car vs. pedestrian 29 41.1 (13.9) Motorcycle vs. pedestrian 7 40.6 (4.3) Car vs. ferences Richmond J, Egol KA, Koval KJ. Management of orthopaedic injuries in polytrauma patients. Bull Hosp Jt Dis. 2001-2002;60:162-7. Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) cyclist 10 40.7 (15.9) Other 6 49.0 (5.4) y Results A total of 541 patients met the inclusion criteria, 378 (69.9%) developed VAP. MVp does not seem to be a RF for VAP because they are longer in vapN than in vapY (mean MVp 5.5 vs. 4.41, P = 0.001). PreHTI (vapY/N: 49.74%/38.65%; OR: 1.57; 95% CI: 1.08 to 2.28), ISS (mean vapY/N: 28.4/25.55; P = 0.0018), BE (mean vapY/N: –3.76/–3.04; P = 0.03) were signifi cantly diff erent between the two groups. In LR only preHTI (OR: 1.47; 95% CI: 1.01 to 2.15) and ISS (OR: 1.03; 95% CI: 1.01 to 1.05) are RF for VAP (HL: P = 0.133). Conclusion In our study MVp are not a RF for VAP in trauma patients, although the whole DMV is longer in patients with VAP (mean DMV vapY/N: 13.57/6.09; P = 0.0001). Further studies could confi rm whether the whole DMV in trauma patients with VAP is a consequence of infection. Conclusion The head is still the most frequently and severely injured region. The severity of injuries increases in the most rigid part of the car. Improving VRUs’ safety devices (active and passive) to reduce impact speed and severity of the primary impact has to be the next step. Reference References 1. Charles MP, et al. Ventilator-associated pneumonia. Australas Med J. 1. Charles MP, et al. Ventilator-associated pneumonia. Australas Med J. 2014;7:334-44. 1. Charles MP, et al. Ventilator associated pneumonia. Australas Med J. 2014;7:334-44. 1. Piantini et al. BMC Emerg Med. 2013;13:3. 2. CDC ventilator-associated event (January 2014). www.cdc.gov. 2. CDC ventilator-associated event (January 2014). www.cdc.gov. P311 Dankook University Hospital, Cheonan City, South Korea Critical Care 2015, 19(Suppl 1):P312 (doi: 10.1186/cc14392) Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) Introduction Severe trauma aff ects the immune system, which in its turn is associated with poor outcome. The mediators driving the immune responses in trauma are largely unknown. The aim of this study was to investigate the role of endogenous microparticles (MPs) in mediating the immune response following severe trauma. Figure 1 (abstract P314). sRAGE kinetics (days). on days 1, 3 and 5. sRAGE was measured by ELISA and cfDNA was measured by UV absorbance after plasma isolation. g p g Methods A prospective, observational substudy of the Acute Coagulopathy and Infl ammation in Trauma (ACIT) II study was performed at our academic level 1 trauma center. Adult multiple trauma patients with an Injury Severity Score of 15 or higher were included between May 2012 and June 2013. Ex vivo whole blood stimulation with lipopolysaccharide was performed on aseptically collected patient plasma containing MPs and in plasma depleted of MPs. Flow cytometry and transmission electronic microscopy were performed on plasma samples to investigate the numbers and cellular origin of MPs. Healthy individuals served as a control group. Results Median ISS was 39 and mortality was 21% (8/38). During the fi rst 5 days after trauma, the median concentration of sRAGE (Figure 1) decreased signifi cantly over time (P <0.0001) while median levels of DNA did not (P = 0.73), and remained elevated compared with normal control. No correlation was found with ISS. Patients initially in shock had lower levels of sRAGE or cfDNA (P <0.05) and had received more fl uid (10.6 l vs. 5.25 l) or blood (6 l vs. 0.5 l). Day 3 and day 5 sRAGE levels were inversely correlated with PRBC received. Medians of sRAGE on days 0 (1,301 vs. 730 pg/ml) and day 1 (925 vs. 760 pg/ml) were signifi cantly higher in nonsurvivors (P <0.01). Finally, day 0 sRAGE was correlated with the maximal (r = 0.44; P = 0.007) and the cumulative renal failure component of the MODS, over the 10 days (r = 0.48; P = 0.005).f Results Ten trauma patients and 10 healthy individuals were included. Trauma patients were signifi cantly injured with a median ISS of 19 (17 to 45). On admission to the hospital, the host response to bacterial stimulation was blunted in trauma patients compared with healthy individuals, as refl ected by decreased production of IL-6, IL-10 and TNFα (P <0.001). In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users Injuries suff ered by each person were related to specifi c impact objects. y g p p g Conclusion In order to prevent misdiagnosis of fractures in patients with severe injury, meticulous physical examination with suspicion of fractures should come fi rst. In addition, obtaining proper radiologic study and thorough evaluation of radiologic images are important to decreasing the rates of missed fracture diagnoses. In addition, thorough surveillance for ipsilateral fractures is important in extremities with identifi ed fractures. i Results The average ISS is 21.5 (SD 10.9). Cars are the most involved in serious urban VRU crashes. Car-to-pedestrian crashes are the most frequent (50%). The impact speed is always over 40 km/hour (Table 1). The head and face are the most frequently injured part (48% of the 571 injuries collected), followed by lower extremities (15%). In terms of maximum AIS (MAIS), the head is the most severely injured region with 42% of MAIS 3+ (Figure 1). i References S111 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P314). sRAGE kinetics (days). 2. Houshian S, Larsen MS, Holm C. Missed injuries in a level I trauma center. J Trauma. 2002;52:715-9. 3. Janjua KJ, Sugrue M, Deane SA. Prospective evaluation of early missed injuries and the role of tertiary trauma Survey. J Trauma. 1998;44:1000-6; discussion 1006-7. 3. Janjua KJ, Sugrue M, Deane SA. Prospective evaluation of early missed injuries and the role of tertiary trauma Survey. J Trauma. 1998;44:1000-6; discussion 1006-7. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients In trauma patients, MP-positive plasma was associated with a signifi cantly higher synthesis of IL-6 and TNFα compared with plasma depleted from MPs (P = 0.047 and 0.002 respectively). Compared with healthy individuals the number of circulating MPs was signifi cantly decreased in trauma patients (P = 0.009). Most MPs originated from platelets. Multiple cellular protrusions, which result in MP formation, were observed in plasma from trauma patients, but not in healthy controls. Conclusion DNA and sRAGE kinetics diff er following trauma. Early elevation of sRAGE predicts mortality in univariate analysis and correlates with subsequent renal failure. P313 P313 Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients N Curry1, K Balvers2, DJ Kleinveld2, AN Boïng2, R Nieuwland2, JC Goslings2, NP Juff ermans2 1John Radcliff e Hospital, Oxford, UK; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) P313 Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients N Curry1, K Balvers2, DJ Kleinveld2, AN Boïng2, R Nieuwland2, JC Goslings2, NP Juff ermans2 1John Radcliff e Hospital, Oxford, UK; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) References 1. Cohen MJ. J Trauma. 2010;68:1273-8. 1. Cohen MJ. J Trauma. 2010;68:1273-8. . Cohen MJ. J Trauma. 2010;68:1273-8. 1. Cohen MJ. J Trauma. 2010;68:1273 8. 2. Simmons JD. Ann Surg. 2013;258:591-6. 2. Simmons JD. Ann Surg. 2013;258:591-6. 2. Simmons JD. Ann Surg. 2013;258:591-6. Clinical decision rule for cervical magnetic resonance imaging in suspected cervical spinal cord injury without bony injury is useful in predicting severity of cervical stenosis T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 1National Center for Global Health and Medicine Hospital, Tokyo, Japan; 2Seirei Hamamatsu General Hospital, Shizuoka, Japan Critical Care 2015, 19(Suppl 1):P315 (doi: 10.1186/cc14395) Clinical decision rule for cervical magnetic resonance imaging in suspected cervical spinal cord injury without bony injury is useful in predicting severity of cervical stenosis p g y T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 Conclusion On admission, trauma patients have a reduced immune response towards endotoxin challenge which is, at least in part, mediated by MPs, which circulate in low numbers and in early stages. Most MPs originate from platelets, which indicates that these cells may be the most important source of MPs involved in initiating an infl ammatory host response post injury. g y T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 1National Center for Global Health and Medicine Hospital, Tokyo, Japan; 2Seirei Hamamatsu General Hospital, Shizuoka, Japan Critical Care 2015, 19(Suppl 1):P315 (doi: 10.1186/cc14395) Introduction Cervical spinal cord injury (CCI) without bony injury (CCIWOBI) is more frequent among Asian than among Caucasian populations and shows various extents of severity. Cervical magnetic resonance imaging (MRI) is useful for detecting intramedullary lesions, ligament injuries and intervertebral disk hernias, but some patients with mild CCIWOBI do not show clinically signifi cant abnormalities on MRI. To date, the cost–benefi t ratio of performing MRI in addition to computed tomography (CT) is unclear. We have developed a clinical decision rule for cervical MRI (MR-CDR), indicating MRI for patients >70 years old with ossifi cation of the posterior longitudinal ligament on CT or injury in a ground-level fall or a fall down stairs. The objective of the present study was to prospectively validate this MR-CDR for cervical MRI in patients with suspected mild CCIWOBI. P314 DNA and sRAGE circulation in the early phase after polytrauma P Joly1, C Massé2, D Dwivedi2, P Liaw2, J Marshall3, Y Berthiaume2, E Charbonney4 1University of Montreal, QC, Canada; 2McMaster University, Hamilton, ON, Canada; 3University of Toronto, ON, Canada; 4CRHSCM-University of Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P314 (doi: 10.1186/cc14394) Introduction Various DAMPS, alarmins are released after trauma. The soluble receptor for advanced glycation endproducts (sRAGE) was reported to be associated with acute renal failure and duration of ventilation [1]. Cell-free DNA (cfDNA) has been associated with prognosis in trauma patients [2]. We studied the kinetics of these two biomarkers over the fi rst 5 days, in a cohort of severely ill trauma patients. Methods We have been conducting a prospective observational study in two institutions in Japan since September 2012, enrolling patients with CCIWOBI among head or neck trauma patients >16  years old brought in by ambulance. We collect data about patient characteristics, injury profi les, neurological fi ndings, results of radiological examinations, and medical courses. We then analyze the sensitivity and specifi city of MR-CDR for detecting intramedullary lesions on MRI and conduct further analysis. Methods Patients who had sustained serious traumatic injury, within 24  hours of trauma, were recruited in a level I trauma center. We collected ISS, baseline demographic characteristics, resuscitation information and daily organ dysfunction (MOD) scores, over 10 days. Blood samples were collected within 24 hours of trauma (day 0) and S112 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results During the study period, 63 patients were brought in with CCIWOBI. Mean age was 60.6  years (standard deviation, 17.9  years) and 76% were male. Forty-fi ve patients presented with mild symptoms (Frankel Grade D). Cervical MRI was performed for 23 patients. Sensitivity and specifi city of MR-CDR in detecting intramedullary lesions on T2- weighted imaging among cases of suspected mild CCIWOBI were 85.7% (95% confi dence interval (CI), 60.1 to 96.0%) and 33.3% (95% CI, 12.1 to 35.4%). Further analysis showed a signifi cant diff erence in minimal spinal canal diameter as measured on sagittal T2-weighted imaging between the MR-CDR-positive and MR-CDR-negative groups (5.0 mm vs. 8.3 mm, P = 0.0003). One patient underwent surgery during hospitalization and no patients experienced exacerbated neurological fi ndings. P316 Accuracy of targeted wire-guided tube thoracostomy in comparison with classical surgical chest tube placement: a clinical study A Protic, I Barkovic, A Sustic University Hospital Rijeka, Croatia Critical Care 2015, 19(Suppl 1):P316 (doi: 10.1186/cc14396) Introduction Chest tube malfunction, after the tube thoracostomy, is often the result of an inappropriate chest tube tip position. The aim of this study was to analyze the precision of chest tube placement using the targeted wire guide technique (TWG technique) with a curve dilator and to compare it with the classical surgical technique (CS technique). Methods In this clinical study 80 patients with an indication for thoracic drainage due to pneumothorax or pleural eff usion were included. The experimental group contained 39 patients whose chest tube was placed using the TWG technique. The control group contained 41 patients whose chest tube was placed using the CS technique. Introduction Chest tube malfunction, after the tube thoracostomy, is often the result of an inappropriate chest tube tip position. The aim of this study was to analyze the precision of chest tube placement using the targeted wire guide technique (TWG technique) with a curve dilator and to compare it with the classical surgical technique (CS technique). Results There is positive signifi cant correlation between T day and LOS of injured patients (P <0.001, Spearman coeffi cient = 0.1672). Statistical analysis by Mann–Whitney test, between groups A and B, showed signifi cant diff erences in ICU LOS (P <0.001); no signifi cant diff erences (P <0.05) were found for age, ISS and outcome (Table 1). p g q q Methods In this clinical study 80 patients with an indication for thoracic drainage due to pneumothorax or pleural eff usion were included. The experimental group contained 39 patients whose chest tube was placed using the TWG technique. The control group contained 41 patients whose chest tube was placed using the CS technique. Conclusion The optimum and early time point of tracheostomy seems to be directly related with LOS in the ICU, independently of the rate of ISS, patient’s age and outcome. These results could account for ICU cost-eff ectiveness, as diminished LOS decreased the overall cost. Reference Results The comparison of the outcomes of the two techniques applied suggests that the TWG technique was signifi cantly more successful, irrespective of patient diagnosis (TWG vs. CS in all patients, 78.4% vs. 36.6%, P <0.001). See Table 1. 1. Alali et al. P317 Evaluating trauma care: comparison of early versus late tracheostomy ICU data outcome on injured patients V Kaldis1, N Mourelatos1, D Markopoulou1, K Venetsanou2, E Diogou1, E Papadaki1, D Chroni1, I Alamanos1 1General Hospital KAT-EKA, Kifi sia Athens, Greece; 2ICU Research Unit KAT, Kifi ssia Athens, Greece Critical Care 2015, 19(Suppl 1):P317 (doi: 10.1186/cc14397) Introduction In the surgical ICU, bedside tracheostomy (T) is one of the most frequently applied surgical techniques for multi-injured patients mainly with TBI [1]. The optimum surgical time decision for T still remains a contradiction in trauma. This retrospective study was designed to register all trauma patients who underwent T, during 60 months of observation (2009 to 2013), in order to identify factors associated with their ICU outcome on the basis of the T day (A <10th day >B) after tracheal intubation. g Conclusion MR-CDR was not validated for predicting the existence of intramedullary lesions on cervical MRI. MR-CDR is useful in predicting the severity of cervical stenosis. Methods Seventy-eight injured patients in the SICU underwent T, from a total of 403 issues; 58 male and 20 female, with mean age 59.3 and 74.7 years respectively. The total length of ICU stay recorded was 2,098 days, nursing time 26.55 (4/93), whereas the T time was adjusted between the 6th and 16th day (mean 11th). Mean ISS score was 22.59 (9 to 50). Classifi cation according to trauma type was TBI (n = 44) followed by thoracic trauma. Thirty-one male survivors were discharged from the ICU, to the ward. The mortality rate amounts to 47 cases due to infectious/non-infectious nosocomial complications and multiorgan dysfunction syndrome. Clinical ISS, the type of injury, ICU length of stay (LOS), T day, demographic (gender, age) data and ICU outcome were registered. Statistical analysis was performed with GraphPad 5.0.i Factors related to sepsis and outcome in multiple trauma patients H Pavlou, E Pappa, M Eforakopoulou KAT-EKA General Hospital, Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) pi Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) Introduction The outcome of multiple trauma patients is related to a number of diagnostic and therapeutic interventions during hospitali- zation. ICU patients with severe trauma are susceptible to sepsis leading to poor outcome. Factors associated with the occurrence of sepsis and the outcome of these patients were investigated. Conclusion Using a curved dilator and the TWG technique for the thoracic drainage procedure we found statistically signifi cant advantage to the TWG technique in comparison with the CS technique regarding precise chest tube placement within the pleural cavity. References Methods We studied retrospectively all trauma patients admitted to the A’ ICU of KAT General Hospital in Athens during the last 3 years and were treated for more than 5 days. Age, gender, the type of injury, the severity of injury (Injury Severity Score), the length of ICU stay, severe sepsis, coexisting diseases, the outcome and the cause of death were recorded. Logistic regression and chi-square tests were used for statistical analysis. 1. Chen F, Yamada T, Aoyama A, et al. Position of a chest tube at video-assisted thoracoscopic surgery for spontaneous pneumothorax. Respiration. 2006;73:329-33. 2. Protic A, Barkovic I, Bralic M, et al. Targeted wire-guided chest tube placement: a cadaver study. Eur J Emerg Med. 2010;17:146-9. P314 No signifi cant diff erences were evident between groups in discharge status, duration of hospitalization, or neurological fi ndings at discharge. P316 Tracheostomy timing in traumatic brain injury: a propensity- matched cohort study. J Trauma Acute Care Surg. 2014;76:70-8. Table 1 (abstract P316) CS technique TWG technique Diagnosis N (% of success) (% of success) P value Pleural eff usion 47 37.5 78.2 0.005 Pneumothorax 31 35.3 78.6 0.029 Total 78 36.6 78.4 <0.001 P318 Factors related to sepsis and outcome in multiple trauma patients H Pavlou, E Pappa, M Eforakopoulou KAT-EKA General Hospital, Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) Ventilator-associated pneumonia in a trauma ICU M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399 Figure 1 (abstract P320). M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Royal London Hospital, London, UK normoxia will create a condition of relative hypoxia, which acts in turn as a stimulus for erythropoietin (EPO) production [1]. Variations in GSH and oxygen free radical (ROS) levels may be involved in this process. We tested the NOP in critically ill patients. y p Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399) Introduction Ventilator-associated pneumonia (VAP) is associated with increased length of ventilation, ICU stay, mortality, cost and antibiotic burden [1]. There is a large variation in reported rates of VAP, partly as a result of inconsistencies in defi nition [2]. We explored a more pragmatic defi nition to describe the VAP rate, antibiotic burden and outcome of VAP in a 44-bed adult critical care unit in a level 1 trauma centre. Methods A prospective observational study on 38 mechanically ventilated (FiO2 <50%) patients with no active bleeding, no blood transfusion needed, and no kidney failure. Eighteen patients underwent a 2-hour period of normobaric hyperoxia (FiO2  = 100%), 20 patients were evaluated as controls (no FiO2 variations). EPO was assayed at baseline (t0), 24 hours (D1) and 48 hours (D2). Serum GSH and ROS were assayed at t0 (baseline), t1 (2-hour FiO2 100%) and t2 (2- hour return to normoxia) in 12 patients in the hyperoxia group. Methods A retrospective review of all adult patients admitted to the ICU at The Royal London Hospital over a 6-month period (February to August 2014). The diagnosis of VAP was based on the Clinical Pulmonary Infection Score. Patients were identifi ed with VAP if they were started on antibiotics for chest sepsis 48  hours after start of mechanical ventilation. Demographic, clinical, microbiological and radiological data were collected to identify risk factors, and compare VAP and non- VAP groups. Chi-squared and ANOVA tests were performed using the SOFA statistics package. Results EPO tended to increase in the hyperoxia group over time (P = 0.05), while it remained stable in the control group (P = 0.53) (Figure 1). ROS levels increased at t1 and decreased at t2, GSH tended to decrease at t1 and increased at t2 in the hyperoxia group. Conclusion Relative hypoxia after a transient period of normobaric hyperoxia induces an increase in GSH levels, thus enhancing ROS scavenging. This may act as a stimulus for EPO production. Table 1 (abstract P317). SICU data Table 1 (abstract P317). SICU data Table 1 (abstract P317). SICU data Outcome ICU data ISS Age ICU LOS (days) Tracheostomy day Survival Mortality A <10 16 ± 87 (16/45) 66 ± 94 (28/87) 602 17.2 ± 21.5 (4/47) 5.5 ± 6.5 (1/10) n = 13 (43.3%) n = 17 (57.7%) B >10 16 ± 25 (9/50) 64 ± 87 (23/93) 1,496 25 ± 34 (9/95) 15 ± 26 (11/23) n = 18 (40%) n = 27 (60%) t test NS NS <0.001 <0.001 NS NS Data presented as median ± IQR S113 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results A total of 106 multiple trauma patients, 85 men and 21 women, met the inclusion criteria. Depending on their age, patients were divided into two groups: <60  years old and >60  years old. In both groups, gender, the type and severity of injuries and the length of ICU stay were not associated with outcome. The length of ICU stay was correlated with severe sepsis and coexisting diseases (P <0.01) in both groups. Mortality was not diff erent in the two groups. The presence of at least one coexisting disease was signifi cantly associated with mortality (P <0.007). Sepsis was signifi cant cause of death in trauma patients >60 years (P <0.05). Figure 1 (abstract P320). Figure 1 (abstract P320). y Conclusion In multiple trauma patients, the length of ICU stay and comorbidities infl uence the occurrence of severe sepsis, comorbidities increase mortality, and sepsis is the leading cause of death in trauma patients >60 years old. Ventilator-associated pneumonia in a trauma ICU M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399 Reference Results A total of 535 mechanically ventilated patients were admitted in the study period, with 281 ventilated for more than 48 hours. The incidence of VAP was 11% in all ventilated patients and 19.6% in those ventilated more than 48 hours. VAP rates were 31% in polytrauma, 25% in neurotrauma and 18% in the neuromedical/surgical cohort. Early and late onset VAP were equal in number. Patients with VAP spent longer on mechanical ventilation (9 ± 9 in no VAP vs. 18 ± 18 days in VAP patients; P <0.001), and had longer ICU and hospital LOS (11 ± 10 vs. 22 ± 20 days; P <0.001). However, APACHE II scores and hospital mortality were unaff ected (VAP 33.3% vs. no VAP 37.6%; P = 0.173). Despite rising infl ammatory markers and secretion load, many patients did not exhibit oxygenation defi cits. Sputum microbiology showed S. aureus, H. Infl uenza, Klebsiella and Enterobacter as predominant pathogens with low rates of Pseudomonas, Acinectobacter and other resistant organisms. Average length of antibiotic use was 6 (3 to 18) days. Conclusion Chest sepsis after 48  hours of mechanical ventilation commonly complicates neurocritical illness and polytrauma requiring signifi cant ICU resources and antibiotic burden. However, it does not aff ect mortality. Further research should focus on pathophysiology and new preventative measures to reduce VAP in the at-risk population. R f 1. Balestra et al. J Appl Physiol. 2006;100:512-8. p References 1. Taylor MD, et al. J Trauma. 2002;53:407-14. 2. Osborn TM, et al. Crit Care Med. 2004;32:2234-40. References References 1. Safdar N, et al. Crit Care Med. 2005;33:2184-93. 2. Nair GB, et al. Intensive Care Med. 2014 [Epub ahead of print]. 1. Safdar N, et al. Crit Care Med. 2005;33:2184-93. 2. Nair GB, et al. Intensive Care Med. 2014 [Epub ahead of print]. Results Mean D-dimer concentration of the control group and of the patient group with PE was 0.28 (95% CI: 0.25 to 0.31)  mg/l and 1.45 (95% CI: 1.23 to 1.72)  mg/l, respectively. Receiver operator characteristics analysis revealed an optimized cutoff value of 0.466 mg/l for the PATHFAST D-dimer assay (AUC = 0.975 (95% CI: 0.938 to 0.993); sensitivity: 95% (95% CI: 86 to 99%); specifi city: 89% (95% CI: 82 to 95%)). Therefore we used a rounded up cutoff value of 0.5 mg/l to examine the diagnostic accuracy of PATHFAST D-dimer to exclude PE. The correlation between PATHFAST and VIDAS results was particularly close for concentrations at or around the critical cutoff value of P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, R Castagnani, N Mininno, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P320 (doi: 10.1186/cc14400) P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, R Castagnani, N Mininno, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P320 (doi: 10.1186/cc14400) P321 Comparison of the PATHFAST D-dimer assay with two POC D-dimer assays E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) Comparison of the PATHFAST D-dimer assay with two POC D-dimer assays E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Methods A total of 272 patients with symptoms of PE and VTE were included. The diagnoses of VTE and PE were established by duplex ultrasound, venography and spiral CT. D-dimer values were determined in the patients and in plasma samples obtained from 102 healthy individuals who served as the control group. Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Methods A total of 272 patients with symptoms of PE and VTE were included. The diagnoses of VTE and PE were established by duplex ultrasound, venography and spiral CT. D-dimer values were determined in the patients and in plasma samples obtained from 102 healthy individuals who served as the control group. Impact of introducing guidelines for thrombolysis of submassive pulmonary embolism at a large UK teaching hospital GP Misselbrook g p p Results The peak action of enoxaparin and rivaroxaban was observed at 4 hours post administration. LPTEG indicators that determine the coagulation state after 4 hours in the fi rst group: CTA was decreased by 72.12% (P <0.05), ICD was decreased by 68.44% (P <0.05), GP was increased by 17.9%; in the second group: CTA was decreased by 76.24% (P <0.05), ICD was decreased by 74.52% (P <0.05), GP was increased by 23.34%. After 12 hours, CTA in the fi rst group decreased by 22.41%, ICD decreased by 5.3%, GP increased by 8.12%, indicating reduction of hypocoagulation eff ect; in the second group, CTA decreased by 39.35% (P <0.05), ICD decreased by 40.24% (P <0.05), GP increased by 18.25%. After 24  hours in the fi rst group LPTEG indicators returned to the original value, and in the second group of patients CTA was decreased by 15.14%, ICD was decreased by 6.62%, GP increased by 14.22%.f University Hospitals Southampton NHS Foundation Trust, Southampton, UK Critical Care 2015, 19(Suppl 1):P324 (doi: 10.1186/cc14404) Introduction Pulmonary embolism (PE) is a signifi cant cause of death with 10% of patients dying within 3 months [1]. Multiple studies now advocate the use of thrombolysis (TPA) in both massive and submassive PE [1,2]. This audit assessed the impact of introducing a guideline allowing for thrombolysis of submassive and massive PE at a large UK teaching hospital. g p Methods Retrospective data collection using notes and imaging to risk-stratify patients. First audit ran from January to June 2012. New guidance was introduced in March 2013 (Figure 1) after which a second cycle ran for a further 6 months. y y y Conclusion Using LPTEG showed the hypocoagulation eff ect of continuous rivaroxaban 24  hours after oral administration compared with enoxaparin, which retains less hypocoagulation eff ect 12 hours after administration. LPTEG indicators in the second group were bigger than in the fi rst group after 12 hours: CTA 43.07%, ICD 69.72%, GP 54.12%. Results Re-audit revealed 46 patients with radiological evidence of massive or submassive PE on CTPA (32% of all PEs). Ten patients had clinical features of submassive PE and nine presented as massive PE. Previous guidelines suggested consideration of TPA in only seven patients in 6  months. TPA was given to two patients; however, six patients had no contraindications to treatment (Table 1). P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study Introduction The normobaric oxygen paradox (NOP) postulates that a period of normobaric hyperoxia followed by a rapid return to S114 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 0.5 mg/l. The correlation between PATHFAST and STRATUS results was particularly close in the patient group with VTE (r = 0.9694), whereas slightly lower results were obtained with STRATUS in the control group. With the widely used cutoff value 0.5 mg/l, PATHFAST demonstrated suitable sensitivity but not STRATUS. ROC analysis indicated that optimal cutoff values could be set at either 0.5 or 0.6 mg/l and at 0.3 or 0.4 mg/l for PATHFAST and STRATUS, respectively. clinical guidelines recommend the administration of pharmacological thromboprophylaxis (PTP) to reduce the risk of developing TEC [2]. However, it is unknown whether delayed PTP initiation increases risk of TEC. We hypothesize that delayed PTP initiation is associated with increased TEC rates. Methods A retrospective chart review (2010 to 2013) was conducted on adult trauma patients that were admitted into a level 1 trauma centre in Toronto. Demographics, date of PTP initiation, date of TEC diagnosis (CT-PE/US Doppler), injury type and severity were collected. A comparison between early and late PTP initiation has been made with regards to TEC development. Student’s t test, univariate and multivariate logistic regression analyses were performed. y Conclusion By use of the PATHFAST D-dimer assay only six of diagnoses were missed at the time of fi rst presentation compared with 10 diagnoses missed by the VIDAS D-dimer Exclusion assay, yielding higher sensitivity of the PATHFAST D-dimer assay compared with the VIDAS assay (90% vs. 83%). The STRATUS assays showed comparable performance and appeared to be suitable for the exclusion of VTE in the emergency room setting, whereas PATHFAST demonstrated superior sensitivity. Moreover, the PATHFAST analyzer allows simultaneous determination of D-dimer and cardiac troponin I within 16 minutes from whole blood samples. Therefore, this method might be useful at the point of care for early diagnostic assessment of patients with symptoms of PE or chest pain admitted to the ER or to the chest pain unit. g g y p Results A total of 1,312 patients received PTP, 821 (62.5%) initiated early PTP (within 48 hours) while 491 (37.5%) initiated after 48 hours. The group that initiated early prophylaxis was younger (mean: 46 vs. Using rivaroxoban in patients with venous thromboembolism I Tyutrin, O Tarabrin, B Todurov, S Shcherbakov, D Gavrychenko, G Mazurenko Odessa National Medical University, Odessa, Ukraine Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P322 (doi: 10.1186/cc14402) Critical Care 2015, 19(Suppl 1):P322 (doi: 10.1186/cc14402) Conclusion Mortality rates in patients with delayed PTP are higher. Our study shows LOS as the only independent predictor for TEC. However, this might not necessarily refl ect causation. Delayed PTP appears not to be an independent predictor to TEC events in trauma patients, which favours current clinical trends when it comes to contraindicating early PTP initiation. Introduction A prospective study was conducted in patients for treatment of venous thromboembolism (VTE) to compare the eff ect of enoxaparin and rivaroxaban using the method of low-frequency piezoelectric thromboelastography (LPTEG) for checking coagulation activation markers. P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study 55, P <0.0005), had lower ISS (mean: 17 vs. 24, P <0.0005), shorter length of stay (LOS) (mean: 11 vs. 23, P <0.0005), more pelvic fractures (19% vs. 13%, P = 0.0058), more head injury (AIS Head ≥3, P <0.0005), less blunt trauma (85% vs. 95%, P <0.0005), lower incidence of TEC (5.3% (44) vs. 8.5% (42), P = 0.023), and lower mortality rate (1.5% vs. 7.5%). Univariate analysis showed LOS (P <0.0005), ISS (P <0.0005), time to PTP initiation (P  = 0.0018) and blunt MOI (P  = 0.0099) signifi cantly associated with TEC events. Multivariate analysis, however, showed TEC events correlated only to LOS (P = 0.0001). Stepwise multiple logistic regression confi rmed LOS as independently associated with TEC events (95% CI = 0.003, 0.006, P <0.0005). P323 P323 Is delaying pharmacological thromboprophylaxis associated with thromboembolic complications? P Padim1, A Alshafai2, S Canestrini3, S Rizoli2, J De Rezende Neto2, A McFarlan2 1Universidade de São Paulo, Ribeirao Preto, Brazil; 2St Michael’s Hospital, Toronto, ON, Canada; 3St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P323 (doi: 10.1186/cc14403) Is delaying pharmacological thromboprophylaxis associated with thromboembolic complications? P Padim1, A Alshafai2, S Canestrini3, S Rizoli2, J De Rezende Neto2, A McFarlan2 1Universidade de São Paulo, Ribeirao Preto, Brazil; 2St Michael’s Hospital, Toronto, ON, Canada; 3St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P323 (doi: 10.1186/cc14403) q g g g Conclusion Delivering a service that off ers TPA to patients with submassive PE signifi cantly increases the need to consider this therapy. Introducing this service is only eff ective if doctors initially assessing these patients are aware of recent changes to guidelines for PE. R f References Methods A total of 60 patients entered the Odessa Clinical Regional Hospital for treatment venous thromboembolism. Patients were divided into two groups. The fi rst group (n  = 30) were receiving enoxaparin in dosage 1.5 mg/kg subcutaneously per day. The second group (n  = 30) were receiving rivaroxaban orally 15  mg/day. For checking the coagulation state we were using such indicators of LPTEG as constant thrombin activity (CTA), intensity of coagulation drive (ICD) and gel point (GP). We performed LPTEG three times per day: 4, 12 and 24 hours after taking the drug to check for changes in the coagulation state in both groups of patients. 1. Geerts W, Code K, Jay R, Chen E, Szalai J. A prospective study of venous thromboembolism after major trauma. N Engl J Med. 1994. 331;1601-6. 1. Geerts W, Code K, Jay R, Chen E, Szalai J. A prospective study of venous thromboembolism after major trauma. N Engl J Med. 1994. 331;1601-6. 2. Rogers FB, Cipolle MD, Velmahos G, et al. Practice management guidelines for the prevention of venous thromboembolism: the EAST Practice Management Guidelines Work Group. J Trauma. 2002;53:142-64. Impact of introducing guidelines for thrombolysis of submassive pulmonary embolism at a large UK teaching hospital GP Misselbrook Limitations to TPA administration were late recognition of submassive PE and inadequate knowledge of changes to guidelines.f p References Introduction Thromboembolic complications (TEC) are very common and lethal in patients suff ering from traumatic injury [1]. The trauma Introduction Thromboembolic complications (TEC) are very common and lethal in patients suff ering from traumatic injury [1]. The trauma Meyer G, et al. N Engl J Med. 2014;370:1402-11. Kearon C, et al. Chest. 2012;141:419-96S. S115 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (P <0.001). The type of thrombosis was directly associated with poor outcome, especially one that resulted from central catheters (P <0.001) and pulmonary embolism (P <0.001). However, no correlations were found with temperature, white blood cells and platelet counts on admission (P > 0.05). (P <0.001). The type of thrombosis was directly associated with poor outcome, especially one that resulted from central catheters (P <0.001) and pulmonary embolism (P <0.001). However, no correlations were found with temperature, white blood cells and platelet counts on admission (P > 0.05). Figure 1 (abstract P324). New local guidelines. Conclusion Thrombosis aff ects the ICU patient’s fi nal outcome. The type of thrombosis contributes to a poor outcome and mainly the occurrence of pulmonary embolism signifi cantly increases the mortality rate. P325 Results A total of 51 patients were included. The median age and BMI were 73 years and 23 kg/m2, respectively; 31% were female and 69% were surgical critical care patients. The median APACHE II and SOFA scores were 20 and 8, respectively. Risk factors associated with DVT were presence of central venous catheter 63%, malignancy 9% and hemodialysis 14%. The rate of DVT was 18.6% and the rate of CRT was 13.7%. All of these were asymptomatic and seen in neck and upper extremities. There was no DVT-associated adverse event (pulmonary embolism, bleeding) during hospital stay. The 28-day all-cause mortality rate was 3.4%. Introduction Despite preventive anticoagulation therapy measures, venous thromboembolic disease is a major cause of morbidity and mortality among patients hospitalized in ICUs. In fact, pulmonary embolism is not only the most serious manifestation of the disease, but also one of the primary causes of sudden death. The aim of this study is to investigate the frequency of thromboembolism and pulmonary embolism in ICU hospitalized trauma and neurosurgical patients. Conclusion While incidence of asymptomatic DVT is relatively high in adult critically ill patients, they were found only in the neck and upper extremities without any adverse event. Further research is needed to evaluate the clinical signifi cance of this type of DVT. Methods One hundred ICU patients, 51 postoperative neurosurgical and 49 trauma, were included in the study. Patients’ demographic data as well as medical history, temperature, white blood cells and platelets counts were recorded on admission, the day of thrombosis diagnosis and the fi nal outcome of their treatment. Statistics were performed with SPSS-19. P <0.05 was considered signifi cant. P326 P326 Incidence and outcome of asymptomatic deep vein thrombosis in critically ill patients: a prospective cohort study R Echigoya, H Okamoto, H Uchino, A Kuriyama, N Tamura, K Sato, T Fukuoka Kurashiki Central Hospital, Okayama, Japan Critical Care 2015, 19(Suppl 1):P326 (doi: 10.1186/cc14406) Figure 1 (abstract P324). New local guidelines. Figure 1 (abstract P324). New local guidelines. Introduction Asymptomatic deep vein thrombosis (DVT) including catheter-related thrombosis (CRT) is an increasingly recognized disease entity in critically ill patients. However, the reported rate and outcome of DVT vary widely depending on study design, patient background and detecting method. The objective of this study is to evaluate the incidence and outcome of DVT in adult critically ill patients. Table 1 (abstract P324). TPA decisions High Intermediate Total risk risk Considered TPA and given 2 2 0 Considered TPA but contraindications 1 1 0 Considered TPA and not given on balance 3 0 3 Considered TPA and not given but fi t 3 2 1 criteria Not considered TPA but contraindicated 4 1 3 anyway Not considered TPA and on balance 5 1 4 would not be given Not considered TPA but fi t criteria 1 0 1 Table 1 (abstract P324). TPA decisions y p Methods This study is a prospective cohort study of patients admitted to a medical and surgical ICU from 1 July 2014 to 15 October 2014. All consecutive patients over 18 years of age and with expected ICU stay over 72 hours were included. Patients who had previous history of DVTs were excluded. We examined internal jugular vein, subclavian vein, axillary vein, brachial vein, femoral vein, superfi cial femoral vein, and popliteal vein, on ICU admission and within 48 hours after ICU discharge. The DVT was diagnosed using compression ultrasonography with color Doppler. Images were interpreted by two independent investigators trained in ultrasonography. All patients received intermittent pneumatic compression and unfractionated heparin twice daily during their IUC stay. Once the DVT was detected, therapeutic anticoagulation was initiated. Contrast-enhanced CT was performed when the patients were suspected to have pulmonary embolism. The primary outcome was the incidence of DVT during the ICU stay. Patients were followed until their hospital discharge. P327 P327 Computed tomographic pulmonary angiographic fi ndings to predict adverse outcomes in acute pulmonary embolism P Tajarernmuang, J Euathrongchit, C Liwsrisakun, A Deesomchok, T Theerakittikul, C Bumroongkit, C Pothirat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2015, 19(Suppl 1):P327 (doi: 10.1186/cc14407) gi Results Thirty-eight out of 100  patients presented thrombosis, 14 trauma and 24 neurosurgical. We examined the correlation of thrombosis development during hospitalization with diagnosis, treatment allocated time and overall patient outcome. It was found that neurosurgical patients developed thrombosis more frequently than trauma patients (P <0.05). In relation to diagnosis, thrombosis was prevalent among patients with brain lesions (P = 0.018). Regarding the type of thrombosis, pulmonary embolism was also commonly apparent among individuals with brain lesion (P = 0.020). In addition, there was a statistically signifi cant correlation in thrombosis occurrence between hospitalization day (P  <0.01) and patients’ outcome on discharge Introduction Computed tomographic pulmonary angiography (CTPA) has been used as a standard tool for diagnosing an acute pulmonary embolism (APE). The right ventricular (RV) strain signs may be used to predict adverse outcomes. However, the results are still controversial. The primary objective of our study was to evaluate the relationship between the RV strain signs and respiratory failure requiring mechanical ventilation or death in APE. The secondary objective was to identify clinical factors which related to those outcomes. Introduction Computed tomographic pulmonary angiography (CTPA) has been used as a standard tool for diagnosing an acute pulmonary embolism (APE). The right ventricular (RV) strain signs may be used to predict adverse outcomes. However, the results are still controversial. The primary objective of our study was to evaluate the relationship between the RV strain signs and respiratory failure requiring mechanical ventilation or death in APE. The secondary objective was to identify clinical factors which related to those outcomes. S116 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P329). Kaplan–Meier analysis of ESRD progression for SC and Ecu treatment for all patients. Methods CTPA and the medical records of patients with suspected APE on admission from June 2011 to March 2013 were reviewed. Mean platelet volume and mean platelet volume/platelet count ratio in risk stratifi cation of pulmonary embolism T Yardan1, M Meric1, C Kati1, Y Celenk2, A Atici1 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) T Yardan , M Meric , C Kati , Y Celenk , A Atici 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) Introduction Recently, mean platelet volume (MPV) was reported to predict early death in acute pulmonary embolism (PE). The aim of this study was to investigate the role of MPV and MPV/platelet count ratio (MPV/P) in risk stratifi cation of patients with acute PE. Conclusion Ecu treatment reduces the number of ESRD events and the rate of progression to ESRD; thus initiation of Ecu early after aHUS diagnosis may prevent cumulative kidney damage and progression to ESRD. i p Methods We retrospectively reviewed the medical records of patients with acute PE admitted to the emergency department. In addition to the clinical evaluation, platelet count and MPV were measured on admission. P328 Mean platelet volume and mean platelet volume/platelet count ratio in risk stratifi cation of pulmonary embolism T Yardan1, M Meric1, C Kati1, Y Celenk2, A Atici1 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) p Reference 1. Ghuysen A, Ghaye B, Willems V, Lambermont B, Gerard P, Dondelinger RF, et al. Computed tomographic pulmonary angiography and prognostic signifi cance in patients with acute pulmonary embolism. Thorax. 2005;60:956-61. p p Results The SC and Ecu treatment phases included 32 and 33 patients, respectively. With SC, during a median (range) of 211 (7 to 745) days, 13 (41%) patients had a total of 16 ESRD events. On Ecu treatment, during a median (range) of 924 (73 to 1,254) days, three (9%) patients had a total of fi ve ESRD events. The ESRD event rate was 92% lower during Ecu treatment versus the SC phase (0.36 vs. 0.07; P = 0.001); the incidence rate ratio was 0.08 (95% CI = 0.02 to 0.37; P = 0.001). HR for progression to ESRD for patients on Ecu versus SC was 0.03 (95% CI <0.01 to 0.34), a 97% reduction (Figure 1). Stratifi cation by baseline CKD stage showed no patients with CKD stage 2 or 3 at baseline progressed to ESRD over 3 years of Ecu treatment. Early initiation of eculizumab treatment in patients with atypical haemolytic uraemic syndrome improves long-term outcomes: a pooled analysis of clinical trials p y J Vande Walle1, Y Delmas2, G Ardissino J Vande Walle1, Y Delmas2, G Ardissino3, J Wang4, J Kincaid4, H Haller5 1University Hospital Ghent, Belgium; 2Centre Hospitalier Universitaire de Bordeaux, France; 3Ospedale Maggiore Policlinico, Milan, Italy; 4Alexion Pharmaceuticals, Cheshire, CT, USA; 5Medical School Hannover, Germany Critical Care 2015, 19(Suppl 1):P330 (doi: 10.1186/cc14410) Introduction Atypical haemolytic uraemic syndrome (aHUS) is a severe, life-threatening disease requiring rapid treatment to inhibit complement-mediated thrombotic microangiopathy (TMA) and avoid irreversible organ damage. Four prospective clinical trials have reported the safety and effi cacy of eculizumab (Ecu) in the treatment of aHUS [1,2]. We report data from a pooled analysis of these trials on renal function in patients starting Ecu within ≤7 days or >7 days after the current aHUS manifestation. Introduction Atypical haemolytic uraemic syndrome (aHUS) is a severe, life-threatening disease requiring rapid treatment to inhibit complement-mediated thrombotic microangiopathy (TMA) and avoid irreversible organ damage. Four prospective clinical trials have reported the safety and effi cacy of eculizumab (Ecu) in the treatment of aHUS [1,2]. We report data from a pooled analysis of these trials on renal function in patients starting Ecu within ≤7 days or >7 days after the current aHUS manifestation. P327 RV dysfunction signs included right ventricular to left ventricular (RV/LV) diameter ratio, interventricular septal shift, main pulmonary artery to ascending aorta (mPA/AA) diameter ratio, IVC contrast refl ux, SVC diameter, IVC diameter, PA diameter and azygos vein diameter. Clinical factors included cardiovascular, respiratory parameter and also time to diagnosis and treatment. g Results There were total of 36 cases with suspected APE on admission. Ten patients required mechanical ventilation (27.8%) and seven patients died (19.4%). Interventricular septal (IVS) shift was a signifi cant risk factor of in-hospital death (85.7% vs. 27.6%, P  = 0.008) and respiratory failure (70% vs. 26.9%, P = 0.026). The sensitivity, specifi city, positive predictive and negative predictive values of IVS shift to predict in-hospital death were 85.7%, 70%, 42.8% and 95.5%, respectively. The sensitivity, specifi city, positive predictive and negative predictive values of IVS shift to predict respiratory failure were 70%, 73.1%, 50% and 86.4%, respectively. The ratios of RV to LV diameter and the ratio of main pulmonary artery to ascending aorta diameter tended to be higher in the nonsurvivor group. The clinical factor that predicted mortality was the PaO2 to FiO2 ratio (P/F ratio). Mean P/F ratio in survivor and nonsurvivor groups was 246.1 ± 94.1 vs. 132.2 ± 78.1, respectively (P = 0.011). P/F ratio ≤150 was the best predictor of mortality (66.7% vs. 8.7%, P = 0.008). Figure 1 (abstract P329). Kaplan–Meier analysis of ESRD progression for SC and Ecu treatment for all patients. with aHUS progress to end-stage renal disease (ESRD) after the fi rst episode [1]. Two prospective clinical trials have assessed the effi cacy of eculizumab (Ecu) in patients with aHUS [2]. We now evaluate data on progression to ESRD before and during Ecu treatment. Methods Patients with chronic kidney disease (CKD) stage 1 to 4 were analysed for progression to an ESRD event (two consecutive glomerular fi ltration rate measurements <15 ml/minute/1.73m2 (CKD stage 5)). ESRD incidence rate ratios during supportive care (SC) and Ecu treatment phases were calculated using a negative binomial regression analysis. Kaplan–Meier analyses were calculated for all patients and stratifi ed by CKD stages 2 to 4 at baseline. Hazard ratios (HR) were calculated from Cox proportional hazard models. Conclusion The IVS shifting from CTPA and P/F ratio ≤150 helps predict poor outcomes in APE. References 1. Fremeaux-Bacchi V, et al. CJASN. 2013;8:554-62. Results One hundred and fi fty-two patients were included. Patients with right ventricular (RV) dysfunction had signifi cantly higher MPV levels and MPV/P than patients without RV dysfunction. Receiver operating characteristic analysis revealed that a MPV cutoff of 7.85 fl provided 53.3% sensitivity and 68.5% specifi city, and a MPV/P cutoff of 0.0339 fl /(109/l) provided 69.6% sensitivity and 65% specifi city for prediction of RV dysfunction. There was a positive correlation between MPV and systolic pulmonary artery pressure (SPAP) and between MPV and RV diameter. There was a positive correlation between MPV/P and SPAP and between MPV/P and RV diameter. The low-risk PE group had lower MPV and MPV/P than the massive PE and submassive PE groups. Conclusion MPV and MPV/P are associated with RV dysfunction and clinical severity in acute PE. Low MPV and MPV/P levels may be an indicator of low risk in patients with acute PE. 2. Legendre C, et al. N Engl J Med. 2013;368:2169-81. Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemiai Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemia A Taha, A Shafi e, M Mostafa, N Syed, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P331 (doi: 10.1186/cc14411) Results We identifi ed 21 eligible studies (16,951 patients). After pooling data from the 20 included cohort studies (16,884 patients), at least around 33% (95% CI: 27 to 39; I2: 97.8%) of patients with TBI in published reports received transfusions at some point during their hospital stay. In a post hoc analysis of one RCT comparing transfusion strategies, 82% of patients were transfused RBCs. Thresholds for transfusion were rarely available and varied from 6 to 10 g/dl. From raw data, Glasgow Coma Scale scores were lower in patients who were transfused than those who were not (three cohort studies; n = 1,371; mean diff erence of 1.38 points (95% CI: 0.86 to 1.89); I2 = 12%). Mortality was not signifi cantly diff erent among transfused and nontransfused patients both in univariate and multivariate analyses. Hospital length of stay was longer among patients who were transfused (three studies; n = 455; mean diff erence 9.58 days (95% CI: 3.94 to 15.22); I2 = 74%). Due to the observational nature of included studies, results should be considered cautiously due to the high risk of confounding. i A Taha, A Shafi e, M Mostafa, N Syed, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P331 (doi: 10.1186/cc14411) Introduction Hemoglobin as the main trigger parameter for blood transfusion usually gives diminutive information about oxygen delivery and consumption. Although central venous oxygen saturation (ScvO2) is an alternative parameter, its changes are unable to detect regional hypoxia. Our aim was to evaluate the quotient of the central venous-to- arterial carbon dioxide gradient (δPCO2) and the arteriovenous oxygen content diff erence (Ca-cvO2) as a valid transfusion trigger parameter in hemodynamically stable anemic patients to reduce the amount of potentially counterproductive erythrocyte transfusions [1].i Methods Forty-fi ve postoperative patients admitted to our cardiac ICU were enrolled between January 2013 and September 2014. Three groups were defi ned according to the trend of blood loss over the surgical drains in the fi rst 24 postoperative hours. Red blood cell transfusion in patients with traumatic brain injury: a systematic review Red blood cell transfusion in patients with traumatic brain injury: a systematic review A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada Critical Care 2015, 19(Suppl 1):P332 (doi: 10.1186/cc14412) minute/1.73 m2 were included. Changes from baseline in eGFR were analysed at study visits using a one-sample t test. y A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada Critical Care 2015 19(Suppl 1):P332 (doi: 10 1186/cc14412) Results Data from 97 patients were analysed: median (range) age at enrolment was 29 (0 to 80) years; 62% of patients were females; median (range) duration of current manifestation to start of Ecu treatment was 23 (1 to 1,447) days; median (range) baseline eGFR was 15.9 (5.6 to 76.1) ml/minute/1.73 m2. Ecu treatment was started in 21 patients in ≤7  days and 76 patients in >7  days after presentation with TMA. Median eGFR was 11 ml/minute/1.73 m2 for the patients started within 7 days and 16 ml/minute/1.73 m2 for those initiating >7 days. The mean change from baseline in eGFR for patients starting Ecu in ≤7 days and in >7 days after presentation with TMA were 57 and 23 ml/minute/1.73 m2 at 1 year, respectively (Figure 1). Critical Care 2015, 19(Suppl 1):P332 (doi: 10.1186/cc14412) Introduction We aimed to evaluate the frequency of red blood cell (RBC) transfusion in patients with traumatic brain injury (TBI) as well as determinants and outcomes associated with RBC transfusion in this population. Methods We conducted a systematic review of cohort studies and trials of patients with TBI. Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemiai Mild blood loss was defi ned as 500 to 1,000  ml/24  hours, moderate (1,000 to 1,500/24  hours) and severe (>1,500  ml/24  hours). In addition to the δPCO2 the following parameters were monitored: CI, CO, SVR, serum lactate, ScvO2 and hemoglobin. Ca-cvO2 was calculated and the δPCO2/ Ca-cvO2 quotient was assessed for a total of 400 paired blood samples. All enrolled patients were hemodynamically stable. A retrospective analysis of this data was performed.i Conclusion RBC transfusion is frequent in patients with TBI, but practices varied widely in cohort studies in this population. The paucity of data precludes defi nitive conclusions and highlights the lack of clinical evidence guiding transfusion strategies in TBI. P329 Progression to end-stage renal disease is reduced with eculizumab in patients with atypical haemolytic uraemic syndrome J Vande Walle1, S Johnson2, E Harvey3, J Kincaid3 1University Hospital Ghent, Belgium; 2Medicus Economics, LLC, Boston, MA, USA; 3Alexion Pharmaceuticals, Cheshire, CT, USA Critical Care 2015, 19(Suppl 1):P329 (doi: 10.1186/cc14409) Methods Data from four phase 2, open-label, single-arm trials including both paediatric and adult patients with aHUS were pooled. Patients with a documented date of onset of current TMA manifestation and a baseline estimated glomerular fi ltration rate (eGFR) of <90  ml/ Introduction Atypical haemolytic uraemic syndrome (aHUS) is associated with severe kidney damage; almost one-half of adults S117 Critical Care 2015, Volume 19 Suppl 1 http://ccforum com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P330). Mean change in eGFR from baseline over 1 year (standard error). hemoglobin capacity decline and signifi cantly improved after erythrocyte transfusions (P <0.005). Figure 1 (abstract P330). Mean change in eGFR from baseline over 1 year (standard error). Conclusion Blood transfusions carry risks of adverse eff ects and should be carried out responsibly. Our fi ndings suggest an additive and easy detectable transfusion trigger parameter (δPCO2/Ca-cvO2) providing physiological information on anemia-related altered oxygen extraction conditions and hence the indication for erythrocyte transfusions. However, additional studies are warranted to confi rm these fi ndings. Reference 1. Mekontso-Dessap A, Castelain V, Anguel N, Bahloul M, Schauvliege F, et al. Combination of venoarterial PCO2 diff erence with arteriovenous O2 content diff erence to detect anaerobic metabolism in patients. Intensive Care Med. 2002;28:272-7. 1. Mekontso-Dessap A, Castelain V, Anguel N, Bahloul M, Schauvliege F, et al. Combination of venoarterial PCO2 diff erence with arteriovenous O2 content diff erence to detect anaerobic metabolism in patients. Intensive Care Med. 2002;28:272-7. y References 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. Red blood cell transfusion in patients with traumatic brain injury: a systematic review We searched Medline, Embase, The Cochrane Library and BIOSIS databases from their inception up to 30 June 2014. We selected cohort studies and RCTs of adult patients with TBI reporting data on RBC transfusions. We extracted data related to demographics, baseline characteristics, blood product use and any relevant clinical patient-oriented outcome. Cumulative incidences of transfusion were pooled through random eff ect models with a DerSimonian approach, after a Freeman–Tukey transformation to stabilize variances. To evaluate the association between RBC transfusion and potential determinants as well as outcomes, we pooled risk ratios or mean diff erences with random eff ect models and the Mantel–Haenszel method. Sensitivity and subgroup analysis were planned a priori.i y p y g Conclusion This pooled analysis indicates that patients treated with Ecu within 7 days of a TMA manifestation had a greater improvement in eGFR over time than patients in whom treatment was delayed. These data show the importance of rapid diagnosis and treatment of aHUS for recovery of renal function. Red blood cell transfusion is associated with an increased mortality in critically ill surgical patients y A Piriyapatsom, O Chaiwat, S Kongsayreepong Siriraj Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P334 (doi: 10.1186/cc14414) Results δPCO2/Ca-cvO2 showed signifi cant correlation with the moderate and severe blood loss groups (P <0.01), while no signifi cant correlation was detected in the mild blood loss group. The abnormality of the δPCO2/Ca-cvO2 was easy detectable and refl ected intracapillary Introduction The aim of this study is to explore the association between red blood cell transfusion (RBCT) and mortality in Thai critically ill surgical patients. Introduction The aim of this study is to explore the association between red blood cell transfusion (RBCT) and mortality in Thai critically ill surgical patients. S118 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods This study was a part of the multicenter, prospective, observational study performed in nine surgical intensive care units (SICUs) across the nation between April 2011 and November 2012 [1]. This study included adult patients admitted to the SICUs after surgery. Patients were categorized into transfusion and no transfusion groups according to whether or not they received RBCT at any time during SICU stay. Demographic data, clinical outcomes as well as SICU and hospital length of stay (LOS) and SICU and hospital mortality were collected. Patients were followed for up to 28 days or until discharge from the SICUs. The primary endpoint was hospital mortality. Data were compared between groups and logistic regression analysis was performed to determine whether RBCT was an independent risk factor of hospital mortality. In addition, patients were matched between groups based on the propensity score of the requirement of RBCT and were then compared. confounders, a Hb level of ≤9 g/dl was associated with a poor neurologic outcome (OR = 2.572, 95% CI = 1.058 to 6.250), whereas an Hb level of ≤10 g/dl was not. A Hb level of ≤10.3 g/dl had a sensitivity of 82% and a specifi city of 62% to predict poor neurologic outcome in TBI patients. Conclusion In TBI patients a Hb level of ≤9 g/dl is associated with poor neurologic outcome. Red blood cell transfusion is associated with an increased mortality in critically ill surgical patients A transfusion threshold of ≤10.3  g/dl may be a reasonable target to be tested in future transfusion trials aimed at improving neurologic outcome of TBI patients.fi Acknowledgements All of the author institutes are affi liated members of the International Trauma Research Network (INTRN) and as such this work represents a combined output resulting from this Network. Microparticles from red blood cell transfusion products induce a strong infl ammatory host response y p q g Results Supernatant from blood bags containing MPs strongly induced production of all cytokines compared with supernatant without MPs, a reaction which equaled that of LPS stimulation. MPs from stored RBC bags induced higher production of TNF (868 (263 to 1,625) vs. 2,596 (407 to 3,040)  pg/ml, P  = 0.049), IL-6 (1,088 (234 to 3,716) vs. 6,952 (1,507 to 21,990), P = 0.042) and IL-8 (1,333 (535 to 3,569) vs. 5,562 (833 to 13,904), P = 0.081) compared with MPs from fresh RBC bags. There was no diff erence in IL-10 responses between groups (8.0 (3.9 to 32.1) vs. 3.9 (3.9 to 22.2), P = 0.390). The host response was dose dependent both for fresh and stored MPs. In addition, the same amount of older MPs induced a stronger host response compared with fresh MPs. Conclusion MPs from RBC transfusion bags induce a strong proinfl ammatory response, which is largely negated when MPs are removed. This MP-mediated response depends both on the amount of MPs as well as on alterations in MPs as a result of storage. Reference Reference Reference 1. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 Suppl 1:S45-54. 1. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 Suppl 1:S45-54. Microparticles from red blood cell transfusion products induce a strong infl ammatory host response Results Overall, 968 of 2,374 (40.8%) patients received RBCT. Trans- fused patients, when compared with those without RBCT, had more frequency of admission after emergency surgery, higher APACHE II score, higher SOFA score, higher number of organ dysfunctions and lower hemoglobin level at admission. When compared with patients without RBCT, those with RBCT had more frequency of all adverse events including infection, AKI, ALI/ARDS and MI, and longer SICU and hospital LOS. Both SICU and hospital mortality were also higher in the transfusion group compared with the no transfusion group (9.4% vs. 1.6% and 13.7% vs. 3.6%, both P <0.001, respectively). The logistic regression analysis showed that RBCT was an independent risk factor of hospital mortality with odds ratio of 1.60 (95% CI 1.05 to 2.45). In the propensity-score matched cohort of 852 patients, when compared with patients without RBCT, transfused patients had more frequency of adverse events including infection and AKI, longer SICU and hospital LOS and higher hospital mortality (7.5% vs. 4.0%, P = 0.027). l M Straat1, M Van Hezel1, A Boing1, R Nieuwland1, R Van Bruggen2, N Juff ermans1 f 1Academic Medical Center, Amsterdam, the Netherlands; 2Sanquin Blood Cell Research, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P336 (doi: 10.1186/cc14416) Introduction Red blood cell (RBC) transfusion is associated with increased morbidity and mortality in the critically ill. Adverse eff ects of transfusion may be mediated by red blood cell storage lesion. In this study, we hypothesized that MPs from stored RBC bags would induce a more pronounced host response than MPs from fresh RBC bags and that this response is dose dependent. p p Methods MPs were isolated by high-speed centrifugation from red blood cell transfusion bags stored for 2 to 7 (fresh) or 25 to 35 (stored) days. Whole blood from healthy volunteers was incubated with supernatant from the bags either containing MPs or depleted from MPs (n = 12 bags per group). Controls were incubated with PBS as a negative and LPS (10 ng/ml) as a positive control. Cytokines in supernatant were measured by ELISA. Data are expressed as medians and interquartile ranges. g y Conclusion This study showed that RBCT was associated with increased morbidity and mortality in critically ill surgical patients. These results supported the restrictive strategy of RBCT suggested by more recent studies. Eff ect of the haemoglobin level on neurologic outcome in patients with severe traumatic brain injury Eff ect of the haemoglobin level on neurologic outcome in patients with severe traumatic brain injury K Balvers1, MR Wirtz1, C Rourke2, S Eaglestone2, K Brohi2, S Stanworth3, C Gaarder4, JC Goslings1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Blizard Institute, Queen Mary University of London, UK; 3John Radcliff e Hospital, Oxford, UK; 4Oslo University Hospital, Oslo, Norway Critical Care 2015, 19(Suppl 1):P335 (doi: 10.1186/cc14415) Introduction Anaemia in patients with severe traumatic brain injury (TBI) may worsen neurologic outcome. The aim of this study was to determine the association of haemoglobin level (Hb) with neurologic outcome and to determine a transfusion threshold which may be used in future transfusion trials aimed at improving neurologic outcome in TBI patients. Value of thromboelastography in managing hypercoagulopathy in intensive care Methods A retrospective audit over 2 months of all blood transfusion forms at St Francis Hospital, Eastern Province, Zambia. Respective patients’ notes were reviewed for: record of observations during transfusion; patient demographics; and length of stay. We surveyed nurses’ attitudes, confi dence and knowledge in relation to blood transfusion standards. J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P340 (doi: 10.1186/cc14420) J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK g p , , Critical Care 2015, 19(Suppl 1):P340 (doi: 10.1186/cc14420 Introduction This aim of this analysis is to explore the use of thromboelastography (TEG) in the management of hypercoagulation in the ICU. TEG allows the assessment of whole blood coagulation and fi brinolysis and hence can identify patients who are hypercoagulable. Methods A prospective audit of TEG tests performed on patients being treated on a general surgical and medical ICU was conducted over a 2-month period. Results In May and June 2014, 457 requests were made for blood, of which 157 (34%) received blood transfusion, of which 108 (69%) had records of observations available. The audit demonstrated that requests were mostly complete (90%), but urgency was indicated in only 32%. The matching of blood to patient by more than one nurse was recorded amongst 66% of cases. Only 2% of transfusions met minimal requirements for transfusion reaction monitoring. Of nurses surveyed (n = 20), most were experienced in their post (mean 7.3 years, range 2 weeks to 20 years). Nurses rated themselves as highly confi dent in handling blood transfusions and identifying and dealing with transfusion reactions. However, 90% believed they could identify all transfusion reactions by measuring temperature alone, and 25% would measure temperature only as a parameter to monitor the transfusion, even in ideal settings. Most knew to check observations before, 15  minutes after the start of transfusion and then hourly thereafter (88%); but only 10% would check at the start, at completion and 4 hours after completing transfusion. The most frequently reported reason for not doing observations was time pressure on the ward (85%). Results Twenty-one out of 78 patients (26.9%) had one or more TEG criteria consistent with hypercoagulopathy. Admission diagnoses included trauma (37%), haemorrhage (23%), postoperative (23%) and sepsis (14.3%). Sixty-two per cent of patients with a primary diagnosis of trauma were in a hypercoaguable state. P337 Improving blood transfusion safety in a low-resource setting: an audit of 1,163 transfusion requests S Kudsk-Iversen1, R Colhoun1, D Chama2, J Mulenga2, MD Bould1, J Kinnear1, D Snell1 1Zambia Anaesthesia Development Project, Lusaka, Zambia; 2Zambia National Blood Transfusion Service, Lusaka, Zambia Critical Care 2015, 19(Suppl 1):P337 (doi: 10.1186/cc14417) Methods A substudy of the prospective multicentre Activation of Coagulation and Infl ammation in Trauma (ACIT) II study was performed on subjects recruited between January 2008 and December 2014. All adult trauma patients admitted to a level 1 trauma centre with severe traumatic brain injury (AIS head ≥3), ICU admission and available Hb levels on admission were selected for analysis. The primary outcome was the cognitive functioning of patients as determined by an estimated Glasgow Outcome Scale (GOS) on discharge. Anaemia was defi ned as a Hb level of ≤9  g/dl (severe anaemia) or ≤10  g/dl (moderate anaemia) within the fi rst 24 hours post injury. Multivariate logistic regression models were used to determine the association between anaemia and neurologic outcome. The receiver operating characteristic curve and the Youden Index were used to determine an optimal transfusion threshold. Introduction Sub-Saharan Africa suff ers from more acute life- threatening indications for blood transfusion compared with high- income countries [1]. The commonest ‘systems failure’ contributing to perioperative death in low-resource settings is the timely availability of correctly cross-matched blood products [2]. Often this is not the result of an absolute shortage of blood products, but failure in the chain of supply and distribution. We audited an early step in this chain, the quality of blood requests, at the University Teaching Hospital (UTH) in Lusaka, Zambia. UTH does not have a formal blood request form, and only the cancer diseases hospital (CDH) has a blood request form developed by the blood bank. Results Of a total of 261 TBI patients, 61 patients (23%) fell below the threshold for severe anaemia and 101 patients (39%) had moderate anaemia within the fi rst 24 hours. In a model adjusted for all relevant S119 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We performed a 1-day retrospective review in June of blood request forms submitted to the cross-match laboratory, followed by a 14-day prospective review in September 2014. Group and save requests were excluded. Inadequate monitoring risks safety of blood transfusion in rural Zambia Results The mean age was 43.5 ± 5.7 years. Of 58 patients (38 male/20 female), 25 (43.1%) had iron defi ciency with outcomes of blood samples used at ICU admission. The overall transfusion rate was 32.8%, being higher in iron-defi ciency patients than in normal iron profi le patients (42.3 vs. 14.9%, P = 0.001). After adjusting for severity of illness and hemoglobin level, iron-defi ciency patients remained signifi cantly associated with transfusion, with a hazard ratio of 4.2 (95% CI, 1.3 to 12.9; P = 0.001). Zambia O Todd1, K Sikwewa1, J Kamp1, I Hodt Rasmussen1, K Mortensen1, S Kudsk-Iversen2 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) O Todd1, K Sikwewa1, J Kamp1, I Hodt Rasmussen1, K Mortensen1, S Kudsk-Iversen2 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) Introduction In Zambia, supply of blood is insuffi cient to meet clinical need, on a national level. Paradoxically, blood is also more often transfused unnecessarily in this setting. The Zambian National Blood Transfusion Service is currently scaling up voluntary blood donation and supply systems, and requires hospitals to improve blood transfusion safety. At a rural district hospital in Zambia, we audited practice and surveyed knowledge amongst staff using standards established in national guidelines. Conclusion Iron defi ciency is common at ICU admission and is associated with higher transfusion requirements. These fi ndings have important implications for transfusion practices in ICU patients. P340 P339 Eff ects of iron defi ciency on transfusion requirements in critically ill patients: a preliminary observational study M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) P339 Eff ects of iron defi ciency on transfusion requirements in critically ill patients: a preliminary observational study M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) Introduction Critically ill patients often need blood transfusion, but no reliable predictors of transfusion requirements are available at ICU admission. We hypothesized that ICU patients admitted with iron defi ciency may be at higher risk for developing anemia, requiring blood transfusion. The aims of this study were to determine the frequency of iron defi ciency in ICU patients at admission and to investigate its relationship with transfusion requirements in ICU patients. p Conclusion The audit revealed an important system failure impacting on effi cacy and safety of transfusion practice at UTH. Full patient identifi ers, as well as vital information such as the indication and urgency, were rarely fi lled in, which are crucial for the blood bank to prioritise the release of blood products. The audit shows that practice may be signifi cantly improved by a cheap intervention such as a standardised blood request form meeting international standards. Acknowledgements UK aid, THET. References 1 L d l T f A h S i 2013 49 416 21 Methods Eighty-fi ve patients admitted to the general ICU were enrolled in the prospective observational study. We studied 58 patients, after excluding those transfused on or before ICU admission. The patients’ age, gender, APACHE II score, diagnosis, severity score, presence of sepsis, ICU complications, ICU treatments, and transfusion-free interval were recorded. Iron defi ciency was assessed on the basis of several parameters, including hemoglobin, hematocrit, levels of serum iron, iron-binding capacity, transferrin saturation, levels of ferritin, soluble transferrin receptor, hepcidin, C-reactive protein, and peripheral blood smear. 1. Lund et al. Transfus Apher Sci. 2013;49:416-21. 2. Ohanaka et al. Turk J Med Sci. 2007;37:219-22. P337 Each form was audited against the American Association of Blood Banks (AABB) minimum standards for content of a blood request form. Analysis was performed with Fisher’s exact test for nominal data and t test for continuous data. Conclusion In this setting, current practice is evidently inadequate to identify and prevent blood transfusion reactions. The survey revealed high confi dence but patchy knowledge amongst nurses of the requirements for safe blood transfusion. Better timing to transfuse at times when nursing staff numbers are higher, alongside compulsory training, may together represent potential low-cost interventions to improve blood transfusion safety. Results A total of 1,163 blood requests were reviewed, 51 from CDH and 1,112 from other wards. Eighteen forms from CDH (35%) and 22 from other wards (2%) met all minimum AABB standards (P <0.0001). The mean number of standards met on the requests from CDH and the rest were 11.25 (SD 0.93) and 8.87 (SD 1.75) respectively (P <0.0001). Considering all blood requests, the standards met in order from least to most were: signature of requesting doctor (36%), urgency of request (43%), hospital number (59%), indication for transfusion (62%), type of product requested (72%), requesting doctor’s name (78%), age or date of birth of patient (84%), gender of patient (89%), quantity of products requested (90%), date form was completed (90%), patient’s ward (95%), and patient’s full name (100%). P339f P342 P342 In trauma, when used in the emergency department, do viscoelastic hemostatic tests decrease mortality? A systematic review J Cousineau, R Daoust, K Doyon, M Marquis, É Piette, JM Chauny, M Clar, D Rose, É Notebaert Hôpital du Sacré Coeur de Montréal, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P342 (doi: 10.1186/cc14422) Introduction This systematic review done in March and updated in November 2014 has been conducted in accordance with the STARD, PRISMA and STROBE recommendations. Retrospective and prospective studies with a comparison group published in English were kept. Methods Two reviewers (JC and ÉN) and two librarians (MC and DR) independently conducted a systematic review and identifi ed abstracts. Full texts were read by two authors (ÉN and ÉP), and data were extracted. The following databases were searched: Cochrane CENTRAL, Medline, Embase, LILACS, Web of Science, Science.gov, SciFinder Scholar, WorldCat, the Transf Evid Lib Database, and proceedings of the congresses of the International Society on Thrombosis and Haemostosis and the American Society of Hematology. Figure 1 (abstract P340). Coagulation modifi cation prior to TEG analysis in hypercoagulable patients. as a result of performing TEG was documented in 14 of these 21 patients. No further blood products were administered in all cases and anticoagulation was commenced or increased in four cases. Conclusion Hypercoagulopathy was present in 27% of patients. One- third of these patients had recently received prothrombotic therapy indicating a possible iatrogenic aetiology. TEG analysis resulted in cessation of prothrombotic drug and blood product administration in all cases. Further research is required to determine whether titrated anticoagulation treatment to normalise the TEG profi le in these patients would be benefi cial. f y y Results We initially kept 2,870 references. In total, 453 articles had mortality in their keywords. After reading the abstracts, 37 papers were analysed, and three articles evaluating mortality in two groups of patients (using and not using a VHT) were identifi ed. Among these three studies, only one had raw data available. We did not succeed in getting this information from the two other authors. We asked the main authors of the 37 selected papers, and renowned authors in the fi eld, if they had studies with new data that could be included in our review. The answers were negative. Thromboelastography may detect hypercoagulation in early sepsis and improve anticoagulation during extracorporeal treatments F T S B R B h AB B ll S M M F l Aurelia and European Hospital, Rome, Italy p p y Critical Care 2015, 19(Suppl 1):P341 (doi: 10.1186/cc14421) Introduction During early sepsis, activation of the infl ammatory response and coagulation occurs. Extracorporeal therapies are used to adsorb mediators, but the coagulation of fi lters is a drawback [1,2]. The aim of this study is to evaluate whether thromboelastography (TEG) may detect hypercoagulation and may improve anticoagulation during extracorporeal treatments. Conclusion With the studies available as of the end of 2014, it is impossible to conclude whether the use of a VHT in the emergency department decreases mortality. Other studies are needed. References 1. Johansson PI, Stensballe J. Eff ect of haemostatic control resuscitation on mortality in massively bleeding patients: a before and after study. Vox Sang. 2009;96:111-8. p Methods Twenty-four patients with early severe sepsis had a TEG monitoring at basal time (T0) and during three diff erent extracorporeal treatments (T1): coupled plasma fi ltration (CPFA) with heparin infusion (Group A), CPFA with citrate infusion (Group B) and RRT with oXiris fi lter – heparin coated – and no heparin infusion (Group C). ANOVA test was used for the statistical analysis. 2. Messenger BM, et al. TEG-guided massive transfusion in trauma patients. Anesth Analg. 2011;112:S-9. 3. Kashuk K, et al. Initial experiences with POC rapid TEG for management of life-threatening postinjuty coagulopathy.Transfusion. 2012;52:23-33. 3. Kashuk K, et al. Initial experiences with POC rapid TEG for management of life-threatening postinjuty coagulopathy.Transfusion. 2012;52:23-33. y Results Table 1 presents the TEG values in early septic patients at T0. At T1, angle and MA decreased and r increased in Group A at diff erence with Group B and Group C (P <0.01). In group C, LY 30 was higher than in Group A and B (P <0.01). Reference 1. Kashuk JL, et al. r-TEG identifi es hypercoagulability and predicts thromboembolic events in surgical patients. Surgery. 2009;146:764-72. 1. Kashuk JL, et al. r-TEG identifi es hypercoagulability and predicts thromboembolic events in surgical patients. Surgery. 2009;146:764-72. P341 Thromboelastography may detect hypercoagulation in early sepsis and improve anticoagulation during extracorporeal treatments F Turani, S Busatti, R Barchetta, AB Belli, S Martini, M Falco Aurelia and European Hospital, Rome, Italy Critical Care 2015, 19(Suppl 1):P341 (doi: 10.1186/cc14421) References Figure 1 (abstract P340). Coagulation modifi cation prior to TEG analysis in hypercoagulable patients. 1. Livigni S, Bertolini G, Rossi C, et al. BMJ Open. 2014;4:e003536. 2. Ronco C, et al. Crit Care. 2014, 18:309. Value of thromboelastography in managing hypercoagulopathy in intensive care Hypercoagulopathy was suggested by an abnormally short R time in 16 patients (76%), an abnormal alpha angle in 17 cases (81%), a maximum amplitude >74 mm in nine cases (43%) and a high LY30 in one case. Procoagulant treatment was given to seven patients and fi ve patients had received no coagulation modifi cation prior to testing (Figure 1). Eight patients were receiving prophylactic anticoagulation and only one was receiving treatment-dose anticoagulation. A change in management S120 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P342 The three studies were: Johansson and Stensballe, total 832 cases, 121 traumas, raw data unavailable for trauma [1]; Messenger and colleagues, prospective study, 50 cases, mortality similar, raw data unavailable [2]; and Kashuk and colleagues, prospective study, 68 cases, mortality 59% control group, 28% VHT group [3]. Role of thromboelastography in the management of haemorrhage: an observational analysis y C Ward, J Aron, A Gibbon, J Ball C Ward, J Aron, A Gibbon, J Ball Table 1 (abstract P341) Control Sepsis r 8 ± 2 6 ± 3 k 4 ± 2 1.7 ± 0.5* Angle 47 ± 15 67 ± 7* MA 55 ± 8 72 ± 5* LYS 30 1.8 ± 1 0.95 ± 0.9 *P <0.01. St George’s Hospital, London, UK g p Critical Care 2015, 19(Suppl 1):P343 (doi: 10.1186/cc14423) Introduction The aim of this analysis is to explore and evaluate the role of thromboelastography (TEG) in aiding the management of patients admitted with haemorrhage to the ICU. TEG has been shown to rationalise and reduce transfusion requirements [1] but the precise role of TEG in the assessment and management of haemostasis in the bleeding patient is uncertain and has not been previously demonstrated. Methods A prospective audit of TEG analyses performed on patients in the general medical and surgical ICU was recorded over a 2-month period. Operators documented the reason for admission, demographic data, indication for TEG, laboratory results, blood gas analysis, TEG results, diagnosis and subsequent action from the TEG result. Only patients who had been admitted with haemorrhage were included in this analysis. Conclusion In early sepsis, TEG monitoring may detect hyper coagu la- bility. CPFA with heparin, but not CPFA with citrate and oXiris, is able to reverse hypercoagulability. OXiris may induce fi brinolysis. TEG detects alterations of coagulation during early sepsis and extracorporeal treatments. S121 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 TEG analysis Both TEG normal/ Standard normal/ abnormal standard abnormal TEG abnormal Clot factor defi cit 9 (11.5) 20 (25.6) 3 (3.8) Platelet defi cit 11 (14.1) 13 (16.6) 2 (2.6) Fibrinogen defi cit 5 (6.4) 4 (5.1) 5 (6.4) Data presented as n (%). Results Seventy-eight audit sheets were completed, of which 31 identifi ed haemorrhage as the reason for admission. The mean age was 59.3 (range 21 to 90) and the mean APACHE II score was 18.23 (range 11 to 37). The main indications for TEG analysis included coagulopathy (64%) and ongoing haemorrhage (45%). As a result of performing TEG analysis, 23 (74%) patients had a documented change in their management. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) M Popescu, D Tomescu M Popescu, D Tomescu Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P345 (doi: 10.1186/cc14425) Introduction Our aim was to assess hemostasis, using ROTEM, in patients with end-stage liver disease (ESLD) undergoing liver transplantation (LT) and to develop a predictive model for patients prone to high intraoperative blood loss. Introduction This review aims to assess the value of thrombo- elastography (TEG) on the general ICU, which has not been previously demonstrated. TEG is a near-patient assessment of whole blood coagulation and fi brinolysis, which reduces transfusion requirements during cardiothoracic surgery and liver transplantation. Methods We retrospectively analyzed 122 patients who underwent LT between January and December 2013 in a single national center. Patients with acute liver failure or incomplete data were excluded. Demographic data, severity of liver disease assessed by MELD score (model for ESLD), presence of portal vein thrombosis, and laboratory data were recorded preoperatively. We performed concomitant ROTEM assay and standard coagulation tests (prothrombin time (PT), International Normalized Ratio (INR), fi brinogen) 1 hour before surgery and 15  minutes after the neohepatic phase. Intraoperative blood loss was recorded. High blood loss was defi ned as loss of one blood volume during surgery. Correlation between recorded data standard ROTEM parameters and derived thrombodynamic ROTEM parameters (potential index (TPI), maximum velocity of clot formation (MaxV), time to MaxV (MaxVt), AUC) were analyzed using SPSS 19.0. Methods A prospective audit of TEG tests performed on patients being treated on a general ICU was conducted over 2 months. Results A total of 332 TEG tests were performed with a failure rate of 29.8%. Seventy-eight audit sheets were collected. Mean patient age was 68.9 years and mean APACHE II score was 18.1. Admissions included trauma (33.0%), perioperative (43.6%), haemorrhage (42.3%) and sepsis (21%). Standard tests of coagulation demonstrated 22 defi cits in coagulation which were not identifi ed as functionally signifi cant with TEG. Of these, 20 had abnormal clotting factor activity as measured by the INR/APTTr and 13 patients were thrombocytopenic. In total, 52.6% documented that the TEG result changed the management of the patient. In 46.8% of these cases no further blood products were required. In 41% there was no documentation. Role of thromboelastography in the management of haemorrhage: an observational analysis Ten patients did not require any further administration of blood products, which they would have received based on conventional laboratory results. The information gained from TEG also resulted in the omission of anticoagulation in three patients, and with a further two patients anticoagulation increased. Conclusion TEG analysis suggested that 22 patients who were identifi ed as coagulopathic with traditional measures of coagulation did not have a functional defi ciency. Over one-half of TEG studies resulted in a change in management and in 46.8% no further transfusions were required. There was a high technical failure rate and a low audit return rate, which may indicate the need for further training. Reference p g Conclusion TEG aids prompt rationalisation of blood products and titration of anticoagulation in the bleeding patient. TEG identifi es a number of patients who required administration of platelets and other procoagulants which would not have been identifi ed by conventional methods. Several patients would have also received inappropriate transfusions which has both cost and resource implications, alongside the potential adverse eff ects on patients. We recognise that further research is needed to clarify the overall effi cacy of TEG in the bleeding patient. f 1. Da Luz et al. Eff ect of TEG and ROTEM on diagnosis of coagulopathy, transfusion guidance and mortality in trauma: descriptive systematic review. Crit Care. 2014;18:518. 1. Da Luz et al. Eff ect of TEG and ROTEM on diagnosis of coagulopathy, transfusion guidance and mortality in trauma: descriptive systematic review. Crit Care. 2014;18:518. Decreased coagulation kinetics is associated with high blood loss in patients with end-stage liver disease undergoing liver transplantation Decreased coagulation kinetics is associated with high blood loss in patients with end-stage liver disease undergoing liver transplantation M Popescu, D Tomescu Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P345 (doi: 10.1186/cc14425) Reference 1. Bollinger D, et al. Principles and practice of thromboelastography in clinical coagulation management and transfusion practice. Transfusion Med Rev. 2012;26:1-13. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) See Table 1 and Figure 1. Table 1 (abstract P344). Summary of concordance with standard tests versus y g Results After applying exclusion criteria, 72 patients were analyzed with mean age of 54.5 years (SD 11.6) and a median MELD score of 17.4 (7 to 34). Preoperative MCE correlated with age (P = 0.044, 95% CI (–7.50, –0.12)) and MELD score (P = 0.009, 95% CI (–37.21, –6.69)), but not with PT (P = 0.557) or INR (P = 0.623). MaxV correlated with fi brinogen level (P = 0.005, 95% CI (0.01, 0.05)) and AUC correlated with age (P = 0.034, 95% CI (–257.74, –11.91)) and MELD score (P = 0.01, 95% CI (–1,233.14, –215.33)). Patients with portal vein thrombosis had an increase in InTEM CFT (P = 0.002, 95% CI (77.98, 317.97)) and MaxVt (P = 0.03, 95% CI (5.53, 105.63)). No correlation was found between preoperative ROTEM parameters and intraoperative blood loss. We calculated ΔMaxV, ΔMaxVt and ΔAUC as the mathematical diff erence between preoperative and intraoperative MaxV, MaxVt and AUC. High blood loss correlated with ΔAUC (P  = 0.005, 95% CI (15.69, 61.03)), ΔMaxV (P = 9=0.002, 95% CI (–20,413, 6,392)) and ΔMaxVt (P = 0.008, 95% CI (15.69, 61.07)). Figure 1 (abstract P344). Change in management due to TEG results. Conclusion MELD score correlated with a decrease in MaxV and AUC on preoperative ROTEM but not with INR. Patients with portal vein thrombosis have increased InTEM CFT and MaxVt. High blood loss was associated with a decrease in thrombodynamic parameters, but no correlations were found between blood loss and standard ROTEM parameters. Figure 1 (abstract P344). Change in management due to TEG results. S122 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P346 time (aPTT), and prothrombin time (PT). In addition, the plasma levels of vitamin K-dependent coagulation factors were determined. P346 Evaluation of fi xed dose four-factor prothrombin complex concentrate for warfarin reversal at a level 1 trauma center H Drone, J Jancik, J Gorlin, M McCarthy Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P346 (doi: 10.1186/cc14426) p g Results Acute coumarin anticoagulation of rats induced a rise in median bleeding time by ≥2-fold from an average of 823 to 1,800  seconds (maximum observation period) compared with untreated animals. Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation E Herzog, F Kaspereit, W Krege, P Niebl, G Dickneite g p g CSL Behring GmbH, Marburg, Germany g g y Critical Care 2015, 19(Suppl 1):P347 (doi: 10.1186/cc14427) Introduction The study was conducted as a head-to-head comparison of a four-factor prothrombin complex concentrate (4F-PCC) and two diff erent three-factor PCCs (3F-PCC) for eff ective reversal of vitamin K antagonist (VKA)-induced anticoagulation using an established rat model of acute bleeding [1]. The 4F-PCC (containing the human coagulation factors II, VII, IX and X) is indicated for the urgent reversal of acquired coagulation factor defi ciency induced by VKA therapy in adult patients with acute major bleeding. In contrast, the 3F-PCCs (containing factors II, IX, X and only minimal VII) are indicated for the prevention and control of hemorrhagic episodes in hemophilia B patients. Nevertheless, the use of 3F-PCC for correcting hemostasis following warfarin overdose has been discussed but the lack of factor VII in these 3F-PCC products has raised questions about effi cacy. Conclusion In conclusion, 4F-PCC treatment eff ectively decreased apixaban-induced hemorrhage at a clinically relevant dose range. References . Herzog et al. Anesthesiology. Forthcoming 2014. 1. Herzog et al. Anesthesiology. Forthcoming 2014. 1. Herzog et al. Anesthesiology. Forthcoming 2014. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. References 1. Varga C, et al. Transfusion. 2013;53:1451-8. 2. Junagade P, et al. Hematology. 2007;12:439-40. g g p Results Dose-dependent increases in time to hemostasis and total blood loss were observed post apixaban administration with maximum bleeding signals seen at 1,200 μg/kg. Treatment with 4F-PCC resulted in a statistically signifi cant reversal in apixaban-induced bleeding time (all doses) and volume (doses ≥12.5 IU/kg). Of the coagulation parameters measured, thrombin generation initiated using phospholipids only was the in vitro coagulation parameter most sensitive to 4F-PCC-mediated bleeding reversal, although statistically signifi cant 4F-PCC-mediated reductions in the prothrombin time and whole blood clotting time were also observed.f P347 Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation E Herzog, F Kaspereit, W Krege, P Niebl, G Dickneite CSL Behring GmbH, Marburg, Germany Critical Care 2015, 19(Suppl 1):P347 (doi: 10.1186/cc14427) p Reference 1. Dickneite. Thromb Res. 2007;119:643-51. f Results The INR was reduced to <2 in 100% of patients in the 4F-PCC group versus 84.6% of patients in the factor IX group (P <0.05). The INR was reduced to <1.6 in 90.8% of patients in the 4F-PCC group versus 50% in the factor IX group (P <0.05). Mean pre-reversal INRs were 3.5 and 4 and ranged from 1.1 to 10 and from 1.3 to 10 in the 4F-PCC and factor IX group respectively (P = 0.29). On average, a medication cost savings of US$802.63 dollars per patient was calculated from using a fi xed 1,500 unit dose over traditional dosing of 4F-PCC. There was a trend toward a shorter mean ICU LOS in the PCC group when compared with the factor IX group (5.8 vs. 2.8 days) and shorter mean hospital LOS (10.7 vs. 5.7  days), although neither outcome was statistically signifi cant. No diff erence in adverse event rates was observed.ii P348 Four-factor prothrombin complex concentrate (Beriplex® P/N) mediated reversal of apixaban-induced bleeding in a rabbit model E Herzog, F Kaspereit, W Krege, J Mueller-Cohrs, B Doerr, P Niebl, G Dickneite CSL Behring GmbH, Marburg, Germany Critical Care 2015, 19(Suppl 1):P348 (doi: 10.1186/cc14428) Introduction This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex®/Kcentra®; CSL Behring) can eff ectively reverse bleeding associated with the direct oral factor Xa inhibitor apixaban in an established in vivo rabbit model [1,2].i Introduction This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex®/Kcentra®; CSL Behring) can eff ectively reverse bleeding associated with the direct oral factor Xa inhibitor apixaban in an established in vivo rabbit model [1,2].i gif Conclusion A fi xed dose of 1,500 units of 4F-PCC was signifi cantly more eff ective at lowering the INR to a threshold of less than either 2 or 1.6 when compared with a combination of factor IX complex and vitamin K with or without FFP. Further research is needed to investigate clinical outcomes and a possible reduction in ICU and hospital LOS. References Methods For dose-fi nding purposes, anesthetized rabbits were treated with a single intravenous dose of apixaban (800 to 1,600  μg/kg). In a subsequent study phase, anesthetized rabbits were treated with apixaban (1,200 μg/kg) followed by 4F-PCC (6.25 to 100 IU/kg). Bleeding signals were quantifi ed following a standardized kidney incision by measurement of the volume of blood loss and time to hemostasis over an observation period of 30 minutes. Blood samples were collected for monitoring of coagulation parameters. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) In parallel, PT and aPTT were prolonged from 8.9 to 29.9  seconds and from 14.5 to 25.5  seconds, respectively. Treatment with 4F-PCC was able to fully and statistically signifi cantly reverse bleeding, achieving average bleeding times of 676 seconds. In parallel, the elevation in PT was reduced to 15.1 seconds. In contrast, the two 3F-PCC products were not or only partially able to reduce coumarin-induced bleeding with average bleeding times of 1,398 and 1,708  seconds post treatment, respectively. This also correlated with inferior reductions in PT which achieved minimum levels of 23.8 and 29.5 seconds, respectively. There was no reduction in aPTT seen for any treatment option.i Introduction FDA-approved dosing of four-factor prothrombin complex concentrate (4F-PCC) in the USA is based on an INR and weight; however, there are data suggesting that a fi xed dose of 4F-PCC may be suffi cient for INR reversal and hemostasis [1,2]. The objective of this study was to assess the effi cacy and safety of a fi xed dose of 1,500 units of 4F-PCC. Historically, warfarin reversal included a combination of factor IX complex, vitamin K, and fresh frozen plasma (FFP). Using a fi xed dose of 4F-PCC may also provide signifi cant cost savings when compared with traditional dosing. y p Conclusion In conclusion, this fi rst direct comparison of 4F-PCC and 3F-PCCs for the reversal of VKA anticoagulation in a rat model of acute bleeding suggests that replenishment of all four vitamin K-dependent coagulation factors including factor VII as achieved using a 4F-PCC may result in superior effi cacy compared with the use of 3F-PCCs. R f Methods This retrospective chart review compared 26 admitted adults who received a fi xed dose of 1,500 units 4F-PCC with 26 patients who received a combination of factor IX complex and vitamin K, with or without FFP, for warfarin reversal from 1 January 2012 to 1 November 2014. Primary outcomes included reversal to an INR of <2 and reversal to an INR of <1.6. Secondary outcomes included ICU and hospital length of stay (LOS), change in INR, INR nadir, potential cost savings from 4F-PCC versus traditional dosing, and major adverse eff ects. Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model M Honickel1, T Braunschweig1, J Van Ryn2, R Rossaint1, O Grottke1 1RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co KG, Biberach, Germany Critical Care 2015, 19(Suppl 1):P351 (doi: 10.1186/cc14431) Results In the ShBl injury strand there was a signifi cant reduction in ATC in Early compared with Late PRBC:FFP treatment (TEG R and K times) in both the prehospital (P = 0.004 and P = 0.003 respectively, ANOVA) and early in-hospital (P = 0.002 and P = 0.005) phases, although clotting was normalised in the Late group within 60  minutes of initiating PRBC:FFP. Prehospital base defi cit (BD) was signifi cantly attenuated in ShBl Early versus Late (9.0 ± 2.1 vs. 14.4 ± 2.2 mM). BD improved in both Early and Late treatment groups during the in-hospital phase but remained greater in the Late group throughout (P <0.001). In the Bl injury strand the trend in coagulation was similar to that seen in the ShBl injury strand (but the diff erences between Early and Late did not attain statistical signifi cance). By contrast, Early versus Late PRBC:FFP treatment did not result in a diff erence in BD in the Bl strand. Finally, there was no diff erence in the total amount of PRBC:FFP used between the two treatments in either injury strand, but in both injury strands the Early treatment groups required signifi cantly less saline (P <0.001). Conclusion Prehospital use of PRBC:FFP may attenuate ATC and improve physiological status. Furthermore the amount of crystalloid may be reduced with potential benefi t of reducing the third-space eff ect and later tissue oedema. M Honickel1, T Braunschweig1, J Van Ryn2, R Rossaint1, O Grottke1 1RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co KG, Biberach, Germany Critical Care 2015, 19(Suppl 1):P351 (doi: 10.1186/cc14431) Introduction The anticoagulant eff ect of dabigatran can be reversed with idarucizumab or PCCs in porcine blood in vitro [1]. However, the impact on clinical parameters such as blood loss is not known. Thus, this study assessed the effi cacy of idarucizumab in comparison with PCC and aPCC in dabigatran-anticoagulated swine following polytrauma on clinically relevant endpoints. Reference 1. Grottke O, et al. Crit Care. 2014;18:R27. p p Results According to LFTEG, polytrauma patients had statistically signifi cant abnormalities in platelet aggregation (intensity of contact coagulation (ICC)), in coagulation (intensity of coagulation drive (ICD), clot maximum density (MA)) and in fi brinolytic activity (index of retraction and clot lysis (IRCL)). ICC in patients with multiple injuries was decreased by 27.51%, ICD was decreased by 34.68%, MA was decreased by 75.16%, IRCL was 91.06% above the norm. Patients of group 1 according to LFTEG had signifi cant changes in all parts of coagulation 24  hours after intensive care. Indicators of platelet hemostasis characterized by persistence of hypoaggregation: ICC was decreased by 24.51%, compared with the norm; parameters of coagulation and fi brinolysis had a reliable trend toward normal and decreasing the activity, the fi brinolysis index reached normal reference values. P350 Results Dabigatran levels were comparable between groups (571  ± 174  ng/ml) and resulted in altered coagulation variables. Blood loss was comparable 12 minutes post trauma between groups (801 ± 49 ml) and increased to 3,816 ± 236 ml in anticoagulated control animals post injury. Idarucizumab treatment reduced total blood loss to 1,086  ± 55 ml (P <0.005 vs. all), aPCC to 1,639 ± 104 ml (P <0.05 vs. control) and PCC to 1,797 ± 80 ml (P <0.05 vs. control) after 5 hours. All animals in the intervention groups survived, whereas control animals died within the observation period (mean survival: 89 minutes, range: 62 to 145 minutes). In histopathology no signs of thromboembolic events were present. Altered coagulation variables returned to baseline levels after idarucizumab application and were also signifi cantly, although inconsistently and to a lesser extent, ameliorated following PCCs. Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model y p Methods After ethical approval, 28 male pigs were administered dabigatran etexilate (30 mg/kg twice daily p.o.) for 3 days. Dabigatran was administered intravenously in anaesthetised animals on day 4 to achieve consistent high concentrations. Animals were randomised to receive idarucizumab (60 mg/kg, n = 7), PCC (50 U/kg; n = 7), aPCC (50 U/kg; n = 7) or placebo (n = 7). Intervention started 12 minutes after bilateral femur fractures and a standardised blunt liver injury. The primary endpoint was blood loss (observation period 300  minutes). Further, histopathology, haemodynamics and several coagulation variables were also assessed. Data were analysed by repeated-measures ANOVA (mean ± SD). Acknowledgements © Crown copyright 2014. Published with the permission of the Dstl on behalf of the Controller of HMSO. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of prehospital versus immediate in-hospital packed red blood cells and fresh frozen plasma (PRBC:FFP) in two models of severe battlefi eld injury. of prehospital versus immediate in-hospital packed red blood cells and fresh frozen plasma (PRBC:FFP) in two models of severe battlefi eld injury. Patients in group 2 had hypoaggregation and hypocoagulation state with increased activity of fi brinolysis: ICC was reduced by 25.62%, ICD decreased by 19.76%, MA was decreased by 22.34%, IRCL was increased by 24.52%. Clinically, patients of group 1 had reduced indicators for infectious complications, reducing the term of mechanical ventilation and reducing the volume of blood transfusions. j y Methods This is a prospective randomised controlled trial using in vivo models of injury conducted in accordance with the Animals (Scientifi c Procedures) Act, 1986. Two injury strands were investigated in 43 terminally anaesthetised Large White pigs: whole body blast exposure (Bl) or no blast (ShBl) plus soft tissue injury and haemorrhage. Thirty minutes later animals were randomly allocated to a 60-minute simulated prehospital hypotensive resuscitation with either PRBC:FFP (1:1 ratio) or 0.9% saline (Early and Late groups respectively). This was followed by 150 minutes of simulated in-hospital resuscitation with a revised normotensive target whereby PRBC:FFP was initiated in the Late group and continued in the Early group.i g Conclusion Patients with multiple injuries have violation in all parts of blood coagulation. The use of prothrombin complex concentrate can reduce the severity of pathological changes in the hemostatic system in patients with polytrauma. Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) g p y O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) Introduction To compare the eff ectiveness of prothrombin complex concentrate and fresh frozen plasma (FFP) in patients with multiple injuries, complicated with coagulopathy bleeding. Introduction To compare the eff ectiveness of prothrombin complex concentrate and fresh frozen plasma (FFP) in patients with multiple injuries, complicated with coagulopathy bleeding. Methods The study involved 51 patients who entered Odessa Regional Hospital with traumatic injuries (concomitant skeletal trauma) complicated with hypocoagulation. Patients were divided into two groups: in the fi rst group (26 patients), as a treatment for coagulopathy, was administered PCC in a dose of 1 ml/kg (25 IU/kg); in the second group (25 patients) was administered FFP in a dose of 15  ml/kg. Evaluation of the functional state of the hemostasis system was carried out using low-frequency thromboelastography (LFTEG) on admission to hospital and 24 hours after the patient’s admission to the ICU. y g Conclusion All medical interventions were associated with reduced blood loss and increased survival. However, idarucizumab, a specifi c antidote to dabigatran, reduced total blood loss more prominently and normalised coagulation parameters to a greater degree as compared with either PCC or aPCC. Reference 1. Grottke O, et al. Crit Care. 2014;18:R27. Reference Benefi cial eff ects of prehospital versus immediate in-hospital blood products during resuscitation in two models of severe military injury S Watts, G Nordmann, C Wilson, A Carter, H Poon, E Kirkman Dstl, Salisbury, UK Critical Care 2015, 19(Suppl 1):P349 (doi: 10.1186/cc14429) Methods Rats received an oral dose of 2.5 mg/kg phenprocoumon. At 15.75 hours post dosing, animals were treated with a single intravenous dose of saline, 4F-PCC (Beriplex® P/N, Kcentra®; CSL Behring) or 3F-PCC (Bebulin® VH; Baxter and Profi lnine® SD; Grifols). Study endpoints included bleeding following tail clip, activated partial thromboplastin Introduction Acute trauma coagulopathy (ATC) is seen in 30 to 40% of severely injured trauma casualties. Early use of blood products is thought to attenuate ATC. This study determined the potential impact S123 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P352 Coagulation support algorithm with rapid TEG and functional fi brinogen TEG in critical bleeding: more results and less time E De Blasio, C Pellegrini, A Federico, V Rocco, M Fumi, Y Pancione, S Sale, D Liberti Hospital G. Rummo, Benevento, Italy Critical Care 2015, 19(Suppl 1):P352 (doi: 10.1186/cc14432) Hospital G. Rummo, Benevento, Italy Critical Care 2015, 19(Suppl 1):P352 (doi: 10.1186/cc14432) Introduction Early coagulation support is essential in massively bleeding patients. A Coagulation Support Algorithm (CSA), integrating rapid TEG (r-TEG) and functional fi brinogen TEG (ff -TEG) could shorten the time to a tailored treatment (Figure 1). Introduction Early coagulation support is essential in massively bleeding patients. A Coagulation Support Algorithm (CSA), integrating rapid TEG (r-TEG) and functional fi brinogen TEG (ff -TEG) could shorten the time to a tailored treatment (Figure 1). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 S124 , pp http://ccforum.com/supplements/19/S1 T d s e s e A e e l , g Table 1 (abstract P353). Results of the cost-eff ectiveness model Total medical Total costs/ Treatment QALYs costs ($) QALY ($) Antibiotics + albumin 2.45 7,628 3,111 Antibiotics 1.48 7,682 5,182 Results Total costs were decreased when using albumin, and the improved survival resulted in an additional QALY for patients on albumin, decreasing the cost per QALY. See Table 1 and Figure 2. Conclusion The use of albumin in the treatment of SPB is cost-eff ective. References 1. Poca M, et al. Clin Gastroenterol Hepatol. 2012;10:309-15. 2. Wells CD, et al. Dig Dis Sci. 2004;49:453-8. P354 Estimation of the latent therapeutic demand for albumin in the USA: a focus on three indications 1 l2 Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 1 (abstract P352). Coagulation Support Algorithm. Figure 1 (abstract P352). Coagulation Support Algorithm. Table 1 (abstract P352). Comparison of time to results Test k-TEG r-TEG r-TEG + ff -TEF SCT r (minutes) 13.8 ± 7.1 2.6 ± 2 – ACT (seconds) 265.7 ± 171.9 – 105.2 ± 46.3 TMA (minutes) 42.6 ± 12.4 25.4 ± 14.1 – CSAT (minutes) 21 ± 7.4 Data presented as mean ± SD. ACT, activated clotting time; CSAT, Coagulation Support Algorithm total time; SCT, standard coagulation tests; TMA, time to maximum amplitude. P352 r: r-TEG versus k-TEG, P = 0.0000003; r-TEG versus SCT, P = 0.000000001; k-TEG versus SCT, P = 0.000000009; TMA: r-TEG versus k-TEG, P = 0.0001; r-TEG versus SCT, P = 0.00000004; k-TEG versus SCT, P = 0.000002; ACT versus r of k-TEG (seconds), P = 0.000004; CSAT versus k-TMA, P = 0.00000005. Table 1 (abstract P352). Comparison of time to results Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Methods A retrospective comparison of the time to available TEG and Standard Coagulation Tests (SCT: INR, aPTTr, fi brinogen level) results in two groups of bleeding and coagulopathic patients using citrate kaolin-TEG (k-TEG) or the CSA protocol (r-TEG/ff -TEG). Statistical analysis was performed with Student’s t test for unpaired samples. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Results Twenty-three patients for each k-TEG and CSA group were compared. The time to available results was shorter using the CSA protocol in comparison with k-TEG (Table  1). The diff erences were both statistically (P  <0.00001) and clinically (mean reduction time 21 minutes) signifi cant. SCT needed the longest time to obtain the fi nal results. Table 1 (abstract P353). Results of the cost-eff ectiveness model Total medical Total costs/ Treatment QALYs costs ($) QALY ($) Antibiotics + albumin 2.45 7,628 3,111 Antibiotics 1.48 7,682 5,182 Conclusion The implementation of a CSA, including r-TEG and ff -TEG, could shorten the time to a targeted treatment in critically bleeding patients. References Results Total costs were decreased when using albumin, and the improved survival resulted in an additional QALY for patients on albumin, decreasing the cost per QALY. See Table 1 and Figure 2. C l i Th f lb i i th t t t f SPB i t ff ti 1. Stensballe J, et al. Curr Opin Anesthesiol. 2014;27:212-18. 3. Kashuk JL, et al. Ann Surg. 2010;251:604-14. g g Conclusion The use of albumin in the treatment of SPB is cost-eff ective. f P353 Use of albumin in spontaneous bacterial peritonitis is cost-eff ective A Farrugia1, M Bansal2, P Caraceni3 P353 Use of albumin in spontaneous bacterial peritonitis is cost-eff ective A Farrugia1, M Bansal2, P Caraceni3 1University of Western Australia, Perth, Australia; 2Thought Semantics LLC, Sterling, VA, USA; 3University of Bologna, Italy Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) g 1University of Western Australia, Perth, Australia; 2Thought Semantics LLC, Sterling, VA, USA; 3University of Bologna, Italy Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) P353 1. Poca M, et al. Clin Gastroenterol Hepatol. 2012;10:309-15. 2. Wells CD, et al. Dig Dis Sci. 2004;49:453-8. Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) Introduction Adequate fl uid therapy is essential to the care of septic patients, aiming to optimize oxygen delivery without compromising microcirculation. In recent years, a few studies have suggested that albumin may be superior when compared with crystalloids in severe cases of septic shock. However, there are no data in the fi rst hours of resuscitation. The aim of this study is to evaluate whether albumin 4% solution compared with lactated Ringer decreases 30-day mortality in cancer patients with septic shock. p p Methods The Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study is a prospective, randomized, double-blind and controlled trial, with 360 patients. Until November 2014, at the Cancer Institute of University of São Paulo, we enrolled 110 patients with cancer and septic shock to receive as resuscitation fl uid in the fi rst 12 hours of ICU an admission bolus of albumin 4% solution or lactated Ringer. The primary outcome was 30-day mortality. Secondary outcomes include ICU mortality, ICU and hospital length of stay, 90-day mortality, daily SOFA score, rates and length of mechanical ventilation, renal replacement, needing of vasopressor drugs, status performance and fl uid balance. Results From 650 eligible patients, 110 patients were included in the study – 50 patients in the albumin group and 60 in the Ringer group. The mean age was 63 (57 to 70) years in the albumin group and 61 (51 to 71) in the Ringer group, P = 0.508. Most patients were male (58% in the albumin group vs. 56.1% in the Ringer group, P = 0.846). The ECOG was similar between the albumin and Ringer groups ((0) 26% vs. 8%, (1) 38% vs. 36.8%, (2) 20% vs. 38.6%, (3) 16% vs. 15.8%, P = 0.05). The SAPS 3 admission score was 51 ± 13 in the albumin group and 49 ± 10 in the Ringer group, P = 0.492. The total amount of administered fl uid in the fi rst 12 hours of resuscitation was 1,000 ml (1,000 to 1,500) in the albumin group and 1,000 ml (1,000 to 1,000) in the Ringer group, P = 0.59. The 12-hour fl uid balance was 1,053 ml (385 to 1,700) in the albumin group and 990 ml (200 to 1,525) in the Ringer group. The 30- day mortality was similar in both groups (60% in the albumin group and 50.9% in the Ringer group, P  = 0.34). Reference Reference 1. Caironi P, et al. N Engl J Med. 2014;370:1412-21. Methods A decision analysis model was constructed using Excel. The model is based on the relationships of the epidemiological and clinical factors shown in the infl uence diagram (exemplifi ed in Figure  1 for sepsis). Data for the individual factors were obtained from the literature. One-way sensitivity analysis was used to generate Tornado diagrams (exemplifi ed in Figure 2 for albumin use in sepsis) to determine the relative contribution of diff erent factors to the LTD. Probabilistic sensitivity analysis was used to generate a probability distribution and calculate a mean level for the LTD of each indication. p g p y p E Scotti, M Ferrari, M Chiodi, F Zadek, I Belloni, L Zazzeron, T Langer, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436) E Scotti, M Ferrari, M Chiodi, F Zadek, I Belloni, L Zazzeron, T Langer, L Gattinoni, P Caironi One-way sensitivity analysis was used to generate Tornado diagrams (exemplifi ed in Figure 2 for albumin use in sepsis) to determine the relative contribution of diff erent factors to the LTD. Probabilistic sensitivity analysis was used to generate a probability distribution and calculate a mean level for the LTD of each indication. Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436) g , , y Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436 Results On average, albumin use was calculated as 104 g per 1,000 inhabitants in severe sepsis, 157 g per 1,000 inhabitants in liver diseases and 61 g per 1,000 inhabitants in CABG. This shows a total LTD of 322 g per 1,000 use of albumin in the US annually. Introduction The induction of ECMO may result in metabolic acidosis [1] due to circuit priming with chloride-rich fl uids, and the sudden decrease in plasma strong ion diff erence (SID). This eff ect can be attenuated using balanced solutions with a SID equal to the patient’s plasma bicarbonate concentration (HCO3 –) [2]. We aimed to compare the eff ects of a novel balanced solution (SID equal to patients’ HCO3 –) with those of commonly employed crystalloids for circuit priming in patients undergoing venovenous ECMO. y Conclusion Albumin consumption in the USA currently averages 479 g per 1,000 population [3]. Hence, the LTD of these three evidence-based indications represents 67% of current usage. Further work is needed to assess the LTD for albumin in other, less well-defi ned areas. R f Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) No signifi cant diff erences in the other secondary outcomes were observed between the two groups. Methods The Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study is a prospective, randomized, double-blind and controlled trial, with 360 patients. Until November 2014, at the Cancer Institute of University of São Paulo, we enrolled 110 patients with cancer and septic shock to receive as resuscitation fl uid in the fi rst 12 hours of ICU an admission bolus of albumin 4% solution or lactated Ringer. The primary outcome was 30-day mortality. Secondary outcomes include ICU mortality, ICU and hospital length of stay, 90-day mortality, daily SOFA score, rates and length of mechanical ventilation, renal replacement, needing of vasopressor drugs, status performance and fl uid balance. Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Figure 2 (abstract P354). Relative importance of inputs into the LTD model for sepsis. g Conclusion In cancer patients with septic shock, resuscitation with albumin 4% as compared with lactated Ringer did not improve the rate of survival at 30 days. allocation, but many of the clinical and epidemiologic variables are subject to uncertainty. Decision analysis [2] may assist in generating an assessment of the demand for albumin. 2. Goel V. CMAJ. 1992;147:413-7. Estimation of the latent therapeutic demand for albumin in the USA: a focus on three indications g y g y Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) Introduction Assessing the cost-eff ectiveness of therapeutic interven- tions is increasingly crucial for health decision-making. Spontaneous bacterial peritonitis (SBP) is one of the major complications of liver cirrhosis. The use of albumin in conjunction with antibiotics has been shown to be eff ective through clinical trials [1]. Introduction The use of albumin in therapeutics is controversial in several areas and requires assessment based on evidence for eff ective resource allocation. Supported indications include sepsis, areas of hepatic diseases and coronary artery bypass grafts (CABG). Latent therapeutic demand (LTD) [1] is the underlying evidence-based demand ensuring ample supplies of drugs are available and aff ordable. Estimating the LTD would assist decision-making and resource Methods A decision tree (TreeAge®) (Figure  1) was populated from published sources for clinical, cost and epidemiologic variables. The perspective taken was that of the US payer. The robustness of the model was checked using one-way and probabilistic sensitivity analyses. The clinical course was followed for 3 months or until death. Total medical costs and quality-adjusted life years (QALYs) [2] were calculated. S125 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury Conclusion During the resuscitation phase of the burn patients, the use of HES (130/0.4/6%) at low doses does not seem to cause more risk or injury according to RIFLE or AKIN criteria than those reported by studies in burn patients resuscitated without HES. However, the need for RRT is associated with a high mortality, although in many cases the display is terminal. Introduction The aim of our study is to investigate the eff ect of intraoperative use of gelatin in living donor liver transplantation on postoperative acute kidney injury (AKI). It has been demonstrated that ischemia and chloride-liberal fl uid management cause AKI in liver transplantation [1]. Gelatin has minimal side eff ects on renal functions [2]; however, it might be a reason for postoperative AKI. g Methods A total of 154 liver transplantation patients were retrospectively evaluated between September 2011 and September 2013, and among these, 128 patients were included in the study. The patients who were under 18 years old, transplanted from cadaveric donors and needed preoperative renal replacement therapy were excluded. The patients were divided into two groups as GI (without gelatin administration) and GII (with gelatin administration). The patient’s age, gender, actual body weight, diagnoses, MELD score, APACHE II score, duration of operation, total clamping time, noradrenalin infusion rate, amount of erythrocyte suspension, fresh frozen plasma (FFP) and thrombocyte suspension used, intraoperative fl uid balance, intraoperative and total clamping diuresis, serum creatinine levels on the postoperative 1st, 2nd, 4th and 7th days, duration of mechanical ventilation, length of ICU and hospital stay, hospital and 1-year mortality rate were recorded. The changes in creatinine levels on the 1st, 2nd, 4th and 7th days were evaluated according to the KDIGO guideline for AKI [3]. Infl uence of anaesthetic factors on skin graft viability in a burns ICU C Isitt, KA McCloskey, A Cabello, P Sharma, MP Vizcaychipi Chelsea and Westminster Hospital, London, UK Critical Care 2015, 19(Suppl 1):P359 (doi: 10.1186/cc14439) Introduction Graft failure is a major cause of morbidity in patients with burns, resulting in increased length of hospital stay and increased number of operations. At our regional burns unit we collated the data from anaesthetic charts of patients admitted to our burns ICU who required skin grafting. The aim was to analyse whether any anaesthetic variables contribute to graft failure. g Methods Thirty-fi ve patients were included in the analysis with a total of 191 operations. References Methods We randomly assigned patients with acute respiratory failure in need of ECMO to receive either NaCl 0.9% (NS, SID = 0), Ringer lactate (RL, SID = 28), or a novel balanced solution (Solution X, SID equal to the patient’s HCO3 –) for circuit priming solution. Arterial blood gases and laboratory parameters were collected at 0, 5, 30, 60, 90, and 1. Stonebraker J, et al. J Clin Immunol. 2014;34:233-44. 2. Goel V. CMAJ. 1992;147:413-7. 3. Market Research Bureau. The plasma proteins market in the USA (2013). http://marketingresearchbureau.com/list-of-reports/ the-plasma-proteins-market-in-the-united-states/. S126 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 120 minutes after pump start. SID, base excess (BE) and total weak acids (Atot) were calculated. total clamping time was longer and amount of blood products used during surgery was more than the other group. Which of these factors is associated with AKI has to be revealed with further studies. 120 minutes after pump start. SID, base excess (BE) and total weak acids (Atot) were calculated. total clamping time was longer and amount of blood products used during surgery was more than the other group. Which of these factors is associated with AKI has to be revealed with further studies. Results We enrolled 20 patients (23 priming procedures – RL, n = 8; NS, n = 8; Solution X, n = 7). ECMO was initiated for ARDS (45%), bridge to lung transplant (25%), acute graft failure after transplant (15%), and acute on chronic respiratory failure (15%). Average priming volume was 10 ± 5 ml/kg; patients’ baseline HCO3 – was 28 ± 6 mEq/l. During the fi rst 2 hours after ECMO initiation, arterial pH raised similarly in all groups (P = 0.39) due to CO2 removal. In contrast, BE decreased starting after 5 minutes in both the NS and RL groups (BE variation, –2.2 ± 1.7 and –1.9 ± 1.3 mEq/l, P <0.001 vs. baseline; P = 0.04 for interaction, two-way ANOVA, 2-hour period). No BE changes were observed in the Solution X group (0.3 ± 0.8 mEq/l). In the NS group, BE reduction was associated with a reduction in SID (from 39 ± 8 to 34 ± 6 mEq/l at 5 minutes, P = 0.008), entirely due to an increase in Cl (103 ± 7 vs. 108 ± 6 mEq/l, P = 0.001). References In the RL group, BE and SID reductions (40 ± 8 vs. 36 ± 8 mEq/l, P  = 0.008) were associated with an increase in both Cl (105  ± 7 vs. 107 ± 7 mEq/l, P = 0.01) and lactate (1.4 ± 0.6 vs. 2.2 ± 1.0 mEq/l, P = 0.008). No changes were observed in other electrolyte concentrations. Dilution did not diff er between groups (P = 0.25 for Atot variation). The acidifying eff ect of NS and RL was amplifi ed in patients with higher baseline HCO3 –. References 1. Nadeem A, et al. Crit Care. 2014;18:625. 2. Eremenko AA, et al. Anesteziol Reanimatol. 2001;3:58-61. 3. KDIGO AKI Work Group. Kidney Int. 2012;2 Suppl:1–138. 1. Nadeem A, et al. Crit Care. 2014;18:625. 2. Eremenko AA, et al. Anesteziol Reanimatol. 2001;3:58-61. 3. KDIGO AKI Work Group. Kidney Int. 2012;2 Suppl:1–138. Incidence of acute kidney injury in critically burned patients resuscitated with crystalloid and colloid according to parameters of transpulmonary thermodilution, diuresis and lactic acid P Extremera Navas, M Sanchez Sanchez, I Pozuelo Echegaray, A Agrifoglio Rotaeche, A Robles Caballero, A García de Lorenzo Hospital Universitario la Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P358 (doi: 10.1186/cc14438) Introduction The purpose was to study the incidence of acute kidney injury (AKI) according to RIFLE and AKIN criteria in critically ill burn patients resuscitated with Ringer’s solution and supplements of lower molecular weight hydroxyethyl starch (HES)130/0.4/6%, and to determine the relationship between RRT indication and mortality. 3 Conclusion As compared with NS and RL, the use of a novel balanced solution with a SID equal to the patient HCO3 – level for ECMO priming uniquely avoids the addition of metabolic acidosis to patients with uncompensated hypercapnia. Methods We studied 165 consecutive patients admitted to the critical care burn unit. Resuscitation was performed using lactated Ringer’s solution and HES at a low dose to achieve urine output, lactate levels, and transpulmonary thermodilution parameters. The contributions of colloids and crystalloids were measured, and renal function was evaluated. Statistical analysis was performed using the Spearman test. Results The average total body surface area (TBSA) burned was 30 ± 15%, and the median of the total volume needed in the fi rst 24 hours was 4.01  ml/kg/% TBSA burned. According to the RIFLE criteria, 10 (6.1%) patients presented with risk, 11 (6.7%) presented with injury, and 11 (6.7%) presented with failure. References 1. Liskaser, et al. Anesthesiology. 2000;93:1170-3. 2. Langer, et al. Intensive Care Med. 2012;38:686-93. 1. Liskaser, et al. Anesthesiology. 2000;93:1170-3. 2. Langer, et al. Intensive Care Med. 2012;38:686-93. References According to the AKIN criteria: 9.7% presented stage I, 3% stage II and 10.3% stage III. Replacement therapy (RRT) was performed in 15 patients (9.1%). In six of these patients RRT was employed in the fi nal stages of multiorgan failure. In the remaining nine patients, for various reasons only one survived. P357 Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury HK Atalan1, B Gucyetmez2, S Aslan1, M Berktas3, KY Polat1 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P357 (doi: 10.1186/cc14437) Association of elevated levels of plasma chloride, in severity and mortality, in adult patients in the ICU M Aguilar Arzapalo Hospital O’Horan, Mérida, Mexico Critical Care 2015, 19(Suppl 1):P360 (doi: 10.1186/cc14440) Introduction For a long time, many investigators have tried to demon- strate increased mortality associated with acid–base distur bances. In this study, we sought to determine the association of hyperchloremia measured at ICU admission and whether this electrolyte disturbance is associated with an increase in morbidity and mortality. Methods A retrospective study was conducted. Patients with a diagnosis of AKI according to KDIGO creatinine criteria and available urinary chemistry at one point during their ICU stay were evaluated. Day 0 was defi ned as the day when SIDu was calculated from urinary spot analysis (SIDu = Na+U + K+U – Cl–U). Patients were followed and staged for AKI in the next 3 days. AKI reversibility was defi ned according to the lack of criteria for AKI. y y Methods Data were retrospectively collected for consecutive adult patients admitted to Agustin O’Horan Hospital ICU, between January 2011 and July 2014, who underwent inpatient medical treatment using electronic fi les. Results In total, 143 critically ill patients with a diagnosis of AKI were included. SIDu at day 0 did not diff er between diff erent AKI stages at day 0. SIDu at day 0 was statistically diff erent between diff erent AKI stages at days 1, 2, 3 (Table 1). SIDu at day 0 was statistically diff erent between reversible and not reversible AKI at days 1, 2, 3 (Table 2). A conventional receiver-operating curve was generated to assess the accuracy of SIDu to predict AKI reversibility at day 1. AUC for SIDu was 0.82 (P <0.0001; 95% CI: 0.75 to 0.88). i Results The dataset consisted of 936 medical fi les and serum chloride concentration values on admission, 853 being eligible. Hyperchloremia (serum chloride >110 mmol/l) is quite common, with an incidence of 47.71%. Patients were propensity matched based on their association with death and hyperchloremia. Of the 853 patients collected, patients with hyperchloremia after admission (n = 446, 52.3%), patients were matched to patients who had normal serum chloride levels after admission. These two groups were well balanced with respect to all variables collected. The hyperchloremic group was at increased risk of mortality at ICU discharge, relative risk ratio = 1.81; 95% confi dence interval, 1.41 to 2.51 risk increase of 25.31%. Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury These were a combination of debridement, split skin grafts (SSG) and change of dressings. All patients were admitted to our burns ICU between January 2009 and October 2013. Exclusion criteria were death prior to discharge and initial surgery at a diff erent hospital. Sixteen patients had good graft viability (Group A) and 19 patients had poor graft viability (Group B). Logistical regression was performed using SPSS (Version 22.0). Hosmer and Lemeshow testing was used to confi rm goodness of fi t. Independent variables were age, sex, preoperative haemoglobin, intraoperative fl uid resuscitation, blood products, inotropes, volatile agents and temperature. Poor graft viability was defi ned as requiring at least one additional skin graft. Analysis was performed on all operations and then by subtype of operation (that is, SSG and debridement, SSG only). Results In total, 128 patients were categorized as GI (58, 45%) or GII (70, 55%). Total clamping time, intraoperative diuresis, intraoperative crystalloid use, intraoperative fl uid balance, operation bleeding, erythrocyte suspension, FFP and thrombocyte suspension use and postoperative lactate levels of GII were statistically signifi cantly higher than GI (P <0.001 for each). According to the KDIGO guideline, AKI in GII on the 1st, 2nd, 4th and 7th days (11.4%; 20%; 24.3%; 17.1%) was statistically signifi cantly higher than GI (P <0.001 for each). Conclusion In patients who received gelatin, kidney dysfunction in the postoperative period was observed more frequently. Also in this group, S127 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results There was no signifi cant diff erence in age, %total burn surface area or Belgian Outcome Burns Injury score between the groups. For all operation data, use of colloids was found to signifi cantly contribute towards poor graft viability (P  = 0.035, 95% CI). When analysis was performed on only SSG and debridement operations, colloids remained signifi cant (P = 0.034, 95% CI) and metarminol use was found to signifi cantly contribute (P = 0.028, 95% CI) to poor graft viability. Overall use of inotropes was not signifi cant between the two groups. Other variables including minimum and maximum temperature, preoperative haemoglobin and blood transfusion were not found to be signifi cant. References 1. Gauthier PM, Szerlip HM. Metabolic acidosis in the intensive care unit. Crit Care Clin. 2002;18:289-308. 1. Gauthier PM, Szerlip HM. Metabolic acidosis in the intensive care unit. Crit Care Clin. 2002;18:289-308. 2. Eisenhut M. Causes and eff ects of hyperchloremic acidosis. Crit Care. 2006;10:413. 2. Eisenhut M. Causes and eff ects of hyperchloremic acidosis. Crit Care. 2006;10:413. 3. Wooten EW. Science review: quantitative acid–base physiology using the Stewart model. Crit Care. 2004:8:448-52. 3. Wooten EW. Science review: quantitative acid–base physiology using the Stewart model. Crit Care. 2004:8:448-52. 36 Urinary strong ion diff erence and acute kidney injury: an early marker of renal dysfunction? i Conclusion Our results suggest that the use of colloids is a contributor to poor graft viability in burns. This was found to be independent of temperature and overall inotrope use; however, the use of metarminol may be a contributing factor. y P Balsorano1, A De Gaudio1, Stefano Romagnoli1, Ipsita Krishnan2 1AOUC Careggi, Florence, Italy; 2Rhode Island Hospital, Providence, RI, USA Critical Care 2015, 19(Suppl 1):P361 (doi: 10.1186/cc14441) Introduction Kidneys play a crucial role in the regulation of electrolytes and acid–base homeostasis. Impaired renal function is associated with greater urinary strong ion diff erence (SIDu) in patients with metabolic acidosis [1]. In critically ill patients, several factors, such as infused fl uids and acid endogenous production, would lead to changes in plasma SID and acid–base homeostasis without renal regulation of urinary electrolytes and SIDu [2]. Hence, AKI can be highlighted as an inability to address acid–base metabolic disturbances, which may be detected before major increases in creatinine or decreases in urine output. We evaluated the eff ects of renal function on urinary strong ion excretion using the Stewart approach to acid–base in critically ill patients with AKI. P360 Association of elevated levels of plasma chloride, in severity and mortality, in adult patients in the ICU Admission hyperchloremia was associated with increased morbidity, mortality and higher scores in severity scales; this association was statistically important. See Figure 1. Conclusion This retrospective cohort trial demonstrates an association between hyperchloremia and poor ICU admission outcome (death). Additional studies are required to demonstrate a causal relationship between these variables. Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission AKI stage 3 2 1 0 P value Day 1 48.1 (21) 46 (22) 37.9 (20) 17.3 (22) <0.001 Day 2 40.2 (23) 45.9 (20) 45 (23) 29 (22) 0.004 Day 3 40.3 (26) 47.2 (18) 53.2 (23) 31 (23) 0.006 Table 2 (abstract P361). SIDu (mEq/l) between reversible versus not reversible AKI at days 1, 2, 3 Reversible Not reversible P value Day1 16.8 (23) 43.9 (21) 0.0001 Day2 28.5 (24) 45.3 (22) 0.0001 Day3 30 (24) 47.3 (21) 0.0001 Conclusion SIDu identifi ed patients with reversible AKI with good accuracy. SIDu can be a promising, simple and cost-eff ective tool in AKI patient evaluation. Further research is needed to assess SIDu capability to early detect patients with renal dysfunction before increases in creatinine or decreases in urine output. References 1. Moviat M, et al. J Crit Care. 2012;27:255-60. 2. Masevicius FD, et al. Crit Care Resusc. 2010;12:248-54. Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission AKI stage 3 2 1 0 P value Day 1 48.1 (21) 46 (22) 37.9 (20) 17.3 (22) <0.001 Day 2 40.2 (23) 45.9 (20) 45 (23) 29 (22) 0.004 Day 3 40.3 (26) 47.2 (18) 53.2 (23) 31 (23) 0.006 Table 2 (abstract P361). SIDu (mEq/l) between reversible versus not reversible AKI at days 1, 2, 3 Figure 1 (abstract P360). Group mortality, high and low chlorine. Reference Reference 1. Ridley SA, et al. Anaesthesia. 2004;59:1193-200. 1. Ridley SA, et al. Anaesthesia. 2004;59:1193-200. P366 Incidence and predisposing factors for the development of disturbed glucose metabolism and diabetes mellitus after intensive care admission: the DIAFIC study S Van Ackerbroeck, K Janssens, P Jorens, T Schepens, W Verbrugghe, V Van Hoof, L Van Gaal, C De Block University Hospital Antwerp, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P366 (doi: 10.1186/cc14446) Low serum 25-hydroxyvitamin D at critical care initiation is associated with sepsis and morbidity in Dutch critically ill patients K De Haan Erasmus MC, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P365 (doi: 10.1186/cc14445) Low serum 25-hydroxyvitamin D at critical care initiation is associated with sepsis and morbidity in Dutch critically ill patients K De Haan Erasmus MC, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P365 (doi: 10.1186/cc14445) Introduction Vitamin D defi ciency may frequently occur in critically ill patients and may be associated with sepsis and increased mortality. We therefore evaluated the prevalence of 25-hydroxyvitamin D defi ciency in a Dutch ICU, and its relationship with sepsis, morbidity and mortality. Methods We conducted a prospective observational study in a 10-bed mixed ICU. A total of 1,372 patients were admitted between July 2011 and June 2013 including 198 readmissions, of which 940 patients were studied. 25-Hydroxyvitamin D levels were determined within 24 hours after admission. 25-Hydroxyvitamin D levels were judged as suffi ciency (>50 nmol/l), insuffi ciency (30 to 50 nmol/l) and defi ciency (<30 nmol/l). Results The prevalence of defi ciency and insuffi ciency was 36% and 38%, respectively. Only 26% of the patients had suffi cient vitamin D levels. Vitamin D defi ciency is associated with sepsis (P <0.001) at ICU admission. Patients with defi cient levels had higher mean APACHE IV scores, 64 versus 52 (P <0.001), and longer length of hospital stay, 12 versus 9 days (P <0.001), respectively, as compared with patients with suffi cient levels. Patients with defi cient vitamin D levels had an odds ratio for in-hospital mortality of 1.4 (95% confi dence interval of 0.84 to 2.29, P = 0.2) relative to patients with suffi cient vitamin D levels.i Conclusion Implementation of i.v. potassium replacement guidelines improved the use of i.v. potassium in the ICU by reducing the requirement for i.v. potassium supplementation and increasing the overall time patients spent without hypokalaemia. Whilst nursing staff found the guideline useful and felt it increased safe use of i.v. potassium, more work is needed to ensure nurse workload is not increased signifi cantly. fi Conclusion 25-Hydroxyvitamin D defi ciency frequently occurs in Dutch critically ill patients. Although relating to sepsis, disease severity and morbidity, vitamin D defi ciency is not an independent predictor of mortality in these patients, which was otherwise relatively low. References 1. Moviat M, et al. J Crit Care. 2012;27:255-60. Masevicius FD, et al. Crit Care Resusc. 2010;12:248-54. S128 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P362 Evaluation of the eff ect of guidelines to reduce intravenous potassium infusions in ICU patients MC Law Min1, RS Bourne1, S Burd2, M Stone2 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2De Montfort University, Leicester, UK Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) P362 P362 Evaluation of the eff ect of guidelines to reduce intravenous potassium infusions in ICU patients MC Law Min1, RS Bourne1, S Burd2, M Stone2 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2De Montfort University, Leicester, UK Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) almost every patient and at every studied time point. Moreover, these levels were signifi cantly higher than in controls or compared with referenced literature. The chronology of exposure was demonstrated: the preoperative urine and serum levels of the DEHP metabolites were often below the detection limit. Medical devices are the source of these chemicals: patients on hemofi ltration, extracorporeal membrane oxygenation or both showed serum levels 100-fold or 1,000-fold higher than the general population or workers in plastic industry. The serum and some of the urinary levels of the DEHP metabolites are the highest ever reported in humans; some at biologically highly relevant concentrations of even ≥10 to 50 μM. almost every patient and at every studied time point. Moreover, these levels were signifi cantly higher than in controls or compared with referenced literature. The chronology of exposure was demonstrated: the preoperative urine and serum levels of the DEHP metabolites were often below the detection limit. Medical devices are the source of these chemicals: patients on hemofi ltration, extracorporeal membrane oxygenation or both showed serum levels 100-fold or 1,000-fold higher than the general population or workers in plastic industry. The serum and some of the urinary levels of the DEHP metabolites are the highest ever reported in humans; some at biologically highly relevant concentrations of even ≥10 to 50 μM. Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) Introduction The aim was to evaluate whether guidelines for intra- venous (i.v.) potassium replacement improved plasma potassium homeostasis in ICU patients. Prompt and eff ective treatment of hypokalaemia is an important intervention in the ICU, but concentrated i.v. potassium solutions may cause serious harm if used inappropriately [1]. There were previously no formalised guidelines on i.v. potassium supplementation in the ICU at Sheffi eld Teaching Hospitals. Practice was reviewed and guidelines were introduced to improve patient safety, plasma potassium homeostasis and reduce i.v. potassium supplementation requirements. Conclusion Adult ICU patients are exposed to plastic softeners, in particular PMs. Despite the continuously tightening regulations, BPA and DEHP are still present in medical devices. Because patient safety is a concern in the ICU, further research into the (possibly toxic and clinical) eff ects of chemicals released from medical devices should be undertaken. pp q Methods A before and after evaluation of plasma potassium homeo- stasis in ICU patients requiring i.v. potassium supplementation was conducted over a period of 8 months (August 2013 to May 2014). Patient data on plasma potassium levels, i.v. and oral potassium supplements administered were obtained from the clinical information system. Clinical appropriateness of i.v. potassium acetate prescriptions, fl uid and chloride intake related to potassium infusions and cost linked to the guidelines were also compared pre/post implementation. Impact of the guidelines on nurses’ practice was assessed using questionnaires. Results Median i.v. potassium replacement dose per patient was signifi cantly reduced in the post-guidelines group from 215 (IQR: 94; 485) to 80 (IQR: 40; 160) mmol; P <0.001. Although the percentage time per group for patients who were hypokalaemic was less in the post group (18.2% vs. 14.8%), there was no diff erence in mean patient values (24.2 (20.3)% vs. 22.1 (17.5)%; P = 0.228). The duration of hyperkalaemia was increased. Prescribing of i.v. potassium acetate was not always appropriate. Median patient fl uid-related dose was increased (107.5 (IQR: 47.1; 242.4) vs. 250 (IQR: 100; 600) ml; P <0.001), whilst chloride doses were reduced (170.7 (IQR: 91.3; 438.3) vs. 110 (IQR: 55; 250) mmol; P <0.009). Nurses were satisfi ed with the new practice, reporting it was safe, eff ective and clinically useful. However, compared with baseline practice, they perceived the guidelines as less eff ective and felt the workload was higher. Associations between the degree of correction of hypoglycemia and ICU mortality g g Results The median glucose in the EndoTool group (141.5  mg/dl) was lower than in the Adult ICU group (159.9  mg/dl) (P  <0.0001). The standard deviation of glucose in the EndoTool group (32.3  mg/ dl) was lower than the Adult ICU group (39.5  mg/dl) (P  = 0.0001). The proportion of patients in each group with 10% or higher of measurements at a severe hyperglycemia level (≥200  mg/dl) in the EndoTool group (35.2%) was lower than the Adult ICU group (64.1%) (P <0.0001). The proportion of patients who had at least one moderate hypoglycemic measurement (<70 mg/dl) was not signifi cantly diff erent between the EndoTool group versus the Adult ICU group (11.73% vs. 9.3%, respectively; P = 0.34). However, there was a higher overall incidence of hypoglycemia in the EndoTool group (5.65 hypoglycemic measurements/100 person-protocol days) compared with the Adult ICU group (3.43/100 person-protocol days) (RR = 1.65, 95% CI = 1.09 to 2.45, P = 0.014). Severe hypoglycemia (<40 mg/dl) was rare, only occurring in 1/179 (0.56%) in the EndoTool group and 4/580 (0.69%) in the Adult ICU group. R Van Hooijdonk1, JM Binnekade1, A Abu-Hanna1, j , , , F Van Braam Houckgeest2, LS Hofstra3, J Horn1, MA Kuiper4, f F Van Braam Houckgeest2, LS Hofstra3, J Horn1, MA Kuiper4, f NP Juff ermans1, HL Van den Oever5, JP Van der Sluijs6, PE Spronk7, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Tergooi Hospitals, Hilversum, the Netherlands; 3Scheper Hospital, Emmen, the Netherlands; 4Medical Centre Leeuwarden, the Netherlands; 5Deventer Hospital, Deventer, the Netherlands; 6Medical Center Haaglanden, The Hague, the Netherlands; 7Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P367 (doi: 10.1186/cc14447) Introduction It is conjectured that transition of hypoglycemia to hyperglycemia may be more harmful than hypoglycemia itself. We investigated the association between the degree of correction of hypoglycemia and ICU mortality in patients under moderately strict to strict glycemic control. Methods This is a retrospective analysis from a pooled cohort from seven ICUs in the Netherlands over 6 years. ICU patients who developed hypoglycemia (<70 mg/dl) were included. We excluded patients who were readmitted, and patients with hypoglycemia in whom no follow- up blood glucose measurement was performed within 8  hours. Marked exposure to the endocrine-disrupting chemicals phthalates and bisphenol A in the ICU J Huygh, P Jorens Antwerp University Hospital, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P364 (doi: 10.1186/cc14444) J Huygh, P Jorens J Huygh, P Jorens Introduction Care for ICU patients has benefi ted from medical devices. Bisphenol A (BPA) and phthalates can leach from the plastic matrix. We hypothesized that ICU patients are exposed to BPA and phthalates through medical devices. Introduction Stress hyperglycaemia (SH) is commonly observed during hospitalisation in the ICU and adversely infl uences outcome [1]. When SH occurs in previously nondiabetic patients, this might refl ect a latent disturbance of glucose metabolism and predict future risk of diabetes. We wanted to assess the incidence of disturbed glucose metabolism (DGM) and identify predictors for future diabetes risk. This could support timely diagnosis, prevention, and early treatment of impending diabetes mellitus (DM). Methods Serum (n = 118) and urinary (n = 102) samples of adult (n = 35) ICU patients were analyzed for total BPA and di(2-ethylhexyl)phthalate (DEHP) and other phthalate metabolites (PMs). We also enrolled patients preoperatively before scheduled thoracic surgery and repeat samples were taken on days 1 to 4 during the ICU stay. Control data came from 44 healthy controls or from referenced literature. Methods In this prospective observational study, we enrolled 338 patients without known DM, who were admitted for at least 36 hours to the ICU of the Antwerp University Hospital between September 2011 and March 2013. A 75  g oral glucose tolerance test was performed Results Our results show that adult ICU patients are continuously exposed to phthalates (that is, DEHP) as well as to BPA, albeit to a lesser extent, resulting in detectable serum and urinary levels in S129 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Not the transition to hyperglycemia, but insuffi cient recovery from hypoglycemia is associated with an increased ICU mortality in patients under moderately strict or strict glucose control with insulin. 6 to 9  months post ICU admission to screen for disturbed glucose metabolism. Furthermore, we examined whether post-discharge glucose disturbances could be predicted by the FINDRISC questionnaire [2], patient demographics, comorbidities, HbA1c at ICU admission, and by parameters related to ICU stay (glucose parameters, insulin need, caloric intake, disease severity). y Results In total, 246 patients (73%) experienced SH during their ICU stay. Eight months post ICU admission, glucose metabolism was disturbed in 119 (35%) subjects. Computer versus paper insulin protocol for managing hyperglycemia in three ICUs A Peckham Oregon Health & Science University, Portland, OR, USA Critical Care 2015, 19(Suppl 1):P368 (doi: 10.1186/cc14448) Computer versus paper insulin protocol for managing hyperglycemia in three ICUs A Peckham Oregon Health & Science University, Portland, OR, USA Critical Care 2015, 19(Suppl 1):P368 (doi: 10.1186/cc14448) Introduction The purpose of this study was to compare a computer protocol against a paper protocol in managing three domains of glucose control. Hyperglycemia is common in critically ill patients, and their risk of death is associated with hyperglycemia, hypoglycemia, and glucose variability. A safe and eff ective insulin protocol must minimize hyperglycemia and glucose variability while also avoiding hypoglycemia. Computer-based insulin protocols promise better performance by adjusting to each individual’s sensitivity to insulin. Conclusion Stress hyperglycaemia is frequent in nondiabetic patients and has a tendency towards future disturbances in glucose metabolism and DM. Glucose metabolism was disturbed in 35% of subjects 8 months post ICU admission, of whom 7% was diagnosed with diabetes mellitus. Predictors of elevated risk included a high FINDRISC score, high SAPS 3 score, and a lower daily caloric intake during ICU stay. R f Methods This is a historical cohort study with 759 patients admitted to three ICUs (medical/cardiac, trauma, and neuroscience) at an academic tertiary care hospital. All adult patients from January 2012 to October 2013 on one of two continuous insulin protocols for at least 8 hours were included. At the start of the study period the paper protocol in use (Adult ICU) had a target glucose of 140 to 180 mg/dl and was used for any patient with a glucose higher than 180 mg/dl. In June 2013 this was replaced by a computer-based insulin protocol (EndoTool) that had the same criteria for initiation and had a target glucose of 150 mg/dl. The primary exposure was the insulin protocol, and the primary outcome was performance in maintaining glucose control. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Schwarz PE, et al. Horm Metab Res. 2009;41:86-97. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Schwarz PE, et al. Horm Metab Res. 2009;41:86-97. Associations between the degree of correction of hypoglycemia and ICU mortality We determined the association between three measures of correction of hypoglycemia within 8 hours after hypoglycemia and ICU mortality: predefi ned ranges of the ‘highest blood glucose level’ (<80 mg/dl; 80 to 110 mg/dl; 110 to 150 mg/dl (reference category); 150 to 180 mg/ dl; and >180  mg/dl); quartiles of the ‘delta glucose’, defi ned as the diff erence between minimum and maximum blood glucose level with the third quartile as reference category; and quartiles of the ‘standard deviation’ of the blood glucose level with the third quartile as reference category. Conclusion Patients on the computer protocol had a lower median glucose, less variability, and less hyperglycemia than patients on the paper protocol. There was a higher risk of moderate but not severe hypoglycemia in the computer group. Marked exposure to the endocrine-disrupting chemicals phthalates and bisphenol A in the ICU J Huygh, P Jorens Antwerp University Hospital, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P364 (doi: 10.1186/cc14444) Of these, 27 (8%) had impaired fasting glucose, 43 (13%) had impaired glucose tolerance, 25 (7%) had impaired fasting glucose and impaired glucose tolerance, and 24 (7%) were diagnosed with DM. A disturbed glucose metabolism tended to be more prevalent in subjects who experienced SH during ICU stay as compared with those without SH (38% vs. 28%, P = 0.065). HbA1c on admission correlated with the degree of SH (r = 0.308, P <0.001). The FINDRISC score (9.5 vs. 11, P = 0.001), SAPS 3 score (median of 42 in both groups, P = 0.003) and daily caloric intake during ICU stay (222 vs. 197, P = 0.011) were associated with a DGM. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf yp g y y Results Of the 1,059 patients admitted with sepsis, 526 (55.8%) had admission glucose levels within the normal range, 270 (25.5%) had mild hyperglycemia and 202 (19.1%) severe hyperglycemia. Patients with severe hyperglycemia were older, had higher APACHE IV scores and were more often diabetics compared with euglycemic patients. Shock on admission was more common in patients admitted with euglycemia. Crude mortality increased with increased admission glucose and a Cox regression analysis showed increased risk for 30- day (HR = 1.67, CI = 1.24 to 2.23), 60-day (HR = 1.42, CI = 1.08 to 1.87) and 90-day (1.31, CI = 1.02 to 1.70) mortality in patients admitted with severe hyperglycemia compared with euglycemia. The association between mortality and severe hyperglycemia on admission was only present in patients without known diabetes but not in patients with a history of diabetes (30-day mortality HR = 1.67, CI = 1.15 to 2.43 vs. 1.84, CI = 0.97 to 3.49). Severe hyperglycemia was associated with a blunted proinfl ammatory cytokine response (IL-6 and IL-8) on admission in patients without, but not in patients with diabetes. g y p R Van Hooijdonk, JH Leopold, T Winters, JM Binnekade, NP Juff ermans, J Horn, JC Fischer, EC Van Dongen-Lases, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P370 (doi: 10.1186/cc14450) Introduction There is a need for continuous glucose monitoring in critically ill patients. The objective of this trial was to determine the point accuracy and reliability of a device designed for continuous monitoring of interstitial glucose levels in ICU patients (Sentrino; Medtronic MiniMed, Northridge, CA, USA). Methods Critically ill patients with an anticipated life expectancy >96 hours were eligible for participation, if the platelet count was >30 × 1012 ml. Device readings were compared with glucose measurements in arterial blood using blood gas analyzers (RapidLab Siemens Healthcare Diagnostics, The Hague, the Netherlands). We used a linear mixed model to determine which factors aff ect point accuracy. In addition, we determined the reliability, including duration of device start-up and calibration, skips in data acquisition, and premature disconnections of sensors. Conclusion Severe hyperglycemia on admission is associated with increased 30-day, 60-day and 90-day mortality in sepsis patients without a history of diabetes mellitus. Results We included 50 patients, aged 65 (56 to 72) years with an APACHE II score of 23 (17 to 26). P370 Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patients R Van Hooijdonk, JH Leopold, T Winters, JM Binnekade, NP Juff ermans, J Horn, JC Fischer, EC Van Dongen-Lases, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P370 (doi: 10.1186/cc14450) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 The number of skips in data acquisition was low, resulting in availability of real-time data during 95 (89 to 98)% of the connection time per sensor. glucose data prove in such routine use?’ Using actual case data, we have shown how comparing the mean absolute relative diff erence (MARD) and integration of the area under the curve (AUC) from the continuous glucose monitoring and intermittent measurement can be used to measure patient risk. Conclusion The point accuracy of the device was relatively low in critically ill patients. The device reliability was relatively good, although sensors were removed prematurely for a variety of reasons. p Methods The analysis used aggregated case data generated from our recent clinical trials, where a GlySure sterile, single-use sensor and dedicated monitoring system was used to measure the blood glucose concentration in patients continuously and in real time. The measurement of risk was compared using the MARD, an accepted error calculation tool, and the AUC was calculated using an AUC analysis software program. g References 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442.i 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442.i 2. Gopal PB, Mulavisala KP. ESICM 2014. Abstract 0264. http://react-profi le.org/ Download/ESICM2014_Abstract_Book_fi nal_version.pdf. P370 Eff ect of admission hyperglycemia in sepsis patients with or without a history of diabetes g Results When MARD from the GlySure sensor and intermittent measurement using the hospital’s existing protocol was compared, the measure of risk to the patient (that is, the uncertainty regarding the patient’s absolute blood glucose status) for the GlySure sensor was 50.5% lower than the intermittent measurement. The results also showed that as the variability of the BG data increases, the benefi t of continuous monitoring increases by signifi cantly reducing patient risk. The continuous monitoring reduces the patient’s risk by 88%, 73%, and 69% respectively in high, medium and low variability situations. Introduction Hyperglycemia is common and often multifactorial in critically ill patients. The association of hyperglycemia with adverse outcome has repeatedly been established in a variety of settings. The objective of this study was to investigate whether hyperglycemia on admission to the ICU impacts presentation and outcome of sepsis patients and whether this eff ect is diff erent for patients with a history of diabetes mellitus. p y g y Conclusion It is more and more evident that continuous glucose technology will be instrumental in driving safe and eff ective glucose management protocols that will support more consistent glycemic management standards within ICUs and across institutions. Methods A two-center, prospective observational cohort study was conducted including all consecutive critically ill patients admitted to the ICU between January 2011 and July 2013. Sepsis patients were identifi ed using strict clinical and diagnostic criteria. The fi rst glucose measurement within a time window of 4 hours before up to 4 hours after ICU admission was categorized into euglycemia (71 to 140 mg/dl), mild hyperglycemia (141 to 200 mg/dl) or severe hyperglycemia (>200 mg/ dl), patients with hypoglycemia were excluded. A multivariable Cox proportional hazard model was used to determine the eff ect of admission hyperglycemia on mortality corrected for covariates. Continuous blood glucose monitoring reduces the risk to ICU patients KP Mulavisala1, J Norrie2, B Crane3, N Barwell3 1CARE Hospitals, Hyderabad, India; 2SumStats Ltd, Edinburgh, UK; 3GlySure Ltd, Abingdon, UK g , Critical Care 2015, 19(Suppl 1):P369 (doi: 10.1186/cc14449) Results In total, 4,516 ICU patients developed at least one episode of hypoglycemia. In three separate multivariate analyses for each of the three measures we adjusted for the respective confounders. The category 80 to 110  mg/dl of the ‘highest blood glucose level’ was associated with increased mortality compared with the reference category (odds ratio (OR) = 1.31, 95% confi dence interval (CI) = 1.06 to 1.61). The lowest quartile of the ‘delta glucose’ (OR = 1.32, 95% CI = 1.03 to 1.69) and the lowest quartile of the ‘standard deviation’ (OR = 1.55, 95% CI = 1.23 to 1.96) were associated with higher ICU mortality than their reference categories. Introduction GlySure Limited (Abingdon, UK) has developed a continuous intravascular glucose monitoring system (CIGMS) to simplify the application of hospital protocols for optimal glucose control at the point of care. We have previously reported on the early results achieved in cardiac patients [1] and MICU patients [2]. This initial success has been sustained and demonstrated in further patient groups. We have now reached the point where we can conjecture upon the regular application of the GlySure CIGMS in day-to-day ICU practice. This in turn prompts the question, ‘How eff ective will continuous blood S130 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P371f Eff ect of admission hyperglycemia in sepsis patients with or without a history of diabetes LA Van Vught1, MA Wiewel1, PM Klein Klouwenberg2, AJ Hoogendijk1, DS Ong2, OL Cremer2, MJ Bonten2, MJ Schultz1, T Van der Poll1 1Academic Medical Center, Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P371 (doi: 10.1186/cc14451) 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf Admission types were medical (62%), elective surgery (22%) and emergency surgery (16%), and 22% had diabetes. For the accuracy analyses we had 929 comparative samples from 100 sensors in 45 patients (11 (7 to 28) samples per patient) during 4,639 hours (46 (27 to 134) hours per patient and 46 (21 to 69) hours per sensor). The Bland–Altman plot showed a bias of –0.6 mg/dl with limit of agreement between –57.2 and 56 mg/dl. Glucose prediction error analysis showed 60% of the glucose values <75  mg/dl within ±15 mg/dl and 75.8% of the glucose values ≥75 mg/dl within 20% of the comparative RapidLab results. Clarke error grid analysis showed 75.3% in zones A and 23.5% of the paired measurements in zones B, 0.3% of the paired measurements in zones C and 0.9% of the paired measurements in zones D. Point accuracy did not meet the ISO14971 standard for dosing accuracy, but improved with increasing numbers of calibrations, and was better in patients who did not have diabetes mellitus. Sixty out of 105 sensors were removed prematurely for a variety of reasons. The device start-up time was 49 (43 to 58) minutes. Acknowledgement This research was performed within the framework the Center for Translational Molecular Medicine (http://www.ctmm.nl), project MARS (grant 04I-201). Point and trend accuracy of continuous glucose monitoring using intravenous microdialysis in critically ill patients JH Leopold, RT Van Hooijdonk, M Boshuizen, T Winters, LD Bos, A Abu-Hanna, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P372 (doi: 10.1186/cc14452) Introduction Insulin infusion in critically ill patients mandates frequent measurements of the blood glucose level [1]. Microdialysis is a well- established technology that off ers the opportunity to sample blood analytes with high accuracy, without the need for drawing blood samples [2,3]. We aimed to determine point and trend accuracy of S131 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 values and arterial glycemia correlated well with 98.6% of data falling in regions A and B of error grid analysis. microdialysis-based continuous glucose monitoring (CGM) (EIRUS®; Maquet Critical Care AB, Solna, Sweden). g g y Conclusion In our study, the use of RT-CGM neither improved glucose control and variability, nor did it reduce hypoglycemic events. y g References 2. Schierenbeck F, et al. Diabetes Technol Ther. 2015;15:26-31. y y p y Results Ninety-nine FP patients were admitted between April 2008 and January 2014. Median age was 73 (IQR 61 to 79), with a female preponderance (53.5%). The median ICU length of stay (LOS) was 5 days (IQR 2 to 16). On admission to critical care, clinical data included (all medians): temperature 36.6°C (IQR 36 to 37.2), systolic blood pressure (BP) 113 mmHg (IQR 104 to 136), diastolic BP 56 mmHg (IQR 49 to 67), lactate 2.3 mmol/l (IQR 1.5 to 3.7), bilirubin 12 μmol/l (IQR 9 to 20), haemoglobin 104 g/l (IQR 93 to 116), haematocrit 31 (IQR 28 to 36), creatinine 88 μmol/l (IQR 66 to 152), prothrombin time 13.1 seconds (IQR 11.9 to 14.4). In 86 patients the initial operation was an emergency laparotomy, with primary perforation in 53 cases. Subsequent anastomotic dehiscence and need for relaparotomy happened in 24 and 33 cases respectively. Forty per cent of patients underwent more than one surgical abdominal intervention. The most common antibiotic used was tazobactam and fl uconazole was the commonest antifungal. The percentages of patients receiving mechanical ventilation, renal replacement therapy and inotropic/vasopressor support during ICU stay were 72.7%, 25.3% and 84.8% respectively. The ICU and hospital mortality rates were 23.5% and 26.1%, respectively, increasing to 26.7% at 28 days, 28.4% at 90 days and 32.2% at 1 year. None of the surgical factors or diabetes infl uenced survival. The strongest independent risk factors associated for ICU mortality were systolic BP on ICU admission (OR = 1.05, 95% CI = 1.01 to 1.09, P = 0.015), acute kidney injury (AKI) within the fi rst 24 hours of ICU admission (OR = 0.15, 95% CI = 0.03 to 0.9, P = 0.026) and lactate on ICU admission (OR = 0.62, 95% CI = 0.39 to 1, P = 0.05). , ; 4. Krouwer JS, et al. J Diabetes Sci Technol. 2010;4:75-83. 5. Clarke WL, et al. Diabetes Care. 1987;10:622-8. 6. Kovatchev, et al. Diabetes Care. 2004;27:1922-8. P373 P373 Real-time continuous glucose monitoring in the ICU J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) P373 Real-time continuous glucose monitoring in the ICU J Gios1, B Manuel-y-Keenoy2, P Rogiers3 J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) J Gios , B Manuel y Keenoy , P Rogiers 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) Introduction Hyperglycemia occurs in 50 to 85% of patients admitted to a medical ICU (MICU) and has been associated with poor prognosis [1,2]. Whether applying intensive insulin therapy to achieve tight glycemic control in critically ill patients is benefi cial remains controversial [2]. Another important observation is a link between glycemic variability and mortality [3]. We performed a pilot study hypothesizing that when implementing intensive insulin therapy, real-time continuous glucose monitoring (RT-CGM) may help to safely achieve tight glucose control, while avoiding hypoglycemia and reducing glycemic variability in MICU patients. Methods This two-center randomized controlled pilot study was performed during a 3-year period. To be included, patients had to be severely ill (APACHE II score ≥20) and CGM monitoring had to be started within 48 hours after admission in the MICU. Thirty-fi ve patients (age 66 ± 10 years; nondiabetic/diabetic patients 27/8; APACHE II score 28 ± 6) were randomly assigned to RT-CGM (n = 16) or to blinded CGM. In both groups a microdialysis-based glucose sensor (GlucoDay®S) was used during a 96-hour period of glucose monitoring. Insulin infusion was performed using a modifi ed Yale protocol. Outcome measures were percentage of time in normoglycemia and in hypoglycemia, glycemic variability, and CGM accuracy. Conclusion In this cohort of critically ill FP patients the ICU and 12-month mortality rates were 23.5% and 32.2%, respectively. The most consistent predictors of mortality across all time points were AKI within 24 hours of ICU admission and admission lactate. fi 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. References 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Kavanagh BP, et al. N Engl J Med. 2010;363:2540-6. 3. Hermanides J, et al. Crit Care Med. 2010;38:838-42. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Kavanagh BP, et al. N Engl J Med. 2010;363:2540-6. 3. Hermanides J, et al. Crit Care Med. 2010;38:838-42. Results Three-hundred and fi fty-four paired samples were obtained from seven patients (66 (59 to 79) years old, APACHE II score 23 (20 to 28), 51 (19 to 77) samples per patient). Point accuracy: 91% of paired values were in zone A, with the remaining 9% of the values in zone B in the Clarke error grid. In the Bland–Altman, bias was 5.4 mg/dl with an upper limit of agreement of 32.5 mg/dl and a lower level of agreement of –21.8 mg/dl. Glucose prediction error analysis showed that 91% of the values ≥75 mg/dl within 20% of the values measured by the blood gas analyzer were within range. Trend accuracy: in the rate error grid of the continuous glucose error grid analysis, 96% of the paired values were in zone A, 3.7% were in zone B and 0.3% were in zone C. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf On the other hand we can state that our insulin infusion protocol already led to overall tight glucose control without a signifi cant hypoglycemia risk, leaving little space for improvement. R f Methods Patients with an expected stay in the ICU of >48  hours needing an arterial catheter and a central venous catheter (CVC) were eligible. For a maximum of 3 days, during 8 hours per day, 125 μl blood was drawn from the arterial line every 15 minutes. Point accuracy was expressed using Clarke error grids, Bland–Altman plots and glucose prediction error analysis [4,5]. Trend accuracy was expressed using continuous glucose error grid analysis [6].i Critically ill patients with faecal peritonitis: a 5-year review in a tertiary centre Critically ill patients with faecal peritonitis: a 5-year review in a tertiary centre V Paul1, A Tridente2, P Kaur1, M Mahmood1, R Mellors1, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454) V Paul1, A Tridente2, P Kaur1, M Mahmood1, R Mellors1, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454) y Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454 Introduction Faecal peritonitis (FP) is a common cause of sepsis and admission to the ICU [1]. We report a review of all patients admitted to our ICU over 5 years with FP. The aim was to defi ne the clinical characteristics, outcomes and risk factors for mortality in ICU patients with FP. Introduction Faecal peritonitis (FP) is a common cause of sepsis and admission to the ICU [1]. We report a review of all patients admitted to our ICU over 5 years with FP. The aim was to defi ne the clinical characteristics, outcomes and risk factors for mortality in ICU patients with FP. Conclusion Point and trend accuracy of the tested microdialysis-based CGM are good in critically ill patients. Acknowledgement Maquet Critical Care AB provided two CGM systems and disposables for the duration of the study, but had no infl uence on study design or study reporting. y Methods Data were extracted retrospectively from electronic case fi les. The primary outcome was ICU mortality. Secondary outcomes were hospital, 28-day, 90-day and 1-year mortality. Logistic regression analysis was used to identify independent risk factors for mortality. P377 Results We included 349 patients: age 67.5 ± 10.8 years, M/F sex ratio 252/97, preoperative left ventricle ejection fraction 58.8  ± 10.6%, bypass/valve ratio 234/154, number of grafts 2.7 ± 0.9, mammal arteries 1.8 ± 0.5. In univariate analyses, bypasses received more anaesthetic drugs (P <0.01), had shorter extracorporeal circulation duration, 67 ± 27 versus 75 ± 24 minutes (P <0.01), and received less blood products (P <0.0001). Bypasses had lower postoperative levels of troponin (3.9 ± 7.6 vs. 8.1 ± 21 pg/ml, P <0.01) and LDH (330 ± 162 vs. 420 ± 175 pg/ ml). In contrast, the intra-abdominal pressure (IAP) was higher and related to the number of grafts at day 0 (Figure 1) and day 1 (P = 0.01 and 0.02 respectively), and to the number of mammal grafts at day 0 and day 1 (P = 0.01 and 0.04 respectively). The TTFE was longer but did not reach signifi cance (P = 0.13) as well as the occurrence of abdominal ischaemia (P = 0.22). The occurrence of pneumonia was higher (P = 0.01). In multivariate analysis, the IAP at day 0 and day 1 was related to propofol quantities only. The predictors of pneumonia were: duration of mechanical ventilation, peak lactate in the postoperative 24 hours, and coronary bypass: OR = 163, 2.6, and 4.2 respectively. P377 Disseminated intravascular coagulation score predicts mortality in critically ill patients with liver cirrhosis A Drolz, T Horvatits, K Rutter, K Roedl, S Kluge, V Fuhrmann University Medical Center Hamburg-Eppendorf, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P377 (doi: 10.1186/cc14457) Introduction The disseminated intravascular coagulation (DIC) score is a predictor of outcome in critically ill patients [1,2]. Yet disturbances of coagulation and hemostasis, as refl ected by the DIC score, are a common fi nding in patients with liver cirrhosis. Thus, it is unclear whether the DIC score has prognostic value in critically ill patients with liver cirrhosis. The aim of this study was to assess the applicability and prognostic impact of the DIC score in critically ill patients with liver cirrhosis. Methods Patients with liver cirrhosis admitted to the medical ICU were analyzed for this study. Detailed laboratory analyses including platelet count, D-dimer, fi brinogen and prothrombin index were performed on admission and the DIC score was calculated. Survival was assessed on site or by contacting the patients or the attending physician. Bowel and related complications after cardiac surgery Results In the RT-CGM group the percentage of time at the target glycemia (80 to 110 mg/dl) was 37 ± 12% versus 34 ± 10% in the control group (NS) and glycemia averaged 119 ± 17 mg/dl versus 122 ± 11 mg/ dl respectively (NS). Time spent in hypoglycemia (<60 mg/dl) was not statistically diff erent between the group assigned to RT-CGM (0.6  ± 1.6% of the time) versus those with blinded CGM registration (2.4 ± 4.3% of the time). Parameters of glucose variability (standard deviation of mean glucose value, coeffi cient of variation, mean amplitude of glucose excursions) did not diff er between the groups. The GlucoDay®S Bowel and related complications after cardiac surgery CK Kerneis, AL Lafarge, LL Larnier, F Scalbert, AB Brusset, PE Estagnasie, PS Squara Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P375 (doi: 10.1186/cc14455) CK Kerneis, AL Lafarge, LL Larnier, F Scalbert, AB Brusset, PE Estagnasie, PS Squara q Clinique Ambroise Paré, Neuilly-sur-Seine, France q Clinique Ambroise Paré, Neuilly-sur-Seine, France Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P375 (doi: 10.1186/cc14455) Introduction Postoperative ileus appears to be underestimated after cardiac surgery. We conducted this study to analyse the incidence, risk factors and outcomes of postoperative ileus. Introduction Postoperative ileus appears to be underestimated after cardiac surgery. We conducted this study to analyse the incidence, risk factors and outcomes of postoperative ileus. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S132 Figure 1 (abstract P375). Figure 1 (abstract P375). Results Eighty-seven patients (69.9%) were females. Mean age was 48.9  years. Primary cancer was colorectal in 42 patients (32.5%), ovarian in 39 (30%), appendiceal in 29 (22%), others in 15.5%. Average operative time was 11 ± 2.1 hours. Average intraoperative crystalloids given were 12,217 ± 4,359 ml, packed RBCs were 2 ± 2.3 units, colloids 1,083 ± 898 ml, average blood loss was 1,108 ± 785 ml. All patients were admitted to the ICU post procedure. The average fl uid balance during the OR was 9,481 ± 4,694 ml. Patients stayed in the ICU for an average of 6 ± 5.3 days. All patients survived the ICU stay. The duration of mechanical ventilation was 57 ± 83 hours, total fl uid balance while in the ICU was 1,467  ± 3,399  ml. Hypomagnesemia was the most frequent electrolyte abnormalities in 79 (61%). 1. Taylor FB, Toh CH, Hoots WK, Wada H, Levi M, Scientifi c Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Towards defi nition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost. 2001;86:1327-30. 2. Angstwurm MWA, Dempfl e C-E, Spannagl M. New disseminated intravascular coagulation score: a useful tool to predict mortality in comparison with Acute Physiology and Chronic Health Evaluation II and Logistic Organ Dysfunction scores. Crit Care Me. 2006;34:314-20; quiz 328. ICU outcome of patients undergoing cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy: a single-center study A Nadeem, A Al-Tarifi King Faisal Specialist Hospital, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P376 (doi: 10.1186/cc14456) g y A Nadeem, A Al-Tarifi King Faisal Specialist Hospital, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P376 (doi: 10.1186/cc14456) overt DIC was 70% compared with 40% in those with a DIC score <5. Conclusion Disturbances in coagulation and hemostasis are found in the majority of cirrhotic patients admitted to the ICU. The DIC score is a suitable predictor of 28-day mortality in critically ill patients with liver cirrhosis. Introduction Peritoneal carcinomatosis (PC) is associated with poor prognosis. The advent of complete cytoreductive surgery (CRRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has shown promise in improved survival for locally advanced intra- abdominal carcinomatosis. Such patients are routinely admitted to the ICU postoperatively. Little is known about the natural course of such patients while in the ICU. P377 Conclusion The number of coronary grafts and of mammal artery used in cardiac surgery is associated with higher IAP and higher risk of pneumonia. However, whether this is due to direct bowel ischaemia or longer anaesthesia remains to be studied in larger trials. Results In total, 150 admissions to the ICU with liver cirrhosis were analyzed. Thirty-nine percent were female. Median age was 56 (IQR 49 to 63) years. The median SOFA score on admission was 9 (6 to 13), median MELD score 26 (IQR 18 to 36). Twenty-eight-day mortality was 59%. Median DIC score on admission was 5 (IQR 4 to 6). Overt DIC (DIC score ≥5) was found in 65%. DIC score was signifi cantly higher in nonsurvivors compared with survivors (5 (IQR 4 to 7) vs. 4 (IQR 3 to 6); P <0.01). AUROC for the DIC score in prediction of 28-day mortality was 0.68 (95% CI = 0.59 to 0.77). Overt DIC on admission was signifi cantly associated with 28-day mortality (OR  = 3.4 (95% CI  = 1.69 to 6.84), P <0.01). The 28-day mortality rate in admissions with cirrhosis and overt DIC was 70% compared with 40% in those with a DIC score <5. P376 Bowel and related complications after cardiac surgery Pleural eff usions in 48 (37%), of which three patients only required drainage, Seven patients (5.6%) developed pneumonia, no patient required renal replacement therapy. Average hospital LOS was 33.7 ± 29 days. Only two patients died in the hospital. When the fi rst 65 patients were compared with the last 64 patients, the duration of MV, ICU LOS and hospital LOS were all signifi cantly shorter in the latter group (72 vs. 43 hours, 6.8 vs. 5.0 and 40 vs. 27 days respectively; P <0.01 for all). Methods In this single-centre observational study we prospectively enrolled all patients undergoing elective cardiac surgery. The primary output was the time to faeces (TTFE) as representing the postoperative ileus. Secondary outputs were the occurrence of ischaemic colitis and pneumonia. Quantitative variables were compared by ANOVA or Wilcoxon tests, qualitative variables by chi-square tests. Multivariate analyses were performed by logistic regression, P  <0.1 for inputs P <0.05 for outputs. Methods In this single-centre observational study we prospectively enrolled all patients undergoing elective cardiac surgery. The primary output was the time to faeces (TTFE) as representing the postoperative ileus. Secondary outputs were the occurrence of ischaemic colitis and pneumonia. Quantitative variables were compared by ANOVA or Wilcoxon tests, qualitative variables by chi-square tests. Multivariate analyses were performed by logistic regression, P  <0.1 for inputs P <0.05 for outputs. Conclusion With proper selection of patients, CRS with HIPEC can be done safely with no major complications. There is a signifi cant reduction in ICU utilization and shorter hospital LOS with more experience in such procedure, suggesting a learning curve as well as better utilization of resources by referring such patients to a high-volume center. P377 References 1. Taylor FB, Toh CH, Hoots WK, Wada H, Levi M, Scientifi c Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Towards defi nition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost. 2001;86:1327-30. Methods The procedure was introduced in our hospital in 2008 as the fi rst regional center performing such therapy. A retrospective chart review of 129 cases of CRS-HIPEC admitted to a 22-bed surgical ICU in a tertiary care academic center between November 2008 and March 2014. Primary outcomes were ICU length of stay (LOS) and duration of mechanical ventilation (MV). Secondary outcomes were hospital LOS and hospital mortality. 2. Angstwurm MWA, Dempfl e C-E, Spannagl M. New disseminated intravascular coagulation score: a useful tool to predict mortality in comparison with Acute Physiology and Chronic Health Evaluation II and Logistic Organ Dysfunction scores. Crit Care Me. 2006;34:314-20; quiz 328. S133 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P378 Conclusion In the present study, it was observed that the sero- prevalences of HBsAg, anti-HCV and anti-HIV were not higher than in our city population. However, taking the safety precautions of the healthcare workers during surgical or invasive procedures such as catheterization, intubation or tracheostomy without any information about the serological test results of the patients will reduce the contamination of these agents. P378 Warm ischemia time, postreperfusion syndrome and initial poor function after liver transplantation: are they connected? E Scarlatescu, G Manga, G Droc, D Tomescu Fundeni Clinical Institute, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P378 (doi: 10.1186/cc14458) Introduction Factors associated with initial poor graft function (IPGF) after liver transplantation are still under debate. Although the initial insult to the graft begins during the cold ischemia time (CIT), recent studies showed that most injuries occur during rewarming. Ischemic– reperfusion (I/R) injuries are present in all grafts and may be responsible for postoperative graft dysfunction. Along with other factors, I/R injury may also play a role in the development of postreperfusion syndrome (PRS) after revascularization of the liver graft. The aim of this study was to assess whether longer warm ischemia time (WIT) is associated with PRS or with IPGF after liver transplant. Outcomes of decompensated chronic liver disease in a UK district general hospital critical care setting E Ahmadnia1, F Manneh2, K Raveendran2 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) , , 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) , , 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) p p Methods This retrospective observational study included 60 liver transplant patients. We excluded from the study group patients with retransplant procedures, and the recipients of divided grafts and of grafts from extended criteria donors. We recorded: demographic data, intraoperative PRS, CIT, WIT, ALT, AST levels and standard coagulation tests on postoperative days (POD) 1 to 5. Statistical analysis was performed using SPSS Statistics v.19.1 with signifi cant P value under 0.05. Results We used the criteria of Nanashima and colleagues for the diagnosis of IPGF (ALT and/or AST level above 1,500 IU/l within 72 hours after OLT). The study group included 33 men (55%) and 27 women. Mean (±SD) age was 50.56 (±13.26) years. WIT longer than 60 minutes correlated signifi cantly with ALT and AST levels in POD 1 to 3 (P <0.0001 for ALT in POD 1 to 3, P = 0.001 for AST in POD 1, P = 0.007 and 0.013 for AST in POD 2 and 3) and with prothrombin time (P = 0.008 in POD 1, P = 0.03 in POD 2 and P = 0.015 in POD 3). We could not fi nd a correlation between PRS and WIT (P = 0.566), CIT (P = 0.439) or transaminase levels on POD 1 to 3. The correlation between WIT >60 minutes and IPGF was confi rmed using the Pearson chi-square test (P <0.0001). The same test was used to correlate IPGF with PRS with nonsignifi cant results (P = 0.876). Introduction Patients with decompensated cirrhosis admitted to the ICU have historically had a very high mortality rate [1]. It has been suggested that improving patient selection can improve ICU outcomes in patients with cirrhosis [2]. The aim of this study was to determine the mortality and evaluate the risk factors that may infl uence the outcome of this group of patients in a large UK district general hospital with a view to introducing selection criteria for future ICU admission of patients with decompensated liver disease. p p Methods A retrospective analysis was performed of all adult patients with decompensated chronic liver disease admitted to a general (nontransplant) critical care unit between January 2012 and December 2013. References The secondary aim was to investigate whether patients with intraoperative PRS have a higher risk for postoperative IPGF. P379 Seroprevalence of hepatitis B, hepatitis C and HIV in ICU patients H Bayir, I Yildiz, E Kocoglu, A Kurt, H Kocoglu Abant Izzet Baysal University, Medical School, Bolu, Turkey Critical Care 2015, 19(Suppl 1):P379 (doi: 10.1186/cc14459) Conclusion Those with decompensated chronic liver disease admitted to the ICU have a signifi cant ICU/hospital mortality, which is increased in alcoholic liver disease. Sepsis and AKI were the most common acute diagnoses in this cohort. Alcoholic liver disease patients requiring organ support have a very high mortality, and the outlook for multiorgan failure requiring RRT in this group is dismal. Introduction Healthcare workers are at risk for infections caused by hepatitis B (HBV), hepatitis C (HCV) and human immunodefi ciency (HIV) viruses that transmit via blood and body fl uids. In the present study, it was aimed to investigate the seroprevalences of HBsAg, anti- HCV and anti-HIV in patients admitted to the ICU. References References 1. Olson JC, et al. Hepatology. 2011;54:1864-72. 2. Sauneuf B, et al. Crit Care. 2013;17:R78. Methods HBsAg, anti-HCV and anti-HIV test results and demographical data of the patients admitted to the Reanimation ICU between January 2012 and December 2014 were evaluated retrospectively. HBsAg, anti-HCV and anti-HIV tests were assayed with a macro-ELISA method (Axsym-Abbott, Architect i2000; Abbott, USA). Statistical analysis was performed with the chi-square test. References 1. Erdem K, Tas T, Tekelioglu UY, Bugra O, Akkaya A, Demirhan A, et al. The hepatitis B, hepatitis C and human immunodefi ciency virus seroprevalence of cardiac surgery patients. SDÜ Tip Fak Derg. 2013;20:14-17 1. Erdem K, Tas T, Tekelioglu UY, Bugra O, Akkaya A, Demirhan A, et al. The hepatitis B, hepatitis C and human immunodefi ciency virus seroprevalence of cardiac surgery patients. SDÜ Tip Fak Derg. 2013;20:14-17 y 2. Tekin A, Deveci O. Seroprevalences of HBV, HCV and HIV among healthcare workers in a state hospital. J Clin Exp Invest. 2010;1:99-103. 2. Tekin A, Deveci O. Seroprevalences of HBV, HCV and HIV among healthcare workers in a state hospital. J Clin Exp Invest. 2010;1:99-103. Outcomes of decompensated chronic liver disease in a UK district general hospital critical care setting E Ahmadnia1, F Manneh2, K Raveendran2 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) Data were collected regarding demographics, ICU mortality, hospital mortality, aetiology of chronic liver disease, severity scores, acute diagnoses, and organ support requirements.i Results Thirty-seven patients were identifi ed, with a median age of 57  years, predominantly male (62%). Seventy-six per cent had alcohol-related cirrhosis. Overall ICU mortality was 29.7% and hospital mortality was 48.6% – these values were higher in the alcoholic group (39.3% and 57.1% respectively). All ICU deaths were in those with alcoholic liver disease. Median scores were: APACHE III 93, SOFA (day 1) 9, Child–Pugh 11, MELD 21. Seventy per cent were treated for sepsis, 22% had a GI bleed, 57% had encephalopathy, 24% had suspected/ confi rmed spontaneous bacterial peritonitis, and 70% had an acute kidney injury. Organ support requirements were: 35% respiratory (non-invasive or invasive ventilation), 38% vasoactive agent support, 24% renal replacement therapy (RRT). Alcoholic liver disease patients requiring respiratory or cardiovascular support had an ICU mortality of 64%, and those requiring RRT had a mortality of 75%. Alcoholic liver disease patients requiring combined respiratory, cardiovascular, and RRT support had 100% mortality. Conclusion Our study showed that PRS is not a risk factor for IPGF after liver transplantation. WIT over 60  minutes does not infl uence the development of PRS, but is associated with IPGF after liver transplantation. Close monitoring of liver tests in the early postoperative period is very important especially in recipients of grafts with WIT over 60 minutes. Further eff orts to decrease WIT may prove useful for the reduction of IPGF in liver transplant patients. First clinical experience with a new type of albumin dialysis: the HepaWash® system First clinical experience with a new type of albumin dialysis: the HepaWash® system First clinical experience with a new type of albumin dialysis the HepaWash® system B Henschel, R Schmid, W Huber TU-München Klinikum rechts der Isar, Munich, Germany Critical Care 2015, 19(Suppl 1):P383 (doi: 10.1186/cc14463) B Henschel, R Schmid, W Huber TU-München Klinikum rechts der Isar, Munich, Germany Critical Care 2015, 19(Suppl 1):P383 (doi: 10.1186/cc14463) Introduction Liver failure (LF) is associated with prolonged hospital stay, increased cost and substantial mortality. With regard to a limited number of donor organs, extracorporeal liver support is an appealing concept to bridge to transplant or to avoid transplant in case of recovery. A new type of albumin dialysis, the HepaWash® system, was recently introduced. The HepaWash® system provides rapid re- generation of toxin-binding albumin by secondary circuits altering binding capacities of albumin by biochemical (changing pH) and physical (changing temperature) modulation of the dialysate.i Results Eight children (fi ve male/three female), 8.6  ± 5.9  years old (range 2 to 15.6 years), BW 32 ± 21 kg, GFR 71 ± 20 ml/minute/1.73 m2, with an uncuff ed double lumen dialysis catheter (8 to 14 Fr Femoralis (n = 6) and 9 Fr Jugularis (n = 2)) were treated according to this protocol. In total, 19 sessions were executed using FX40 (n = 13), FX50 (n = 3), and FX60 (n = 3) dialysers during 6.5 ± 0.9 hours. Blood fl ow was 149 ± 45  ml/minute, albumin fl ow 226  ± 49  ml/minute, and ultrafi ltration fl ow 432 ± 517 ml. RRs were 70 ± 15% (urea), 34 ± 14% (Crea), 44 ± 16% (bili), and 36 ± 10% (NH3). Primary survival rate was 100%. Four patients were transplanted (bridge to transplant) of which, however, one died within 30 days after discharge from the ICU. The fi fth patient died due to primary disease 9 months after treatment, and the remaining three patients fully recovered (bridge to recovery). y y Methods We evaluated the fi rst 14 patients treated with the HepaWash® system with regard to safety and effi cacy. Seven patients were treated in the context of the run-in phase of the studies (HEPATICUS 1 and HEPATICUS 2) and seven patients were treated since the HepaWash® system received the CE certifi cate in July 2013. P381 Prometheus® liver therapy in children with acute liver failure J Vande Walle, S Claus, E Snauwaert, J De Rudder, A Raes, M Dick, A Prytula, W Van Biesen, S Eloot Ghent University Hospital, Gent, Belgium Critical Care 2015, 19(Suppl 1):P381 (doi: 10.1186/cc14461) Results The records of 462 patients admitted to our ICU were reviewed. The results of 36 patients could not be reached, so 426 patients were evaluated in the study. Among 426 patients, 169 (39.7%) were female and 257 (60.3%) were male. The mean age was 63.7 ± 18.7. HBsAg was positive in nine (2.1%) patients; all of these nine were male. Anti-HCV was positive in four (0.9%) patients; among these, three were male and one was female. Only one patient was positive for anti-HIV. Introduction The Fractionated Plasma Separation and Adsorption System Prometheus® (Fresenius Medical Care, Germany) aims at being S134 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Close observation and treatment of coagulopathy and electrolyte disturbances is essential when treating patients with MARS. MARS can reduce and stabilize ammonium and other biomedical markers in patients listed for urgent liver transplantation with high MELD score and liver encephalopathy. It seems that, in some cases and with our settings, the detoxifi cation properties of MARS may be insuffi cient. a supportive therapy as a bridge to transplantation or recovery in adults with liver failure. The system off ers specifi c challenges when applied in children due to the large extracorporeal volume (700 to 750 ml). We therefore developed an adapted protocol for the application in children. Methods Priming of the blood circuit is performed using 2 l isotonic saline, whereas the plasma circuit, containing both adsorption devices, is fi lled with 2 U fresh frozen plasma or 400 ml stabilized solution of human plasma proteins. Next, for children with body weight (BW) <25 kg, a solution of 60 to 65% packed cells (PC) is infused in the inlet blood line at 40  ml/minute. The volume of PC needed is calculated based on the circuit priming volume and the maximum allowed extracorporeal blood volume of the child (= 8 ml/kg × BW). After the priming phase, blood and plasma fl ow are increased to at least 100 ml/ minute and 200 ml/minute, respectively, and dialysate fl ow is set at 300 ml/minute. P381 Regional citrate anticoagulation is done with a calcium- free dialysate, while, eventually, heparin is added to the priming solution. Post treatment, the circuit volume is either not reinfused (BW <25 kg) or reinfused using isotonic saline (BW >25 kg), with a volume depending on the hydration status and the originally infused volume of PC. Reduction ratios (RRs, %) of urea, creatinine (Crea), bilirubin (bili), and ammonia (NH3) were calculated from pretreatment and posttreatment serum concentrations. Primary and secondary patient outcome was evaluated.i P383 Molecular adsorbent recirculating system treatment in 69 patients listed for liver transplantation P Oli H H Molecular adsorbent recirculating system treatment in 69 patients listed for liver transplantation P Olin, H Haugaa Oslo University Hospital, Oslo, Norway Critical Care 2015, 19(Suppl 1):P382 (doi: 10.1186/cc14462) Introduction The molecular adsorbent recirculating system (MARS) is used to remove circulating albumin-bound toxins in patients with liver failure. However, the application of MARS has not demonstrated improved survival in randomized clinical trials and the clinical utility has not been fi nally established. In our department, the use of MARS is now restricted to the most critically ill patients with acute or acute on chronic liver failure. We aimed to explore MARS effi cacy in removing toxicity parameters and the safety of the system. Conclusion So far the HepaWash® system has proven to be a safe and feasible procedure to eff ectively eliminate water and protein-bound toxins in humans with LF. First clinical experience with a new type of albumin dialysis: the HepaWash® system Patients treated suff ered under acute on chronic LF (n  = 9) or secondary LF which resulted from nonhepatic diseases such as sepsis (n  = 5). Primary endpoint: comparison of serum bilirubin, creatinine and serum BUN before and after the fi rst treatment with the HepaWash® system. Statistics: IBM SPSS Statistics version 22. The Wilcoxon test for paired samples was used to detect signifi cant treatment eff ects. gif Results A total of 254 treatments (1 to 101 per patient) were performed in 14 patients (six female, eight male). Mean age 54 ± 13. MELD score 33.7 ± 7.0, CLIF-SOFA 14.6 ± 2.7. Main underlying disease: nine acute- on-chronic LF; fi ve secondary LF. While bilirubin did not change signifi cantly on the day before HepaWash® treatment (26.2 ± 15.4 vs. 26.0 ± 15.4 mg/dl; P = 0.116), serum bilirubin levels were signifi cantly decreased by the HepaWash® procedure (26.0 ± 15.4 vs. 17.7 ± 10.5 mg/ dl; P = 0.001). Similarly, serum creatinine (2.2 ± 0.8 vs. 1.6 ± 0.7 mg/dl; P = 0.005) and serum BUN (49.4 ± 23.3 vs. 31.1 ± 19.7 mg/dl; P = 0.003) were signifi cantly lowered by the HepaWash® procedure. There were no serious adverse events observed in conjunction with the HepaWash® treatment. Conclusion This adapted Prometheus® protocol is promising for the treatment of children with liver failure. P382 Intensive care referral and admission: do the criteria for liver disease match? J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P384 (doi: 10.1186/cc14464) to c ty pa a ete s a d t e sa ety o t e syste . Methods Since 2005, we have treated 69 patients (30 males/39 females with median age of 39 years ranging from 1 month to 70 years) listed for liver transplantation with MARS. The median Model of End Stage Liver Disease (MELD) score in patients older than 12 years of age (n = 56) was 33 (interquartile range 26 to 39). The fl ow rate was 35 to 40 ml/ kg/hour and treatment kits were changed every 8 to 12  hours. The patients were treated for a median of 31 hours (range 1 to 240 hours). Results Fifty-fi ve patients (79%) were successfully bridged to transplantation. Nine died before they could be transplanted, and fi ve patients recovered without liver transplantation. Forty-four (81%) of the transplanted patients were alive 30 days after transplantation. Ammonium decreased modestly from a median of 148 to 124 μM (P  = 0.03) during MARS treatment. We detected worsening of coagulopathy with signifi cant decreases in platelet count and fi brinogen concentrations, and increase in International Normalized Ratio. Phosphate and magnesium decreased signifi cantly during MARS treatment. J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow UK J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Introduction Hospital admission and mortality rates for patients with cirrhosis in the UK are rising [1]. Cirrhotic patients are physiologically challenged and at increased risk of sepsis and death [2]. Mortality rates for cirrhosis in nontransplant ICUs are up to 37% [3]. Increased availability of medical therapies and public expectation places pressure on limited intensive care resources. There is a lack of research into factors used to decide which patients to admit or refer to the ICU Introduction Hospital admission and mortality rates for patients with cirrhosis in the UK are rising [1]. Cirrhotic patients are physiologically challenged and at increased risk of sepsis and death [2]. Mortality rates for cirrhosis in nontransplant ICUs are up to 37% [3]. Increased availability of medical therapies and public expectation places pressure on limited intensive care resources. There is a lack of research into factors used to decide which patients to admit or refer to the ICU. Methods A prospective survey was sent to all consultant gastroenterologists and consultant intensivists in Scotland. Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? g A Vaz, M Eusebio, A Antunes, A Sousa, P Perez, R Ornelas, C Granja, H Guerreiro Centro Hospitalar do Algarve, Faro, Portugal Critical Care 2015, 19(Suppl 1):P385 (doi: 10.1186/cc14465) Methods A prospective study of 132 consecutive patients admitted to the Critical Burn Unit between October 2008 and October 2011. In all of them resuscitation was performed by objectives: blood pressure (>65  mmHg), hourly diuresis (0.5 to 1  cm3/kg), lactic acid clearance and thermodilution transpulmonary parameters (CI >2.5 l/minute/m2, ITBI: 600 ml/m2). We performed measurements of IAP with a bladder catheter every 8 hours in the fi rst 72 hours. Introduction Cirrhotic patients admitted to the ICU are usually regarded as having a particularly poor prognosis when compared with other groups of critically ill patients. The aim of our study was to evaluate the prevalence, case mix and outcomes of patients with cirrhosis admitted to the general ICU of a nontransplant center. yi Results Ninety-eight men and 34 women were studied. Mean age 48 ± 18 years and a TBSA of 35 ± 22%. The fl uid provided by %TBSA in the fi rst 8 hours was less than predicted by Parkland (4.05 ml/kg), although the total contribution in the fi rst 24 hours was similar. The evolution of the intra-abdominal pressure was: admission 9.7 mmHg, 8 hours 11, 16 hours 10.5, 24 hours 12.1, 32 hours 12.0, 40 hours 12.0, 48 hours 11.1, 56 hours 10.3, 64 hours 10.0 and 72 hours 10.0. A total of 44 patients (33.3%) had a determination higher than 12  mmHg, distributed: 15 patients between 12 and 15 mmHg (IAHT I grade), 14 between 16 and 20 mmHg (II), nine between 21 and 25 mmHg (III) and six >25 mmHg (IV). See Figures 1 and 2.l g p Methods Data were collected from a running ICU database. We studied cirrhotic patients admitted to the ICU between January 2013 and November 2014. Results A total of 30 patients with cirrhosis were admitted, accounting for 3% of total ICU admissions. Mean age was 54.5 years, with a male preponderance (76.7%). The main cause for cirrhosis was alcohol (53.3%), followed by alcohol plus chronic hepatitis C virus (HCV) infection (20%) and HCV virus infection alone (13.3%). The most common causes for admission were sepsis/septic shock (26.7%), surgical (23.4%), gastrointestinal bleeding and hepatic encephalopathy (16.7% each). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 decision. Recipients listed additional criteria used in their own practice and asked whether they would admit or refer individual grades of Child–Pugh cirrhosis with either a gastrointestinal bleed or sepsis.i decision. Recipients listed additional criteria used in their own practice and asked whether they would admit or refer individual grades of Child–Pugh cirrhosis with either a gastrointestinal bleed or sepsis.i C in the Child–Pugh score; mean Acute Physiology and Chronic Health Evaluation (APACHE II) 29.2 ± 8.7 and new Simplifi ed Acute Physiology Score (SAPS II) 62.7  ± 29. Regarding organ failure at admission, the mean Sequential Organ Failure Assessment score was 12.8 ± 4.5. The ICU mortality of these patients was 43.3% and hospital mortality was 53.3%. The variables at admission that related signifi cantly with ICU mortality were: all scores described except for Child–Pugh score, bilirubin, the International Normalized Ratio, creatinine, bicarbonate, lactate, pH and the use of renal replacement therapy during the ICU stay (P  <0.05). The mortality rate of cirrhotic patients was superior to the general ICU mortality (43% vs. 26%). However, patients with cirrhosis presented signifi cantly higher severity scoring systems (APACHE II; SAPS II) at admission compared with noncirrhotics, with high prevalence of organ dysfunction as assessed by SOFA score. Results Thirty-fi ve consultant gastroenterologists and 65 intensive care consultants responded, representing a response rate of 34% and 45% respectively. The only criterion given an average rating of 5 by both gastroenterologists and intensivists was Child–Pugh score when stable. Presence on the transplant list, referral secondary to bleeding varices, recent discharge from the ICU, abstinence from alcohol, nutritional status, age under 30 and more than one additional organ failure all scored 4 or 5 from both groups. Sex, employment, smoking or drug use, deprivation and positive virology status did not infl uence the decision to refer or admit patients. Clinicians reported compliance with medication and outpatient appointments plus an obvious precipitant factor as important features in their decision. The majority of respondents would refer or admit all grades of Child–Pugh cirrhosis with gastrointestinal bleeding. Most would refer or admit Child–Pugh A or B with sepsis. A total 76.5% of gastroenterologists would refer Child–Pugh C cirrhosis with sepsis but only 33.3% of intensivists would accept. P385 Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? A Vaz, M Eusebio, A Antunes, A Sousa, P Perez, R Ornelas, C Granja, H Guerreiro Centro Hospitalar do Algarve, Faro, Portugal Critical Care 2015, 19(Suppl 1):P385 (doi: 10.1186/cc14465) Introduction The aim was to study the evolution and incidence of intraabdominal hypertension in critical burn patients using a slightly restrictive fl uid therapy protocol based on monitoring transpulmonary thermodilution and lactic acid. Intraabdominal pressure in critical burn patients PM Millan 2. Foreman MG, et al. Chest. 2003;124:1016-20. , ; 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. Hospital Universitario La Paz, Madrid, Spain Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P386 (doi: 10.1186/cc14466) p , , p Critical Care 2015, 19(Suppl 1):P386 (doi: 10.1186/cc14466) Intensive care referral and admission: do the criteria for liver disease match? J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P384 (doi: 10.1186/cc14464) Each recipient rated the signifi cance of 18 physiological and social criteria on their decision to refer or admit a patient to intensive care from 1 to 5, with 1 being no infl uence and 5 denoting signifi cant impact on the Results Fifty-fi ve patients (79%) were successfully bridged to transplantation. Nine died before they could be transplanted, and fi ve patients recovered without liver transplantation. Forty-four (81%) of the transplanted patients were alive 30 days after transplantation. Ammonium decreased modestly from a median of 148 to 124 μM (P  = 0.03) during MARS treatment. We detected worsening of coagulopathy with signifi cant decreases in platelet count and fi brinogen concentrations, and increase in International Normalized Ratio. Phosphate and magnesium decreased signifi cantly during MARS treatment factors used to decide which patients to admit or refer to the ICU. Methods A prospective survey was sent to all consultant gastroenterologists and consultant intensivists in Scotland. Each recipient rated the signifi cance of 18 physiological and social criteria on their decision to refer or admit a patient to intensive care from 1 to 5, with 1 being no infl uence and 5 denoting signifi cant impact on the S135 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The high severity of disease in conjunction with the high mortality rate observed in this group of patients should make us consider the possible benefi ts of earlier referring/admission to the ICU, ideally before multiorgan failure arises. On the other hand, in nontransplant centers where cirrhotic patients constitute a small percentage of total ICU admissions, the complexity and peculiarities of the management of these patients should prompt their early transfer to a specialized center. Conclusion Referral and admission decisions for patients with cirrhosis are multifactorial. Child–Pugh status when stable appears to be of greatest signifi cance. The diff erence in opinion of admission of patients with Child–Pugh C with sepsis requires further evaluation. References 1. Thomson SJ, et al. Alcohol Alcohol. 2008;43:416-22. 2. Foreman MG, et al. Chest. 2003;124:1016-20. 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. 1. Thomson SJ, et al. Alcohol Alcohol. 2008;43:416-22. P387 Intraabdominal hypertension in burn patients Results are depicted as mean values ± SD in Tables 1 and 2. y y Results Twenty patients were enrolled in the study. The mean age was 36 ± 13 years. There were 14 males and six females. The average TBSA was 44 ± 17%. Screening and monitoring of IAP were applied by: oliguria (42%), abdominal distension (31.5%) and gastrointestinal trouble (21%). IAH occurred between day 2 and day 3 after early burn resuscitation, respectively in 52% and 63%. IAH was observed in 69% of cases in patients admitted to the ICU with a delay of 1.6 days post burn injury. IAH was noted in 13 patients; of these, fi ve patients developed an abdominal compartment syndrome. The mean IAP was 16 ± 7 mmHg. Patients were assigned into two groups: G1 (IAH+; n = Conclusion Both sepsis and IAH have negative eff ects on respiratory mechanics. However, their combination has even more detrimental eff ects, which do not ameliorate after deinsuffl ation. Table 1 (abstract P388). P387 Intraabdominal hypertension in burn patients Methods Sixteen pigs were divided into two groups of eight (G-A/ G-B). All animals received general anesthesia and were mechanically ventilated. Parameters recorded included respiratory system, chest wall and lung compliance (CRS, CCW, CL) and respiratory system and chest wall inspiratory and expiratory resistances (RRSisp, RRSexp, RCWisp, RCWexp). After baseline measurements (0  minutes), intra- abdominal pressure IAP was raised by helium insuffl ation to 25 mmHg in both groups and remained at that level for the whole study. In G-B, sepsis was induced 60 minutes after IAP increase, by i.v. administration of Escherichia coli endotoxin. Parameters were recorded every 20 minutes. The last measurement was made at 180 minutes, right after deinsuffl ation, and IAP return to baseline levels.i Intraabdominal hypertension in burn patients A Mokline, I Rahmani, L Gharsallah, A Hachani, S Tlaili, R Hammouda, B Gasri, A Ksontini, AA Mesadi Trauma and Burn Centre of Tunis, Tunisia Critical Care 2015, 19(Suppl 1):P387 (doi: 10.1186/cc14467) Introduction Intra-abdominal hypertension (IAH) is frequent in the ICU and has been associated with adverse outcomes and worse prognosis. The purpose of our study was to assess risk factors for IAH and prognosis of major injured patients during burn resuscitation. Introduction Intra-abdominal hypertension (IAH) is frequent in the ICU and has been associated with adverse outcomes and worse prognosis. The purpose of our study was to assess risk factors for IAH and prognosis of major injured patients during burn resuscitation. j j p g Methods Adult burned patients with a burn injury exceeding 20% of total body surface area, from 1 April to 30 November 2013, were included. IAP was measured when IAH was suspected, according to the Kron method via the Foley catheter. Monitoring of IAP was performed every 6 hours during 5 days until normalization. fl Results CRS decreased statistically signifi cantly in both groups after IAP increase and increased after deinsuffl ation only in G-A. Similarly, CCW decreased in both groups but returned to baseline values in both groups after deinsuffl ation. CL decreased more signifi cantly in G-B and returned to baseline values only in G-A. RRSisp increased only in G-B and did not decrease after deinsuffl ation, whereas RRSexp increased in both groups, in a more signifi cant manner in G-B, and decreased only in G-A after deinsuffl ation. RCWisp and RCSesp did not show any alterations during the study period. P388 Eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension Figure 2 (abstract P386). IAH classifi cation. Introduction The aim of our study was to investigate the eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension (IAH). aSepsis induction. Comparison with baseline: *P <0.05, ** P<0.01. Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? At admission, these patients presented an average Model for End- Stage Liver Disease score of 23.5 ± 10.4 with 70% classifi ed as grade Conclusion IAH incidence when a slightly restrictive fl uid protocol used is less than expected. Figure 1 (abstract P386). Intraabdominal pressure. S136 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 2 (abstract P386). IAH classifi cation. Figure 2 (abstract P386). IAH classifi cation. 13) and G2 (IAH–; n = 7). Comparative study of the two groups shows that HIA increases signifi cantly body weight gain within the fi rst 5 days after injury: 8 kg for G1 versus 2 kg for G2 (P = 0.04), occurrence of ARDS (70% for G1 vs. 16.7% for G2, P = 0.02), respiratory failure (77% for G1 vs. 28.5% for G2, P = 0.06), shock (70% for G1 vs. 16.7% for G2, P 0.02) and mortality (61.5% vs. 50%). 13) and G2 (IAH–; n = 7). Comparative study of the two groups shows that HIA increases signifi cantly body weight gain within the fi rst 5 days after injury: 8 kg for G1 versus 2 kg for G2 (P = 0.04), occurrence of ARDS (70% for G1 vs. 16.7% for G2, P = 0.02), respiratory failure (77% for G1 vs. 28.5% for G2, P = 0.06), shock (70% for G1 vs. 16.7% for G2, P 0.02) and mortality (61.5% vs. 50%). Conclusion IAH was frequent in early burn resuscitation of major injured patients. It seems to be associated with fl uid overload in burns and contributes to organ damage. P388 P388 Eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension B Fyntanidou, K Kotzampasi, M Kyparissa, G Stavrou, E Oloktsidou, X Mpesi, K Papapostolou, V Grosomanidis Aristotle Medical School, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P388 (doi: 10.1186/cc14468) aSepsis induction. Comparison with baseline: *P <0.05, ** P<0.01. P387 Intraabdominal hypertension in burn patients Compliance alterations during the study CRS (ml/cmH2O) CCW (ml/cmH2O) CL (ml/cmH2O) Minutes A B P value A B P value A B P value 0 35 ± 8 31 ± 4 NS 58 ± 20 46.1 ± 11 NS 99 ± 22 98 ± 14 NS 20 14 ± 4** 12 ± 3** NS 20 ± 6** 17.7 ± 3** NS 52 ± 21** 35 ± 11** NS 40 13 ± 4** 12 ± 1** NS 19 ± 6** 16.8 ± 3** NS 47 ± 23** 36 ± 13** NS 60a 12 ± 3** 11 ± 2** NS 20 ± 5** 18.4 ± 4** NS 41 ± 20 28 ± 7** NS 80 13 ± 3** 10 ± 1** NS 20 ± 6 18.2 ± 44 NS 41 ± 21** 24 ± 6** <0.05 100 12 ± 3** 10 ± 2** NS 21 ± 8** 19.9 ± 5** NS 37 ± 20** 19 ± 4** <0.05 120 12 ± 3** 10 ± 1** NS 20 ± 6** 21.9 ± 7** NS 37 ± 20** 17 ± 3** <0.05 140 12 ± 3** 9 ± 1** NS 21 ± 7** 21.8 ± 6** NS 37 ± 20** 17 ± 5** <0.05 160 12 ± 3** 9 ± 1** NS 21 ± 7** 22.3 ± 7** NS 37 ± 19** 16 ± 4** <0.05 180 33 ± 7** 16 ± 5** <0.001 50 ± 18 77.0 ± 18** <0.05 81 ± 20* 21 ± 7** <0.001 aSepsis induction. Comparison with baseline: *P <0.05, ** P<0.01. Table 1 (abstract P388). Compliance alterations during the study S137 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 2 (abstract P388). P389 P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G Tsaousi1, EJ Giamarellos-Bourboulis2 1Aristotle University of Thessaloniki, Greece; 2 University of Athens Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P390 (doi: 10.1186/cc14470) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G Tsaousi1, EJ Giamarellos-Bourboulis2 1Aristotle University of Thessaloniki, Greece; 2 University of Athens Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P390 (doi: 10.1186/cc14470) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery Introduction CD73/ecto-5’-nucleotidase is an enzyme that generates adenosine, which dampens infl ammation and improves vascular barrier function in several disease models. CD73 also circulates in a soluble form in the blood [1]. We studied whether levels of soluble form of CD73 and cytokines/chemokines predict the development of organ failure in acute pancreatitis [2,3]. Introduction Heterogeneous published results led us to conduct a clinical trial to assess the effi cacy of a new formulation of four probiotics (P) as prophylaxis for complications after colorectal surgery. p Methods Altogether, 161 patients with acute pancreatitis (107 were subclassifi ed according to the revised Atlanta criteria into mild, 29 into moderately severe and 25 into severe forms) were studied. Serum and blood cell samples were collected at admission. Protein levels of soluble form of CD73 in serum were determined using a novel enzyme- linked immunosorbent assay, activity of soluble form of CD73 using radioactive enzyme assays, and CD73 messenger RNA levels from leukocytes using quantitative PCR. Serum levels of 48 cytokines and growth factors were determined using Bio-Plex Pro Human Cytokine Assay 21-plex and 27-plex magnetic bead suspension panels. y g y Methods A double-blind, placebo-controlled randomized study was conducted enrolling patients undergoing colorectal cancer surgery. Placebo or a formulation of L. acidophilus, L. plantarum, B. lactis and S. boulardii was administered starting 1  day before operation and continuing for 15  days post operation. Patients were followed-up for 30 days with the development of postoperative complications as the primary outcome. PAXGene tubes and serum were collected on postoperative day 4 for measurement of gene expression and serum cytokines (ClinicalTrials.gov NCT02313519). 1. Salmi M, Jalkanen S. Semin Immunopathol. 2014;36:163-76. 2. Maksimow M, et al. Crit Care Med. 2014; 42:2556 3. Nieminen A, et al. Crit Care. 2014 [Epub ahead of print]. P387 Intraabdominal hypertension in burn patients Respiratory system resistance alterations during the study RRSisp (cmH2O/l/minute) RRSexp (cmH2O/l/minute) Minutes A B P value A B P value 0 8.1 ± 0.8 8.3 ± 0.7 NS 13.6 ± 4.1 15.5 ± 3.7 NS 20 7.8 ± 0.6 8.1 ± 0.7 NS 17.1 ± 5.2** 19.3 ± 2.2** NS 40 7.8 ± 0.8 7.6 ± 0.9 NS 18.1 ± 5.4** 20.6 ± 3.1** NS 60a 7.6 ± 0.9 7.5 ± 1.1 NS 18.9 ± 4.4** 19.9 ± 4.7** NS 80 7.8 ± 1.2 8.2 ± 1.2 NS 18.6 ± 4.2** 21.1 ± 4.12** NS 100 7.8 ± 0.9 7.9 ± 0.9 NS 18.2 ± 4.5** 22.7 ± 4.5** NS 120 8 ± 0.6 9.1 ± 1.1 <0.05 18.6 ± 4.3** 20.9 ± 2.2** NS 140 7.7 ± 0.6 9.5 ± 1.2 <0.01 18.8 ± 3.5** 22.9 ± 3.2** <0.05 160 7.3 ± 0.7 9.6 ± 1.5* <0.01 17.7 ± 3.5** 22.2 ± 2.3** <0.01 180 8.1 ± 0.8 9.7 ± 1.2* <0.01 14.9 ± 3.3 18.9 ± 2.3* <0.05 aSepsis induction. Comparison with baseline: *P <0.05, **P <0.01. Table 2 (abstract P388). Respiratory system resistance alterations during the study P389 Severity markers in acute pancreatitis S Jalkanen University of Turku, Finland Critical Care 2015, 19(Suppl 1):P389 (doi: 10.1186/cc14469) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G T i1 EJ Gi ll B b li 2 P389 y g Results Administration of P signifi cantly decreased the rate of all postoperative major complications (28.6% vs. 48.8% of placebo, P = 0.010, odds ratio: 0.42). Major benefi t was found in the reduction of the postoperative pneumonia rate (2.4% vs. 11.3%, P = 0.029), of wound infections (7.1% vs. 20.0%, P = 0.020), of anastomotic leakage (1.2% vs. 8.8%, P = 0.031) and of the need for mechanical ventilation (20.2% vs. 35.0%, P = 0.037). The time until hospital discharge was shortened as Results Activity and protein concentration of soluble form of CD73 and messenger RNA level of CD73 all decreased along with the disease severity (P ≤0.01 for all). The activity of soluble form of CD73 at admission predicted the development of severe pancreatitis in diff erent groups of the patients. Especially, activity of soluble form of CD73 was better than C-reactive protein or creatinine in predicting the severity of pancreatitis in the group of patients without any signs of organ failure at admission. In subgroup analyses of patients with severe pancreatitis and without organ dysfunction upon admission, IL- 8, hepatocyte growth factor and granulocyte colony-stimulating factor (G-CSF) levels predicted the development of severe pancreatitis, with G-CSF being the most accurate cytokine. Figure 1 (abstract P390). Figure 1 (abstract P390). y Conclusion Activity of soluble form of CD73 and levels of certain cytokines at admission to the hospital have prognostic value in predicting the development of the severe form of acute pancreatitis. The possibility that combining them with other prognostic markers might improve prognostic accuracy requires further studies. References 3. Nieminen A, et al. Crit Care. 2014 [Epub ahead of print]. Figure 1 (abstract P390). S138 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 well. Gene expression of SOCS3 was positively related with circulating IL-6 in the P group but not in the placebo group (Figure 1). Conclusion The studied P formulation signifi cantly decreased the risk of postoperative complications, namely mechanical ventilation, infections and anastomotic leakage. Modulation of the gene expression of SOCS3 is one suggested mechanism. thiamine 200  mg or placebo i.v. twice/day for 7  days. The primary outcome was lactate levels at 24 hours. Secondary outcomes included the SOFA score at 24 hours and mortality. Role of ultrasonography in detection of the localization of the nasoenteric tube R Dagli1, H Bayir2, Y Dadali1, T Tokmak1, Z Erbesler1 1Ahi Evran University Education and Research Hospital, Kirsehir, Turkey; 2Abant Izzet Baysal University, Medical School, Bolu, Turkey Critical Care 2015, 19(Suppl 1):P391 (doi: 10.1186/cc14471) Introduction In this study, we aimed to determine the success rate of nasoenteric tube (NET) insertion into the postpyloric area by ultrasonography (USG) and compare it with the commonly used method, direct abdominal radiography. g p y Methods Patients admitted to an adult ICU between April and July 2014 with an indication for NET insertion for enteral feeding were included in the study after informed consent was given from patients’ relatives. Nasoenteric feeding tubes were placed using the blind bedside method by a single anesthesiologist. Any motility stimulant agent was not used. The outside of the polyurethrane 8 F with unweighted NET (Bexen, Spain) and its guiding wire were lubricated with gel. The NET was inserted into the nostril after determination of the mouth– posterior ear–xiphoid distance and pushed on at least such a distance. Followed by auscultation of the gastric area and air infusion of 30 to 50 ml into the tube, the patient was positioned on their right side and the tube was advanced 20 to 30 cm more. Then the guiding wire inside the NET was removed. The patient was then brought to the supine position and NET was visualized by two radiologists simultaneously by M5 portable USG (Mindray, PRC), with a 3.5 MHz convex probe whether it passes through the postpyloric area or not. Localization of the tube was confi rmed with abdominal radiography in all patients. During the fi rst insertion of the NET, the ratios for inaccurate localization and correct placements through the postpyloric area were recorded and results were compared with abdominal radiography. Conclusion Thiamine defi ciency is prevalent in septic shock. Thiamine did not decrease overall median lactate levels at 24  hours. In the patients with thiamine defi ciency, there were statistically signifi cant lower lactate levels at 24  hours in the thiamine group and a large, although nonsignifi cant, diff erence in mortality. P392 Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial M Donnino1, LW Andersen1, M Chase1, KM Berg1, TA Giberson1, H Smithline2, M Tidswell2, R Wolfe1, M Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Critical Care 2015, 19(Suppl 1):P392 (doi: 10.1186/cc14472) Unraveling the link between malnutrition and adverse clinical outcomes: association of acute and chronic malnutrition measures with blood biomarkers from diff erent pathophysiological systems S Felder, N Braun, A Kutz, M Batschwaroff , P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P393 (doi: 10.1186/cc14473) Introduction Malnutrition is common in hospitalized medical patients and is associated with poor clinical outcomes. Whether malnutrition has a direct link to adverse outcomes or is rather a mirror of the severe patient condition remains debated. Our aim was to study the association of acute and chronic malnutrition status with blood biomarkers from diff erent pathophysiological concepts to better understand the underlying mechanisms of malnutrition. Results In this study, the bedside blind method was used for NET insertion into 34 patients. Eleven of the tubes were detected passing through the postpyloric area by USG. In one case the NET could not be seen in the postpyloric area by USG, but it was detected in the postpyloric area by control abdominal radiography. In 22 patients, NETs were detected in the stomach with control abdominal radiography. Success for NET placement with the bedside blind method and USG imaging was 35% versus 91.6%, respectively. Methods We prospectively followed consecutive adult medical inpatients hospitalized between February 2013 and October 2013 in a tertiary care Swiss hospital. Nutritional risk was assessed using the Nutritional Risk Screening (NRS 2002) score, which incorporates acute and chronic measures of malnutrition. Multiadjusted regression models were used to investigate the associations between acute and chronic nutritional risk and biomarkers mirroring infl ammation (CRP, PCT, proADM, leucocytes), stress (copeptin), renal dysfunction (creatinine, urea), nutritional status (vitamin D25, albumin, calcium, glucose), and hematological function (platelets, INR, Hb, RDW). Biomarker levels were transformed into deciles due to skewed distributions. Conclusion The success rate of the bedside blind method in the NET placement was low. It is clear that if any other placement techniques with high success rate will be applied, USG will be useful in a higher number of patients reducing the need for abdominal radiography. P389 Lactate levels at 24 hours were compared between groups using the Wilcoxon rank-sum test and categorical variables were compared using the Fisher’s exact test. Lactate values at 24 hours, for those who died before 24 hours, were imputed according to a predefi ned plan. We performed a preplanned analysis in those with baseline thiamine defi ciency (≤7 nmol/l). well. Gene expression of SOCS3 was positively related with circulating IL-6 in the P group but not in the placebo group (Figure 1).i thiamine 200  mg or placebo i.v. twice/day for 7  days. The primary outcome was lactate levels at 24 hours. Secondary outcomes included the SOFA score at 24 hours and mortality. Lactate levels at 24 hours were compared between groups using the Wilcoxon rank-sum test and categorical variables were compared using the Fisher’s exact test. Lactate values at 24 hours, for those who died before 24 hours, were imputed according to a predefi ned plan. We performed a preplanned analysis in those with baseline thiamine defi ciency (≤7 nmol/l). Conclusion The studied P formulation signifi cantly decreased the risk of postoperative complications, namely mechanical ventilation, infections and anastomotic leakage. Modulation of the gene expression of SOCS3 is one suggested mechanism. yi y ( ) Results We enrolled 88 patients; 43 received thiamine and 45 placebo. Baseline characteristics were similar between groups. We found no overall statistical signifi cant diff erence in 24-hour lactate levels between thiamine and placebo groups (2.5 (IQR: 1.5 to 3.4) vs. 2.6 (IQR: 1.6 to 5.1), P = 0.40). Fewer patients in the thiamine group had lactate levels >4 mmol/l at 24 hours (21% vs. 38%, P = 0.10) and this was statistically signifi cant if only evaluating survivors at 24 hours (7% vs. 33%, P = 0.03), although our preplanned analysis was to impute data. We found no diff erence in 24-hour SOFA score or mortality. A total of 28 (35%) patients were thiamine defi cient. Of the defi cient patients, those receiving thiamine had statistically signifi cant lower lactate levels at 24 hours (2.1 (IQR: 1.4 to 2.5) vs. 3.1 (IQR: 1.9 to 8.3), P = 0.03) and more patients in the placebo group had a lactate >4 mmol/l (38% vs. 7%, P = 0.07). Mortality in the thiamine and placebo groups was 13% and 46%, respectively (P = 0.10).i Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial We considered target calories to be 25 kcal/kg/day and target proteins to be 1.5 g/kg/day. Introduction The provision of nutrition in the critical care unit (CCU) has shifted from nutrition support to nutrition therapy, and the potential benefi ts derived from this in the recovery of the critically ill is being explored [1]. We audited the management of nutrition in the CCU in a South African Hospital against the American Society of Parenteral and Enteral Nutrition Guidelines. Furthermore, we reviewed the knowledge and confi dence of healthcare providers in the management of nutrition in the CCU. g y g p g g y Results Patients received more calories (78.3% vs. 59.1%) and more proteins (70.2% vs. 54.6%) post implementation. The mean percentage of patients in the post group who achieved >70% of required calories was 80.1% versus 30.9% in the pre group. The mean percentage of patients who achieved >70% of required proteins was 58.3% versus 32.1% in the pre group. p g p Conclusion The multipronged approach of the quality improvement methodology helped to increase the provision of calories and proteins in our population of critically ill surgical patients. However, there is still room for improvement in terms of achieving optimal enteral nutrition targets early in our population. There is also a need to look into sustaining such results. Methods Retrospective data collection of patients admitted to a four- bed CCU over a 4-month period in 2013. A survey was distributed to diff erent disciplines involved in patient nutrition in the CCU. Results Seventy-two patients were admitted to the CCU during this time period, and notes were able for 44. Three paediatric patients were excluded. Twenty-nine patients stayed for 2 or more days (the audit population). The median age of the audit population was 38, 19 were female. Sixteen were postoperative admissions. The median APACHE II score of the patients with suffi cient available data (n = 16) was 14 (range 6 to 34). The audit found that 21 of the patients had nutrition started in the CCU, with 15 having nutrition started within 48 hours. Only eight patients had a nutritional assessment done. A total of 45 responded to the survey: eight anaesthetists, 25 from surgical disciplines, seven CCU nurses, and fi ve dieticians. All agreed that nutrition should be started in the fi rst 48 hours, except from the surgeons only 14 (56%) agreed. Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial The average self-rating of knowledge of nutrition management in the CCU (1 = lowest, 5 = highest) was 2.1 with the dieticians and CCU nurses showing the highest confi dence with 3.4 and 2.6, respectively. The anaesthetists rated their knowledge at 1.9 and the surgeons rated themselves at 1.8. P397 Introduction Optimizing enteral nutrition early has been shown to be benefi cial in critically ill patients. However, underfeeding is still a common problem. The critically ill surgical patient often presents with additional challenges to optimal enteral feeding. The objective of this study was to improve enteral feeding practices in a surgical ICU. P395 P395 Quality improvement project to optimize enteral nutrition in a tertiary hospital’s surgical ICU J Li, LY Koh, JH Yang, C Khoo, T Ter, BH Tan National University Health System, Singapore Critical Care 2015, 19(Suppl 1):P395 (doi: 10.1186/cc14475) g p g Conclusion Perioperative optimal nutrition support for at least 7 days could modulate the infl ammatory status and clinical outcome of severe malnourished surgical neoplasic patients. Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial Results A total of 529 patients (mean age 72 years, 57.1% male) were included. Overall, there was a signifi cant association of NRS and most biomarkers of infl ammation, stress, renal function, nutrition and the hematological system (coeffi cient and 95% CI): CRP 0.021, P = 0.0021, PCT 0.28, P = 0.003, proADM 0.4, P <0.001, copeptin 0.44, P <0.001, urea 0.28, P = 0.002, vitamin D25 –0.23, P = 0.012, albumin –0.6, P <0.001, hemoglobin –0.5, P <0.001, RDW 0.46, P <0.001. These associations remained robust after adjustment for sociodemographics (model 1), comorbidities (model 2) and main medical diagnosis (model 3). Subgroup analysis suggested that mainly the acute part of malnutrition and not chronic malnutrition was associated with an increase in biomarker levels. H Smithline2, M Tidswell2, R Wolfe1, M Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Introduction The objective was to determine whether the administration of thiamine mitigates elevated lactate levels in patients with septic shock. Thiamine is essential for aerobic metabolism and we have found that thiamine levels are low and inversely correlated with lactate levels in patients with sepsis. Conclusion Acute malnutrition was associated with a pronounced infl ammatory response and an increase in biomarkers from diff erent pathophysiological systems which may partly explain the link between malnutrition and adverse medical outcomes. However, interventional trials are needed to prove causal relationships. Methods We performed a randomized, double-blind, placebo- controlled, two-center trial from January 2010 to October 2014. We enrolled patients with septic shock, elevated lactate (≥3 mmol/l) and no obvious competing cause of lactate elevation. Patients received S139 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P394 Evaluation of the provision of nutrition in a South African provincial hospital S Kudsk-Iversen, R Matos-Puig, R Naidoo, S Moorad Stanger Provincial Hospital, Stanger, South Africa Critical Care 2015, 19(Suppl 1):P394 (doi: 10.1186/cc14474) interventions were conducted with lectures to physicians and nurses. Visual aids in the form of screensavers at each bedside computer served as reminders to the team to optimize feeding. A subsequent audit was then conducted to determine the improvement in achieving the desired outcomes, namely the amount of calories and proteins received as well as the proportion of patients who achieved >70% of their target calories and proteins. Could preoperative and postoperative optimal nutrition support modulate the infl ammatory response and clinical outcome of severe malnourished surgical patients with gastrointestinal neoplasia? p L Mirea, D Pavelescu, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P396 (doi: 10.1186/cc14476) p L Mirea, D Pavelescu, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P396 (doi: 10.1186/cc14476) p L Mirea, D Pavelescu, I Grintescu Introduction Our aim was to assess whether perioperative and postoperative optimal 7-day nutrition support could modulate the infl ammatory status and clinical outcome of severe malnourished patients with surgery for gastrointestinal neoplasia. Methods A prospective randomized study of 64 patients with gastrointestinal neoplasia, severe malnourished BMI <18.5, albumin level <3 g/dl, BW loss >10%, NRS >3, scheduled for surgery, allocated into two groups. Group A: 32 patients, minimal enteral nutrition in the postoperative period according to tolerance, medium 500  kcal/day. Group B: 32 patients received optimal parenteral nutrition support (25 kcal/kg/day) 3 days before surgery and continued for at least 4 days postoperatively. We measured CRP, fi brinogen, IL-6, TNF, albumin level preoperative and at 96 hours, the incidence of complications, and the length of ICU stay.i Conclusion We found that there is poor management of nutrition in the CCU. This is paired with limited knowledge and low confi dence in management amongst the attending staff . Evidence would suggest that the development and dissemination of clear hospital guidelines could improve rates of correct management [2]. However, the lack of uniform guidance based on strong evidence from the leading global authorities on nutrition suggests that, in order to improve implementation of adequate nutrition, more research is urgently required. References 1. McClave S, et al. JPEN J Parenter Enteral Nutr. 2009;33:277-316. 2. Martin CM, et al. CMAJ. 2004;170:197-204. Results There was a signifi cant decrease in the values of CRP, IL-6, TNF, albumin at 96  hours in group B. No diff erence in fi brinogen. A signifi cantly lower rate of complications and a shorter time of ICU stay were observed in group B. See Figures 1 and 2 (overleaf). 1. McClave S, et al. JPEN J Parenter Enteral Nutr. 2009;33:277-316. 2. Martin CM, et al. CMAJ. 2004;170:197-204. Does discontinuation of the use of hydroxyethyl starches in the critically ill cardiac surgery patient have an impact on caloric intake? E De Waele, K De Bondt, S Mattens, J Czapla, J Nijs, M La Meir, D Nguyen, PM Honoré, H Spapen Universitair Ziekenhuis Brussel, Brussels, Belgium Critical Care 2015, 19(Suppl 1):P397 (doi: 10.1186/cc14477) Methods The Clinical Practice Improvement Programme is a local quality improvement initiative involving a multidisciplinary team aimed at identifying and improving defi ciencies in the process of delivery of care. A team led by an intensivist, consisting of doctors, surgeons, nurses and a pharmacist, was formed to improve enteral feeding practices in a surgical ICU. The quality improvement methodology was employed. An audit was carried out to determine the problem of underfeeding in the unit. Root cause analyses were conducted and team members identifi ed key barriers to optimal feeding and areas for improvement. Protocols were developed to standardize and encourage early enteral feeding as well as to reduce the time feeds are interrupted for patients who were going for surgeries or for various other reasons. Educational Introduction After research revealed unwanted eff ects of the use of starches in critically ill patients, its use in the immediate postoperative period of cardiac surgery patients came to an abrupt ending. However, they constitute an important source of non-intended calories, providing 4 calories per gram. We investigated whether this phenomenon (involuntary) attributed to an increase in caloric debt for this critically ill patient population. Methods We retrospectively searched a database of 417 elective cardiac surgery patients, representing 5,004 observation-days. Caloric S140 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 Figure 1 (abstract P396). Results 1. Figure 2 (abstract P396). Results 2. Figure 1 (abstract P396). Results 1. Figure 1 (abstract P396) Results 1 Figure 1 (abstract P396). Results 1. Figure 2 (abstract P396). Results 2. Figure 2 (abstract P396). Results 2. by aggressive nutrition therapy, in the general population of an ICU. Cancer patients are more prone to be at nutritional risk due to the disease and treatment complications. Our aim was to characterize NUTRIC score behavior in the population of patients admitted to an oncologic ICU. intake was evaluated in the group of patients before and after the cessation of starch use. Results Patient characteristics and caloric needs were comparable: 2,054 ± 395 kcal/day and 2,056 ± 347 kcal/day. Does discontinuation of the use of hydroxyethyl starches in the critically ill cardiac surgery patient have an impact on caloric intake? The 140 patients who in the immediate postoperative period had volume resuscitation without the use of starches had a mean non-intended fl uid caloric intake of 69 (± 36.3) kcal/day. The group of 277 patients who received starches in the postoperative period had a mean non-intended fl uid caloric intake of 105 (± 100.2) kcal/day. g Methods Between January and June 2014 we applied the NUTRIC score to all patients, age >18 years, without cerebral death criteria and with a length of stay (LOS) >72  hours. Data were collected and analyzed using SPSS v20.0. To evaluate the impact on mortality we used logistic regression. y Conclusion Withdrawal of the use of starches resulted in a 34% decrease of non-intended caloric intake by fl uids, contributing to caloric debt. Whether outcome is infl uenced and/or whether these fi ndings are clinically relevant needs further research. g Results Sixty-nine patients were included, 23 women (33.3%) and 46 men (66.7%). Most patients were aged between 50 and 75 years (72.5%) and had normal range weight 58% (n = 40). The mean LOS was 11.56 (minimum: 3 to maximum: 69). The most common motive for admission was sepsis (7.7%, n = 26). APACHE II score was above 15 in 77% of the patients (n = 53) and SOFA score was superior to 6 in 56.5% (n = 30). The NUTRIC score was low risk in 42% (n = 29) of the patients and high in 58% (n = 40). Twenty-eight-day mortality was 26.1% (n = 18). A high NUTRIC score corresponded to a 22-fold increased odds of dying in the fi rst 28 days (P <0.001). Both APACHE II and SOFA were mortality predictors alone, with an increase of 1 point in APACHE score corresponding to an increase of 14% (P = 0.002) and an increase of Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) Conclusion The NUTRIC score is a good tool in cancer patients to predict 28-day mortality. Nevertheless, the only compounds of the NUTRIC score that correlated independently with mortality were APACHE II and SOFA scores. Further investigation towards the inclusion of other categories such as tumor staging and the type of tumor could be useful to develop a specifi c prognostic tool for this population. Introduction Critically ill patients experience hypermetabolism in- creas ing substrate utilization, especially glucose oxidation. Glycogen is the main source of glucose in the body, being 85% and 15% stored in skeletal muscle and liver respectively. Since glycogen stores are limited we evaluated the hypothesis that critical illness could be associated with glycogen depletion leading to skeletal muscle catabolism for gluconeogenesis and eventually resulting in cachexia, an important determinant of future ICU survival and ICU-acquired weakness. Methods Nine critically ill patients (58.75  ± 25 to 75  years old) with an ICU stay from 1  day to 5  weeks were evaluated for skeletal muscle glycogen content using a rapid, non-invasive high-frequency ultrasound methodology (MuscleSound®, Denver, CO, USA). Scans were obtained from the rectus femoris and vastus lateralis muscles. Glycogen content was measured with a score from 0 to 100 according to the MuscleSound® scale. Patients had a variety of primary diagnoses including septic shock (n  = 3), hemorrhagic shock/abdominal hypertension (n = 1), hypovolemic shock/post major oncologic surgery (n = 1), trauma (n = 3), and burn injury (n = 1). Early calorie-dense immune nutrition in haemodynamically compromised cardiac patients S Efremov, V Lomivorotov Research Institute of Circulation Pathology, Novosibirsk, Russia Critical Care 2015, 19(Suppl 1):P399 (doi: 10.1186/cc14479) Introduction The aims of present study were to test the hypothesis that early enteral nutrition (EN) with calorie-dense food supplemented with glutamine improves recovery of nutritional status in critically ill cardiac patients and to evaluate their resting energy expenditure (REE). Results Six out of nine patients had no glycogen present in the muscle (score = 0). The other three patients had glycogen scores between 5 and 15 which are well below scores of healthy individuals (reference 50 to 70). Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) As a comparison we collected post-competition levels in competitive athletes, which decrease their glycogen stores (score 15 to 25) but are well above those of most critically ill patients we have studied. p g gy p Methods A prospective randomised study of 40 adult cardiac patients undergoing elective cardiopulmonary bypass surgery no more than 24  hours before eligibility assessment, complicated with acute heart failure syndrome. Patients were randomised to receive either standard isocaloric isonitrogenic early EN (standard group, n  = 20) or immunomodulating early EN (immune group, n  = 20). The daily energy target was set using REE measured by indirect calorimetry (CCM Express; Medgraphics, St. Paul, MN, USA). Serum prealbumin, transferrin, C-reactive protein, blood lactate and clinical characteristics were analysed. Conclusion This is the fi rst time that muscle glycogen stores have been evaluated in critical illness. Our data show severe glycogen depletion in ICU patients which probably leads to muscle catabolism necessary for gluconeogenesis, eventually resulting in cachexia. This fi nding poses severe metabolic challenges for ICU patients in which interfering with recovery can contribute to poor survival. In light of our fi ndings, re-evaluation of nutritional protocols and potential anabolic/anti- catabolic therapy to decrease muscle catabolism may improve survival. Diff erent therapeutics that may prevent hypermetabolism (such as beta-blockers) should be re-evaluated along with anabolic agents (that is, oxandrolone) which could counteract the severe catabolic response in critical illness. Monitoring of muscle glycogen repletion could signal the transition from the catabolic to anabolic phase. y Results The actual REE was an average of 6.8 and 7.5  kcal/kg/day higher than the REE calculated using the Harris–Benedict equation and empiric approach (25 kcal/kg/day), respectively (Figure 1). Early EN with immune formula was associated with higher levels of prealbumin concentration on the 14th day (0.13 ± 0.01 g/l and 0.21 ± 0.1 g/l; P = 0.04) and transferrin on the 3rd, 5th, 7th, and 14th day (P <0.05) after surgery. Conclusion Haemodynamically compromised cardiac patients have increased REE, which in the absence of indirect calorimetry should be set at 30 kcal/kg/day. Early EN using a calorie-dense immune formula leads to better recovery of nutritional status as assessed by serum protein levels. Plasma glutamine after acute or elective admission on the ICU H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) NUTRIC score in oncologic patients A Patrão, L Bei, F Coelho A Patrão, L Bei, F Coelho Instituto Português de Oncologia – Porto, Portugal g g , g Critical Care 2015, 19(Suppl 1):P398 (doi: 10.1186/cc14478) Introduction The NUTRIC score is a tool designed to quantify the risk of critically ill patients developing adverse events that may be modifi ed S141 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 1 point in SOFA corresponding to an increase in the odds of being dead at 28 days (P = 0.002). Body mass index, age, number of comorbidities, and days in the ICU did not correlate with mortality. Conclusion The NUTRIC score is a good tool in cancer patients to predict 28-day mortality. Nevertheless, the only compounds of the NUTRIC score that correlated independently with mortality were APACHE II and SOFA scores. Further investigation towards the inclusion of other categories such as tumor staging and the type of tumor could be useful to develop a specifi c prognostic tool for this population. 1 point in SOFA corresponding to an increase in the odds of being dead at 28 days (P = 0.002). Body mass index, age, number of comorbidities, and days in the ICU did not correlate with mortality. Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis y p Methods Sepsis was induced by E. coli LPS. GLN was infused: i.v. through the femoral vein (0.5 g/kg); enterally (E) through jejunostomy (0.5 g/kg); and i.v. + E. Blood was drawn continuously from the femoral artery and the portal vein for GLN plasma levels in systemic-S and portal-P circulation. P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) p Results In healthy swine, GLN levels remained stable, both in S and P; i.v. infusion, and even more i.v. + E signifi cantly increased GLN in the S circulation (P <0.001), whereas E infusion failed to do so (P = 0.4). On the contrary, GLN P levels were signifi cantly increased after i.v. + E infusion, as well as after E infusion (P <0.001) and to a lesser extent, after i.v. (P <0.001). In sepsis, both S and P GLN levels decreased signifi cantly. Introduction Intravenous fi sh oil (FO) lipid emulsions (LEs) are rich in ω-3 polyunsaturated fatty acids, which exhibit anti-infl ammatory and immunomodulatory eff ects. We previously demonstrated that FO- containing emulsions may be able to decrease mortality and ventilation days in the critically ill. Over the last year, several additional randomized controlled trials (RCTs) of FO-based LEs have been published. Therefore, the purpose of this meta-analysis was to update our systematic review aimed to elucidate the effi cacy of FO-based LEs on clinical outcomes in the critically ill. Figure 1 (abstract P404). Figure 1 (abstract P404). y Methods We searched computerized databases from 1980 to 2014. Overall mortality was the primary outcome and secondary outcomes were infections, ICU and hospital length of stay (LOS), and mechanical ventilation (MV) days. We included RCTs conducted in critically ill adult patients that evaluated FO-based LEs in parenteral nutrition (PN) or enterally fed patients. We analyzed data using RevMan 5.1 (Cochrane IMS, Oxford, UK) with a random eff ects model. f Results A total of 10 RCTs (n  = 733), including four trials published over the last year, met inclusion criteria. Plasma glutamine after acute or elective admission on the IC H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) Figure 1 (abstract P399). Indirect calorimetry measured signifi cantly higher resting energy expenditures. Introduction Low plasma glutamine concentrations are associated with unfavourable outcome at acute ICU admission. We questioned whether there is a diff erence in plasma glutamine level after acute or elective ICU admission. Methods We performed a single-centre prospective observational study in a 22-bed mixed ICU. Exclusion criteria were age <18 years and total parental nutrition at admission. Patients were divided into two groups: elective surgery and acute admissions. Blood samples were taken at ICU admission and daily at 6.00 a.m. Glutamine levels were measured using the Bioprofi le 100 plus analyser (Nova Biomedical UK, Cheshire, UK). A Mann–Whitney U test was used to detect diff erences between groups and a Bonferroni method to correct for multiple comparisons. Results We included 88 patients after elective surgery (76 cardiac and 12 general surgery) and 90 patients after acute admission (27 sepsis, 17 acute surgery, two trauma and 44 medical). Baseline characteristics are presented in Table 1. Plasma glutamine levels at admission were signifi cantly lower in acute patients compared with elective surgery, 0.25  mmol/l (IQR 0.09 to 0.37) versus 0.43 mol/l (IQR 0.33 to 0.55) Figure 1 (abstract P399). Indirect calorimetry measured signifi cantly higher resting energy expenditures. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S142 Table 1 (abstract P402) Elective Acute Age (years) 68 ± 10 59 ± 17* APACHE IV 46 ± 14 67 ± 29* LOS ICU 2 (2 to 2) 4 (2 to 7)* Survival (n) 88 85* Mean ± SD or median (IQR). *P <0.01. 0.17, heterogeneity I2 = 0%), and hospital LOS (WMD –4.06; 95% CI, –10.14 to 2.03; P = 0.19, I2 = 89%, P <0.00001), without eff ect on ICU LOS. See Figure 1. Conclusion FO-based LEs may be associated with a reduction in infections, as well as clinically important reductions in duration of ventilation, and hospital LOS. Nevertheless, according to current literature there is inadequate evidence to give a fi nal recommendation on the routine use of FO-containing emulsions in PN and/or as a pharmaconutrient strategy in enterally fed critically ill patients. Further large-scale RCTs which should aim to consolidate potential positive treatment eff ects are warranted. (P <0.001). There appeared to be a signifi cant correlation between the APACHE IV score and glutamine levels (R = 0.52, P <0.001). P403 P403 Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) P404 Glutamine administration in sepsis: enteral, parenteral or both? Experimental study in swine G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) Plasma glutamine after acute or elective admission on the IC H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) Moreover, in a backward linear regression analysis this correlation was independently associated with APACHE IV scores and the presence of infection, but not with the type of admission.i Glutamine administration in sepsis: enteral, parenteral or both? Experimental study in swine G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) yp Conclusion Plasma glutamine levels were signifi cantly lower after acute admission compared with elective surgery. In both groups a considerable amount of patients had decreased glutamine levels, but this was not independently associated with the type of admission. In contrast to previous studies we found that glutamine levels were determined by severity of illness and the presence of an infection. p Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) Introduction Glutamine (GLN) is recommended in the critically ill for i.v. administration but enteral use is not quite clarifi ed. We decided to measure GLN plasma levels in healthy and septic swine after GLN given by i.v., enteral or both routes, over a 3-hour period. P405 Methods The SALOMON algorithm is based on 53 common presentation fl owcharts using specifi c discriminators to triage calls into four categories according to the level of care required: emergency medical services, nonemergent visit to local emergency department, PCP home visit or PCP delayed consultation. Using an appropriate statistical test, we assessed the accuracy of presentation fl owchart and triage category selections attributed to 130 clinical scenarios, by 10 diff erent nurses, in comparison with references established by a local team of experts, at two diff erent time periods: immediately after training (T0) and 3 to 6 months after algorithm practice (T1).l Prehospital factors associated with an ICU admission from the emergency department P406 P406 Reliability of a new French-language triage algorithm for out-of- hours primary care calls: the SALOMON rule E Brasseur1, A Ghuysen1, AF Donneau2, V D’Orio1 1C.H.U. of Liège, Belgium; 2University of Liège, Belgium Critical Care 2015, 19(Suppl 1):P406 (doi: 10.1186/cc14486) Figure 2 (abstract P404). Introduction Because of increased workloads associated with primary care physician (PCP) shortage, many western countries have been facing the diffi cult challenge of optimizing their out-of-hours primary care services. PCPs initially gathered in small rotation groups, and then further collaborations led to larger-scale cooperatives. In such models, implementation of patient call triage is mandatory to increase the effi ciency, quality and safety of care [1]. Organization models diff er, from the PCP performing all patient calls to nurses and nurse assistants answering calls and performing triage, but no validated triage algorithm has been reported to date. We developed a specifi c French- language triage algorithm called SALOMON (Système Algorithmique Liégeois d’Orientation pour la Médecine Omnipraticienne Nocturne) in order to guide nurse triage PCP out-of-hours calls. The present study tested this algorithm reliability. As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P  = 0.08). See Figures 1 and 2. As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P  = 0.08). See Figures 1 and 2. Conclusion In our experimental early sepsis model, a combination of E and i.v. GLN seems to be the most appropriate; this results in high GLN levels for the functional needs, including those of the gut mucosa. Reference 1. Crit Care. 2014;18:R76. Reference Reference 1. Huibers L, et al. Scand J Prim Health Care. 2011;29:198-209. 1. Huibers L, et al. Scand J Prim Health Care. 2011;29:198-209. Prehospital factors associated with an ICU admission from the emergency department TA Williams1, J Finn1, D Fatovich2, D Brink3, KM Ho2, H Tohira4 1Curtin University, Bentley, Australia; 2Royal Perth Hospital, Perth, Australia; 3St John Ambulance – WA, Belmont, Australia; 4 Curtin University Health Sciences, Bentley, Australia Critical Care 2015, 19(Suppl 1):P405 (doi: 10.1186/cc14485) Introduction This study aimed to describe the patient characteristics and prehospital factors associated with an ICU admission from the ED. There is a paucity of information about the early recognition of critical illness by paramedics; especially in the Australian prehospital setting. Methods A retrospective cohort study, July 2012 to June 2014, conducted in the Perth metropolitan area, which is served by a single ambulance service. Adult patients were included if transported to a public hospital ED that used the ED information system (EDIS) (seven of eight EDs) and were admitted to the ICU from the ED (ED-ICU group). Patients aged <16  years, those from rural areas or transfers were excluded. We used existing ambulance clinical data linked to EDIS data. Prehospital cohort characteristics are described using univariate statistical techniques. Logistic regression was conducted with admission to the ICU from the ED (critical illness surrogate) as the outcome variable. Variables included in regression models were age, sex, paramedic-identifi ed urgency, that is the time patients should be seen by a doctor based on the Australasian Triage Scale, paramedic- identifi ed patient problem and the time taken from the ambulance service receiving the call to hospital arrival. Physiological variables: systolic blood pressure (SBP), heart rate (HR), respiratory rate (RR), temperature, oxygen saturation, and GCS were included in the logistic models. Results Overall selection of fl owcharts was accurate for 94.1% of scenarios at T0 and 98.7% at T1. Triage category selection was correct for 93.3% of scenarios at T0 and 98.4% at T1. Both fl owchart selection and triage category were correct in of 89.5% case in T0 and at 97.5% T1. When an incorrect fl owchart was used, triage category remained accurate in 64.9% and 70.5% respectively. Both fl owchart and triage selection accuracy improved signifi cantly from T0 to T1 (P <0.0001). y gi y Conclusion The results of the present study revealed that using the SALOMON algorithm is reliable for out-of-hours PCP call triage by nurses. Validity of this rule may be further evaluated through its actual implementation in real-life conditions. Reference Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis There was considerable heterogeneity in interventions tested in these trials. No eff ect on overall mortality was found. When the results of fi ve RCTs that reported infections were aggregated, FO-containing emulsions signifi cantly reduced infections (RR 0.64; 95% CI, 0.44 to 0.92; P = 0.02, heterogeneity I2  = 0%). Furthermore, FO-based LEs were associated with a trend toward a reduction in MV days (WMD, –1.41; 95% CI, –3.43 to 0.61; P = Figure 1 (abstract P404). Figure 1 (abstract P403). Eff ects of parenteral fi sh oil lipid emulsions on infections. S143 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P  = 0.08). See Figures 1 and 2. Conclusion In our experimental early sepsis model a combination of Figure 2 (abstract P404). (all P <0.001) except the time taken from receiving the call to hospital arrival (P = 0.48). Figure 2 (abstract P404). Conclusion Three-quarters of the ED-ICU patients were transported to the ED with high urgency. Currently no prehospital severity of illness or early warning system (EWS) is used in our ambulance service. Given the small proportion of ED-ICU patients who presented with abnormal observations, it is unlikely that introducing an EWS would alter practice or patient outcome. P407 Adequacy of trained assistance, emergency equipment and drugs at emergency calls in the ICU and in remote areas of the hospital I Kolic1, S Unell2, A Watts2, A Barry2, J Short2 1South London Healthcare NHS Trust, London, UK; 2Lewisham and Greenwich NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P407 (doi: 10.1186/cc14487) Results Of the 142,448 eligible patients transported by ambulance, 1,076 (0.75%) were admitted to the ICU from the ED: the ED-ICU group was younger (mean 53 vs. 61 years, P <0.001). Seventeen percent of ICU patients were transported as Urgency 1 (resuscitation/immediate) and 58% as Urgency 2 (within 10 minutes) while 70% of non-ICU patients were transported as Urgency 3 to 5 (P <0.001). Thirteen percent of ICU patients had a SBP <90 mmHg, 15% had a HR ≥130 and 19% had a RR >30. Drug overdose (21%) and respiratory conditions (18%) were the most common ICU conditions identifi ed by paramedics for the ED-ICU group. All variables entered into the logistic models were signifi cant Introduction We aimed to identify the adequacy of assistance provided and to assess correct anaesthetic equipment and drug availability at emergency calls made in the ICU and in remote areas of the hospital. Emergency calls often involve managing critically ill patients with the highest mortality results. The importance of a clinical team with the necessary competencies and the right level of resources are paramount. Methods We undertook a prospective survey of all adult patients with emergency calls put out to the anaesthetic team in a London district S144 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 general hospital over a 6-week period. We performed a snapshot audit of equipment in resuscitation trolleys across each ward and in the radiology department. We compared the data collected on available equipment with the standard set by the Resuscitation Council (UK) Recommended Minimum Equipment Checklist [1]. The survey addressed the availability, clinical competency and appropriate duration of stay of the anaesthetic assistant at the emergency calls. Further qualitative data were collected on the availability of required emergency drugs. general hospital over a 6-week period. We performed a snapshot audit of equipment in resuscitation trolleys across each ward and in the radiology department. Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P408 (doi: 10.1186/cc14488) g g p J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P408 (doi: 10.1186/cc14488) J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK f Results The intervention signifi cantly increased satisfaction amongst staff regarding: identifi cation of the team leader and other key staff members at the response; and time out eff ectiveness in reducing repetition and improving staff understanding of the patient’s status and medical issues. We found no signifi cant change in staff perceptions regarding the clarity of the ongoing treatment plan at the end of the MET call response. Introduction There is widespread concern regarding excess mortality for patients admitted to hospital out of hours. We introduced an electronic track and trigger system (Patientrack) with automated alerts in a large university teaching hospital between 2010 and 2012. The system computes the patient’s early warning score and alerts medical staff via a pager. It is operational 24 hours a day, 7 days a week and could be an eff ective tool to reduce variations in mortality throughout the working week. Conclusion Utilising a low-cost intervention in a regional setting, we were successful in improving staff perceptions of role allocation and communication within our MET call responses. The intervention also led to signifi cantly increased overall satisfaction with the MET call system. Through our surveys we have identifi ed other facets of the MET call response that also require attention. Given our encouraging results we are designing a follow-up intervention incorporating structured multidisciplinary training in MET call scenarios. g Methods We extracted hospital outcome data for all admissions during the fi nancial years between 2007 and 2014. We identifi ed variables that predicted mortality and incorporated them into a multivariate logistic regression model to assess risk of death for admissions in hours (9:00 am to 5:00 pm, Monday to Friday) versus out of hours (all other times). P409 Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) P409 Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) Introduction Medical emergency team (MET) calls are quickly becoming an integral part of the response to a deteriorating patient in Australia. Conceptually the MET call response incorporates a structured approach, but in practice this can quickly disintegrate. This collapse of method can leave patients without clear treatment plans and staff disenfranchised. We sought to improve the process of the MET call response at our regional hospital by introducing targeted interventions focused on teamwork, communication, leadership and role allocation. Introduction Medical emergency team (MET) calls are quickly becoming an integral part of the response to a deteriorating patient in Australia. Conceptually the MET call response incorporates a structured approach, but in practice this can quickly disintegrate. This collapse of method can leave patients without clear treatment plans and staff disenfranchised. We sought to improve the process of the MET call response at our regional hospital by introducing targeted interventions focused on teamwork, communication, leadership and role allocation. Methods We invited junior doctors and nurses to complete a survey designed by a multidisciplinary MET Call Working Group; 138 staff (40% of population) completed the survey. Based on analysis of responses, a focused three-pronged intervention was formulated and implemented hospital wide. The arms of the intervention were: identifi cation of the name and role of each staff member using highly visible labels; role allocation according to policy written through a multidisciplinary working group; and a time out during the response allowing a structured synopsis of the patient’s current status to be communicated to the team. The intervention was preceded by extensive staff education, and 175 staff (50%) completed the survey 6 months later to assess its success. Results The intervention signifi cantly increased satisfaction amongst staff regarding: identifi cation of the team leader and other key staff members at the response; and time out eff ectiveness in reducing repetition and improving staff understanding of the patient’s status and medical issues. We found no signifi cant change in staff perceptions regarding the clarity of the ongoing treatment plan at the end of the MET call response. Conclusion Emergency calls require standards to be met involving the competency of responding team members and adequate resources. This leads us to question whether guidelines should exist regarding the clinical competency and timeliness of the assistant available to the physician at emergency calls. Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia C l C (S l ) P (d / ) 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Methods We invited junior doctors and nurses to complete a survey designed by a multidisciplinary MET Call Working Group; 138 staff (40% of population) completed the survey. Based on analysis of responses, a focused three-pronged intervention was formulated and implemented hospital wide. The arms of the intervention were: identifi cation of the name and role of each staff member using highly visible labels; role allocation according to policy written through a multidisciplinary working group; and a time out during the response allowing a structured synopsis of the patient’s current status to be communicated to the team. The intervention was preceded by extensive staff education, and 175 staff (50%) completed the survey 6 months later to assess its success.i Reference 1. Resuscitation Council (UK). Recommended minimum equipment for in hospital adult resuscitation (October 2004). https://www.resus.org.uk/pages/ eqipIHAR.html. P407 We compared the data collected on available equipment with the standard set by the Resuscitation Council (UK) Recommended Minimum Equipment Checklist [1]. The survey addressed the availability, clinical competency and appropriate duration of stay of the anaesthetic assistant at the emergency calls. Further qualitative data were collected on the availability of required emergency drugs. sex, unplanned admission and admission from supportive care. Risk of death for out-of-hours admissions was not signifi cantly diff erent to in- hours for any year (1.01 (0.92 to 1.11), P = 0.784). There was a signifi cant fall in risk of death over the 7-year period compared with baseline values in 2007/08 (Table 1). Conclusion In our cohort there was no evidence of increased mortality for patients admitted out of hours compared with in hours. This remained true after adjustment for age, sex, emergency admissions and admission source. Our data demonstrated an overall fall in risk of death over the study period. The introduction of Patientrack could have contributed to this reduction in mortality. Results During the study period 44 emergency calls were attended. Twenty-three (52%) of these calls were in the accident and emergency department, and four (9%) in the ICU. Survey results demonstrated two cases where no anaesthetic assistant arrived at the emergency call put out to them. In cases where timely assistance was available, the assistant did not have the adequate clinical and anaesthetic skills required by the attending physician. In 6% of cases where skilled assistance was required (n = 2), it was felt that the assistant did not stay for the clinically required length of time. Emergency drugs required were found to not be available in 11% of cases (n = 5) and in 17% of cases (n = 6) the necessary emergency equipment was not available. Data were collected on equipment from 17 resuscitation trolleys. The inadequacies identifi ed were the oxygen cylinders were fi lled less than 75% full in 41% cases (n = 7) and end-tidal capnography was identifi ed to be absent. P409 Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital P410 S Results Data were available for 1,180,268 hospital admissions, of which 7,264 (0.6%) died. Predictors for hospital mortality included: age, male P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) Among true cardiac arrest events, 92.6% was due to pulseless electrical activity/asystole and 7.7% was due to ventricular fi brillation (VF)/pulseless ventricular tachycardia (VT); both of these did not have any diff erence on the initial outcome. But having an initial rhythm of VF/pulseless VT had 90% more chance for discharge from the hospital, with P  = 0.04. Although arrival time of the CBT did not have any infl uence on the fi nal outcome, duration of resuscitation ≤20 minutes had an odds ratio of 10.6 with P <0.001 favoring return of spontaneous circulation over death after controlling for age. Of the 203 patients who had true cardiac arrest events, 43 (21.2%) were discharged from the hospital. Good neurological outcome at discharge was seen among 22 (10.8%) of the patients based on Cerebral Performance Category Score. Conclusion Our experience shows that out of every 1,000 patients admitted to our hospital, about fi ve sustained cardiac arrest, of whom only 11.3% survived to hospital discharge with good neurological recovery. Variation in the eff ectiveness of the cardiopulmonary resuscitation quality in comparison with world data could be due to the inherent diff erence in the severity of the primary illness in the patients and diversity in the reported data. P411 Evaluation of emergency call Code Blue over a 5-year period N Bakan, G Karaoren, S Tomrk, S Keskin Istanbul Umraniye Training and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P411 (doi: 10.1186/cc14491) Introduction Code systems are the emergency call and management systems for rapid response in healthcare institutions. The main aim of these systems is to provide common institutional understanding of what is necessary to be done immediately at the time of an event. Code Blue (CB), which is used throughout the world and was described in the 2008 service quality standards of Turkey, defi nes the necessary emergency intervention in cases of respiratory or cardiac arrest. This study aimed to evaluate the clinical and application data of patients for whom a CB call was made between 2009 and 2013. Methods After approval of local ethics committee, retrospective examination was made of CB forms. P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. Methods All Code Blue activations from 1 January 2012 to 31 December 2012 were analyzed retrospectively. Data were gathered from the Code Blue form and fi ner details of individual patients were linked through their medical records. Code Blue was activated only for events that happened outside the medical and surgical ICUs. Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. f Methods All Code Blue activations from 1 January 2012 to 31 December 2012 were analyzed retrospectively. Data were gathered from the Code Blue form and fi ner details of individual patients were linked through their medical records. Code Blue was activated only for events that happened outside the medical and surgical ICUs. Conclusion The MET has been successfully implemented, with demand for its services having increased by 32.7% in 1 year. The unadjusted immediate mortality rate of patients for whom a MET/cardiac arrest call is activated is 3.64%. Response time had no infl uence on mortality, most probably due to the rapid response time. Immediate mortality was low, probably as a result of early adequate intervention. Further evaluation of overall hospital mortality is warranted for future studies. pp g Results A total of 260 Code Blue activations were made, out of which there were 203 true cardiac arrest events among 40,168 in-patients; the cumulative incidence of the same was 0.51%. Mean (SD) duration of arrival of the Code Blue Team (CBT) to the scene was 64.5 (27.7) seconds. Cardiovascular illness was the predominant baseline morbidity but none of the baseline illness showed increased risk of mortality in this group. P410 Successful implementation of a medical emergency team: 2-year experience in a teaching hospital A Tridente, J Elmore, R Varia, T Mahambrey St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P410 (doi: 10.1186/cc14490) Table 1 (abstract P408). Overall risk of death ratios compared to 2007/08 Year ROD 95% CI 2008/09 1.01 0.73 to 1.5 2009/10 1.03 0.74 to 1.5 2010/11 0.46* 0.33 to 0.7 2011/12 0.31* 0.22 to 0.4 2012/13 0.27* 0.2 to 0.39 2013/14 0.26* 0.2 to 0.37 *P <0.001. Table 1 (abstract P408). Overall risk of death ratios compared to 2007/08 Introduction A medical emergency team (MET) was introduced in our institution in January 2012 to provide timely response to the needs of acutely ill inpatients and cardiac arrest calls. The MET assesses the patient and prescribes a management plan for the responsible team to follow; promptly stabilising and transferring patients to a place of safety where required. We aimed at evaluating the eff ects of introducing the MET on clinically relevant processes and outcomes. Methods Prospective data were analysed using STATA 10.1. The primary outcome measure was immediate mortality (defi ned as mortality at S145 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The time taken to reach patients conformed with the global standard mean 2 to 3  minutes [1]. The rates of erroneous CB and time to reach patients reduced each year due to more staff experienced and knowledgeable in CB and the structuring of the emergency clinic. Conclusion The time taken to reach patients conformed with the global standard mean 2 to 3  minutes [1]. The rates of erroneous CB and time to reach patients reduced each year due to more staff experienced and knowledgeable in CB and the structuring of the emergency clinic. conclusion of the MET intervention); the secondary outcome measures were admission to critical care after a MET call and cardiac arrest. conclusion of the MET intervention); the secondary outcome measures were admission to critical care after a MET call and cardiac arrest. Results A total of 5,763 MET calls were made between 9 January 2012 and 4 March 2014, of which 5,310 (92.1%) were MET calls, 349 (6.1%) cardiac arrest calls, 36 (0.6%) false alarms and 68 (1.2%) unclassifi ed. The number of calls increased by 32.7% from 2,255 in 2012 to 2,993 in 2013, with all month-specifi c comparisons showing signifi cant increases in MET activity (ranging from 0.5% to 103.6% increases). P410 Successful implementation of a medical emergency team: 2-year experience in a teaching hospital A Tridente, J Elmore, R Varia, T Mahambrey St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P410 (doi: 10.1186/cc14490) MET activity displayed cyclical yearly changes, with the winter months and the month of August (junior doctors’ changeover period) being particularly busy. Median response time (interquartile range) was 1 (1 to 2) minutes, with 99.1% calls attended to within 3 minutes. There were 210 (3.64%) immediate deaths (with no signifi cant diff erences between years), 112 (1.9%) patient transfers to critical care, 233 (4%) patients were transferred to other locations (other than critical care) while 4,697 (81.5%) patients remained on the ward of origin. In 408 cases (7.1%) a do-not-resuscitate order was instituted. On multiple logistic regression analyses, when the type of call was taken into consideration, the response time had no infl uence on primary (mortality OR = 0.83, 95% CI = 0.63 to 1.09, P = 0.18) and secondary outcomes (admission to critical care OR = 0.85, 95% CI = 0.62 to 1.17, P = 0.33; subsequent cardiac arrest OR = 0.57, 95% CI = 0.27 to 1.2, P = 0.14). References 1. Abella BS, et al. JAMA. 2005;293:305-10. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. 1. Abella BS, et al. JAMA. 2005;293:305-10. 1. Abella BS, et al. JAMA. 2005;293:305 10. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. Results From CB calls for a total of 1,195 patients over the 5-year period, 1,035 (86.6%) were evaluated. The rate of erroneous CB was 36.9%. Patients comprised 413 (39.9%) females and 622 (60.1%) males with a mean age of 59.73 ± 23.13 years (range, 0.1 to 102 years). The distribution of total cases over the 5 years (2009 to 2013) was 15.5%, 25.2%, 26.5%, 19% and 13.8% respectively. Distribution according to clinic was emergency internal (37.5%), internal (16.5%) and emergency surgical (9.5%). Clinical diagnosis was cardiac 28.8%, neurological 15.6% and end-stage cancer 13.5%. A total 19.9% of the patients were those discharged from intensive care. The total survival rate was 59.6%. The duration of CPR in survivors was statistically longer than in nonsurvivors (P <0.01). There was no statistically signifi cant relationship between the duration of CPR and age (P >0.05). The overall mean time taken to reach the patient was 102.71 ± 22.47 seconds, which reduced to 93.64  ± 19.91  seconds in 2013. The APACHE II–PRISM scores and mortality rates were low in the cases of erroneous CB (P <0.05). P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) The age and gender of the patient, diagnosis, department to which admitted, time of CB call, reason for CB, whether or not CB was appropriate, whether or not CPR was applied, duration of CPR if applied, APACHE II and PRISM scores and predicted mortality were recorded from the hospital automated record system and the CB form. Patients who refused treatment or who could not reach the necessary parameters for the calculation of APACHE II and PRISM scores were excluded. References Reference 1. Nolan JP, et al. European Resuscitation Council guidelines for resuscitation 2010 section 1. Executive summary. Resuscitation. 2010;81:1219-76. P412 P413 Are we failing to teach cardiopulmonary resuscitation (CPR) in schools? A pilot study to assess CPR and automated external defi brillator training in London schools J Salciccioli, D Marshall, M Sykes, A Wood, S Joppa, M Sinha, PB Lim Imperial College London, UK Critical Care 2015, 19(Suppl 1):P413 (doi: 10.1186/cc14493) TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest Methods We conducted a registered audit of CPR and AED training in London schools. Secondary schools were identifi ed via web links for each of the London Borough Councils. Telephone interviews with school staff familiar with CPR and AED training practices were conducted prospectively using a standardized web-based survey. All survey response data were captured electronically. We defi ned universal training as any programme which delivers CPR and AED training to all students in the school. We used simple descriptive statistics to summarise the results. J Callerova1, R Skulec2, J Knor3, V Cerny3 1Emergency Medical Service of Central Bohemian Region, Beroun, Czech Republic; 2Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015, 19(Suppl 1):P416 (doi: 10.1186/cc14496) J Callerova1, R Skulec2, J Knor3, V Cerny3 1Emergency Medical Service of Central Bohemian Region, Beroun, Czech Republic; 2Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015 19(Suppl 1):P416 (doi: 10 1186/cc14496) Introduction Implementation of point-of-care ultrasound examination into cardiopulmonary resuscitation (CPR) may increase diagnostic accuracy for determining the cause of cardiac arrest. However, current protocols either do not refl ect all causes detectable by ultrasound or are too complicated for prehospital use. Thus, we decided to construct and validate a new protocol TRACE (ThoRacic and Abdominal sonography in Cardiac arrEst) for ultrasound examination during out-of-hospital cardiac arrest (OHCA). Results A total of 51 schools completed the survey covering an estimated student population of 54,037. There were four (8%) schools that provide universal training programmes and an additional 23 (45%) off er optional training programmes for students. There were 16 (31%) schools which have an AED available on the school premises. The most common reasons for not having a universal CPR training programme is the requirement for additional class time (15/51; 29%) and that funding is unavailable for such a programme (12/51; 24%). There were three students who died from sudden cardiac arrest over the period of the past 10 years. Methods We designed a new protocol for ultrasound examination during OHCA to increase the success rate for the establishment of OHCA cause. Body position during transport in a refractory cardiac arrest porcine model p Methods Data were prospectively collected from January 2011 to January 2012 from 336 female medical and pharmacy students undergoing CPR training at the Lithuanian University of Health Sciences. During the training process, the instructors performed a simple 5-second intervention (Andrew’s manoeuvre) with all of the rescuers in the study group. The instructor pushed 10 times on the shoulders of each trainee while she performed CCs to achieve the maximal required compression depth. Immediately after training, the participants were asked to perform a 6-minute BLS test on a manikin that was connected to a PC with SkillReporter™ System software (Laerdal, Norway); the quality of the participants’ CPR skills was then evaluated. Body position during transport in a refractory cardiac arrest porcine model J Belohlavek1, M Mlcek2, M Huptych3, T Boucek1, T Belza2, P Krupickova2, O Kittnar2 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) Introduction Cardiac arrest patients are not transported only supine. The eff ect of body position on resuscitability and cerebral perfusion in a 30° and 60° incline is not known.i Results The CC depth in the study group increased by 6.4 mm (P <0.001) compared with the control group (52.9 vs. 46.6 mm). A regression analysis showed that Andrew’s manoeuvre increased the depth of the CCs among women by 14.87 × (1 – 0.01 × weight) mm.i Methods Twenty-fi ve female pigs were subjected to a simulated cardiac arrest (3  minutes no fl ow, 5  minutes mechanical CPR). Next, animals were randomly assigned to one of the three groups: GROUP 60 (n  = 8), 60° incline for 3  minutes to simulate transport in space restricted elevator; GROUP 30 (n  = 8), 30° incline for 8  minutes to simulate staircase transport; and GROUP 0, with no incline. During subsequent standard CPR including rescue ECMO, resuscitability and cerebral perfusion were assessed. Conclusion Andrew’s manoeuvre during CPR training signifi cantly improved the performance of the female rescuers and helped them achieve the CC depth required by 2010 resuscitation guidelines. It is most eff ective among the women with the lowest body weight. Are we failing to teach cardiopulmonary resuscitation (CPR) in schools? A pilot study to assess CPR and automated external defi brillator training in London schools Introduction Mortality from cardiac arrest remains high [1]. Bystander cardiopulmonary resuscitation (CPR) and the use of automated external defi brillators (AED) are two of the most important factors favouring survival [2]. CPR/AED training in schools is a recommended intervention for signifi cantly improving training rates across a large population [3]. The current practice for CPR/AED training in London schools is unknown. The primary aim of this study was to assess current Introduction Mortality from cardiac arrest remains high [1]. Bystander cardiopulmonary resuscitation (CPR) and the use of automated external defi brillators (AED) are two of the most important factors favouring survival [2]. CPR/AED training in schools is a recommended intervention for signifi cantly improving training rates across a large population [3]. The current practice for CPR/AED training in London schools is unknown. The primary aim of this study was to assess current S146 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 practices relating to CPR and AED training in London secondary schools. 1. Krikscionaitiene A, et al. Magical manoeuvre: a 5-s instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth. Eur J Emerg Med. 2014;21:424-8. Magical manoeuvre: a 5-second instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth Magical manoeuvre: a 5-second instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth A Krikscionaitiene, A Pranskunas, M Dambrauskiene, N Jasinskas, Z Dambrauskas, E Vaitkaitiene, J Vencloviene, D Vaitkaitis Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P415 (doi: 10.1186/cc14495) A Krikscionaitiene, A Pranskunas, M Dambrauskiene, N Jasinskas, Z Dambrauskas, E Vaitkaitiene, J Vencloviene, D Vaitkaitis Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P415 (doi: 10.1186/cc14495) Conclusion Implementation of the TRACE protocol to the CPR process was feasible, required minimal interruption of cardiac compressions and resulted in a high recognition rate for the cause of OHCA. Introduction Adequate chest compression (CC) depth is crucial for resuscitation outcomes. Lightweight rescuers, particularly women, are often unable to achieve the required 5 to 6  cm CC depth. This nonrandomised cohort study investigated new strategies to improve CC performance. TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest The subcostal view was performed during planned rhythm check to assess the presence of cardiac tamponade and size of the right and left ventricle and inferior caval vein. Thereafter, during ongoing cardiac compressions, Morrison’s pouch and right pleural space were investigated to exclude intraperitoneal and inrapleural free fl uid. The same procedure was applied on the left side of the body. Finally, the anterior thoracic view was done to exclude pneumothorax. Working diagnosis was compared with the fi nal in-hospital diagnosis or autopsy. Conclusion CPR and AED training rates in London secondary schools are low. The majority of schools do not have an AED available on premises. The most common reason for not providing CPR training is the requirement for additional class time. These data highlight an opportunity to vastly improve CPR training rates in a large population. Future studies should assess programmes which are cost-eff ective and which do not require signifi cant amounts of additional class time. References 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87. 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87. Results We examined 40 consecutive OHCA patients. Correct cause of OHCA during CPR was recognised in 38 patients (95%). Leading causes were acute coronary syndrome (55.0%), pulmonary embolism (15.0%) and complication of chronic heart failure (10.0%). Incorrect recognition was performed in one patient with respiratory cause, originally considered as pulmonary embolism, and in another with pulmonary embolism, considered as respiratory cause. One rhythm check was suffi cient to perform TRACE in 31 patients, in the other two interruptions of cardiac compressions were required. Return of spontaneous circulation was achieved in 15 (37.5%) patients, favourable neurological outcome at hospital discharge in eight (20%) patients. Specifi c therapy to aff ect the cause of OHCA was applied during OHCA in 12 (30%) patients. 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. P417 Body position during transport in a refractory cardiac arrest porcine model J Belohlavek1, M Mlcek2, M Huptych3, T Boucek1, T Belza2, P Krupickova2, O Kittnar2 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Ch 1 ZM Li 1 JH T 1 S S h 1 M H i Bi I k d 1 B L 2 Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Chua1, ZM Lin1, JH Tan1, S Surentheran1, M Haris Bin Iskander1, B Leong2 1National University of Singapore, Singapore; 2National University Hospital, Singapore Critical Care 2015 19(Suppl 1):P418 (doi: 10 1186/cc14498) Introduction Despite several large studies concerning out-of-hospital cardiac arrests in recent years, it is not clear whether their in-hospital counterparts have benefi ted from advances in resuscitation as well as post-resuscitation care. Introduction Despite several large studies concerning out-of-hospital cardiac arrests in recent years, it is not clear whether their in-hospital counterparts have benefi ted from advances in resuscitation as well as post-resuscitation care. p Methods We identifi ed all cases of in-hospital cardiac arrest (IHCA) occurring in the National University Hospital in Singapore from 1 June 2008 to 31 May 2009. Patients for which IHCA occurred but where no resuscitation was attempted were excluded. Key outcomes were classifi ed as primary (survival to discharge) and secondary (return of spontaneous circulation). Additionally, various arrest characteristics were analysed to identify predictive factors for survival to discharge with level of signifi cant set at P <0.05. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. Figure 2 (abstract P419). Comparison complications of CPR between the OHCA group and the IHCA group. gi Results Among 353 unique cases of IHCA analysed, 63 patients (17.8%) had a shockable rhythm (ventricular fi brillation and pulseless ventricular tachycardia) of which 17 (27.0%) survived to discharge. While 290 (82.2%) patients presented with nonshockable rhythm (asystole or pulseless electrical activity), only 32 patients (11%) survived to discharge. For patients who survived to discharge, univariate analysis showed that event location (P = 0.016), nationality (P = 0.035), paying class (P = 0.038), use of ECG monitoring (P = 0.048), initial cardiac rhythm (P = 0.000) and presence of a house offi cer (P = 0.005) were statistically signifi cant. Multivariate analysis showed that patients with shockable rhythms were 2.52 times more likely to survive but other factors were not signifi cant. For patients who attained ROSC, univariate analysis showed that time of day (P = 0.006), event location (P = 0.000), and number of adrenaline doses administered (P = 0.000) were statistically signifi cant. 9 Comparison of complications secondary to cardiopulmonary resuscitation between out-of-hospital cardiac arrest and in-hospital cardiac arrest M Seung Y Park M Seung, Y Park Yonsei University Severance Hospital/Yonsei University College of Medicine, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P419 (doi: 10.1186/cc14499) M Seung, Y Park Yonsei University Severance Hospital/Yonsei University College of Medicine, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P419 (doi: 10.1186/cc14499) Introduction Chest compression during cardiopulmonary resuscitation (CPR) could bring out unintended complications which are mainly composed of chest injuries. The aim of this study was to assess whether there was a signifi cant diff erence in the complications of CPR between out-of-hospital cardiac arrest (OHCA) and in-hospital cardiac arrest (IHCA) survivors using multidetector computed tomography (MDCT). g p g p y Methods We performed a retrospective cohort study in the emergency departments of two academic tertiary care centres from January 2009 to May 2014. We enrolled both OHCA and IHCA patients who underwent successful CPR. The enrolled patients had undergone a chest CT within 48 hours after ROSC. We evaluated the MDCT fi ndings of the CPR-related chest injuries and compared complications between OHCA and IHCA patients. Figure 1 (abstract P417). Figure 1 (abstract P417). (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1. (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1. p Results A total of 148 patients were fi nally enrolled in this study, OHCA were 89 (60.1%) and IHCA were 59 (39.8%). The mean CPR time, both in-hospital and total, was longer in OHCA survivors. Rib fractures were Conclusion Positional changes during simulated refractory cardiac arrest in this experimental model signifi cantly aff ect resuscitability and brain perfusion. Animals subjected to shorter time in a more inclined (GROUP 60) position were more easily resuscitated; however, cerebral blood fl ow was better preserved in GROUP 30. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. P418 Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Chua1, ZM Lin1, JH Tan1, S Surentheran1, M Haris Bin Iskander1, B Leong2 1National University of Singapore, Singapore; 2National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P418 (doi: 10.1186/cc14498) Body position during transport in a refractory cardiac arrest porcine model Reference Results Attainment of ROSC (3, 5, 5 in respective groups, P = 0.021), time to ROSC (15:24 (13:26; 16:02) vs. 19:19 (18:28; 19:37) vs. 9:10 minutes (8:28; 9:41), respectively, P = 0.005) signifi cantly diff ered. Changes in carotid blood fl ow according to the respective periods of the protocol 1. Krikscionaitiene A, et al. Magical manoeuvre: a 5-s instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth. Eur J Emerg Med. 2014;21:424-8. S147 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1 Figure 1 (abstract P417). Figure 1 (abstract P417). Impact of intra-arrest fl uid loading with diff erent doses of crystalloid infusion on hemodynamics in experimental cardiac arrest R Skulec1, A Truhlar1, R Parizkova1, Z Turek1, D Astapenko1, J Dudakova2, V Cerny3 R Skulec1, A Truhlar1, R Parizkova1, Z Turek1, D Astapenko1, J Dudakova2, V Cerny3 y 1Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 2Emergency Medical Service of Central Bohemian Region, Kladno, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic l ( l ) (d ) 1Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 2Emergency Medical Service of Central Bohemian Region, Kladno, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015, 19(Suppl 1):P420 (doi: 10.1186/cc14500) Introduction Fluid loading during cardiopulmonary resuscitation for nonhypovolemic cardiac arrest remains controversial. Thus, we conducted an experimental study comparing the impact of two diff erent doses of balanced crystalloid infusion on hemodynamics in a porcine model of ventricular fi brillation.i Conclusion Although our study has a number of methodological limitations, these results about incidence in cardiac arrest survivors corroborate previous retrospective reports. It is possible that every cardiac arrest survivor has had to live a NDE, regardless of brain mechanisms associated with experience, but only some patients remember it. If some chronic medications, such as benzodiazepine, may decrease memorization, the role of the elements of the clinical context about NDE during resuscitation is speculative References i Methods Ventricular fi brillation was induced for 15  minutes in 19 anesthetized domestic pigs. Before induction, the animals were randomized to receive either 1,000  ml (34  ± 3  ml/kg, group A, n  = 7) or 500  ml (16  ± 2  ml/kg, group B, n  = 7) of balanced crystalloid solution or to undergo no fl uid loading during CPR (group C, n = 5). After spontaneous circulation (ROSC) was restored, the animals were observed for 90 minutes. 1. Van Lommel P, van Wees R, Meyers V, Elff erich I. Near-death experience in survivors of cardiac arrest: a prospective study in the Netherlands. Lancet. 2001;358:2039-45. Results In all groups, signifi cant increase of intracranial pressure followed by its decrease after ROSC was observed. While in groups B (from 12 ± 2 to 18 ± 2 mmHg, P <0.05) and C (from 13 ± 1 to 18 ± 3 mmHg, P <0.05) it was comparable (P >0.05), the rise of intracranial pressure in group A was signifi cantly higher (from 12  ± 3 to 23  ± 3 mmHg, P <0.05). Impact of intra-arrest fl uid loading with diff erent doses of crystalloid infusion on hemodynamics in experimental cardiac arrest Whereas coronary perfusion pressure was lower in group A than in control group C during volume loading, fl uid infusion induced its mild increase in group B (group A: 12.1  ± 2.4, group B: 16.0 ± 2.6, group C: 13.6 ± 2.8 mmHg, P = 0.043). Decrease of cerebral perfusion pressure was equal in all groups. Cardiac index 10 minutes after ROSC signifi cantly diff ered among all groups (group A: 8.9 ± 2.2, group B: 7.1 ± 1.3, group C: 4.9 ± 1.9 l/minute/m2, P = 0.007) and the dose of crystalloid infusion during cardiac arrest positively correlated with cardiac index increase (r = 0.815, P <0.001).i 2. Parnia S, Spearpoint K, de Vos G, Fenwick P, Goldberg D, Yang J, et al. AWARE – AWAreness during REsuscitation – a prospective study. Resuscitation. 2014;85:1799-805. 3. Greyson B. The near-death experience scale. Construction, reliability, and validity. J Nerv Ment Dis. 1983;171:369-75. 3. Greyson B. The near-death experience scale. Construction, reliability, and validity. J Nerv Ment Dis. 1983;171:369-75. f References 1. Kim EY, Yang HJ, Sung YM, et al. Multidetector CT fi ndings of skeletal chest injuries secondary to cardiopulmonary resuscitation. Resuscitation. 2011;82:1285-8. Kim EY, Yang HJ, Sung YM, et al. Multidetector CT fi ndings of skeleta Methods In a retrospective study from 2005 to 2012, 295 consecutive cardiac patients who were successfully resuscitated after cardiac arrest were enrolled. In total, 204 (69%) were alive during the research period (mean delay: 55 months). A total of 118 (40%), over 18 years, able to answer a short standardized interview were included in the study when they accepted to participate. Demographic, medical, pharmacological and psychological data were recorded and we used the Greyson NDEs scale to identify and characterize NDEs. Descriptive and unifactorial analysis was performed using the Jacknife method and Wald test according to low event frequency. 2. Kim MJ, Park YS, Kim SW, et al. Chest injury following cardiopulmonary resuscitation: a prospective computed tomography evaluation. Resuscitation. 2013;84:361-4. g y Results From our 118 reports, 20 described a core experience and 18 (15.3%) met the criteria for NDEs (Greyson NDEs total score >6/32 (7 to 19)). Only one patient recounted a negative experience. Regarding the risk factors for NDEs, using univariate analysis, we found for demographic data: woman (CI: 1.11 (1.10 to 1.12), P  <0.0001), age under 60 (CI: 1.23 (1.21 to 1.24), P <0.0001), prior knowledge of NDEs (CI: 1.97 (1.95 to 1.99)) and previous NDE (CI: 5.82 (4.19 to 8.08)). According to the history of previous disease, we found an increased risk for pulmonary disease (CI: 1.75 (1.73 to 1.77)), rheumatic disease (CI: 3.79 (3.75 to 3.84)), endocrine disease (CI: 1.45 (1.43 to 1.46)), and a decrease for cardiac disease (CI: 0.65 (0.64 to 0.66)), psychiatric disease (CI: 0.71 (0.69 to 0.72)) and digestive tract disease (CI: 0.71 (0.69 to 0.72)). For previous pharmacological treatment we found a decrease of risk for all classes and particularly when two drugs were simultaneously given (CI: 0.37 (0.36 to 0.38)). Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Centre Hospitalier Universitaire, Reims Cedex, France Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Centre Hospitalier Universitaire, Reims Cedex, France Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) p Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) Introduction Near-death experiences (NDEs) are increasingly being reported as a clear reality of clinical signifi cance. Previous studies, essentially, have been trying to estimate their incidence in various populations, notably after cardiac arrest resuscitation, and to understand the implication of resuscitation characteristics [1,2]. Using the Greyson NDE scale [3], the present retrospective study aimed at exploring cardiac arrest survivors and the correlations between NDE and physiological, medical, psychological and pharmacological context. i Conclusion Frequency of rib fractures and multiple rib fractures were higher in OHCA survivors. Further investigation is needed into the relation between the location of CPR and the CPR-related injuries, eff orts to reduce the complications after CPR. Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Ch 1 ZM Li 1 JH T 1 S S h 1 M H i Bi I k d 1 B L 2 Multivariate analysis showed that an arrest occurring in the ICU setting was 2.9 times more likely to attain ROSC (95% CI: 1.02 to 5.59, P = 0.044). Conclusion The results of this study have described some key predictive factors regarding positive outcomes in IHCA in Singapore. These are vital in understanding important features regarding IHCAs and will aid in developing policies to help improve care and survival in this group of patients. S148 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P421 detected more in OHCA survivors. Frequency of multiple rib fractures was higher in OHCA survivors. Frequency of sternum fractures was higher in OHCA survivors, showing no signifi cant diff erence. In lung injuries, lung contusion and pneumothorax account for the large part, and OHCA survivors had higher incidence in both complications but statistically insignifi cant. Major complications occurred in eight cases in OHCA survivors and three cases in IHCA survivors during the study period. After adjusting for the time factor in multiple logistic regression analysis, rib fractures and multiple rib fractures became statistically signifi cant in OHCA survivors. See Figures 1 and 2. Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest Eighty-three percent (n = 45) of survivors admitted to the ICU went to the cardiac cathlab before ICU admission compared with 53% (n  = 39) of nonsurvivors. Forty-three percent of survivors had PPCI compared with 26% of nonsurvivors. Eighty- one percent (n  = 44) of survivors received therapeutic hypothermia compared with 62% (n = 48) of nonsurvivors. Introduction Mild hypothermia and fever control have been shown to improve neurological outcomes post cardiac arrest. Common methods to induce hypothermia include body surface cooling and intravascular cooling; however, a new approach using a catheter placed into the esophagus has recently become available. p g y Methods We report the fi rst three cases of temperature control using an esophageal cooling device (ECD). The ECD was placed orally in a similar fashion to orogastric tubes. Temperature reduction was achieved by connecting the ECD to a commercially available heat exchange unit (Blanketrol II or III). Conclusion Over 3 consecutive years our annual case mix, ICU and hospital mortalities for OOHCA patients admitted to the ICU have remained stable, while our annual pre-ICU cathlab and PPCI utilisation have risen consistently in both survivors and nonsurvivors. ICU survivors were more likely to have had a shockable rhythm, been to the cathlab, and received PPCI and TH, but all may simply refl ect selection bias. Any benefi t these conferred to cardiac patients may have been off set by our liberal ICU admission policy to OOHCAs with nonshockable rhythms. Access to 24–7 PPCI in this group may determine survival and we suggest that OOHCA patients should be taken directly to regional heart attack centres. Results The fi rst patient, a 59-year-old male (73 kg), was admitted after successful resuscitation from a protracted out-of hospital cardiac arrest. His initial temperature was 35°C, which is within our current institutional protocol of 34 to 36°C. Several hours after arrival, his temperature slowly increased to 35.8°C despite application of a cooling blanket and ice packs to the groin and axilla. The ECD was inserted and a reduction of temperature to 34.8°C was achieved within 3  hours. The patient expired on day 3. The second patient, a 54-year-old female (95 kg), was admitted after resuscitation from an out-of-hospital PEA arrest. Despite initiating our cooling protocol with surface-cooling blankets and cold intravenous saline, she mounted a fever peaking at 38.3°C shortly after admission. Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, V Achan, M Davies, M Peck Frimley Park Hospital, Frimley, UK y p y Critical Care 2015, 19(Suppl 1):P422 (doi: 10.1186/cc14502) y p y Critical Care 2015, 19(Suppl 1):P422 (doi: 10.1186/cc14502) Conclusion Fluid loading during CPR had signifi cant impact on hemodynamics in our experimental model. While a high dose led to unintentional increase of intracranial pressure and decrease of coronary perfusion pressure, a low dose did not aff ect intracranial pressure and was associated with mild increase of coronary perfusion pressure during cardiac arrest. Introduction Our 700-bed hospital has a 24–7 cathlab service that routinely investigates patients with indications prior to ICU admission following out-of-hospital cardiac arrest (OOHCA). Our aim was to compare survivors and nonsurvivors and evaluate utilisation and S149 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 nonshockable rhythms. However, access to 24–7 PPCI may determine survival and we suggest that all OHCA patients should be taken directly to regional heart attack centres. nonshockable rhythms. However, access to 24–7 PPCI may determine survival and we suggest that all OHCA patients should be taken directly to regional heart attack centres. prognostic impact of angiography and primary percutaneous coronary intervention (PPCI) in this patient group. Methods A retrospective analysis using Trust electronic databases (Symphony, WardWatcher, PICIS, PRISM) of all OOHCA patients admitted to our ICU over 3 consecutive years between 1 November 2011 and 31 October 2014. P424 Targeted temperature management after cardiac arrest and fever control with an esophageal cooling device A Hegazy, D Lapierre, E Althenayan University of Western Ontario, London, ON, Canada Critical Care 2015, 19(Suppl 1):P424 (doi: 10.1186/cc14504) Results A total of 351 patients presented to our emergency department (ED) following OOHCA in this period, and of these 50% died in the ED, 37% were admitted to the ICU and 13% were admitted elsewhere. Of the 129 patients admitted to the ICU, median age was 66 (range 18 to 93), 71% were male, 68% had a shockable presenting rhythm, median ICU LOS was 3.75 (range 1 to 34 days) and ICU and hospital mortalities were 50% and 60% respectively. Eighty-nine percent (n = 48) of OOHCA survivors admitted to the ICU had a shockable rhythm compared with 55% (n = 41) of nonsurvivors. P425 P425 Is selective nasopharyngeal brain cooling detrimental to neuroprotection? M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P425 (doi: 10.1186/cc14505) P423 Utilisation and prognostic impact of angiography and primary percutaneous coronary intervention prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, M Davies, V Achan, M Peck Frimley Park Hospital, Frimley, UK Critical Care 2015, 19(Suppl 1):P423 (doi: 10.1186/cc14503) P423 Utilisation and prognostic impact of angiography and primary percutaneous coronary intervention prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, M Davies, V Achan, M Peck Frimley Park Hospital, Frimley, UK Critical Care 2015, 19(Suppl 1):P423 (doi: 10.1186/cc14503) Introduction Our 700-bed hospital has a 24–7 cathlab service that routinely investigates patients with indications prior to ICU admission following out-of-hospital cardiac arrest (OHCA). Our aim was to compare ICU survivors and nonsurvivors and evaluate the utilisation and prognostic impact of angiography and primary percutaneous coronary intervention (PPCI) in this patient group. f Conclusion The ECD is a novel technology that can be used for temperature management post cardiac arrest and for fever control in critically ill patients. Despite patients mounting a febrile response, temperature control was achieved and maintained successfully. The device was reported as being easy to use, by both physicians and nurses. y p g p Methods A retrospective analysis using Trust electronic databases (Symphony, WardWatcher, PICIS, PRISM) of all OHCA patients admitted to our ICU over 3 consecutive years between 1 November 2011 and 31 October 2014. Results A total of 351 patients presented to our hospital following OHCA in this period, and of these 50% died in the ED, 37% were admitted to the ICU and 13% elsewhere. Of the 129 patients admitted to the ICU, median age was 66 (range 18 to 93), 71% were male, 68% had a shockable presenting rhythm, median ICNARC score was 31 (range 10 to 66), median ICU LOS was 3.75 (range 1 to 34 days) and ICU and hospital mortality were 50% and 60% respectively. ICU survivors were more likely to have had a shockable rhythm (89 vs. 55%), been to the cathlab (83 vs. 53%), received PPCI (43 vs. 26%) and TH (82 vs. 62%) and had lower median ICNARC scores (26 vs. 35) than nonsurvivors. Over the 3 consecutive years, pre-ICU cathlab and PPCI utilisation increased annually in ICU survivors (73 vs. 86 vs. 89% and 45 vs. 54 vs. 59% respectively) and nonsurvivors (40 vs. 38 vs. 50% and 20 vs. 27 vs. 31% respectively). However, our annual ICU and hospital mortality remained unchanged (46 vs. 51 vs. 51% and 60 vs. 57 vs. 62% respectively). Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest After ECD insertion and confi rming the external heat exchanger connection, her temperature gradually dropped to 35.7°C over a period of 4 hours. She subsequently made a favorable neurological recovery and was discharged home at day 23. The third patient, a 47-year-old male patient (86 kg) presented with an ongoing fever secondary to necrotizing pneumonia in the postoperative period after coronary artery bypass grafting. His fever was unresponsive to empiric antibiotic therapy, antipyretics, cooling blankets, and ice packs. ECD insertion resulted in a decrease in temperature from 39.5°C to 36.5°C in less than 5 hours. The patient eventually made a full recovery and was discharged home after 59 days. In all three patients, placement of the device occurred in less than 3  minutes and ease of use was reported as excellent by nursing staff and physicians. P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) Introduction Uncontrolled shivering may have negative consequences by increasing metabolic demand and subsequently neutralize the benefi ts of therapeutic normothermia [1]. Previous anti-shivering protocols that utilize the least sedation have been described in therapeutic temperature modulation (TTM) [2]. Our aim is to describe and evaluate an anti-shivering protocol that emphasizes the least sedating regimen with the least number of pharmacologic agents for patients undergoing therapeutic normothermia. brain temperature, intracranial pressure (ICP), core temperatures and vital signs were continuously recorded. Cooling was terminated once the brain reached 32°C and the animals were allowed to passively rewarm. Results In the SNBC group, the brain target temperature was reached in 54 ± 11 minutes. The mean ICP dropped precipitously to a nadir of –15 mmHg. TCD showed signifi cant vasospasm in the MCA, compared with the internal carotid artery (ICA), during the entire cooling phase (Table 1). Upon termination of cooling, the brain temperature spontaneously rewarmed to core temperature in 13  ± 4  minutes. Rewarming was associated with hyperemia and elevation of ICP. In group 2, there was no cerebral vasospasm or hyperemia during cooling and rewarming respectively.i Methods This retrospective chart review includes patients with neurologic injury who underwent TTM from March 2013 to November 2014 and were evaluated for the following outcomes: percentage of total patient hours in each score of the Bedside Shivering Assessment Scale (BSAS) at 72  hours, 168  hours, and total duration of TTM; percentage of total patient days in each tier of the anti-shivering protocol at 72  hours, 168  hours, and total duration of TTM; de- escalation of anti-shivering agents with or without the necessary need for re-escalation; ICU and hospital length of stay (LOS); rescue agents utilized; and hospital mortality. Conclusion SNBC is associated with signifi cant vasospasm of the MCA. In addition, spontaneous and rapid rewarming of the brain, vasodilation, rapid reperfusion, and rebound elevation of ICP, all occurring minutes after termination of SNBC, are likely to be detrimental to an already ischemic brain. y Results Evaluation of 47 patients who underwent TTM resulted in a total of 505 patient-days of TTM with 6,967 BSAS hours. Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) 2 yg Methods A prospective, observational study during therapeutic hypothermia (24 hours –33°C) in 82 post-CA patients monitored with near-infrared spectroscopy. Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) y Results Forty-three patients (52%) survived in CPC 1 to 2 until 180 days post CA. The mean MAP range associated with maximal survival was 76 to 86 mmHg (OR = 2.63, 95% CI (1.01; 6.88), P = 0.04). The mean SVO2 range associated with maximal survival was 67 to 72% (OR  = 8.23, 95% CI (2.07; 32.68), P  = 0.001). In two separate multivariate models, a mean MAP (OR = 3.88, 95% CI (1.22; 12.33), P = 0.02) and a mean SVO2 (OR = 8.79, 95% CI (1.69; 18.36), P = 0.01) in the optimal range persisted as independently associated with increased survival after correction for presence of early bystander CPR and presenting shockable rhythm. Based on more than 1,625,000 data points, we found a strong linear relation between SVO2 (range 40 to 90%) and average cerebral saturation (R2 = 0.86) and between MAP and average cerebral saturation for MAPs between 40 and 87 mmHg (R2 = 0.70). Based on our hemodynamic model, the optimal MAP and SVO2 were determined to be 87 mmHg and 72%. Introduction Therapeutic hypothermia (TH) is the international standard of care for all comatose patients after cardiac arrest, but criticism focuses on poor outcomes. We sought to develop criteria to identify American urban patients more likely to benefi t from TH. Introduction Therapeutic hypothermia (TH) is the international standard of care for all comatose patients after cardiac arrest, but criticism focuses on poor outcomes. We sought to develop criteria to identify American urban patients more likely to benefi t from TH. Methods A retrospective chart review of 107 consecutive adults under- going TH in downtown New Orleans from 2010 to 2014 yielded records for 99 patients with all 44 survivors or families contacted up to 4 years. Results Sixty-nine males and 38 females with a mean age of 60.2 years showed 63 dead (58%) and 44 survivors (42%). Presenting cardiac rhythm was divided into shockable (pulseless ventricular tachycardia, ventricular fi brillation) and nonshockable (pulseless electrical activity, asystole). Presenting in shockable rhythms with ROSC <20  minutes were 21 patients with 15 (71%) survivors (P = 0.001). P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) Overall, patients spent 85.5% of total hours at BSAS 0, 11.4% of total hours at BSAS 1, 2.5% of total hours at BSAS 2, and 0.6% of total hours at BSAS 3. Patients were in tier 0 of the anti-shivering protocol 33.1% of the time, in tier 1 of the anti-shivering protocol 20.6% of the time, in tier 2 of the anti-shivering protocol 43.6% of the time, and in tier 3 of the anti- shivering protocol 2.8% of the total duration of TTM. There were 487 rescue doses of fentanyl and 243 rescue doses of meperidine that were required for shivering. Patients had a mean ICU LOS of 19 days, mean hospital LOS of 21 days, and a mortality rate of 23.4%.fi P426 Conclusion This study demonstrates a high level of effi cacy of our protocol and the feasibility of de-escalation to limit the number of pharmacologic interventions. With our patient population spending a large percentage of time without shivering, it would suggest that this protocol could be revised further by utilizing rescue agents more frequently in order to prevent escalation of therapy to the next tier. References Hemodynamic targets during therapeutic hypothermia after cardiac arrest: a prospective observational study K Ameloot, I Meex, C Genbrugge, W Boer, F Jans, B Ferdinande, W Mullens, M Dupont, C Dedeyne, J Dens ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P426 (doi: 10.1186/cc14506) 1. Badjiatia N, et al. Crit Care Med. 2009;37:S250-7. 2. Choi HA, et al. Neurocrit Care. 2011;14:389-94. 1. Badjiatia N, et al. Crit Care Med. 2009;37:S250-7. 2. Choi HA, et al. Neurocrit Care. 2011;14:389-94. Introduction In analogy with sepsis, current postcardiac arrest (post- CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVO2 above 70%. This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival. Introduction In analogy with sepsis, current postcardiac arrest (post- CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVO2 above 70%. This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival. Methods A prospective, observational study during therapeutic hypothermia (24 hours –33°C) in 82 post-CA patients monitored with near-infrared spectroscopy. P428 Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) Is selective nasopharyngeal brain cooling detrimental to neuroprotection? M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P425 (doi: 10.1186/cc14505) M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Introduction Clinical outcomes vary depending on the method used to induce therapeutic hypothermia following stroke or cardiac arrest. In swine, we tested the hypothesis that selective nasopharyngeal brain cooling (SNBC), in contrast to systemic hypothermia, adversely impacts cerebral perfusion. Methods In two groups of animals (34 to 35  kg), blood fl ow in the right middle cerebral artery (MCA) was measured using transcranial Doppler (TCD). In group 1, SNBC was initiated using perfl uorohexane aerosol (1  ml/kg/minute) and oxygen (1  l/kg/minute) through a nasopharyngeal catheter atomizer. In group 2, the animals were body surface cooled using water-circulating blankets (4°C). In both groups, Conclusion ICU survivors were more likely to have had a shockable rhythm, been to the cathlab, received PPCI and TH and been less sick than nonsurvivors, but these may simply refl ect selection and other biases. Any benefi t these factors did confer to cardiac patients may have been off set by our liberal ICU admission policy to OHCAs with S150 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 brain temperature, intracranial pressure (ICP), core temperatures and vital signs were continuously recorded. Cooling was terminated once the brain reached 32°C and the animals were allowed to passively rewarm. Results In the SNBC group, the brain target temperature was reached in 54 ± 11 minutes. The mean ICP dropped precipitously to a nadir of –15 mmHg. TCD showed signifi cant vasospasm in the MCA, compared with the internal carotid artery (ICA), during the entire cooling phase (Table 1). Upon termination of cooling, the brain temperature spontaneously rewarmed to core temperature in 13  ± 4  minutes. Rewarming was associated with hyperemia and elevation of ICP. In group 2, there was no cerebral vasospasm or hyperemia during cooling and rewarming respectively. Conclusion SNBC is associated with signifi cant vasospasm of the MCA. In addition, spontaneous and rapid rewarming of the brain, vasodilation, rapid reperfusion, and rebound elevation of ICP, all occurring minutes after termination of SNBC, are likely to be detrimental to an already ischemic brain. Is selective nasopharyngeal brain cooling detrimental to neuroprotection? P426 Hemodynamic targets during therapeutic hypothermia after cardiac arrest: a prospective observational study K Ameloot, I Meex, C Genbrugge, W Boer, F Jans, B Ferdinande, W Mullens, M Dupont, C Dedeyne, J Dens ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P426 (doi: 10.1186/cc14506) Introduction In analogy with sepsis, current postcardiac arrest (post- Table 1 (abstract P425). Cerebral vasospasm during SNBC MCA fl ow ICA fl ow velocity velocity Lindegaard Pulsatility ICP Intervention (cm/second) (cm/second) ratio ratio (mmHg) Baseline 62 41 1.51 0.77 13 SNBC cooling 128 52 2.46 0.48 –15 SNBC rewarming 96 44 1.74 1.12 21 Table 1 (abstract P425). Cerebral vasospasm during SNBC Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest p g C Endisch, C Storm, CJ Ploner, C Leithner C Endisch, C Storm, CJ Ploner, C Leithner Methods With IRB approval, we prospectively measured rSO2 during ALS in consecutive OHCA patients. One sensor of the Equanox™ 7600 (NONIN) was applied on the patient’s forehead’s right side when the medical emergency team arrived in an OHCA. ROSC was defi ned as ROSC >20 minutes. Charité Universitätsmedizin Berlin, Germany Charité Universitätsmedizin Berlin, Germany Critical Care 2015, 19(Suppl 1):P431 (doi: 10.1186/cc14511) y Critical Care 2015, 19(Suppl 1):P431 (doi: 10.1186/cc14511) Introduction Electrical median nerve stimulation elicits a burst of high-frequency oscillations (HFOs) superimposing onto the cortical short-latency potential. Digital fi ltering of somatosensory evoked potentials (SSEPs) enables non-invasive analysis of these HFOs. The late HFO component following the cortical N2O peak is ascribed to spiking activity of cortical neurons. Results We included 88 prehospital cardiac arrest patients between December 2011 and October 2014 with eight (9%) patients with CPC 1 or 2. Twenty-seven patients of the nonsurvivors had ROSC >20 minutes and one patient had CPC 3 at hospital discharge. We observed no signifi cant diff erence between both groups in age (P  = 0.161), time between emergency call and start of ALS (P  = 0.788) and duration of basic life support performed by bystanders, general practitioners or paramedics (P  = 0.649). The initial rhythm was asystole in one (12.5%) survivor and in 50 (62.5%) nonsurvivors (P = 0.009), ventricular fi brillation in six (75%) survivors and 13 (16%) nonsurvivors (P = 0.001), and pulseless electrical activity in one (12.5%) survivor and 17 (21%) nonsurvivors (P = 1.00). The cardiac arrest was witnessed in all survivors (100%) and in 49 (61%) nonsurvivors (P = 0.046). First measured rSO2 was 38% (27 to 67) in the survivor group compared with 22% (8 to 32) in the nonsurvivor group (P = 0.004). Also a signifi cant diff erence was found in mean rSO2 until 1  minute before ROSC between survivors and nonsurvivors, respectively 46% (40 to 74) and 34% (22 to 42). We observed no signifi cant diff erence in increase of rSO2 until 1 minute before ROSC between survivors 12.5% (5 to 21) and nonsurvivors 11% (5 to 18) (P = 0.719). Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) Time >20 minutes until ROSC in shockable rhythms had fi ve patients with three survivors (78%, P  = 0.001). Presenting in nonshockable rhythms with ROSC <20  minutes were 54 patients with 18 survivors (33%, P  = 0.001). ROSC >20 minutes in nonshockable rhythms had 19 patients with two survivors (8%, P = 0.001). Survivors of shockable rhythms showed 19 (100%) living post TH. Fifteen survivors (79%, n = 19, P = 0.001) had CPC g Conclusion The optimal SVO2 (72%) and MAP (87  mmHg) derived from our hemodynamic model matched with the observed SVO2 (67 to 72%) and MAP (76 to 86 mmHg) associated with maximal survival. Prospective interventional studies to reach or maintain these targets are needed to confi rm these fi ndings. S151 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of this study were to investigate the association between hemoglobin, cerebral oxygenation (SctO2) and outcome in post-CA patients. score 1 or 2 with four survivors (21%, n = 19) having a CPC score of 3. A total of 25 survived nonshockable rhythm. Acute survival of patients with nonshockable rhythm showed 18 expired <72 hours (72%, n = 25) with long-term survival of four patients (5%, n = 74) and CPC scores of 1 or 2 (P = 0.001). Interestingly, patients with time to ROSC <20 minutes exhibiting more than one loss of sustained ROSC showed 100% mortality (P = 0.001). Patients presenting with shockable >20 minutes ROSC had overall survival of 70% (P = 0.001), but those undergoing >3 cardiac rhythm changes had 100% mortality (P = 0.001). yg 2 Methods A prospective observational study in 82 post-CA patients during hypothermia in the fi rst 24 hours of ICU stay. Hemoglobin was determined hourly together with a corresponding SctO2 by NIRS and SVO2 in patients with a pulmonary artery catheter (n = 62). 2 Results Based on 2,099 paired data, we found a strong linear relationship between hemoglobin and average SctO2 (SctO2 = 0.70 × hemoglobin + 56 (R2 = 20.84, P = 10–6)). Given the previously suggested SctO2 target between 66 and 68%, hemoglobin levels below 10  g/dl generally resulted in suboptimal brain oxygenation. Forty-three patients (52%) had a good neurological outcome (CPC 1 to 3) at 180 days post CA. P432 P432 One-size-fi ts-all or patient-tailored hemodynamic targets in post-cardiac arrest patients: an observational near-infrared spectroscopy study on cerebral autoregulation C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) P432 One-size-fi ts-all or patient-tailored hemodynamic targets in post-cardiac arrest patients: an observational near-infrared spectroscopy study on cerebral autoregulation C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest y Methods We retrospectively studied late HFO components of median nerve SSEPs obtained 24  hours to 4  days after cardiac arrest in patients treated with mild hypothermia (33°C for 24 hours). Cortical average recordings were digitally fi ltered at 450 to 750 Hz and noise- corrected maximum peak-to-peak amplitudes of the cortical late HFO bursts determined. Outcome upon ICU discharge was dichotomized according to the Cerebral Performance Category (CPC) scale. CPC 1 to 3 was classifi ed as good outcome, CPC 4 to 5 (unresponsive wakefulness syndrome and death) as poor outcome. Results Of 307 consecutive patients, 153 (50%) achieved good outcome (CPC 1 to 3) and 154 (50%) had poor outcome. Late HFO bursts were present in 102 (33%) recordings. Among patients with late HFO amplitudes above 0.1 μV, 26 had CPC 1 to 3, none had CPC 4 and eight died. Case review indicated causes of death other than hypoxic encephalopathy in all patients who died despite HFO amplitudes above 0.1 μV. Conclusion We found cortical late HFO bursts, obtained by digital fi ltering of standard SSEP recordings, in a signifi cant proportion of patients after cardiac arrest treated with mild hypothermia. Our data indicate that the presence of late HFO bursts with amplitudes above 0.1 μV may confi rm absence of severe hypoxic encephalopathy early after cardiac arrest with high specifi city. Conclusion We observed a signifi cant diff erence in fi rst measured rSO2 and mean rSO2 until 1 minute before ROSC between patients with good neurological outcome (CPC 1 or 2) at hospital discharge and patients with worse neurological outcome or nonsurvivors (CPC 3 or 4 or 5). However, no signifi cant diff erence was observed in the increase between both groups. Larger studies are necessary to confi rm these results. Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) There was a signifi cant association between average hemoglobin above 12.3 g/dl and good neurological outcome (OR = 2.88, 95% CI = 1.02; 8.16, P = 0.04). In a multivariate model, this association persisted after correction for comorbidities and age by the modifi ed Charlson score (OR = 2.99, 95% CI = 1.05; 8.53, P = 0.03). This association was entirely driven by results obtained in patients with an average SVO2 below 70% (OR = 17.55, 95% CI = 1.67; 184.41, P = 0.01). Conclusion Patients presenting with shockable rhythms undergoing TH had overall acute survival of 70% followed by long-term survival of 100% after 4 years. In contrast, patients presenting with nonshockable rhythm had long-term survival of 5%. TH is not recommended. Diff erence in cerebral saturation during cardiopulmonary resuscitation between survivors with favorable neurological outcome and compromised neurological outcome at hospital discharge C Genbrugge1, W Boer1, I Meex2, F Jans1, C Deyne1, J Dens1 1ZOL, Genk, Belgium; 2Hasselt University, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P429 (doi: 10.1186/cc14509) C Genbrugge , W Boer , I Meex , F Jans , C Deyne , J Dens 1ZOL, Genk, Belgium; 2Hasselt University, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P429 (doi: 10.1186/cc14509) Conclusion There is a steep linear relationship between hemoglobin and SctO2 in post-CA patients with hemoglobin levels below 10 g/dl generally resulting in cerebral desaturation. Average hemoglobin below 12.3 g/ dl was independently associated with worse neurological outcome 180  days post CA. An interventional trial is necessary to investigate whether maintaining higher hemoglobin would improve outcome. Introduction During out-of-hospital cardiac arrest (OHCA) monitoring possibilities are limited. Recently, the role of cerebral oximetry, using near-infrared spectroscopy, during ALS was investigated. In this study we determined whether the magnitude of increase in cerebral saturation (rSO2) or mean rSO2 during prehospital ALS was associated with good neurological outcome at hospital discharge (Cerebral Performance Category (CPC) 1 or 2). P431 Response of regional oxygen saturation technologies during hypoxia UB Borg, AM Neitembach Covidien, Boulder, CO, USA Critical Care 2015, 19(Suppl 1):P433 (doi: 10.1186/cc14513) Results Thirty-four simplifi ed EEG samples were analysed. According to standard EEG, 11 patients showed a DS pattern, three had CI, six showed BS, four showed PEDs and 10 had an SE. Neurophysiologists interpreted all samples with a high accuracy. Mean sensitivity was 82.12% and mean specifi city was 91.88%. Only one SE was missed by one neurophysiologist. Unfortunately, only one PED was confi rmed by both neurophysiologists. Interobserver reliability was high (κ  = 0.843). High correlations were found for the comparison of full and simplifi ed EEG for both neurophysiologists (r = 0.809). Further, the two inexperienced physicians identifi ed SE with a sensitivity of 85% and specifi city of 98%. Introduction The purpose of this study was to determine the rate and magnitude of response to hypoxia for three diff erent regional oxygen saturation (rSO2) devices. rSO2 technologies are focused on absolute accuracy without consideration of response characteristics. Current rSO2 technologies assume that the oxygen saturation is a fi xed ratio of arterial and venous blood. Cerebral arteries have an oxygenation- related vasoactivity that may change the arterial/venous ratio during hypoxia. Thus, absolute rSO2 accuracy may be less important compared with sensitivity to changes in cerebral rSO2. pi y Conclusion Simplifi ed EEG monitoring, using BIS, resulted in high accuracy of a simple classifi cation system in post-CA patients. Not only neurophysiologists, but also treating physicians were capable to identify SE, which may play an important role in the early detection of SE. We suggest using BIS as a screening tool in post-CA patients to save valuable time in the detection of SE, without replacing the need for full EEG monitoring for confi rmation. 2 Methods Ten subjects completed the study and are included in the analysis. One INVOS sensor (SAFB-SM) was placed on the left side and one Equanox (8000CA) or Foresight (1 July 2007 or 1 July 2005) sensor (alternated between subjects) was placed on the right side of the forehead for bilateral monitoring. Desaturation was induced by adjusting the inspiratory gas mixture of O2/N2. Desaturation was titrated from room air to achieve a plateau of 70% arterial oxygen saturation (SpO2). Resaturation was induced by rapid change in FiO2 to 1.0. Diff erences in cerebral saturation measured during prehospital advanced life support, between patients with presumed cardiac origin and noncardiac origin of cardiac arrest C Genbrugge1, W Boer1, K Anseeuw2, I Meex3, F Jans1, J Dens1, C De Deyne1 2 g p Results The rate of rSO2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was signifi cantly faster for INVOS (42 ± 16) compared with Foresight (57 ± 20) (P <0.05). There was signifi cant diff erence in total rSO2 change between INVOS (23 ± 4%) and Equanox (15 ± 4%) during desaturation and resaturation (P <0.005) and between INVOS compared with Foresight (20 ± 5%) (P <0.05). Conclusion All rSO2 devices followed the SpO2 slope during desaturation as expected. The diff erences between the devices in terms of total rSO2 change reached statistical signifi cance. There were also signifi cant diff erences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change 2 Results The rate of rSO2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was signifi cantly faster for INVOS (42 ± 16) compared with Foresight (57 ± 20) (P <0.05). There was signifi cant diff erence in total rSO2 change between INVOS (23 ± 4%) and Equanox (15 ± 4%) during desaturation and resaturation (P <0.005) and between INVOS compared with Foresight (20 ± 5%) (P <0.05). Introduction During out-of hospital cardiac arrest (OHCA) cerebral saturation may provide relevant information on cerebral oxygenation. In this study we examined the time course in cerebral saturation (rSO2) during prehospital ALS comparing patients with a presumed cardiac origin (survivor = Sc, nonsurvivor = NSc) of arrest and noncardiac origin (survivor = Snc, nonsurvivor = NSnc) of arrest. Conclusion All rSO2 devices followed the SpO2 slope during desaturation as expected. The diff erences between the devices in terms of total rSO2 change reached statistical signifi cance. There were also signifi cant diff erences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change Methods With IRB approval, we prospectively measured rSO2 from the start of ALS in consecutive OHCA patients. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients (4) The time under the individual optimal MAP was negatively associated with survival (OR = 0.97, 95% CI (0.96; 0.99), P = 0.02). The time under previously proposed fi xed targets (65, 70, 75, 80 mmHg) was not associated with a diff erential survival rate. Introduction Assessment of prognosis in postcardiac arrest (post- CA) patients became very challenging since the introduction of therapeutic hypothermia (TH). Continuous EEG monitoring has been proposed to improve prognostication; however, its use is limited due to diffi culties in ready interpretation. Thus emerges the need for a simple EEG montage. The bispectral index (BIS) monitor is a simplifi ed EEG system, mainly calculating an index ranging from 0 (isoelectric EEG) to 100 (full consciousness) to provide information on hypnotic depth of anaesthesia. The aim of the study was to validate the accuracy of simplifi ed EEG monitoring in a CA setting. pi g g Methods BIS monitoring (BIS VISTATM) was applied to collect frontotemporal data in TH-treated CA patients. A standard 19-channel EEG was performed after return to normothermia. Afterwards, small EEG frames coincident with the time of full EEG registration were extracted from the BIS monitor. We asked two neurophysiologists to indicate the presence of status epilepticus (SE), cerebral inactivity (CI), burst suppression (BS), periodic epileptiformic discharges (PEDs) or a diff use slowing pattern (DS). In addition, these samples were analysed by two inexperienced physicians, who were asked to indicate the presence of SE. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients g , yg neurologic outcome in postcardiac arrest patients I Meex, K Ameloot, C Genbrugge, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P430 (doi: 10.1186/cc14510) I Meex, K Ameloot, C Genbrugge, M Dupont, B Ferdinande, J Dens C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P430 (doi: 10.1186/cc14510) p py y g C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) Introduction The safety of a restrictive transfusion threshold of 7 g/dl applied in all critically ill patients can be questioned in postcardiac arrest (post-CA) patients since these are phenotypically clearly distinct. The aims Introduction A subgroup of post-CA patients with disturbed cerebral autoregulation might benefi t from higher mean arterial pressures S152 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 in seconds and the magnitude of absolute change may result in better resolution to detect physiological changes. Clinical studies are required to elucidate the clinical relevance of the diff erences. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. Methods A prospective observational study in 51 post-CA patients monitored with near-infrared spectroscopy. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. p Methods A prospective observational study in 51 post-CA patients monitored with near-infrared spectroscopy. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients P434 P434 Use of bispectral index EEG monitoring for a fast and reliable detection of epileptic activity in postcardiac arrest patients J Haesen1, L Desteghe1, I Meex2, C Genbrugge2, J Demeestere2, J Dens2, L Ernon2, C De Deyne2 1Universiteit Hasselt, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium Critical Care 2015, 19(Suppl 1):P434 (doi: 10.1186/cc14514) p py Results (1)  In multivariate analysis, patients with preserved auto- regulation (33.65%) had a signifi cant higher 180-day survival rate (OR = 4.62, 95% CI (1.06; 20.06), P = 0.04). (2) Phenotypically, a higher proportion of patients with disturbed autoregulation had pre- CA hypertension (31  ± 47 vs. 65  ± 49%, P  = 0.02) suggesting that right shifting of autoregulation is caused by chronic adaptation of cerebral blood fl ow to higher blood pressures. Based on an index of autoregulation (COX), the average COX-predicted optimal MAP was 85 mmHg in patients with preserved and 100 mmHg in patients with disturbed autoregulation. (3)  An individual optimal MAP could be determined in 33/51 patients. (4) The time under the individual optimal MAP was negatively associated with survival (OR = 0.97, 95% CI (0.96; 0.99), P = 0.02). The time under previously proposed fi xed targets (65, 70, 75, 80 mmHg) was not associated with a diff erential survival rate. Conclusion Cerebral autoregulation was shown to be disturbed in 35% of post-CA patients of which a majority had pre-CA hypertension. Disturbed cerebral autoregulation within the fi rst 24 hours after CA is associated with a worse outcome. In contrast to uniform MAP goals, the time spent under a patient-tailored optimal MAP, based on an index of autoregulation, was negatively associated with survival. Results (1)  In multivariate analysis, patients with preserved auto- regulation (33.65%) had a signifi cant higher 180-day survival rate (OR = 4.62, 95% CI (1.06; 20.06), P = 0.04). (2) Phenotypically, a higher proportion of patients with disturbed autoregulation had pre- CA hypertension (31  ± 47 vs. 65  ± 49%, P  = 0.02) suggesting that right shifting of autoregulation is caused by chronic adaptation of cerebral blood fl ow to higher blood pressures. Based on an index of autoregulation (COX), the average COX-predicted optimal MAP was 85 mmHg in patients with preserved and 100 mmHg in patients with disturbed autoregulation. (3)  An individual optimal MAP could be determined in 33/51 patients. Response of regional oxygen saturation technologies during hypoxia After 5  minutes of SpO2 100%, the process was repeated by desaturation to SpO2 70% and rapid return to SpO2 100%. Cerebral and pulse oximetry data were recorded during the study and the time of each FiO2 change and plateau was recorded. rSO2 levels at 10, 20, 40, 60 and 80% of the total SpO2 response were calculated for each device to assess the rate of rSO2 change. The rate of rSO2 change in seconds and total rSO2 change were compared. P436 Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) C Storm, CJ Ploner, C Leithner Introduction Bilaterally absent cortical somatosensory evoked potentials (SSEPs) predict poor outcome after cardiac arrest. A threshold for the amplitude of early cortical SSEPs above which patients may survive with good outcome has not been determined. Thus, tolerable noise levels for the interpretation of cortical SSEPs are poorly defi ned. Furthermore, it has not been systematically investigated whether high amplitudes of cortical SSEPs may exclude severe hypoxic encephalopathy incompatible with re-awakening. Conclusion In patients with cardiac arrest and targeted temperature management at 33°C, an NSE serum concentration of >90 μg/l strongly indicates poor outcome. NSE producing tumors, acute brain diseases, severe hematologic diseases, use of an intra-aortic balloon pump and blood transfusions need to be considered as potential confounders. An NSE serum concentration of <17  μg/l largely excludes hypoxic encephalopathy incompatible with re-awakening. p p y p g Methods We prospectively studied SSEPs after median nerve stimulation obtained 24 hours to 4 days after cardiac arrest in patients treated with targeted temperature management at 33°C for 24 hours. Amplitudes of cortical SSEPs were determined, if at least two peripheral, spinal and cortical recordings per side were available, spinal potentials were bilaterally reproducible and cortical noise level was below 0.25 μV. Cortical SSEP amplitude was defi ned as largest amplitude of a reproducible cortical SSEP of four cortical recordings (two per side) within 50 milliseconds after stimulation. Outcome was assessed upon ICU discharge using the Cerebral Performance Category (CPC) scale. CPC 1 to 3 was defi ned as good outcome, CPC 4 to 5 as poor outcome. Results Of 318 consecutive patients examined, 293 had complete SSEP recordings with reproducible spinal potentials and cortical noise levels below 0.25 μV. Of those, 137 (47%) had good outcome and 156 (53%) had poor outcome. The lowest amplitude of the early cortical SSEPs in a survivor with good outcome was 0.62 μV. All 79 patients with amplitudes below this threshold had poor outcome. None of 27 patients who survived with CPC 4 (unresponsive wakefulness syndrome) had cortical SSEP amplitudes above 2.5 μV. Twenty-four patients with amplitudes above this limit died. Detailed case review indicated a cause of death other than hypoxic encephalopathy in these patients. P436 Results Of 601 included patients, 309 (51%) had good outcome. An NSE serum concentration threshold of 90  μg/l predicted poor outcome with a positive predictive value of 0.98 and a sensitivity of 0.51. Three patients survived with good outcome despite an NSE serum concentration >90 μg/l. In two of these patients NSE elevations had been documented prior to cardiac arrest. One patient had a neuroendocrine tumor of the pancreas, the other patient suff ered from encephalitis of unknown etiology and an osteomyelofi brosis. Potential confounders in the third patient were an ovarian carcinoma, the use of an intra-aortic balloon pump and blood transfusions shortly after cardiac arrest. Only 16 of 205 patients with NSE <17  μg/l had poor outcome, the majority of these patients died from causes other than hypoxic encephalopathy. Diff erences in cerebral saturation measured during prehospital advanced life support, between patients with presumed cardiac origin and noncardiac origin of cardiac arrest One sensor (Equanox™ 7600; Nonin) was applied on the patient’s forehead’s right side when S153 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 the medical emergency team arrived at the OHCA setting. ROSC was defi ned as ROSC >20 minutes. Retrospectively, included patients were divided into two groups with respect to their presumed origin of arrest. Results Between December 2011 and October 2014, 113 OHCA patients were included. We observed a signifi cant diff erence in asystole and VF as initial rhythm between NSc and NSnc, respectively (P = 0.035 and P = 0.001). In both groups of NS, duration of ALS was signifi cant longer compared with the two S groups (P  = 0.001 in both comparisons). We observed no signifi cant diff erence in fi rst measured rSO2, mean rSO2 until 1 minute before ROSC and increase in rSO2 until 1 minute before ROSC (respectively P = 0.123, P = 0.501, P = 0.265) between Sc and Snc. However, when we compare the nonsurvivors of cardiac with noncardiac origin, we observed a signifi cant diff erence in mean rSO2 until 1 minute before ROSC, 35% (27 to 44) in the NSc group and 27 (21 to 34) in the NSnc group (P = 0.026). First measured rSO2 was 24.5% (13 to 34) in the NSc group and 14 (4 to 28) in the NSnc group (P = 0.069) trending to be signifi cantly diff erent. No signifi cant diff erence was observed in increase until 1 minute before ROSC between both groups of NS (P = 0.920). Signifi cant diff erences was observed in mean rSO2 until 1 minute before ROSC and increase in rSO2 between Snc and NSnc (P = 0.033; P <0.001) and between Sc and NSc (P = 0.001; P <0.001).i Prognostic value of neuron-specifi c enolase after cardiac arrest and targeted temperature management Prognostic value of neuron-specifi c enolase after cardiac arrest and targeted temperature management g p g K Streitberger, C Leithner, CJ Ploner, C Storm Charité Universitätsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P437 (doi: 10.1186/cc14517) K Streitberger, C Leithner, CJ Ploner, C Storm Charité Universitätsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P437 (doi: 10.1186/cc14517) Introduction The serum concentration of neuron-specifi c enolase (NSE) has been established as a highly specifi c predictor of poor outcome after cardiac arrest. Recent studies have indicated that patients treated with targeted temperature management at 33°C for 24 hours may have good outcome despite NSE serum concentrations considerably higher than the cutoff established for normothermic patients. The threshold above which survival with good outcome becomes very unlikely, its positive predictive value and sensitivity for prediction of poor outcome have not been established in this patient group. Furthermore, a threshold below which hypoxic encephalopathy may be largely excluded has not been determined. Methods From 2006 through 2014 we prospectively included in- hospital and out-of-hospital cardiac arrest patients treated with targeted temperature management at 33°C for 24  hours. The NSE serum concentration was determined 3 days after cardiac arrest and the outcome was assessed according to the Cerebral Performance Category (CPC) upon ICU discharge. CPC 1 to 3 was defi ned as good outcome and CPC 4 to 5 as poor outcome. Individual case review was performed in patients with good outcome despite very high NSE serum concentration and in patients with poor outcome despite very low NSE serum concentration. Conclusion We can conclude that NSc have a signifi cant higher mean rSO2 and trend to have a signifi cant diff erence in fi rst measured rSO2 compared with NSnc. However, no signifi cant diff erence was observed between Sc and Snc. P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522 Results One hundred and ninety-three patients (110 males, 83 females, mean age 48.2  ± 18.3  years) were studied. The mean time interval between collapse and onset of resuscitation was 2.3  ± 2.1  minutes. A total of 65.3% arrests were witnessed. Sustained ROSC occurred in 36.8% patients and the SOHD was 24.9%. The initial rhythm recorded during CPR was asystole in 133 patients, pulseless electrical activity in 21 patients and ventricular fi brillation/tachycardia (VF/VT) in 39 patients. SOHD for these rhythms was 8.3%, 33.3% and 76.9%, respectively. On univariate analysis, type of rhythm, witnessed arrests and time to resuscitation were associated with sustained ROSC and SOHD. On multivariate analysis, only type of rhythm, VF/VT (P = 0.000) and PEA (P  = 0.017), were signifi cantly associated with SOHD, while witnessed arrest and time to resuscitation were not. Introduction The association between blood alcohol level (BAL) on mortality and neurologic outcome in patients with polytrauma and head injury is not clear and the data in the literature are sometimes confl icting. Some studies suggest a possible neuroprotective eff ect of alcohol and increased survival while others show the opposite. The rationale for this study was to investigate whether BAL has any impact on presentation, neurologic outcome and survival in patients with combination of polytrauma and head injury. y j y Methods This is a retrospective study of 43 polytraumatized patients with head injury who were intubated and treated by the prehospital unit and transported to the trauma center. Patients were grouped according to their BAL into BAL+ (>0.5  mg/l) and BAL– (≤0.5  mg/l). Inclusion criteria were age ≥18, Injury Severity Score ≥16 and head Abbreviated Injury Scale (AIS) ≥3. Physiological parameters and outcome with respect to survival to hospital discharge (STHD) and functional outcomes were analyzed. Severity of injuries was measured using the Trauma–Injury Severity Score (TRISS) and head injury using AIS. Functional outcome was measured using the Glasgow Outcome Scale (GOS), Cerebral Performance Category (CPC) and Glasgow Coma Conclusion Sustained ROSC occurred in 36.8% patients and the SOHD was 24.9%. P440 Outcome after CPR: when we cannot save lives, we can save organs C Caestecker, J Froyman, P Lormans, W Stockman AZ Delta, Roeselare, Belgium Critical Care 2015, 19(Suppl 1):P440 (doi: 10.1186/cc14520) Introduction Patients resuscitated after cardiac arrest (CA) who suff er bad neurologic outcome or become brain dead might become organ donors (OD) when well managed and identifi ed. We report on the cohort of patients resuscitated after in-hospital or out-of-hospital CA becoming OD in a tertiary community hospital with an intensive donor identifi cation program. i g Methods Data from our 28-bed mixed medical/surgical adult ICU in a 900-bed tertiary hospital were analyzed from 2010 to 2014. y y Results Our ICU admitted 2,320 patients/year. Overall ICU mortality in this period was 7.4%. A summary of the results is presented in Table 1. Of the 219 patients admitted after CA, 21 (10%) became OD. This resulted in 55 successfully transplanted organs (28 kidneys, 17 livers, seven lung pairs, three hearts). Of note, good outcome (CPC 1 and 2) was achieved in 55 patients (25%). frequently suff ered cardiogenic shock, had more organ dysfunctions and died more frequently, respectively, with hospital mortality of 79.5% versus 29.1%, P <0.001; see also Figure 1. Table 1 (abstract P440) Organ Donors ICU admission Year donors after CA DCD/DBD after CA 2010 11 3 0/3 40 2011 9 5 3/2 41 2012 18 4 3/1 47 2013 17 5 4/1 53 2014 13 4 1/3 38 Total 68 21 (31%) 11/10 219 Conclusion Ten percent of patients resuscitated after CA and admitted to the ICU become OD, consisting of up to 31% of the total number of OD in our center. Patients resuscitated after CA who suff er severe irreversible brain damage or are brain dead can thus substantially expand the donor pool. This justifi es extensive resuscitation eff orts, if not to save lives, then to save organs. This might be reassuring for families, staff and the community. Table 1 (abstract P440) Organ Donors ICU admission Year donors after CA DCD/DBD after CA 2010 11 3 0/3 40 2011 9 5 3/2 41 2012 18 4 3/1 47 2013 17 5 4/1 53 2014 13 4 1/3 38 Total 68 21 (31%) 11/10 219 Conclusion In patients hospitalized for acute heart failure, both prehospital and postadmission resuscitated cardiac arrest is a severe complication associated with signifi cantly morbidity and mortality. Outcome of cardiopulmonary resuscitation in cancer patients in an Indian tertiary cancer center Conclusion Ten percent of patients resuscitated after CA and admitted to the ICU become OD, consisting of up to 31% of the total number of OD in our center. Patients resuscitated after CA who suff er severe irreversible brain damage or are brain dead can thus substantially expand the donor pool. This justifi es extensive resuscitation eff orts, if not to save lives, then to save organs. This might be reassuring for families, staff and the community. Introduction Cardiopulmonary resuscitation (CPR) after cardiac arrest in cancer patients is often discouraged as it is associated with poor outcome. In our 700-bed tertiary cancer hospital in Mumbai, India, the ICU runs an in-hospital cardiac arrest team (CAT). We reviewed our data to determine outcome from CPR, identify factors associated with improved outcomes and justify the presence of a CAT in our cancer hospital. p Methods All in-hospital patients from November 2012 to November 2014 (2-year period) with unanticipated cardiorespiratory arrests were included. Data were recorded prospectively using the Utstein template. Only patients with cardiac arrest rhythms were included. Patients with anticipated progression towards arrest, those with seizures, hypotension without dysarrythmias or other medical emergencies were excluded. The outcomes studied were return of spontaneous circulation (ROSC) and survival on hospital discharge (SOHD). Binary logistic regression analysis was performed to determine factors associated with ROSC and SOHD. P436 Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) P438 38 Resuscitated cardiac arrest in patients admitted with acute heart failure: analysis of a large prospective AHEAD network registry J Parenica1, J Belohlavek2, J Spinar1, G Dostalova2, S Havranek2, A Linhart2 R Miklik1, L Dusek3, J Jarkovsky3 1University Hospital Brno, Czech Republic; 2General University Hospital, Prague, Czech Republic; 3Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P438 (doi: 10.1186/cc14518) Introduction Heart failure is a frequent cause of cardiac arrest and vice versa, cardiac arrest frequently complicates acute heart failure episodes. We aimed to characterize the infl uence of cardiac arrest on outcome of acute heart failure patients admitted to hospital. Methods The AHEAD registry includes patients hospitalized for acute heart failure from 10 PCI and fi ve non-PCI centers in the Czech Republic. The data were collected from September 2006 to October 2012. Conclusion Our data indicate that the prognostic value of SSEP after cardiac arrest extends beyond a mere absent–present dichotomy. Bilaterally absent as well as very low amplitude SSEPs predict poor outcome with high positive predictive value. SSEPs should not be used for prognostication, if noise in cortical recordings could mask critically low amplitudes. High amplitudes of early cortical SSEPs strongly argue against severe hypoxic encephalopathy incompatible with re-awakening. Results In the respective period, a total of 6,242 patients were enrolled into the registry. Resuscitated cardiac arrest occurred in 313 patients prehospitally and in 484 after admission; the remaining 5,445 patients were not resuscitated during their index hospitalization. Patients resuscitated after admission in comparison with prehospitally resuscitated were older, had lower left ventricle ejection fraction, more S154 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P438). Overall mortality (including hospitalization). Total n = 6,242. Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Endothelial dysfunction in acute brain injury and the development of cerebral ischemia S Van Ierssel1, VM Conraads1, EM Van Craenenbroeck1, Y Liu2, AI Maas1, PM Parizel1, VY Hoymans1, CJ Vrints1, PG Jorens1 1University Hospital Antwerp, Edegem, Belgium; 2The Third Xiangya Hospital, Changsha, China Critical Care 2015, 19(Suppl 1):P443 (doi: 10.1186/cc14523) Introduction Since most patients with acute brain injury are treated head-up at 30 to 45°, there can be a height diff erence of up to 15 cm between the heart and the ear canal. This causes a diff erence between mathematical CPP and true CPP of up to 11 mmHg depending on the zero reference level used and the body length of the patient (Figure 1). We conducted a survey to analyze the current practice on CPP targets and zero reference levels in diff erent ICUs. Introduction Cerebral ischemia (CeI) is a major complicating event after acute brain injury (ABI) in which endothelial dysfunction is a key player. p y Methods We studied cellular markers of endothelial dysfunction and peripheral reactive hyperemia index (RHI) in 26 patients with ABI at admission and after 6 and 12 days, and compared these with healthy volunteers (n = 15). CeI was determined clinically or using computer tomography.i f Methods Neuro-ICU physicians were invited to participate in a survey on ICP and CPP targets and the level of measurement. Results The results of 30 centers are summarized in Table 1. Most centers (83.3%) use head-up elevation of 30 to 45° and consider an ICP of 20 mmHg as the threshold to start treating ICP (80%). More variation is noted in minimal and maximal CPP threshold. All centers measure ICP at the ear canal. Twenty-seven centers (90%) measure MAP at the heart, three centers measure MAP at the ear canal. These three centers use >50, 60 and 65 mmHg as minimal CPP and raise the bed to 30 to 45°. The two centers using minimal CPP >60 mmHg do not apply an upper limit. g p y Results In patients with ABI, RHI at admission was signifi cantly reduced compared with healthy subjects (P = 0.003), coinciding with a decrease in circulating endothelial progenitor cells (EPC) (P = 0.002) (Table 1). The RHI recovered in eight patients without development of CeI (Figure 1, black), but failed to fully recover by day 12 in three out of four patients that developed CeI (Figure 1, red). P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) A reduced response time, witnessed arrest and type of rhythm are associated with ROSC and improved SOHD. The type of rhythm was independently associated with SOHD, with VF/VT and PEA having improved survival while asystolic patients had a poor outcome. These considerations justify the presence of a CAT in our cancer hospital. S155 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P443). Evolution in time of markers of endothelial dysfunction after acute brain injury Scale (GCS) at discharge. Diff erences among groups were analyzed using Student’s t test, the Mann–Whitney U test and the chi-square test. Results BAL did not have a signifi cant eff ect on presenting physiological parameters. There was a signifi cant diff erence in the age of the groups, showing that patients in the BAL+ group were younger (32.6 vs. 56.8 years; 95% confi dence level; P = 0.000). BAL had a lasting eff ect on functional measures of neurological outcome which showed better results in the BAL+ group; it had signifi cantly better GOS (4.7 vs. 3.9; 95% confi dence level; P = 0.027), and GCS at discharge was on the border of statistical signifi cance (15 vs. 14; 95% confi dence level; P = 0.05). Other variables (TRISS, AIS, STHD, CPC) did not show statistically signifi cant diff erences among groups. gif g g p Conclusion Presence of alcohol in the blood had a positive eff ect on neurological outcome but there was no signifi cant diff erence in survival. However, the positive results in neurologic outcome in the BAL+ group may also be due to the fact that this group was younger. The small number of patients in the study is another limiting factor of the interpretation. Therefore, the neuroprotective role of alcohol needs further clarifi cation. Conclusion Patients with ABI exhibit impaired microvascular endo thelial function measured as RHI and a decreased circulating level of EPC. P444 P444 P444 Survey on ICP, target CPP and MAP measurement level in patients with severe acute brain injury in diff erent ICUs M Van Laer1, K Deschilder2, W Stockman2 1UZ Brussel, Brussels, Belgium; 2AZ Delta, Roeselare, Belgium Critical Care 2015, 19(Suppl 1):P444 (doi: 10.1186/cc14524) Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Despite recovery Figure 1 (abstract P443). y Conclusion Considering the infl uence of position on CPP, the variations among centers and the narrow range of CPP thresholds, future studies and guidelines should describe where MAP is measured. Alternatively, we propose a new formula for CPP: true CPP = MAP(heart) – ICP(ear) – height diff erence (heart to ear canal (cm)) × 0.73. Conclusion Considering the infl uence of position on CPP, the variations among centers and the narrow range of CPP thresholds, future studies and guidelines should describe where MAP is measured. Alternatively, we propose a new formula for CPP: true CPP = MAP(heart) – ICP(ear) – height diff erence (heart to ear canal (cm)) × 0.73. Figure 1 (abstract P443). Figure 1 (abstract P444). Table 1 (abstract P444) ICP threshold (mmHg); n (%) >15; 2 (6.7%) >18; 1 (3.3%) >20; 24 (80%) >25; 3 (10%) Minimal CPP (mmHg); n (%) >50; 6 (20%) >55; 4 (13.3%) >60; 16 (53.3%) >65 to 70; 4 (13.3%) Maximal CPP (mmHg); n (%) <70 to 75; 8 (26.7%) <80; 8 (26.7%) <85; 2 (6.7%) No limit; 12 (40%) Figure 1 (abstract P444). Figure 1 (abstract P444). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S156 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P445 with traumatic brain injuries defi ned as a Glasgow Coma Score <8 upon admission. Patients included were 18 to 59  years of age and were mechanically ventilated with intracranial pressure monitoring for greater than 72 hours. The primary outcome evaluated was the number of patients treated for confi rmed infections. Secondary outcomes included the antibiotic length of therapy (LOT), antibiotic days of therapy (DOT), number of cultures, and ICU and hospital length of stay (LOS). DOT was defi ned as the sum of days on which each antibacterial drug was administered. P445 Comparison of 15oxygen positron emission tomography and near-infrared spectroscopy for measurement of cerebral physiology J Simpson1, N Sudhan1, H Hare2, J Donnelly1, X Liu1, F Aigbirhio1, T Fryer1, G Stocks-Gee1, P Smielewski1, D Bulte2, J Coles1 1University of Cambridge, UK; 2University of Oxford, UK Critical Care 2015, 19(Suppl 1):P445 (doi: 10.1186/cc14525) Introduction The gold standard technique for imaging cerebral blood fl ow (CBF) and metabolism is 15oxygen positron emission tomography (15O PET). g References 1. Zweifel C, et al. Stroke. 2010;41:1963-8. 1. Zweifel C, et al. Stroke. 2010;41:1963-8. 2. Coles JP, et al. JCBFM. 2004;24:202-11. 3. Culver JP, et al. JCBFM. 2003;23:911-24. f Results Sixty-four treatments (32 HTS, 32 Man) were studied among 26 patients (19 TBI, seven SAH; age 42  ± 17  years, time from injury to treatment 2.6 ± 1.9 days). Man and HTS eff ectively decreased ICP (coeffi cient = –2.5 mmHg, 95% CI = –3.2 to –1.8 mmHg and –2.9 (–3.8 to –2.0) respectively; both P < 0.0001). No signifi cant diff erence was found in CMD lactate, pyruvate, glucose and PbtO2 after HTS or Man treatment. CMD glutamate decreased signifi cantly after Man (–0.73 ( –1.41 to –0.052); P <0.05), but not after HTS. Eff ect of osmotherapy with mannitol or hypertonic saline on cerebral oxygenation and metabolism in patients with intracranial hypertension after severe brain injury y j T Suys, H Quintard, C Patet, M Oddo Introduction Osmotherapy with mannitol (Man) or hypertonic saline (HTS) is currently used to treat elevated intracranial pressure after severe acute brain injury (sABI); however, their eff ect on cerebral oxygenation and metabolism has not been extensively evaluated. Methods A retrospective analysis of a cohort of patients with sABI after traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) monitored with cerebral microdialysis (CMD), brain oxygen (PbtO2) and ICP, who were treated with Man (20%, 0.5 g/kg) or HTS (7.5%, 100 ml) for ICP >25 mmHg. Osmotherapy was administered over 20 minutes and each patient’s individual response to intervention was analyzed up to 120 minutes following infusion. Only episodes where no other hypertonic solute was administered within 2  hours before or after treatment were selected. Variables analyzed included CMD lactate, pyruvate, glucose, glutamate, lactate/pyruvate ratio, and main brain physiologic variables ICP, PbtO2, CPP. Analysis was conducted using mixed-eff ects multilevel regression. Introduction Osmotherapy with mannitol (Man) or hypertonic saline (HTS) is currently used to treat elevated intracranial pressure after severe acute brain injury (sABI); however, their eff ect on cerebral oxygenation and metabolism has not been extensively evaluated. Conclusion We found no relationship between NIRS and baseline physiology as determined by 15O PET. Further studies should assess the dynamic response of NIRS to a measured change in physiology in patients. Further confi nes of NIRS include its limited and focal brain coverage. Conclusion We found no relationship between NIRS and baseline physiology as determined by 15O PET. Further studies should assess the dynamic response of NIRS to a measured change in physiology in patients. Further confi nes of NIRS include its limited and focal brain coverage. Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Continuous near-infrared spectroscopy (NIRS) has been used to assess adequacy of cerebral oxygenation following stroke, traumatic brain injury and subarachnoid haemorrhage [1], and measurements have been compared with jugular oxygen saturation. In this study we compared NIRS with 15O PET within healthy volunteers. g Results A total of 23 patients treated with normothermia and 119 patients in the control group were evaluated between January 2009 and September 2014. The number of patients treated for confi rmed infections was similar between groups (normothermia: 73.9%, control: 80%, P  = 0.803). Empiric antibiotic therapy was more commonly utilized in the normothermia group at 34% versus 20.5% (P = 0.173). Antibiotic LOT and DOT were 13.8 versus 10.8 days (P = 0.157) and 18.3 versus 16.2 days (P = 0.575) in the normothermia versus control groups, respectively. Total culture rate was lower in the normothermia group with 13.2 versus 8.78 (P = 0.0002) cultures per patient. No signifi cant diff erence in hospital LOS (normothermia: 23 days, control: 18 days, P = 0.158) or ICU LOS (normothermia: 17 days, control: 15 days, P = 0.185) was demonstrated.i y Methods Fifteen healthy subjects were recruited (12 male, average age 38 years); PET precluded females of reproductive age. Steady-state 15O PET with arterial sampling was performed to measure CBF, cerebral metabolic rate of oxygen (CMRO2), oxygen extraction ratio (OEF) and cerebral blood volume (CBV) [2]. Simultaneously, NIRS data were collected using a Hamamatsu NIRO 200 with sensors on either side of the forehead. NIRS OEF was calculated from tissue oxygen saturation, SaO2 and an assumed arterial/venous blood volume ratio of 30/70 [3]. Results The frontal region 15O PET CBF, CMRO2, OEF and CBV were mean (SD) 44.9 (10) ml/100 ml/minute, 158.7 (24.7) μmol/100 ml/minute, 45.8 (7.3)%, and 2.8 (0.8)  ml respectively, and there was no relationship between NIRS and 15O PET (Figure 1). Conclusion Rates of confi rmed infections and number of antibiotic days were similar between the normothermia and control groups, suggesting that normothermia does not increase infection risk. However, the number of cultures obtained in the control group was signifi cantly greater than the normothermia group with a trend toward increased empiric antibiotic use. Figure 1 (abstract P445). Linear correlation between NIRS and PET OEF. Reference Figure 1 (abstract P445). Linear correlation between NIRS and PET OEF. Reference 1. Greer DM, et al. Stroke. 2008;39:3029-35. 1. Greer DM, et al. Stroke. 2008;39:3029-35. Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate C Patet1, H Quintard1, T Suys1, L Pellerin2, P Magistretti3, M Oddo1 1CHUV – Lausanne University Hospital, Lausanne, Switzerland; 2University of Lausanne, Switzerland; 3Brain Mind Institute, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P448 (doi: 10.1186/cc14528) Introduction In patients with acute brain injury (ABI), increased cerebral energy demand is frequent, potentially leading to cerebral glucose depletion (GD) and poor outcome. In this setting, lactate may act as supplemental fuel. We examined dynamics of cerebral lactate supply during prolonged GD in ABI. Results ICP registrations were made parallel with all TCD measurements in 51 patients. Intraparenchymal ICP monitoring was inserted within the fi rst 3 hours after trauma and there was no complication (infections, intracranial hemorrhage, or technical failure) related to invasive ICP monitoring. Increased ICP was noted upon transducer insertion in 38 children with male prevalence (10 girls, 28 boys). Median GCS was 6, indicating the magnitude of injury in this group of patients. The overall results of the 38 patients showed a strong correlation between the ICP and PI and during outbursts of ICP with a correlation coeffi cient of r = 0.89 (ICP >20 mmHg) and r = 0.90 (ICP <20 mmHg). The relation between ICP and PI was estimated by the linear regression equation: ICP = 22,299 × PI – 10,705 (ICP >20 mmHg) and ICP = 38,592 × PI – 16,972 (ICP <20 mmHg). The CPP and PI were correlated signifi cantly during the changes in intracranial pressure. However, a better correlation was found when ICP >20 mmHg and PPC <50 mmHg (PI was 2.4 ± 0.89 when CPP was 35.96 ± 4.48 with a correlation coeffi cient of Pearson r = 0.80) than when ICP <20 mmHg and CPP >50 mmHg (PI was 0.78 ± 0.14 when CPP was 57.11 ± 9.62 with a correlation coeffi cient of Pearson r =0.76). y Methods We retrospectively analyzed severe ABI (18 TBI, eight SAH) monitored with brain oxygen and cerebral microdialysis (CMD) to measure hourly levels of cerebral extracellular glucose, lactate, pyruvate and lactate/pyruvate ratio (LPR). Variations of CMD variables were analyzed as a function of GD (defi ned as spontaneous decreases of CMD glucose from normal to low (<1.0 mM), at least 2 hours) and increased cerebral energy demand (LPR >25). Results During GD (60 episodes; 26 patients), we found an increase of CMD lactate (4 ± 2.3 vs. 1. Paolin A, Manuali A, Di Paola F, Boccaletto F, Caputo P, Zanata R, et al. Reliability in diagnosis of brain death. Intensive Care Med. 1995;21:657-62. 2. Gan TJ, Glass PS, Windsor A, Payne F, Rosow C, Sebel P, et al. Bispectral index monitoring allows faster emergence and improved recovery from propofol, alfentanil, and nitrous oxide anesthesia. Anesthesiology. 1997;87:808-15. Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury g Conclusion The BIS is a noninvasive, simple, and easy to interpret method, showing a perfect correlation with the other diagnostic methods. BIS can be used in severely comatose patients as an assessment of brain death. j y H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate 5.4 ± 2.9 mM) and LPR (27 ± 6 vs. 35 ± 9; all P <0.005) while brain oxygen and blood lactate remained normal. Dynamics of lactate and glucose supply were studied by analyzing the relationship between blood and CMD samples. No correlation between blood and brain lactate was found when brain glucose and LPR were normal (r = –0.12, P = 0.48; Figure 1), while this correlation became linear during GD, progressively rising to r = 0.53 (P <0.0001) when energy demand increased, suggesting increased cerebral lactate availability. The correlation between blood and brain glucose changed in the opposite direction, decreasing from r = 0.78 to 0.37 (P <0.0001) during GD and at LPR >25. Conclusion The absolute value of the PI is a reliable noninvasive indicator of ICP in pediatric severe TBI. A strong correlation between PI and ICP was demonstrated. Therefore, PI may be of guiding value in the invasive ICP placement decision in the neurointensive care patient when ICP monitoring is not systematically performed. In particular, ICP monitoring remains as grade C in the latest guidelines of management of STBI in children published in 2012. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. P449 Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Figure 1 (abstract P448). Correlations between blood and brain lactate. Figure 1 (abstract P448). Correlations between blood and brain lactate. P450 P449 P449 Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Evaluation of infection risk and antibiotic exposure in traumatic brain injury patients treated with therapeutic normothermia h k , , Hennepin County Medical Center, Minneapolis, MN, USA Hennepin County Medical Center, Minneapolis, MN, USA p y p Critical Care 2015, 19(Suppl 1):P446 (doi: 10.1186/cc14526) Introduction The purpose of this study is to assess the rate of confi rmed infections, antibiotic exposure, and monitoring practices with normothermia protocol utilization for traumatic brain injury patients. Treatment and prevention of fever is a focus of therapy for patients with severe neurological injury as fever has been identifi ed as an independent risk factor for morbidity and mortality [1]. Conclusion Osmotherapy with Man and HTS treatment had no eff ect on cerebral oxygenation and metabolism. Man, but not HTS, favorably reduced brain glutamate. These fi ndings support further investigation to test the value of alternative osmotic agents (such as hypertonic lactate) that may reduce ICP and at the same time improve cerebral metabolism after sABI. Methods The retrospective chart review analyzed outcomes of maintaining normothermia at 36.5°C versus a similar population without temperature control as a standard of care in patients admitted Acknowledgements Supported by grants from the Swiss National Science Foundation and the Novartis Foundation for Biomedical Research. S157 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P448 Methods A prospective and descriptive study conducted in our PICU from June 2011 to December 2013. We investigated 51 children with severe trauma brain injury (TBI). The TCD measurements were routinely performed bilaterally on the middle cerebral artery parallel to the ICP registration. The ICP and CPP data were correlated to PI and the correlation coeffi cient calculated. To control the linear correlation, the residuals were tested for normal distribution around the regression line. P448 Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate C Patet1, H Quintard1, T Suys1, L Pellerin2, P Magistretti3, M Oddo1 1CHUV – Lausanne University Hospital, Lausanne, Switzerland; 2University of Lausanne, Switzerland; 3Brain Mind Institute, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P448 (doi: 10.1186/cc14528) Bispectral index as a predictor of unsalvageable traumatic brain injury Introduction The aim was to evaluate the accuracy of bispectral index (BIS) monitoring for the diagnosis of brain death in severely comatose patients. We aimed to determine the utility of the BIS as a tool for clinical evaluation of the moment of brain death. Methods A prospective observational study with waiver of consent was conducted in the trauma ICU for 2 years from October 2012 to September 2014. Monitoring of BIS occurred during patient stay in the ICU. Population: 62 severely comatose patients (Glasgow Coma Score less than 6) admitted to the ICU mainly because of intracerebral hemorrhage, head injury, or postanoxic coma. BIS was recorded continuously during the hospitalization in the ICU. Where necessary, clinical brain death was confi rmed by EEG or brain stem test. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Results Twenty-nine patients were already clinically brain dead at the time of admission, and their individual BIS values were 0. Twenty-four patients were not clinically brain dead at the time of admission, and their individual BIS values were between 20 and 30. These patients became brain dead, and their individual BIS values dropped to 0 in a few hours to a few days. Seventeen patients who did not become brain dead during their hospitalization in the ICU had persistent electrocerebral activity on EEG, and their average BIS values remained above 31. References Introduction This study was designed to see whether there is a correlation between the transcranial Doppler (TCD) parameters and the CCP and intracranial pressure (ICP) monitoring during the cerebral hemodynamic changes and to evaluate ICP indirectly by TCD. S158 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P451 P451 Neuromonitoring of patients with severe traumatic brain injury at the bedside M Aries1, JG Regtien1, M Czosnyka2, J Donnelly2, P Smielewski2 1UMCG, Groningen, the Netherlands; 2Addenbrookes Hospital, University of Cambridge, UK Critical Care 2015, 19(Suppl 1):P451 (doi: 10.1186/cc14531) PbtO2 decreased and the microdialysis showed continuous signs of ischemia and cellular degradation. Conclusion This experimental study documents that during protracted pronounced hemorrhagic shock, cerebral energy metabolism was severely compromised and exhibited a biochemical pattern typical of ischemia and cellular degradation. After retransfusion this pattern continued. From intravenous microdialysis in the sagittal sinus, it is possible to achieve semiquantitative information of the intracerebral redox state. Accordingly, it might be possible to monitor the global cerebral energy state continuously with a strictly extracerebral technique. This technique might be valuable in various severe conditions during critical care when cerebral energy metabolism may be compromised; for example, resuscitation after cardiac standstill, open heart surgery, multitrauma and so forth. Interestingly the study also showed that after reinfusion of blood other parts of the body recovered, evaluated by microdialysis, but the brain showed signs of damage, making the brain the limiting organ in hemorrhagic shock. Introduction Measurement of intracranial pressure (ICP) and arterial blood pressure is used to derive cerebral perfusion pressure (CPP) and to guide targeted therapy of severe traumatic brain injury (TBI) necessitating ICU admission. In this review we discuss the evidence for ICP monitoring, CPP calculation, and ICP/CPP-guided therapy after severe TBI. Despite its widespread use, there is currently no class I evidence that ICP/CPP-guided therapy improves outcomes. Similarly, no class I evidence can currently advise the ideal CPP. y Methods A review of current literature with special focus on autoregulation (PRx)-guided CPP treatment in TBI patients.i Results Optimal CPP is probably patient, time, and pathology specifi c and related to cerebral autoregulation status. P453 P453 Amantadine sulfate treatment in cases with brain injury in the ICU: a retrospective clinical trial S Goksu Tomruk, N Bakan, G Karaören, A Salvarcý Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P453 (doi: 10.1186/cc14533) P452 y Results The patients’ diagnoses included two post-CPR, fi ve intracranial and one subdural hematoma, one CVA, one postoperative aneurysm, one subarachnoid hemorrhage and one brain contusion. Table  1 (overleaf) presents the fi ndings. The AS therapy was initiated between days 3 and 33 of admission in all patients other than Patients 2 and 8. A dramatic improvement was observed in a patient with both GCS and JFK- CRS score of 5 when AS therapy was initiated in month 5 and the patient was discharged for home care. In Patient 9, AS therapy was withdrawn on day 5 due to persistent thrombocytopenia (TP) despite exclusion of other reasons; subsequently, improvement was observed in TP. The complications were relatively less severe with average acceptability. Amantadine sulfate treatment in cases with brain injury in the ICU: a retrospective clinical trial S Goksu Tomruk, N Bakan, G Karaören, A Salvarcý Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P453 (doi: 10.1186/cc14533) Introduction Improvement of recovery is a challenging process in cases with varying degrees of severe brain injury (BI) requiring intensive care. Amantadine sulfate (AS) is recommended for use in cases with brain injury. The Coma Remission Scale (JFK-CRS) consists of auditory–visual– motor–mouth–tongue functions, communication and awareness scales; provides a score between 0 and 23; and shows numeric recovery from coma. The aim of this study was to evaluate outcomes and eff ects of AS used for neurorecovery on the Glasgow Coma Scale (GCS) and JFK-CRS in our ICUs during the last 5 months. Conclusion The management of TBI is likely to become increasingly based on a more comprehensive monitoring and management approach rather than relying on absolute numbers of ICP and CPP in isolation. This will allow individual optimization of perfusion and therefore of oxygen and energy substrate delivery. We await further robust, high-quality evidence to support the benefi ts of using more sophisticated monitoring tools like the autoregulation- guided CPP concept during the ICU management of TBI. For the near future, more important is a greater understanding of the underlying pathophysiology. Methods After approval of the Ethics Committee, we recruited 12 patients with brain injury resulted from trauma or hemorrhage who had initial GCS of ≤9 and received AS (500 mg, twice daily over 10 days) during the recovery period. In all subjects, age, gender, diagnosis, initial APACHE II score, time of initiation of AS therapy, JFK-CRS and GCS scores, aspartate aminotransferase, alanine aminotransferase, BUN, creatinine, platelet count, electrocardiography fi ndings, electrolyte values and arterial blood gas values on days 1, 6, 10 and 14 were recorded.i p p y References References 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2. Jaeger M, et al. Crit Care Med. 2010;38:1343-7. 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2. Jaeger M, et al. Crit Care Med. 2010;38:1343-7. 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2 Jaeger M et al Crit Care Med 2010;38:1343-7 Technique for continuous bedside monitoring of the global cerebral energy state gy R Jakobsen1, A Granfeldt2, TH Nielsen1, P Toft1, CH Nordström1 1Odense University Hospital, Odense, Denmark; 2Regional Hospital of Randers, Denmark Critical Care 2015, 19(Suppl 1):P452 (doi: 10.1186/cc14532) Introduction In the present experimental study we explore whether cerebral venous lactate/pyruvate ratio (LP ratio) measured by intra- vascular microdialysis during induced hemorrhagic shock may be used as a surrogate marker for compromised cerebral oxidative metabolism. Methods Six female pigs were anesthetized and vital parameters was recorded. Microdialysis catheters were placed in cerebral hemisphere parenchyma, the superior sagittal sinus and femoral artery. Brain tissue oxygenation (PbtO2) and intracranial pressure (ICP) was recorded. Hemorrhagic shock was achieved by bleeding the animals to a mean arterial pressure (MAP) of approximately 35 mmHg. Animals were kept at a MAP of about 30 to 40 mmHg for 90 minutes. The animals were resuscitated with reinfusion of shed blood followed by 3 hours of observation. Conclusion We suggest that an AS dose of 1,000 mg/day (over 10 days) seems to improve neurorecovery in BI patients with good tolerability. Prospective controlled studies with large, homogeneous BI populations will better defi ne the role of AS for recovery and complications. P454 References The fact that optimal CPP and autoregulation status varies between individual patients and over time makes it an attractive bedside tool to serve as a (simplifi ed) model to investigate the use of autoregulation (PRx) status to fi ne tune or feedback clinical treatments in individual sedated TBI patients (optimal CPP concept) [1]. Evidence is emerging for the role of other monitors (representing (local) metabolism, oxygen supply/use, perfusion, neuronal functioning) that enable the intensivist to employ an individualized multimodality monitoring approach in TBI [2]. P453 Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) P454 Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Results In the cerebral hemisphere, hemorrhagic shock caused a marked increase in the LP ratio, while a signifi cant but minor increase was observed in the sagittal sinus. The LP ratio increased and continued doing so to a very high level. In the femoral artery, the shock period was associated with a slight increase of the LP ratio. The increase in the LP ratio in the sagittal sinus was markedly and signifi cantly higher than in the arterial blood. Further, the dynamic changes in the LP ratio in the sagittal sinus followed that of the parenchyma, not the arterial blood. After infusion of blood ICP increased, cerebral perfusion pressure and M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Introduction Traumatic brain injury (TBI) prognostication is a developing fi eld striving to identify indicators, notably neurochemical S159 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P453). Findings of 12 patients receiving amantadine sulfate Table 1 (abstract P453). P454 The main purpose of this study was to determine the impact of CB2R inhibition on leukocyte activation within the microcirculation following endotoxin challenge in an experimental stroke model. Methods This was a prospective, randomized animal study. Five experimental groups (male C57BL/6 mice, age: 6 to 8  weeks) were subjected to the following treatments: control; endotoxemia (LPS 5 mg/kg, i.v.); transient cerebral hypoxia–ischemia (HI) + endotoxemia; HI + endotoxemia + CB2R antagonist (AM630 2.5 mg/kg, i.v.). HI was induced by unilateral carotid artery occlusion, followed by 50-minute exposure to a low oxygen atmosphere (8% O2). The CB2R antagonist was given 15 minutes prior to LPS administration. Intravital microscopy was carried out 2  hours after LPS administration. Brains were then extracted and stained with tetrazolium chloride to calculate the infarct volume. The primary outcome measurement in this study regarding the immune response after stroke was the quantifi cation of leukocyte adhesion following endotoxin challenge in submucosal venules of the gut – an important organ in the development of multiorgan failure in endotoxemia and sepsis. Methods The MEDLINE, Embase, The Cochrane Library and BIOSIS electronic databases, conference abstracts and existing narrative reviews were searched from their inception to July 2013. Cohort studies including patients with moderate or severe TBI having evaluated the prognostic value of UCHL-1 according to mortality or the Glasgow Outcome Scale (GOS) were considered. Data concerning patients, outcomes, study methods, and laboratory methods were abstracted. Pooled results were planned to be presented using mean diff erences and analyzed using random eff ect models, as well as sensitivity analyses to explain potential heterogeneity.i Results Our search strategy yielded 2,257 articles, of which fi ve studies corresponded to our inclusion criteria (n  = 730). All studies were performed by the same group of researchers. Five studies reported mortality (n = 515), two studies reported GOS (n = 58). Results from all included studies observed that UCHL-1 was a signifi cant predictor of mortality. However, only two studies represented a unique study population, thus precluding a meta-analysis. Results Compared with endotoxemic animals without CNS injury, mice subjected to HI displayed reduced leukocyte activation in intestinal submucosal venules indicative of CIDS. Administration of the CB2R antagonist in animals with CIDS challenged with endotoxin restored peripheral leukocyte recruitment without a detrimental impact on infarct size. P455 Cannabinoid 2 receptor antagonism reverses central nervous system injury-induced immune defi ciency syndrome IB Burkovskiy, J Zhou, G Robertson, C Lehmann Dalhousie University, Halifax, NS, Canada Critical Care 2015, 19(Suppl 1):P455 (doi: 10.1186/cc14535) P454 Findings of 12 patients receiving amantadine sulfate Initiation of Patient number Sex/age (years) APACHE II GCS A/B/C AS therapy (day) CRS score D/E/F/G Complications Outcome 1 M/14 24 3/5/7 6 0/4/5/6 H, J Still in ICU 2 F/78 33 8/8/10 84 0/0/0/0 K Ex 3 F/75 35 5/6/4 4 1/1/0/Ex K, L Ex 4 M/24 29 3/3/3 6 0/0/Ex K, L Ex 5 M/27 27 4/4/15 6 1/13/18/22 L, M, J Discharged 6 F/70 45 3/3/3 20 0/0/0/0 – Ex 7 F/72 22 5/5/12 3 4/7/11/17 K, L, I Discharged 8 F/53 27 3/5/10 150 5/7/10/14 K Discharged 9 F/38 34 4/4/4 33 0/0/1/1 H, J Still in ICU 10 M/50 26 4/6/13 8 0/7/18/19 L, J Discharged 11 M/63 24 8/10/12 14 6/8/8/12 – Discharged 12 M/50 31 4/6/11 6 2/5/7/9 L, J Still in ICU F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, convulsion. F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, l i F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, convulsion. biomarkers, of long-term outcomes. Among these, ubiquitin carboxy- terminal hydrolase L1 (UCH-L1) is currently being investigated to defi ne its potential prognostic value. The objective of this systematic review is to determine the ability of UCH-L1 to predict prognosis following a moderate or severe TBI. CIDS. P454 Conclusion In this systematic review, we observed that all published studies on UCHL-1 were conducted by the same group of investigators and presented results from an intersecting cohort of patients. Due to the paucity of data, we could not perform a pooled analysis and conclude on the association of this biomarker with long-term prognosis. Assays using UCHL-1 were only recently developed and further studies done by diff erent research teams will be needed to determine the reproducibility and validity of UCH-L1 as a potential prognostic tool. Conclusion CB2R-related modulation of leukocyte activation is involved in the impaired immune response following CNS injury. Future studies will explore the CB2R pathway in order to develop novel therapies to improve the immune response in CIDS. P456 Implementation process of a large multicenter study in trauma AF Turgeon, TBI-Prognosis Team in collaboration with the CCCTG CHU de Québec, QC, Canada Critical Care 2015, 19(Suppl 1):P456 (doi: 10.1186/cc14536) Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Results Overall, 33.4  weeks (95% CI  = 24.8 to 42.1) were required on average from the start-up package mailing to centers and the start of the screening process. Data sharing and fi nancial agreement were mainly responsible for this delay with an average of 28.6 weeks (95% CI = 20.4 to 36.7) needed to complete the agreement with the coordinating center. REB approval was obtained on average 17.5 weeks (95% CI = 13.8 to 21.2) from the shipping of the study package to the participating centers. Eighty percent of the REBs had members with prior experience in multicenter clinical research, and more than half with specifi c clinical expertise in critical care medicine or neurology/ neurosurgery. A standardized electronic REB submission process was used in most REBs (60%). All centers had experience in implementing multicenter clinical studies. PIs had experience conducting from zero to 40 prior clinical studies and from 3 to 24  years of research experience with protected research time ranging from 5 to 75%. RCs had managed from zero to more than 50 clinical studies. Most centers (87%) organized specifi c presentation of the study to the critical care medical staff (93%), while some (60%) presented the study to other medical teams. Agreements from other departments such as radiology (87% of centers), electrophysiology (80%) and clinical imaging (73%) were requested in most centers. p L Tsentsiper, E Kondratyeva, S Kondratyev, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P458 (doi: 10.1186/cc14538) p L Tsentsiper, E Kondratyeva, S Kondratyev, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P458 (doi: 10.1186/cc14538) Introduction Currently, the most common way to predict the outcome of acute brain damage is to study the level of protein S-100 in the serum. This method lacks specifi city as the concentration of protein S-100 signifi cantly increases with age, more for men than women, and there are no data on prognostically signifi cant changes in the level of S-100 after removal of the tumor and cerebral hemorrhages. Endothelins, vasopressin, some cytokines, excess sodium or calcium in serum, activation of the sympathoadrenal system, and tachycardia are the stimulants of brain natriuretic peptide production. Levels of N-terminal pro-brain natriuretic peptide in brain injury patients The rise of the natriuretic peptide level in cases of acute brain damage has a functionally adaptive nature, based on vasodilation, diuretic action peptide and ability to reduce sympathoadrenal system activity. Thus, we can suppose that the more severe the damage, the higher the stimulation of natriuretic peptide. In this study we investigate the level of N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with severe brain damage and fi nd correlation between the level of peptide and outcome. Endothelins, vasopressin, some cytokines, excess sodium or calcium in serum, activation of the sympathoadrenal system, and tachycardia are the stimulants of brain natriuretic peptide production. The rise of the natriuretic peptide level in cases of acute brain damage has a functionally adaptive nature, based on vasodilation, diuretic action peptide and ability to reduce sympathoadrenal system activity. Thus, we can suppose that the more severe the damage, the higher the stimulation of natriuretic peptide. In this study we investigate the level of N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with severe brain damage and fi nd correlation between the level of peptide and outcome. Conclusion The implementation of a large Canadian multicenter and multifaceted clinical study in the trauma population involved a signifi cant amount of time and energy from both the coordinating and the participating sites. The variable experience of participating sites and teams, as well as the involvement of diff erent medical disciplines, may have had an impact on study implementation time. Delays for REB approval but also for data sharing and fi nancial agreement must be taken into consideration in the timeline for implementing large multicenter clinical studies in trauma. Methods We studied 110 patients having brain injuries of various origins. All patients were divided into four groups. All patients were 20 to 72 years old, 58 men and 52 women. Group 1 (n = 17) – acute TBI, group 2 (n = 29) – patients operated on for the brain tumor, group 3 (n = 36) – hemorrhagic stroke, group 4 (n = 28) – vegetative state. We measured the level of brain natriuretic peptide on days 1 to 3, and then every 7 days for 21 days. Results All patients with severe acute brain damage (groups 1, 2, 3) had a level of NT-proBNP higher than normal (normal 0 to 125 pg/ml). Signifi cant diff erence in values between the groups was not observed. P458 the ongoing Pan-Canadian TBI-Prognosis Study. Descriptive statistics were used. the ongoing Pan-Canadian TBI-Prognosis Study. Descriptive statistics were used. P457 Optic nerve sheath diameter by bedside ultrasound is a reliable screening test for cerebral edema in the comatose ICU patient A Mohamed, N Alharbi, N Salahuddin, I Hussain, O Solaiman King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P457 (doi: 10.1186/cc14537) Conclusion In cases of acute severe brain damage the level of NT- proBNP signifi cantly increased. Correlation between the level of NT-proBNP and etiology of acute brain damage was not observed. If the level of NT-proBNP is above 700 pg/ml and/or in the absence of its reduction to normal, then poor outcome of the disease – severe disability or death  – can be predicted. Level of NT-proBNP cannot be used as an indicator for the severity of the status for patients in a vegetative state in contrast to patients with acute brain damage. Introduction ICU patients may remain comatose after resolution of critical illness. Frequently this is due to delayed sedative clearance but may also result from increases in intracranial pressure (ICP) and cerebral edema. We proposed that measurement of the optic nerve sheath diameter (ONSD) is a rapid, bedside screening test that can be used to quickly identify patients with cerebral edema and increased ICP. y y Methods This was a prospective, observational study carried on consecutive patients admitted to a multidisciplinary medical and surgical ICU. Stable patients with unexplained coma and scheduled for brain imaging were included. Patients with obvious ocular trauma or on sedative, narcotic infusions were excluded. ONSD was measured using a 7.5 to 10 MHz linear array ultrasound transducer probe placed on the closed eye in the transverse axis. The ONSD was measured at a predefi ned point 3 mm posterior to the globe. Both eyes were measured and the mean value used. The study protocol was approved by the Hospital Research Ethics Committee (RAC Prop No. 2141 103). Statistical analysis was carried out using SPSS version 20.0. Cannabinoid 2 receptor antagonism reverses central nervous system injury-induced immune defi ciency syndrome IB Burkovskiy, J Zhou, G Robertson, C Lehmann Dalhousie University, Halifax, NS, Canada Critical Care 2015, 19(Suppl 1):P455 (doi: 10.1186/cc14535) Introduction Our purpose was to evaluate the time from shipping of the study start-up package to study screening, as well as conditions that may impact this process, in the context of a large-scale multifaceted and multicenter clinical study in trauma. Introduction Our purpose was to evaluate the time from shipping of the study start-up package to study screening, as well as conditions that may impact this process, in the context of a large-scale multifaceted and multicenter clinical study in trauma. Introduction Central nervous system (CNS) injury, such as stroke, is known to increase susceptibility to infections that adversely aff ect clinical outcome. This impaired immune response to infection is termed CNS injury-induced immune defi ciency syndrome (CIDS). Activation of the cannabinoid 2 receptor (CB2R) suppresses immune function suggesting that antagonism of this receptor may overcome Methods We designed a survey questionnaire based on four domains: REB characteristics and process, centers’ characteristics, experience of the study and clinical teams, and center-specifi c implementation approaches. The questionnaire was self-administered to all lead research coordinators of the 17 level 1 participating trauma centers in S160 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Level of NT-proBNP above 700  pg/ml and/or the absence of its reduction to normal dynamic indicators was marked by an unfavorable outcome of the disease – severe disability (n = 25) or death (n = 18). For patients from group 4 regardless of their age, sex, severity of condition and treatment results in a level of NT-proBNP below 250 pg/ml. P459 C b Cerebral oximetry monitoring in pediatric seizure patients in the emergency department T Abramo1, B Schnieder1, E Storm1, N Hobart-Porter1, Z Hu1, N Todd1, L Crawley1, M Meredith2, S Godbold1 1University of Arkansas School of Medicine, Little Rock, AR, USA; 2University of Tennessee School of Medicine, Memphis, TN, USA Critical Care 2015, 19(Suppl 1):P459 (doi: 10.1186/cc14539) Introduction During ictal/post-ictal events, altered cerebral physiology occurs: increased neuronal activity causes signifi cant increase in cerebral metabolism with changes in ipsilateral cerebral blood fl ow. Standard PED seizure monitoring is by O2SAT and ETCO2 which yield no direct data about regional cerebral oxygenation/physiology (rSO2). Signifi cant abnormal hemispheric cerebral physiology resulting in neurological injury can occur without knowing because the current monitoring system could not detect the abnormal hemispheric abnormality. Cerebral oximetry can provide a rapid, non-invasive detection of each hemisphere’s cerebral physiologic changes during ictal/post-ictal phases. The aim was to study left and right rSO2 values in patients in the pre and post seizure periods and in nonseizing controls. Methods An observational study of seizing and nonseizing patient’s left and right rSO2 readings compared with nonseizure patients. Results ONSD was measured in 43 patients; mean age was 62  ± 19.2 years, 48% (n = 20) were female. Admitting diagnosis was sepsis in 24% (n = 10), intracranial vascular event in 21% (n = 9), cardiac arrest in 12% (n = 5), hepatic encephalopathy in 7% (n = 3), malignancy with metastases in 7% (n = 3) and other causes in 28% (n = 12). The ONSD measured correlated highly between eyes, Spearman’s ρ  = 0.799, P ≤0.001. The area under the ROC curve for detecting cerebral edema was 0.812 (95% CI  = 0.667 to 0.957). Using a 0.58  cm cutoff ONSD diameter, the sensitivity was 86%, specifi city 74%, negative predictive value 96% and the positive likelihood ratio = 3.3. Conclusion This study suggests that bedside measurement of ONSD by ultrasound performs well as an initial screening test for cerebral edema. The identifi ed cutoff value of 0.58 cm can be used to detect cerebral edema with reasonable accuracy. S161 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P459). Results No diff erence for ictal left and right rSO2 readings across ages. See Figure 1. of our institute. In post-anoxic comatose patients we recorded EEG during hypothermia to assure burst suppression. P460 Neurophysiological tests in the neuro ICU Neurophysiological tests in the neuro ICU A Marudi1, Y Valzani2, F Valzania1, M Carpentiero1, C Parenti1, S Scacchetti1, S Baroni1, E Bertellini1 1Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy; 2University of Modena and Reggio Emilia, Modena, Italy Critical Care 2015, 19(Suppl 1):P460 (doi: 10.1186/cc14540) P459 C b In post-traumatic brain-injured patients with a persistent comatose state we use EEG to detect nonconvulsive states which potentially can increase secondary brain injury if untreated. In malignant epilepticus status we use EEG to monitor the eff ect of therapy and to modify it. In patients who present profound weakness of legs and hands we performed EMG to distinguish primary peripheral myopolyneuropathy (Guillian Barrè, miastenia gravis) from secondary illness acquired in the ICU (critical polyneuropathy, critical myopathy) [3]. Conclusion We have demonstrated abnormal hemispheric cerebral physiology during focal or generalized ictal activity. In patients with generalized seizures, the left and right rSO2 values were signifi cantly decreased. In patients with focal seizures, the ipsilateral rSO2 values were signifi cantly diff erent from the contralateral rSO2 readings and correlated to the hemisphere experiencing the focal seizure. In certain patients, during the ictal phase their rSO2 readings rose and stayed or rose then dropped. Overall, cerebral oximetry has shown great monitoring potential for actively seizing patients in the emergency department. Conclusion NPT can improve management of patients admitted to the neuro ICU. The data provided can modify therapeutic strategies and improve outcome in these settings of patients. R f References 1. Guerit JM. Curr Opin Crit Care. 2010;16:98-104. 2. Bednarik J, et al. Intensive Care Med. 2003;29:1505-14. 3. Pohhmann-Eden B, et al. Intensive Care Med. 1997;23:301-8. References 1. Guerit JM. Curr Opin Crit Care. 2010;16:98-104. 2. Bednarik J, et al. Intensive Care Med. 2003;29:1505-14. 3. Pohhmann-Eden B, et al. Intensive Care Med. 1997;23:301-8. Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery Patients were randomized into two groups: MAP group, in which MAP S162 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 was maintained above 70 mmHg or within 20% from baseline (n = 78); or CPP group, in which CPP was maintained above 60 mmHg or within 20% from baseline (n = 54). ICP, MAP and CPP were assessed every hour of anesthesia. Time of recovery of consciousness, incidence of POD and length of stay in the ICU and in the hospital were also evaluated. ischemia-operated animals with normothermia (normothermia  + ischemia group); sham-operated animals with hyperthermia (hyperthermia  + sham group); and ischemia-operated animals with hyperthermia (hyperthermia  + ischemia group). Hyperthermia (39.5 ± 0.2°C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30  minutes before and during ischemia–reperfusion.i y Results Initial ICP was 14 ± 3 mmHg in the MAP group and 15 ± 2 mmHg in the CPP group. During the anesthesia it was stable without any signifi cant change. Decreasing of MAP after induction of anesthesia was similar in two groups and it was stable during the anesthesia. The frequency of use of vasopressors and infusion rate was higher in the CPP group. Time of recovery of consciousness in the MAP group was higher (28 ± 7 minutes vs. 18 ± 5 minutes (P <0.05)). The incidence of postoperative delirium was higher in the MAP group (18% vs. 11% in the CPP group (P <0.05)). There were no signifi cant diff erences between two groups in other complications. Total length of stay in the ICU and in the hospital was higher in the MAP group (6 ± 2 days vs. 4 ± 2 (P <0.05) and 15 ± 3 days vs. 12 ± 2 in the N group (P <0.05)). Results In the normothermia + ischemia group, a signifi cant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5  days after ischemia–reperfusion. In the hyperthermia  + ischemia group, neuronal death in the SP of the CA1 occurred at 1  day post ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2  days post ischemia. In addition, we examined activation of astrocytes and microglia using immunohistochemistry for anti-glial fi brillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). Hydroquinone shows neuroprotective potential in an experimental ischemic stroke model via attenuation of blood–brain barrier disruption CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P464 (doi: 10.1186/cc14544) JH Cho, CW Park, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P462 (doi: 10.1186/cc14542) Introduction Therapeutic exercise is an integral component of rehabilitation for patients with stroke. Despite the high prevalence of cerebral ischemia in the older population, the mechanisms linking restorative exercise to memory recovery from ischemic stroke have not been completely understood in aged animals. In this study, we investigated eff ects of long-term exercise on neuronal death and memory recovery in the aged gerbil hippocampus after transient cerebral ischemia. We also investigated changes in gliosis, ischemia- induced myelin repair, microvessels, neurogenesis, and growth factor immunoreactivity in the hippocampus to study possible mechanisms of restorative exercise in memory recovery. Introduction Hydroquinone (HQ), a major benzene metabolite, occurs naturally in various plants and food, and is also manufactured for commercial use. Although many studies have demonstrated the various biological eff ects of HQ, the neuroprotective eff ects of HQ following ischemic stroke have not been investigated. Therefore, in this study, we fi rst examined the neuroprotective eff ects of HQ against ischemic damage in a focal cerebral ischemia rat model. Methods It was proven that pre and post treatment with 100  mg/ kg HQ protects from ischemia-induced cerebral damage, which was confi rmed by evaluation of neurological defi cit, positron-emission tomography and 2,3,5-triphenyltetrazoliumchloride staining. Methods The gerbils were divided into four groups (n = 12 in each group): the sham-operated group (Sham), 4-week sedentary group following ischemia (SD4), 1-week treadmill group following ischemia (TR1) and 4-week treadmill group following ischemia (TR4). Treadmill exercise was stared at 5 days after ischemia/reperfusion (I/R) and lasted for 1 or 4 weeks, and the animals were sacrifi ced 31 days after ischemia. Results In this study, 4 weeks of treadmill exercise facilitated memory recovery despite neuronal damage in the CA1 region after I/R. On the other hand, the long-term treadmill exercise alleviated the increased gliosis in the CA1 region, and increased the myelin repairing and microvessels in the CA1 region and DG, and enhanced the ischemia- induced cell proliferation, neuroblast diff erentiation, neuronal maturation of the newly generated cells, and BDNF expression in the ischemic DG of the aged gerbil. Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery Introduction Neurophysiological tests (NPTs) are important prog- nostic and diagnostic tools for patients admitted to the modern neuro ICU (NICU) [1]. Electroencephalography (EEG), somasensorial evoked potentials (SSEP), auditory brainstem response (ABR) and electromyography (EMG) complete clinical examination and radio- logical fi ndings in patients suff ering from post-traumatic brain injury, post-anoxic brain injury, refractory male epiletticus status, and neuromuscular illness. We evaluate the spread of NPTs in our NICU. Methods We collected data from patients admitted to our NICU from January 2014 to November 2014. We recorded the admission diagnosis and the NPT applied. y j I Zabolotskikh, N Trembach I Zabolotskikh, N Trembach Kuban State Medical University, Krasnodar, Russia Kuban State Medical University, Krasnodar, Russia Critical Care 2015, 19(Suppl 1):P461 (doi: 10.1186/cc14541) y Critical Care 2015, 19(Suppl 1):P461 (doi: 10.1186/cc14541) Introduction Increased intracranial pressure (ICP) adversely aff ects anesthesia due to a disturbed cerebral blood fl ow. In older patients this disturbance may increase the incidence of postoperative delirium (POD) and may lead to a poor outcome [1]. The standard hemodynamic protocol involves maintaining the mean arterial blood pressure (MAP), but in patients with intracranial hypertension it may not be enough to maintain adequate cerebral perfusion. The purpose of this study was to evaluate the protocol of maintaining cerebral perfusion pressure (CPP) in the prevention of postoperative delirium in older patients in abdominal surgery. Methods We collected data from patients admitted to our NICU from January 2014 to November 2014. We recorded the admission diagnosis and the NPT applied. Results From January 2014 to November 2014 we performed 521 EEG, 45 SSEP/ABR and 10 EMG. In post-anoxic and post-traumatic brain- injured comatose patients we performed EEG, SSEP and ABR 24 to 48 hours after the admission to predict later prognosis and expected neurological defi cit [2]. In the presence of a benign pattern no further evaluation was performed; in the presence of a malignant pattern the NPTs were repeated every 48 to 72 hours according to the protocol Methods A total of 132 ASA 3 patients, undergoing major abdominal surgery (duration 5.2 (4.3 to 6.5) hours) with ICP >12 mmHg evaluated by a venous ophthalmodynamometry [2], were included in our research. Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia  + ischemia group than those in the normothermia  + ischemia group.i y g p Conclusion A goal-directed hemodynamic protocol of maintaining CPP can decrease the incidence of POD in older patients with intracranial hypertension after major abdominal surgery compared with a protocol of maintaining MAP. Conclusion Based on our fi ndings, we suggest that experimentally hyperthermic precondition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus. Hydroquinone shows neuroprotective potential in an experimental ischemic stroke model via attenuation of blood–brain barrier disruption y y Results In addition, pre and post treatment with 100  mg/kg HQ signifi cantly attenuated ischemia-induced Evans blue dye extra- vasation, and signifi cantly increased the immunoreactivities and protein levels of SMI-71 and glucose transporter-1 (GLUT-1), which were well known as useful makers of endothelial cells, in ischemic cortex compared with a vehicle-treated group.l Conclusion Briefl y, these results indicate that pre and post treatment with HQ can protect from ischemic damage induced by transient focal cerebral ischemia, and the neuroprotective eff ects of HQ may be closely associated with the prevention of BBB disruption via increase of SMI-71 and GLUT-1 expressions. P462 P464 Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P464 (doi: 10.1186/cc14544) References 1. Zabolotskikh I, et al. Eur J Anesth. 2013;30:114-5. 1. Zabolotskikh I, et al. Eur J Anesth. 2013;30:114-5. 2. Firsching R, et al. J Neurosurg. 2011;115:371-4. 2. Firsching R, et al. J Neurosurg. 2011;115:371-4. P463 Impact of hyperthermia before and during ischemia reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P463 (doi: 10.1186/cc14543) Conclusion These results suggest that, in the aged gerbil, long-term treadmill exercise after ischemic stroke could restore the impaired short-term memory function through the cumulative eff ects of multiple neurorestorative processes. Prognostic value of blood lactate and glucose levels after aneurysmal subarachnoid hemorrhage y g S Dijkland1, K Van Donkelaar2, W Van den Bergh2, J Bakker1, D Dippel1, M Nijsten2, M Van der Jagt1 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) j g 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) Results A total of 307 patients were included in the validation cohort. The observed 60-day case fatality rate was 30.6%. Discrimination was good, and was considerably better for the model with WFNS grade at treatment decision (AUC = 0.89) compared with the model with WFNS grade on admission (AUC  = 0.82). Calibration was poor, with mean predicted probabilities of 17.0% for the model with WFNS grade on admission and 17.7% for the model with WFNS grade at the time of treatment decision. Introduction In critically ill patients, blood lactate on admission is associated with outcome, but in patients with aneurysmal sub- arachnoid hemorrhage (SAH) this has not been investigated. We studied the association of early circulating lactate and glucose with unfavorable disease course. The prognostic role of both lactate and glucose was studied, hypothesizing that both may be increased due to sympathetic activation after SAH [1]. y p Methods In this retrospective cohort study we included consecutive patients with aneurysmal SAH admitted to the ICUs of two university hospitals in the Netherlands between November 2006 and December 2011. Exclusion criteria were: nonaneurysmal SAH, ICU admission >24 hours after ictus, death ≤48 hours after admission and no lactate measurement <24 hours after admission. Maximum blood lactate and glucose levels within the fi rst 24 hours after SAH were used for analyses. The outcomes were DCI, defi ned as a new hypodensity on brain CT due to DCI, and poor outcome, defi ned as a modifi ed Rankin Scale of 4, 5 or death 3 to 6 months after the ictus. We performed proportional hazard analyses to assess the associations of lactate and glucose with DCI, and logistic regression was used to assess the associations with poor outcome. Association between high arterial oxygen survival after intracerebral hemorrhage Introduction Hyperthermia can exacerbate the brain damage pro- duced by ischemia. In the present study, we investigated eff ects of hyperthermia before and during ischemia–reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofl uorescence staining. Methods The animals were randomly assigned to four groups: sham- operated animals with normothermia (normothermia + sham group); M Fallenius1, R Raj1, M Reinikainen2, S Bendel3, MB Skrifvars1 1Helsinki University Hospital, Helsinki, Finland; 2North Karelia Central Hospital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2015, 19(Suppl 1):P465 (doi: 10.1186/cc14545) M Fallenius1, R Raj1, M Reinikainen2, S Bendel3, MB Skrifvars1 1Helsinki University Hospital, Helsinki, Finland; 2North Karelia Central Hospital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2015, 19(Suppl 1):P465 (doi: 10.1186/cc14545) Introduction Liberal use of oxygen after brain insults remains controversial [1,2]. We studied whether high arterial oxygen tension S163 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Maximum lactate in the acute phase after aneurysmal SAH is associated with both DCI-related infarction and poor outcome. Once glucose was considered, early lactate remained independently associated with poor outcome, while glucose, instead of lactate, was associated with DCI. These routinely available laboratory measurements may improve identifi cation of patients at risk for complications or poor outcome after SAH. Confi rmation of the pathophysiological signifi cance of lactate and glucose in prospective research is warranted. Reference (PaO2) is associated with decreased long-term survival in patients with spontaneous intracerebral hemorrhage (ICH) treated in the ICU. (PaO2) is associated with decreased long-term survival in patients with spontaneous intracerebral hemorrhage (ICH) treated in the ICU. Methods Data on primary admissions for adult patients (>18  years) treated for ICH in Finnish ICUs between 2003 and 2012 were collected from a nationwide ICU database. Patients were divided into three groups according to the PaO2 value associated with the lowest measured PaO2/ FIO2 ratio during the fi rst 24 hours after ICU admission. High arterial oxygen tension was defi ned as PaO2 >19.9 kPa; intermediate as PaO2 13 to 19.9 kPa; and low as PaO2 <13 kPa. The primary outcome was 6-month mortality. 1. Kaukonen K, et al. Crit Care Med. 2014;42:1379-85. Association between high arterial oxygen survival after intracerebral hemorrhage Results Of the 3,033 patients, 63% (n = 1,923) had low PaO2, 29% (n = 892) intermediate PaO2, and 7% (n = 218) high PaO2. Forty-nine percent of the patients died during the 6-month follow-up. Of these, 75% died before discharge from hospital. Univariate analysis showed that 6-month mortality was higher in the high PaO2 group (61%) compared with the intermediate and low PaO2 groups (52% and 46% respectively, P  <0.001). Multivariate analysis, however, showed no statistically signifi cant correlation between high PaO2 and mortality (with the low PaO2 group as the reference category, odds ratio for death (OR) for high PaO2 = 1.10, 95% confi dence interval (CI) = 0.76 to 1.58 and for intermediate PaO2 = 0.96, 95% CI = 0.78 to 1.17). P467 Prediction of 60-day case fatality after aneurysmal subarachnoid hemorrhage: external validation of a prediction model S Dijkland, B Roozenbeek, P Brouwer, H Lingsma, D Dippel, L Vergouw, M Vergouwen, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P467 (doi: 10.1186/cc14547) Introduction Aneurysmal subarachnoid hemorrhage (SAH) is a devastating disease with substantial morbidity and mortality. Prognostic modeling is an important instrument to identify high- risk patients in both clinical practice and research settings. Recently, a prognostic model to predict 60-day case fatality after aneurysmal SAH was developed with data from the International Subarachnoid Aneurysm Trial (ISAT) [1]. Our aim was to externally validate this model in a retrospective cohort of consecutive SAH patients. 2 Conclusion High PaO2 is not predictive of 6-month mortality in patients treated for spontaneous ICH in the ICU. Therefore, targeting higher PaO2 values appears to be a safe approach in order to avoid hypoxemia. References 1. Rincon F, Kang J, Maltenfort M, et al. Association between hyperoxia and mortality after stroke. Crit Care Med. 2014;42:387-96. 1. Rincon F, Kang J, Maltenfort M, et al. Association between hyperoxia and mortality after stroke. Crit Care Med. 2014;42:387-96. p p Methods We included consecutive aneurysmal SAH patients admitted to one university hospital between October 2007 and October 2011. Exclusion criteria were: age <18  years, hospital admission >28  days after SAH, nonaneurysmal SAH, explicit objection by the patient to view the medical data and missing data on 60-day case fatality. The model’s predictors were age, maximum lumen size of the aneurysm, Fisher grade and World Federation of Neurological Surgeons (WFNS) grade. Two versions of the model were validated: one with WFNS grade scored on admission and the other with WFNS grade assessed at the time of treatment decision, as a proxy to WFNS grade at randomization used in the ISAT. The outcome was 60-day case fatality. Model performance was assessed by studying discrimination, expressed by the area under the receiver operating characteristic curve (AUC), and calibration. 2. de Jonge E, Peelen L, Keijzers PJ, et al. Association between administered oxygen, arterial partial oxygen pressure and mortality in mechanically ventilated intensive care unit patients. Crit Care. 2008;12:R156. P467 P467 Prediction of 60-day case fatality after aneurysmal subarachnoid hemorrhage: external validation of a prediction model S Dijkland, B Roozenbeek, P Brouwer, H Lingsma, D Dippel, L Vergouw, M Vergouwen, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P467 (doi: 10.1186/cc14547) Reference 1. Risselada R, et al. Eur J Epidemiol. 2010;25:261-6. P468 Prognostic value of blood lactate and glucose levels after aneurysmal subarachnoid hemorrhage Multivariable analyses were adjusted for established predictors for DCI and poor outcome.i Conclusion Our results indicate that the ISAT prediction model is generalizable, since the model showed adequate performance in an independent, unselected cohort of aneurysmal SAH patients. The model discriminated well between patients who died and those who survived the fi rst 60 days after SAH. Additionally, use of WFNS grade at the time of treatment decision of the ruptured aneurysm improved model performance. However, since predicted probabilities were lower than observed probabilities, the ISAT prediction model needs to be adapted before use in clinical practice. Cortical spreading depolarizations in patients with intracerebral hemorrhage: preliminary datal Results Two hundred and eighty-fi ve patients were included in the analyses. DCI occurred in 84 patients (29%) and 106 patients (39%) had poor outcome. Lactate was independently associated with DCI (adjusted HR = 1.16, 95% CI = 1.04 to 1.30) and poor outcome (adjusted OR = 1.53, 95% CI = 1.25 to 1.94). Maximum lactate and glucose were strongly related (Spearman’s ρ  = 0.55, P  <0.001). In multivariable analyses including both lactate and glucose as independent variables, only lactate was independently related to poor outcome (OR = 1.42, 95% CI = 1.11 to 1.81), and only glucose was independently associated with DCI (HR = 1.10, 95% CI = 1.02 to 1.19). AJ Schiefecker1, R Beer1, M Kofl er1, B Pfausler1, I Unterberger1, P Lackner1, G Broessner1, P Rhomberg1, F Sohm1, M Mulino1, C Thome1, M Fabricius2, E Schmutzhard1, R Helbok1 1Medical University of Innsbruck, Austria; 2Rigshospitalet, Copenhagen, Denmark Critical Care 2015, 19(Suppl 1):P468 (doi: 10.1186/cc14548) Introduction Perihematomal edema (PHE) expansion contributes to increased morbidity and mortality after spontaneous intracerebral S164 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 positivity on admission is an independent prognostic predictor in acute ischemic stroke. Reference Figure 1 (abstract P469). hemorrhage (ICH). Pathophysiology of PHE is incompletely understood. Recently, the role of cortical spreading depolarizations (CSDs) in secondary brain injury was established in subarachnoid and traumatic brain injury patients. However, the value of CSDs after ICH is not known. Methods Patients with ICH fulfi lling the inclusion criteria were prospectively enrolled in the observational COSBID study (Co-operative Study on Brain Injury Depolarisations). g.BSamp (g.tec, Austria) connected to PowerLab and LabChart software (Adinstruments) was used for electrocorticography (EcoG). Electrocardiogram patches at the patient’s shoulder and bed served as groundings, and a surface reference electrode was glued on the mastoid. The duration of EcoG depressions was defi ned as the time between depression onset and start of EcoG recovery in the integral of power calculations (0.5 to 45 Hz; 60 seconds time constant decay). Brain tissue oxygen tension (PbtO2), cerebral blood fl ow (CBF), cerebral metabolism and intracranial pressure were monitored in the PHE region. Data are presented as median and interquartile range. q g Results Eighteen patients with ICH (ICH volume: 54 (33 to 69)  ml) were analyzed. Hematoma evacuation was performed in 17 patients, one subject underwent craniectomy only. Troponin level as a predictor of prognosis in patients with acute ischemic stroke H Bayir1, R Dagli2, H Kaymaz2, I Yildiz1, H Kocoglu1 1Abant Izzet Baysal University, Medical School, Bolu, Turkey; 2Ahi Evran University Education and Research Hospital, Kirsehir, Turkey Critical Care 2015, 19(Suppl 1):P469 (doi: 10.1186/cc14549) H Bayir1, R Dagli2, H Kaymaz2, I Yildiz1, H Kocoglu1 1Abant Izzet Baysal University, Medical School, Bolu, Turkey; 2Ahi Evran University Education and Research Hospital, Kirsehir, Turkey Critical Care 2015, 19(Suppl 1):P469 (doi: 10.1186/cc14549) Methods The data from 1 million National Health Insurance bene fi ciaries were utilized. All adult benefi ciaries were followed from 1 January 2005 until 31  December 2012 to identify those who developed ischemic stroke. Meanwhile, each identifi ed patient with burn injury was matched with 100 unexposed patients based on the high-dimensional propensity score. Cox regression models were applied to compare the hazards of ischemic stroke in the matched cohorts. Introduction The aim of this study was to identify the association between troponin level and the outcome in patients with acute ischemic stroke. Introduction The aim of this study was to identify the association between troponin level and the outcome in patients with acute ischemic stroke. Methods We retrospectively investigated 152 patients admitted to our reanimation ICU for cerebrovascular accident between 1 January 2013 and 31 December 2013. Inclusion criteria were as follows: patients with acute ischemic stroke, measurement of serum troponin level and electrocardiography performed within 24  hours of admission. Not included were patients with intracerebral hemorrhage, no brain imaging or electrocardiography, previous myocardial infarction, stable or unstable angina pectoris before admission, previous coronary angioplasty or coronary bypass surgery. Figure 1 (abstract P470). Patient fl owchart of enrollment. g p y y yp g y Results Of 152 patients, 51 patients were excluded from the study because of the exclusion criteria. The serum troponin level was elevated in 81 patients. The patients were divided into two groups; patients in group 1 (n = 81) with serum troponin level >0.01, and those in group 2 (n = 20) with serum troponin level ≤0.01. For 1-month follow-up results of patients, death had occurred in 50.6% (n = 41) of patients in group 1 and in 25% (n = 5) of patients in group 2. There was a signifi cant positive correlation between the increase in troponin level and death within 1 month (r = 0.205; P = 0.040). P469 Introduction The results of studies attempting to assess the risks of ischemic stroke in patients with burn injury have been confl icting. We investigated the risks of ischemic stroke in hospitalized burn injury patients in Taiwan to evaluate whether the risk is higher compared with the general population.i Cortical spreading depolarizations in patients with intracerebral hemorrhage: preliminary datal Patients were 60 (55 to 67) years old and 38% female. Monitoring time per patient was 10 (6 to 14) days. A total of 129 CSDs with 16 (10 to 29) minutes of EcoG depression were observed. Eighty-four percent (n  = 15) of patients showed expansion of PHE by 25 (10 to 50) ml within 3 to 6 days after bleeding. Neuromonitoring probes were 35 (23 to 58) mm distant from the EcoG strip. CSDs occurred in 73% (n  = 11) of patients with PHE expansion. The interval between CSDs was 98  minutes (25 to 308). CSDs were associated with a signifi cant decrease of PbtO2 (–4 mmHg (–3; –7); duration 10 (5 to 23) minutes) in 68% (52/77), CBF changes in 95% (19/20) and metabolic derangement in 80% (80/100) of CSDs. PHE expansion was observed in all patients with spreading convulsions (n = 2) and patients with repetitive CSDs occurring as clusters (n = 3). positivity on admission is an independent prognostic predictor in acute ischemic stroke. positivity on admission is an independent prognostic predictor in acute ischemic stroke. Reference 1. Whiteley W, Chong WL, Sengupta A, Sandercock P. Blood markers for the prognosis of ischemic stroke: a systematic review. Stroke. 2009;40:e380-9. P470 Increased risk of ischemic stroke in patients with burn injury: a nationwide cohort study in Taiwan TY Hung1 YC Su2 Conclusion CSDs are common in ICH patients and associated with perihematomal PbtO2 decreases and metabolic derangement. Especially, clusters of CSDs might be associated with detrimental metabolic changes of the perihematomal brain tissue. TY Hung , YC Su 1Zhongxing Branch of Taipei City Hospital, Taipei, Taiwan; 2Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan Critical Care 2015, 19(Suppl 1):P470 (doi: 10.1186/cc14550) TY Hung , YC Su 1Zhongxing Branch of Taipei City Hospital, Taipei, Taiwan; 2Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan Critical Care 2015, 19(Suppl 1):P470 (doi: 10.1186/cc14550) Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patientsf R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil fi y Results Ninety-nine of 204 included patients developed ICU-AW. Patients with ICU-AW had higher APACHE IV and SOFA scores, a longer duration of mechanical ventilation, longer ICU stay, and died more often on the ICU compared with ICU patients without ICU-AW. Maximal levels of IL-1β, IL-6, IL-8, IL-10, TNFα, IFNγ, fractalkine and sICAM-1 were higher in patients who developed ICU-AW compared with patients who did not develop ICU-AW (univariable analysis). PC 1 to 4 derived from maximal levels explained >69% of the total variance in the data. Multivariable logistic regression models showed that PC 1 (mainly loaded by IL-6, IL-8 and IL-10) and PC 4 (mainly loaded by sP-selectin) were signifi cantly higher in patients with ICU-AW compared with patients without ICU-AW (OR of 1.27 (95% CI = 1.02 to 1.60) and 1.55 (1.06 to 2.27) respectively). Introduction Coronary artery bypass grafting (CABG) surgery usually improves myocardial contractility, reducing cardiovascular events. However, it is a high-risk procedure associated with signifi cant neurological complications, including stroke, delirium and cognitive impairment. The pathophysiology of these complications is not very well known, and may include low fl ow state after surgery, low cardiac output, embolism and reperfusion lesion. The aim of this study is to prospectively evaluate the cerebral hemodynamics through transcranial color and spectral Doppler sonography in high-risk patients undergoing cardiac surgery with a pump. y Methods This was a prospective, single-center study, performed at the Heart Institute from University of São Paulo. From May to November 2014 we included 35 patients in the study, aged older than 18 years old, submitted to CABG with a pump, with EuroSCORE higher than 6 or left ventricular ejection fraction lower than 40%. Transcranial color and spectral Doppler sonography was performed 48 hours before surgery (T0), 7 days (T1) and 6 months after surgery (T2). We used a probe of 2.5 to 2  MHz (Doppler Box DWL/Compumedics, Singen, Germany). All recordings were taken with the patient in a supine position. P471f Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patients R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P471 (doi: 10.1186/cc14551) P472 ncreased early systemic infl ammation in patients with ICU-acquired weakness k h d ll h k E Witteveen, L Wieske, C Verhamme, T Van der Poll, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P472 (doi: 10.1186/cc14552) Introduction Infl ammation may be important in the pathogenesis of ICU-acquired weakness (ICU-AW) since SIRS, sepsis and multiple organ failure are the main risk factors. Local infl ammation has been found in muscle and nerve tissue of patients with ICU-AW, but little is known about the association with systemic infl ammation. We hypothesized that systemic infl ammation is increased in patients who develop ICU- AW compared with patients who do not develop ICU-AW. Figure 2 (abstract P470). Burn injury patients have higher adjusted hazard ratio of ischemic stroke. Results A total of 743,237 patients were enrolled. After matching, 1,763 burn injury patients and 176,300 unexposed patients were selected. The adjusted hazard ratio of ischemic stroke was signifi cantly increased in burn injury patients (1.84; 95% CI, 1.43 to 2.36). Such phenomenon remained signifi cantly after 12 months (1.54; 95% CI, 1.11 to 2.13). See Figures 1 and 2. p p p Methods Newly admitted ICU patients ≥48  hours on mechanical ventilation were included. Daily plasma samples were collected from leftover plasma. Muscle strength was evaluated as soon as patients were awake and attentive. ICU-AW was defi ned by a mean Medical Research Council score <4. IL-1β, IL-6, IL-8, IL-10, IL-13, TNFα, IFNγ, fractalkine, GM-CSF, sICAM-1, sE-selectin and sP-selectin were measured in plasma samples of days 0, 2 and 4 using cytometric bead arrays and FACS. Diff erences of maximum levels between patients with and without ICU- AW were calculated using Mann–Whitney U tests. Principal component (PC) analysis was used to avoid multicollinearity and to reduce the set of mediators into a smaller set of PCs. To investigate whether diff erent infl ammatory profi les are associated with development of ICU-AW, we used multivariable logistic regression models of selected PCs, corrected for a priori selected variables, being age, gender, BMI, sepsis, SOFA score, APACHE IV score, immune insuffi ciency and corticosteroids. Conclusion The risk of ischemic stroke is higher in patients hospitalized with burn injury than in the general population, and the eff ects may be extended longer than expected previously. f 1. Cho SJ, Minn YK, Kwon KH. Stroke after burn. Cerebrovasc Dis. 2007;24:261-3. Troponin level as a predictor of prognosis in patients with acute ischemic stroke The best cutoff point revealed by the ROC curve of troponin was 0.291 mg/l; at which the sensitivity was 73% and the specifi city was 79% when used for prediction of death within 1 month (area = 0.319, CI = 0.214 to 0.423, P = 0.021; Figure 1). Conclusion These results suggest that increased serum troponin g Conclusion These results suggest that increased serum troponin level at admission is associated with higher mortality rate. Troponin Figure 1 (abstract P470). Patient fl owchart of enrollment. S165 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 2 (abstract P470). Burn injury patients have higher adjusted hazard ratio of ischemic stroke. at T0, 0.85 ± 0.24 at T1 and 0.91 ± 0.25 at T2, P = 0.146). There was a signifi cant diff erence in the levels of hemoglobin (13.19 ± g/dl 1.97 at T0 and 9.64 ± 1.48 g/dl at T1, P = 0.002). However, this diff erence was not maintained at T2 (12.7 ± 2.02 g/dl at T2, P = 0.252). There were no diff erences regarding PETCO2 at the time points. f g g 2 p Conclusion After cardiac surgery with a pump in high-risk patients, improvement of cerebral hemodynamic occurs, perhaps due to the optimization of cardiovascular function. These fi ndings must be better investigated. Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patientsf We measured the middle cerebral artery mean fl ow velocity and pulsatility index. The end-expiratory pressure of CO2 (PETCO2) was measured with infrared capnography attached to a face mask. Blood pressure, hematocrit and axillary temperature was also recorded. Conclusion Development of ICU-AW is associated with increased systemic infl ammation in the fi rst days after ICU admission. li Acknowledgement This research was supported by the Center for Translational Molecular Medicine (MARS). P473f P472 P472 Increased early systemic infl ammation in patients with ICU-acquired weakness E Witteveen, L Wieske, C Verhamme, T Van der Poll, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P472 (doi: 10.1186/cc14552) Eff ect of prolonged critical care admissions on upper and lower limb muscle architecture P Turton1, R Hay1, JK Taylor2, J Little2, J McPhee3, I Welters4 1Royal Liverpool and Broadgreen University Hospitals, Liverpool, UK; 2Warrington and Halton Hospitals NHS Trust, Warrington, UK 3Manchester Metropolitan University, Manchester, UK; 4University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P473 (doi: 10.1186/cc14553) P Turton1, R Hay1, JK Taylor2, J Little2, J McPhee3, I Welters4 1Royal Liverpool and Broadgreen University Hospitals, Liverpool, UK; 2Warrington and Halton Hospitals NHS Trust, Warrington, UK 3Manchester Metropolitan University, Manchester, UK; 4University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P473 (doi: 10.1186/cc14553) Results The mean age of patients was 64 years; most patients were male (74%). Middle cerebral artery mean fl ow velocity increased signifi cantly after cardiac surgery. It was 53.89 ± 17.23 m/second at T0, 61.48 ± 15.18 m/second at T1 and 59.27 ± 16.12 m/second at T2 (P = 0.029). The pulsatility index was similar at all time points (0.88 ± 0.25 Introduction Muscle wasting is a common consequence of long- term stay in the critical care environment, which may slow down the rehabilitation of survivors. Previous ultrasound studies have S166 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (35.6% vs. 15.2%, P <0.001). This association remained signifi cant after correction for weakness and other risk factors (OR: 2.463 (95% CI: 1.113 to 5.452), P = 0.026). Also among conscious/cooperative patients without weakness, reduced CMAP was independently associated with a higher likelihood of death within 1 year (HR: 2.818 (95% CI: 1.074 to 7.391), P = 0.035). (35.6% vs. 15.2%, P <0.001). This association remained signifi cant after correction for weakness and other risk factors (OR: 2.463 (95% CI: 1.113 to 5.452), P = 0.026). Also among conscious/cooperative patients without weakness, reduced CMAP was independently associated with a higher likelihood of death within 1 year (HR: 2.818 (95% CI: 1.074 to 7.391), P = 0.035). demonstrated a loss of cross-sectional area of lower limb muscles during a 10-day intensive care stay. In this study, we have looked at how markers of muscle architecture (muscle thickness, pennation angle and fascicle length) change in the lower limb, as well as looking at changes in muscle thickness in the upper limb. demonstrated a loss of cross-sectional area of lower limb muscles during a 10-day intensive care stay. Predictive value for weakness and 1-year mortality of screening electrophysiology tests in the ICU g Results A total of 115 ICU patients were included. The average age was 61 years and 67% of the patients were male. ICU admission diagnoses were 53% acute surgery, 14% elective surgery and 33% were admitted for medical nonsurgical reasons. Inter-rater reliability of the DEMMI was high: intraclass correlation coeffi cient (ICC) ranging from >0.91 (range 0.85 to 0.94) at ICU admission, >0.98 (range 0.96 to 0.99) at the MICU and >0.97 (range 0.96 to 0.98) at the general ward. Internal consistency reliabilities (Cronbach α) of the DEMMI were 0.84, 0.87 and 0.98 at the ICU, MICU and hospital ward respectively. Validity coeffi cients (Spearman’s rank correlations) with BI, Katz-ADL and MMT were 0.63, –0.45 and 0.62. Introduction Muscle weakness in long-stay ICU patients contributes to 1-year mortality [1]. Whether electrophysiological screening is an alternative diagnostic tool also in unconscious/uncooperative patients remains unknown. The aims of this study were to determine the diagnostic properties of abnormal compound muscle action potential (CMAP), sensory nerve action potential (SNAP) and spontaneous electrical activity (SEA) for Medical Research Council (MRC)-sum score defi ned weakness and their predictive value for 1-year mortality. i p y y Methods Data were prospectively collected during the EPaNIC trial (ClinicalTrials.gov NCT00512122) [2] from October 2008 to November 2010. From day 8 onwards, nerve conduction studies and electromyography were performed weekly in 642 long-stay and 88 randomly selected short-stay patients and muscle strength was assessed in cooperative patients using the MRC-sum score. The electrophysiologist was blinded for the clinical assessments of the physiotherapists and vice versa. The two primary outcomes were: sensitivity, specifi city, positive and negative predictive values of abnormal CMAP, SNAP and SEA for weakness (MRC-sum score <48); and the predictive value for 1-year mortality of abnormal fi ndings on fi rst electrophysiological screening. This association was assessed by univariate and multivariate analyses correcting for weakness and other risk factors, including baseline risk factors, comorbidities, illness severity and ICU exposures. Conclusion The DEMMI is a reliable, responsive and feasible measure ment instrument for the assessment of mobility in critically ill ICU patients. R f 1. Stevens RD. Intensive Care Med. 2007;33:1876-91. 2. de Morton NA, et al. Health Qual Life Outcomes. 2008;6:63. 2. de Morton NA, et al. Health Qual Life Outcomes. 2008;6:63. Eff ect of prolonged critical care admissions on upper and lower limb muscle architecture In this study, we have looked at how markers of muscle architecture (muscle thickness, pennation angle and fascicle length) change in the lower limb, as well as looking at changes in muscle thickness in the upper limb. Methods Following ethical approval, patients who were intubated and ventilated in one of two critical care departments were assented to take part in the study by their next of kin. B-mode ultrasound scans of the right biceps, vastus lateralis and the medial head of gastrocnemius were performed on days 1, 5 and 10. Scans were not performed in patients once they were free of sedation. Muscle thickness (MT) was measured in all three muscles, with pennation angle (PA) being measured in the lower limb muscles. Fascicle length (FL) was derived from PA and MT. Conclusion The diagnostic properties of electrophysiological screening vary over time. Abnormal CMAP documented early during critical illness carries information about longer-term outcome, which should be further investigated mechanistically. Acknowledgement HVM and GH contributed equally. Acknowledgement HVM and GH contributed equally. References 1. Hermans G, et al. Am J Respir Crit Care Med. 2014;190:410-20. 2. Casaer M, et al. N Eng J Med. 2011;365:506-17. 1. Hermans G, et al. Am J Respir Crit Care Med. 2014;190:410-20. 2. Casaer M, et al. N Eng J Med. 2011;365:506-17. g Results Twenty patients were recruited, of which 15 were scanned on day 5, and eight were scanned on day 10. In the biceps, there were no alterations in MT over 5 or 10 days. MT of the vastus lateralis signifi cantly decreased on day 5 (1.77 ± 0.06 mm muscle loss, P = 0.03) and day 10 (5.58 ± 0.09 mm muscle loss, P = 0.01). There was also a signifi cant loss in PA over 5 days (1.48 ± 0.63°, P = 0.01) and 10 days (2.96 ± 0.72°, P = 0.01). However, FL was unchanged over time. There was a signifi cant relationship between size of PA and percentage loss of PA and FL in over 5 days. Loss of MT and PA (MT: 3.21 ± 2.08 mm lost, PA: 2.19 ± 1.64°) was observed in the medial gastrocnemius over 10 days, but did not approach signifi cance. Large fascicles on day 1 were associated with greater percentage loss of FL on day 5 (P = 0.012). P475 P474 y Methods The inter-rater reliability and validity were determined in a prospective observational study. Patients were included and assessed by two independent raters until hospital discharge. Reliability was expressed using the intraclass correlations (ICC). To evaluate the validity, the DEMMI scores were compared with the Barthel Index (BI), Katz-ADL and manual muscle testing (MMT). Predictive value for weakness and 1-year mortality of screening electrophysiology tests in the ICU H Van Mechelen1, G Hermans1, F Bruyninckx2, T Vanhullebusch1, B Clerckx1, P Meersseman2, Y Debaveye1, MP Casaer1, A Wilmer2, PJ Wouters1, I Vanhorebeek1, R Gosselink1, G Van den Berghe1 1KU Leuven, Belgium; 2UZ Leuven, Belgium Critical Care 2015, 19(Suppl 1):P474 (doi: 10.1186/cc14554) Psychometric properties of the de Morton Mobility Index in ICU patients J Sommers1, T Vredeveld2, J Horn1, RH Engelbert2, R Lindeboom1, M Vd Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2Amsterdam School of Health Professions (ASHP), University of Applied Sciences, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P475 (doi: 10.1186/cc14555) Conclusion In the lower limb, we have shown that MT and PA alterations occur in the fi rst 10 days. Patients with larger PA and FL appear to lose a greater percentage of angle and fascicle length in the fi rst 5 days. In contrast, we have demonstrated a sparing eff ect on the muscles of the upper limb compared with the lower limb. These fi ndings may have implications for rehabilitation and interventions to preserve muscle mass. Introduction Many ICU patients develop ICU-acquired muscle weak- ness (ICU-AW) due to inactivity and critical illness. ICU-AW is associated with short-term and long-term physical impairments and impaired functional status [1]. The de Morton Mobility Index (DEMMI) was developed to measure changes in mobility across clinical settings and proved to be reliable, feasible and sensitive to small but clinically relevant changes in functioning [2]. Our aim was to evaluate the psychometric properties of the DEMMI in ICU patients. Prevalence of psychiatric disorders in trauma patients: results from a major trauma unit M Adlam, A Feehan, V Metaxa Methods This was a prospective observational study of trauma patients from two critical care trauma centers. We excluded patients who had ICU stay <48  hours and those with severe traumatic brain injury (TBI) (GCS ≤8). Patients were followed until ICU discharge, resolution of delirium, death or ICU length of stay >28 days. Delirium was assessed daily using the Confusion Assessment Method for the ICU until the end of the follow-up period. Demographic and admission data, daily consumption of medications, and environmental factors (that is, presence of clock, TV/radio, and so forth) were collected daily. Univariate analysis was performed using Cox regression analysis to identify risk factors for delirium. The independent eff ect of modifi able risk factors was assessed using multivariate Cox regression analysis adjusting for severity of illness and nonmodifi able risk factors. King’s College Hospital, London, UK g g p Critical Care 2015, 19(Suppl 1):P477 (doi: 10.1186/cc14557) Introduction Mental illness has been recognised as a potential risk factor both for intentional and unintentional injury. About 50% of patients presenting with self-infl icting injuries in emergency departments had previous known psychiatric disorder (PD) [1,2], whereas individuals with mental illness were admitted for unintentional injury twice as often as those without [3]. We aimed to assess the prevalence of PD in trauma patients being admitted to a major trauma ICU and compare it with the nontrauma population. Methods We retrospectively reviewed all admissions from January 2010 to December 2013 in a tertiary, mixed ICU that serves a London major trauma centre (MTC) hospital. Data obtained were age, APACHE II score, reason for admission, length of stay (LOS), mortality and a diagnosis of depression, bipolar, self-harm, psychosis, schizophrenia and suicide attempt. j g yi Results We enrolled 150 trauma patients resulting mostly from falls (40%) and motor vehicle accidents (28.7%) over 14 months. Patients with TBI accounted for 56.7% while polytrauma patients without TBI accounted for 43.3%. Mean ICU length of stay was 8.1 ± 7.1 days, 69.3% required mechanical ventilation, 14.7% required a tracheostomy. Delirium developed in 58 patients (38.7%) (mean age 62.9 ± 15.7, mean APACHE score 15.4 ± 6.1, mean ISS score 23.4 ± 9.1). Univariate analysis revealed that delirium was signifi cantly associated with the following nonmodifi able risk factors: age (per 10-year range), APACHE II score (per 10-point increase), need of mechanical ventilation, presence of TBI and pre-existing diabetes. j References 1. Schecter WP, et al. Arch Surg. 2005;140:90. 1. Schecter WP, et al. Arch Surg. 2005;140:90. 2. Colman I, et al. Acad Emerg Med. 2004;11:136. 3 Wan JJ et al J Tra ma 2011 61 1299 2. Colman I, et al. Acad Emerg Med. 2004;11:136. 3. Wan JJ, et al. J Trauma. 2011;61:1299. B-type natriuretic peptide and estimated glomerular fi ltration rate at ICU admission as a predictor of delirium Introduction Delirium in the ICU is a predictor of mortality and cognitive impairment at hospital discharge. Although several pathways for delirium have been described, it is very diffi cult to predict the occurrence of delirium. In this study, we examined plasma biomarkers in delirious and nondelirious patients at admission and whether the biomarkers can predict onset of delirium. y Results A total of 730 patients were electrophysiologically screened, of which 432 were tested for weakness. On day 8, only normal CMAP excluded weakness with a high negative predictive value (80.5%). By day 15, abnormal SNAP and the presence of SEA revealed a high positive predictive value (91.7% and 80.0%, respectively). On day 8, only a reduced CMAP was associated with higher 1-year mortality S167 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We targeted 103 ICU patients in Okayama University Hospital between April 2013 and February 2014. Delirium was diagnosed using the Confusion Assessment Method  – ICU. On admission, blood was obtained for biomarker analysis. Patients with severe head injury and under 16 years old were excluded. P <0.05 was considered statistically signifi cant. Conclusion Trauma patients with PD have increased mortality and LOS. MTCs provide a unique opportunity to identify mental illness during hospitalisation through screening and intervention programmes. Integration of mental health services into ICU care should be examined, as it might provide an effi cient and cost-eff ective way of decreasing the risk of reinjury. gi Results Thirty-seven delirious and 66 nondelirious patients were included. We found that delirious patients tented to have higher B-type natriuretic peptide (BNP) levels and to have lower estimated glomerular fi ltration rate (eGFR) (BNP: delirious patients 188.6 pg/ml, nondelirious patients 78.2  pg/ml (P  = 0.001); eGFR: delirious patients 58.6  ml/ minute/1.73  m2, nondelirious patients 81.3  ml/minute/1.73  m2 (P  = 0.020)). Procalcitonin (PCT) and D-dimer were almost the same between delirious and nondelirious patients (PCT: delirious patients 0.202 ng/ml, nondelirious patients 0.150 ng/ml (P = 0.613); D-dimer: delirious patients 5.25 ng/ml, nondelirious patients 5.35 ng/ml (P = 0.714)). Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study Conclusion BNP and eGFR in ICU admission was associated with delirium. PCT and D-dimer in ICU admission was not associated with delirium. BNP and eGFR might evaluate a predictor of delirium in ICU. References 1. Ely EW, et al. JAMA. 2004;291:1753-62. 1. Ely EW, et al. JAMA. 2004;291:1753-62. Introduction Delirium is associated with signifi cant morbidity and mortality in critically ill medical and surgical patients. However, patients suff ering from trauma are generally excluded from these studies. Our objectives were to assess the incidence of delirium and identify modifi able risk factors associated with delirium among critically ill trauma patients. y 2. van den Boogaard M, et al. Crit Care. 2011;15:R297. P478i Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study MA Duceppe1, A Elliott1, M Para1, MC Poirier1, M Delisle1, AJ Frenette2, D Deckelbaum1, T Razek1, M Desjardins2, JC Bertrand2, F Bernard2, P Rico2, L Burry3, D Williamson2, MM Perreault1 1The Montreal General Hospital, Montreal, QC, Canada; 2Hôpital du Sacré- Coeur de Montreal, QC, Canada; 3Mount Sinai Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P478 (doi: 10.1186/cc14558) Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study MA Duceppe1, A Elliott1, M Para1, MC Poirier1, M Delisle1, AJ Frenette2, D Deckelbaum1, T Razek1, M Desjardins2, JC Bertrand2, F Bernard2, P Rico2, L Burry3, D Williamson2, MM Perreault1 1The Montreal General Hospital, Montreal, QC, Canada; 2Hôpital du Sacré- Coeur de Montreal, QC, Canada; 3Mount Sinai Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P478 (doi: 10.1186/cc14558) Prevalence of psychiatric disorders in trauma patients: results from a major trauma unit In a multivariate analysis when adjusting for the nonmodifi able risk factors, opioids (adjusted HR = 0.37, 95% CI (0.14 to 1.0)), presence of a TV/radio in the room (adjusted HR = 0.28, 95% CI (0.12 to 0.67)), and number of hours mobilized (adjusted HR = 0.77, 95% CI (0.68 to 0.88)) had a protective eff ect on delirium; whereas use of physical restraints (adjusted HR = 2.20, 95% CI (1.11 to 4.35)) and active infection (adjusted HR = 2.08, 95% CI (1.16 to 3.71)) remained strongly associated with delirium. Results Of 978 trauma patients admitted to the ICU, 68 (7%) had a known PD. Their diagnoses are shown in Figure 1. Median APACHE II score and unadjusted mortality were 13 and 18% respectively in the PD group (15 and 12% in the entire cohort, P >0.05). Patients suff ering from more than one diagnosis or self-harm alone had increased median LOS (6 vs. 4 days in the entire cohort, P >0.05). Figure 1 (abstract P477). Conclusion Considering the long-term consequences of delirium, steps should be implemented to prevent its development in trauma and include optimizing opioids and mobilizing patients while limiting use of physical restraints. References References 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. References 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. P479 Evaluation of the PRE-DELIRIC delirium prediction tool on a general ICU J Hanison, S Umar, K Acharya, D Conway Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P479 (doi: 10.1186/cc14559) Introduction Delirium is a frequently occurring complication of critical care, occurring in approximately 45% of unplanned UK ICU admissions Introduction Delirium is a frequently occurring complication of critical care, occurring in approximately 45% of unplanned UK ICU admissions Figure 1 (abstract P477). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S168 Figure 1 (abstract P479). Range of PRE-DELIRIC scores. Methods A literature study via MEDLINE and Embase search with keywords ‘PRIS’, ‘lactic acid’, ‘propofol’ and ‘sedation’. All cases in English, French and Spanish were indentifi ed. Exclusion criteria were onset >48 hours, unclear description of time pattern and dose. p p Results Twenty-two cases of early PRIS were found. These concerned 10 pediatric versus 12 adult patients. Eleven were identifi ed in the ICU versus 11 in the operating room. The survival rate of early PRIS was 95.5%, and morbidity was restricted to four patients. In the adult subgroup, the mean propofol dose was 4.9  mg/kg/hour. Triggering factors such as use of catecholamines and corticosteroids were found in 36.4% and 45% of patients. In total, 3/22 cases matched Bray’s defi nition of PRIS. In 14/22 cases, lactic acidosis was interpreted as onset of PRIS. Conclusion Compared with a review by Fudickar [1], we found signifi cant diff erences in critical dose, risk factors, symptomatology and morbidity/mortality between PRIS and early PRIS cases. As criticisms are off ered, a question is whether these cases really are the onset of the fatal syndrome PRIS. Therefore, we completed diff erential diagnosis of lactic acidosis and found that not all possible causes (for example, hyperglycemia, ketonemia, pharmacologic confounders as biguanides, epinephrine) were ruled out in most cases. This is important since PRIS is an exclusion diagnosis. The existence of early PRIS should indeed be questioned and investigated by large, multicenter observational trials. Reference [1]. The presence of delirium in critical care is an independent risk factor for mortality; for every day of delirium, there is an additional 10% relative risk of death at 1 year [2]. A delirium prediction tool PRE- DELIRIC has been recently developed and calibrated in a multinational project [3]. This study aimed to determine the utility of PRE-DELIRIC on our ICU. Propofol sedation reduces contraction and motion of diaphragm in humans: preliminary results p y G Ranieri, M Luigi, F Belsito, M Rocco, RA De Blasi Sant’Andrea, Rome, Italy Results The PRE-DELIRIC scores predicted a mean rate of delirium of 39%. PRE-DELIRIC risk scores ranged from 4 to 93% (Figure 1). Six (15%) patients developed delirium in the fi rst 24 hours following extubation. Fifteen (37%) of patients were predicted 20% or less probability of delirium. Twelve (29%) patients developed delirium at any point during their ICU stay. This resulted in 36 total delirium bed-days. Introduction Among drugs used for sedation, propofol has a primary role [1]. Despite propofol being described to exert a relaxant eff ect on skeletal muscle, no data showing its action on diaphragm are reported. The aim of this observational study on humans is to apply ultrasound to assess propofol’s eff ect on diaphragmatic contraction and motion during endoscopic procedures. y y Conclusion Our observation that <30% of patients experienced delirium is less than the reported prevalence in similar settings and our own audits. This study demonstrates that there is some agreement between recorded rates of delirium and predicted rates using PRE- DELIRIC. We suggest that PRE-DELIRIC can be used in quality/audit work on UK ICUs in order to assess attempts to improve the management of delirium. Further work is required to assess the utility of PRE-DELIRIC as a risk assessment tool in individual patients. Methods We investigated seven consecutive patients undergoing gastroscopy or colonoscopy in the endoscopy unit of our hospital. Patients received propofol at a dose able to induce and maintain sedation to level 6 of the Ramsay Sedation Scale during the procedure. Measurements were obtained on right side of the thorax in millimeters; diaphragmatic motion (DM) and diaphragmatic motion at maximal inspiration (DM forced) were measured in M-Mode with a 3.5  MHz array convex probe placed on the midclavicular line using the liver acoustic window. Thickness at end inspiration (TEI) and thickness at end expiration (TEE) were measured in M-Mode with a 10 MHz vascular probe. The thickening fraction (TF) was calculated: (TEI – TEE) / TEE [2]. Time points of measurements were taken when the patient arrived in the surgery room (Baseline), 1 minute after level 6 of the Ramsey Sedation Scale was obtained (Sedation) and 5 minutes after the patient had a recovery to level 1 on the Ramsey Sedation Scale (Awakening). q Reference 1. Fudickar A. Propofol infusion syndrome in anaesthesia and intensive care medicine. Curr Opin Anaesthesiol. 2006;19:404-10. Methods This study prospectively investigated 41 patients. Medical and surgical general ICU patients were included after 24 hours of sedation and mechanical ventilation. The researchers calculated PRE-DELIRIC scores for each patient. PRE-DELIRIC involves recording 10 variables, submitted into an online algorithm that estimates the percentage risk of delirium. We diagnosed delirium with the CAM-ICU which was performed 12 hourly [4]. P484 Prolonged dexmedetomidine infusion and drug withdrawal in critically ill children A Haenecour, A Goodwin, W Seto, C Urbain, P Laussen, C Balit The Hospital for Sick Children, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P484 (doi: 10.1186/cc14564) Prolonged dexmedetomidine infusion and drug withdrawal in critically ill children A Haenecour, A Goodwin, W Seto, C Urbain, P Laussen, C Balit The Hospital for Sick Children, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P484 (doi: 10.1186/cc14564) Results One hundred and twenty-six of 174 respondents indicated that they practice in the ICU setting. Sixty-six per cent were specialists and mainly anaesthesiologists (42%), whilst 32% were critical care subspecialists. The respondents indicated that on average 30 ± 20% of their patients experience delirium. Eighty per cent of the respondents indicated that delirium impacts signifi cantly negatively on patient outcomes whilst 1% indicated that there was no such association. Delirium screening is achieved mainly by clinical assessment (77%). Twenty-four per cent utilise an objective tool to screen for delirium and amongst them the CAM-ICU is utilised by 80%. Amongst delirious patients the sedative of choice is dexmedetomidine in the majority. However, 20% prescribe midazolam as a fi rst choice in this setting. Conclusion The fi ndings are comparable with reports of similar surveys conducted in other regions. The delirium screening method is inadequate as the vast majority do not utilise an objective method. Introduction We investigated the incidence, symptoms and risk factors for withdrawal associated with prolonged dexmedetomidine use. Dexmedetomidine is an α2-adrenergic receptor agonist, with anxiolytic, analgesic and sedative properties. Intended for short-term use, there is increasing literature describing prolonged use for sedation. However, this raises the potential of withdrawal syndrome and there is no recommendation for the discontinuation of dexmedetomidine. Other goals included determining the hemodynamic eff ects of discontinuation of dexmedetomidine and role of clonidine in patients with prolonged dexmedetomidine use. g Methods A retrospective review of patients admitted to the critical care unit who had exposure to dexmedetomidine for longer than 48  hours, between 1 January 2014 and 15 July 2014. Data included patient demographics, dexmedetomidine exposure (bolus dose, total cumulative dose, duration), other sedative exposure, withdrawal symptoms measured by WAT-1 score, nursing subjective assessment and treatment given for withdrawal. Each potential withdrawal episode was reviewed by two reviewers. Hemodynamic parameters were analyzed to assess hemodynamic changes associated with discontinuation of dexmedetomidine. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. i Results During propofol administration TEI reduced 19% whereas after awakening it increased 14.5% but did not reach baseline. Conversely TEE did not change during the study. During propofol sedation, TF decreased 34% and returned to baseline after recovery. DM showed 29% reduction during propofol administration whereas the forced diaphragmatic motion tested when patients were conscious (forced DM) did not evidence any change. y g Conclusion In this observational study, ultrasound assessed that propofol causes a reduction of diaphragmatic contraction and motion during endoscopic procedures. P482 Delirium knowledge and assessment by ICU practitioners in South Africa: results of a national survey S Chetty, F Paruk University of the Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P482 (doi: 10.1186/cc14562) Delirium knowledge and assessment by ICU practitioners in South Africa: results of a national survey S Chetty, F Paruk University of the Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P482 (doi: 10.1186/cc14562) p g p Conclusion These results are consistent with the hypothesis of 2 days reduction of the median weaning time in the loxapine group, but the diff erence was not statistically signifi cant. Loxapine reduces the need for resuming sedation during weaning from MV. Given the quality of the data and methodology, these results may be useful in future meta-analyses. Introduction Delirium recognition in critically ill patients is considered to be important taking into account the poor outcomes associated with its occurrence. The purpose of this study was to evaluate knowledge pertaining to delirium as well as the implementation of screening practices. This study constituted a component of a survey that explored current sedation-related practices in South African ICUs. Methods Following approval from the University Human Research ethics committee, a validated questionnaire was distributed electronically to physician members of various medical databases in South Africa as South Africa does not have a formal registry of critical care practitioners. P484 Descriptive statistics were used with t test and chi-square test. Median and interquartile range (IQR) are reported. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. Results The trial was discontinued after 101 patients had been randomized because of insuffi cient enrollment. Fifteen patients withdrew consent, leaving 86 patients for analysis. Forty-seven patients were assigned to the loxapine group and 39 to the placebo group. Median time to successful extubation was 3.2  days in the loxapine group and 5 days in the placebo group (RR = 1.2, 95% CI = 0.75 to 1.88; P = 0.45). During the fi rst 24 hours, sedation was more frequently resumed in the placebo group (44% vs. 17%, P = 0.01). One patient had a transient seizure in the loxapine group. Data analyzed are reported in Table  1 and expressed as mean (SD). *ANOVA was used to compare data for repeated measurements. Post hoc statistical comparison with Bonferroni’s test was used to identify signifi cant variations. than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. g References p g p yp Results The trial was discontinued after 101 patients had been randomized because of insuffi cient enrollment. Fifteen patients withdrew consent, leaving 86 patients for analysis. Forty-seven patients were assigned to the loxapine group and 39 to the placebo group. Median time to successful extubation was 3.2  days in the loxapine group and 5 days in the placebo group (RR = 1.2, 95% CI = 0.75 to 1.88; P = 0.45). During the fi rst 24 hours, sedation was more frequently resumed in the placebo group (44% vs. 17%, P = 0.01). One patient had a transient seizure in the loxapine group. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande University Hospitals Leuven, Belgium Critical Care 2015, 19(Suppl 1):P480 (doi: 10.1186/cc14560) University Hospitals Leuven, Belgium University Hospitals Leuven, Belgium y g Critical Care 2015, 19(Suppl 1):P480 (doi: 10.1186/cc14560) Table 1 (abstract P481) Variable Baseline Sedation Awakening P value Thickness end inspiration (mm) 3.25 (0.21) a2.66 (0.20) b,c3.22 (0.30) <0.001* Thickness end expiration (mm) 2.11 (0.20) d2.00 (0.15) 2.07 (0.15) 0.112* Thickening fraction 0.54 (0.07) e0.36 (0.10) f,g0.49 (0.11) <0.001* Diaphragmatic motion (mm) 18.66 (2.23) h13.27 (4.93) 15.31 (0.40) 0.055 Diaphragmatic motion forced 54.61 (19.34) – 56.34 (13.75) 0.298 (mm) aP <0.001 versus baseline. bP = 0.043 versus baseline. cP <0.001 versus sedation. dP = 0.030 versus baseline. eP =0.012 versus baseline. fP =0.353 versus baseline. gP = 0.041 versus sedation. hP = 0.022 versus baseline. Introduction Propofol infusion syndrome (PRIS) is a rare propofol complication, leading to cardiac failure. It was fi rst described in critically ill children and in adults with traumatic brain injury. Pathophysiology is unknown although common factors are the prolonged (>48 hours) use of high-dose (>5 mg/kg/hour) propofol combined with elevated levels of catecholamines and corticosteroids. Recently, case reports of early- onset PRIS during anesthesia and in the early postoperative setting were published. In many of these, lactic acidosis is interpreted as onset of PRIS. Criticism off ers that it might concern a poor diff erential diagnostic approach or an observational bias. Also, lactic acidosis is not an obligate PRIS symptom and incidence of lactic acidosis during propofol sedation is unknown. To gain insight into the incidence and characteristics of early PRIS, we performed a systematic review on early PRIS cases. S169 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. P487 Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients Introduction Recently, several animal studies observed that dexmedetomidine (DEX), a new sedative and α2-adrenoceptor agonist, inhibited the infl ammatory responses [1-3]. Moreover, DEX was reported to have anti-infl ammatory eff ects in patients [4,5]. However, these studies were the small-sized studies and there are few studies about the eff ects of long-term administration of DEX in severe septic patients. The present study evaluated the eff ects of long-term administration of DEX on infl ammatory responses and severity in severe septic patients. We hypothesize that the administration of DEX has benefi cial eff ects for severe septic patients. y y g y Methods We investigate the role of DEX as a sedative agent used during recovery from deep sedation and weaning from extracorporeal support in patients on vv-ECMO. From May 2014 to October 2014 we prospectively enrolled seven patients aff ected by ARDS of diff erent etiologies treated with vv-ECMO. The mean age was 53.7 ± 7.9 years and the mean ICU stay was 21.4 ± 11.5 days. Initially, all patients were sedated with association of opioids and GABA receptor agonists, following the internal protocol. At the time of weaning from ECMO, ruled out cardiovascular instability, we started the administration of DEX (0.7 μg/kg/hour, without initial bolus) with progressive decrease of the dose of other sedative drugs. if Methods In 66 patients (M/F 44/22, mean age 66 years) with severe sepsis, who were administered propofol (0.5 to 4.0  mg/kg/hour) only for sedation, 42 patients (M/F 28/14, mean age 67  years) were administered DEX (0.2 to 0.7  μg/kg/hour) for more than 24  hours in addition to propofol (DEX group). Twenty-four patients were not administered DEX (Control group). Primary outcome were changes in infl ammatory responses at 48 hours after the administration of DEX or none, and secondary outcomes were changes in APACHE II and SOFA scores at 48 hours after the administration of DEX or none. Results The mean duration of DEX infusion was 6.1 ± 4.8 days. Loxapine to control agitation during weaning from mechanical ventilation: a randomized controlled trial S Gaudry1, B Sztrymf2, R Sonneville3, B Megarbanne4, C Clec’h5, J Ricard1, D Hajage3, D Dreyfuss1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Béclère, Clamart, France; 3Hôpital Bichat, Paris, France; 4Hôpital Lariboisière, Paris, France; 5Hôpital Avicennes, Bobigny, France Critical Care 2015, 19(Suppl 1):P483 (doi: 10.1186/cc14563) Results A total of 53 patients accounted for 69 unique dexmedetomidine treatment courses. Median age at the time of dexmedetomidine infusion was 5  months (range 1  day to 3  years). Dexmedetomidine dose ranged from 0.1 to 2  μg/kg/hour with a median cumulative dose of 87  μg/kg (IQR 53, 156). Median duration of exposure to dexmedetomidine was 124  hours (IQR 76, 178) with a maximum duration of 466 hours. We identifi ed 24 separate episodes of withdrawal (incidence 35%). Most common symptoms were agitation (100%), fever (67%), vomiting/retching (46%), loose stools (29%) and decreased sleep (20%). Statistical analysis showed that factors signifi cantly associated Introduction Weaning from prolonged mechanical ventilation (MV) in the ICU may be impeded by the occurrence of agitation. Loxapine had the ability to control agitation without aff ecting the effi cacy of spontaneous ventilation in an observational study, justifying the implementation of a randomized controlled trial. Methods We conducted a multicenter, placebo-controlled, parallel group, randomized trial at fi ve French ICUs between November 2011 and November 2013. Patients (aged >18  years) under MV for more S170 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 to help tolerate intubation and reduce pain and anxiety. Propofol (Pr) is a widely used option, but other viable alternatives for short-term sedation (STS; that is, <24 hours) include benzodiazepines (BDZ) and dexmedetomidine (Dx). We aimed at pooling all available evidence on the comparative eff ects of Pr in terms of awakening and recovery times after STS in mechanically ventilated ICU patients. with withdrawal were cumulative dose (P = 0.01) and duration of use of dexmedetomidine (P = 0.02). Duration of opioids exposure prior to dexmedetomidine wean was also a risk factor for withdrawal (P = 0.01). Use of clonidine as a transition from dexmedetomidine did not protect against withdrawal (P = 0.59). g Conclusion This study showed that withdrawal syndrome is associated with prolonged infusion of dexmedetomidine. Patients with higher cumulative doses and longer duration of exposure were more at risk. P486 P486 Short-term sedation of mechanically ventilated ICU patients with propofol, benzodiazepines, or dexmedetomidine: systematic review and meta-analysis on awakening and recovery times A Feuersenger1, L Pradelli2, A Aliano2, JF Baron3, M Westphal1 1Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany; 2AdRes, Torino, Italy; 3Fresenius Kabi ELAMA, Paris, France Critical Care 2015, 19(Suppl 1):P486 (doi: 10.1186/cc14566) Conclusion In the present study, the long-term administration of DEX has benefi cial eff ects of infl ammatory responses and severity for severe septic patients. P485 Weaning from extracorporeal membrane oxygenation: experience with dexmedetomidine in seven adult ARDS patients M Cozzolino, A Franci, A Peris, L Tadini Buoninsegni, B Loriga A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P485 (doi: 10.1186/cc14565) Introduction Sedation in the ICU is a basic therapeutic procedure to increase tolerance of invasive treatments and reduce discomfort. Extracorporeal membrane oxygenation (ECMO) is a highly invasive treatment and prolonged sedation may be required. Patients undergoing ECMO represent a challenge with respect to sedation. Initially, deep sedation may be required to optimize ventilation and circuit–patient fl ows and to minimize oxygen consumption. The other critical phase is represented by weaning from ECMO support. Optimal sedation is not clearly defi ned, moreover there are no data on sedation practices with dexmedetomidine (DEX) in adult patients undergoing ECMO. In contrast to other sedatives, DEX has analgosedative eff ects without respiratory depression, and could be useful to facilitate spontaneous respiratory activity during recovery from sedation. i Conclusion In conclusion, Pr is associated with shorter awakening and recovery times after STS than BDZ, while no diff erence could be shown when Pr was compared with Dx. P487f P487 Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) Except for one patient, who received DEX as a single drug after suspension of other sedatives, a low-dose infusion of another sedative (<50% compared with initial dose) was maintained. Three patients presented adverse events: two bradycardia and one hypotension. In four patients DEX was discontinued after recovery of respiratory function; in two patients deeper sedation for ventilatory dyssynchrony was needed so other sedative drugs were started. Only in one patient was the drug suspended for extreme bradycardia, resolved after suspension. Results The administration of DEX occurred for a mean 130 hours (24 to 433  hours) in the DEX group. White blood cell counts, C-reactive protein (CRP) and procalcitonin (PCT) in both groups signifi cantly decreased after the administration of DEX or none. However, CRP and PCT in the DEX group were signifi cantly lower than those in the control group: CRP 7.7 (5.0) versus 13.6 (7.9)  mg/dl; P  <0.05, PCT 7.6 (11.7) versus 18.6 (11.6) ng/ml; P <0.05, mean (SD). APACHE II and SOFA scores in both groups decreased after the administration of DEX or none, but APACHE II and SOFA scores in the DEX group were lower than those in the control group: APACHE II 10.8 (4.8) versus 15.2 (5.1); P <0.05, SOFA 3.6 (2.0) versus 5.8 (2.9); P <0.05, mean (SD). p y p Conclusion In our study, DEX allowed the reduction of doses of other sedative drugs during weaning from vv-ECMO; this may lead to a cooperative sedation, promoting spontaneous breathing. Side eff ects described and the cost–benefi t ratio must still be verifi ed extensively in patients during weaning from ECMO. Loxapine to control agitation during weaning from mechanical ventilation: a randomized controlled trial Our results suggested that clonidine use is not protective for withdrawal from dexmedetomidine. y Methods We planned a systematic literature review searching Medline and Scopus and performed a meta-analysis on direct comparisons reporting on weaning time (Tw), duration of mechanical ventilation (Tmv), time to extubation (Tex) and length of stay in the ICU (Ticu). The primary analysis considered only data from RCTs, while in a secondary analysis observational studies were also included.i y Results The literature search identifi ed 15 relevant RCTs, of which 11 versus BDZ, and a further fi ve observational studies, of which one versus BDZ. When compared with BDZ, Pr associated with signifi cantly reduced Tw (–1.6 hours, 95% CI: –2.5 to –0.8), Tmv (–2.0 hours, 95% CI: –3.7 to –0.2), and Ticu (–5.0 hours, 95% CI: –8.5 to –1.4); no statistically signifi cant diff erence resulted when comparing Pr and Dx. When nonrandomized evidence was included, results did not change signifi cantly. P490 Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) Results A total of 77 children with a median age of 15 (4 to 84) months, weight of 10 (5.7 to 20) kg and length of ICU stay of 8 (5 to 14) days received DEX, with a mortality rate of 9%. Indications were: weaning from mechanical ventilation (32.5%), neurosurgical postoperative (NCI) and upper airway surgery (VAS) (24.7%), non-invasive ventilation (13%), refractory tachycardia (6.5%) and other indications (23.3%). There was no diff erence between the initial and maximum doses and infusion length. There was a signifi cant decrease in MAP and HR after 6 hours infusion of DEX in the total group; however, no signifi cant diff erence occurred between groups when analyzing MAP and HR 24 hours after the start of infusion (P = 0.798 and 0.379, one-way ANOVA, respectively). In six patients (8%) DEX was suspended for possible side eff ects. Introduction To determine which of three methods of pain manage- ment provided the best pain control in severe ASA III older patients in the surgical ICU (SICU). As technology improves, more older patients benefi t from surgery and need SICU care. Older surgery patients frequently present two medical problems. First is unspecifi c symptoms and decreased pain sensation resulting in delayed diagnosis. Second, they usually are not given enough perioperative pain relief. Optimal pain management results in perioperative stable hemodynamic status and decreased morbidity, mortality, length of stay and medical costs. Methods A retrospective cohort study chart review of 1,872 all-cause patients in a 16-bed SICU during April 2011 to September 2012. Un- consciousness, uncooperative, ASA <III and <65-year-old patients were excluded. The primary point was to compare eff ectiveness of three diff erent methods of pain management: P.R.N. i.v. Demerol/NSAID (D/N), i.v. patient-controlled analgesia (PCA) and patient-controlled epidural analgesia (PCEA), in three diff erent conditions: rest, movement and coughing, with visual analogue scales (VAS 0 to 100). Secondary point was patient satisfaction. Introduction To determine which of three methods of pain manage- ment provided the best pain control in severe ASA III older patients in the surgical ICU (SICU). As technology improves, more older patients benefi t from surgery and need SICU care. Older surgery patients frequently present two medical problems. P490 Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) First is unspecifi c symptoms and decreased pain sensation resulting in delayed diagnosis. Second, they usually are not given enough perioperative pain relief. Optimal pain management results in perioperative stable hemodynamic status and decreased morbidity, mortality, length of stay and medical costs. Methods A retrospective cohort study chart review of 1,872 all-cause patients in a 16-bed SICU during April 2011 to September 2012. Un- consciousness, uncooperative, ASA <III and <65-year-old patients were excluded. The primary point was to compare eff ectiveness of three diff erent methods of pain management: P.R.N. i.v. Demerol/NSAID (D/N), i.v. patient-controlled analgesia (PCA) and patient-controlled epidural analgesia (PCEA), in three diff erent conditions: rest, movement and coughing, with visual analogue scales (VAS 0 to 100). Secondary point was patient satisfaction. f Conclusion Increased DEX indications have been observed in the pediatric population. In this study DEX was demonstrated to be a safe and tolerable drug with few side eff ects, especially related to the cardiovascular system. References 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 2. Afonso J RF. Dexmedetomidina: Papel Atual em Anestesia e Cuidados Intensivos. Rev Bras Anestesiol. 2012;62:118-33. 3. Panzer O, Moitra V, et al. Pharmacology of sedative-analgesic agents: dexmedetomidine, remifentanil, ketamine, volatile anesthetics, and the role of peripheral mu antagonists. Crit Care Clin. 2009;25:451-69. 4. Hsu CD CJ. Pharmacologic agents for pediatric procedural sedation outside of the operating room. UpToDate2014. http://www.uptodate.com/contents/ pharmacologic-agents-for-pediatric-procedural-sedation. 5. Tobias JD. Dexmedetomidine: applications in pediatric critical care and pediatric anesthesiology. Pediatr Crit Care Med. 2007;8:115-31. p p Results A total of 1,292 patients were excluded. VAS results are presented in Table  1 as mean  ± SD. At rest, the PCEA group is signifi cantly better than the other two groups. When at movement, there is no diff erence between the PCEA group and the D/N group but both are better than the PCA group. While coughing, the PCEA group is worse than the D/N group, although there is no diff erence between the PCEA group and the PCA group. The PCEA group gets the best grades in patient satisfaction. 2. Afonso J RF. Dexmedetomidina: Papel Atual em Anestesia e Cuidados Intensivos. Rev Bras Anestesiol. 2012;62:118-33. 3. Panzer O, Moitra V, et al. Pharmacology of sedative-analgesic agents: dexmedetomidine, remifentanil, ketamine, volatile anesthetics, and the role of peripheral mu antagonists. Crit Care Clin. 2009;25:451-69. 4. Hsu CD CJ. Pharmacologic agents for pediatric procedural sedation outside of the operating room. UpToDate2014. http://www.uptodate.com/contents/ pharmacologic-agents-for-pediatric-procedural-sedation. 5. Tobias JD. Dexmedetomidine: applications in pediatric critical care and pediatric anesthesiology. Pediatr Crit Care Med. 2007;8:115-31. Psychometric comparison of three behavioral scales for the assessment of pain in critically ill patients unable to self-report p y p G Papakitsos1, A Kapsali1, T Papakitsou2 p , p , p 1GHA, Arta, Greece; 2General Hospital Messologi, Greece p g Critical Care 2015, 19(Suppl 1):P489 (doi: 10.1186/cc14569) Introduction Pain assessment is associated with important out omes in ICU patients but remains challenging, particularly in non communicative patients. Use of a reliable tool is paramount to allow any implementation of sedation/analgesia protocols in a multidisciplinary team. This study compared psychometric properties (inter-rater agreement primarily; validity, responsiveness and feasibility secondarily) of three pain scales: Behavioural Pain Scale (BPS/BPS-NI, that is BPS for non-intubated patients), Critical Care Pain Observation Tool (CPOT) and Non-Verbal Pain Scale (NVPS), the pain tool routinely used in this 16-bed medical ICU. Conclusion PCEA provided better pain control at rest than the other two methods, whereas P.R.N. Demerol/NSAID and PCEA were somewhat better than PCA when patients were moving. While coughing, P.R.N. Demerol/NSAID provided the best pain control. However, patient satisfaction was signifi cantly better with PCEA. p p References References 1. Taniguchi T, et al. Crit Care Med. 2004;32:1322-6. 2. Qiano H, et al. Crit Care. 2009;13:R136. 3. Wu Y, et al. Mediators Infl amm. 2013;2013:562154. 4. Pandharipande PP, et al. Crit Care. 2010;14:R38. 5. Ji F, et al. Circulation. 2013;127:1576-84. 1. Taniguchi T, et al. Crit Care Med. 2004;32:1322-6. 2. Qiano H, et al. Crit Care. 2009;13:R136.l Introduction Sedation in the ICU is crucial in reduction of patients’ discomfort, in particular in patients undergoing mechanical ventilation Introduction Sedation in the ICU is crucial in reduction of patients’ discomfort, in particular in patients undergoing mechanical ventilation S171 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P488 P488 Characteristics of the use of dexmedetomidine in critically ill children: a Brazilian study PL Lago, C Andreolio, J Piva, E Baldasso Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2015, 19(Suppl 1):P488 (doi: 10.1186/cc14568 FRAP values. We found that FRAP values were inversely correlated with APACHE II score (r = –0.266, P <0.01) suggesting that, in trauma patients, increased antioxidant response, as measured by the FRAP assay, could be a pathophysiological response to stress. Albumin and uric acid concentrations reproduced the FRAP trend with severity. Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, P <0.002) and head trauma (β = –0.219, P <0.045). These results accen- tuate the infl uence of trauma location and severity in TAC changes. The TAC response in ED patients reinforces the need for adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. Table 1 (abstract P490) Demerol/NSAID PCA PCEA (n = 449) (n = 62) (n = 69) P value VAS (rest) 21 ± 14 * 19 ± 9* 12 ± 10 <0.001 VAS (movement) 35 ± 20 41 ± 12* 33 ± 11 0.02 VAS (coughing) 40 ± 24* 58 ± 14 51 ± 14 <0.001 *Signifi cant diff erence. P488 Characteristics of the use of dexmedetomidine in critically ill children: a Brazilian study Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, P <0.002) and head trauma (β = –0.219, P <0.045). These results accen- tuate the infl uence of trauma location and severity in TAC changes. The TAC response in ED patients reinforces the need for adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. Introduction To describe the main indications, doses, infusion length and side eff ects of dexmedetomidine (DEX) administered to children and adolescents admitted to the pediatric ICU (PICU). p Methods A retrospective observational study including children (<18 years) admitted to a Brazilian PICU who received DEX between November 2011 and June 2014. Demographic data, indications, initial dose, maximum dose and time of infusion of DEX, side eff ects and impact on heart rate (HR) and mean arterial pressure (MAP) 6 and 24 hours after the start of infusion. P491 P491 Subanesthetic xenon increases erythropoietin levels in humans and remains traceable in the fi rst 24 hours after exposure: a randomized controlled trial J Ney, C Stoppe, M Brenke, A Goetzenich, S Kraemer, G Schaelte, A Fahlenkamp, R Rossaint, M Coburn RWTH Aachen University, Aachen, Germany Critical Care 2015, 19(Suppl 1):P491 (doi: 10.1186/cc14571) Methods In a prospective observational study of ED polytraumatized patients (n = 23, mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score of 11 ± 6) we measured (in the fi rst 24 hours) plasma TAC by the ferric reducing activity/antioxidant power (FRAP). For control subjects, we used age-matched and gender-matched volunteers (n = 32). We also evaluated the contribution of antioxidant molecules (uric acid, bilirubin, and albumin) to these values.f J Ney, C Stoppe, M Brenke, A Goetzenich, S Kraemer, G Schaelte, A Fahlenkamp, R Rossaint, M Coburn RWTH Aachen University, Aachen, Germany Critical Care 2015, 19(Suppl 1):P491 (doi: 10.1186/cc14571) Introduction The noble gas xenon was recently amended to the list of prohibited substances by the World Anti-Doping Agency as Introduction The noble gas xenon was recently amended to the list of prohibited substances by the World Anti-Doping Agency as Results Polytraumatized patients show diff erences in TAC with reference to control subjects. ED polytraumatized patients show high S172 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. system (Pall Medical), a new device for sedation in ICU patients [2]. The system delivers volatile anesthetics in mechanically ventilated patients. An open reservoir scavenger and a dedicated gas fi lter avoid residual volatile anesthetic halogenate escaping into the room air. g p g Methods Ten mechanically ventilated patients electively admitted for ICU postoperative monitoring were sedated with sevofl urane delivered with the MIRUS™ system. Two patients were excluded from the analysis because inclusion criteria had been lost during the study period. Analgesia was obtained with morphine sulfate: bolus 0.1 mg/ kg i.v. at the end of surgery and 0.2 to 0.4 mg/kg/24 hours. The primary endpoint was to achieve predefi nite levels of sedation (Riker scale 4). Secondary endpoints were the assessment of hemodynamic stability (MAP and HR), blood lactates, any type of side eff ects, and sevofl urane consumption. Data were collected at the following times: admission to the ICU (T1), 1 hour after initiation of sedation (T2), and 1 hour after sedation withdrawal (T3). i References Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. it is supposed to trigger the production of HIF-1α and subsequently erythropoietin. Subsequently, researchers and clinicians started a scientifi c discussion about the potential clinical benefi t in support of humans exposed to high demand, such as critically ill patients. The objective of this study was therefore to evaluate the eff ect of xenon on serum levels of erythropoietin in healthy volunteers. P491 Results were expressed as median (IQR) or mean (SD), where appropriate. Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. pp p Results The local ethical board approved the protocol. Median duration of sedation was 4 (5.5 to 2) hours. Predefi nite levels of sedation were achieved in all patients with a median MAC of sevofl urane of 0.5 (0.5 to 0.3)% and with a median gas consumption of 9.9 (14.3 to 5.3) ml/hour. MAP and HR values at T1 were 86.5 (97 to 80.8) mmHg and 81.5 (103.8 to 65) bpm, respectively; at T2, 74.5 (89 to 69.5) mmHg and 74 (82 to 58.3) bpm, respectively; and at T3 92.5 (101 to 76.8) mmHg and 74 (88.5 to 66.3) bpm, respectively. Lactates were always normal. Mechanical ventilation was interrupted 5.4 (3.1) minutes after withdrawal of sevofl urane and respiratory parameters always were within normal values. Finally, no side eff ects were registered at any phase of the study. Conclusion This pilot study shows that MIRUS™ is eff ective and safe in delivering sevofl urane for sedation at a predefi nite target level in postsurgical patients, without side eff ects. Further data with a larger number of patients and for a longer duration of sedation are required to confi rm these positive, preliminary observations. References Bispectral index-guided anesthesia on time to tracheal extubation after onpump cardiac surgery Bispectral index-guided anesthesia on time to tracheal extubation after onpump cardiac surgery E Kaval, P Zeyneloglu, A Camkiran, A Sezgin, A Pirat, G Arslan Baskent University Faculty of Medicine, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P493 (doi: 10.1186/cc14573) y y Methods This is a monocenter, randomized, blinded, crossover trial, which was registered at ClinicalTrials.gov (NCT01285271). Healthy study test persons were spontaneously breathing randomly 1  hour of xenon 30% (Xe/O2 30%/65%) or control gas (N2/O2 30%/65%). The primary outcome parameter was the erythropoietin level 24  hours after exposure. Secondary outcome parameters are xenon’s elimination kinetics measured in blood and exhalation samples. Introduction Electroencephalographic-based cerebral monitors such as the bispectral index (BIS) have been used for titration of both inhalational and intravenous anesthetics during general anesthesia. Titration of anesthetics using these monitors may facilitate an earlier recovery from general anesthesia and less consumption of anesthetics. The primary aim of this study was to investigate whether BIS-guided anesthesia would reduce time to tracheal extubation when compared with minimum alveolar concentration (MAC)-guided anesthesia in patients undergoing onpump cardiac surgery. Results The application of xenon increases erythropoietin levels with a maximum 24  hours after exposure (1.32 (0.99 to 1.66) P  = 0.033) compared with the baseline values and compared with control values (0.87 (0.68 to 1.05) P = 0.012, Figure 1). Xenon was gas chromatographically traceable in blood and exhalation probes up to 24 hours after exposure. Methods Fifty patients undergoing elective coronary artery bypass grafting surgery from a single tertiary referral university hospital were randomized to BIS-guided anesthesia (Group BIS, n  = 25) and MAC-guided anesthesia (Group MAC, n = 25). The inspired desfl urane concentration was titrated to maintain a BIS value of 40 to 60 in Group BIS and an age adjusted minimum alveolar concentration of 0.7 to 1 was used in Group MAC. Time to tracheal extubation across the two groups was the primary outcome measure. Secondary outcomes included intraoperative desfl urane consumption, postoperative complications, and lengths of stay in the ICU and hospital. p Conclusion One hour of a subanesthetic level of xenon increases erythropoietin levels in healthy study test persons and remains gas chromatographically traceable in blood and exhalation probes 24 hours after exposure. These fi ndings may stimulate larger studies to confi rm these results and to open new avenues for the therapeutic use of xenon in critically ill patients. P492 MIRUS™, a new system for sedation with halogenates in the ICU: a preliminary study of feasibility in postsurgical patients P Mancinelli, S Romagnoli, C Chelazzi, G Zagli, E Bonicolini, A Belardinelli, AR De Gaudio Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P492 (doi: 10.1186/cc14572) Results Demographic features, logistic EuroSCOREs, duration of cardiopulmonary bypass and surgery were similar in both groups. Mean desfl urane consumption was signifi cantly lower in Group BIS (11.9 ± 1.7 ml/hour) compared with Group MAC (13.4 ± 3.0 ml/hour) (P = 0.031). Time to tracheal extubation was not signifi cantly diff erent between the groups (13.3 ± 9.6 hours vs. 17.0 ± 22.4 hours) (P = 0.68). Incidences of postoperative complications were similar and lengths of stay in the ICU and hospital were 2.4 ± 0.7 days versus 3.2 ± 2.7 days Introduction Sedation is standard practice in the ICU [1]. The aim of this study was to investigate the effi ciency and safety of the MIRUS™ Introduction Sedation is standard practice in the ICU [1]. The aim of this study was to investigate the effi ciency and safety of the MIRUS™ S173 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The study was approved by the appropriate institutional review board. After written informed consent, 63 ASA I to III patients undergoing elective hip or knee replacement surgery under general anaesthesia were included. Patients were randomly organised into three groups (20 to 22 each). Anaesthesia was induced with intravenous anaesthetics. After tracheal intubation MIRUS™ automatically adjusted the end-tidal VA concentration to 1.0 MAC. Patients were ventilated with the Puritan Bennett 840 ICU ventilator. After 1 hour of anaesthesia with 1.0 MAC the ventilator mode was switched from SIMV VC+ (totally controlled ventilation, passive patient, with a tidal volume of 8 ml/IBW) to proportional assist ventilation with 50% support (active patient). At the end of surgery the MIRUS™ system was stopped (MAC set to 0.0) and recovery times were measured. and 5.3 ± 1.2 days versus 6.5 ± 3.1 days in Group BIS and Group MAC respectively (P >0.05 for all). and 5.3 ± 1.2 days versus 6.5 ± 3.1 days in Group BIS and Group MAC respectively (P >0.05 for all). Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) y Results Patients were comparable in age, height, weight and operation time. In 60/63 patients a MAC of 1.0 was reached by MIRUS™. Therefore, ISO 11.2 ± 3.3 ml/hour, SEVO 24.3 ± 4.8 ml/hour or DES 41.7 ± 7.9 ml/ hour (mean ± SD; t test: P <0.001) were used during passive ventilation. During patients’ active ventilation, mean VA consumptions of ISO 9.6 ± 5.1 ml/hour, SEVO 19.4 ± 9.6 ml/hour or DES 35.5 ± 23.0 ml/hour were detected (NS between passive and active patients). ISO was the cheapest VA (€2.70  ± 3.10/hour passive patient, €1.90  ± 2.30 active patient), followed by SEVO (€8.40 ± 3.70 passive patient and €6.8 ± 3.8 active patient) and DES (€9.6 ± 4.1 passive patient and €8.6 ± 6.5 active patient). Recovery times were signifi cantly shorter after SEVO and DES compared with ISO (minutes:seconds; ISO 9:31 ± 6:04, SEVO 6:19 ± 2:56, DES 5:27 ± 1:59). Introduction The aim of this study is to present our experience with sevofl urane in the ICU, outline which patients were sedated with sevofl urane and present the safety profi le. Sevofl urane has some potential advantages over intravenous sedation: rapid elimination and few interactions. The optimal role of sevofl urane in ICU is not known. Methods We performed a retrospective study on adult patients who were sedated with sevofl urane in the medical ICU. The decision to use sevofl urane was left to the attending physician. Institutional ethics committee approval was obtained. The target mean alveolar concentration in all patients was 0.5 to 1%. The AnaConDa® device (Sedana Medical, Uppsala, Sweden) was used along with the Anastasia® (Sedana Medical) gas monitor. Data were obtained from patients’ medical records. P495 Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia V Vinnikov, D Drees, J Herzog-Niescery, P Gude, H Vogelsang, B Cevik, T Weber, M Bellgardt St. Josef-Hospital, Ruhr-Universität, Bochum, Germany Critical Care 2015, 19(Suppl 1):P495 (doi: 10.1186/cc14575) P492 Conclusion Intraoperative use of BIS monitoring in patients undergoing onpump cardiac surgery reduced desfl urane requirement but BIS- guided anesthesia did not facilitate time to extubation and lengths of stay in the ICU and hospital. P494 Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery JL Iribarren, JJ Jimenez, N Perez, M Brouard, R Perez, O Gonzalez, A Arbesu, R Martinez, ML Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P496 (doi: 10.1186/cc14576) Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery JL Iribarren, JJ Jimenez, N Perez, M Brouard, R Perez, O Gonzalez, A Arbesu, R Martinez, ML Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P496 (doi: 10.1186/cc14576) Introduction The use of etomidate is a risk factor for relative adrenal insuffi ciency in patients undergoing cardiopulmonary bypass (CPB) [1]. The objective was to determine the possible interaction between Conclusion We identifi ed sevofl urane as an appropriate sedation agent in a diverse group of patients. Sevofl urane advantages over intravenous sedation could be more pronounced in some patient groups (for example, successful resuscitation after cardiac arrest). The safety profi le of sevofl urane sedation was comparable with intravenous sedation [1]. Reference Figure 1 (abstract P496). 1. Mesnil et al. Intensive Care Med. 2011;37:933-41. 1. Mesnil et al. Intensive Care Med. 2011;37:933-41. Reference 1. Bomberg H, et al. Anaesthesia. 2014;69:1241-50. 1. Bomberg H, et al. Anaesthesia. 2014;69:1241-50. P496 Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Introduction The aim of this study is to present our experience with sevofl urane in the ICU, outline which patients were sedated with sevofl urane and present the safety profi le. Sevofl urane has some potential advantages over intravenous sedation: rapid elimination and few interactions. The optimal role of sevofl urane in ICU is not known. l Methods We performed a retrospective study on adult patients who were sedated with sevofl urane in the medical ICU. The decision to use sevofl urane was left to the attending physician. Institutional ethics committee approval was obtained. The target mean alveolar concentration in all patients was 0.5 to 1%. The AnaConDa® device (Sedana Medical, Uppsala, Sweden) was used along with the Anastasia® (Sedana Medical) gas monitor. Data were obtained from patients’ medical records. Conclusion This study showed that MIRUS™ could automatically control end-tidal VA concentrations in ventilated and spontaneously breathing patients. Using ISO reduces costs. Further studies must be taken to analyse feasibility, costs and recovery times of ISO, SEVO and DES used for sedation in an ICU setting. f Results We included 61 adult patients who were admitted from April 2012 to November 2014. Mean age was 62.6 ± 14.9 years, 39 (63.9%) were male. ICU mortality was 41%, hospital mortality was 43%. Mean duration of sevofl urane use was 3.56 ± 2.31 days. Admission diagnoses were: successful resuscitation after cardiac arrest (44.2%), sepsis (37.7%), cardiogenic shock (4.9%), pancreatitis (3.3%) and liver failure, acute exacerbation of COPD, asthma, tetanus and intracerebral hemorrhage (1.6% each). During treatment with sevofl urane, no patients developed malignant hyperthermia, new hyperkalemia or QT prolongation. In three (4.9%) patients, worsening liver function tests prompted sevofl urane discontinuation. Ischemic hepatitis was considered an alternative in all three patients. Seven (11.4%) patients developed renal failure while receiving sevofl urane. Sevofl urane was continued in all patients and renal failure was attributed to alternative diagnoses. No self-extubations were recorded. In seven (11.4%) patients, sevofl urane was discontinued because of worsening ventilation. In six (9.8%) patients, unexpected awakening occurred. Eight patients (13.1%) had symptoms of delirium after sevofl urane inhalations were concluded.il Reference Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia l V Vinnikov, D Drees, J Herzog-Niescery, P Gude, H Vogelsang, B Cevik, T Weber, M Bellgardt St. Josef-Hospital, Ruhr-Universität, Bochum, Germany Critical Care 2015, 19(Suppl 1):P495 (doi: 10.1186/cc14575) Introduction The new MIRUS™ system as well as the established AnaConDa® system uses a refl ector to conserve volatile anaesthetics (VA) [1]. Both systems act with commercially available ICU ventilators. In contrast to AnaConDa®, MIRUS™ includes an automated control of end-tidal VA concentrations. In this study we compared feasibility, costs and recovery times after anaesthesia with isofl urane (ISO), sevofl urane (SEVO) or desfl urane (DES) in ventilated and spontaneously breathing patients. S174 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of more signifi cant impact than medication errors. We assessed the incidence and characteristics of operation-related MEs to improve patient safety in such patients. etomidate and beta tumoral necrosis factor (TNFβ) polymorphism on hemodynamics after CPB. Methods A prospective cohort study on CPB patients who received etomidate or not during anesthetic induction during 2008 to 2011. Demographic and postoperative variables were collected. We tested the Hardy–Weinberg equilibrium in order to avoid selection bias. V18 SPSS was used. Methods The Japan Adverse Event (JET) study was a prospective cohort study which had evaluated AEs and MEs at two tertiary care hospitals. We included all adult patients aged ≥15 years old who had operations over a 2-month period. The primary outcome of this study was the operation-related MEs, defi ned as any deviation from appropriate process of an operation or perioperative care. Trained nurses placed at each participating hospital reviewed all charts daily on weekdays, along with laboratories, incident reports, and prescription queries to collect any potential event. They also collected the characteristics of the patients in the cohort. Some operation-related MEs are associated with operation-related AEs, which are operation-related preventable AEs. After those suspected events were collected, physician reviewers independently evaluated them and classifi ed them as operation- related MEs, AEs, or rule violations. Physician reviewers assessed and rated operation-related AEs according to the symptom and the severity of injury. Results We studied 433 patients undergoing CPB, 285 (65.8%) men and 148 (34.2%) women, 66 ± 6 years, EuroSCORE I 5.3 ± 4%. TNFβ was in Hardy–Weinberg equilibrium (χ2: 0.6; P = 0.42). Visualising patients’ dynamics in the ICU and predicting mortality in real-time using big data MK Komorowski, AF Faisal MK Komorowski, AF Faisal Imperial College London, UK Critical Care 2015, 19(Suppl 1):P499 (doi: 10.1186/cc14579) , Imperial College London, UK Critical Care 2015, 19(Suppl 1):P499 (doi: 10.1186/cc14579) Introduction As informatisation of hospitals continues to spread, increasing amounts of healthcare related data are being collected, and the ICU is no exception. Large datasets are being made available to the scientifi c community, and off er the potential to answer clinical questions and to develop the next generation of clinical tools. A demonstration of such a tool is presented here, built using data from the Multiparameter Intelligent Monitoring in Intensive Care II (MIMIC-II) open database. Methods All of the adult patients who died during their stay in the ICU were included, as well as a matched cohort of patients who survived for more than 28 days after discharge. Data regarding their vital signs, laboratory tests and demographics were collected. Using Matlab, a graphical method involving principal component analysis was developed. The expected mortality was computed using the k-nearest neighbours’ method and compared with several classifi cation algorithms (logistic regression, random forest, support vector machine, Gaussian mixture models). g Conclusion The fi ndings are comparable with reports of sedation surveys conducted in other countries. There is an evidence–practice gap that needs to be addressed. Sedation practices in South African ICUs: results of a national survey F Paruk, S Chetty University of Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P497 (doi: 10.1186/cc14577) Introduction There has been a paradigm shift in the approach to sedation of critically ill patients. The purpose of this study was to evaluate current sedation-related practices in South African ICUs. Conclusion Ninety-three percent of operation-related MEs resulted in operation-related AEs and 21% of them resulted in life-threatening events. Prevention of operation-related MEs should improve the mortality of surgical patients. p Methods A validated questionnaire was distributed electronically to physician members of various medical databases in South Africa as South Africa does not have a formal registry of critical care practitioners. Results One hundred and twenty-six of 174 respondents indicated that they practice in the ICU setting. Sixty-six per cent were specialists and mainly anaesthesiologists (42%), whilst 32% were critical care subspecialists. The public and private-sector representation was 64% and 46% respectively. A written sedation guideline is implemented by 42%. Forty-three per cent utilise a sedation scale, with the Ramsey Sedation Scale being the commonest in use. However, 38% of sedation scale users do so infrequently. Daily interruption of sedation is practiced by 75%. Light sedation is targeted by 42% and 14% do not follow any sedation targets. Upon admission and on subsequent days, sedation targets are achieved most of the time by 48% and 69% of the respondents respectively. Whilst a wide variety of sedatives are prescribed, midazolam constitutes the most commonly prescribed agent. Dexmedetomidine is the agent of choice for postcardiac surgery patients with cardiovascular comorbidities, delirious patients, during weaning and for non-invasive ventilation. Propofol is the agent of choice amongst neurological patients. The respondents indicated that there is a need for local sedation guidelines.i Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia A total of 254 (58.7%) patients received etomidate, 152 out of them required vasoactive drugs. Homozygous G was defi ned as unfavorable TNFβ versus the A allele [2]. Using the general linear model after adjusting for sex and amines dose at 4  hours, an independent association was observed between the systemic vascular resistance index (SVRI) at 4 hours and the use of etomidate (F: 18; P <0.001): 1,849 (95% CI: 1,673 to 2,024) versus 2,493 (95% CI: 2,258 to 2,729) dinas.seg/cm5.m2, the presence of homozygous G (F: 6.5; P  = 0.01), and also showed a signifi cant etomidate–homozygous G interaction (F: 22.8: P <0.001): 1,687 (95% CI: 1,350 to 2,023) versus 3,041 (95% CI: 2,589 to 3,492) dinas seg/cm5. m2 (Figure 1). y Results This study included 389 patients with 6,624 patient-days. The median age of patients was 69 years and 224 (58%) were male. Among these 389 patients, 31 patients had 46 operation-related MEs during their hospital stay and the incidence of operation-related MEs was 12 per 100 patients. Operation-related AEs occurred in 29 patients with 43 events. The most frequent symptoms for operation-related MEs were skin (26%), bleeding (21%), and central nervous system (14%). Among 46 operation-related MEs, 43 (93%) were not intercepted, and they resulted in operation-related AEs that were considered as preventable operation-related AEs. Nine of preventable operation-related AEs (21%) were fatal or life-threatening: fi ve were nerve injury during operation and stroke after neurosurgical operation, and one biliary peritonitis after gastrectomy and cholecystectomy, and tension pneumothorax after lung lobectomy, and two unexpected massive bleeding due to vessels injury. Conclusion Etomidate use is associated with lower postoperative SVRI which is increased in the presence of G homozygosity for TNFβ polymorphism. P498 Epidemiology of operation-related medical errors in inpatients in Japan: the JET study y References 1. Iribarren JL, et al. J Cardiothorac Surg. 2010;5:26. 1. Iribarren JL, et al. J Cardiothorac Surg. 2010;5:26. 2. Iribarren et al. Interact Cardiovasc Thorac Surg. 2008;7:1071-4. 2. Iribarren et al. Interact Cardiovasc Thorac Surg. 2008;7:1071-4. Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study y M Soares1, JM Kahn2, FA Bozza1, T Lisboa3, LP Azevedo4, W Viana5, L Brauer6, PE Brasil1, DC Angus2, JI Salluh1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Sta. Casa de Porto Alegre, Brazil; 4Hospital Sírio Libanês, São Paulo, Brazil; 5Hospital Copa DOr, Rio de Janeiro, Brazil; 6Hospital Sao Luiz Itaim, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P500 (doi: 10.1186/cc14580) Figure 1 (abstract P501). HEWS prior to critical event. Methods We conducted a prospectively identifi ed, retrospectively gathered cohort study at two hospitals of consecutively admitted medical and surgical patients over a 6-month period. One hospital had a rapid response team (RRT) and used HEWS with a trigger of 5 while the other was undergoing implementation of the HEWS without a RRT. HEWS was calculated for each patient on the fi rst day of admission and for the 3 days prior to inpatient arrest or death. A study investigator reviewed all events for accuracy. Our outcome of interest was a composite of inpatient cardiac arrest and hospital mortality. Introduction The aim was to investigate the impact of organizational factors on patient outcomes in a large sample of Brazilian ICUs. Introduction The aim was to investigate the impact of organizational factors on patient outcomes in a large sample of Brazilian ICUs. Methods A retrospective cohort study of 59,483 patients admitted to 78 ICUs in 51 hospitals during 2013. We retrieved demographic, clinical and outcome data from an electronic ICU quality registry (Epimed Monitor System). We surveyed ICUs using a standardized questionnaire regarding hospital and ICU structure, organization, staffi ng patterns, process of care, and family care policies. We used multilevel logistic regression analysis to identify characteristics associated with hospital mortality. y Results There were 7,138 patients admitted over 6 months. We found 0.5% of patients suff ered an inpatient arrest and 3.6% of patients died. Moreover, 66% of patients who died or arrested were admitted to the hospital without a RRT. Patients who arrested or died had more comorbidities defi ned by the Charlson Comorbidity Index (CCI) of 6.0 and 8.2 respectively compared with the general population, which had a CCI of 5.2. Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Results A total of 6,084 patients were included in the analyses, adding up to more than 12 million data points. Using this multidimensional dataset, a 3D representation of the clusters of survivors and nonsurvivors was built, showing how their trajectories diverge through time. Patterns in the evolution of individuals or subgroups of patients can be identifi ed using this approach. For example, the evolution of a new patient can be visualised, progressing through the clusters as his severity changes. His expected mortality can be predicted at any point in time, with an AUC ROC constantly above 0.85. Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) p y Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) y Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) Introduction Operation therapy is more invasive than medication therapy and then operation-related medical errors (MEs) might be Introduction Operation therapy is more invasive than medication therapy and then operation-related medical errors (MEs) might be S175 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P501). HEWS prior to critical event. Conclusion Machine learning tools off er an appealing mathematical framework for modelling complex medical situations. This proof of concept demonstrates that the application of computational sciences to high-quality data such as the MIMIC-II database has the potential to lead to the development of meaningful tools which will ultimately be capable of assisting physicians in making the right decision at the right time for an individual patient. P500 P500 Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study M Soares1, JM Kahn2, FA Bozza1, T Lisboa3, LP Azevedo4, W Viana5, L Brauer6, PE Brasil1, DC Angus2, JI Salluh1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Sta. Casa de Porto Alegre, Brazil; 4Hospital Sírio Libanês, São Paulo, Brazil; 5Hospital Copa DOr, Rio de Janeiro, Brazil; 6Hospital Sao Luiz Itaim, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P500 (doi: 10.1186/cc14580) Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Only tight cooperation between clinicians and data scientists can help close the gap that currently separates these two worlds, for the ultimate benefi t of patients. Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study The median and mean HEWS at time of admission was 1 and 1.7 for the general population, 2 and 2.4 for patients who suff ered an inpatient arrest and fi nally 3 and 3.8 for those who died. There was a rise in median HEWS from 2 to 5 in the 24 hours prior to in patient arrest or death. See Figure 1. y Results ICUs were mostly medical or medical–surgical (62.79%) and located in private hospitals (67.86%). Approximately half (40.51%) had critical care training programs. Median physician and nurse staff – bed ratios were 0.15 (IQR, 0.12 to 0.19) and 0.71 (0.61 to 0.84); board- certifi ed intensivists were present 24/7 in 16 (21%) of ICUs. Routine clinical rounds occurred in 67 (86%) and daily clinical checklists were used in 36 (46%) ICUs. Most frequently implemented protocols focused on sepsis management and VAP and CLABSI prevention. Median number of patients per center was 898 (IQR 585 to 1,715) and there were 67% medical admissions; 18% patients received mechanical ventilation (MV). Median SAPS 3 score was 41 (33 to 52) points. ICU and hospital mortality rates were 9.6% and 14.3%, respectively. Adjusting for relevant patients’ characteristics (SAPS 3 score, diagnostic admission category, chronic health status, comorbidities, MV use), case-volume and type of ICU, the ICU size (OR = 1.50 (95% CI, 1.45 to 1.95), for 11 to 20 beds; OR = 2.02 (1.40 to 2.92), for >20 beds) and ≥2 clinical protocols (OR  = 0.65 (0.42 to 0.99)) were the organizational characteristics associated with mortality. g Conclusion We found that a 2.5-fold increase in HEWS occurred 24 hours prior to critical events. Similar to previous studies, a RRT in conjunction with HEWS is the best system to reduce unanticipated adverse events. An absolute HEWS of 5 and/or a rapidly increasing HEWS should trigger rapid assessment and treatment to reduce preventable inpatient deaths and arrests. Reference 1. McNeill, Bryden. Resuscitation. 2013;84:1652-67. 1. McNeill, Bryden. Resuscitation. 2013;84:1652-67. Conclusion In a large sample of Brazilian ICUs, the implementation of clinical protocols was associated with better outcomes. Conversely, mortality was higher in larger ICUs. Ethnicity and trial recruitment h y g g Acknowledgements Funded by IDOR, CNPq and FAPERJ. Endorsed by BRICNet. HL Cronshaw, SW Scott, S Bowrey, JP Thompsoni HL Cronshaw, SW Scott, S Bowrey, JP Thompson Leicester Royal Infi rmary, Leicester, UK , , y, Leicester Royal Infi rmary, Leicester, UK yi y Critical Care 2015, 19(Suppl 1):P502 (doi: 10.1186/cc14582 Introduction Surrogate consent and time-sensitive recruitment in critical care research is challenging, yet low enrolment numbers or omitting ethnic groups skew results and conclusions. Patients from diff erent ethnic groups may respond diff erently to therapeutics [1]. There are few data about the eff ect of ethnicity on recruitment into ICU trials. Our ICU recruits to national trials and serves an increasingly non- White British population (24% of population). We undertook this study to determine whether ethnicity aff ects ICU consent rates. Validation of an electronic early warning score using decision tree analysis: proposal M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) Introduction Decision tree analysis uses an algorithm to classify data items by recursively posing a series of questions about items within a dataset. Each question leads to another node and potentially more questions until a predefi ned end condition is reached or no more questions can be asked (Figure 1). We hypothesize that scores generated using the decision tree method will improve upon our existing Hamilton Early Warning Score (HEWS) for a composite endpoint of cardiac arrest, unplanned ICU admission or death. Introduction Timely availability of a kidney specialist poses a formidable challenge in ICUs located in tier II and tier III cities of the developing world. Renal replacement therapy (RRT) is often required in the ICU for acute renal failure patients but availability of a nephrologist/ specialist is scarce, leading to unnecessary and risky transfer to higher centers in metropolitans or even worse to death. We explored whether a remotely monitored ICU  – an electronic ICU (eICU)  – would help mitigate this demand–supply gap. Methods A database of 156,642 electronically captured vital signs from 6,757 consecutively admitted patients to eight medical and surgical wards will be used to train and test the decision tree early warning score. One-third of the data will be withheld from the algorithm for use as a testing set. The algorithm will look for signifi cant changes in vitals 72 hours prior to an outcome and develop the score based upon the resulting relative risk of the composite endpoint happening given a certain vital sign. The scores and predictions generated by the decision tree analysis will then be compared with that of the inception HEWS cohort. y Methods This retrospective study was conducted at four Critinext affi liates where the eICU was being used to provide 24 × 7 support on 89 ICU beds from a remote command center with intensivist and other requisite staff . The eICU had complete access to the patient’s real-time vitals, hemodynamic parameters, imaging, laboratory values, audiovisuals and appropriately engineered smart alerts. The eICU model was further extended in initiating and getting RRT done in patients whenever deemed necessary in times of unavailability of a specialist at the same site. P501 Hamilton Early Warning Score: predict, prevent and protect B Tam, M Xu, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P501 (doi: 10.1186/cc14581) Introduction This study determined the pattern of decline prior to an inpatient arrest. We implemented the Hamilton Early Warning Score (HEWS) within our electronic vital signs documentation to track and trigger care for deteriorating patients. Other EWS have been described in the literature with varying success [1]. In contrast to previous observational studies, we chose to implement a score modifi ed from published EWS using the consensus opinion of a steering committee and evaluate the score in real time. f Methods We performed a retrospective review of screening logs from three national UK trials (PROMISE, BALTI-P, GAiNS) and one local trial (Nociceptin in Sepsis). We analysed consent rates of eligible patients by ethnicity, age, sex, interventional or observational trial, and ethnicity of the researcher seeking consent. We performed chi-squared analysis, and entered signifi cant values into a logistic regression model using SPSS v22. S176 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results The planned analysis for determining the discriminatory and predictive ability of the decision tree HEWS will be conducted with area under the receiver operating characteristic curves. We will test whether the current HEWS has the appropriate sensitivity and specifi city when compared with that of the decision tree score. The AUROC will be calculated for both the training set of data as well as the separate population of additional medical and surgical patients. The two scores will also be plotted along an effi ciency curve, comparing the percentage of vitals that precede a critical event with the percentage of vitals that produce a EWS value greater than or equal to a given EWS value. Results We identifi ed 332 eligible patients across all trials, of whom 37 (11%) were not White British (nWB). Analysis demonstrated consent/assent refusal being signifi cantly associated with: nWB (14, 38%, P <0.001), interventional trial (21, 25%, P = 0.003) and diff erent researcher–patient ethnicities (P <0.001). P504 P503 Validation of an electronic early warning score using decision tree analysis: proposal M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) P504 Can an electronic ICU support timely renal replacement therapy in resource-limited areas of the developing world S Gupta1, A Kaushal1, S Dewan2, A Varma1 1Fortis Escorts Heart Institute, New Delhi, India; 2Fortis Memorial Research Institute, Gurgaon, India Critical Care 2015, 19(Suppl 1):P504 (doi: 10.1186/cc14584) . Lilly CM, et al. Chest. 2014;145:500-7. 1. Bjornsson TD, et al. J Clin Pharmacol. 2003;43:943-67. P501 Logistic regression analysis confi rmed these as independent factors (nWB OR = 4.5, 95% CI = 2.1 to 9.8, P <0.001; interventional trial OR = 2.7, 95% CI = 1.4 to 5.2, P = 0.003; data points missing for researcher–patient ethnicity so variable excluded).f Conclusion This initial study suggests that ethnicity may aff ect assent/ consent to ICU research, with patients from diff erent ethnicities being four times less likely to be recruited. Whilst data are incomplete for researcher–patient ethnicity, our data suggest that this may be an important factor and may infl uence future consent processes. We believe that the role of ethnicity warrants further investigation, not only in clinical trials but also in areas such as organ donation. Reference Conclusion Decision tree analysis methodology with real-life vital signs can produce an EWS superior to previous observational studies. Using a decision tree, especially one that composites all vitals, may show that certain vitals are more predictive of critical events than others. Data will be used to further improve our current HEWS score. 1. Bjornsson TD, et al. J Clin Pharmacol. 2003;43:943-67. Validation of an electronic early warning score using decision tree analysis: proposal Patient baseline demographics, including risk factors, severity score, all-cause mortality at 30 days, transfers to higher center for RRT and its prevention were recorded. Descriptive analysis was performed. Between-group comparison was performed by applying the chi-squared statistic, signifi cance was assumed at a value of P <0.05. Out of a total of 5,146 admissions, 752 inpatient fi les with acute kidney injury/acute renal failure were reviewed, January to July 2013 (n = 373) and July 2013 to January 2014 (n = 379) pre and post eICU implementation respectively.i Figure 1 (abstract P503). Sample heart rate decision tree. p p y Results While baseline demographics and the patient profi le in the two groups did not show statistically signifi cant diff erence, mean APACHE II score was 14.25 ± 1.94 and 14.65 ± 1.76 pre and post eICU respectively; there was a statistically signifi cant diff erence in all-cause mortality at 30 days which decreased from 31 (8.3%) to 16 (4.2%) pre and post eICU respectively, a reduction of >49% (P = 0.030) and transfer out for RRT came down by >77%, from 15 (4%) to two (0.5%) post eICU implementation (P <0.002). Conclusion Over the years there is now broad consensus over the benefi ts of eICU intervention in deprived areas [1]. There is now a need for a paradigm shift to elevate specialized care to improve outcomes. Our small study has clearly indicated the benefi ts in outcome and economics even while providing intervention in such remote areas. An eICU as a bridge to the demand–supply gap needs to be explored and utilized further to its full potential in the emerging world. Reference Reference Figure 1 (abstract P503). Sample heart rate decision tree. Figure 1 (abstract P503). Sample heart rate decision tree. 1. Lilly CM, et al. Chest. 2014;145:500-7. S177 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P506). Mean change in systolic and diastolic BP and HR in relation to ICU work experience Table 1 (abstract P506). Mean change in systolic and diastolic BP and HR in relation to ICU work experience P505 Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study M Soares1, DC Angus2, JI Salluh1, AB Cavalcanti3, F Colombari4, R Costa5, E Silva6, A Japiassu7, JM Kahn2, FA Bozza1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Hospital do Coracao, São Paulo, Brazil; 4Hospital Alemao Oswaldo Cruz, São Paulo, Brazil; 5Hospital Quinta DOr, Rio de Janeiro, Brazil; 6Hospital Israelita Albert Einstein, São Paulo, Brazil; 7Rede Amil de Hospitais, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P505 (doi: 10.1186/cc14585) Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study Group 1 Group 2 P value SBP (mmHg) 3.22 –0.40 0.053 DBP (mmHg) 2.08 –0.27 0.061 HR (bpm) 4.46 1.16 0.048 M Soares1, DC Angus2, JI Salluh1, AB Cavalcanti3, F Colombari4, R Costa5, E Silva6, A Japiassu7, JM Kahn2, FA Bozza1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Hospital do Coracao, São Paulo, Brazil; 4Hospital Alemao Oswaldo Cruz, São Paulo, Brazil; 5Hospital Quinta DOr, Rio de Janeiro, Brazil; 6Hospital Israelita Albert Einstein, São Paulo, Brazil; 7Rede Amil de Hospitais, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P505 (doi: 10.1186/cc14585) Results The mean duration of working in an ICU was 7.3 ± 5.32 years (from 2 to 18 years). The nurses were grouped according to experience – Group A: 17 nurses with <10  years of experience (mean: 4.4  years), Group B: six nurses with >10 years (mean: 15.6 years). There were 640 BP–HR measurements. The mean systolic BP, diastolic BP and HR did not diff er between the two groups (systolic BP: 111.2 ± 10.9 vs. 113.7 ± 14.1 mmHg, P = 0.654; diastolic BP: 72.8 ± 8.2 vs. 71.9 ± 8.1 mmHg, P = 0.835; HR: 81.4 ± 7.2 vs. 78.1 ± 8.4 bpm, P = 0.365). Nevertheless, the mean change in BP and HR during the shift did diff er between the two groups, with the more experienced nurses showing a trivial reduction in systolic and diastolic BP and minor increase in HR whereas the less experienced ones showed slight increase in both BP and HR measurements (Table  1), reaching almost statistical signifi cance. For the less experienced nurses in Group 1, it was noted that the mean changes were bigger in night shifts although the limited number of measurements did not allow robust statistical analysis. Introduction The aim was to evaluate outcomes and resource use and to investigate the association between organizational factors and effi cient resource use in a large sample of Brazilian ICUs. Methods A retrospective cohort study in 59,483 patients (medical admissions: 39,734 (67%)) admitted to 78 ICUs (private hospitals, n = 67 (86%); medical or medical–surgical, n = 62 (79%)) during 2013. We retrieved demographic, clinical and outcome data from an electronic ICU quality registry (Epimed Monitor System). P506 Blood pressure and heart rate changes during shifts in ICU nurses in relation to their work experience A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) y y Methods We included all adult patients (≥18 years) with a fi rst-time ambulance transport to the ED at Odense University Hospital in the period 1 April 2012 to 30 September 2013. Ambulance personnel recorded vital signs and other clinical fi ndings on a structured form on paper during the ambulance transport. Each contact was linked to information from population-based healthcare registers in order to identify comorbid conditions and information on mortality. Demographic factors and fi rst registered vital sign were analysed by univariate logistic regression analysis, with 7-day mortality as outcome. Results In total, 18,572 fi rst-time ambulance contacts were identifi ed in the period of inclusion. Overall 7-day mortality was 4.3% (95% CI = 4.0 to 4.6). Univariate analysis showed increasing age, Charlson Comorbidity Index ≥2, vital parameters outside the normal reference range and summoned physician-assisted mobile emergency care units to be associated with 7-day mortality. Further analyses are currently being carried out. Prehospital transported patients: a resource for accessing prognostic risk factors p g C Bech1, M Brabrand2, A Lassen1 1Od U i it H it l Od C Bech1, M Brabrand2, A Lassen1 1Odense University Hospital, Odense, Denmark; 2Sydvestjysk Sygehus Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P507 (doi: 10.1186/cc14587) Conclusion We observed a great variability in outcome and resources in a large sample of Brazilian ICUs. Implementation of clinical protocols and nursing staffi ng patterns can be targets to improve the effi ciency in resource use in emerging countries such as Brazil. Critical Care 2015, 19(Suppl 1):P507 (doi: 10.1186/cc14587) Introduction The survival of patients transported by ambulance to the emergency department (ED) depends on clinical conditions, patient-related factors and organisational prehospital set up. Data and information concerning patients in the prehospital system could form a valuable resource for assessing potential risk factors associated with adverse outcomes and mortality. Our aim was to describe ambulance transports to the ED and identify prognostic factors accessible in the prehospital phase and associated with 7-day mortality.i Acknowledgements Funded by IDOR, CNPq and FAPERJ. Endorsed by BRICNet. Reference 1. Rothen et al. Intensive Care Med. 2007;33:1329-36. 1. Rothen et al. Intensive Care Med. 2007;33:1329-36. Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study We surveyed ICUs using a standardized questionnaire regarding hospital and ICU structure, organization, staffi ng patterns, process of care and family care policies. Effi cient resource use was assessed by estimating standardized mortality rates (SMR) and standardized resource use (SRU) adjusted for the severity of illness according to the SAPS 3 score, as proposed by Rothen and colleagues [1]. y Conclusion The less experienced ICU nurses, with <10 years of ICU work experience, tended to increase their BP and HR levels during the shift, a fi nding probably heightened during night shifts. Further research, including not only cardiovascular parameters, is warranted to uncover the eff ects of shift-work pattern in ICU nurses, taking into account this specifi cally stressful work environment. Results The median admissions per center was 898 (IQR 585 to 1,715) and SAPS 3 score was 41 (33 to 52) points. Median ICU length of stay was 2 (1 to 5) days, and ICU and hospital mortality rates were 9.6% and 14.3%, respectively. Estimated SMR and SRU were 0.97 (0.72 to 1.15) and 1.06 (0.89 to 1.37), respectively. There were 28 (36%) most effi cient (ICUs with both SMR and SRU <median), 28 (36%) least effi cient (ICUs with both SMR and SRU >median), 11 (14%) overachieving (ICUs with low SMR and high SRU) and 11 (14%) underachieving (ICUs with high SMR and low SRU) ICUs. Most effi cient ICUs were usually located in private and accredited hospitals, with step-down units and training programs in critical care. In univariate analyses comparing most and least effi cient ICUs, ≥2 clinical protocols (OR  = 7.22 (95% CI, 1.41 to 36.97)) and graduated nurse/bed ratio >0.25 (OR = 4.40 (1.04 to 18.60)) were associated with effi cient resource use. Daily checklists also tended to be associated with effi cient resource use (OR = 2.89 (0.95 to 8.72), P = 0.057). P508 Contribution of medical senior house offi cers to a medical referral in the emergency department i g Conclusion In 2011 the Royal College of Physicians emphasized the impact that the quality of the care provided within the fi rst 48 to 72 hours had on clinical outcomes. An evaluation of consultant input into acute admissions management revealed that hospitals in which two or more ward rounds of all acute medical unit patients were performed daily had a lower adjusted case fatality rate for patients with hospital stays over 7 days. Despite twice-daily consultant ward rounds of all new acute admissions and the addition of a third 4:00 pm round from Monday to Friday, only 62% of patients were seen by a consultant within 12  hours. With 67% of patients being admitted between the hours of 12:00 am and 12:00 pm, it is possible that the substitution of an evening round for one of the afternoon rounds would help increase the number of patients seen within the target time frame. This would require a change in the working pattern of the acute medicine consultants. f Introduction In Irish hospitals, the medical senior house offi cer (SHO) is the most junior fully qualifi ed doctor on the medical on-call team. After a patient has been seen by an emergency department doctor of any level, they are almost always referred directly to the medical SHO. This process has been shown to delay a patient’s ward admission by 3 hours 30 minutes [1]. We attempted to quantify the additional benefi t for the patient of being seen by the on-call medical SHO, in terms of patients discharged, new diagnoses reached, and new treatments initiated. g g Methods The emergency department notes and clinical charts of 182 patients were assessed. This constituted a random sample of patients referred by emergency department doctors to the medical team on call over a 2-month period (November to December 2011). Results Discharged: 3/182 (1.6%) of patients referred to the medical team were discharged directly by the medical SHO. Diagnosed: medical SHOs suggested a diagnosis which was diff erent from, or additional to, the ED doctor, in 52/182 cases (28.6%). However, the medical consultant only agreed with this diagnosis in 25 cases (13.7%). This means an incorrect new diagnosis was reached more often than not (14.9%). Treatment: the majority of cases (116/182 (63.7%)) saw no new treatment initiated by the medical SHO. P508 Contribution of medical senior house offi cers to a medical referral in the emergency department Of the rest, only 31 (17%) had a new treatment initiated by the medical SHO which was continued on by the medical consultant through the admission. P510 Interhospital critical care transfer delays result from organisational not geographical factors: secondary analysis of deteriorating ward patients in 49 UK hospitals DJ Wong1, SK Harris2 1King’s College Hospital, London, UK; 2University College London, UK Critical Care 2015, 19(Suppl 1):P510 (doi: 10.1186/cc14590) Conclusion Few direct discharges, new diagnoses, or key new treatments were initiated by the medical SHO in the emergency department. A paper from our hospital shows that more patients referred in by GPs to ED are admitted compared with those referred in to the acute medical assessment unit, with comparable disease severity (43% vs. 12.5) [2]. That paper highlighted the fact that the junior level of the medical NCHDs who see patients in the ED may contribute to their lack of discharging/decision-making zeal. Our survey further illustrated this feature. Our study provided no evidence that a formal medical assessment should delay a patient progressing to the medical ward. Additional genuine urgent OPD appointment slots could be another benefi cial measure. Introduction Critically ill patients may require interhospital transfer for specialist care or because of a lack of local ICU capacity. Harm is assumed from delays that result, but it is not clear whether these delays are due to transfer distances or defi ciencies in the organisation of care. Methods In total, 151 of 15,602 deteriorating ward patients in the (SPOT)light study [1] were transferred rather than admitted locally. We defi ned delay as the time from critical care assessment in the fi rst hospital to arrival in critical care in the second hospital. We used hospital postcodes to derive latitude and longitude, and calculated both geodesic (straight-line) distances (Figure 1) and road distances between the sites using R version 3.1.1 [2]. We compared daytime versus overnight (7:00  pm to 7:00  am) transfer durations assuming traffi c would contribute less to delay overnight. Mapping and visualisation was performed on Quantum GIS version 2.4 [3]. i References 1. Gilligan P, et al. The referral and complete evaluation time study. Eur J Emerg Med. 2010;17:349-53. 1. Gilligan P, et al. The referral and complete evaluation time study. Eur J Emerg Med. 2010;17:349-53. 2. Watts M, et al. Acute medical assessment units: an effi cient alternative to in-hospital acute medical care. Ir Med J. 2011;104:47-9. 2. Watts M, et al. Acute medical assessment units: an effi cient alternative to in-hospital acute medical care. Ir Med J. 2011;104:47-9. Results The median delay to admission was 22  hours (range 41 to 167  hours). The median geodesic distance was 18  km (range 1 to 141 km), and road distance was 24 km (range 2 to 180 km). Correlations between time delay and geodesic/road distances were weak (Figure 2, Blood pressure and heart rate changes during shifts in ICU nurses in relation to their work experience A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) Introduction The aim of the study was to assess the blood pressure (BP) and heart rate (HR) changes during shifts in ICU nurses in relation to their work experience. Our hypothesis was that less experienced nurses, in comparison with more experienced ones, would be subjected to more work stress and this could be demonstrated by higher changes in BP and HR during shifts. Methods We enrolled 23 nurses working in an 8-hour shift schedule at a general adult ICU. Demographic and clinical data were obtained by completing a short questionnaire. The nurses were invited to measure their BP and HR at the beginning, in the middle and at the end of their shift. An ESH/BSH-certifi ed automatic device was used for the BP and HR measurements. Conclusion We found that several prehospital-registered vital signs recorded by ambulance personnel at fi rst contact with the patient were prognostic factors of 7-day mortality. S178 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P508 P508 Contribution of medical senior house offi cers to a medical referral in the emergency department GF Fitzpatrick University Hospital, Limerick, Ireland Critical Care 2015, 19(Suppl 1):P508 (doi: 10.1186/cc14588) of patients were fi rst seen by a consultant within 12 hours of admission, with a range from 23 minutes to 26 hours. When looking at patients admitted during the weekdays, 63% of them were seen within 12 hours; for those admitted at the weekend the fi gure was 57%. Reference 1. Royal College of Physicians. Acute medical care: the right person in the right setting-fi rst time. Report of the Acute Medicine Task Force. London: RCP; 2007. References 1. Gantner D, et al, Intensive Care Med. 2014;40:1528-35. 2. Flower M, et al, Crit Care. 2011;15:P464. R2 = 0.015 and 0.011, respectively). Transfer delays in the daytime and overnight were similar (Wilcoxon rank sum, P = 0.6).i P512 g Conclusion Interhospital transfers are subject to clinically signifi cant delays, and substantial travel distances. Delays are only weakly correlated to distances travelled and may refl ect delays resulting from organisational ineffi ciencies. We infer that eff orts to improve the effi ciency of transfer should focus on local organisational issues. There was no diff erence in the duration taken for overnight versus daytime transfers. R f Achieving a time to fi rst consultant review of under 12 hours for acutely ill medical patients y B Greatorex, EC Colley B Greatorex, EC Colley Figure 1 (abstract P510). Map of transfers. y Great Western Hospital, Swindon, UK p , , Critical Care 2015, 19(Suppl 1):P509 (doi: 10.1186/cc14589) Critical Care 2015, 19(Suppl 1):P509 (doi: 10.1186/cc14589) Introduction In 2007, the Acute Medicine Task Force made recommendations about the operation and staffi ng of acute medical units (AMU). Consultant-led care was seen as critical to ensuring high standards of care for patients and maintaining effi cient patient fl ow [1]. It also recommended that during the hours when the AMU is staff ed by a consultant, all new patients should be seen within 6 to 8 hours. Patients admitted overnight should have a consultant review within 12 to 14 hours. Following the introduction of a 4:00 pm consultant ward round of newly admitted acute medical patients to the existing 8:00 am and 2:00 pm rounds, it was our intention to establish whether our trust was meeting those recommendations. Methods We conducted a prospective survey of all new acute medical admissions over a 2-week period. Data collected included date and time of admission to the hospital, location on arrival, time of fi rst medical clerking, and time of fi rst consultant review. i Results Data were collected for 420 admissions. Sixty-seven percent of patients were admitted to the hospital between 12:00 am and 12:00 pm with a peak occurring between 4:00 pm and 6:00 pm. Sixty-two percent Figure 1 (abstract P510). Map of transfers. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S179 R2 = 0.015 and 0.011, respectively). Transfer delays in the daytime and overnight were similar (Wilcoxon rank sum, P = 0.6). Conclusion Interhospital transfers are subject to clinically signifi cant delays, and substantial travel distances. Delays are only weakly correlated to distances travelled and may refl ect delays resulting from organisational ineffi ciencies. We infer that eff orts to improve the effi ciency of transfer should focus on local organisational issues. There was no diff erence in the duration taken for overnight versus daytime transfers. References 1. Harris SK, et al. Intensive Care Med. 2014;40 Suppl 1:127. 2. www.r-project.org. 3 i Figure 2 (abstract P510). (a) Time duration versus geodesic distance (R2 = 0.015). (b) Time duration versus road distance (R2 = 0.011). in those patients who are discharged out of hours is nearly twice that of those discharged during the day [1]. Evaluation of patients with wild mushroom poisoning in the emergency department Introduction Wild mushroom poisoning (MP) is an important medical emergency that may have bad clinical outcome. We aimed to evaluate the clinical and laboratory features of patients with wild MP admitted to our emergency department in the Central Black Sea Region and to inform the emergency department physicians about early diagnosis and management of wild MP in the light of obtained data.i Achieving a time to fi rst consultant review of under 12 hours for acutely ill medical patients These results have been replicated in our institution with a mortality of 8.7% (discharged 22:00 to 06:59) versus 4.8% (discharged 07:00 to 21:59). In the UK, NICE CG50 advised that transfer from critical care to the ward out of hours should be avoided and documented as an adverse event. We postulated that one important factor in our hospital is the decreased medical and nursing cover overnight and so looked at the delay from discharge to fi rst medical review and to outreach review. in those patients who are discharged out of hours is nearly twice that of those discharged during the day [1]. These results have been replicated in our institution with a mortality of 8.7% (discharged 22:00 to 06:59) versus 4.8% (discharged 07:00 to 21:59). In the UK, NICE CG50 advised that transfer from critical care to the ward out of hours should be avoided and documented as an adverse event. We postulated that one important factor in our hospital is the decreased medical and nursing cover overnight and so looked at the delay from discharge to fi rst medical review and to outreach review. Methods The case notes of 100 consecutive patients discharged to the ward between September 2013 and October 2013 were examined to identify the time of discharge from the ICU and the subsequent fi rst review by the receiving medical team and the Critical Care Outreach team. The grade of the doctor reviewing the patient was recorded. Results Of these 100 patients, 22 were discharged between 22:00 and 07:59. From the 100 case notes requested, only 50 were available for examination. Forty patients were discharged to the wards, with only 37 having further documented medical reviews in the notes. Only 62% of patients were reviewed by a consultant following intensive care, with over 20% of patients waiting more than 24 hours for any medical review. During this time 18% of patients received a review by the nurse- led outreach team. See Figure 1. Conclusion It is clear that a highly vulnerable group of patients who are recovering from critical illness [2] receive inadequate early follow-up within the hospital. We postulate that the delay in medical review and the lack of senior review may be caused by over 40% being discharged overnight and contribute to the increased mortality seen in our institution and the ANZICS study [1] with nighttime discharges. References Delayed ICU discharges and medical follow-up: a cause of increased mortality? The BASIC Patient Safety course was only administered to staff from ICU1, and safety culture was assessed in both units before and after, using a survey based on the Hospital Survey on Patient Safety Culture [2]. Relative risk (95% CI) of improvement: baseline to follow-up in hospitals in patient safety domains, adjusted for duration of work in the unit (≤10  years vs. >10  years), was calculated. Responses were coded according to the Survey User’s Guide, and positive response percentages for each patient safety domain were compared with the 2012 Agency for Healthcare Research and Quality (AHRQ) ICU sample of 36,120 respondents. organizational culture to improve patient safety culture is considered important. We conducted a prospective, controlled study to assess the impact of a standardized patient safety course on an ICU’s patient safety culture, using a validated patient safety culture assessment tool. Methods Staff from two ICUs – ICU1 (tertiary referral hospital) and ICU2 (district hospital) – in Hong Kong were recruited to compare changes in the measured safety culture before and after a patient safety course. The BASIC Patient Safety course was only administered to staff from ICU1, and safety culture was assessed in both units before and after, using a survey based on the Hospital Survey on Patient Safety Culture [2]. Relative risk (95% CI) of improvement: baseline to follow-up in hospitals in patient safety domains, adjusted for duration of work in the unit (≤10  years vs. >10  years), was calculated. Responses were coded according to the Survey User’s Guide, and positive response percentages for each patient safety domain were compared with the 2012 Agency for Healthcare Research and Quality (AHRQ) ICU sample of 36,120 respondents. Conclusion Wild MP can cause bad clinical outcome. The public should be informed about the probable hazards of wild mushroom ingestion because collection and consumption of wild mushrooms from nature is common. Public health units should take protective precautions against wild MP. Education of health personals regarding MP will lead to successful results in patient management. Results Preintervention and postintervention period response rates for ICU1 were 88.1% (37/42) and 79.3% (23/29); and for ICU2 63% (20/32) and 63% (15/24). Delayed ICU discharges and medical follow-up: a cause of increased mortality? Methods This study was designed retrospectively by examining fi les of the patients with wild MP who were admitted to Ondokuz Mayis University Emergency Department, between January 2008 and December 2012. Patients older than 18 years were included in the study. Patients were evaluated according to gender, age, location, duration between mushroom intake and the start of clinical symptoms, time of application to hospital, clinical features and fi ndings and treatment method. The number of patients has been compared with the regional distribution of population, monthly temperature and average annual rainfall. y p , , Critical Care 2015, 19(Suppl 1):P511 (doi: 10.1186/cc14591) Introduction Discharge from intensive care is a potentially vulnerable time for patients who are recovering from critical illness. Recent data from the ANZICS group have highlighted that the mortality diff erence Figure 1 (abstract P511). S180 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results A total of 420 patients poisoned by wild mushrooms were studied. The male/female ratio was 1/1.5. The age of patients changed from 18 to 92 and mean age was 46 years. MP constituted 13.3% of all intoxication cases. The time when the fi rst symptom occurred after mushroom intake was a mean 2 (0.17 to 2.15) hours. Of the patients, 47.6% lived in villages, 38.6% in towns and 13.8% in city centers. Admissions were mostly made in autumn, with 57.6%. Eighty-six percent of intoxications happened because of wild mushrooms collected in nature. The most frequent symptoms were nausea (93.8%), and vomiting (87.1%). Increase in liver function tests in 47 patients was observed. Two of these patients died while 10 patients were transferred to further centers for liver transplantation. The remaining patients were discharged from the hospital. organizational culture to improve patient safety culture is considered important. We conducted a prospective, controlled study to assess the impact of a standardized patient safety course on an ICU’s patient safety culture, using a validated patient safety culture assessment tool. Methods Staff from two ICUs – ICU1 (tertiary referral hospital) and ICU2 (district hospital) – in Hong Kong were recruited to compare changes in the measured safety culture before and after a patient safety course. P513 Triage after drug overdose: eff ect of the introduction of a medical psychiatry unit on the allocation of ICU beds D Kleinloog, F Braam Houckgeest, D Sierink Tergooiziekenhuizen, Hilversum, the Netherlands Critical Care 2015, 19(Suppl 1):P513 (doi: 10.1186/cc14593) y y D Kleinloog, F Braam Houckgeest, D Sierink y y D Kleinloog, F Braam Houckgeest, D Sierink Tergooiziekenhuizen, Hilversum, the Netherlands g Critical Care 2015, 19(Suppl 1):P513 (doi: 10.1186/cc14593) Introduction Many patients with drug overdose are sedated, but do not have medical reasons to warrant ICU admission. Historically, monitoring behavior and suicide risk was done in the ICU, until the patient was awake enough for psychiatric consultation. Conclusion The study provides supportive evidence that a structured, reproducible short course on patient safety is associated with a general improvement in the ICU’s patient safety culture, measured with a validated safety culture assessment tool. Methods A medical psychiatry unit (MPU) was instituted as part of the Department of Clinical Psychiatry. For all patients with drug overdose in the emergency department, a risk assessment was made by the intensivist. Those without ICU indication (such as cardiac or respiratory monitoring) were admitted to the MPU. Alternatively, when awake enough, they were seen by the psychiatrist immediately. We performed an analysis of all patients with drug overdose, admitted to our ICU (before MPU n = 88, after MPU n = 191). We used the Welch t test for comparisons. Delayed ICU discharges and medical follow-up: a cause of increased mortality? Post intervention, compared with ICU2, ICU1 showed signifi cantly improved perceptions of teamwork within the hospital unit, RR (95% CI for diff erence between ICUs) 1.55 (1.10 to 2.19, P = 0.01); and overall perception of safety, 1.94 (1.11 to 3.37, P = 0.02); but not increased frequency of reporting mistakes, 0.90 (0.33 to 2.49, P = 0.84). Overall, ICU1 demonstrated a greater improvement in positive responses in fi ve safety culture domains than staff from ICU2. Patient safety culture indices were generally poorer in the two ICUs than the average ICU in the AHRQ database. Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Before MPU Since MPU (18 months) (51 months) Diff erence 95% CI P value Admissions per month 4.9 3.5 –1.4 –2.7 to –0.1 0.04 APACHE II score 8.4 12.5 +4.0 +1.8 to +6.2 0.0004 APACHE III score 30.9 40.3 + 9.4 +2.6 to +16.2 0.0069 Length of stay 0.8 1.3 + 0.5 –0.0 to +1.0 0.05 Conclusion Introduction of an MPU was associated with reduced numbers of patients with drug overdose admitted to the ICU. Those admitted to the ICU after the institution of the MPU were sicker, probably indicating more appropriate use of ICU beds. P514 Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Ling, G Joynt, A Lee, W Samy, H Fung, CD Gomersall The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) Introduction It is estimated that about one in 10 patients may be harmed by adverse events during their hospital stay [1]. Transforming Before MPU Since MPU (18 months) (51 months) Diff erence 95% CI P value Admissions per month 4.9 3.5 –1.4 –2.7 to –0.1 0.04 APACHE II score 8.4 12.5 +4.0 +1.8 to +6.2 0.0004 APACHE III score 30.9 40.3 + 9.4 +2.6 to +16.2 0.0069 Length of stay 0.8 1.3 + 0.5 –0.0 to +1.0 0.05 Methods Patients admitted to the ICU between 21 September 2014 and 2 October 2014 were included. If an investigation did not involve ionising radiation or was not requested by intensive care clinicians it was excluded. The indication for imaging was noted, and patient notes were analysed no less than 48 hours after the imaging was reported. Figure 1 (abstract P515). Conclusion Introduction of an MPU was associated with reduced numbers of patients with drug overdose admitted to the ICU. Those admitted to the ICU after the institution of the MPU were sicker, probably indicating more appropriate use of ICU beds. References 1. de Vries EN, et al. Qual Saf Health Care. 2008;17;216-23. 2. Nieva VF, et al. Qual Saf Health Care. 2003;12 Suppl 2:ii17-23. Audit of imaging documentation on an ICU l N Arora, SJ Glover p Results After institution of the MPU, there was a 28% reduction in the number of patients with drug overdose per month, admitted to the ICU. Also, patients admitted to the ICU were sicker and stayed longer (see Table 1). There were no patients admitted to the ICU after initial MPU admission. p pi Critical Care 2015, 19(Suppl 1):P515 (doi: 10.1186/cc14595) Introduction The Ionising Radiation (Medical Exposure) Regulations 2001 recommend to ‘ensure that a clinical evaluation of the outcome of each medical exposure is recorded’ [1]. This audit looked at whether ICU documentation of investigations involving ionising radiation could be improved. Anticipated benefi ts would be improved communication between the multidisciplinary team and better- informed decision-making. Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU P514 P514 Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Ling, G Joynt, A Lee, W Samy, H Fung, CD Gomersall The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) P516 Barriers to the implementation of checklists in the ICU: a survey on the perceptions of 314 Brazilian critical care nurses and physicians J Salluh1, W Viana2, F Machado3, A Cavalvanti4, F Bozza1, M Soares1 1IDOR, Rio de Janeiro, Brazil; 2Hospital Copa D’OR, Rio de Janeiro, Brazil; 3UNIFESP, São Paulo, Brazil; 4Hcor, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P516 (doi: 10.1186/cc14596) Introduction Checklists have been used increasingly in the ICU aiming at mitigating avoidable adverse events, but their content is variable and little is known about barriers to their full implementation. This survey reported practices on the use of checklists and perceptions regarding barriers on its implementation. Methods A web-based survey was conducted among a convenience sample of Brazilian ICU nurses and physicians between August and October 2014. Standard descriptive statistics were used. Results A total of 314 professionals, 104 nurses (33.1%) and 210 physicians (66.9%), responded. The majority (82.4%) had more than 5  years of experience in intensive care. Checklists were applied every day, including weekends, in only 49.8% (n  = 227). When we compared the barriers perceived by those working in smaller versus larger ICUs (<10 beds vs. >20 beds), the absence of a more comprehensive checklist content (93.6% vs. 81.9%, P = 0.026), the absence of specialized software or app (80.8% vs. 63.8%, P = 0.014), low availability of mobile devices (87.2% vs. 72.4%, P = 0.019), Internet unavailability (83.3% vs. 67.6%, P = 0.017), and the limited number of computers (88.5% vs. 76.2%, P = 0.0366) were the most often barriers to implementation. Checklists were applied with similar frequencies (<3 times a week, three to fi ve times a week, and every day, including on weekends, P  >0.05) regardless of the ICU size. When the type of tool used (paper vs. electronic) was considered, the main barrier highlighted was the lack of 100% Internet availability in the ICU (64.8% vs. 100%, P = 0.009). Users of paper form had higher demands for more comprehensive checklist content (84.3% vs. 63.6%, respectively, P = 0.037) and experienced more barriers to team adherence (98.1% vs. 86.4%, respectively, P = 0.034) as compared with those using specialized software. Conclusion Although checklists are recognized as valuable tools for the adherence to best practice in the ICU, it is diffi cult to ensure the uniformity of their daily use. Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Li G J A L W S H F CD G ll g y y g The Chinese University of Hong Kong, Shatin, Hong Kong y g g g g Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) Introduction It is estimated that about one in 10 patients may be harmed by adverse events during their hospital stay [1]. Transforming Figure 1 (abstract P515). S181 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 to concentrate, multitasking and the need to frequently change priorities in patient care-related procedures. It is therefore a continuous challenge to communicate and implement plans in an early, effi cient and reliable way within the team and to constantly keep track about tasks that are completed, postponed or that are about to be forgotten. To facilitate every caregiver’s workfl ow and to early and effi ciently communicate, we implemented the After Round Comprehensive Plan Summary (ARCoPS) tool. to concentrate, multitasking and the need to frequently change priorities in patient care-related procedures. It is therefore a continuous challenge to communicate and implement plans in an early, effi cient and reliable way within the team and to constantly keep track about tasks that are completed, postponed or that are about to be forgotten. To facilitate every caregiver’s workfl ow and to early and effi ciently communicate, we implemented the After Round Comprehensive Plan Summary (ARCoPS) tool. Results As shown in Figure 1, imaging requests were generally poorly documented (61%). In total, 17/26 (65%) chest X-rays (CXRs) were documented. A total of 0/2 CT scans were documented, despite one showing acute changes. In total, 17/20 (85%) CXRs requested following procedures carried out on ITU (such as insertion of central venous catheters) were documented, and the three not documented had no signifi cant fi ndings. The six other CXRs were requested to investigate worsening respiratory function. None were documented. Five had signifi cant fi ndings. y Methods Most of our patient care-related therapeutic and diagnostic decisions are made during the morning round consisting of intensivists, nursing team, surgeons, respiratory therapists, dieticians, clinical pharmacists and physiotherapists. P516 Resource limitations in smaller ICUs and the absence of comprehensive digital tools, mobile devices and Internet availability preclude full compliance at the bedside. P518 P518 Multicenter Thai university-based surgical ICU study (Thai-SICU study): adverse events and outcome in the SICU S Kongsayreepong, K Chittawatanarat Siriraj Hospital, Mahidol University, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P518 (doi: 10.1186/cc14598) Introduction The aim of this Thai-SICU was to study the incidence and outcome of adverse events in nine SICU university-based hospitals in Thailand. Introduction The aim of this Thai-SICU was to study the incidence and outcome of adverse events in nine SICU university-based hospitals in Thailand. Methods This multicenter prospective observational study was done in >18 years old, admission >6 hours surgical patients admitted to the large, postgraduate medical training university-based SICU during April 2011 to January 2012. Patient data were divided into three main phases as admission, discharge data and daily CRFs during the ICU stay. The patients were followed until they were discharged from the ICU or up to 28 days of their ICU admission and up to 28 days following discharge from the ICU if they survived. Results Following a 19.7-month recruitment period, a total of 4,654 patients (17,579 ICU-days) were included in the analysis process. Admitted patients had the median age of 64 years. Most of the patients were admitted directly from the OR for postoperative monitoring with median APACHE II score 10, 23% were admitted with priority I who needed aggressive hemodynamic resuscitation and respiratory support. ICU mortality and 28-day mortality were 9.61% and 13.80%. Each day of ICU increment was associated with a 1.38-day increase of hospital stay (95% CI, 1.24 to 1.53; P <0.01). On the surveillance periods, the six most common adverse events were sepsis (19.5%), AKI (16.9%), new cardiac arrhythmias (6.17%), respiratory failure (5.83%), cardiac arrest (4.86%) and delirium (3.5%) respectively. The other events including reintubation within 72 hours, intraabdominal hypertension, acute MI, unplanned extubation, upper GI hemorrhage, pneumohemothorax, seizure, drug error and pulmonary aspiration were <3% each. The risk Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Li G J A L W S H F CD G ll Immediately thereafter, all plans for the next 24-hour period are again discussed, summarized, confi rmed, prioritized and organized by the multidisciplinary team under aspects of optimal patient care and workfl ow effi ciency by entering the plan data into a fl at screen visualized template connected to our intranet immediately accessible at every physician and nursing caregiver workstation. ii Conclusion Investigations following procedures were generally well documented, but investigations seeking pathology were not documented at all, regardless of the fi ndings. This may have infl uenced the management of the patient and compromised patient safety. As such, the audit was presented at a departmental meeting to emphasise the importance of imaging documentation. A place for investigations was added to the ICU patient list to improve communication between the team, and a second audit is planned to assess the impact of this. Reference Results Twelve months after implementation of the ARCoPS tool we conducted caregiver interviews and identifi ed the following eff ects: increase in treatment confi dence through standardized multidisciplinary decision-making; reduced loss of information through immediate plan summarization by the whole team; reduction of ambiguity and misunderstanding about care plans through early written documentation; higher level of security not to forget procedures or tasks; positive acceptance of the tool to fl exibly change priorities of care procedures; positive acceptance of the tool to mark accomplished tasks providing visual feedback about the care plan status; individual caregiver workfl ow economization through permanent treatment plan availability; and higher job satisfaction throughout the caregiver team. Conclusion ARCoPS tool utilization has become a daily routine in our ICU. It functions as an eff ective communication and workfl ow tool and has helped us to reduce patient care-related misunderstandings and delays. It also enhanced the economics of our work sequence, which also highly contributes to a better level of patient safety. Furthermore, it has markedly contributed to an improved level of quality of work for caregivers. 1. The ionising radiation (medical exposure) regulations 2000. London: Stationery Offi ce; 2000. 1. The ionising radiation (medical exposure) regulations 2000. London: Stationery Offi ce; 2000. P517 After Round Comprehensive Plan Summary tool: an effi ciency approach for the ICU R Marktanner, M Mostafa, A Shafei, H Hon, R Ashraf, P Sreedhara, N Syed, D Gharaibeh, A Taha Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P517 (doi: 10.1186/cc14597) Introduction ICU workfl ow for physicians, nurses and other healthcare providers is often complicated by distractions, interruptions, diffi culties Introduction ICU workfl ow for physicians, nurses and other healthcare providers is often complicated by distractions, interruptions, diffi culties S182 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We performed an observational study in a 17-bed, mixed clinical and surgical emergency department observation. Before the year 2012, the staff were composed of fi rst-year internal medicine and surgical residents. There were no senior physicians specifi cally assigned to this unit, and residents were supervised by the members of the emergency department team, who changed shifts on a daily basis. In February 2012, two senior physicians (one cardiologist and one intensive care doctor) were specifi cally assigned to supervise the internal medicine residents and provide horizontal care for medical patients during the day, from Monday to Friday. There was no change in assistance for surgical patients. The schedule for nights and weekends remained unchanged. Mortality and length of stay for medical and surgical patients were measured in 2011, 2012 and 2013.i of adverse events on 28-day mortality were signifi cant on cardiac arrest (RR = 9.45; P <0.01), ARDS (RR = 4.58; P <0.01), AKI (RR = 4.18; P <0.01), sepsis (RR = 3.62; P <0.01), iatrogenic pneumohemothorax (RR = 3.23; P <0.01), seizure (RR = 3.12; P <0.01), upper GI hemorrhage (RR = 2.97; P <0.01), cardiac arrhythmia (RR = 2.91; P <0.01), ALI (RR = 2.71; P <0.01), delirium (RR  = 2.13; P  <0.01), MI (RR  = 2.12; P  <0.01), unplanned extubation (RR = 2.06; P <0.01), abdominal hypertension (RR = 1.75; P <0.01) and reintubation within 72 hours (RR = 1.51; P = 0.02). Conclusion This is the largest systemic surveillance observation in the SICU. The study results are the reference for future research and also provide information for patient and relative advice when confronted with adverse events during SICU admission. P521 Results There were 6,203 surgical admissions during the 8-year study period. For both ICU and in-hospital mortality; the median LOS in days for survivors was 2.2 (IQR: 1.2 to 4.9) and that of nonsurvivors was 3.2 (IQR: 1.5 to 7.9). At 28 days, 1 year, and 2 years, the respective values were 2.2 (1.2 to 4.9) and 3.3 (1.5 to 6.7); 2.1 (1.2 to 4.7) and 3.1 (1.5 to 7.3); and 2.1 (1.2 to 4.6) and 3.0 (1.5 to 7.0), all P <0.0001. The greatest median LOS was found in neurosurgery and cardiothoracic surgery; 3.3 days (IQR: 1.7 to 9.5) and 3.1 days (IQR: 1.5 to 8.0) respectively. They corresponded to the specialities with the greatest percentage ICU (9.7% and 10.2%) and 2-year mortality (27.9%, and 35%). The greatest mortality and median LOS was found in ventriculostomy cases; 40.8% at 2 years and 10.6 days (IQR: 4.8 to 18.2). Retrospective observational cohort study of mortality and length of stay for surgical ICU admissions Introduction A signifi cant number require a postoperative ICU stay which may be prolonged [1]. Very limited data exist to characterise mortality and ICU length of stay (LOS) in diff erent surgical specialties [2,3]. We aim to describe this relationship in a large cohort of surgical patients. Conclusion Emergency department observation units are an alternative to alleviate emergency room overcrowding when there are no intensive care beds available. However, patients end up staying in those units for days and horizontal care by senior doctors may improve outcomes. p Methods We performed a retrospective observational cohort study of adult surgical admissions to the ICU of a large academic tertiary medical centre. The primary endpoint was 28-day mortality. We used simple descriptive statistics to characterise the population. The Wilcoxon rank- sum test or Kruskall–Wallis test was used, as appropriate, for tests of continuous data. Rejection for ICU admission: analysis and results of this modality of limitation of therapeutic eff ort Williams TA, et al. Br J Anaesth. 2010:104:459-64. P517 Results In the fi rst year after the implementation of the clinic intensive care team, mortality in internal medicine patients decreased from 47% to 34% in 2012 and 33% in 2013. Although other changes happened in this period (the number of beds decreased from 24 to 17, nurses and physical therapists were hired and trained specifi cally for this unit), we believe the horizontal staff was critical, because mortality between surgical patients remained almost unchanged in the same period of time (23% in 2011, 22% in 2012 and 23% in 2013), in spite of the structural improvements that equally aff ected those patients. Length of stay decreased from 6.35 days in 2011 to 3.43 in 2012 and 3.15 in 2013 in medical patients and from 3.9 days on average in 2011 to 3.2 in 2012 and 2.8 in 2013 in surgical patients. Rejection for ICU admission: analysis and results of this modality of limitation of therapeutic eff ort pf F Acosta, O Moreno, M Muñoz, R Fernandez, M Colmenero Hospital Universitario San Cecilio, Granada, Spain Critical Care 2015, 19(Suppl 1):P521 (doi: 10.1186/cc14601) Critical Care 2015, 19(Suppl 1):P521 (doi: 10.1186/cc14601) Introduction The aim was to know the frequency, criteria and implications of rejection for ICU admission to our ICU unit, a second- level hospital (18 beds). Introduction The aim was to know the frequency, criteria and implications of rejection for ICU admission to our ICU unit, a second- level hospital (18 beds). Methods An observational retrospective study in a time interval of 6  months (January to June 2013). We retrospectively registered all patients rejected for admission to our unit, analyzing the clinical rejection report used in our hospital. From this report we extracted diff erent variables: demographical (age, sex), provenance (emergency room, hospital), clinical (comorbidity, functional situation, diagnosis, reason of requesting admission), rejection motive (‘too good’, ‘too bad’, futility, lack of beds, patient rejection), whether it was defi nitive or conditional, whether the patient was admitted afterwards, and the state at hospital discharge. We realized a descriptive analysis (frequencies) and multivariant analysis of the factors related to futility rejection. Conclusion There is an association between postoperative ICU LOS and mortality that persists for at least 2 years after admission. Neurosurgery and cardiothoracic surgery patients appear to have a worse prognosis and also a more prolonged LOS. Our results may provide a more objective basis for clinical decisions, the use of limited resources, and inform on appropriate expectations of treatment. 1. Pearse RM, et al. Lancet. 2012:380:1059-65. y y j Results There were 165 rejections, which represents 25% of total ICU patients evaluated for admission. A total of 59.4% were male. Mean age was 69 ± 7 years (19 to 98). In total, 53.9% had more than two comorbidities (pluripathological) and 31.5% moderate to severe functional disability. The cause of rejection was in 55.2% of situations that the patient was ‘too good’, 37.6% related to qualitative futility, 4.8% was ‘too bad’ and in 1.2% a mix of lack of space (beds) and patient rejection. In the multivariant analysis the signifi cant variables related to futility rejection were age (by years) with an OR of 1.05 (1.02 to 1.08), severe functional disability, OR of 4.35 (2.09 to 9.06), and the hospital provenance with an OR of 2.82 (1.1 to 7.2). 2. Timmers TK, et al. Ann Surg. 2011:253:151-7. 3. References 1. Komindr A, et al. Int J Emerg Med. 2014;5:1-6. 1. Komindr A, et al. Int J Emerg Med. 2014;5:1-6. 2. Baugh C, et al. Health Care Manag Rev. 2011;36:28-37. 2. Baugh C, et al. Health Care Manag Rev. 2011;36:28-37. P519 Retrospective observational cohort study of mortality and length of stay for surgical ICU admissions M Hameed1, M Maruthappu2, D Marshall3, M Pimentel1, L Celi4, J Salciccioli3, J Shalhoub3 1University of Oxford, UK; 2National Institute for Health and Care Excellence, London, UK; 3Imperial College London, UK; 4Massachusetts Institute of Technology, Cambridge, MA, USA Critical Care 2015, 19(Suppl 1):P519 (doi: 10.1186/cc14599) P522 Impact of age, physiological status and APACHE score on acceptance of patients to the ICU Introduction Evidence suggests that age, chronic health status and acute illness severity aff ect the decision-making of clinicians regarding admission to the ICU (ITU) [1-3]. This prospective service review assesses the impact of age, APACHE II score and WHO functional score towards admission acceptance or refusal to ITU in a tertiary-level facility. p Results Following implementation of the new transfer process, the average wait time decreased by 58 minutes, process time decreased by 9 minutes, and lead time decreased by 66.5 minutes. The handoff error rate decreased by 1.3 errors/patient and fi rst-time quality rate increased by 67%. Staff satisfaction improved from 48% to 73%. By elimination of the PACU stay, the costs involved in admission to the PACU were deferred. admission acceptance or refusal to ITU in a tertiary-level facility. Methods Design: a planned prospective review of all referrals over a 14- day period. Data collection: review (LT, DP, SP) of case notes of patients referred to ITU with the following variables collected: age, sex, APACHE II scores, WHO functional status score, grade of referrer and source of referral. Data were collected on 37 patients: 22 accepted to ITU and 15 refused admission. Statistics: data were analyzed using GraphPad 6.05. Categorical variables were expressed as mean and standard error of mean. For unpaired variables, statistical signifi cance is determined using unpaired t test. P <0.05 is considered statistically signifi cant. Results The WHO functional status was the most signifi cant variable aff ecting admission (P <0.001). The APACHE score of patients admitted to ITU was signifi cantly lower than refused patients (P = 0.039). Patient age did not aff ect admission status (P = 0.15). See Table 1. Methods Design: a planned prospective review of all referrals over a 14- day period. Data collection: review (LT, DP, SP) of case notes of patients referred to ITU with the following variables collected: age, sex, APACHE II scores, WHO functional status score, grade of referrer and source of referral. Data were collected on 37 patients: 22 accepted to ITU and 15 refused admission. Statistics: data were analyzed using GraphPad 6.05. Categorical variables were expressed as mean and standard error of mean. For unpaired variables, statistical signifi cance is determined using unpaired t test. P <0.05 is considered statistically signifi cant.i Conclusion A single, multidisciplinary bedside handoff process between the OR and ICU leads to cost and time savings. Improved interprofessional communication, handover and ward rounds in critical care (ICARUS) Introduction There is growing evidence that optimising communication and patient assessment practices including ward rounds and handoff s can improve clinical, safety and operational outcomes, particularly in the critical care setting [1,2]. Here we describe the design and baseline phases of a 5-year project utilising improvement sciences to optimise the quality of interprofessional communication, handoff s and rounding in one of the largest critical care units in the UK. Conclusion This study was performed to assess decision-making for admission to the ITU in times of increased demand and limited availability. We want to validate our fi ndings from this short pilot study in a larger prospective study. Multivariate regression analysis will be used to identify signifi cant features that aff ect clinician decision- making in critical care admission. Methods We obtained institutional ethical and research approvals. We used mixed methods including interviews of opinion leaders and a representative cross-section of staff , roundtables, a survey targeting the whole critical care team (n = 546) and a Delphi exercise to generate a baseline consensus for the need to improve and a set of novel quality improvement interventions and tools. We tested two of these in a pilot plan–do–study–act (PDSA) cycle. g References References 1. Reignier J, et al. Crit Care Med. 2008;36:2076-83. 2. Garrouste-Orgeas M, et al. Crit Care Med. 2005;33:750-5. 3. Azoulay E, et al. Crit Care Med. 2001;29:2132-6. References 1. Reignier J, et al. Crit Care Med. 2008;36:2076-83. 2. Garrouste-Orgeas M, et al. Crit Care Med. 2005;33:750-5. 3. Azoulay E, et al. Crit Care Med. 2001;29:2132-6. Results Baseline consensus for the need and potential to improve was obtained (97.4% and 94.5%). Despite a large degree of heterogeneity of perceptions around current communication and rounding practices, it was possible to develop a set of interventions based on consensus that could be applied in this complex setting. These included a handoff bundle, an operational touch-base, a unit-level safety briefi ng, a unit- level safety check, a lean rounding bundle and board rounds. These core interventions were supported by several more detailed resources describing the evidence base around best handoff and rounding practices; and a feedback document that described all outputs and recommendations from the ICARUS project. A pilot PDSA cycle demonstrated a 55.3% and 76.3% improvement in key information transfer using a safety briefi ng and board round summary. P522 Impact of age, physiological status and APACHE score on acceptance of patients to the ICU By elimination of redundant, nonvalue-added processes, less opportunity for medical errors occurred, with substantial improvements in fi rst-time quality. Such a process can be successfully attained while aff ecting staff satisfaction positively. g p y gi Results The WHO functional status was the most signifi cant variable aff ecting admission (P <0.001). The APACHE score of patients admitted to ITU was signifi cantly lower than refused patients (P = 0.039). Patient age did not aff ect admission status (P = 0.15). See Table 1. Table 1 (abstract P522) Accepted Refused P value Mean age 56.5 ± 4.4 65.3 ± 3.6 0.15 Sex 73% male 56% male N/A WHO 0.45 ± 0.2 2.21 ± 0.3 <0.001 APACHE 13.5 ± 1.6 18.8 ± 2.0 0.039 5 Improved interprofessional communication, handover and ward rounds in critical care (ICARUS) P Hopkins, A Chan, S Rasoli, C Bell, K Peters, A Feehan, J Dawson King’s Health Partners AHSC, London, UK Critical Care 2015, 19(Suppl 1):P524 (doi: 10.1186/cc14604) Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness f SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) Introduction This Six Sigma project was initiated to evaluate and improve the transfer of care of patients from the OR to the ICU. Medical errors are responsible for billions of dollars in increased healthcare spending. Miscommunication among healthcare providers is a major contributor to these errors, with handoff s a particularly vulnerable period in the care process. At our institution, surgical patients with scheduled admissions to the ICU are fi rst recovered in the postanesthesia care unit (PACU). With this process, multiple, unstructured, and individual handoff s occur in parallel between providers, which may lead to communication errors, diff erential information sharing, content variability, care delays, and ineffi ciency. Conclusion Despite wide heterogeneity in baseline beliefs, opinions and perceptions around inter-professional communication, rounding and handoff s, we were able to develop a novel set of feasible quality improvement interventions, targeting these areas, that can be applied in a large complex critical care setting. Furthermore, they can be driven by improvement science methodology and tested for eff ectiveness using qualitative and quantitative measures. We now plan to use these interventions to deliver quality improvements in communication practices in parallel with the planning and implementation phases of a new critical care facility and electronic clinical information system. R f Implementation of a horizontal intensive care team can impact effi ciency in an emergency observation unit in Brazil S Ribeiro, C Petrini, L Marino, R Brandao-Neto Hospital das Clinicas School of Medicine, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P520 (doi: 10.1186/cc14600) fi y g y S Ribeiro, C Petrini, L Marino, R Brandao-Neto Hospital das Clinicas School of Medicine, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P520 (doi: 10.1186/cc14600) Introduction Emergency department observation units are often used to monitor critical patients in a situation of constant emergency department overcrowding and lack of intensive care beds. However, those units are often understaff ed and might not have enough personnel and equipment to provide the same quality of care as a conventional ICU. Conclusion Rejection for admission to ICU units is a frequent medical activity in our day-to-day job. The type of patient most rejected is cardiologic, mostly evaluated for thoracic pain probably ischemic but with low risk. In second place we found patients for which we decide rejection based on subjective qualitative futility, related mostly to age, prior functional disability and provenance. S183 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P522 were conducted, which began by defi ning the current process via direct observation and value stream mapping of orthopedic and neurosurgical patients. A new process was then introduced, including direct transfer of the patient to the ICU and a single, structured, bedside report between all care providers. A standardized handoff tool was implemented. We used process times, wait times and information content as process measures and handoff errors as outcome measures. A 10-point satisfaction score was also measured. were conducted, which began by defi ning the current process via direct observation and value stream mapping of orthopedic and neurosurgical patients. A new process was then introduced, including direct transfer of the patient to the ICU and a single, structured, bedside report between all care providers. A standardized handoff tool was implemented. We used process times, wait times and information content as process measures and handoff errors as outcome measures. A 10-point satisfaction score was also measured. P522 Impact of age, physiological status and APACHE score on acceptance of patients to the ICU L Terry, S Passey, D Porter, F Clark, R Matsa Royal Stoke University Hospital, Stoke on Trent, UK Critical Care 2015, 19(Suppl 1):P522 (doi: 10.1186/cc14602) P523 P523 Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) P523 Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) P526 Out-of-hours discharge from critical care: does it matter? M Ahmed, G Lumley, S Nourse, A Hurding, A Thomas, M Healy The Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P526 (doi: 10.1186/cc14606) Introduction The aim of this audit was to assess the clinical impact of out-of-hours (OOH) discharge from the adult critical care unit (ACCU) in a tertiary care hospital. Discharging patients from critical care OOH has been associated with both higher mortality and increased rate of readmissions [1,2]. As a result, national guidance stipulates that OOH discharges from critical care should be avoided [3]. Results A total of 2,248 emergency admissions were analysed. The majority of patients were admitted within 1 week of hospital admission (n = 1,897). They were younger and had lower APACHE II scores (15 vs. 19; P <0.001). APACHE II scores were the same in all other groups (groups 2 to 5). Patients admitted to the ICU 3 weeks following hospital admission had signifi cantly higher hospital mortality (up to 50%; P <0.001) and ICU length of stay (12 ± 15 vs. 8 ± 10 days; P <0.001). ICU mortality remained the same in all groups (20 to 28%). ICNARC and SOFA scores were equal between the groups. The post-ICU lengths of stay were signifi cantly longer in groups 3 to 5. In-hospital CPR prior to admission to ICU was lower in patients from groups 4 and 5, probably signifying appropriate DNAR decisions made on the ward. Methods A retrospective snapshot analysis of patients was conducted at least 48  hours after discharge from the ACCU in October 2014. Patients discharged OOH (20:00 to 07:59) were compared with those discharged in hours (IH; 08:00 to 19:59). Analysis included: patient demographics, APACHE II score, handover procedure prior to discharge, follow-up by the receiving team, appropriate and timely drug prescribing, recognising and acting upon clinical deterioration and readmission rates. Conclusion Prolonged pre-ICU hospital admission is associated with higher hospital but not ICU mortality. Commonly measured scores do not refl ect this higher mortality in patients admitted after prolonged stay in hospital. Further research into parameters that may refl ect changes in physiological reserves may strengthen these scores for such patients. Results A total of 161 patients were discharged from the ACCU in October 2014. Of these, 46% of the patients were discharged OOH. P526 Forty-one (of 74) OOH and 19 (of 87) IH discharges were sampled for further analysis. The baseline demographics and APACHE II score were similar between both groups. The majority of discharges were delayed (>4 hours, 90% IH vs. 93% OOH). More patients had nursing handover completed if discharged IH (88% IH vs. 61% OOH) and doctors’ summaries were less frequently completed OOH (83% OOH vs. 94.4% IH). A management plan for the ward was outlined in 94% Pre-ICU length of hospital stay is a predictor of hospital but not ICU mortality y K Flavin, D Hall, G Marshall, P Zolfaghari g Royal London Hospital, London, UK Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P527 (doi: 10.1186/cc14607) Introduction Prolonged hospital stay prior to admission to the ICU was previously shown to be independently associated with poorer outcome [1,2]. This is probably due to slow deterioration of physiological function in hospital and infl uenced by processes leading to critical care admission [2]. We investigated whether commonly measured severity scoring systems (Acute Physiology and Chronic Health Evaluation (APACHE) II, Intensive Care National Audit and Research Centre (ICNARC) and Sequential Organ Failure Assessment (SOFA) scores) are signifi cantly diff erent in patients admitted with prolonged pre-ICU hospital length of stay, and describe mortality and hospital length of stay. Conclusion There is a higher prevalence of adverse events in readmitted patients with similarity in the type of adverse eff ects despite readmission. g References 1. Priestap F, et al. Crit Care Med. 2006;34:2946-51. p 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 3. Acutely ill patients in hospital. NICE; 2007 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 3 Acutely ill patients in hospital NICE; 2007 gii Results The readmission rate was 15% (n = 392), particularly among males (55% (n  = 214)). A 14.3% adverse event rate was observed among the readmitted patients (9.5% among those who were not readmitted). The readmitted patients were older (median (md): 68.0 (95% CI: 65.7 to 67.3) vs. md: 71.0 (95% CI: 67.1 to 71.3); P <0.05) and remained hospitalized for a longer period of time (md: 5.0 (95% CI: 13.2 to 20.7) vs. md: 11.0 (95% CI: 17.0 to 33.2); P <0.05), but not necessarily in the CICU (P = 106).The most prevalent adverse events in readmitted patients were pressure ulcers (n = 16 (4.1%)), drug administration error (n = 13 (3.3%)) and enteral feeding tube (n = 10 (2.6%)). Meanwhile, among the nonreadmitted, phlebitis due to peripheral vein access and pressure ulcers (n = 42 (1.9%)), drug administration error (n = 41 (1.9%)) and enteral feeding tube (n = 31 (1.4%)). There was a tendency (P = 0.71) that readmitted patients were presented with higher prevalence of pressure ulcers (n = 16 (4.1%) vs. n = 42 (1.9%)). 3. Acutely ill patients in hospital. NICE; 2007 Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) Introduction Security policies for the patient are of interest to any health professional. The rates of adverse events in hospitals reach values ranging between 3.7 and 16.6%, being the highest range (40 to 70%) considered preventable or avoidable [1]. The objective of this study is to assess the prevalence and types of adverse eff ects in intensive care patients according to readmission. g p Conclusion This audit compares with current evidence suggesting harm from OOH discharge despite most discharges being delayed. Discharging patients is a complex hospital process, but focus needs to be on discharging patients IH. Therefore, areas for improvement include targeting forward fl ow of patients throughout the hospital, completion of handover documents IH and publication of guidance for receiving teams. Methods During 4 years the data from 2,582 patients admitted to the coronary ICU (CICU) were analyzed in the coronary care unit of the city of Presidente Prudente, Brazil. We analyzed the rate of readmissions, length of stay and the mainly detected adverse events. We considered signifi cant P <0.05 two-tailed and confi dence intervals at 95% (CI). p Reference 1. Forster AJ, et al. CMAJ. 2004;170:345-9. 1. Forster AJ, et al. CMAJ. 2004;170:345-9. y Methods A retrospective analysis of prospectively collected data of all emergency admissions in the ICNARC database of a 44-bed adult critical care unit within a major trauma centre over a 2-year period. Demographic data, APACHE II score, SOFA score, ICNARC model score, mortality, and length of hospital stay prior to and after ICU admission were collected. Five groups of patients were defi ned as follows: those admitted to ICU within 1 week of hospitalization (group 1), within 8 to 14 days (group 2), within 15 to 21 days (group 3), within 22 to 28 days (group 4), and more than 28 days (group 5). Chi-squared and ANOVA tests were performed using the SOFA statistics package. References Methods A multidisciplinary QI project was initiated with input from the ICU, anesthesia and surgical services. A series of PDSA cycles Lane D. Crit Care Med. 2013;41:2015. Starmer AJ. N Engl J Med. 2014;371:1803. S184 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of IH versus 65.% of OOH discharges. Seventy-eight per cent OOH versus 95% IH patients discharged were reviewed by a doctor within 24 hours. Twenty-nine per cent OOH versus 67% IH patients discharged were reviewed by a consultant within 24 hours. Following discharge a management plan was followed in 94% of IH patients versus 44% OOH, patients had drug charts correctly charted in 100% of IH cases versus 66% OOH and missed/delayed doses were documented in 11.1% of IH cases versus 61% OOH. There was no diff erence between the groups in incidence of clinical deterioration and recognition and follow-up of clinical deterioration. Two patients were readmitted within 48 hours from the OOH group. of IH versus 65.% of OOH discharges. Seventy-eight per cent OOH versus 95% IH patients discharged were reviewed by a doctor within 24 hours. Twenty-nine per cent OOH versus 67% IH patients discharged were reviewed by a consultant within 24 hours. Following discharge a management plan was followed in 94% of IH patients versus 44% OOH, patients had drug charts correctly charted in 100% of IH cases versus 66% OOH and missed/delayed doses were documented in 11.1% of IH cases versus 61% OOH. There was no diff erence between the groups in incidence of clinical deterioration and recognition and follow-up of clinical deterioration. Two patients were readmitted within 48 hours from the OOH group. P525 Adverse events in patients in a Brazilian intensive care unit according to readmission GM Plantier1, CE Bosso1, AC Correa2, BN Azevedo3, JS Alves2, CD Monteiro2, SV Ferreira3, GE Valerio3, JV Moraes3, V Raso3 1Instituto do Coração de Presidente Prudente, Brazil; 2Santa Casa de Presidente Prudente, Brazil; 3Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) Higgins TL, et al. Crit Care Med. 2003;31:45-51. Goldhill DR, et al. Intensive Care Med. 2004;30:1908-13. P525 Adverse events in patients in a Brazilian intensive care unit according to readmission Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) F Kavanagh, I Browne g National Maternity Hospital, Dublin, Ireland g National Maternity Hospital, Dublin, Ireland y p Critical Care 2015, 19(Suppl 1):P530 (doi: 10.1186/cc14610) y p Critical Care 2015, 19(Suppl 1):P530 (doi: 10.1186/cc14610) Introduction Pregnancy and labour usually progress uneventfully; however, serious complications can occur and develop rapidly, necessitating critical care admission and support. Successive confi dential enquires have highlighted defi ciencies in this area and suboptimal care leading to increased morbidity and mortality [1]. The National Maternity Hospital is the largest maternity hospital in the Republic of Ireland where a total of 8,954 babies were delivered in 2013. It is a stand-alone institution and onsite facilities include a two- bed dedicated anaesthesia lead high-dependency unit. Introduction Hospital costs are a constant concern within health, especially in the ICU. Hospital admissions and average life expectancy have been growing gradually mainly in older and critical patients. This study is aimed to observe the direct costs of patients admitted to an ICU and their relation to the SAPS 3, length of stay in the ICU and fi nal outcome. Methods A retrospective observational study in which the direct costs were studied (materials, medicines, oxygen therapy and hospital fees) for 1,790 ICU patients from November 2013 to November 2014. The readmissions within 48 hours were excluded and also 10% of patients who had the highest and lowest costs. The remaining 1,401 patients were divided by age groups. Methods A retrospective observational study was carried out from January 2011 to January 2014 especially looking at the following parameters: admitting diagnosis, demographics, length of stay and the number of admissions requiring transfer for tertiary-level care. y g g Results Of the patients studied, 54.6% were male. Average age was 57.8 years (18 to 105 years). The biggest ICU average cost was in the group of patients 81 to 90 years old (US$793.00), as well as longest ICU stay (9.25 days), highest SAPS 3 (53.96) and higher ICU and in-hospital mortality (14.29% and 19.25% respectively). This study shows that the direct cost of the ICU stay for older patients was higher than for younger patients. The diff erence was explained by the higher severity measured by SAPS 3 in the older age groups (Figure 1), and the required greater length of stay in the ICU (Figure 2). As might be expected, the mortality in the group of older patients was also signifi cantly higher. References Higgins TL, et al. Crit Care Med. 2003;31:45-51. Goldhill DR, et al. Intensive Care Med. 2004;30:1908-13. S185 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P528 Reference 1. Special issue: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–2008. The Eighth Report of the Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG. 2011;118 Suppl S1:1-203. 1. Special issue: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–2008. The Eighth Report of the Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG. 2011;118 Suppl S1:1-203. P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) q g y Results In total, 29,344 deliveries occurred. A total of 376 HDU admissions were recorded in this period, representing 1.28% of all admissions. The average age of patients admitted to the HDU was 34 years. The predominant reasons for admission were hypertensive disease of pregnancy (49.7%), haemorrhage (antepartum/postpartum) (36.4%), sepsis (4.2%) and other reasons (11.1%) including cardiac rhythm disturbances, neurological complications and pre-existing medical disease. In 2013 the average length of stay was 2 days. A total 6.1% of those admitted to HDU required transfer for tertiary-level ICU care in other centres during the study period, this represented 0.07% of all deliveries.i Conclusion This study showed that greater age is associated with higher severity measured by SAPS 3, higher direct costs, and higher mortality both in the ICU and in-hospital environment. Conclusion There is signifi cant demand within our institution for HDU care for our patients, with the number of admissions increasing in 2013. The main admitting diagnoses are hypertensive disease of pregnancy and haemorrhage with an increase in the number of patients being admitted for management of sepsis in 2013. This highlights the increasing awareness, recognition and management of this condition in pregnancy. The increased number of HDU admissions in 2013 could also be explained by the recent introduction of an early warning score for the deteriorating patient in our hospital but this would require further evaluation. The low number of transfers of patients to other tertiary centres underpins the importance of an anaesthesia lead service. Figure 1 (abstract P528). Figure 2 (abstract P528). Figure 1 (abstract P528). Figure 1 (abstract P528). Reference P530 Critical care in a maternity hospital: a retrospective observational study P530 Critical care in a maternity hospital: a retrospective observational study P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) P531 Post-traumatic stress symptoms in intensive care staff working in adult and paediatric settings G Colville1, J Hammond1, L Perkins-Porras2 1St George’s Hospital, London, UK; 2St George’s University of London, UK Critical Care 2015, 19(Suppl 1):P531 (doi: 10.1186/cc14611) P534 P534 Mortality in a French military burn centre: a 12-year retrospective study analysing seasonal variations M Boutonnet1, C Dussault2, N Donat1, P Laitselart1, A Cirodde1, J Schaal1, C Hoff mann1, P Jault1, T Leclerc1 1HIA Percy, Clamart, France; 2IRBA, Brétigny sur Orge, France Critical Care 2015, 19(Suppl 1):P534 (doi: 10.1186/cc14614) f Methods After local ethics committee approval, a total of 1,004 patients (2012, n = 492; 2013, n = 454; 2014, n = 58) treated in the ICU in a 2-year period were reviewed. This study included for evaluation 135 patients determined with traumatic or nontraumatic brain damage, with a more than 24-hour stay in the ICU. Reasons for brain damage were determined as brain damage associated with head trauma (Group HT), head trauma accompanying general body trauma (Group HT  + GBT) and spontaneous haemorrhage (Group SH). The type of brain damage was defi ned from the radiological diagnosis (CT and/or MRI) as subarachnoid haemorrhage, intracranial haemorrhage (ICH), subdural haematoma (SDH), epidural haematoma (EDH), skull fracture, brain contusion or a combination of these (COM). Operations were evaluated as performed by the brain surgery department. Introduction Factors associated with mortality in severely burned patients are well known. We aimed to assess the seasonal variations of the mortality of patients admitted to the main French military intensive care burn unit (ICBU) and to determine their relations to seasonal variations of severity or other factors (for example, staffi ng). Methods We performed a retrospective study analysing the medical records of all patients admitted to the ICBU of Percy Military Hospital (France) between January 2000 and December 2011. Statistical analysis was performed with R version 3.1.2. We fi rst conducted univariate analyses with simple logistic regression, then a simple periodic regression for seasonal variations (with and without harmonics for robustness), and fi nally a multivariate logistic regression with periodic terms in order to adjust seasonal variations for known severity factors. Results A total of 1,913 patients were admitted for acute burn injury during the study period, with a male to female ratio of 2.34. Mean total body surface area burned (TBSA) was 23.2% (IQR: 10 to 30) and mean full thickness total body surface area burned (FTBSA) was 13.4% (IQR: 0 to 15). Association with amount of registration and outcome in pediatric severe trauma patients S Ohnishi, N Saito, T Yagi, Y Konda, Y Hara, H Matsumoto Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan Critical Care 2015, 19(Suppl 1):P533 (doi: 10.1186/cc14613) Figure 1 (abstract P531). Main themes of staff descriptions of traumatic events on ICU. Introduction Unexpected trauma is a one of the most major causes of death for children in the world. However, pediatric severe trauma patients are rare and scattered. Although there is a strong association between patient’s volume of trauma center volume and outcomes in adults, such a report is less in children. In this study, we aimed to clarify the relationship of the amount of registration and outcome in pediatric trauma patients. most commonly endorsed: patient death (and particularly untimely deaths on adult units); handling distressed families; and concerns about the quality of care and dealing with staff confl ict (see Figure 1). Conclusion A signifi cant minority of staff reported clinically signifi cant levels of post-traumatic stress related to their work. The facts that post- traumatic stress levels were higher on paediatric units despite their lower rates of mortality and that untimely deaths were frequently mentioned by adult unit staff suggest it may be that untimely deaths are particularly hard to deal with. More research is needed on the prevalence of distress in staff working in these settings. Methods This retrospective study analyzed data of the Japan Trauma Data Bank from 2004 to 2012. We registered pediatric patients aged younger than 12  years with severe multiple or torso trauma (maximum Abbreviated Injury Scale score ≥3 or Injury Severity Score ≥9, and excluded patients with cardiopulmonary arrest on arrival, burn, isolated head or limb injury, lack of data). We divided the facilities into six groups according to every 10 patients registered and compared mortality between each group. Results A total of 1,015 patients from 105 facilities were included in the study. Number of registrations: 0 to 10 patients: 673 patients/68 facilities, 11 to 20: 381/28, 21 to 30: 141/6, 31 to 40: 101/3, 41 to 50: 45/1, 51 to 60: 58/1. Victims of blunt trauma accounted for 98.1%. Median age was 7 (interquartile range: 5 to 10) years, median injury severity score (ISS) was 17 (10 to 26). P533 P533 Association with amount of registration and outcome in pediatric severe trauma patients S Ohnishi, N Saito, T Yagi, Y Konda, Y Hara, H Matsumoto Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan Critical Care 2015, 19(Suppl 1):P533 (doi: 10.1186/cc14613) Distribution and mortality factors of cases with traumatic and nontraumatic brain damage treated in ICU S G k T k N B k G K ö E Ak S K ki Distribution and mortality factors of cases with traumatic and nontraumatic brain damage treated in ICU S Goksu Tomruk, N Bakan, G Karaören, E Akcay, S Keskin Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P532 (doi: 10.1186/cc14612) S Goksu Tomruk, N Bakan, G Karaören, E Akcay, S Keskin Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P532 (doi: 10.1186/cc14612) Introduction Cases of traumatic and nontraumatic brain damage have high rates of morbidity and mortality. In this study of cases being treated in the ICU for a diagnosis of brain damage, it was aimed to evaluate the relationship between mortality and the distribution of reason for and resulting type of brain damage and to determine other factors aff ecting mortality. i Conclusion There was a strong association between amount of registration and outcome. Post-traumatic stress symptoms in intensive care staff working in adult and paediatric settings Figure 2 (abstract P528). Figure 2 (abstract P528). Introduction The objectives of this survey were to establish the prevalence of symptoms of post-traumatic stress in mixed staff groups working in adult and paediatric intensive care settings and to examine the main themes in staff descriptions of the most traumatic event they had experienced at work. p Methods A total of 355 health professionals working on three adult and four paediatric units at two centres were asked to rate their current level of post-traumatic stress symptoms on the Trauma Screening Questionnaire (TSQ).f Results Paediatric/neonatal intensive care staff were more likely to score above the clinical cutoff point for post-traumatic stress symptoms on the TSQ in relation to an incident at work than adult intensive care staff in this sample (PICU n = 33/193 (17%) vs. AICU n = 13/162 (8%), P <0.001). For the 172 staff who provided a description of the most traumatic event they had experienced, the following themes were S186 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P531). Main themes of staff descriptions of traumatic events on ICU. (P <0.01). No statistical diff erence was determined in mortality in terms of age, gender, duration in ICU, surgical treatment or not, or reasons for brain damage (P >0.05). Conclusion There is considerable variation in causes of head injury. From this retrospective study it can be suggested that mortality of neurological/neurosurgical patients, regardless of management method, depends on APACHE II, arrival GCS, number of ventilator-days and type of brain damage. Association with amount of registration and outcome in pediatric severe trauma patients Multivariate analysis adjusted for age, revised trauma score and ISS revealed that the amount of registration (every 10 patients) was independently associated with survival (odds ratio: 1.55, 95% confi dence interval: 1.06 to 2.26, P = 0.023). P534 Simple periodic regression showed a biannual seasonal eff ect on mortality, documented with a 1-year periodic (P <0.01) and a 6-month periodic (P = 0.01) dependency. Multivariate analysis with or without periodic terms identifi ed age, past medical history, TBSA, FTBSA, inhalation, intoxication and admission date as the only factors independently associated with mortality. Conclusion Predictive factors for mortality in our ICBU are in line with the literature. The documented seasonal variations in mortality are fully explained by variations in these severity factors. Complementary analyses are under way to further study a nonlinear age eff ect. Results Baseline liver function was assessed in 1,565 patients. Of those, 522 (33%) exhibited liver dysfunction at baseline. No relationship was found with mortality according to baseline liver dysfunction status at day 28 (HR, 0.847 (95% CI, 0.647 to 1.109); P = 0.2267), day 90 (HR, 0.883 (95% CI, 0.701 to 1.112); P = 0.2892) and day 180 (HR, 0.885 (95% CI, 0.710 to 1.103); P = 0.2761). A total of 403 (26%) patients developed a new liver dysfunction (257/1,043, 25%) or worsened liver dysfunction (146/522, 28%) in the 28-day post-baseline period. The overall median time to develop or to worsen liver dysfunction was 2 (1 to 4) days. Signifi cant relationships between the new onset or the worsening of liver dysfunction post baseline and mortality at day 28 (HR, 1.67 (95% CI, 1.26 to 2.21); P = 0.0004), day 90 (HR, 1.65 (95% CI, 1.30 to 2.09); P <0.0001) and day 180 (HR, 1.57 (95% CI, 1.26 to 1.97); P <0.0001) were found. P534 Inhalation injury was present in 441 (23.1%) cases, intoxication (CO, CN) was present in 88 (4.6%) cases, and associated traumatisms also y g y Results The patients comprised 73.3% males and 26.7% females with a mean age of 40.58 ± 24.14 years (range, 1 to 87 years), mean APACHE II score of 19.07 ± 10.19 (range, 2 to 41), mean GCS of 9.17 ± 4.42 (range, 3 to 15) and 68.1% of whom were discharged and 31.9% were exitus. When the causes of brain damage were evaluated according to the type, the most frequently seen in the HT and HT + GBT groups were COM (37.3%, 42.9%), followed by EDH (13.6%, 28.6%). In the SH group, the most common reason was ICH (43.9%) followed by SDH (24.4%). Directly proportionally, only an increase in APACHE II score increased the mortality risk 1.3-fold (logistic regression analyses, coeffi cient 0.658) but the duration of intubation and ICH ratio was statistically signifi cantly high and GCS was low in the exitus cases, and rates of EDH were determined as high in discharged cases compared with exitus S187 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods All of the patients enrolled in the PROWESS-SHOCK were included. Liver dysfunction at baseline was defi ned by a liver Sequential Organ Function Assessment (SOFA) score >0. The onset of a liver dysfunction post baseline was defi ned as any post-baseline increase of the hepatic SOFA score from 0. The worsening of liver dysfunction post baseline was defi ned as any increase of the hepatic SOFA score from the baseline assessment. The post-baseline period studied extended from study drug infusion to day 28. The main outcome was the mortality at day 180, and the secondary outcomes were the mortality at day 28 and at day 90. Cox proportional hazard models were used to estimate the hazard ratio of death. in 88 (4.6%) cases. The mortality rate was 10.9%. The following factors were associated with mortality in univariate logistic regression: age, body mass index, past medical history, TBSA, FTBSA, intoxication (CO, CN), inhalation injury, fl ame burns, self-infl icted burns (all P <0.0001), sex (P <0.001), and admission date (P <0.01). Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Results One patient was admitted twice during this period. The sample comprised 11 males and 11 females (50%, respectively). Featuring an average age of 52 years, 78% of them were admitted to the ICU because of respiratory failure, 50% were due to diff use alveoli hemorrhage, 36% due to sepsis, 4% hypovolemic shock and fi nally 4% because of tuberculosis. According to the BVAS/WG, 20 patients corresponded to severe disease, one to limited diseases and one to persistent disease. The average ICU length of stay was 20.6 days and as inpatients 43 days. While comparing the SOFA score between alive and deceased patients there was a 0.5-point diff erence (P = 0.077), 63% of the alive patients were diagnosed while they were in the ICU. Plasmapheresis was found to be a protector factor (P <0.05).if Introduction In cancer patients, sepsis is the main cause of admission to the ICU and is associated with elevated mortality rates and healthcare costs. In this population, specifi c factors such as poor functional status and immunosuppression secondary to malignancy and/or antineoplastic treatment contribute to decreased survival. The aim of this study is to identify predictors of mortality in cancer patients admitted to the ICU with septic shock. Methods This is a retrospective study that analyzed predictors of 30- day mortality in 269 patients admitted to the ICU of the Institute of Cancer of State of São Paulo, Brazil, from February 2012 to November 2014. Conclusion The BVAS/WG score was signifi cantly diff erent between alive and deceased patients. Plasmapheresis was found to be a survival predictor. This study has shown that both SOFA and APACHE II scores have no prognostic value in these patients. Results From 1,250 patients admitted to the ICU, 269 patients had the admission diagnosis of septic shock and were analyzed. The mean age was 62 ± 14 years and 152 patients (56.5%) were male. Most patients had solid cancer (93.6%), and 87 patients (34.5%) had gastrointestinal neoplasm. Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Upon admission, the median SOFA score was 4 (IQR: 2 to 7), median SAPS 3 score was 55 (IQR: 48 to 68) and median lactate level was 1.88 mmol/l (IQR: 1.22 to 3.21). After 48 hours of ICU admission, acute kidney injury (AKI) was diagnosed according to AKIN classifi cation as follows: 202 patients (75.1%) had grade 0, 49 (18.2%) grade 1, seven (2.6%) grade 2 and 11 (4.1%) had grade 3. The 30-day mortality rate was 24.9%. In the univariate analysis, associated variables with 30-day mortality were age, urea and creatinine levels at admission, SOFA score at admission, SAPS 3 score and 48-hour AKI. In multivariate analysis, the predictive factors for 30-day mortality were SOFA score at admission (OR = 1.12; 95% CI: 1.04 to 1.21, P = 0.002) and 48-hour AKI defi ned as AKIN grades 1, 2 and 3 (OR = 2.69; 95% CI: 1.45 to 4.97, P = 0.002). P534 P535 ICU outcomes in patients suff ering granulomatosis with polyangitis C Hernandez-Cardenas1, GL Lugo-Goytia1, MS Sierra Beltran2, G Dominguez Cherit3 1Instituo Nacional de Enfermedades Respiratorias, Mexico DF, Mexico; 2Medica Sur, Mexico DF, Mexico; 3Instituo Nacional de Ciencias Medicas y Nutrición Salvador Zubirán, Mexico DF, Mexico Critical Care 2015, 19(Suppl 1):P535 (doi: 10.1186/cc14615) Introduction This study aims to describe both the clinical course and prognostic factors of patients suff ering granulomatosis with polyangitis (Wegener granulomatosis) (GP) who were admitted to the Salvador Zubirán National Medical Sciences and Nutrition ICU.f Introduction This study aims to describe both the clinical course and prognostic factors of patients suff ering granulomatosis with polyangitis (Wegener granulomatosis) (GP) who were admitted to the Salvador Zubirán National Medical Sciences and Nutrition ICU.f Conclusion The new onset or the worsening of liver dysfunction during the course of septic shock impacts strongly on long-term mortality. Septic patients with liver dysfunction need long-term follow-up. Future research should focus on developing specifi c septic liver therapeutics and new tools for earlier detection of septic liver dysfunction. Reference Methods Twenty-two patients suff ering GP admitted to the ICU, between January 2002 and December 2012, were included. The Acute Physiology and Chronic Health Evaluation (APACHE) II prognostic score scale was used in order to assess the severity of illness on the fi rst ICU day. The Sequential Organ Failure Assessment (SOFA) score was used to measure organ dysfunction, and the Birmingham vasculitis activity score for Wegener granulomatosis (BVAS/WG) was used to assess vasculitis activity. The outcome measurements taken into account were ICU mortality and ICU length of stay. 1. Ranieri et al. N Engl J Med. 2012;366;2055-64. Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Introduction Knowledge of the impact of liver dysfunction on mortality during septic shock is limited. However, the liver appears to play a key role during septic illness. To better explore this issue, we investigated the data collected during the Prospective Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis and Septic Shock (PROWESS-SHOCK) trial in which a cohort of 1,697 septic shock patients were constituted [1]. The study aimed to assess the relationship of liver dysfunction at the onset and during the course of septic shock on short-term and long-term mortality. Conclusion In cancer patients with septic shock, SOFA score at admission and acute kidney injury at 48  hours of admission were predictors of 30-day mortality. These fi ndings reinforce the needing of early strategies of diagnosis and therapy in this subset of patients. S188 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 disease remission and 31 (11.2%) had newly diagnosed disease. The ICU mortality rate was 26%, hospital mortality was 35.7% and 6-month mortality was 55.2%. The median number of organ dysfunction was 3 (IQR 2 to 4) and respiratory failure was the leading dysfunction, being present in 209 patients (75.5%). During the ICU stay, 21 patients needed hemodialysis (8%), 69 (25%) needed mechanical ventilation, 162 (58%) used vasoactive agents and 22 (8%) had a decision for limitation of medical treatment. On univariate analysis, risk factors for hospital mortality were acute myeloid leukemia, hospital stay prior to ICU admission >4 days, number of organ dysfunction ≥2, colonization and infection by a multidrug-resistant (MDR) agent, use of mechanical ventilation, use of vasoactive agents and renal replacement therapy. I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar Institute of Cancer of State of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P539 (doi: 10.1186/cc14619) Introduction In recent decades, therapeutic advances resulted in increased survival of patients with hematologic malignancies. These patients are increasingly admitted to the ICU due to infections, treatment toxicity and decompensation of chronic diseases. The aim of this study is to evaluate ICU, hospital and 6-month mortality in patients with hematological malignancies admitted to the ICU and to identify predictors of ICU mortality. y g Conclusion In our study, ICU and hospital mortality were lower than the pooled mortalities seen in a recent large systematic review [3] – despite our study excluding planned postoperative admissions (who are known to have better outcomes). The 180-day mortality was signifi cantly lower than in a previous study at our own institution [4]. Our study looked at a number of factors that might reasonably be expected to be associated with short-term and long-term outcomes and identifi ed several that were independent predictors of death by 180 days. Methods We performed a retrospective study of 277 consecutive patients with hematological malignancies admitted to the ICU of the Institute of Cancer of State of São Paulo, Brazil, from January 2010 to December 2013. Patient clinical and laboratory characteristics, evaluation of organ dysfunctions and need for hemodialysis, mechanical ventilation and vasoactive agents in the ICU were collected. The primary outcome was ICU mortality. Data were analyzed with univariate and multivariate logistic regression. Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 Multivariate analysis revealed that renal replacement therapy (OR  = 6.35 (95% CI: 1.5 to 25.92), P = 0.010), SOFA score (OR = 1.69 (95% CI: 1.38 to 2.06), P <0.001), RDW (OR = 1.27 (95% CI: 1.11 to 1.46), P = 0.001), lactate (OR = 1.04 (95% CI: 1.02 to 1.06), P <0.001), colonization of MDR agent (OR = 10.73 (95% CI: 2.13 to 53.96), P = 0.004) and hospital stay prior to ICU admission >4 days (OR = 4.72 (95% CI: 1.8 to 12.3), P = 0.002) were predictive factors of ICU mortality. Factors associated with short-term and long-term mortality in solid cancer patients admitted to the ICU R Fi h 1 C D i 1 S C i h 2 S Sl 1 L S 1 Factors associated with short-term and long-term mortality in solid cancer patients admitted to the ICU R Fisher1, C Dangoisse1, S Crichton2, S Slanova1, L Starsmore1, T Manickavasagar1, C Whiteley1, M Ostermann1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2King’s College London, UK Critical Care 2015, 19(Suppl 1):P540 (doi: 10.1186/cc14620) Introduction Despite multiple reports demonstrating an improvement in outcomes of critically ill cancer patients admitted to ICUs over the last two decades [1], there is concern that admission policies for patients with cancer are overly restrictive [2]. The purpose of this study was to identify factors associated with mortality in the 180  days following unplanned ICU admission in patients with nonhaematological malignancy. Introduction Despite multiple reports demonstrating an improvement in outcomes of critically ill cancer patients admitted to ICUs over the last two decades [1], there is concern that admission policies for patients with cancer are overly restrictive [2]. The purpose of this study was to identify factors associated with mortality in the 180  days following unplanned ICU admission in patients with nonhaematological malignancy. Conclusion The ICU mortality of critically ill patients with HM is high, particularly in the group on MV. Diff erent factors were independent predictors of mortality, but 46.4% of admitted patients survived because of adequate support possibilities and were transferred back to hematology ward. The ICU admission with organ support is according results important for life saving in this extremely high-risk patient group. g p References Methods We carried out a retrospective analysis of all patients with solid tumours admitted to the Guy’s Critical Care Unit (13-bed level 3 ICU) as an emergency between August 2008 and July 2012. Data were collected regarding patient demographics, type of cancer, reason for referral and organ support required during the ICU stay. References 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 2. Grgic Medic M, et al. Hematologic malignancies in the medical intensive care unit – outcomes and prognostic factors. Hematology. 2014 [Epub ahead of print]. y Results During the 4-year study period there were 356 unplanned admissions of patients with solid cancer (8.3% of all admissions). Three hundred individual patients were admitted and 180-day survival data were available for 293 of these. Mean age at fi rst admission was 65.2  years, 115 (38.3%) were female. The most frequently present malignancies were lung (42.7%), head and neck (17.3%) and renal (6.7%). ICU, hospital and 180-day mortality were 19.1%, 31.0% and 52.2% respectively. Those factors found to be independently associated (in multivariate analysis) with increased risk of 180-day mortality include: metastases (OR = 4.0, 95% CI = 2.1 to 7.6); sepsis on admission (OR = 2.2, 95% CI = 1.2 to 4.1); APACHE II score on admission (OR = 1.06, 95% CI = 1.004 to 1.12); need for inotropes/vasopressors during admission (OR = 2.3, 95% CI = 1.05 to 4.8); and need for renal replacement therapy during admission (OR = 4.65, 1.7 to 12.8). References 1. Mokart D, et al. Intensive Care Med. 2014;40:1570-2. 2. Azoulay E, et al. Ann Intensive Care. 2011;1:5. 3. Puxty K, et al. Intensive Care Med. 2014;40:1409-28. 4. McGrath S, et al. QJM. 2010;103:397-403. P538 Is admission of hematologic malignancies in the ICU justifi ed? VG Gasparovic, MM Grgic Medic, IG Gornik KBC Zagreb, Croatia Critical Care 2015, 19(Suppl 1):P538 (doi: 10.1186/cc14618) P538 Is admission of hematologic malignancies in the ICU justifi ed? VG Gasparovic, MM Grgic Medic, IG Gornik KBC Zagreb, Croatia Critical Care 2015, 19(Suppl 1):P538 (doi: 10.1186/cc14618) Introduction The admission of malignant hematology patients to the ICU is combined with poor prognosis [1]. A young population on one side and serious prognosis on the other are the main characteristics. Do we help? We are analyzing continuously clinical characteristics, treatment, and outcomes of critically ill patients with hematologic malignancies admitted to the medical ICU to identify predictors of adverse outcome [2]. Methods Demographic characteristics, hematologic diagnosis, reasons for ICU admission, transplant status, the presence of neutropenia, and APACHE II and SOFA scores were analyzed. Predictors of ICU mortality were evaluated using univariate analysis. g y Results There was a total of 194 patients (103 male), APACHE II score by admission was 27 ± 8, SOFA 9 ± 3. Acute leukemia (L) in 81 patients (41.8%), chronic L in 19 patients (9.8%), lymphoma in 58 patients (29.9%), and multiple myeloma in 28 patients (14.4%) were the etiology. Respiratory insuffi ciency, hemodynamic instability, AKI and CNS disturbances were responsible for the admission of 169 patients (87.1%) from the hematology ward to the ICU. In total, 127 patients (59.7%) were mechanically ventilated; 93 required invasive mechanical ventilation (MV). Non-invasive ventilation started in 34 patients and was successful in 14 (6.5%). The ICU mortality rate was 104 patients (53.6%), and the mortality of MV patients was 98 (77.2%). Need for vasopressors at admission, MV, neutropenia, and APACHE II and SOFA scores were identifi ed as independent predictors of fatal outcome. Overall mortality of admitted patients was 53.6% (104 patients), and in ventilated patients was 77.2% (98 patients). Conclusion Patients from our institution have survival rates comparable with data from the literature. Our study suggests that mortality is associated with late ICU admission and colonization of MDR bacteria. P540 P540 Outcomes of patients with hematologic malignancies admitted to the ICU I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar Institute of Cancer of State of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P539 (doi: 10.1186/cc14619) y References References 1. Mokart D, et al. Intensive Care Med. 2014;40:1570-2. 2. Azoulay E, et al. Ann Intensive Care. 2011;1:5. 3. Puxty K, et al. Intensive Care Med. 2014;40:1409-28. 4. McGrath S, et al. QJM. 2010;103:397-403. Results The median age of the population was 57  years and 144 patients (52%) were male. Upon admission, 15 patients (5.4%) had S189 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P541 Characteristics and outcomes of lung cancer patients requiring ventilatory support: results from a multinational study AC Toff art1, JF Timsit1, J Salluh2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, B Almeida7, E Azoulay8, M Soares9 1Hopital A. Michalon CHU Grenoble, France; 2Post-graduation Program – Inst. Nacional de Cancer, Rio de Janeiro, Brazil; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton Hospital, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Hopital Saint Louis, Paris, France; 9DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P541 (doi: 10.1186/cc14621) P541 using Hosmer–Lemeshow goodness of fi t test. Data will be reported as median (range) or proportions (95% confi dence interval). P541 Characteristics and outcomes of lung cancer patients requiring ventilatory support: results from a multinational study AC Toff art1, JF Timsit1, J Salluh2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, B Almeida7, E Azoulay8, M Soares9 1Hopital A. Michalon CHU Grenoble, France; 2Post-graduation Program – Inst. Nacional de Cancer, Rio de Janeiro, Brazil; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton Hospital, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Hopital Saint Louis, Paris, France; 9DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil Critical Care 2015 19(S ppl 1) P541 (doi 10 1186/cc14621) Results A total of 2,769 patients were included, median age 65 (18 to 100) years and 52% female. Thirty-day mortality was 4.5% and 2.2% were admitted to ICU. Median time to ICU admission was 1 (0 to 70) day. Nursing staff assessed 65% and physicians 26% of admissions. NEWS could be calculated for 85%. Nursing staff had a discriminatory power of 0.865 (0.786 to 0.944) with little variation with experience. Calibration was acceptable, except for the least experienced nurses (<5  years). References 1. Fuchs L, et al. Intensive Care Med. 2012;38:1654-61. 2. Nielsson MS, et al. Acta Anaesthesiol Scand. 2014;58:19-26. Hospital of South West Jutland, Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P542 (doi: 10.1186/cc14622) Introduction Not all patients in need of critical care arrive in clinical distress and some deteriorate after arrival. Identifying these patients early in their clinical course could potentially improve outcome. The present study was performed with the aim of assessing whether nursing and physician staff were able to identify patients in need of critical care using only clinical judgment and to compare this with the National Early Warning Score (NEWS). y References Physicians had a discriminatory power of 0.789 (0.641 to 0.937), with little variation with experience. Calibration was very good, regardless of experience. NEWS had a discriminatory power of 0.809 (0.727 to 0.891) and poor calibration. There was no signifi cant diff erence in discriminatory power between the three assessments.f Critical Care 2015, 19(Suppl 1):P541 (doi: 10.1186/cc14621) Introduction The aim was to evaluate clinical characteristics and outcomes of patients with lung cancer requiring ventilatory support. Methods Secondary analysis of a prospective multicenter study including patients requiring either invasive (IMV) or non-invasive (NIV) mechanical ventilation for >24 hours admitted to 22 ICUs in six countries from Europe and South America during 2011. We used shared frailty models to identify factors associated with 6-month survival. f y p Conclusion Both nursing staff and physicians were as good as NEWS at identifying patients at increased risk of ICU admission after admission to a medical admission unit. However, both nursing staff and physicians had better calibration (accuracy) than NEWS. P543 5 3 Clinical characteristics in surviving and nonsurviving older patients admitted to the ICU Results Out of 449 patients admitted to the ICUs, 239 (small-cell (SCLC) = 34; non-SCLC = 205) required ventilatory support (NIV = 104; IMV = 135). Out of NIV patients, 31 (30%) were subsequently intubated for IMV. Main reasons for ventilatory support were sepsis (n  = 119; among them, 102 patients had pneumonia), airway involvement by tumor (n = 79), ARDS (n = 47) and coma (n = 18). Mean SAPS II score was 54 ± 20 and median SOFA score was 7 (4 to 12) points. Hospital and 6-month mortality rates were 55% and 67%; 94 (39%) patients received treatment limitations in the ICU. Mortality according to ventilatory strategy was 56% for NIV only, 77% for NIV followed by IMV, and 70% for IMV only. In the multilevel model, adjusting for the hospital size, presence of step-down units, type of admission and treatment limitation, performance status (PS) 3 to 4 (HR = 2.25 (95% CI, 1.52 to 3.34)), metastasis (HR = 1.66 (1.18 to 2.33)) and the ventilatory strategy compared with NIV only (HR  = 1.73 (1.02 to 2.92), for NIV followed by IMV; HR = 2.25 (1.51 to 3.35), for IMV only) were associated with increased mortality. Conversely, patients with sepsis had higher survival (HR = 0.67 (0.46 to 0.96)). Introduction In recent years the proportion of older people admitted to the ICU has increased. A variety of clinical and physiological factors are associated with outcome in these patients. The aim of this study is to determine the clinical characteristics associated with survival of ICU mechanically ventilated older patients. Introduction In recent years the proportion of older people admitted to the ICU has increased. A variety of clinical and physiological factors are associated with outcome in these patients. The aim of this study is to determine the clinical characteristics associated with survival of ICU mechanically ventilated older patients. Methods We retrospectively studied 74 patients, aged >65  years, admitted to the ICU who underwent mechanical ventilation. Standard demographic, clinical, and physiologic data were recorded. We examined the signifi cant diff erences in clinical characteristics between survivors and nonsurvivors using the Student t and chi-square tests. Methods We retrospectively studied 74 patients, aged >65  years, admitted to the ICU who underwent mechanical ventilation. Standard demographic, clinical, and physiologic data were recorded. Admission to intensive care can be reliably predicted using only clinical judgment Admission to intensive care can be reliably predicted using only clinical judgment M Brabrand Hospital of South West Jutland, Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P542 (doi: 10.1186/cc14622) M Brabrand 5 3 Clinical characteristics in surviving and nonsurviving older patients admitted to the ICU We examined the signifi cant diff erences in clinical characteristics between survivors and nonsurvivors using the Student t and chi-square tests. Results The mean age of patients studied (43 men and 31 women) was 79 ± 6.4 years. The type of admission was surgical 18%, trauma 26% and medical 57%. The ICU mortality of these patients was 57% and it was not associated with gender and cause of admission to ICU. Patients who survived had lower Charlson Comorbidity Index (P <0.05) and shorter duration of mechanical ventilation (P <0.01). The episodes of ventilation-associated pneumonia, sepsis and renal failure were less frequently in survivors (P <0.05). Also, in addition serum iron and cholesterol levels were signifi cantly lower in nonsurvivors (P <0.01). Conclusion The mortality of ICU older patients is high. VAP, sepsis and renal failure are frequent complications in nonsurvivors. Pre-existing comorbidities considerably aff ect mortality. R f su o s a d o su o s us g t e Stude t t a d c squa e tests. Results The mean age of patients studied (43 men and 31 women) was 79 ± 6.4 years. The type of admission was surgical 18%, trauma 26% and medical 57%. The ICU mortality of these patients was 57% and it was not associated with gender and cause of admission to ICU. Patients who survived had lower Charlson Comorbidity Index (P <0.05) and shorter duration of mechanical ventilation (P <0.01). The episodes of ventilation-associated pneumonia, sepsis and renal failure were less frequently in survivors (P <0.05). Also, in addition serum iron and cholesterol levels were significantly lower in nonsurvivors (P <0 01) Conclusion In a multinational study, 6-month survival in lung cancer patients requiring ventilatory support is better than perceived a priori. Palliative care should be preferred in patients with poor PS. p p p Acknowledgements Funded by INCA, CNPq and FAPERJ. cholesterol levels were signifi cantly lower in nonsurvivors (P <0.01). Conclusion The mortality of ICU older patients is high. VAP, sepsis and renal failure are frequent complications in nonsurvivors. Pre-existing comorbidities considerably aff ect mortality. References Frailty predicts increased resource use and postoperative care requirements after revision hip surgery P546 Emergency laparotomy clinical outcome according to patient characteristics, level of postoperative care and time of surgery T Banerjee1, M Templeton2, C Gore2 1St Mary´s Hospital, London, UK; 2Imperial College Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P546 (doi: 10.1186/cc14626) Critical Care 2015, 19(Suppl 1):P546 (doi: 10.1186/cc14626) Introduction Emergency laparotomies have poor outcomes with variable postoperative critical care provision [1-3]. All patients requiring an emergency laparotomy with an estimated risk of death of >10% should go to critical care. Time of surgery should not aff ect standard of care [3,4]. In advance of the National Emergency Laparotomy Audit (NELA) results [2], our objective was to see whether the level of postoperative care and time of surgery aff ect outcome. Results A total of 389 patients with a mean age of 68.7  years were identifi ed. Frail patients were signifi cantly more likely to need vasopressors postoperatively (P  = 0.012). Each increase in frailty score was associated with 0.16 increase in length of stay on the HDU (P  = 0.025). Analysis of patient location at 30  days shows that frail patients stay in hospital longer (P = 0.00). Frail patients also bleed more intraoperatively (P = 0.00 with a coeffi cient value of 239; that is, for every point increase in frailty, average blood loss increases by 239 ml). For each increase by unit of blood transfused, the length of stay increased by 5.3 days (P = 0.000). The use of epidural is not associated with increased need for postoperative vasopressors (P  = 0.598). See Figure 1. g yf Methods Retrospective data were collected across the Imperial NHS trust for all emergency laparotomies over 3 months in 2014: length of stay in days (LOS); mortality; age; ASA; surgery time and postoperative care level, that is ward (L1), high dependency (L2), or ICU (L3). Statistical tests: Mann–Whitney, Pearson correlation (PCC) and multilinear regression analysis. g y Results Seventy-one patients underwent surgery. Overall mortality was 13% and 70% of patients went to a L2/3 bed. More ASA 1/2 patients went to L1 and all ASA 4/5 went to L2/3. Median (IQR) for age was 61 (44 to 67) for L1, 65 (48 to 73) for L2/3 (P = 0.11), LOS was 10 (7 to 16) for L1, 19 (12 to 57) for L2/3 (P = 0.002), and mortality (%) was 0 for L1 and 18 for L2/3. g p References 1. Saunders D, et al. Br J Anaesth. 2012;109:368-75. 1. Saunders D, et al. Br J Anaesth. 2012;109:368-75. 1. Saunders D, et al. Br J Anaesth. 2012;109:368 75. 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. , ; 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. 2. NELA project team. Executive summary, fi rst organisational report of the N ti l E L t A dit L d RC A 2014 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. Methods We prospectively collected data on 339 adult patients admitted to two ICUs in Rwanda between August 2013 and July 2014. We described demographic and presenting characteristics and outcomes. We assessed the discrimination and calibration of the MPMo-III model. Using stepwise selection, we then developed a new logistic model for mortality prediction, the R-MPM. Frailty predicts increased resource use and postoperative care requirements after revision hip surgery A Panickar1, N Singatullina1, J Stubbs1, C Johnson1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Leicester Hospitals, Leicester, UK Critical Care 2015, 19(Suppl 1):P544 (doi: 10.1186/cc14624) A Panickar1, N Singatullina1, J Stubbs1, C Johnson1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Leicester Hospitals, Leicester, UK Critical Care 2015, 19(Suppl 1):P544 (doi: 10.1186/cc14624) y Methods This was a prospective cohort study of all adult patients with a fi rst-time admission to a medical admission unit at a 450-bed regional teaching hospital over a 3-month period in 2010. All subspecialties of internal medicine are present as well as a level 2 ICU. Upon fi rst contact with the patient after arrival, nursing staff and physicians were asked to report their estimation of the probability of ICU admission (0 to 100%). Survival status was extracted from the Danish Civil Registry. All administrative details (including transfers to other hospitals, wards and ICUs) were extracted from the National Patient Registry, both ensuring complete follow-up. The discriminatory power (ability to identify patients at increased risk) was estimated using area under the receiver- operating characteristics curve. Calibration (accuracy) was assessed Introduction There is increasing demand for revision hip surgery in older patients with poor frailty. Our previously submitted work demonstrated that frailty predicts the need for medical review [1]. We reviewed patients for a further 16 months to see whether frailty impacts on care [2]. This is the largest reported study reviewing frailty and the need for organ supports and outcomes in complex orthopaedic surgery. Methods A retrospective note review of all patients from January 2012 to April 2014 undergoing revision hip surgery. Data collected included frailty, comorbidities, operative blood loss, anaesthetic technique and level of organ supports and patient location at 30 days. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S190 Figure 1 (abstract P544). Frailty versus length of stay. of 8.881. The MPMo-III predicted mortality with area under the curve of 0.720 and Hosmer–Lemeshow chi-square statistic of 16.391, indicating that the predictive risk scores of the MPMo-III were not well calibrated to the Rwandan data. Figure 1 (abstract P544). Frailty versus length of stay. Conclusion The MPMo-III had modest risk prediction capacity in a population of Rwandan ICU patients. The R-MPM is an alternative severity score with fewer and more accessible variables that provides better predictive power. This model needs to be validated in other ICUs. References References 1. Singatullina et al. Abstract. Crit Care. 2014;18 Suppl 1:P49N. 2. Rockwood K, et al. Can Med J. 2005;173:489-95. 1. Singatullina et al. Abstract. Crit Care. 2014;18 Suppl 1:P49N. 2. Rockwood K, et al. Can Med J. 2005;173:489-95. Frailty predicts increased resource use and postoperative care requirements after revision hip surgery For surgery between 08:00 and 17:59, 14 went to L1, 28 to L2/3. Mortality was 5% and LOS 15 (9 to 24). Between 18:00 and 21:59, two went to L1, 10 to L2/3. Mortality was 17% and LOS 22 (8 to 34). Between 22:00 and 07:59, fi ve went to L1, 12 to L2/3. Mortality was 29% and LOS 15 (9 to 36). ASA strongly predicted mortality (P = 0.006, PCC 0.32). There was a negative correlation between postoperative destination and mortality with all deaths happening in those who went to L2/3 (P = 0.038 and PCC –0.25); however, sicker patients may have gone here. There was a strong correlation between mortality and time of surgery, night surgery being a strong predictor of mortality (PCC 0.31, P = 0.008). LOS can be predicted by a combination of ASA, age and care level (P = 0.027); the postoperative care regression coeffi cient was negative (–18.04, SE 10.41) with prolonged LOS in patients admitted to L2/3, which could also be explained by illness severity. g Conclusion Frailty is associated with increased intraoperative resource use and postoperative care requirements independent of choice of anaesthetic technique. This type of surgery should be subject to health economic analysis as demand amongst the frailer surgical population increases. P545 P545 Predicting outcomes in critically ill patients in a resource-poor setting: the Rwanda Mortality Probability Model T Twagirumugabe1, E Riviello2, R Fowler3, V Novack4, A Mueller2, W Kiviri1, V Banner-Goodspeed2, D Talmor2 1University of Rwanda, Kigali, Rwanda; 2Beth Israel Deaconess Medical Center, Boston, MA, USA; 3University of Toronto, ON, Canada; 4Ben-Gurion University of the Negev, Beer Sheva, Israel Critical Care 2015, 19(Suppl 1):P545 (doi: 10.1186/cc14625) Predicting outcomes in critically ill patients in a resource-poor setting: the Rwanda Mortality Probability Model g y y T Twagirumugabe1, E Riviello2, R Fowler3, V Novack4, A Mueller2, W Kiviri1, V Banner-Goodspeed2, D Talmor2 p y y Conclusion Trust mortality is similar to that in the Emergency Laparotomy Network audit [1]. Higher ASA patients are appropriately going to L2/3 care. Baseline health status and time of surgery are the strongest predictors of mortality in emergency laparotomy patients. References Introduction ICU mortality prediction models provide robust tools for research and benchmarking in the developed world, but an ICU mortality prediction model has not been validated in a resource-poor setting. We sought to validate the Mortality Probability Admission Model, version III (MPMo-III) in two public ICUs in Rwanda and to develop a simplifi ed Rwanda Mortality Probability Model (R-MPM) for use in developing countries. P549 P549 Disturbed circadian rhythm in ICU patients as indicated by melatonin levels: a prospective pilot study K Kiss1, I Földesi1, L Kemény1, V Csernus2, Z Molnár1, J Singer3 1University of Szeged, Hungary; 2University of Pécs, Hungary; 3Hungarian Society for Clinical Biostatistics, Hungary Critical Care 2015, 19(Suppl 1):P549 (doi: 10.1186/cc14629) pi y Results Age, platelets, ALT and APACHE II were selected to be included in the new laboratory-based score. The AUC for the score was 0.714, which was higher than each of the individual laboratory parameters. The AUC was increased further to 0.781 by including all 14 variables (age, lactate, FiO2, urea, creatinine, ALT, APACHE II, platelet, bicarbonate, haemoglobin, pH, ionised Ca, carboxyhaemoglobin and albumin), although this improvement was not considered signifi cant as the confi dence intervals of the two scores (4 and 14 variables) overlapped. Conclusion A laboratory-based score was successfully established in ICU patients, revealing an AUC of 0.714 which is comparable with established scores in a similar population. The compilation of the variables to produce a laboratory-based score showed greater prognostic power than individual variables. Model developers require an AUC of >0.7 to be termed useful; however, in order to be used in a clinical setting the AUC must be at least 0.75. Further research including internal and external validation studies must be performed to optimise the model before clinical implementation. Introduction The aim of this prospective pilot study was to test how melatonin levels follow the circadian cycle in ICU patients. There is strong evidence that changes of circadian rhythm are refl ected in melatonin levels with peak levels at dawn and low levels during daytime [1]. The ICU stay is accompanied by disturbed circadian rhythm [2], which could potentially be the result of artifi cial lighting conditions. Methods Melatonin levels were determined in eight medical ICU patients on mechanical ventilation, without brain injury or infection. Arterial blood samples were taken on the day of admission at 18:00 (TE0) and 03:00 in the morning (TM0), then at the same time points 48  hours later (TE1, TM1). Blood samples were centrifuged at 3,000 rpm for 8  minutes, and then serum samples were stored at –80°C. Measurements were performed using a US-CEA908Ge melatonin ELISA kit. For statistical analysis, a binary (yes/no) variable was created from the pairs for each day, assigning ‘Yes’ if the TE values were greater than TM (melatonin peak reversion). Developing a laboratory-based score to predict mortality in patients admitted to the ICU A Iqbal1, I Welters2, R Kolamunnage-Dona3, C Toh3, C Downey2 1Institute of Ageing and Chronic Disease, Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P547 (doi: 10.1186/cc14627) A Iqbal1, I Welters2, R Kolamunnage-Dona3, C Toh3, C Downey2 1Institute of Ageing and Chronic Disease, Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P547 (doi: 10.1186/cc14627) g y p Results Patient median age was 34 (IQR 26 to 49) years; 48.7% were male. Mortality was 50.3%. The variables most predictive of mortality in univariate analyses were: age, sepsis within 24 hours of ICU admission, hypotension or shock at ICU admission, Glasgow Coma Scale score at ICU admission, and heart rate (beats per minute) at ICU admission. Using these fi ve variables, the R-MPM predicted mortality with area under the curve of 0.829 and Hosmer–Lemeshow chi-square statistic Introduction Scoring systems can be used to predict mortality in patients admitted to the ICU. They are produced using variables that are associated with an increased risk of mortality such as patient Introduction Scoring systems can be used to predict mortality in patients admitted to the ICU. They are produced using variables that are associated with an increased risk of mortality such as patient S191 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The citizens surveyed valued outcomes associated with quality of life and intact neurological function. Mortality and other indices of duration of critical illness were valued less. These preferences need to be confi rmed in larger groups with greater diversity. Future research also needs to further understand these outcome preferences in survivors of critical illness, clinicians, decision-makers, and researchers. Understanding the outcome preferences of pertinent stakeholders and whether these preferences are congruent is imperative to inform the design of future critical care trials. Conclusion The citizens surveyed valued outcomes associated with quality of life and intact neurological function. Mortality and other indices of duration of critical illness were valued less. These preferences need to be confi rmed in larger groups with greater diversity. Future research also needs to further understand these outcome preferences in survivors of critical illness, clinicians, decision-makers, and researchers. Understanding the outcome preferences of pertinent stakeholders and whether these preferences are congruent is imperative to inform the design of future critical care trials. Patient preferences for outcomes in critical care trials (OPTICS): preliminary resultsf p y J Muscedere1, F Lamontagne2, G Boyd1, M Herridge3, S Fleury1, T Sinuff 3 1Queen’s University, Kingston, ON, Canada; 2University of Sherbrooke, QC, Canada; 3University of Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P548 (doi: 10.1186/cc14628) Results Melatonin levels were not normally distributed and were ranging between 9.2 and 23.7  pg/ml at T0 and 11.3 and 17.9  pg/ ml at T1. The median diff erences of the morning and evening serum melatonin levels were: TM0 – TE0 = –2.8 (–3.8 – (–0.2)); TM1 – TE1 = –1.2 (–2.5 – (–0.4)) presented as median (IQR). The proportion of subjects with peak reversals was 92.9% (80.8 to 100.0%). Introduction Although patient-centered outcomes are important to inform therapeutic choices for critically ill patients, patient preferences for outcomes in critical care studies are unknown. It is also unknown whether outcome preferences diff er between researchers, decision- makers and those who have never been critically ill (citizens). The aim of the OPTICS Program is to investigate these preferences. Herein we report the preliminary results for outcomes preferences in citizens. Conclusion Our preliminary data suggest that circadian rhythm disturbances may occur in critically ill patients within 48 hours after admission, and can be detected by inversion of melatonin peaks. Despite the limitations of this study, it may justify the need for larger observational and randomized trials on the eff ect of light on melatonin levels and on outcomes in ICU patients. Methods We recruited and surveyed lay public members without a history of critical illness as to their preferences for outcomes in critical care trials. After an in-person educational session, citizens were asked to rank 11 potential critical care trial outcomes in order of personal preference. Each outcome was also rated for importance on a 7-point Likert scale. Participants were then asked to indicate their agreement with potential tradeoff s between potential outcomes. References 1. Pevet P, et al. J Physiol. 2011;105:170-82. 1. Pevet P, et al. J Physiol. 2011;105:170-82. 2. Castro R, et al. Annual update in Intensive Care and Emergency Medicine. J.-L. Vincent, editor. Berlin: Springer-Verlag; 2011. p. 766-80. 2. Castro R, et al. Annual update in Intensive Care and Emergency Medicine. J.-L. Vincent, editor. Berlin: Springer-Verlag; 2011. p. 766-80. pf p Results The in-person session was attended by 31 citizens whose had a mean age (SD) of 71.6 (5.9) years and 1.5 (1.4) chronic health conditions; 25 (81%) had partially or fully completed post-secondary school education. Of the 11 potential outcomes, the three outcomes ranked of highest importance were: permanent brain dysfunction, quality of life, and requirement for long-term institutional care. The three outcomes ranked of least importance were: duration of hospitalization, death after a prolonged illness, and occurrence of delirium. When rated on a 7-point Likert scale the results were similar. Of the participants, 24/27 (89%) indicated that they would be willing to receive a therapy which was associated with a higher mortality rate but resulted in an improved quality of life in survivors. Conversely, 16/27 (59%) indicated that they would not be willing to receive a therapy which increased the chances of survival but was associated with a reduced quality of life in the survivors. Developing a laboratory-based score to predict mortality in patients admitted to the ICU demographics, physiological measurements and coexisting conditions and can be used to evaluate ICU performance, to stratify patients in clinical trials and to assist in-hospital and healthcare decisions such as resource allocation. The aim of the project was to determine whether a general score derived from routine laboratory parameters could be used to predict mortality rates in patients admitted to the ICU in the UK. Methods P values were calculated using the t test, Mann–Whitney U test and chi-squared test, depending on distribution of data, in order to determine which variables were signifi cantly diff erent in the survivors and nonsurvivors of critical illness. Signifi cant variables were categorised into subgroups according to medically relevant landmarks and univariately analysed by assessing the correlation with mortality. Forward logistic regression models were used to choose the parameters to include in our score. ROC curves illustrated the sensitivity and specifi city of selected variables via their AUC. P549 The proportion of reversals and their 95% confi dence intervals were estimated using a GEE model for repeated binary data, assuming a binomial distribution and log link, and accounting for subject as a repetition factor. All calculations were done in SAS 9.4. P548 Patient preferences for outcomes in critical care trials (OPTICS): preliminary results J Muscedere1, F Lamontagne2, G Boyd1, M Herridge3, S Fleury1, T Sinuff 3 1Queen’s University, Kingston, ON, Canada; 2University of Sherbrooke, QC, Canada; 3University of Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P548 (doi: 10.1186/cc14628) Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay Both devices can be used to eff ectively monitor and characterize sleep in the ICU environment. g y Methods In this multicenter follow-up study of the Dexamethasone for Cardiac Surgery (DECS) trial, validated self-report questionnaires were sent 1.5 to 4 years after cardiac surgery and ICU treatment to assess symptoms of PTSD and depression, in relation to cumulative life stress (that is, childhood trauma, major stressful life events) and trait anxiety as determinants of psychopathology. Data were available for 1,125 out of 1,244 (90.4%) eligible participants. Mediating and moderating analyses were performed with multivariable linear regression to assess the eff ect of trait anxiety. Subgroup analyses were performed for both sexes. P552 P552 Hospital anxiety and depression after ICU survival: results of a post-ICU aftercare program D Ramnarain, C Slobbe, W Schapendonk, J Van Gorp, I Gnirrip, S Voermans, A Rutten, G Van der Nat, N Van der Lely St.Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P552 (doi: 10.1186/cc14632) Hospital anxiety and depression after ICU survival: results of a post-ICU aftercare program Introduction Although the ICU survival rate has increased in the last decade, the negative eff ects on mental health and related quality of life become more clear. In the literature the prevalence of anxiety and depressive symptoms post ICU ranges from 10 to 43% [1]. Early recognition and treatment of anxiety and depressive symptoms is important because depression caries a risk for suicide, limited quality of life, and delayed return to work. We studied hospital anxiety and depression (HAD) symptoms after ICU discharge. Results Trait anxiety partially mediates the relationship between cumulative life stress and PTSD (β-value reduction from 0.325 to 0.068; P  = 0.000 to P  = 0.003) and fully mediates the association between cumulative life stress and depression (β-value reduction from 0.282 to 0.015; P = 0.000 to P = 0.507). Trait anxiety was not a moderating factor between cumulative life stress and psychopathology. Full mediation of trait anxiety was found in female patients (n = 247), whereas only partial mediation was seen in male patients (n = 878) with regard to PTSD symptoms. As for depression, full mediation was present in both female and male patients. p y p g Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013 for more than 5 days were invited to visit our post-ICU aftercare clinic. Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay L Kok1, M Sep1, D Veldhuijzen2, S Cornelisse1, A Nierich3, J Van der Maaten4, P Rosseel5, J Hofl and6, J Dieleman1, C Vinkers1, L Peelen1, M Joëls1, D Van Dijk1, M Hillegers1 L Kok1, M Sep1, D Veldhuijzen2, S Cornelisse1, A Nierich3, J Van der Maaten4, P Rosseel5, J Hofl and6, J Dieleman1, C Vinkers1, L Peelen1, M Joëls1, D Van Dijk1, M Hillegers1 1University Medical Center Utrecht, the Netherlands; 2Leiden University, the Netherlands; 3Isala Clinics, Zwolle, the Netherlands; 4University Medical Center Groningen, the Netherlands; 5Amphia Hospital, Breda, the Netherlands; 6Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P550 (doi: 10.1186/cc14630) Introduction ICU survivors are at risk for post-traumatic stress disorder (PTSD) and depression. The development of psychopathology depends partially on stable personality factors such as trait anxiety. Introduction ICU survivors are at risk for post-traumatic stress disorder (PTSD) and depression. The development of psychopathology depends partially on stable personality factors such as trait anxiety. S192 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 distribution was light sleep 62.2% (PAT) and 74.1% (PSG); REM 13.0% (PAT) and 10.7% (PSG); deep sleep 14.5% (PAT) and 7.9% (PSG). Conclusion Both wristwatch-style PAT and miniature PSG devices successfully recorded sleep in ICU patients. Although the simple PAT device overestimated TST, sleep stage times were generally in agreement, especially REM time which correlated strongly. Both devices can be used to eff ectively monitor and characterize sleep in the ICU environment. distribution was light sleep 62.2% (PAT) and 74.1% (PSG); REM 13.0% (PAT) and 10.7% (PSG); deep sleep 14.5% (PAT) and 7.9% (PSG). Among ICU patients a high level of trait anxiety is relatively common and associated with intrusions, a symptom of PTSD. Independently, childhood trauma and stress exposure throughout life have been associated with depression. In cardiac surgery patients admitted to the ICU postoperatively, the eff ect of trait anxiety on the relationship between cumulative life stress and stress-related psychopathology remains unknown. Therefore we aimed to assess the mediating or moderating role of trait anxiety in this at-risk patient population. Conclusion Both wristwatch-style PAT and miniature PSG devices successfully recorded sleep in ICU patients. Although the simple PAT device overestimated TST, sleep stage times were generally in agreement, especially REM time which correlated strongly. Sleep monitoring in ICU patients Sleep monitoring in ICU patients S Namba1, K Hayashi1, T Hirayama1, T Hirayama1, Y Namba1, M Terado1, P Easton2, Y Ujike1 1Okayama University Hospital, Okayama, Japan; 2University of Calgary, AB, Canada Critical Care 2015, 19(Suppl 1):P551 (doi: 10.1186/cc14631) Sleep monitoring in ICU patients S Namba1, K Hayashi1, T Hirayama1, T Hirayama1, Y Namba1, M Terado1, P Easton2, Y Ujike1 1Okayama University Hospital, Okayama, Japan; 2University of Calgary, AB, Canada Critical Care 2015, 19(Suppl 1):P551 (doi: 10.1186/cc14631) Introduction Sleep disruption and deprivation is a continuing problem in the ICU. Strategies to improve sleep are confounded by diffi culties in monitoring and measuring sleep in the ICU; traditional polysomnography cannot be utilized. Practical, non-intrusive diagnostic monitoring of sleep is required. The aims were to test two new ambulatory sleep diagnostic devices to monitor sleep in the ICU, compare sleep data generated by the diff erent devices, and characterize sleep in the ICU. p Methods The devices were: Watch PAT 200 (Itamar), simple wristwatch style, employing peripheral arterial tone and actigraphy to evaluate sleep time and sleep stage by an automatic algorithm (PAT device); and ALICE PDx (Respironics Philips), miniature polysomnographic device utilizing EEG and EMG recordings, requiring post-study sleep technician scoring (PSG device). Nineteen ICU patients provided informed consent (mean age 37 years, two female). Diagnosis of most patients was trauma. Device technical problems terminated one ALICE PDx study and three Watch PAT study; one patient revoked consent. Therefore, 14 patients were recorded successfully in a private room in the ICU, while simultaneously wearing both devices, from 20:00 to 06:00. No patient received sedation. Subjective sleep quality was estimated by the visual analog scale. i References 1. Myhren H, et al. Crit Care. 2010;14:R14. 1. Myhren H, et al. Crit Care. 2010;14:R14. 2. Zimond M, et al. Behav Res Ther. 2003;41:1489-96. 2. Zimond M, et al. Behav Res Ther. 2003;41:1489-96. Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay Six weeks after discharge they received a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale (HADS) questionnaire [2]. Patients were asked to return the questionnaire prior to their visit. All data were analyzed and if the HADS score indicated a clinically signifi cant anxiety or depression, patients were referred to a psychologist for further analyses and treatment. All patient data were analyzed retrospectively. Results Seventy-nine patients, 54 men and 43 women, mean age 57 years. Median APACHE II and IV was 18 and 60 respectively. Median ICU and hospitals days were 9 and 20 respectively. Seventy-six percent were mechanically ventilated with a median of 5 days. Median time after ICU discharge to aftercare visit was 165 days. Patients were divided into three categories: 1, no HAD (45.4%); 2, possible HAD (9.3%); and 3, clinically signifi cant HAD (45.4%). Women compared with men showed signifi cantly more HAD symptoms (26.8% vs. 18.6%, P <0.05). Patients with subarachnoid hemorrhage, neurotrauma and multitrauma patients showed more HAD symptoms. Pain, fatigue, muscle weakness, impairment of daily activity dyspnea, and hoarseness were signifi cantly associated with clinically signifi cant HAD. No association between age and HAD was found. Diagnosis at ICU admission, length of stay, severity of illness, delirium and use of sedatives were not associated with HAD. Conclusion Prevalence of clinically signifi cant post-ICU HAD was 45.4%. Female sex and post-ICU physical complaints – pain, fatigue, muscle weakness, impairment in daily activities, hoarseness and dyspnea  – were signifi cantly associated with HAD. Conclusion In cardiac ICU patients, trait anxiety mediates the infl uence of cumulative life stress on the occurrence of PTSD and depression symptoms. Further prospective research is necessary to establish these factors as reliable measures for the early identifi cation of ICU patients at risk for stress-related psychopathology. Somatic complaints after ICU survival: results of a post-ICU aftercare program p g D Ramnarain, W Schapendonk, I Gnirrip, G Van der Nat, A Rutten, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P554 (doi: 10.1186/cc14634) p g D Ramnarain, W Schapendonk, I Gnirrip, G Van der Nat, A Rutten, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P554 (doi: 10.1186/cc14634) Introduction Critical illness today is well recognized as being associated with new or worsening physical impairment, diminished mental health and cognitive dysfunction. We studied the scope of somatic complaints in ICU survivors 4 to 6 months after ICU treatment. Introduction Critical illness today is well recognized as being associated with new or worsening physical impairment, diminished mental health and cognitive dysfunction. We studied the scope of somatic complaints in ICU survivors 4 to 6 months after ICU treatment. yp yp p Conclusion Irrespective of defi nition using PCL-S or DSM-IV mapping, PTSD was identifi ed in no more than one in 10 survivors of critical illness at either 3 or 12 months post ICU, which is still nearly double the US population past-year PTSD prevalence. In ICU survivors with moderate probability PTSD by PCL-S, the CAPS gold-standard interview is challenging to complete and adds only a small number of diagnoses. However, two in fi ve ICU survivors will develop PTSD subtypes of avoidance or hyperarousal, which both occur twice as frequently as the intrusion subtype. Targeting predominant PTSD subtypes may help optimize treatment strategies for the ICU survivor, such as prolonged exposure and eye movement desensitization and reprocessing for those with the avoidance subtype, and pharmacologic antidepressants targeting the sympathetic nervous system to produce anxiolysis for those with the hyperarousal subtype. Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013, for 5 or more days, were invited to visit our ICU aftercare clinic. Six weeks after ICU discharge a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale questionnaire [1] and Impact of Event Scale Revised questionnaire [2], was sent. Patients were asked to return the questionnaires before visiting our clinic. The main purpose of the post- ICU aftercare was to screen for somatic complaints, mental health and cognitive dysfunction. If necessary, further examination or treatment was advised. All data were retrospectively analyzed. y y Results Ninety-seven patients visited our aftercare program in 2013. P553 Post-traumatic stress disorder after ICU discharge: results of a post-ICU aftercare program D Ramnarain, I Gnirrip, W Schapendonk, A Rutten, G Van der Nat, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P553 (doi: 10.1186/cc14633) Results Both devices calculated total sleep time (TST), but the results were signifi cantly diff erent (P <0.05), with mean TST reported as 443.07 and 270.8 minutes for PAT and PSG devices. VAS correlated tightly with TST calculated by the PSG device (r  = 0.559, P <0.05). Both devices were able to successfully discern diff erent sleep stages, summarized as light sleep, deep sleep, and REM. Measurements of sleep stage were generally in agreement between the two devices; REM sleep time correlated strongly between PAT and PSG devices (P <0.05). Sleep stage Introduction Patients who survive ICU treatment may experience psychological distress for some time after discharge from the ICU. In the literature the reported prevalence of post-traumatic stress disorder (PTSD) ranges from 5 to 64% [1]. We studied PTSD symptoms in relation to ICU factors, demographic data and physical complaints reported by patients 4 to 6 months after ICU discharge. Introduction Patients who survive ICU treatment may experience psychological distress for some time after discharge from the ICU. In the literature the reported prevalence of post-traumatic stress disorder (PTSD) ranges from 5 to 64% [1]. We studied PTSD symptoms in relation to ICU factors, demographic data and physical complaints reported by patients 4 to 6 months after ICU discharge. S193 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013 for more than 5  days were invited to visit our ICU aftercare clinic. Six weeks after discharge a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale questionnaire and Impact of Event Scale (IES-R) questionnaire, was sent to the patient. Patients were asked to return the questionnaires prior to visiting the aftercare clinic. All data were analyzed and if the IES-R score indicated a possible PTSD, patients were referred to a psychologist for further analyses and treatment. All patient data were analyzed retrospectively. y Referencefi Referencefi 1. Griffi ths J, et al. Intensive Care Med. 2007;33:1506-18. , yp g Results Of the 180 eligible participants at 3  months, PTSD was identifi ed in 10 (6%) using PCL-S scores and 15 (8%) using DSM-IV mapping of the PCL-S. Of the 160 eligible participants at 12 months, PTSD was identifi ed in two (1%) using PCL-S scores and 10 (6%) using DSM-IV mapping of the PCL-S. Of those eligible for CAPS assessments, at 3 months only 13 of 24 (54%) interviews were completed resulting in three extra PTSD diagnoses, and at 12 months only six of 22 (27%) interviews were completed resulting in two extra PTSD diagnoses. At 3 and 12 months, the intrusion subtype was present in 25 (14%) and in 25 (16%), the avoidance subtype was present in 78 (43%) and 60 (38%), and the hyperarousal subtype was present in 82 (46%) and 71 (44%). Conclusion Irrespective of defi nition using PCL-S or DSM-IV mapping, PTSD was identifi ed in no more than one in 10 survivors of critical illness at either 3 or 12 months post ICU, which is still nearly double the US population past-year PTSD prevalence. In ICU survivors with moderate probability PTSD by PCL-S, the CAPS gold-standard interview is challenging to complete and adds only a small number of diagnoses. However, two in fi ve ICU survivors will develop PTSD subtypes of avoidance or hyperarousal, which both occur twice as frequently as the intrusion subtype. Targeting predominant PTSD subtypes may help optimize treatment strategies for the ICU survivor, such as prolonged exposure and eye movement desensitization and reprocessing for those with the avoidance subtype, and pharmacologic antidepressants targeting the sympathetic nervous system to produce anxiolysis for those with the hyperarousal subtype. Results Of the 180 eligible participants at 3  months, PTSD was identifi ed in 10 (6%) using PCL-S scores and 15 (8%) using DSM-IV mapping of the PCL-S. Of the 160 eligible participants at 12 months, PTSD was identifi ed in two (1%) using PCL-S scores and 10 (6%) using DSM-IV mapping of the PCL-S. Of those eligible for CAPS assessments, at 3 months only 13 of 24 (54%) interviews were completed resulting in three extra PTSD diagnoses, and at 12 months only six of 22 (27%) interviews were completed resulting in two extra PTSD diagnoses. y Referencefi At 3 and 12 months, the intrusion subtype was present in 25 (14%) and in 25 (16%), the avoidance subtype was present in 78 (43%) and 60 (38%), and the hyperarousal subtype was present in 82 (46%) and 71 (44%).i P553 The Pearson chi-squared test was used to compare groups and Cramer’s V analyses was used to examine strength of the association between groups. activity, pain and hoarseness were associated signifi cantly with PTSD and HAD. There was no signifi cant diff erence in somatic complaints between men and women. Conclusion Somatic complaints after ICU discharge are frequently reported in our post-ICU aftercare patients, infl uencing daily performance and quality of life. Patient-centered research and treatment focusing on somatic complaints is of great importance. References 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. Results Seventy-nine patients, 54 male and 43 women, with mean age 57 years. Median APACHE II and APACHE IV were 18 and 60 respectively. Median ICU days and hospital days were 9 and 20 respectively. Seventy- six percent of patients were mechanically ventilated with a median of 5 days. Median time of ICU discharge to aftercare visit was 165 days. Delirium occurred in 22 (22.7%) patients during ICU treatment. The prevalence of PTSD was 43.3% and was most seen in patients after subarachnoid hemorrhage (SAH) (28.6%). Pain, muscle weakness, fatigue, impairment in daily activity, dyspnea, and hoarseness reported during the ICU aftercare clinic visit were signifi cantly associated with PTSD. There was no signifi cant diff erence in men and women. Sedation, opiates, benzodiazepine, inotropic medication and delirium during ICU treatment were not associated with higher prevalence of PTSD. None of the other demographic data analyzed were signifi cantly associated with PTSD. Post-traumatic stress disorder prevalence and subtypes among survivors of critical illness Post-traumatic stress disorder prevalence and subtypes among survivors of critical illness M Patel1, J Jackson1, A Morandi2, T Girard1, C Hughes1, A Kiehl1, J Thompson1, R Chandrasekhar1, E Ely1, P Pandharipande1 1Vanderbilt University, Nashville, TN, USA; 2Ancelle Hospital, Cremona, Italy Critical Care 2015, 19(Suppl 1):P555 (doi: 10.1186/cc14635) M Patel1, J Jackson1, A Morandi2, T Girard1, C Hughes1, A Kiehl1, J Thompson1, R Chandrasekhar1, E Ely1, P Pandharipande1 1Vanderbilt University, Nashville, TN, USA; 2Ancelle Hospital, Cremona, Italy Critical Care 2015, 19(Suppl 1):P555 (doi: 10.1186/cc14635) Introduction Among North American survivors of critical illness, we aim to describe the prevalence of post-traumatic stress disorder (PTSD), and its subtypes of intrusion, avoidance, and hyperarousal. Methods In this prospective, observational, multicenter cohort study from 2009 to 2010, we screened adults (age ≥18  years) with new- onset respiratory failure, cardiogenic shock, or septic shock, who were admitted to medical and surgical ICUs in four facilities. At 3-month and 12-month follow-ups, high probability of PTSD was defi ned by 17-symptom PTSD Checklist – Event Specifi c Version (PCL-S) score ≥50. Also, PCL-S responses were mapped onto DSM-IV criteria for PTSD. To augment PTSD identifi cation, those with a moderate probability of post-ICU PTSD (PCL-S score ≥35) were further confi rmed with the Clinician Administered PTSD Scale (CAPS) structured interview. Moderate or greater symptoms for each PTSD subtype of intrusion, avoidance, and hyperarousal were categorized. Conclusion Prevalence of PTSD was 43.3% and most seen in patients after SAH, refl ecting the majority of patients treated in our ICU. PTSD was associated signifi cantly with pain, muscle weakness, fatigue, dyspnea, hoarseness and impairment of daily activity after a median 165 days post ICU treatment. Somatic complaints after ICU survival: results of a post-ICU aftercare program Median time after ICU discharge and visit to our after care clinic was 165  days. Twenty-fi ve patients died after ICU discharge. Fifty-four patients were excluded because of various reasons; that is, language barrier, psychiatric illness, mental handicap, hospital admittance elsewhere, great distance. Seventy patients (81.4%) had somatic complaints infl uencing daily performance and quality of life. Fatigue (74.4%), muscle weakness (48.8%), dyspnea (34.9%), impairment of daily activity (81.4%), pain (38.4%) and weight loss (33.3%) were the most frequently reported complaints. Pain was most reported in patients with subarachnoid hemorrhage (27.3%), multitrauma (15.2%) and pneumonia (12.1%). Pain was most localized in the head (15.6%), one or both legs (15.6%), back (10.9%), shoulder (9.3%), hip (9.3%) and thorax (6.3%). Muscle weakness, fatigue, dyspnea, impairment of daily Physiotherapy in the ICU: an evidence-based, expert-driven, practical statement J Sommers1, R Engelbert2, D Dettling1, R Gosselink3, P Spronk4, J Horn 1, F Nollet1, M Van der Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2School of Health, Amsterdam, the Netherlands; 3KU Leuven, Belgium; 4Gelre Hospital, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P558 (doi: 10.1186/cc14638) J Sommers1, R Engelbert2, D Dettling1, R Gosselink3, P Spronk4, J Horn 1, F Nollet1, M Van der Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2School of Health, Amsterdam, the Netherlands; 3KU Leuven, Belgium; 4Gelre Hospital, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P558 (doi: 10.1186/cc14638) g q y Results A total of 1,743 papers were retrieved, of which 18 studies were eligible for inclusion in the review. Studies had a combined population of 1,970 patients admitted to 38 ICUs from Europe, Asia and North America. Eleven studies were randomized controlled trials (RCTs). Interventions were classifi ed as four groups – music; therapeutic touch; diary and psychotherapeutic interventions. Ten studies found that music interventions were eff ective in the short term; however, follow- up results were limited and some studies were low quality. There was moderate quality evidence from three studies for the eff ectiveness of diary interventions, with medium-term follow-up results. There was mixed-quality evidence for therapeutic touch interventions in the short term from three studies. The two psychotherapeutic interventions studied were of moderate quality, and one showed promising results at 12-month follow-up.fi Introduction Evidence-based, expert-driven, practical statements improve quality and eff ectiveness of the diagnostic and therapeutic process of patient care. Although the eff ectiveness of physiotherapy treatment strategies in ICU patients has been described, statements or guidelines of physiotherapy for ICU patients are not available [1]. Guidelines on safety management and on the diagnostic and therapeutic process may support and guide clinical decision-making leading towards evidence-based tailored care. The aim of this study was to develop an evidence-based statement for the physiotherapy treatment of ICU patients with recommendations for eff ective and safe diagnostic assessment and intervention strategies. Conclusion The evidence for the effi cacy of nonpharmacological interventions to reduce short-term or long-term stress in intensive care patients was of low to moderate quality. Studies included mainly short- term and medium-term follow-up. This highlights the need for larger- scale, better-quality RCTs with longer-term outcome measurement. However, the results indicate that nonpharmacological, including psychological, approaches are likely to be benefi cial for reducing short- term or long-term stress in intensive care patients. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 particularly those of working age. More work is required to understand optimal rehabilitation pathways in this patient group. References particularly those of working age. More work is required to understand optimal rehabilitation pathways in this patient group. References effi cacy of such interventions. Previous work has shown that intensive care patients undergo many stressful experiences, which can aff ect their long-term psychological well-being. Studies have demonstrated a high prevalence of depression, anxiety or post-traumatic stress disorder after intensive care admissions. Methods A systematic review was carried out according to the Prisma statement. A search was conducted of Medline, Embase and Psychinfo databases. Inclusion criteria included studies of populations of adult patients in mixed or general ICUs. No study designs were excluded, but studies that focused on specifi c disease states were excluded. Included studies were assessed for risk of bias, using a quality checklist. P557 P557 Utilisation of existing community rehabilitation services by critical care survivors CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) Utilisation of existing community rehabilitation services by critical care survivors CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) l Results Three expert-based relevant clinical questions were formulated within the physiotherapy clinical reasoning process and were classifi ed according to the International Classifi cation of Functioning, Disability and Health. In a systematic literature search, 129 studies were identifi ed and assessed for methodological quality and classifi ed according to the level of evidence. The fi nal Evidence Statement consisted of recommendations for physiotherapy in ICU patients including safety criteria, a core set of instruments to assess impairments and activity restrictions and eff ective interventions. CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) Introduction Patients recovering from critical illness suff er many physical and psychological problems during their recovery, including muscle weakness, fatigue, signs and symptoms of PTSD, anxiety and depression [1]. At present, specialist intensive care follow-up and rehabilitation is inconsistent and in many geographical areas is nonexistent. As a result, many survivors of critical illness will require using existing community rehabilitation services [2]. The aim of this present service evaluation was to understand the utilisation of community rehabilitation services by critical care survivors. f Conclusion The Evidence Statement for physiotherapeutic diagnostics and intervention in ICU patients will contribute to the quality of clinical practice by supporting the clinical decision-making process. References 1. Kayambu G, Boots R, Paratz J. Physical therapy for the critically ill in the ICU: a systematic review and meta-analysis. Crit Care Med. 2013;41:1543-54. 2. Burgers JS, van Everdingen JJE. Evidence-based richtlijnontwikkeling in Nederland: the EBRO-platform. Ned Tijdschr Geneeskd. 2004;148:2057-9. Methods A database of acute referrals to community rehabilitation services was retrospectively analysed from 1 May 2014 to 31 October 2014. Age, referring specialty and reason for referral for rehabilitation were documented. This database was cross-checked with the critical care database in Glasgow Royal Infi rmary to identify which individuals had been admitted to critical care during their admission. Physiotherapy in the ICU: an evidence-based, expert-driven, practical statement Methods For the development of this evidence statement, we used the EBRO method, as recommended by the Dutch Evidence Based Guideline Development Platform [2]. This method consists of the identifi cation of clinically relevant research questions, followed by a systematic literature search, quality assessment, and summary of the evidence eventually leading to establishing of concept and fi nal recommendations based on feedback from experts. The fi nal recommendations were prepared according to this methodical approach and summarized in fi gures, fl owcharts and appendices. Nonpharmacological interventions to reduce short-term or long-term psychological stress in ICU patients: a systematic review D Wade1, Z Moon2, S Windgassen2, J Weinman2 1University College Hospital, London, UK; 2Kings College London, UK Critical Care 2015, 19(Suppl 1):P556 (doi: 10.1186/cc14636) Introduction A systematic review was performed of studies of nonpharmacological interventions aiming to reduce short-term or long-term stress in intensive care patients, as little is known about the S194 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) i Results A total of 697 hospitals records were analyzed. Patients had on average (SD) 67.8 (13.1) years and the majority of them were males (57%). Our results revealed that 65% of patients in the CICU received phase 4 and 43% of patients in the CICU received phase 5 of the early mobilization protocol. No diff erences in the proportion of patients receiving phase 4 or 5 were found among arrhythmia, coronariopathies and congestive heart failure groups. The only diff erence found was between congestive heart failure group and other cardiovascular pathologies (P  <0.001). The congestive heart failure group was mobilized 5.6 times (95% CI: 2.7 to 11.5) and 3.2 times (95% CI: 1.7 to 5.7) more than the other cardiovascular pathologies group in phase 4 and 5, respectively. Introduction Increasing cost is an important issue in critical care medicine. We tried to analyze in a level 3 care ICU in Kolkata of a tertiary care hospital whether the diff erent payment options (self-paying vs. insurance/corporate paying) do affect the outcome in the critically ill insurance/corporate paying) do aff ect the outcome in the critically ill. Methods Our prospective study included 1,520 patients admitted consecutively to a level 3 care ICU for a period of 20 months. Readmitted patients during the same period were excluded. Payment method was documented for all and divided into two groups as self-paying and insurance/corporate paying. Outcome assessment was done using the APACHE IV model for all cases. Demographic data, number of observed deaths, predicted mortality rate (PMR), standardized mortality ratio (SMR), average length of stay (ALOS), predicted length of stay, and number of discharge against medical advice (DAMA) were documented for each group. Statistical analysis was carried out using unpaired Student t test and P <0.05 was considered signifi cant. Conclusion A considerable proportion of patients was mobilized without any serious complications in the CICU. Our fi ndings suggest that patients diagnosed with arrhythmia, coronariopathies and congestive heart failure can be equally mobilized in an ICU. p gi Results Of 1,520 patients, 995 (65.46%) cases were self-paying while 525 (34.54%) cases were insurance/corporate paying. P559 E l Early mobilization according to diagnosis in a Brazilian coronary ICU GS Zavanelli1, SA Padulla1, MR Franco1, RZ Pinto1, LL Faccioli1, DN Barbosa1, DT Neves2, CE Bosso2 1Universidade Estadual Paulista – UNESP, Presidente Prudente, Brazil; 2Instituto do Coração de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P559 (doi: 10.1186/cc14639) g Results Over this 6-month period 769 patients were referred from their parent specialty for community rehabilitation in North East Glasgow. Thirty-three of the 769 patients (4.3%) referred had a critical care stay during their admission. Of these, eight patients were referred for rehabilitation by orthopaedics, eight by medicine for the older patients, 11 from acute medicine and the remaining six from other specialties. Six of the 769 patients who had a critical care admission were of working age (<1%). Two individuals were admitted to critical care following trauma whilst four had complex social needs prior to their critical care admission. This included an individual with a high body mass index. None of the individuals of working age were referred as a consequence of their critical care stay. Introduction Early mobilization has been advocated to improve muscle function and, consequently, the patient quality of life after discharge. Nevertheless, few studies have explored it in a coronary ICU (CICU). The aims of the present study were to describe the use of an early mobilization protocol in a CICU and to investigate whether diff erent groups of diagnoses respond similarly to this protocol. Introduction Early mobilization has been advocated to improve muscle function and, consequently, the patient quality of life after discharge. Nevertheless, few studies have explored it in a coronary ICU (CICU). The aims of the present study were to describe the use of an early mobilization protocol in a CICU and to investigate whether diff erent groups of diagnoses respond similarly to this protocol. Conclusion This service evaluation demonstrates that very few critical care survivors are referred to community rehabilitation services, Methods This is a retrospective observational study conducted in a medium-sized hospital located in the city of Presidente Prudente, S195 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Brazil. P559 E l The early mobilization protocol consists of fi ve phases: 1  – passive exercises for the unconscious patient; 2  – active exercises associated with respiratory exercises (patient lying on the bed); 3  – phase 2 exercises with the patient sitting on the bed; 4  – phase 2 exercises with the patient sitting on a chair or in a standing position; 5 – phase 4 exercises plus walking. All hospital records from patients, between September 2013 and August 2014, were included in this study. Data extracted from hospital records were: age, gender, diagnosis (arrhythmia, coronariopathies, congestive heart failure and other pathologies), length of stay, number of discharge and number in each phase of the early mobilization protocol. Pearson chi-square test was used to compare the number of mobilizations (phase 4 and 5) per group of diagnoses. Odds ratios were calculated for those comparisons found to be statistically signifi cant (P <0.05). with mortality for pulmonary artery catheter, platelet transfusion and vasoactive drug infusion (one drug) (P >0.05). Conclusion In this large retrospective multicenter study, the TISS item associated with the highest risk of death was cardiac arrest and/or countershock. Unexpectedly, the independent eff ect of emergency admission was of comparable magnitude in terms of impact on hospital mortality. Of these, in-ICU cardiac arrest might be amenable to preventive measures and should be studied further. P561 P560 Need for therapeutic interventions as a predictor of mortality in intensive care Need for therapeutic interventions as a predictor of mortality in intensive care I Ef dij 1 R R j1 S H 2 MB Sk if 1 M R i ik i 3 Need for therapeutic interventions as a predictor of mortality in intensive care I Efendijev1, R Raj1, S Hoppu2, MB Skrifvars1, M Reinikainen3 1HUS, Helsinki, Finland; 2TAYS, Tampere, Finland; 3PKKS, Joensuu, Finland Critical Care 2015, 19(Suppl 1):P560 (doi: 10.1186/cc14640) I Efendijev1, R Raj1, S Hoppu2, MB Skrifvars1, M Reinikainen3 1HUS, Helsinki, Finland; 2TAYS, Tampere, Finland; 3PKKS, Joensuu, Finland Critical Care 2015, 19(Suppl 1):P560 (doi: 10.1186/cc14640) Introduction Various therapeutic interventions needed in critical care may refl ect a high risk of death. We evaluated associations between commonly used interventions and hospital mortality in Finnish ICU patients. Methods We retrieved data on adult patients treated in Finnish ICUs between 2003 and 2013 from the Finnish Intensive Care Consortium database. We used the Therapeutic Intervention Scoring System (TISS-76) for categorizing ICU interventions and the Simplifi ed Acute Physiology Score (SAPS II) for quantifying severity of illness. We excluded readmissions, patients with missing outcome, SAPS II and TISS data. We also excluded very common interventions (arterial line, bolus intravenous medication), very rare ones (prevalence <1%), and interventions only applicable in specifi c populations (intracranial pressure monitoring, intra-aortic balloon assist). We grouped several TISS categories when applicable. We performed a backward stepwise binary logistic regression analysis with TISS items to assess the impact of each intervention on hospital mortality (expressed as odds ratio (OR) with 95% confi dence intervals (CIs)). Age, admission type, and SAPS score (minus age and admission type scores) were adjusted for in the multivariate analysis. P562 P561 Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) Payment options: do they aff ect outcome in the critically ill A Kar, A Datta In the self-paying group, mean age was 59.65 years ± 17.26 SD (median 62), APACHE IV score mean was 62.50 ± 33.61 SD (median 57), average LOS 4.67 days ± 4.29 SD (median 3), PMR was 22.71, 226 observed deaths, 85 cases of DAMA, and SMR was 1.00 (CI  = 0.87 to 1.14). In the insurance/ corporate-paying group, mean age was 61.75 years ± 17.19 SD (median 65), APACHE IV score mean was 58.53 ± 32.94 SD (median 54), average LOS was 5.64 days ± 5.61 SD (median 4), PMR was 21.26, 113 observed deaths, six cases of DAMA, and SMR was 1.01 (CI = 0.83 to 1.21). In the two compared groups, predicted mortality and SMR were not statistically signifi cant (P = 0.2808); however, ALOS in the insurance/ corporate paying group was signifi cantly higher than the self-paying group (P  = 0.0002), mean age of the insurance/corporate paying group was signifi cantly higher than the self-paying group (P = 0.02), and incidence of DAMA is signifi cantly higher in the self-paying group (8.54%) as compared with insurance/corporate paying group (1.14%). Root-cause analysis showed DAMA cases are mostly fi nancial (>95%). Conclusion Statistically signifi cant diff erences in ALOS and DAMA in the two groups are probably due to cost of healthcare not aff ordable to all. Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P562 (doi: 10.1186/cc14642) Introduction Source of admission to the ICU is of importance. We tried to identify the diff erent sources of ICU admission to a level 3 ICU of a tertiary care hospital in Kolkata and analyze whether the overall patient outcome is aff ected by the diff erent sources of admission. Results We identifi ed 161,134 patients eligible for analysis. The multivariate analysis showed that the highest risk for hospital mortality in all patients was associated with cardiac arrest and/or countershock, OR 2.58 (95% CI = 2.43 to 2.73), SAPS II emergency admission, OR 2.52 (95% CI = 2.32 to 2.74), vasoactive drug infusion (>1 drug), OR 1.66 (95% CI = 1.59 to 1.73) and blood transfusion (a combined TISS item), OR 1.53 (95% CI = 1.44 to 1.63). TISS items associated with the lowest risk of mortality in general population were: active anticoagulation, OR 0.51 (95% CI = 0.49 to 0.53), induced hypothermia, OR 0.68 (95% CI = 0.62 to 0.74) and measurement of cardiac output by any method, OR 0.87 (95% CI = 0.83 to 0.91). All aforementioned associations were statistically signifi cant (P  <0.001). There was no notable association ff Methods Our prospective study included 2,056 patients admitted to a level 3 care ICU over a period of 2 years. Numbers of readmissions were not considered. ICU outcome was analyzed using the APACHE IV model and source of admission to the ICU was documented as either from emergency (ER), from the fl oor or from other hospital. Analysis was carried out between diff erent groups based on admission using unpaired Student t test and P  <0.05 was considered signifi cant. Number of ventilations and the mortality rate in each group were also documented. S196 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Of 2,056 admissions, 1,223 cases (59.48%) were from ER, 809 cases (39.35%) were from fl oor and 24 cases (1.16%) were from other hospitals. Association between out-of-hours discharge and mortality in adult patients leaving critical care Association between out-of-hours discharge and mortality in adult patients leaving critical care S Edie, K Burt, J Paddle Royal Cornwall Hospital, Truro, UK Critical Care 2015, 19(Suppl 1):P564 (doi: 10.1186/cc14644) Introduction Out-of-hours (OOH) discharge from critical care is associated with a signifi cantly increased mortality rate in Australasia [1]. In the UK, daytime discharges from critical care are considered a core standard [2]. We sought to assess the impact of OOH discharge from critical care on mortality in a large general ICU, where operational pressures appear to have led to a high rate of OOH discharges. Methods Retrospective data for all patients admitted to our ICU from April 2007 to September 2014 were recorded, using routinely collected data from our databases. Adult patients (>15 years) discharged from their fi rst ICU admission during each hospital stay (episode) were included. Patients that died on the unit and those discharged for palliative care were excluded. Patients transferred to other centres were no longer subject to discharge within our control and were therefore also excluded. Patients discharged directly home from ICU were excluded. We defi ned OOH discharges as those occurring between 22:00 and 06:59, a standard defi nition in UK practice. Mortality status at the time of hospital discharge for each episode was used. We also recorded the readmission rate to ICU. The relative risk (RR) for OOH mortality and readmission was calculated. Statistical signifi cance was accepted at P <0.05. Conclusion The severity of illness index in patients admitted to the ICU from fl oors is signifi cantly higher than emergency admissions. Overall outcome for patients transferred to the ICU from the fl oor is worse based on mortality rate, SMR, and ALOS when compared with the emergency group. Readmission to the ICU: is it a big concern? An analysis A Ahmed, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P563 (doi: 10.1186/cc14643) Readmission to the ICU: is it a big concern? An analysis A Ahmed, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P563 (doi: 10.1186/cc14643) Results Of 4,476 index cases, 714 died on the unit and 80 were discharged for palliative care. A total of 490 patients were excluded for transfer to other centres and discharge directly home. Data were missing for three patients, which left 3,189 records for analysis. In total, 2,711 patients were discharged during daytime hours, of which 145 (5.35%) died. A total of 478 patients were discharged at night, 40 died (8.37%). The RR for OOH mortality was 1.56 (95% CI = 1.12 to 2.19, P = 0.0091). Readmission rate was 5.2% by day, 6.1% at night. The RR for readmission was 1.17 (95% CI = 0.79 to 1.72, P = 0.436). Introduction Readmission to the ICU is an important quality indicator of ICU care. We conducted a prospective study in a level 3 care ICU in Kolkata of a tertiary care hospital to analyze whether there are overall outcome diff erences when comparing the readmission group with the entire group. Conclusion Our data demonstrate an association between critical care discharge time and mortality, to a statistically signifi cant level. Due to the retrospective observational nature of the study, causation cannot be assumed; however, a number of factors may contribute to the increased risk of harm to patients discharged from the ICU at night. Further work will focus on annual OOH mortality trends, thereby gaining an insight into whether bed occupancy demands impact on the necessity for nighttime discharges. Methods Our prospective study included 2,140 patients admitted to a level 3 care ICU over a period of 1 year. The number of readmissions (n = 85) during the same period was also documented. Readmission was defi ned as all patients who were transferred back to the ICU prior to hospital death/discharge during the above period. ICU outcome was calculated using the predictive APACHE IV model. Payment methods were documented as either self-paying or corporate/insurance paying. A comparison analysis between the entire group with the readmission group was done using unpaired Student t test and P  <0.05 was considered statistically signifi cant. Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed In the ER group, mean APACHE IV was 55.03  ± 31.49 SD (median 50), PMR 16.26, observed deaths 198, ALOS 4.78 days ± 4.83 SD (median 3), SMR 0.995 (CI = 0.86 to 1.14), mean age 60.52 years ± 17.63 SD (median 63), 323 ventilations. In the fl oor group, mean APACHE IV was 65.17 ± 34.40 SD (median 60), PMR 27.03, observed deaths 234, ALOS 5.23 days ± 5.22 SD (median 3), SMR 1.07 (CI = 0.94 to 1.21), mean age 61.38 years ± 15.72 SD (median 64), 302 ventilations. In the other hospital group, mean APACHE IV was 55.29 ± 29.82 SD (median 50), PMR 18.0, observed deaths 2, ALOS 6 days ± 5.85 SD (median 3), SMR 0.46 (CI: 0.23 to 0.88), mean age 56.08 years ± 17.79 SD (median 56.5), six ventilations. During analysis, the other hospital group was omitted because of inadequate sample size. There was statistically signifi cant diff erences in APACHE IV (fl oor >ER, P  <0.0001), PMR (fl oor >ER, P <0.0001), ALOS (fl oor >ER, P = 0.04) noted between the fl oor and ER groups. Number of ventilations (37.33% vs. 26.4%), SMR (1.07 vs. 0.995), and mortality rate (28.92% vs. 16.19%) were also signifi cantly higher for patients admitted from the fl oor. No signifi cant statistical diff erence was observed in age between two groups (P = 0.26). y References 1. Gantner D, et al. Int Care Med. 2014;40:1528-35. 1. Gantner D, et al. Int Care Med. 2014;40:1528-35. 2. Faculty of Intensive Care Medicine and Intensive Care Society. Core standards for intensive care units (2013). http://www.fi cm.ac.uk/standards. 2. Faculty of Intensive Care Medicine and Intensive Care Society. Core standards for intensive care units (2013). http://www.fi cm.ac.uk/standards. y gi Results In the entire group (n = 2,140), mean APACHE IV was 50.34 ± 31.54 SD (median 42), PMR 15.49, observed deaths 327, ALOS 4.05 days ± 4.55 SD (median 3), SMR 0.99 (CI = 0.88 to 1.1), mean age 60.55 years ± 15.68 SD (median 63), 490 ventilations, 72.71% of patients were self-paying while 27.29% of patients were corporate/insurance paying. In the readmission group (n = 85), mean APACHE IV was 77.16 ± 33.72 SD (median 73), PMR 38.89, observed deaths 42, ALOS 5.23 days ± 4.18 SD (median 4), SMR 1.27 (CI = 0.95 to 1.67), mean age 64.79 years ± 14.40 SD (median 67), 43 ventilations, 75.3% of patients were self- paying while 24.7% of patients were corporate/insurance paying. During comparison between the two groups there were statistically signifi cant diff erences, with the readmission group having signifi cantly higher APACHE IV (P <0.0001), PMR (P <0.0001), ALOS (P = 0.002), age (P = 0.005), and SMR (1.27 vs. 0.99) compared with the entire group. Percentage of patients requiring ventilation (50.59% vs. 22.90%) and mortality rate (49.11% vs. 15.28%) were also signifi cantly higher in the readmission group. Readmission was signifi cantly higher in the self- paying group. Root-cause analysis showed most readmissions were due to deteriorating conditions (desaturation, hypotension, sepsis, arrhythmias); however, it was also associated with cases where transfer policy from the ICU was not followed by stakeholders and fi nancial issues were a cause of early transfer. P566 Determination of brain death for adult patients with ECMO I Ceylan, R Iscimen, E Cizmeci, N Kelebek Girgin, F Kahveci Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P566 (doi: 10.1186/cc14646) f Results The CFA showed a good fi t indicating factorial validity (CFI: 0.97), reliabilities were from α 0.79 to 0.93 and ICCs were signifi cant (~0.20, P <0.001). HLM revealed that unit-level IL of nurses and residents was positively related to PS (b = 0.34, P <0.001). Being a resident and working in a smaller unit also predicted PS. As expected, unit-level PS was negatively related to individual PFC (b = –0.38, P = 0.025). Further predictors of higher PFC were: being a nurse, having more than 5 years of job experience and higher workload. PS mediated the relationship between unit-level IL and individual PFC (indirect eff ect: –0.13, P <0.001). Additional analyses revealed that attendings’ PFC was negatively related to their perception of residents PS (b = –0.44, P = 0.019). Introduction ECMO support in ARDS is an emerging strategy when conventional treatment modalities fail. ECMO has advantages on oxygenation and circulation but also it has some unfavorable eff ects. The most serious complication is brain death due to cerebrovascular hemorrhage. An apnea test is the most important component in confi rming brain death. For patients supported by ECMO, apnea testing remains challenging. Brain-death diagnosis is often made without an apnea test. p Methods We present two cases who receive V-V ECMO support after progression to ARDS. After initiation of ECMO we used sedation to prevent movement and improve adaptation to mechanical ventilation. Also we used anticoagulation with heparin to prevent thromboembolic events and ECMO circuit occlusion. On daily follow-up we noticed that patients had lost their pupil reactions to light. Their sedation was ceased and a computed brain tomography was performed. Both patients had intracerebral hemorrhage. We decided to determine brain death with apnea tests. We increased ECMO blood fl ow and fi O2 and then decreased sweep gas fl ow and disconnected the patient from mechanical ventilation respectively. In one patient we did not see any spontaneous breathing eff orts after carbon dioxide retention. We concluded that the apnea test was successful and confi rmed brain death. P566 On the other hand, we confi rmed the brain death of the other patient with cerebral angiography due to the occurrence of hypoxia and hypotension during apnea testing. p p Conclusion A sense of PS in an ICU team might reduce futile care by increasing the safety of speaking-up behavior of nurses and residents. PS can be enhanced by attending physicians who practice inclusive leadership behavior to foster autonomy and participation of residents and nurses. References 1. Manthous CA, et al. Am J Respir Crit Care Med. 2011;184:17-25. 2. Nembhard IM, et al. J Organ Behav. 2006;27:941-66. End-of-life decisions: how do patients die in the ICU? Introduction One of the main goals of intensive care medicine is to reduce the mortality of critically ill patients. However, it is essential to recognize end-of-life care as an integral component of critical care. Besides survival, the success of intensive care should also include the quality of lives preserved and the quality of dying. The objective of this study was to evaluate the incidence and type of end-of-life decisions (ELD) in critical patients that died in an ICU. y Results We experienced some challenges while determining brain death in patients under ECMO support for ARDS. It is challenging to conduct the apnea testing during ECMO support. Auxiliary tests are required for patients who cannot tolerate the changes needed to conduct the apnea test. With increasing use of ECMO therapies, clinicians may come face to face with more complicated life-ending decisions. p Methods Analysis of all patients included in an ICU running database and who died from 1 November 2013 to 31 October 2014. The following variables were evaluated: age, gender, reason for admission, SAPS II, length of ICU stay and type of ELD. To classify ELD, four concepts were defi ned: ‘Comfort care’, a change from curative therapy to comfort care therapy; ‘Limited therapy’, maintenance of curative therapy but without escalating it (for example, no renal substitution); ‘Decision not to resuscitate’, not to perform advanced life support if cardiac arrest occurs; and ‘Without previous end-of-life decisions’, when there was no prior decision regarding the ELD. Conclusion Current guidelines do not include brain death criteria using supportive therapies such as ECMO and therefore should be updated. References 1. Goswami S, et al. J Cardiothor Vasc Anesth 2013;27:312-4. 2. Jarrah RJ, et al. Pediatr Crit Care Med. 2014;15:38-43. 3. Marasco SF, et al. Heart Lung Circ 2008;17 Suppl:S41–7. 4. Farrah JM, et al. Arch Neurol. 2011;68:1543-9. P567 Results A total of 507 patients were admitted to the ICU and 132 died (26%). Reasons for admission in those who died were septic shock (47%), post cardiac arrest (13%), cardiogenic shock (8%), and nontraumatic brain bleeding (8%). Fifty-three patients (40%) died after a ‘Comfort care’ decision, 28 patients (21%) after ‘Decision not to resuscitate’ and 14 (11%) after a ‘Limited therapy’ decision. Thirty-seven patients died ‘Without previous end-of-life decisions’. However, specifi cally in this group, when looking for individual records, 32 patients died (86%) in the fi rst 48 hours after the admission and four (11%) had evidence of brain death and were organ donors, which leaves one patient (3%) in whom there was no ELD. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU D Schwarzkopf1, J Felfe2, CS Hartog1, F Bloos1 1Jena University Hospital, Jena, Germany; 2Helmut Schmidt University, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P567 (doi: 10.1186/cc14647) Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) , Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) Introduction Severe critical illness requiring treatment in the ICU may have a serious impact on patients and their families. However, optimal follow-up periods are not defi ned and data on health-related quality of life (HRQOL) before ICU admission as well as those beyond 2-year follow-up are limited. The aim of our study was to assess the impact of ICU stay up to 5 years after ICU discharge. Methods We performed a long-term prospective cohort study in patients admitted >48 hours to a medical–surgical ICU. The Short-Form 36 was used to evaluate HRQOL before admission (by proxy within 48 hours after admission of the patient), at ICU discharge and at 1, 2 and 5 years following ICU discharge (all by patients). Changes in HRQOL were assessed using linear mixed modeling. Conclusion Readmission to the ICU was associated with worse outcome in our study group. Lack of adherence to transfer policy by concerned stakeholders was a concern as well as increasing cost of healthcare. Results We included a total of 749 patients (from 2000 to 2007). At 5  years after ICU discharge, 234 patients could be evaluated. After S197 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 correction for natural decline in HRQOL, the mean scores of four dimensions – physical functioning (P <0.001), physical role (P <0.001), general health (P <0.001) and social functioning (P = 0.003) – were still signifi cantly lower 5  years after ICU discharge compared with their preadmission levels, although eff ect sizes were small (<0.5). Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study Conclusion Five years after ICU discharge, survivors still perceived a signifi cantly lower HRQOL than their preadmission HRQOL (by proxies), and that of an age-matched general population. Importantly however, after correction for natural decline, the eff ect sizes were small suggesting that patients regain their age-specifi c HRQOL 5 years after their ICU stay. correction for natural decline in HRQOL, the mean scores of four dimensions – physical functioning (P <0.001), physical role (P <0.001), general health (P <0.001) and social functioning (P = 0.003) – were still signifi cantly lower 5  years after ICU discharge compared with their preadmission levels, although eff ect sizes were small (<0.5). report less perceived futile care (PFC). We also expected that attending physicians’ inclusive leadership (IL), which invites nurses’ and residents’ participation [2], would decrease PFC and that PS mediates this relationship. Methods The hypotheses were tested in a cross-sectional, multicenter paper-and-pencil survey addressing medical staff on participating ICUs. A total of 22 ICUs and four intermediate care units were included in the sample and 73 attendings, 147 residents and 659 nurses participated in the study (52% participation). Psychometric properties were tested by confi rmatory factor analysis (CFA), Cronbach’s α and intraclass correlations (ICC). A series of hierarchical linear models (HLM) were conducted to test the study hypotheses separately among nurses/residents and attendings. IL and PS were entered as unit-level predictors (mean values per unit). Covariates were demographics, working hours per week, workload and unit size (number of staff ). Mediation eff ects were tested.i gf Conclusion Five years after ICU discharge, survivors still perceived a signifi cantly lower HRQOL than their preadmission HRQOL (by proxies), and that of an age-matched general population. Importantly however, after correction for natural decline, the eff ect sizes were small suggesting that patients regain their age-specifi c HRQOL 5 years after their ICU stay. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU D Schwarzkopf1, J Felfe2, CS Hartog1, F Bloos1 1Jena University Hospital, Jena, Germany; 2Helmut Schmidt University, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P567 (doi: 10.1186/cc14647) Introduction Psychological safety (PS), for example safety of speaking up, fosters team learning and prevents treatment errors on the ICU [1]. Since speaking up might also prevent excessive and inappropriate (futile) care for patients, we hypothesized that teams with higher PS Conclusion In this study, ‘Comfort care’ was the main ELD, which is in line with the concept that ELD are essential to ensure that care provided is S198 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 examine the association of palliative care screening criteria with adverse patient outcomes. consistent with quality of life and death. The apparent large proportion of patients ‘Without previous end-of-life decisions’ was due to patients who died in the fi rst 48 hours after ICU admission corresponding to conditions refractory to treatment. Additionally, this study also draws our attention to better plan ICU admissions and hospital outreach in order to reduce early ICU mortality. consistent with quality of life and death. The apparent large proportion of patients ‘Without previous end-of-life decisions’ was due to patients who died in the fi rst 48 hours after ICU admission corresponding to conditions refractory to treatment. Additionally, this study also draws our attention to better plan ICU admissions and hospital outreach in order to reduce early ICU mortality. Methods We performed an observational unicentric study on a 12-bed, medical emergency department intensive care unit (EDICU). A three- item palliative care screen was developed from consensus reports. A senior critical care physician screened patients upon admission using these questions during a 10-week period. The questions were: does this patient suff er from a life-limiting disease (end-stage lung, liver, heart or kidney disease, severe neurological disability, extreme frailty, locally advanced or metastatic cancer, advanced-stage AIDS). If the answer to the fi rst question is yes, we proceed to the next one: do you believe this patient will survive to hospital discharge? Answers to those questions were recorded, SAPS III was calculated and all patients were followed until death, discharge or transfer to another center. Diff erences in mortality and SAPS III score between groups were examined using a Student’s t test. Proportions were compared using chi-square test. References Table 1 (abstract P569). Respondents advocating withdrawal for the patient Withdrawal self (%) Withdrawal family (%) Day 3 71 67 Day 7 83 76 Day 28 96 88 Day 42 98 97 Table 1 (abstract P569). Respondents advocating withdrawal for the patient 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. Parents’ return to the hospital after the death of their children: importance of palliative care after death p p G Halal, PL Lago, J Piva, M Halal p g Critical Care 2015, 19(Suppl 1):P572 (doi: 10.1186/cc14652 Introduction To analyze the perception of parents regarding their return to the hospital where their children died to participate in a conversation with doctors and to analyze the feelings of parents about their participation in a study evaluating the care provided in the moments leading up to the death of children. p Withdrawal self (%) Withdrawal family (%) Day 3 16 10 Day 7 23 15 Day 14 25 19 Day 42 42 29 Methods A descriptive exploratory qualitative study. The study sites were the pediatric ICUs of the Hospital São Lucas and Hospital de Clinicas de Porto Alegre. Fifteen parents of children who died in the PICUs studied participated in the study. Data collection occurred in 2010 and was conducted through semistructured interviews. Data were analyzed using thematic content analysis. The research was approved by the research ethics committees of both hospitals. Conclusion Of the ICU physicians who would withdraw care for their patient, the majority would also want the same for themselves. The disparity between decision to continue to treat the patients versus treating self or family increased with increasing length of stay. Reference Results The ability to return to the hospital and talk to medical assistants was considered by parents as a positive and enlightening opportunity. Parents who participated in the study understood this moment as an opportunity to be heard and demonstrated the intention to contribute with their experiences in order to improve care in the hospitals studied. Conclusion We conclude that there is a need to implement measures to provide palliative care to parents after the death of their children. It is necessary to consider the possibility of providing families with follow-up meetings with the multidisciplinary team after the death of children. 1. Korones DN. What would you do if it were your kid? N Engl J Med. 2013;369:1291-3. 1. Piva J, Lago P, Othero J, Garcia PC, Fiori R, Fiori H, et al. Evaluating end of life practices in ten Brazilian paediatric and adult intensive care units. J Med Ethics. 2010;36:344-8. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU P <0.05 was considered statistically signifi cant. P569 Do intensivists prognosticate patients diff erently from themselves or their loved ones? Do intensivists prognosticate patients diff erently from themselves or their loved ones? S Gupta, C Green, R Tiruvoipati, J Botha S Gupta, C Green, R Tiruvoipati, J Botha Peninsula Health, Frankston, Australia Critical Care 2015, 19(Suppl 1):P569 (doi: 10.1186/cc14649) Introduction There is a paucity of data about whether our treatment philosophy is diff erent for our patients as compared with what we would have wanted for ourselves, or while acting as surrogate decision- makers for our loved ones. y gi Results During the period, 191 patients were admitted to the EDICU, from which 151 had complete data and follow-up. A total of 63 patients (41.7%) suff ered from a life-limiting disease and were evaluated as having a high probability of death in 1 year. This group was further divided between 35 patients who in the moment of initial screening were expected to die in this hospital admission and 28 patients who were believed to survive to discharge. Comparison between these two groups showed patients believed to die at this hospital admission had higher SAPS III scores (66.9 vs. 59, P = 0.010) and hospital mortality (48.6% vs. 10.7%, P = 0.001). Methods An anonymous survey was sent to all the members of Australia and New Zealand Intensive Care Society and the College of Intensive Care Medicine (CICM). The fi rst section comprised a hypothetical case scenario spanning over 6 weeks of ICU stay for a patient. At four diff erent stages of the ICU stay, responders were requested to answer multiple- choice questions regarding the philosophy of treatment, based on their perceived prognosis of the patient at that particular time. The following two sections contained the same set of questions with the hypothetical scenario of responders acting as surrogate decision-makers for the patient and that of responders being patients themselves, in the same situation. The responses were compared amongst three sections at each stage using the chi-square test. Conclusion A high percentage of patients admitted to our EDICU have life-limiting disease and might benefi t from palliative care. These patients can be identifi ed using simple screening questions at admission and positive answers to those questions can be associated with worse outcomes. Results A total of 115 responses were received from the fellows of CICM. The results are presented in Tables 1 and 2. P573 How readable are our Patient Information Sheets? L Strachan, M Booth Glasgow Royal Infi rmary, Glasgow, UK Critical Care 2015, 19(Suppl 1):P573 (doi: 10.1186/cc14653) Results Sixty-eight knowledge to care gaps were proposed, rated and revised by the committee over three rounds of review, resulting in 13 priorities for improvement. Then, 1,103 providers (62% response rate) representing nurses, respiratory therapists, allied health professionals and physicians evaluated the priorities and rated nine as necessary. In multivariable logistic regression analyses, provider (profession, experience and teaching status of ICU) and knowledge to care gap characteristics (strength of supporting evidence, potential to benefi t the patient, potential to improve patient/family experience, and potential to decrease costs) were associated with priorities rated as necessary. After disseminating the results to all network members, 627 responded (35% response rate) and indicated that the priorities were reasonable choices for quality improvement initiatives (87%), that they were highly supportive of working on initiatives targeting the priorities (61%) and would be willing to act as local champions for the initiatives (n = 92 individuals). Introduction We often need to obtain consent for clinical studies in the ICU. Participant Information Sheets (PIS) can be diffi cult to understand. A recent French publication [1] supports our hypothesis that PIS have poor readability scores. Methods Protocols submitted for ethics approval between 2008 and 2009 were obtained with permission from the Scotland A Ethics Research Committee. Ethical approval was not required for this observational study. All header, footers, diagrams and tables were removed. Readability scoring was performed using the Flesch Reading Ease and Flesch–Kincaid (FK) grades. Statistical analysis using Excel and MiniTab was then performed. The readability of these documents was compared with everyday documents – newspaper articles, politicians’ speeches [2] and standard contract agreements. p g Results A total of 104 protocols containing 209 PIS were reviewed. Of these, 99 (47%) were written for patients, 56 (27%) for GPs, 26 (12%) for relatives, 17 (8%) for carers, fi ve (2%) for legal representatives and six (3%) were summary sheets only. Sixty-seven (64%) of these protocols were submitted by academic institutions (for example, university or health boards) and 37 (36%) by pharmaceutical companies. Results are expressed as the median and 25th and 75th percentiles. P573 The word count and number of pages were higher for those PIS submitted by pharmaceutical companies compared with academic institutions: 1,561 (471; 5,167) versus 1,177 (626.5; 1,559.8) with P <0.05 and 4 (2; 10) versus 3 (2; 4) with P <0.05 respectively. The Flesch Reading Ease (63 (56; 69) vs. 60 (52.6; 65.4)) and FK grades (3 (5.4; 7.2) vs. 6.8 (6; 7.6)) were similar for both groups. Further subanalysis demonstrated that PIS designed for GPs had a lower word count, lower Flesch and higher FK grade compared with those for patients – the diff erence in Flesch and FK grade were compared using a Mann–Whitney test and were statistically signifi cant. Conclusion Our research approach engaged a diverse group of stakeholders to identify nine priorities for improving the quality and value of care provided to critically ill patients. This methodology can be used to engage stakeholders and identify priorities for quality improvement in other healthcare systems and domains. Additional work is required to reconcile provider/decision-maker and patient/ family priorities. p H Stelfox1, E McKenzie1, S Bagshaw2, M Gill1, P Oxland1, D Boulton1, D Oswell1, M Potestio1, D Niven1 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P575 (doi: 10.1186/cc14655) y gi Conclusion The FK grade is equivalent to US school grade level. The US government advises all policies produced should have a FK grade of <9. Our study suggests that protocols submitted to the ethics committee are easy to read, comparing favourably with broadsheet journalism and standard contract, for example loan contract. However, the average reading age in the UK is 9 years [3], suggesting participants may struggle with the information provided. References Introduction With increasing emphasis on patient and family-centred care, it follows that patients and their family members should be included when priorities for improving care are established. We therefore used a novel methodology that employs former patients and family members as researchers to describe the experiences of critically ill patients and their families with ICUs and to identify opportunities for improvement. journalism and standard contract, for example loan contract. However, the average reading age in the UK is 9 years [3], suggesting participants may struggle with the information provided. References p Methods Using the patient engagement framework developed by Marlett and Emes, we engaged four former patients and family members trained in qualitative research methods to conduct and analyse semistructured focus groups and interviews with adult patients who had recovered from critical illness and family members of both surviving and deceased patients. Participants were recruited from 13 ICUs in Alberta, Canada. Focus groups and interviews were recorded, transcribed and analysed using phenomenological reduction. Data collection continued until thematic saturation was reached. 1. Menoni V. The readability of information and consent forms in cl research in France. PLos One. 2010;5:e10576. Menoni V. The readability of information and consent forms in clinic research in France. PLos One. 2010;5:e10576. 2. http://www.britishpoliticalspeech.org/speech-archive.l 3. Gillies K. Patient information leafl ets for UK randomised controls. Trial. 2014;15:62. References 1. Piva J, Lago P, Othero J, Garcia PC, Fiori R, Fiori H, et al. Evaluating end of life practices in ten Brazilian paediatric and adult intensive care units. J Med Ethics. 2010;36:344-8. Introduction A high percentage of patients admitted to ICUs fulfi ll one or more criteria for palliative care. There are currently few comprehensive studies in critical care settings that have set out to 2. Lago PM, Nilson C, Pedro Piva J, Vieira AC, Halal MG, de Carvalho Abib GM, et al. Nurses’ participation in the end-of-life process in two paediatric intensive 2. Lago PM, Nilson C, Pedro Piva J, Vieira AC, Halal MG, de Carvalho Abib GM, et al. Nurses’ participation in the end-of-life process in two paediatric intensive S199 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 care units in Brazil. Int J Palliat Nurs. 2011;17:264, 267-70. 3. Abib El Halal GM, Piva JP, Lago PM, El Halal MG, Cabral FC, Nilson C, et al. Parents’ perspectives on the deaths of their children in two Brazilian paediatric intensive care units. Int J Palliat Nurs. 2013;19:495-502. Methods Using a modifi ed RAND/UCLA Appropriateness Methodology, a committee of 38 providers and decision-makers representing a population-based clinical network of adult (n = 14) and pediatric (n = 2) medical–surgical ICUs in Alberta, Canada (population 4 million) serially proposed, rated and revised potential knowledge to care gaps as priorities for improvement. The priorities developed by the committee were sent to the network’s 1,790 frontline providers to rate their importance. The fi nal list of priorities that were rated as important was disseminated to all network members for feedback. Using patient researchers to understand patient and family experiences in ICUs p H Stelfox1, E McKenzie1, S Bagshaw2, M Gill1, P Oxland1, D Boulton1, D Oswell1, M Potestio1, D Niven1 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P575 (doi: 10.1186/cc14655) Survey of visiting hours in critical care units in English trauma centres One hundred per cent were able have questions answered satisfactorily. Linked to the FS-ICU, we have seen marked improvements in decision-making and satisfaction. Methods A telephone survey on visiting times was conducted in 53 adult critical care units in trauma centres in England. A list of trauma centres was obtained from the NHS England website. All critical care units (other than obstetric high dependency units and coronary care units, unless part of a cardiothoracic critical care unit) within each hospital were surveyed. Each respondent was asked about the visiting hours, whether children were allowed to visit and how many visitors to a bed space. Conclusion We have shown progressive improvement over 3  years across all domains. Marked improvement in information provision and decision-making support from 53% to 96% over 3  years since introducing the FWR correlates with the improved overall satisfaction (Figure 1). Interestingly FWR is more helpful than relatives anticipated. The FWR was very well received and our results suggest an unrecognised need is being met. Because this was a pragmatic study, we feel this is a true representation of family satisfaction. It is encouraging that communication, information and decision-making support continue to improve. They have become embedded in the fabric of our critical care practice and lead to marked improvement in satisfaction for families. Reference Results Fifty-three units with between four and 75 beds and covering the whole of England were surveyed: there was a 100% response rate. Visiting hours varied between hospitals and between units within the same hospital. Nine units (17%) had open visiting hours, although most gave advice on times to avoid such as nursing handover. The majority of units (44.83%) operated restricted visiting with a median (range) of 6 (2 to 9) hours. All units allowed a maximum of two visitors to the bedspace. Children were allowed in nine units without restriction, the remaining units advised that it may not be appropriate for children to visit and it was at the discretion of the parents and medical staff . 1. Wall et al. Refi nement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit Care Med. 2007;35:271-9. f Conclusion The majority of adult critical care units in England, including our own, have restricted visiting policies. Survey of visiting hours in critical care units in English trauma centres Figure 1 (abstract P577). Trends in family satisfaction. E Taylor, N Bunker E Taylor, N Bunker The Royal London Hospital, London, UK round (FWR), to enhance and standardise communication and improve satisfaction. Following introduction of the FWR we have audited family satisfaction using the validated FS-ICU questionnaire [1]. round (FWR), to enhance and standardise communication and improve satisfaction. Following introduction of the FWR we have audited family satisfaction using the validated FS-ICU questionnaire [1]. Methods This was a prospective study of relatives’ satisfaction for patients completing their critical care episode. The questionnaire was completed anonymously and data collected. This was a pragmatic study, no changes were made to communication strategies. Results There is a high degree of satisfaction across all domains of the FS-ICU including treatment of family and provision of information (Figure 1). One hundred per cent found FWR to be helpful, only 55% had anticipated this. Fifteen per cent changed their perception of critical care. It enabled 15% to raise new concerns. One hundred per cent were able have questions answered satisfactorily. Linked to the FS-ICU, we have seen marked improvements in decision-making and satisfaction. Introduction The purpose of this study was to assess the visiting restrictions placed on families visiting adult patients on critical care units within trauma hospitals in England. Whilst it is well recognised that high-quality care for patients is of paramount importance, we should also be aware that supporting patients’ families off ers long- term benefi ts for patient, family and hospital. In our own unit we are reviewing whether we could adopt a more fl exible attitude to visiting times and assessing how to provide a more welcoming environment to relatives. To inform our own review and in order to develop a best practise approach, we surveyed all of the major trauma centres in England. Methods This was a prospective study of relatives’ satisfaction for patients completing their critical care episode. The questionnaire was completed anonymously and data collected. This was a pragmatic study, no changes were made to communication strategies. Results There is a high degree of satisfaction across all domains of the FS-ICU including treatment of family and provision of information (Figure 1). One hundred per cent found FWR to be helpful, only 55% had anticipated this. Fifteen per cent changed their perception of critical care. It enabled 15% to raise new concerns. Survey of visiting hours in critical care units in English trauma centres Visiting policies are a source of debate amongst staff in intensive care with concerns about open visiting including increased workload and interruptions to normal routine [1]. This is consistent with the views of staff at our own unit who, in appreciative enquiry, have expressed mixed opinions about extending visiting times. Extending visiting times is only part of a wider project to improve the way relatives experience intensive care whilst ensuring both medical and nursing staff feel supported, creating an environment for optimal communication. P578 Bereavement care in UK ICUs: a national survey M Berry1, E Brink2, V Metaxa2 1Imperial Healthcare Trust, London, UK; 2King’s College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P578 (doi: 10.1186/cc14658) Introduction For the families of critically ill patients, the death of a loved one in the ICU is often an unexpected and traumatic event, characterised by diffi cult decisions regarding withholding or withdrawing life-sustaining therapy. Increasingly the importance of bereavement care (BC) in the ICU is being acknowledged, although reports continue to highlight the inadequacies around end-of life care in the critical care environment. In 1998, the Intensive Care Society (ICS) published guidelines mapping out BC in the ICU [1]. We aimed to compare BC in ICUs across England against the recommendations set out by the ICS. 1. Berti D, et al. Intensive Care Med. 2007;33:1060-5. 1. Berti D, et al. Intensive Care Med. 2007;33:1060-5. P574 Stakeholder engagement to identify priorities for improving the quality and value of care provided to critically ill patients H Stelfox1, D Niven1, S Bagshaw2, E McKenzie1, M Potestio1, F Clement1, D Zygun2 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P574 (doi: 10.1186/cc14654) Results Thirty-two participants including patients (n = 11) and family members (n = 21) participated in fi ve focus groups (n = 23 participants) and eight interviews (n  = 9 participants). Participants articulated themes refl ecting important components of care organised across three phases of the ICU experience; admission to ICU, daily care in ICU and after ICU discharge. Admission to ICU comprised three themes: patient and family transition into ICU, patient and family disorientation upon admission to ICU and preferred staff actions to help patients/ family adapt to the ICU. The daily care phase of ICU consisted of fi ve themes: honouring patient’s voices, needing to know, making decisions, culture in ICU and medical care. The experience after ICU discharge comprised two themes: transition from ICU to a hospital ward and long-term eff ects of critical illness. Participants identifi ed fi ve Critical Care 2015, 19(Suppl 1):P574 (doi: 10.1186/cc14654) Introduction Healthcare systems do not make optimal use of evidence, which results in suboptimal patient care. Large amounts of scientifi c evidence are generated but not implemented into patient care (knowledge to care gap). We sought to identify and prioritize knowledge to care gaps in critical care medicine as opportunities to improve quality and value in care. S200 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P577). Trends in family satisfaction. priorities for improvement: provide families with a guide/navigator; educate providers about the fragility of family trust; improve provider communication skills; inform patients about the long-term eff ects of critical illness; and develop strategies to facilitate continuity of care between providers. p Conclusion Patients and family members are an untapped resource and engaging them as researchers is a viable strategy to identify opportunities for quality improvement that are patient and family centred. P577 y Methods All adult ICUs in England were contacted over a 2-week period, using a standardised questionnaire based on the nine domains identifi ed by the ICS. All answers were collected anonymously using SurveyMonkey®. An 80% compliance rate was deemed acceptable.i y Methods All adult ICUs in England were contacted over a 2-week period, using a standardised questionnaire based on the nine domains identifi ed by the ICS. All answers were collected anonymously using SurveyMonkey®. An 80% compliance rate was deemed acceptable. Results From the 148 ICUs identifi ed, 113 answered the questionnaire (76%). Forty-three per cent of the responders had access to training in BC and in communication skills, and 54% had a named member of staff responsible for training, writing, auditing and developing the R Handslip, A Molokhia Introduction Patient satisfaction is a crucial part of clinical care and there is now increasing recognition of the importance of family involvement and satisfaction in the provision of care for the critically ill. Since 2012 our unit has introduced a consultant-led family ward Results From the 148 ICUs identifi ed, 113 answered the questionnaire (76%). Forty-three per cent of the responders had access to training in BC and in communication skills, and 54% had a named member of staff responsible for training, writing, auditing and developing the S201 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion This is the fi rst national audit of BC in the ICU since the initial ICS guideline publication. Even though most ICUs provided relatives with information around the time of death, training, auditing and adequate facilities do not meet the recommended standards. The lack of adherence is defi nitely multifactorial and requires further research. In the meantime, vigorous implementation of these guidelines is warranted in order to ensure optimal care for the bereaved families. Reference BC policy. When asked about the presence of a written BC policy only 45% responded positively, and even less (19%) had provisions for audit and development of the service. Information to staff about cultural and religious rites around the time of death, and to relatives on what to do after a death was available in 81% and 96% respectively. The general practitioner was informed of the deaths taking place in the ICU in 77% of the cases. . Intensive Care Society (1998). http://www.ics.ac.uk/ics-homepage/ guidelines-and-standards/. P577 In more than 70% of the participating ICUs, eff orts were made to ensure privacy of the grieving relatives and to have dedicated follow-up facilities for the bereaved. Even though staff support programmes were recognised as paramount, only 54% of the ICUs had formal ones set up.
https://openalex.org/W4225552082
http://www.jci.org/articles/view/156119/files/pdf
English
null
Sensing of RNA stress by mTORC1 drives autoinflammation
˜The œJournal of clinical investigation/˜The œjournal of clinical investigation
2,022
cc-by
2,762
Sensing of RNA stress by mTORC1 drives autoinflammation Min Ae Lee-Kirsch Min Ae Lee-Kirsch Min Ae Lee-Kirsch Department of Pediatrics, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. IRE-1 during endoplasmic reticulum stress (10), thereby limiting type I IFN–depen- dent immune activation. However, the role of the SKIV2L RNA exosome for immune homeostasis in the absence of ER stress remains poorly understood. Loss-of-function mutations in SKIV2L underlie trichohepatoenteric syndrome (THES2), a rare inborn error of immunity characterized by diarrhea, skin lesions, brittle hair, and immunodeficiency. SKIV2L is part of a multiprotein complex required for exosome-mediated RNA surveillance through RNA decay. In this issue of the JCI, Yang et al. delineate a mechanism underlying autoinflammatory skin disease in Skiv2l-deficient mice. Thus, a lack of SKIV2L activates mTORC1 signaling in keratinocytes and T cells, impeding skin barrier integrity and T cell homeostasis. Interestingly, treatment with the mTOR inhibitor rapamycin improves skin symptoms in Skiv2l-deficient mice, suggesting a possible therapeutic avenue for patients with THES2. RNA stress due to SKIV2L deficiency activates mTORC1 signaling Loss-of-function mutations in SKIV2L cause trichohepatoenteric syndrome (THES2), a rare inborn error of immuni- ty characterized by intrauterine growth retardation, early-onset chronic diarrhea, brittle hair with trichorrhexis nodosa, skin lesions, and immunodeficiency (11, 12). In this issue of the JCI, Yang et al. (13) set out to dissect the functional consequenc- es of Skiv2l deficiency in mice. To bypass embryonic lethality of mice with complete Skiv2l knockout, they turned to mice with tamoxifen-inducible whole-body deletion of Skiv2l. These mice developed inflamma- tory skin lesions due to impaired epidermal stratification with loss of epidermal barrier integrity. Skin-specific deletion of Skiv2l also led to epidermal hyperplasia with defective hair morphogenesis, recapitulat- ing the human disease phenotype. Inter- estingly, these phenotypic changes were not accompanied by activation of the type I IFN axis, as shown by a lack of expression of IFN-stimulated genes in Skiv2l-defi- cient epidermis or primary keratinocytes. Moreover, mice with myeloid-specific Skiv2l knockout did not show any skin pathology or signs of inflammation, sug- gesting that skin pathology in Skiv2l defi- ciency occurs independent of the hema- topoietic system. Together, these findings indicate a cell-intrinsic mechanism by which the SKI-associated RNA exosome regulates keratinocyte function and which is required for skin barrier integrity inde- pendent of type I IFN signaling. T i i l fili f id flammation or autoimmunity (5). Sensing of viral RNA by receptors of the innate immune system is an essential strategy in antiviral immunity (6). A central chal- lenge for the host cell is to discriminate between harmful foreign RNA and self- RNA. Ligand specificity of RNA sensors, such as the cytosolic RNA helicases RIG-I and MDA5, relies largely on unique struc- tural properties of viral RNA (5, 6). Thus, while RIG-I senses 5′-triphosphate-RNA or 5′-diphosphate-RNA, MDA5 recognizes long, double-stranded RNA (7–9). Engage- ment of these RNA sensors triggers type I interferon (IFN) signaling, resulting in the activation of numerous antiviral transcrip- tional programs (5, 6). The processes of RNA metabolism and RNA sensing must be tightly regulated to avoid accumula- tion of potentially immunostimulatory self-RNA and to prevent inappropriate innate immune activation. The SKIV2L RNA exosome has been shown to degrade immunostimulatory self-RNA arising as a cleavage product of the endonuclease C O M M E N TA R Y The Journal of Clinical Investigation RNA metabolism and RNA sensing The human genome is transcribed to generate an extraordinary diversity of RNA, which is subject to complex regu- lation to maintain cellular homeostasis. The turnover and quality control of ribo- some-associated mRNA are controlled by the cytoplasmic RNA exosome, an RNA degradation machinery acting in concert with the super-killer (SKI) complex (1). The SKI complex consists of the SKIV2L helicase, two subunits of the WD-repeat protein WDR61, and the tetratricopeptide repeat motif containing protein TTC37 (2). It associates with the 80S ribosome and extracts mRNA that is no longer needed or recognized as being faulty to initiate ribo- nucleolytic degradation from the 3′ end by the RNA exosome (Figure 1) (3, 4). RNA metabolism is also critical for immune homeostasis, as self-RNA occur- ring in the wrong place at the wrong time can turn into a danger signal that triggers immune responses leading to autoin- Related Article: https://doi.org/10.1172/JCI146176 Conflict of interest: The author has declared that no conflict of interest exists. Copyright: © 2022, Lee-Kirsch et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Reference information: J Clin Invest. 2022;132(2):e156119. https://doi.org/10.1172/JCI156119. Related Article: https://doi.org/10.1172/JCI146176 Conflict of interest: The author has declared that no conflict of interest exists. Copyright: © 2022, Lee-Kirsch et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Reference information: J Clin Invest. 2022;132(2):e156119. https://doi.org/10.1172/JCI156119. The Journal of Clinical Investigation Figure 1. A model for mTORC1 signaling in SKIV2L deficiency that leads to uncontrolled proliferation and immune activation. (A) The turnover and quality control of ribosome-associated mRNA are controlled by the cytoplasmic RNA exosome, which acts as a molecular shredder by degrading mRNA from the 3′ end. The RNA exosome is assisted by the SKI complex. The SKIV2L helicase unwinds RNA and, together with the TPR motif–con- taining protein TTC37 and two subunits of the WD-repeat protein WDR61, initiates ribonucleolytic degradation. The mTORC1 complex senses nutrient deprivation, which induces activation of downstream targets S6K and 4E-BP1 by phosphorylation. mTORC1 signaling promotes a broad range of anabolic processes, including cell growth and proliferation, which require a sufficient supply of deoxyribonucleotides for DNA synthesis. (B) Aberrant mTORC1 signaling in SKIV2L deficiency may be triggered by sensing changes in cellular concentrations of deoxyribonucleotides, which are produced from ribonucleotides by the action of ribonucleotide reductase (RNR). Unabated mTORC1 activity in keratinocytes and T cells leads to hyperproliferation with secondary inflammatory responses. Rapamycin inhibits the protein kinase mTOR, core component of the mTORC1 complex, and may therefore be of therapeutic benefit in patients with SKIV2L deficiency. Figure 1. A model for mTORC1 signaling in SKIV2L deficiency that leads to uncontrolled proliferation and immune activation. (A) The turnover and quality control of ribosome-associated mRNA are controlled by the cytoplasmic RNA exosome, which acts as a molecular shredder by degrading mRNA from the 3′ end. The RNA exosome is assisted by the SKI complex. The SKIV2L helicase unwinds RNA and, together with the TPR motif–con- taining protein TTC37 and two subunits of the WD-repeat protein WDR61, initiates ribonucleolytic degradation. The mTORC1 complex senses nutrient deprivation, which induces activation of downstream targets S6K and 4E-BP1 by phosphorylation. mTORC1 signaling promotes a broad range of anabolic processes, including cell growth and proliferation, which require a sufficient supply of deoxyribonucleotides for DNA synthesis. (B) Aberrant mTORC1 signaling in SKIV2L deficiency may be triggered by sensing changes in cellular concentrations of deoxyribonucleotides, which are produced from ribonucleotides by the action of ribonucleotide reductase (RNR). Unabated mTORC1 activity in keratinocytes and T cells leads to hyperproliferation with secondary inflammatory responses. Rapamycin inhibits the protein kinase mTOR, core component of the mTORC1 complex, and may therefore be of therapeutic benefit in patients with SKIV2L deficiency. The Journal of Clinical Investigation a patient with THES2 who presented with failure to thrive, diarrhea, trichorrhexis nodosa, erythematous rash, elevated liver enzymes, and glomerulonephritis, indi- cating activation of the mTORC1 pathway also in human SKIV2L deficiency. Finally, the authors demonstrated that both sys- temic and topical treatment of Skiv2l-defi- cient mice with the mTOR inhibitor rapa- mycin ameliorated epidermal hyperplasia and skin inflammation. ment of genes acting in the mTORC1 sig- naling pathway. The mTORC1 complex, a multiprotein assembly consisting of the serine/threonine protein kinase mTOR, raptor, mLST8, PRAS40, and DEPTOR, acts as a central hub that integrates nutri- ent signaling pathways to promote cell growth (14). The authors confirmed acti- vation of mTORC1 signaling in Skiv2l- deficient keratinocytes by demonstrating enhanced phosphorylation of ribosom- al S6 kinase (S6K) and 4E-BP1, the key downstream effectors of the mTORC1 pathway (14), along with increased glob- al protein synthesis. Skiv2l-deficient skin lesions showed T cell infiltrates and these T cells were chronically activated as shown by increased proliferation in response to ex vivo stimulation with anti- CD3/CD28. Similar to keratinocytes, T cell hyperactivation was accompanied by increased phosphorylation of S6K and 4E-BP1, consistent with an mTORC1- dependent, cell-intrinsic role for Skiv2l in T cell homeostasis. Interestingly, increased epidermal phospho-S6K stain- ing was also observed in lesional skin of with THES2 develop reactive hemophago- cytic syndrome, a hyperinflammatory state caused by hyperactivation of T cells and macrophages (12, 15). Moreover, the findings by Yang et al. indicate that a lack of cytoplasmic RNA quality control due to dysfunction of the SKI-associated RNA exosome is sensed by mTORC1, although the exact nature of the metabolites that are actually sensed by mTORC1 under these circumstances is still unknown. ment of genes acting in the mTORC1 sig- naling pathway. The mTORC1 complex, a multiprotein assembly consisting of the serine/threonine protein kinase mTOR, raptor, mLST8, PRAS40, and DEPTOR, acts as a central hub that integrates nutri- ent signaling pathways to promote cell growth (14). The authors confirmed acti- vation of mTORC1 signaling in Skiv2l- deficient keratinocytes by demonstrating enhanced phosphorylation of ribosom- al S6 kinase (S6K) and 4E-BP1, the key downstream effectors of the mTORC1 pathway (14), along with increased glob- al protein synthesis. Skiv2l-deficient skin lesions showed T cell infiltrates and these T cells were chronically activated as shown by increased proliferation in response to ex vivo stimulation with anti- CD3/CD28. Related Article: https://doi.org/10.1172/JCI146176 Conflict of interest: The author has declared that no conflict of interest exists. Copyright: © 2022, Lee-Kirsch et al. This is an open access article published under the terms o Commons Attribution 4.0 International License. Transcriptional profiling of epider- mal tissue of mice with keratinocyte- specific Skiv2l deletion revealed enrich- Reference information: J Clin Invest. 2022;132(2):e156119. https://doi.org/10.1172/JCI156119. 1 C O M M E N TA R Y C O M M E N TA R Y Perturbations of the mTORC1 path- way have been implicated in a variety of human diseases, including common auto- immune diseases such as systemic lupus erythematosus (22). Given the genetic and phenotypic heterogeneity of these com- plex diseases, mTORC1 hyperactivation may represent a useful endotype, enabling further stratification of patients based on mechanistic insight. the recycling of the nucleotide pool in the cell (Figure 1). However, whether chang- es in cellular nucleotide concentrations underlie mTORC1 signaling in SKIV2L deficiency remains to be investigated. mediated recognition of RNA bearing 5’-diphos- phates. Nature. 2014;514(7522):372–375. mediated recognition of RNA bearing 5’-diphos- phates. Nature. 2014;514(7522):372–375. 9. Kato H, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA virus- es. Nature. 2006;441(7089):101–105. 10. Eckard SC, et al. The SKIV2L RNA exosome limits activation of the RIG-I-like receptors. Nat Immunol. 2014;15(9):839–845. Interestingly, the authors describe a lack of type I IFN activation in skin and blood of Skiv2l-deficient mice, arguing against a pathogenetic role of RIG-I– dependent innate immune activation by unprocessed self-RNA in SKIV2L defi- ciency. This result contrasts with work by Eckard et al., which demonstrates an IFN signature in the blood of two patients with THES2, but not in three patients with THES1 who carried mutations in the SKI complex component TTC37 (10, 21), despite having indistinguishable clinical features. The findings by Yang et al. (13) are also in line with a clinical report, demon- strating absence of an IFN signature in a patient with THES2 (15). Nonetheless, a moderately increased expression of MX1, an IFN-regulated gene, was found in skin lesions of the patient studied by Yang et al. (13). The type I IFN activation observed in patients with THES2 might act as a permis- sive factor rather than as the primary cause of inflammation. This notion is also sup- ported by the therapeutic efficacy of rapa- mycin in Skiv2l-deficient mice. Patients with THES develop intractable diarrhea commonly leading to failure to thrive (11, 12). Although mice with Skiv2l deficien- cy do not exhibit intestinal symptoms, it is possible that a loss of intestinal barrier integrity due to aberrant mTORC1 signal- ing may account for intestinal dysfunction in patients with THES2. As such, rapamy- cin may provide a promising and potential- ly curative therapy for these patients. 11. Fabre A, et al. SKIV2L mutations cause syndrom- ic diarrhea, or trichohepatoenteric syndrome. Am J Hum Genet. 2012;90(4):689–692. 12. C O M M E N TA R Y Vély F, et al. Combined immunodeficiency in patients with trichohepatoenteric syndrome. Front Immunol. 2018;9:1036. 4. Zinoviev A, et al. Extraction of mRNA from stalled ribosomes by the ski complex. Mol Cell. 2020;77(6):1340–1349. 3. Schmidt C, et al. The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex. Sci- ence. 2016;354(6318):1431–1433. The Journal of Clinical Investigation Similar to keratinocytes, T cell hyperactivation was accompanied by increased phosphorylation of S6K and 4E-BP1, consistent with an mTORC1- dependent, cell-intrinsic role for Skiv2l in T cell homeostasis. Interestingly, increased epidermal phospho-S6K stain- ing was also observed in lesional skin of Cell growth and proliferation requires increased DNA replication, which depends on a sufficient supply of nucleotides (deoxyribonucleotides), the building blocks of DNA synthesis (14). An increased demand for nucleotides is also controlled downstream of mTORC1 through stimu- lation of de novo nucleotide biosynthesis (16–19). However, the major pathway for biosynthesis of DNA precursors is medi- ated by ribonucleotide reductase, which generates deoxyribonucleotides from ribonucleotides (20), the end product of the RNA exosome. Thus, while RNA deg- radation is an inherent step in RNA quality control mechanisms, it also contributes to Conclusions and implications The mTORC1 pathway promotes cell growth through activation of anabolic processes, including the biosynthesis of proteins, lipids, and nucleotides, as well as through cell-cycle acceleration (14). Thus, enhanced mTORC1 signaling observed in keratinocytes and T cells of Skiv2l-defi- cient mice provides a mechanistic expla- nation for uncontrolled hyperprolifera- tion, which is initiated cell-autonomously, resulting in the loss of skin barrier integrity and T cell homeostasis (Figure 1). The T cell phenotype described in Skiv2l-deficient mice is intriguing, because some patients 2 J Clin Invest. 2022;132(2):e156119 https://doi.org/10.1172/JCI156119 1. Houseley J, et al. RNA-quality control by the exo- some. Nat Rev Mol Cell Biol. 2006;7(7):529–539. 5. Schlee M, Hartmann G. Discriminating self from non-self in nucleic acid sensing. Nat Rev Immu- nol. 2016;16(9):566–580. The Journal of Clinical Investigation C O M M E N TA R Y 6. Rehwinkel J, Gack MU. RIG-I-like receptors: their regulation and roles in RNA sensing. Nat Rev Immunol. 2020;20(9):537–551. Acknowledgments 13. Yang K, et al. Cytoplasmic RNA quality control failure engages mTORC1-mediated autoinflam- matory disease. J Clin Invest. 2022;132(2):e146176. MLK is supported by grants from the Deut- sche Forschungsgemeinschaft (KFO249 160548243 and CRC237 369799452/B21). 14. Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol. 2020;21(4):183–203. Address correspondence to: Min Ae Lee-Kirsch, Department of Pediatrics, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Fet- scherstr. 74, 01307 Dresden, Germany. Phone: 49.351.4586887; Email: minae. lee-kirsch@uniklinikum-dresden.de. 15. Hiejima E, et al. Tricho-hepato-enteric syn- drome with novel SKIV2L gene mutations: a case report. Medicine (Baltimore). 2017;96(46):e8601. 16. Robitaille AM, et al. Quantitative phosphoproteomics reveal mTORC1 acti- vates de novo pyrimidine synthesis. Science. 2013;339(6125):1320–1323. 17. Ben-Sahra I, et al. Stimulation of de novo pyrimidine synthesis by growth signal- ing through mTOR and S6K1. Science. 2013;339(6125):1323–1328. 1. Houseley J, et al. RNA-quality control by the exo- some. Nat Rev Mol Cell Biol. 2006;7(7):529–539. 1. Houseley J, et al. RNA-quality control by the exo- some. Nat Rev Mol Cell Biol. 2006;7(7):529–539. 18. Ben-Sahra I, et al. mTORC1 induces purine synthesis through control of the mito- chondrial tetrahydrofolate cycle. Science. 2016;351(6274):728–733. 2. Halbach F, et al. The yeast ski complex: crystal structure and RNA channeling to the exosome complex. Cell. 2013;154(4):814–826. 3. Schmidt C, et al. The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex. Sci- ence. 2016;354(6318):1431–1433. 19. Düvel K, et al. Activation of a metabolic gene regulatory network downstream of mTOR com- plex 1. Mol Cell. 2010;39(2):171–183. 20. Reichard P. Ribonucleotide reductase and deoxyribonucleotide pools. Basic Life Sci. 1985;31:33–45. 21. Hartley JL, et al. Mutations in TTC37 cause trichohepatoenteric syndrome (phenotyp- ic diarrhea of infancy). Gastroenterology. 2010;138(7):2388–2398. 22. Sharabi A, Tsokos GC. T cell metabolism: new insights in systemic lupus erythematosus pathogenesis and therapy. Nat Rev Rheumatol. 2020;16(2):100–112. 7. Hornung V, et al. 5’-Triphosphate RNA is the ligand for RIG-I. Science. 2006;314(5801):994–997. 8 Goubau D et al Antiviral immunity via RIG-I- 7. Hornung V, et al. 5’-Triphosphate RNA is the ligand for RIG-I. Science. 2006;314(5801):994–997. 8. Goubau D, et al. Antiviral immunity via RIG-I- J Clin Invest. 2022;132(2):e156119 https://doi.org/10.1172/JCI156119 3
https://openalex.org/W2327496382
https://hal-pasteur.archives-ouvertes.fr/pasteur-03516029/document
English
null
Characterization of a Genogroup I Sapovirus Isolated from Chimpanzees in the Republic of Congo
Genome announcements
2,014
cc-by
1,578
To cite this version: Illich M. Mombo, Nicolas Berthet, Christiane Bouchier, Joseph N. Fair, Bradley S. Schneider, et al.. Characterization of a Genogroup I Sapovirus Isolated from Chimpanzees in the Republic of Congo. Genome Announcements, 2014, 2 (4), pp.e00680-14. ￿10.1128/genomeA.00680-14￿. ￿pasteur-03516029￿ Characterization of a Genogroup I Sapovirus Isolated from Chimpanzees in the Republic of Congo Illich M. Mombo, Nicolas Berthet, Christiane Bouchier, Joseph N. Fair, Bradley S. Schneider, François Renaud, Eric M. Leroy, Virginie Rougeron Characterization of a Genogroup I Sapovirus Isolated from Chimpanzees in the Republic of Congo Illich M. Mombo, Nicolas Berthet, Christiane Bouchier, Joseph N. Fair, Bradley S. Schneider, François Renaud, Eric M. Leroy, Virginie Rougeron Distributed under a Creative Commons Attribution 4.0 International License Characterization of a Genogroup I Sapovirus Isolated from Chimpanzees in the Republic of Congo Illich M. Mombo,a,b Nicolas Berthet,a,b Christiane Bouchier,c Joseph N. Fair,d Bradley S. Schneider,d François Renaud,b,e Eric M Leroy a b Virginie Rougerona b Illich M. Mombo,a,b Nicolas Berthet,a,b Christiane Bouchier,c Joseph N. Fair,d Bradley S. Schneider,d François Renaud,b,e Eric M. Leroy,a,b Virginie Rougerona,b International Center for Medical Research of Franceville, Franceville, Gabona; Laboratoire MIVEGEC UMR 224–5290 CNRS-IRD-UM1-UM2, IRD Montpellier, Montpellier, Franceb; Centre National de la Recherche Scientifique, UMR3569, Paris, Francec; Metabiota, San Francisco, California, USAd; CHRU de Montpellier, Montpellier, Francee E.M.L. and V.R. contributed equally to this work. E.M.L. and V.R. contributed equally to this work. Sapoviruses, which are members of the Caliciviridae family, are small nonenveloped viruses known to infect a large spectrum of mammalian hosts. We report here the first complete genome sequences of two genogroup I sapoviruses isolated from fecal sam- ples from chimpanzees living in the Tchimpounga sanctuary, Republic of Congo. Sapoviruses, which are members of the Caliciviridae family, are small nonenveloped viruses known to infect a large spectrum of mammalian hosts. We report here the first complete genome sequences of two genogroup I sapoviruses isolated from fecal sam- ples from chimpanzees living in the Tchimpounga sanctuary, Republic of Congo. Received 13 June 2014 Accepted 26 June 2014 Published 17 July 2014 Received 13 June 2014 Accepted 26 June 2014 Published 17 July 2014 Citation Mombo IM, Berthet N, Bouchier C, Fair JN, Schneider BS, Renaud F, Leroy EM, Rougeron V. 2014. Characterization of a genogroup I sapovirus isolated from chimpanzees in the Republic of Congo. Genome Announc. 2(4):e00680-14. doi:10.1128/genomeA.00680-14. Received 13 June 2014 Accepted 26 June 2014 Published 17 July 2014 Copyright © 2014 Mombo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Illich M. Mombo, mombo.illich@gmail.com. and a VP1 at residue 1740 (PPG). Based on the deduced amino acid sequences of the capsid gene (4,530 to 6,843 nt), Cpz-IJC04 and Cpz-IJC09 are 100% identical, cluster within the human GI clade, and are closest (99.7% identity) to the GI human enteric calicivirus Plymouth isolate (GenBank accession no. X86559) (7). S apoviruses (SaVs), which are members of the Caliciviridae family, are small nonenveloped viruses known to infect a large spectrum of mammalian hosts, such as humans, minks, sea lions, swine, dogs, and bats. SaVs are responsible of gastroenteritis in humans (1). Downloaded from https://journals.asm.org/journal/genomea on 14 January 2022 by 157.99.174.131. Downloaded from https://journals.asm.org/journal/genomea on 14 January 2022 by 157. Since this is the first detection of sapoviruses in nonhuman primates living in close contact with humans, we investigated their full genomes. Unbiased deep sequencing employing an Illumina HiSeq platform was conducted as follows: extracted RNA was treated with Turbo DNase (Life Technologies), reverse tran- scribed (RT) using random hexamers (Life Technologies), and amplified using Phi29 enzyme (4). Contigs were assembled with ABySS software (5) and the CAP3 program (6) to construct the viral genomes. The ORFs were identified using the ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/gorf.html). Nucleotide sequence accession numbers. The sequenced ge- nomes of Cpz-IJC04 and Cpz-IJC09 have been submitted to Gen- Bank under accession no. KJ858686 and KJ858687, respectively. Downloaded from https://journals.asm.org/journal/genomea on 14 ACKNOWLEDGMENTS We thank the Gabonese Government and Total Gabon for their financial support. This work benefitted from financial contributions from the PRE- DICT project of the United States Agency for International Development (USAID) Emerging Pandemic Threats Program. This work was supported by the fellowship BSTD of IRD France. Despite repeated analysis, the 5= untranslated region (UTR) (10 nucleotides [nt]) and 3= UTR [28 nt] were not obtained. The assembled genomes consist of 7,320 nt and contain three pre- sumptive ORFs: nucleotide positions 1 to 6837 (ORF1), 6840 to 7319 (partial ORF2), and 5168 to 5653 (ORF3). An examination of the 2,280 amino acids of ORF1 revealed that these genomes contain motifs that are characteristic of caliciviruses: a 2C-like NTPase at residue 480 (GAPGIGKT), VPg at residues 951 (KG- KTK) and 962 (DEYDE), a protease at residue 1167 (GDCG), RNA polymerase at residues 1503 (GLPSG) and 1551 (YGDD), Downloaded from https://journals.asm.or We thank the personnel of the Tchimpounga Sanctuary in the Repub- lic of Congo who collected the fecal samples. We also thank all people of the research unit MIVEGEC (IRD, France) and of the Institut Pasteur (France) for discussions. July/August 2014 Volume 2 Issue 4 e00680-14 HAL Id: pasteur-03516029 https://pasteur.hal.science/pasteur-03516029v1 Submitted on 7 Jan 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License genomea.asm.org 1 1. Hansman GS, Saito H, Shibata C, Ishizuka S, Oseto M, Oka T, Takeda N. 2007. Outbreak of gastroenteritis due to sapovirus. J. Clin. Microbiol. 45:1347–1349. http://dx.doi.org/10.1128/JCM.01854-06. Characterization of a Genogroup I Sapovirus Isolated from Chimpanzees in the Republic of Congo Their genome, a nonsegmented positive-sense single-strand RNA, is 7.5 to 8.5 kb in length. It contains two or three open reading frames (ORFs) (2). Based on the complete capsid gene, SaVs are classified into 14 genogroups (GI to GXIV) (3). Currently, full-length genomes are available for GI to GVII and GXIV SaVs, all obtained from human fecal samples isolated from several parts of the world, except Africa. Here, we report the complete genome of SaVs (Cpz-IJC04 and Cpz-IJC09) identified in two chimpanzee (Pan troglodytes troglodytes) fecal samples from primates living in the Tchimpounga sanctuary (Republic of Congo) that displayed no gastrointestinal symptoms. The clustering of these two genomes within the human GI clade and the knowledge that these chimpanzees were living in close contact to humans (in the sanctuary) suggest recent cross- species transmission of these viruses from humans to chimpan- zees. However, further analysis is required to confirm this hypoth- esis. It is important to investigate the prevalence of these viruses in wild chimpanzee populations in order to obtain a better under- standing of their evolution and adaptation in these animals. The acquisition of these new SaV genomes will facilitate the design of new specific primer panels for reverse transcription-PCR (RT- PCR) assays, allowing a better understanding of the epidemiology and potential pathogenicity of these SaVs. p g 4. Berthet N, Reinhardt AK, Leclercq I, van Ooyen S, Batéjat C, Dickinson P, Stamboliyska R, Old IG, Kong KA, Dacheux L, Bourhy H, Kennedy g 3. Scheuer KA, Oka T, Hoet AE, Gebreyes WA, Molla BZ, Saif LJ, Wang Q. 2013. Prevalence of porcine noroviruses, molecular characterization of emerging porcine sapoviruses from finisher swine in the United States, and unified classification scheme for sapoviruses. J. Clin. Microbiol. 51: 2344–2353. http://dx.doi.org/10.1128/JCM.00865-13. 2 genomea.asm.org 2. Clark I, Estes M, Green K, Hansman G, Knowles N, Koopmans M, Matson D, Meyers G, Neill J, Radford A. 2012. Family Caliciviridae, p 977–986. In King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, Virus taxonomy: Ninth Report of International Committee on Taxonomy of Vi- ruses. Elsevier Academic, London, United Kingdom. July/August 2014 Volume 2 Issue 4 e00680-14 REFERENCES 1. Hansman GS, Saito H, Shibata C, Ishizuka S, Oseto M, Oka T, Takeda N. 2007. Outbreak of gastroenteritis due to sapovirus. J. Clin. Microbiol. 45:1347–1349. http://dx.doi.org/10.1128/JCM.01854-06. Genome Announcements genomea.asm.org Mombo et al. GC, Korfhage C, Cole ST, Manuguerra JC. 2008. Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR. BMC Mol. Biol. 9:77. http://dx.doi.org/10.1186/1471-2199-9-77. 2. Clark I, Estes M, Green K, Hansman G, Knowles N, Koopmans M, Matson D, Meyers G, Neill J, Radford A. 2012. Family Caliciviridae, p 977–986. In King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, Virus taxonomy: Ninth Report of International Committee on Taxonomy of Vi- ruses. Elsevier Academic, London, United Kingdom. 5. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I. 2009. ABySS: a parallel assembler for short read sequence data. Genome Res. 19:1117–1123. http://dx.doi.org/10.1101/gr.089532.108. y p q 19:1117–1123. http://dx.doi.org/10.1101/gr.089532.108. g g 6. Huang X, Madan A. 1999. CAP3: a DNA sequence assembly program. Genome Res. 9:868–877. http://dx.doi.org/10.1101/gr.9.9.868. 7. Liu BL, Clarke IN, Caul EO, Lambden PR. 1995. Human enteric calici- viruses have a unique genome structure and are distinct from the Norwalk- like viruses. Arch. Virol. 140:1345–1356. http://dx.doi.org/10.1007/ BF01322662. p g 4. Berthet N, Reinhardt AK, Leclercq I, van Ooyen S, Batéjat C, Dickinson P, Stamboliyska R, Old IG, Kong KA, Dacheux L, Bourhy H, Kennedy ded from https://journals.asm.org/journal/genomea on 14 January 2022 by 157.99.174.131. Genome Announcements Genome Announcements 2 genomea.asm.org
https://openalex.org/W3198189088
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0256934&type=printable
English
null
Prevalence of unrecognized or “silent” myocardial ischemia in chronic kidney disease patients: Protocol for a systematic review and meta-analysis
PloS one
2,021
cc-by
5,235
PLOS ONE PLOS ONE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 This is a Registered Report and may have an associated publication; please check the article page on the journal site for any related articles. REGISTERED REPORT PROTOCOL Prevalence of unrecognized or “silent” myocardial ischemia in chronic kidney disease patients: Protocol for a systematic review and meta-analysis Christophe Dongmo Fokoua-MaximeID1,2☯*, Armel Jackson SeukepID3☯, Yahia Bellouche4☯, Takeude Erwan Cheuffa-KarelID5☯, Dickson Shey Nsagha3☯, Franc¸ois Folefack Kaze5,6☯¤* 1 University of New York State—University at Albany School of Public Health, Albany, NY, United States of America, 2 New York State Department of Health, Albany, NY, United States of America, 3 Faculty of Health Sciences, University of Buea, Buea, Cameroon, 4 Brest University Hospital, Brest, France, 5 Faculty of Medicine and Biomedical Sciences, University of Yaounde´ I, Yaounde´, Cameroon, 6 Yaounde´ University Teaching Hospital, Yaounde´, Cameroon ☯These authors contributed equally to this work. ¤ Current address: Yaounde´ University Teaching Hospital, Yaounde´, Cameroon * f_kaze@yahoo.fr (FFK); fokouamaxime@yahoo.fr (CDFM) ☯These authors contributed equally to this work. ¤ Current address: Yaounde´ University Teaching Hospital, Yaounde´, Cameroon * f_kaze@yahoo.fr (FFK); fokouamaxime@yahoo.fr (CDFM) This is a Registered Report and may have an associated publication; please check the article page on the journal site for any related articles. This is a Registered Report and may have an associated publication; please check the article page on the journal site for any related articles. Introduction Citation: Fokoua-Maxime CD, Seukep AJ, Bellouche Y, Cheuffa-Karel TE, Nsagha DS, Kaze FF (2021) Prevalence of unrecognized or “silent” myocardial ischemia in chronic kidney disease patients: Protocol for a systematic review and meta-analysis. PLoS ONE 16(9): e0256934. https:// doi.org/10.1371/journal.pone.0256934 Chronic kidney disease (CKD) patients are at an extremely high risk of silent myocardial ischemia (SMI). However, there is a dearth of evidence on the worldwide prevalence of this very lethal and yet unrecognizable complication of CKD. The proposed systematic review and meta-analysis aims to estimate the global prevalence of SMI among CKD patients. PROSPERO registration number CRD42020211929 Strengths and limitations of this study The intended systematic review and meta-analysis will fill the knowledge gap on the global prevalence of silent myocardial ischemia (SMI) in CKD patients. The eligible studies will be identified through a methodic literature search followed by a rigorous screening process; we will then use robust meta-analysis tools to pool the data and provide reliable estimates of the global prevalence of SMI in CKD patients. Two major limitations could be: the predomi- nance of clinical trials that might limit the generalizability of the findings, given that some informative patients might have been sidelined by the strict inclusion criteria of these stud- ies; the high probability of type 1 error originating from the important number of subgroup and sensitivity analyses. PLOS ONE PLOS ONE Silent myocardial ischemia and chronic kidney disease will further investigate the potential sources of heterogeneity. Finally, sensitivity analyses will be performed to measure the impact of low-quality studies on the results of the meta- analysis, and power calculations will determine the probability that we will detect a true effect if it does exist. Funding: The author(s) received no specific funding for this work. Funding: The author(s) received no specific funding for this work. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Dongmo Christophe Fokoua Maxime (the first author) is currently working on a research project with Simeon Pierre Choukem who is an Academic Editor for PLOS ONE journal. This does not alter our adherence to PLOS ONE policies on sharing data and materials. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 Methods and analyses Editor: Carmine Pizzi, University of Bologna, ITALY Received: April 19, 2021 Accepted: August 18, 2021 Published: September 2, 2021 This protocol was conceived according to the preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) statement. The systematic review will involve all observational studies and clinical trials published until April 30, 2021, and reporting on the prevalence of SMI in CKD patients. Electronic sources including MEDLINE, Embase, Web of Science, and Cochrane database of systematic reviews will be perused for potentially eli- gible studies, restricted to only studies published in English or French. Two investigators will independently select studies and use a pre-pilot tested form to extract data. Further, they will independently perform a qualitative assessment of the risk of bias and overall quality of the selected studies, followed by a quantitative assessment using funnel plots and Egger’s tests. The heterogeneity between studies will be assessed with the Cochrane’s Q statistic, and the I2 statistic will measure the percentage of variation across studies that is due to their heterogeneity rather than chance; the I2 will decide if a meta-analysis can be conducted. In case it cannot be conducted, a descriptive analysis will be performed. Otherwise, study-spe- cific estimates will be pooled using either a fixed-effects or a random-effects model, depend- ing on the value of the I2 statistic. Subgroup and random effects meta-regression analyses Copyright: © 2021 Fokoua-Maxime et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The search strategy for MEDLINE (via PUBMED) is available in the supplementary materials (S1 Appendix). Further, the data that will support the findings of this study will also be available in the supplementary materials of the final report of the systematic review and meta-analysis. 1 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 4-Methods The present protocol has been drafted in line with the preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) statement [16]. This protocol will be regis- tered in the international prospective register of systematic reviews (PROSPERO) network. The anticipated systematic review and meta-analysis will agree with this protocol, and will be reported based on the meta-analyses of observational studies (MOOSE) guidelines [17] and the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) guidelines [18]. 2-Objective To determine the global prevalence of silent or unrecognized myocardial ischemia (SMI) among patients with CKD. 4.1-Search methods for the identification of eligible studies A trained and experienced medical librarian will perform a systematic and comprehensive search in the following databases from inception until July 31, 2021: MEDLINE/ PubMed (1947 toJuly 31, 2021), Embase (1973 to July 31, 2021), Web of Science (1985 to July 31, 2021) and Cochrane Central Register of Controlled Trials (1991 to July 31, 2021). Key terms that will be included in the search are: “silent myocardial ischemia”, “silent ischemic heart disease”, “silent coronary artery disease”, “silent coronary heart disease”, “chronic kidney disease”, “chronic renal disease”, “chronic kidney failure”, “chronic renal failure”, “chronic kidney dis- order”, “chronic renal disorder”. The search strategy for MEDLINE (via PubMed) is presented in the S1 Appendix. Following the search in the databases, we will peruse the reference lists of all selected articles to identify potential supplementary data sources. The search will be repeated prior to the publication of the systematic review in order to include any potential eligible study that could have been published since the end of the initial electronic search. 3-Review question What is the prevalence of silent or unrecognized myocardial ischemia among patients with CKD? 1-Introduction Chronic kidney disease (CKD) is a global public health concern [1]. In 2017, the world counted 697.5 million CKD cases and registered 1.2 million deaths attributable to CKD [2]. CKD is an independent risk factor of cardiovascular disease (CVD) [1]. In turn, CVD is the leading cause of morbidity and mortality among patients with CKD [3]. Coronary artery dis- ease (CAD) is responsible for around 50 percent of the deaths of patients with CKD [4–6]. Research has highlighted the uniqueness of CAD in patients with CKD—as compared with the general population—because of an early onset, a more rapid progression and atypical symp- toms [7]. CKD patients are at a high risk of silent myocardial ischemia (SMI) because like dia- betes, CKD is associated with neuronal disturbances [8] that can alter the patients’ perception of pain. Another hypothesis proposes that SMI among CKD patients is due to the CKD- induced microinflammatory state [9] which disrupts the balance between proinflammatory and anti-inflammatory cytokines that is needed for the perception of chest pain [10]. Finally, SMI is well-documented in dialysis [11–14]; indeed, the hemodialysis procedure itself pro- vokes regional wall motion abnormalities and disruptions in the myocardial perfusion [15]. Thus, CKD is a major risk factor for SMI. In 2017, the number of deaths due to CKD plus the number of deaths of CVD patients imputable to impaired kidney function represented 46% of the total death toll in the world [2]. Many of these deaths were probably induced by silent myocardial ischemia that went unnoticed and so were not diagnosed, consequently, they did not receive proper care and ulti- mately resulted in earlier deaths. Therefore, it is urgent to take appropriate preventive mea- sures to quell the deleterious effects of this highly lethal and imperceptible complication of CKD. A key pre-requisite for this task is to know the proportion of CKD patients in the world who suffer from SMI, as well as the potential factors which contribute to the late or non-diag- nosis of SMI in CKD patients. To date, no study has attempted to provide such information. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 2 / 10 PLOS ONE Silent myocardial ischemia and chronic kidney disease The proposed systematic review and meta-analysis aims to fill this gap in the literature by pro- viding a reliable estimate of the global prevalence of SMI among patients with CKD. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 4.2-Eligibility criteria for study selection 4.2.1-Study design. The review will include all cross-sectional, cohort, case-control stud- ies and clinical trials published until April 30, 2021. The search will be supplemented by hand searches in the reference lists of included studies and systematic searches in the gray literature for additional relevant articles. 4.2.2-Participants. Study subjects in the eligible studies must be individuals diagnosed with any stage of CKD. There will be no restrictions based on sex, age, race/ethnicity, socioeco- nomic status, history of previous myocardial infarction, or geographic region. 4.2.3-Clinical outcomes. CKD described in the eligible studies must have been identified by a physician and the diagnosis must have been made based on the criteria from the Improv- ing Global Outcomes (KDIGO) Consensus Conference which defines CKD as kidney damage (albumin-to-creatinine ratio > 30 mg/g in two of three spot urine specimens) or glomerular 3 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 PLOS ONE Silent myocardial ischemia and chronic kidney disease filtration rate (GFR) < 60 mL/min/1.73m2 for 3 months or more, irrespective of the cause [19–21]. Silent myocardial ischemia (SMI) must have been diagnosed by a physician and diag- nosed in patients who display no symptoms during an exercise or pharmaceutical stress test but who exhibit transient ST-segment changes, perfusion defects, or reversible regional wall motion abnormalities [22], or patients who display no symptoms and a myocardial ischemia is later diagnosed based on myocardial imaging evidence or pathological findings on autopsy [23, 24]. 4.2.4-Outcome measure. The outcome of focus of this review will be the prevalence of SMI in CKD patients. 4.2.4-Outcome measure. The outcome of focus of this review will be the prevalence of SMI in CKD patients. 4.2.5-Language. The search will be restricted to only studies published in English or French. 4.2.5-Language. The search will be restricted to only studies published in English or French. 4.2.6-Exclusion criteria. We will exclude book chapters, (systematic) reviews and meta- analyses, case-series, fact sheets, white papers, conference proceedings, letters to the editor, commentaries, editorials, and studies without primary data and/or with unachieved methods description. For search leading to similar publications (duplicates), only the most comprehen- sive report including the largest sample size, a complete methods section, and an entire results report will be included. 4.2-Eligibility criteria for study selection Also, Kin relationships, defined as multiple publications describing the same or overlapping series of patients, will be identified; in this instance, only the study with the largest sample size, a comprehensive methods description, and a complete results section will be selected. The complete bibliography of the validated and included as well as the rejected studies will be available by request to the corresponding author. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 4.3-Data collection and analysis 4.3.1-Data compilation. Search results will be imported into EndNote X9.3.3 (Clarivate Analytics, Philadelphia, USA). Duplicate articles will be removed, and the remaining refer- ences will be alphabetically ordered according to the first authors’ names. 4.3.2-Selection of studies. Two authors (DCFM, AJS) will independently screen titles and abstract records that were imported after the literature search. Consecutive to the initial screening, the full texts of records considered eligible will be retrieved and further evaluated for inclusion by the same researchers. Discrepancies in the list of selected articles will be resolved by consensus or by a third reviewer (YB) if necessary. In line with the PRISMA guide- lines [16], a flow diagram will summarize the entire study selection process (Fig 1). The antici- pated start date for the selection of articles is August 1, 2021 and the expected completion date is August 31, 2021. 4.3.3- Data extraction and management. Two authors (DCFM, AJS) will use a pre-pilot tested and systematized data extraction form to collect data on: • Study identification: first author’s name, year of publication, country and/or region • Study identification: first author’s name, year of publication, country and/or region • Study characteristics: study design (cross-sectional, cohort, case-control study, or random- ized control trial), setting (hospital- or community-based), duration of follow-up for cohort studies and clinical trials, number of controls per cases, and type of matching (if present) for case-control studies. • Study population: sample size, mean or median age, age range, sex ratio, race/ethnicity distribution. • Primary exposure: stage of CKD • Primary outcome: SMI • Primary outcome: SMI PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 4 / 10 PLOS ONE Silent myocardial ischemia and chronic kidney disease Fig 1. PRISMA flow diagram of the selection of studies to include in a systematic review. Fig 1. PRISMA flow diagram of the selection of studies to include in a systematic review. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 • Epidemiological measure: prevalence. We will contact the corresponding author(s) of articles from which we could not extract important information during the retrieval process. Finally, two other authors (YB, TECK) will randomly select 5 studies each and will extract the data one more time to validate that the data extraction process was performed accurately. 4.3.4-Quality assessment and risk of bias of selected studies. Two authors (DCFM, ELY) will separately appraise the methodological quality of each included study, using the tool conceived by Hoy et al for prevalence studies [25]. Each item will be assigned a score of 1 (yes) or 0 (no), and scores will be summed across items to generate an overall quality score that will range from 0 to 10. According to the overall scores, each of the two authors (DCFM, AJS) will classify studies as having a low (>8), moderate (6–8), or high (5) risk of bias. Further, the non-weighted Cohen’s kappa statistic will be used to assess the level of agreement between the reviewers. In case of substantial disagreement between the two reviewers (DCFM, AJS), a third reviewer (YB) will be solicited for arbitration. Following this evaluation, authors of publica- tions containing confusing results and/or results prone to multiple interpretations will be reached out by email to request some clarification or supplemental information. In case a study is excluded, the reasons will be explicitly presented. If more than 10 eligible studies are found, then publication bias will be evaluated by the symmetry of funnel plots, supplemented by a quantitative analysis through an Egger’s test. 4.3.5-Data synthesis. The data of the included studies will be summarized in ad hoc tables. The heterogeneity between studies will be assessed with the Cochrane’s Q statistic. Further, the I2 statistic will be used to measure the percentage of variation across studies that is due to their heterogeneity rather than chance [19, 20]. The value of the I2 statistic will be classified as small if 0I225%, medium if 25%<I250%, and large if I2>50% [26, 27]. The category of the I2 statistic will determine whether a meta-analysis is possible. If the I2 statistic is large, then a meta-analysis will be considered not possible, and a descriptive analysis will be performed. Oth- erwise, a meta-analysis will be deemed feasible, and the category of the I2 statistic will further decide the type of statistical model to be used to pool the study-specific estimates. 4.3-Data collection and analysis • Covariates: glomerular filtration rate, creatinine, albuminuria, urine albumin-to-creatinine ratio, type of treatment received (medicine, dialysis, or both), method used to detect the SMI (ECG, medical imaging, or autopsy), duration since the diagnosis of CKD, mean or median age at diagnosis of CKD, proportion of patients with a history of 1 or more acute complica- tions of CKD (edema, gout, metabolic acidosis, hyperkalemia), proportion of patients with 1 or more chronic complications of CKD (anemia, renal osteodystrophy, left ventricular hyper- trophy, dyslipidemia, malnutrition), body mass index (BMI), proportion of patients smoking, proportion of patients with diabetes, proportion of patients with hypertension, proportion of patients with other cardiovascular diseases (heart failure, arrhythmias, heart valve disease, car- diomyopathies, pericarditis, aorta diseases, stroke), proportion of patients with 1 or more other major comorbidities (HIV/AIDS, cancer, chronic obstructive pulmonary disease). 5 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 PLOS ONE Silent myocardial ischemia and chronic kidney disease • Epidemiological measure: prevalence. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 • Epidemiological measure: prevalence. If the I2 statis- tic is small then a fixed-effects model analysis will be conducted, otherwise, a random-effect model analysis [28] will be performed, after stabilizing the variance of individual studies with the Freeman-Tukey double arc-sine transformation [29]. R software version 3.6.1 (R Core Team, Vienna, Austria) will be used to combine data, along with 95% confidence intervals. 4.3.6. Sources of heterogeneity. The potential sources of heterogeneity will be investi- gated by subgroup and meta-regression analyses [26]. Subgroup analyses will be performed by stage of CKD, dialysis status, sex, menopause status, race, obesity status, diabetes status, and hypertension status. If more than 10 studies are included in the quantitative synthesis, then subgroup analyses will be supplemented by random effect meta-regression analyses which will allow the effects of multiple factors (called effect modifiers) to be simultaneously investigated [30]. The potential effect modifiers considered will be the following: sex, race, obesity status, diabetes status, hypertension status, and type of study (observational vs experimental). We will use the model F value and its statistical significance to assess whether there is evidence for an association between any of the covariates and the outcome; all the covariates with p-value <0.2 in bivariate models will be added to the multivariable model, in which a p-value <0.05 will be considered statistically significant. The model fit will be assessed using the proportion of the between-study variance explained by the covariates (adjusted R2) [31]. To control for the risk of type I error when performing meta-regression with multiple covariates, we will perform Monte Carlo permutation tests to calculate P values adjusted for type 1 error and we will check if there is a change in statistical significance [31, 32]. 6 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 PLOS ONE Silent myocardial ischemia and chronic kidney disease 4.3.7. Robustness of the results. The robustness of the results will be assessed by perform- ing sensitivity analyses to measure the impact of low-quality studies (identified through their risk of bias). Low-quality studies will be removed one at a time and the meta-analysis will be performed again; we will then compare the results of the meta-analysis with and without the study being assessed, while also accounting for the study sample size, strength of evidence, and impact on aggregated effect size. However, if all the selected studies are at a high risk of bias, we will not conduct sensitivity analyses. 4.3.8. • Epidemiological measure: prevalence. Power analyses. Power analyses will measure the probability that we will detect a true effect if it does exist. Assuming a normal distribution of the effect estimates, the power will be: Power ¼ 1β ½33Š; ð1Þ with β ¼ F ðCa λÞ F ðCa λÞ; ð2Þ Power ¼ 1β ½33Š; ð1Þ ð1Þ with β ¼ F ðCa λÞ F ðCa λÞ; ð2Þ ð2Þ where β is the probability of type II error or false negative rate, where β is the probability of type II error or false negative rate, Cα represents the critical value of a Z-distribution, F is the standard normal density function obtained through the formula F ¼ 1ffiffiffiffi 2p p eZ2=2, F is the standard normal density function obtained through the formula F ¼ 1ffiffiffiffi 2p p eZ2=2, λ i th t l d fi d l 1fifififi ith b i th t ff t i d V it i fifififi λ is the true value defined as l ¼ 1ffiffiffiffi Vϙ p , with ϙ being the true effect size and Vϙ its variance. fifififi Under the assumption that the heterogeneity between the selected studies will be moderate, Vϙ will be calculated according to the method of Hedges and Pigott: Vϙ = 1.67 x Vy/k [34], with k being the number of included studies and Vy being the between-study variance. The power calculations will be performed with the power.analysis function contained in the dmetar package of the statistical software R version 4.0.4. Under the aforementioned assump- tions, if the true effect is 0.4 and 10 studies are included in the final analyses, then our study will have an 85% power. 4.3.9. Ethics and dissemination. This systematic review was considered exempt from Institutional Review Board approval since our analyses will only include previously published non-identifiable data. The anticipated systematic review and meta-analysis will be reported fol- lowing the meta-analyses of observational studies (MOOSE) guidelines and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The results of the systematic review and meta-analysis will be made available through conference proceed- ings and peer-reviewed publications. S1 Checklist. PRISMA-P 2015 checklist. (DOCX) S1 Appendix. MEDLINE(PubMed) search strategy. (DOCX) Author Contributions Conceptualization: Christophe Dongmo Fokoua-Maxime, Franc¸ois Folefack Kaze. Methodology: Christophe Dongmo Fokoua-Maxime, Dickson Shey Nsagha, Franc¸ois Folefack Kaze. Methodology: Christophe Dongmo Fokoua-Maxime, Dickson Shey Nsagha, Franc¸ois Folefack Kaze. Supervision: Christophe Dongmo Fokoua-Maxime, Dickson Shey Nsagha. Validation: Christophe Dongmo Fokoua-Maxime, Armel Jackson Seukep, Yahia Bellouche, Takeude Erwan Cheuffa-Karel, Dickson Shey Nsagha, Franc¸ois Folefack Kaze. Writing – original draft: Christophe Dongmo Fokoua-Maxime. Writing – original draft: Christophe Dongmo Fokoua-Maxime. Writing – review & editing: Christophe Dongmo Fokoua-Maxime, Armel Jackson Seukep, Yahia Bellouche, Takeude Erwan Cheuffa-Karel, Dickson Shey Nsagha, Franc¸ois Folefack Kaze. 5- Strengths and limitations of this study The intended systematic review and meta-analysis will fill the knowledge gap on the global prevalence of silent myocardial ischemia (SMI) in CKD patients. The eligible studies will be identified through a methodic literature search followed by a rigorous screening process; we will then use robust meta-analysis tools to pool the data and provide reliable estimates of the global prevalence of SMI in CKD patients. Two major limitations could be: the predominance of clinical trials that might limit the generalizability of the findings, given that some informa- tive patients might have been sidelined by the strict inclusion criteria of these studies; the high probability of type 1 error originating from the important number of subgroup and sensitivity analyses. 7 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 PLOS ONE Silent myocardial ischemia and chronic kidney disease 6-Conclusion CKD is a major contributor to the mortality and morbidity in the world. Coronary artery dis- ease (CAD) is responsible for around half of the deaths of CKD patients, yet many of these cases are silent or unrecognized because of the biochemical and morphological changes that accompany CKD; thus, these cases are not medically attended and subsequently lead to earlier deaths. Therefore, it is urgent to take appropriate preventive measures to quell the deleterious effects of this highly lethal and imperceptible complication of CKD. However, there is a gap in our knowledge about the worldwide prevalence and risk factors of silent myocardial ischemia (SMI) in CKD patients. This systematic review will fill this gap by estimating the pooled global prevalence of SMI in CKD patients. It will provide key evidence that will help the worldwide scientific community in its efforts to quell the deleterious effects of this highly lethal and yet unrecognizable complication of CKD. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 Supporting information S1 Checklist. PRISMA-P 2015 checklist. (DOCX) S1 Checklist. PRISMA-P 2015 checklist. (DOCX) References 1. Sarnak Mark J. et al., “Kidney Disease as a Risk Factor for Development of Cardiovascular Disease,” Circulation, vol. 108, no. 17, pp. 2154–2169, Oct. 2003, https://doi.org/10.1161/01.CIR.0000095676. 90936.80 PMID: 14581387 2. Bikbov B. et al., “Global, regional, and national burden of chronic kidney disease, 1990–2017: a system- atic analysis for the Global Burden of Disease Study 2017,” The Lancet, vol. 395, no. 10225, pp. 709– 733, Feb. 2020, https://doi.org/10.1016/S0140-6736(20)30045-3 PMID: 32061315 3. Sarnak M. J. et al., “Chronic Kidney Disease and Coronary Artery Disease: JACC State-of-the-Art Review,” Journal of the American College of Cardiology, vol. 74, no. 14, pp. 1823–1838, Oct. 2019, https://doi.org/10.1016/j.jacc.2019.08.1017 PMID: 31582143 4. “IDF Atlas 9th edition and other resources.” https://www.diabetesatlas.org/en/resources/?gclid= Cj0KCQjwy 6T1BRDXARIsAIqCTXpGlzUYrak0ExBMo5qjcxj8t1nzxaLeeYNLQMYpcDRxp6S d5Bg9dbIaAhVbEALw_wcB (accessed Apr. 29, 2020). 5. “Excerpts from the United States Renal Data System 2000 Annual Data Report: Atlas of End-Stage Renal Disease in the United States—American Journal of Kidney Diseases.” https://www.ajkd.org/ article/S0272-6386(14)70070-8/fulltext (accessed Jun. 20, 2020). 8 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 PLOS ONE Silent myocardial ischemia and chronic kidney disease 6. Tyroler H. A., “Nutrition and coronary heart disease epidemiology,” Adv. Exp. Med. Biol., vol. 369, pp. 7–19, 1995, https://doi.org/10.1007/978-1-4615-1957-7_2 PMID: 7598018 7. “Clinically silent myocardial infarctions in the CKD community | Nephrology Dialysis Transplantation | Oxford Academic.” https://academic.oup.com/ndt/article/27/9/3387/1860696 (accessed Jun. 20, 2020). 8. National Kidney Foundation, “K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification,” Am. J. Kidney Dis., vol. 39, no. 2 Suppl 1, pp. S1–266, Feb. 2002. 9. Carrero J. J. and Stenvinkel P., “Persistent inflammation as a catalyst for other risk factors in chronic kidney disease: a hypothesis proposal,” Clin J Am Soc Nephrol, vol. 4 Suppl 1, pp. S49–55, Dec. 2009, https://doi.org/10.2215/CJN.02720409 PMID: 19996005 10. “Silent Myocardial Ischemia May Be Related to Inflammatory Response—PubMed.” https://pubmed. ncbi.nlm.nih.gov/14962636/ (accessed Jun. 20, 2020). 11. Mohi-ud-din K., Bali H. K., Banerjee S., Sakhuja V., and Jha V., “Silent myocardial ischemia and high- grade ventricular arrhythmias in patients on maintenance hemodialysis,” Ren Fail, vol. 27, no. 2, pp. 171–175, 2005. PMID: 15807181 12. Narula A. S., Jha V., Bali H. K., Sakhuja V., and Sapru R. P., “Cardiac arrhythmias and silent myocardial ischemia during hemodialysis,” Ren Fail, vol. 22, no. 3, pp. 355–368, May 2000, https://doi.org/10. 1081/jdi-100100879 PMID: 10843246 13. McIntyre C. W. References et al., “Hemodialysis-induced cardiac dysfunction is associated with an acute reduction in global and segmental myocardial blood flow,” Clin J Am Soc Nephrol, vol. 3, no. 1, pp. 19–26, Jan. 2008, https://doi.org/10.2215/CJN.03170707 PMID: 18003765 14. Dasselaar J. J. et al., “Haemodialysis is associated with a pronounced fall in myocardial perfusion,” Nephrol. Dial. Transplant., vol. 24, no. 2, pp. 604–610, Feb. 2009, https://doi.org/10.1093/ndt/gfn501 PMID: 18775808 15. Assa S. et al., “Hemodialysis-induced regional left ventricular systolic dysfunction: prevalence, patient and dialysis treatment-related factors, and prognostic significance,” Clin J Am Soc Nephrol, vol. 7, no. 10, pp. 1615–1623, Oct. 2012, https://doi.org/10.2215/CJN.00850112 PMID: 22822014 16. Shamseer L. et al., “Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation,” BMJ, vol. 349, no. jan02 1, pp. g7647–g7647, Jan. 2015, https://doi.org/10.1136/bmj.g7647 PMID: 25555855 17. Stroup D. F. et al., “Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group,” JAMA, vol. 283, no. 15, pp. 2008–2012, Apr. 2000. https://doi.org/10.1001/jama.283.15.2008 PMID: 10789670 18. Liberati A. et al., “The PRISMA statement for reporting systematic reviews and meta-analyses of stud- ies that evaluate healthcare interventions: explanation and elaboration,” BMJ, vol. 339, Jul. 2009, https://doi.org/10.1136/bmj.b2700 PMID: 19622552 19. Levey A. S. et al., “Nomenclature for Kidney Function and Disease: Executive Summary and Glossary From a Kidney Disease: Improving Global Outcomes (KDIGO) Consensus Conference,” Kidney Int Rep, vol. 5, no. 7, pp. 965–972, Jun. 2020, https://doi.org/10.1016/j.ekir.2020.03.027 PMID: 32647753 20. Levey A. S. et al., “Nomenclature for kidney function and disease: report of a Kidney Disease: Improving Global Outcomes (KDIGO) Consensus Conference,” Kidney Int, vol. 97, no. 6, pp. 1117–1129, Jun. 2020, https://doi.org/10.1016/j.kint.2020.02.010 PMID: 32409237 21. Levin A. et al., “International consensus definitions of clinical trial outcomes for kidney failure: 2020,” Kidney Int, vol. 98, no. 4, pp. 849–859, Oct. 2020, https://doi.org/10.1016/j.kint.2020.07.013 PMID: 32998816 22. Conti C. R., Bavry A. A., and Petersen J. W., “Silent Ischemia: Clinical Relevance,” Journal of the Amer- ican College of Cardiology, vol. 59, no. 5, pp. 435–441, Jan. 2012, https://doi.org/10.1016/j.jacc.2011. 07.050 PMID: 22281245 23. Turkbey E. B. et al., “Prevalence and Correlates of Myocardial Scar in a US Cohort,” JAMA, vol. 314, no. 18, pp. 1945–1954, Nov. 2015, https://doi.org/10.1001/jama.2015.14849 PMID: 26547466 24. de Torbal A. et al., “Incidence of recognized and unrecognized myocardial infarction in men and women aged 55 and older: the Rotterdam Study,” Eur. Heart J., vol. 27, no. 6, pp. PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 References 729–736, Mar. 2006, https:// doi.org/10.1093/eurheartj/ehi707 PMID: 16478749 25. Hoy D. et al., “Assessing risk of bias in prevalence studies: modification of an existing tool and evidence of interrater agreement,” J Clin Epidemiol, vol. 65, no. 9, Art. no. 9, Sep. 2012, https://doi.org/10.1016/j. jclinepi.2011.11.014 PMID: 22742910 26. Higgins J. P. T. and Thompson S. G., “Quantifying heterogeneity in a meta-analysis,” Stat Med, vol. 21, no. 11, pp. 1539–1558, Jun. 2002, https://doi.org/10.1002/sim.1186 PMID: 12111919 27. Higgins J. P. T., Thompson S. G., Deeks J. J., and Altman D. G., “Measuring inconsistency in meta- analyses,” BMJ, vol. 327, no. 7414, pp. 557–560, Sep. 2003. https://doi.org/10.1136/bmj.327.7414.557 PMID: 12958120 PLOS ONE | https://doi.org/10.1371/journal.pone.0256934 September 2, 2021 9 / 10 PLOS ONE Silent myocardial ischemia and chronic kidney disease 28. DerSimonian R. and Laird N., “Meta-analysis in clinical trials,” Control Clin Trials, vol. 7, no. 3, pp. 177– 188, Sep. 1986. https://doi.org/10.1016/0197-2456(86)90046-2 PMID: 3802833 29. Higgins J. P. T. and Thompson S. G., “Quantifying heterogeneity in a meta-analysis,” Stat Med, vol. 21, no. 11, Art. no. 11, Jun. 2002, https://doi.org/10.1002/sim.1186 PMID: 12111919 30. Thompson S. G. and Sharp S. J., “Explaining heterogeneity in meta-analysis: a comparison of meth- ods,” Stat Med, vol. 18, no. 20, pp. 2693–2708, Oct. 1999, https://doi.org/10.1002/(sici)1097-0258 (19991030)18:20<2693::aid-sim235>3.0.co;2-v PMID: 10521860 31. Kelley G. A. and Kelley K. S., “Statistical models for meta-analysis: A brief tutorial,” World J Methodol, vol. 2, no. 4, pp. 27–32, Aug. 2012, https://doi.org/10.5662/wjm.v2.i4.27 PMID: 25237614 32. “Controlling the Risk of Spurious Findings From Meta-Regression—PubMed.” https://pubmed.ncbi.nlm. nih.gov/15160401/ (accessed Jun. 29, 2020). 33. M. H. M.Sc.1, P. D. P. Cuijpers2, P. D. T. A. Furukawa3, and A. P. D. D. D. Ebert2, Doing Meta-Analysis in R. Accessed: Mar. 17, 2021. [Online]. Available: https://bookdown.org/MathiasHarrer/Doing_Meta_ Analysis_in_R/ 34. Hedges L. V. and Pigott T. D., “The power of statistical tests for moderators in meta-analysis,” Psychol Methods, vol. 9, no. 4, pp. 426–445, Dec. 2004, https://doi.org/10.1037/1082-989X.9.4.426 PMID: 15598097 10 / 10
https://openalex.org/W4365804640
https://www.scielo.br/j/cr/a/WFzsnYvpw3WschYhzLFj8Cs/?lang=en&format=pdf
Portuguese
null
Genetic identification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil
Ciência rural
2,023
cc-by
5,430
Identificação genética, aspectos clínicos e epidemiológicos de um foco de anemia infecciosa equina no Estado do Rio Grande do Sul, Brasil RESUMO: O estudo descreve a identificação genética, as características clínicas e epidemiológicas de um foco de Anemia Infecciosa Equina que ocorreu no Estado do Rio Grande do Sul, Brasil. Três equinos criados na região periurbana da cidade de Uruguaiana testaram positivos pela prova sorológica de IDGA. O exame foi realizado como requerimento para trânsito dos animais. Nenhum animal apresentava sinais clínicos da infecção, um cavalo foi necropsiado e os outros dois foram roubados. Na necropsia não obsevou-se nenhuma alteração e microscopicamente foi constatada hemosiderose discreta em fragmento do fígado e baço. A infecção foi confirmada pela amplificação e sequenciamento de um segmento do genoma pró-viral do EIAV de amostras do sangue, baço e linfonodo mesentérico. A análise filogenética do primeiro EIAV sequenciado no Estado do RS indica similaridade com outras amostras que circulam no Brasil. O resultado confirma a presença do vírus no rebanho equino da região e descreve características clínicas e epidemiológicas que contribuem para a manutenção e disseminação do vírus no rebanho. Palavras-chave: equinos, vírus da anemeia infecciosa equina, epidemiologia molecular, análise filogenética, doença transfronteiriça. p g q p ç ç : equinos, vírus da anemeia infecciosa equina, epidemiologia molecular, análise filogenética, doença transfronteiriça. Genetic identification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil José Conrado dos Santos Jardim1 Paula Fonseca Finger2 Grazielle Vieira Cristofari3 Bruno Leite Anjos1,2 Carolina Kist Traesel2 Mário Celso Sperotto Brum1,2* José Conrado dos Santos Jardim1 Paula Fonseca Finger2 Grazielle Vieira Cristofari3 Bruno Leite Anjos1,2 Carolina Kist Traesel2 Mário Celso Sperotto Brum1,2* 1Programa de Pós-graduação em Ciência Animal, Universidade Federal do Pampa (UNIPAMPA), 97508-000, Uruguaiana, RS, Brasil. E-mail: mariobrum@unipampa.edu.br. *Corresponding author. @ p p p g 2Curso de Medicina Veterinária, Universidade Federal do Pampa (UNIPAMPA), Uruguaiana, RS, Brasil. 3Secretaria do Estadual de Agricultura, Pecuária e Desenvolvimento Rural (SEAPDR), Inspetoria de Defesa Agropecuária (IDA), Uruguaiana, RS Brasil 3Secretaria do Estadual de Agricultura, Pecuária e Desenvolvimento Rural (SEAPDR), Inspetoria de Defesa Agropecuária (IDA), Uruguaiana, RS, Brasil. Abstract: The study describes the genetic identification, clinical, and epidemiological characteristics of an outbreak of equine infectious anemia occurring in the state of Rio Grande do Sul, Brazil. Three animals kept in the periurban region of Uruguaiana city tested positive for the AGID test. The serology was performed as a requirement for transit. None of the animals showed clinical signs of infection, one animal was necropsied, and the others were stolen. In the post-mortem examination, no macroscopic changes were observed, and microscopically, discrete hemosiderosis was detected in fragments of the liver and spleen. Amplifying and sequencing a proviral DNA fragment in blood, spleen, and mesenteric lymph node samples confirmed EIAV infection. Phylogenetic analysis of the first sequenced EIAV sample from the Rio Grande do Sul State indicates a high similarity with other Brazilian samples. Results confirmed the viral presence in the state’s herds and described epidemiological and virological characteristics of EIA that contribute to the maintenance and dissemination of the virus in herds. Key words: horses, equine infectious anemia virus, molecular epidemiology, phylogenetic analysis, transboundary disease. Ciê Received 09.12.22 Approved 01.19.23 Returned by the author 03.24.23 CR-2022-0506.R2 Editor: Rudi Weiblen Genetic identification, clinical and epidemiological aspects of an equ Ciência Rural, Santa Maria, v.53:11, e20220506, 2023 Genetic identification, clinical and epidemiological aspects of an equ Ciência Rural, Santa Maria, v.53:11, e20220506, 2023 break in the Rio Grande do Sul State, Brazil.1 ISSNe 1678-4596 http://doi.org/10.1590/0103-8478cr20220506 MICROBIOLOGY Genetic identification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil Introduction Orthoretrovirinae, and genus Lentivirus (COOK et al., 2013, LEROUX et al., 2004). During the process of infection of the host (horses, donkeys, mules, and zebras), the viral RNA genome is reverse transcribed into DNA and inserted into the cell genome, giving rise to the proviral DNA, especially in monocytes and macrophage cells. Thus, the animals develop persistent infection and are considered the primary source of the virus to susceptible animals (COOK et al., 2013; LEROUX et al., 2004). EIAV is transmitted by blood transfer between animals, with the iatrogenic route or mechanical vectors (Tabanus Equine infectious anemia (EIA), also known as swamp fever, is a significant viral disease in horses worldwide (JARA et al., 2020). The first description of the condition occurred in 1843 in France, and in 1904 there was the definition of viral etiology (COOK et al., 2013). In Brazil, with an equine herd of more than 4 million animals, the infection was confirmed in 1968 (MAPA, 1988; IBGE, 2017). Equine infectious anemia virus (EIAV) belongs to the Retroviridae family, subfamily Ciê Received 09.12.22 Approved 01.19.23 Returned by the author 03.24.23 CR-2022-0506.R2 Editor: Rudi Weiblen Jardim et al. 2 spp. and Stomxys spp.) being the most common ways of dissemination (COOK et al., 2013, ISSEL & FOIL, 2015). The clinical presentation associated with infection can be classified as acute, chronic, or not apparent. The clinical signs are anemia, edema of the ventral region, mucosal hemorrhages, and progressive weight loss (COOK et al., 2013). Laboratory diagnosis (gold standard) is made by serological agar gel immunodiffusion test (AGID) performed in Brazil by private laboratories accredited by the Official Veterinary Service (OVS) (BRASIL, 2004; COOK et al., 2013). The owner of a positive horse may request a retest, performed from a new blood collection by the OVS and tested in the reference laboratory of MAPA (Ministério da Agricultura, Pecuária e Abastecimento) (BRASIL, 2004). in identifying clinical cases, lack of preventive examinations, and even illegal cross-border movement of animals (BARZONI et al., 2018; MACHADO et al., 2021). The present study described the clinical, epidemiological, and virological characteristics observed in an Equine Infectious Anemia outbreak. Results assist in understanding the maintenance of EIAV in the western region of RS and can serve as a model to understand other situations. Case description Three equines, two males and one mare, mixed breed, were diagnosed positive for EIA by AGID. The animals were on a property located in the peri-urban perimeter of Uruguaiana; the Rio Grande do Sul, Brazil (Figure 1). The clinical and epidemiological data were obtained from the official forms for disease investigation; at the time of the first serological examination, no animal presented clinical signs of infection. In 1981, the Brazilian authorities (MAPA) instituted the official control of diagnosis and notification of positive cases, which followed the recommendations of the World Organization for Animal Health (WOAH) (BRASIL, 2004). Infection is widespread throughout the territory at varying levels, with the Pantanal (25%) and northern regions (46%) showing the highest prevalence; conversely, southern states show low levels (> 1%) (ALMEIDA et al., 2006; BORGES et al., 2013; CRUZ et al., 2020; TIGRE et al., 2017). In Rio Grande do Sul (RS), located in the extreme south of Brazil, the estimated prevalence of EIAV is close to 0.3% (SEAPA, 2014; BARZONI et al., 2018; REBELATTO et al., 1992). RS has the State Equine Health Program (PESE - SEAPDR), which follows the guidelines of the national program and is suitable for the state’s epidemiological situation (SEAPI, 2018). All properties and animals must be registered in the Agricultural Defense System (SDA-SEAPDR). The movement of equines should only happen upon the presentation of a negative AGID test for EIA and the issuing of an animal transport certificate (GTA) by the OVS. The property that presents at least one positive animal is considered an outbreak; the seropositive horses are sacrificed, following the interdiction and testing of all other susceptible animals on the property (SEAPI, 2018). Hematology and pathology Blood samples were collected for hematological analysis. An autopsy was performed on one horse when fragments of different tissues (kidney, lung, liver, spleen, and lymph nodes) were collected for anatomopathological and viral analysis. According to routine protocols, the tissue fragments were fixed in 10% formalin, processed and stained with Hematoxylin and Eosin, and analyzed by light microscopy. DNA extraction Whole blood (500μL) and tissue (100mg) samples were subjected to digestion with proteinase K, then extracted total DNA by the phenol-chloroform method. Total DNA was precipitated with sodium acetate (3M) and 2.2 volumes of ethanol (100%) (OAKS et al., 1998). After centrifugation, the pellets were washed with 70% alcohol, solubilized in 32 μl of TE buffer, and stored at -80 °C. DNA concentration and purity were determined by spectrophotometry (GE NanoVue Plus). The western border region of Rio Grande do Sul is characterized by having a significant population of horses used in cattle and sheep management, breeding, and sports (rodeo, racing, and horse riding) (COSTA et al., 2013). This region corresponds to the extensive Brazil-Argentina-Uruguay triple border area and concentrates a considerable portion of the EIA outbreaks in the state (BARZONI et al., 2018; Cardenas et al., 2022; MACHADO et al., 2021). These outbreaks are associated with the illegal movement of animals between properties, difficulty DNA Amplification by Nested-PCR Total DNA (250 - 500 ng) from each sample was subjected to amplification of proviral DNA by Nested-PCR reaction within the LTR region, extending to the trans-activator gene (tat) (DONG et al., 2012). In the first reaction, the primers 5’-GTAATTGGGCGCTAAGTCTAG-3’ and 5’- CCTCTAATAAATCTTGCTGTC-3’ were used, generating a 246 bp product. We used the primers DNA Amplification by Nested-PCR Total DNA (250 - 500 ng) from each sample was subjected to amplification of proviral DNA by Nested-PCR reaction within the LTR region, extending to the trans-activator gene (tat) (DONG et al., 2012). In the first reaction, the primers 5’-GTAATTGGGCGCTAAGTCTAG-3’ and 5’- CCTCTAATAAATCTTGCTGTC-3’ were used, generating a 246 bp product. We used the primers Ciência Rural, v.53, n.11, 2023. entification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil.3 Figure 1 - Location (●) of the equine infectious anemia (EIA) outbreak in horses kept in the periurban perimeter of the city of Uruguaiana, Rio Grande do Sul (RS), Brazil, border with Argentina. Figure 1 - Location (●) of the equine infectious anemia (EIA) outbreak in horses kept in the periurban perimeter of the city of Uruguaiana, Rio Grande do Sul (RS), Brazil, border with Argentina. Ciência Rural, v.53, n.11, 2023. Results and Discussion 5’-TGGGTGAATACCATACAGACA-3’ 5’-CCAGTGGAGCATTCGGTAA-3’ in the second round, generating an amplicon between 198 and 203 bp. The products of the reactions were resolved by 1.5% agarose gel electrophoresis and stained with non-mutagenic reagents (Sybr Green Invitrogen) for identification. Positive (DNA from leukocytes from a serologically positive animal) and negative (water) controls were included in all tests. 5’-CCAGTGGAGCATTCGGTAA-3’ in the second round, generating an amplicon between 198 and 203 bp. The products of the reactions were resolved by 1.5% agarose gel electrophoresis and stained with non-mutagenic reagents (Sybr Green Invitrogen) for identification. Positive (DNA from leukocytes from a serologically positive animal) and negative (water) controls were included in all tests. Equine infectious anemia virus infection in the state of Rio Grande do Sul has a low occurrence (BARZONI et al., 2018; Cardenas et al., 2022; REBELATTO et al., 1992). However, in the last five years, there has been a moderate increase in the notification of cases of infection. At the beginning of the 2010s, there were 2 to 4 outbreaks/per year, and by the end of the decade, there were more than ten outbreaks per year. This situation has drawn the attention of producers and animal health authorities (BARZONI et al., 2018; Cardenas et al., 2022; MACHADO et al., 2021). Results presented here described the specific characteristics of an outbreak that occurred in the state region that concentrates the most significant record of EIAV infections. This outbreak represents a unique situation that combines clinical and epidemiological aspects that can contribute to the maintenance and dissemination of the virus among animals and herds. Furthermore, knowledge of the different elements involved contributes to understanding the epidemiology of infection. It demonstrated possibilities that should be considered in investigating outbreaks in the region and serve as an alert for other situations. Sequencing and phylogenetic analysis Thus, identifying regional characteristics of equine breeding and the occurrence of outbreaks contributes to understanding the factors that influence viral transmission in a given population. and the transit of equines is prohibited (PESE) (SEAPI 2018). The counter-test (using the same serum sample) conducted by the accredited laboratory confirmed the previous result. The producer requested the retest during the interdiction (day 11). Retesting is allowed by the legislation, and the AGID test is performed in the official laboratory. However, the OVS performs the new blood collection (SEAPI, 2018). The result of the retest (day 28) confirmed the previous findings. On the day the OVS communicated the final retest results, the owner informed thus that the three animals had been stolen and were missing. The disappearance of animals diagnosed with EIAV is a fact described and contributes to the maintenance of a carrier animal in the population (BARZONI et al., 2018; CRUZ et al., 2020). The animals of this outbreak were identified by AGID serological test for issuing the animal transport permit (GTA). These animals would participate in agglomeration event with more than 5,000 horses (parade), which would have the presence of the inspection of the OVS. For participation, the horses must be moved and undergo the serological examination for EIA, which is mandatory for issuing the GTA. Most EIA diagnoses in Rio Grande do Sul are registered in August, September, and October. This is due to a large number of cultural and sporting events with the presence of horses that exist in this period. Therefore, in this period, the highest number of diagnoses occurs (BARZONI et al., 2018; MACHADO et al., 2021). Thus, passive surveillance plays an essential role in identifying infected animals since the report of clinical cases of EIA is relatively infrequent (BARZONI et al., 2018; REBELATTO et al., 1992). The breeding and keeping horses in the peri-urban region may contribute to the maintenance and dissemination of EIAV (BARZONI et al., 2018; CRUZ et al., 2020). In this situation, animals are raised on small properties, with inadequate nutritional and sanitary conditions, and without regular veterinary assistance. The eventual contact of these animals with other equids raised on farms or stud farms may contribute to the maintenance of the virus. On day 78, one of the stolen horses was found and euthanized. At the time of sacrifice, the animal was in good body condition and healthy. Sequencing and phylogenetic analysis Sequencing and phylogenetic analysis Phylogenetic analysis was performed with the program Molecular Evolutionary Genetics Analysis (MEGA) version X 20, using the Joining Tree methodology with bootstrap values calculated using 1000 replicates. Positive PCR amplified samples were purified (PCR Products Purification Kit Mebep Bioscience) and sequenced by the Sanger method using a sequencer AB-3500 (Applied Biosystems) (ACTGene Análises Molecular Ltda, Alvorada, Rio Grande do Sul, Brazil). The BioEdit Sequence Alignment Editor Software suite evaluated and edited the sequences 7.0.5.3 (http:// www.mbio.ncsu.edu/bioedit/bioedit.html) to obtain the consensus. Subsequently, the consensus sequences were submitted to the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/BLAST/) to compare sequences already deposited in GenBank. In Brazil, the infection presents varying prevalence levels among the different states and Ciência Rural, v.53, n.11, 2023. Jardim et al. 4 and the transit of equines is prohibited (PESE) (SEAPI 2018). The counter-test (using the same serum sample) conducted by the accredited laboratory confirmed the previous result. The producer requested the retest during the interdiction (day 11). Retesting is allowed by the legislation, and the AGID test is performed in the official laboratory. However, the OVS performs the new blood collection (SEAPI, 2018). The result of the retest (day 28) confirmed the previous findings. On the day the OVS communicated the final retest results, the owner informed thus that the three animals had been stolen and were missing. The disappearance of animals diagnosed with EIAV is a fact described and contributes to the maintenance of a carrier animal in the population (BARZONI et al., 2018; CRUZ et al., 2020). regions (ALMEIDA et al., 2006; BORGES et al., 2013; CRUZ et al., 2020; TIGRE et al., 2017). The variations are associated with regional factors such as equine population, the form of breeding, occupation of animals, climate, temperature, the occurrence of mechanical vectors, cultural aspects, and control legislation (BARZONI et al., 2018; Cardenas et al., 2022). Thus, identifying regional characteristics of equine breeding and the occurrence of outbreaks contributes to understanding the factors that influence viral transmission in a given population. regions (ALMEIDA et al., 2006; BORGES et al., 2013; CRUZ et al., 2020; TIGRE et al., 2017). The variations are associated with regional factors such as equine population, the form of breeding, occupation of animals, climate, temperature, the occurrence of mechanical vectors, cultural aspects, and control legislation (BARZONI et al., 2018; Cardenas et al., 2022). Ciência Rural, v.53, n.11, 2023. Sequencing and phylogenetic analysis The hematological examination showed mild leukocytosis (14,500 leukocytes/µL - 5,000 - 11,000) associated with neutrophilia (7,380 cells/µL – 2,200 – 6,100). No macroscopic changes were detected during the necropsy. The absence of clinical signs, hematological changes, and visible lesions are compatible with carrier status and are described in the literature as the most common form of infection (BUENO et al., 2020; COOK et al., 2013). Animal-to-animal transmission of EIAV occurs mainly through blood transfer between equids by iatrogenic route or mechanical vectors (Tabanus spp. and Stomxys spp.) (ISSEL & FOIL, 2015). The form and timing of infection of these animals have not been identified. However, horseflies are present in the region, and sharing injectable material is common (BARZONI et al., 2018). The region’s climatic conditions, with cold winter and hot summer, are associated with the seasonality of tabanids, which may limit the role of these insects in the transmission of the agent, different from that observed in the Brazilian Pantanal (CURSINO et al., 2018). Histopathological evaluation of the spleen revealed moderate hemosiderosis in the splenic parenchyma, both free and in the cytoplasm of reticulo-phagocytic cells. The other organs evaluated showed no noteworthy changes. The spleen and liver are recognized as the main reservoirs of the virus due to the significant presence of macrophages (BUENO et al., 2020). This microscopic change is associated with the accumulation of ferric pigment, especially in resident macrophages, resulting from hemoglobin metabolism triggered after intra and extravascular hemolysis associated with the viral infection and expected in these diseases (BUENO et al., 2020; COOK et al., 2013). The other two stolen horses have remained missing and are probably still being transported illegally. Animals in this situation serve as a source of infection for susceptible equines as The property where the horses were raised is located in the periurban region of the city of Uruguaiana (Figure 1). The herd consisted of only three horses, and none had clinical signs suggestive of the infection. The animals were slightly thin, which was associated with poor feed quality. The owner did not inform the origin of the animals, but it is believed to be rural (farm). After the positive serology result for EIA (day 0), the OVS interdicted the property (day 11). During the interdiction period, the owner is responsible for the isolation and maintenance of positive animals, Ciência Rural, v.53, n.11, 2023. Sequencing and phylogenetic analysis entification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil.5 they stay in the herd (BARZONI et al., 2018; CRUZ et al., 2020). in RS samples is impossible. However, further genetic identification studies must be performed to characterize the infection’s epidemiology. The sequenced region refers to the terminal portion of the LTR (long terminal repeat) and the tat (trans- activator) gene (DONG et al., 2012; LEROUX et al., 2004). Despite the high variability of lentiviruses associated with geographic distribution, the LTR region has highly conserved elements, similar to the tat gene (DONG et al., 2012; LEROUX et al., 2004, MALOSSI et al., 2020). Thus, the amplified region can be used in phylogeny studies for epidemiological research. However, other regions of the genome or the whole genome may be more suitable for this purpose (BUENO et al., 2020; CURSINO et al., 2018; MALOSSI et al., 2020; OAKS et al., 1998; TIGRE et al., 2017). Nested-PCR detected proviral DNA in blood, lung, and spleen samples, and a DNA fragment (~ 203bp) corresponding to that described by DONG et al. (2012) was amplified. Amplifying proviral DNA in tissue samples confirms the serology result and demonstrates that the virus is present in organs with a high concentration of macrophages (BUENO et al., 2020; OAKS et al., 1998). The PCR products were sequenced and the sequences obtained (Submission ID: ON639609) showed 98% to 100% identity with other EIAV samples (Figure 2). Several genetic analyses of EIA viruses present in Bahia, Rio Grande do Norte, and Pantanal have been performed (BUENO et al., 2020; CURSINO et al., 2018; TIGRE et al., 2017). The Rio Grande do Sul is the southernmost state in Brazil, and this is the first time that an EIAV sample from this region has been partially identified by sequencing. Thus, comparison with other viruses circulating The highest concentration of EIAV outbreaks in RS is located in the western border region (BARZONI et al., 2018; Cardenas et al., 2022). This region is characterized by extensive cattle and Ciência Rural, v.53, n.11, 2023. Figure 2 - Phylogenetic analysis of region LTR (long terminal repeat) and the tat (trans-activator) gene of the equine infectious anemia virus sample (EIAV LV 49/17 URG) present in the lung, spleen, and blood. DECLARATION OF CONFLICT OF INTEREST The authors declare no conflict of interest. Funding sponsors had no role in the study design, collection, analysis, and data interpretation during the writing this manuscript and in the decision to publish the results. The voluntary testing of animals is relatively low. This may be due to the absence of clinical suspicion, the costs of the examination, and the fear that owners have about having an infected horse sacrificed. The health certificate requirement for transportation and the presence of inspection in events forces producers to perform health examinations, thus increasing the number of tests and; consequently, identifying outbreaks (MACHADO et al., 2021). Therefore, passive surveillance plays a crucial role in identifying infected animals (BARZONI et al., 2018; CRUZ et al., 2020; MACHADO et al., 2021). Another aspect that should be reinforced is building a relationship of trust between the producer and OVS. This type of relationship facilitates epidemiological investigation, clarifying the origin of the infection. It enables measures to prevent new cases and adopt actions that can reduce the circulation of the agent. Sequencing and phylogenetic analysis Most likely, the animals originated from a property located in the interior of the municipality and were transported without examination. Rio Grande do Sul opens the way for the phylogenetic study of samples from other foci, helping to determine the origin of the samples present in the State and can assist in establishing epidemiological links in situations of unavailability of information. Acknowledgments JCJS is recipient of scholarship of doctoral from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This work was financed with resources from Laboratório de Virologia/UNIPAMPA, Pró-Reitoria de Pós-Graduação, Pesquisa e Inovação, Universidade Federal do Pampa (PROPPI/UNIPAMPA), and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil - Finance code 001. The authors thank the Secretaria de Agricultura, Pecuária e Desenvolvimento Rural do Estado of the State of Rio Grande do Sul (SEAPDR/RS), Brazil, for providing data and information. BIOETHICS AND COMMITTEE APPROVAL For all due purposes, the authors of the article “Genetic identification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil” declare that the project that gave rise to the data of the same was not submitted for evaluation to the Ethics Committee of the Universidade Federal do Pampa. However, we are aware of the content of the resolutions of the National Council for the Control of Animal Experimentation - CONCEA “http://www.mct.gov.br/ index.php/content/view/310553.html” if it involves animals. Thus, the authors assume full responsibility for the data presented and are available for possible questioning, should they be required by the competent bodies. Conclusion The results described the characteristics of an outbreak of equine infectious anemia that occurred in a region of Rio Grande do Sul, Brazil. A moderate increase in the registration of cases has been observed in the last five years. The conditions of absence of clinical signs and carrier of the infection, associated with the form of breeding and disappearance of two positive animals, are characteristics that contribute to the maintenance and dissemination of EIAV among animals in the region. These factors alone or together create opportunities for EIAV to perpetuate itself in herds. The molecular identification of a sample circulating in the BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL Sequencing and phylogenetic analysis Figure 2 - Phylogenetic analysis of region LTR (long terminal repeat) and the tat (trans-activator) gene of the equine infectio anemia virus sample (EIAV LV 49/17 URG) present in the lung, spleen, and blood. Figure 2 - Phylogenetic analysis of region LTR (long terminal repeat) and the tat (trans-activator) gene of the equine infectious anemia virus sample (EIAV LV 49/17 URG) present in the lung, spleen, and blood. Ciência Rural, v.53, n.11, 2023. Jardim et al. 6 sheep production and concentrates the largest equine herd in the state (~149,000 animals, 14% of the state’s herd) (COSTA et al., 2013). In this region, horses are used for work on the farms and eventually participate in sporting events and parades. Besides the animals used for work, there are pure breed’s farms (PSI and Crioulo) that move animals to other regions of the state and Brazil (COSTA et al., 2013; IBGE, 2017). On the outskirts of cities, many draft horses and animals are raised on small farms, which EIAV can eventually infect (CRUZ et al., 2020; MACHADO et al., 2021). The illegal movement of animals is frequently reported in the region (BARZONI et al., 2018; CRUZ et al., 2020). The animals diagnosed with EIA were kept on the outskirts of the city; however, the owner did not inform the origin of the animals, nor did he suspect the moment of infection. Most likely, the animals originated from a property located in the interior of the municipality and were transported without examination. sheep production and concentrates the largest equine herd in the state (~149,000 animals, 14% of the state’s herd) (COSTA et al., 2013). In this region, horses are used for work on the farms and eventually participate in sporting events and parades. Besides the animals used for work, there are pure breed’s farms (PSI and Crioulo) that move animals to other regions of the state and Brazil (COSTA et al., 2013; IBGE, 2017). On the outskirts of cities, many draft horses and animals are raised on small farms, which EIAV can eventually infect (CRUZ et al., 2020; MACHADO et al., 2021). The illegal movement of animals is frequently reported in the region (BARZONI et al., 2018; CRUZ et al., 2020). The animals diagnosed with EIA were kept on the outskirts of the city; however, the owner did not inform the origin of the animals, nor did he suspect the moment of infection. AUTHORS’ ConTRIBtribuTIontis JCJS (execution, results analysis, writing, review), PFF, GVC, CKT, BLA (execution, results analysis, review), MCSB (conception, results analysis, writing, review, coordinator). All authors critically revised the manuscript and approved of the final version. entification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil.7 Available from: <http://www.scielo.br/scielo.php?script=sci_ arttext&pid=S0103-84782018000600451&lng=en&tlng=en>. Accessed: May, 04, 2020. Available from: <http://www.scielo.br/scielo.php?script=sci_ arttext&pid=S0103-84782018000600451&lng=en&tlng=en>. Accessed: May, 04, 2020. gov.br/agro/2017/templates/censo_agro/resultadosagro/pecuaria. html?localidade=0&tema=75665>. Accessed: Apr. 20, 2020. gov.br/agro/2017/templates/censo_agro/resultadosagro/pecuaria. html?localidade=0&tema=75665>. Accessed: Apr. 20, 2020. ISSEL, C. J.; FOIL, L. D. Equine infectious anaemia and mechanical transmission: man and the wee beasties. Revue Scientifique et Technique, v.34, n.2, p.513–523, 2015. Available from: <https://doi. org/10.20506/rst.34.2.2376>. Accessed: May, 4, 2020. BORGES, A. M. C. M. et al. Prevalence and risk factors for equine infectious anemia in Poconé municipality, northern Brazilian Pantanal. Research in Veterinary Science, v.95, n.1, p.76–81, 2013. Available from: <https://linkinghub.elsevier.com/retrieve/ pii/S0034528813000659>. Accessed: May, 04, 2020. BORGES, A. M. C. M. et al. Prevalence and risk factors for equine infectious anemia in Poconé municipality, northern Brazilian JARA, M. et al. Phylogeography of equine infectious anemia virus. Frontiers in Ecology and Evolution, v.8, p.000127, 2020. Available from: <https://doi.org/10.3389/fevo.2020.00127>. Accessed: May, 05, 2020. BRASIL. Normas para prevenção e o controle da anemia infecciosa equina - A.I.E. Instrução Normativa nº45, de 15 de julho de 2004. Secretaria de Defesa Agropecuária, Ministério da Agricultura, Pecuária e Abastecimento. Diário Oficial da República Federativa do Brasil, 2004. Available from: <http:// www.agricultura.rs.gov.br/upload/arquivos/201701/05142347- pese-in-45-2004.pdf>. Accessed: Jan. 07, 2016. LEROUX, C. et al. Equine infectious anemia virus (EIAV): what has HIV’s country cousin got tell us? Veterinary Research, v.35, n.4, p.485–512, 2004. Available from: <https://doi.org/10.1051/ vetres:2004020>. Accessed: Nov. 11, 2021. BUENO, B. L. et al. Molecular detection, histopathological analysis, and immunohistochemical characterization of equine infectious anemia virus in naturally infected equids. Archives of Virology, v.165, n.6, p.1333–1342, 2020. Available from: <https:// doi.org/10.1007/s00705-020-04616-4>. Accessed: Feb. 12, 2021. MACHADO, G. et al. Impact of changes of horse movement regulations on the risks of equine infectious anemia: a risk assessment approach. Preventive Veterinary Medicine, v.190, p.e105319, 2021. Available from: <https://linkinghub.elsevier. com/retrieve/pii/S0167587721000635>. Accessed: May, 19, 2021. Cardenas, N. C. et al. Coupling spatial statistics with social network analysis to estimate distinct risk areas of disease circulation to improve risk-based surveillance. Transboundary and Emerging Diseases, p.1–12, 2022. Available from: <https:// doi.org/10.1111/tbed.14627>. Accessed: Jun. 12 2022. MALOSSI, C. D. et al. High genomic variability in equine infectious anemia virus obtained from naturally infected horses in Pantanal, Brazil: an endemic region case. Viruses, v.12, n.2, p.207-222, 2020. Available from: <https://www.mdpi.com/1999- 4915/12/2/207>. Accessed: Nov. 12, 2022. COOK, R. F. et al. Equine infectious anemia and equine infectious anemia virus in 2013: a review. Veterinary Microbiology, v.167, n.1–2, p.181– 204, 2013. Available from: <https://www.sciencedirect.com/science/ article/pii/S0378113513004707?via%3Dihub>. REFERENCES ALMEIDA, V. M. A. et al. Equine infectious anemia: prevalence in working equids of livestock herds, in Minas Gerais, Brazil. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.58, n.2, p.141–148. 2006. Available from: <http://www.scielo.br/scielo. php?script=sci_arttext&pid=S0102-09352006000200001&lng=pt &tlng=pt>. Accessed: May, 04, 2020. BARZONI, C. S. et al. Equine infectious anemia in the western region of Rio Grande do Sul, Brazil. Ciência Rural, v.48, n.6, 2018. Ciência Rural, v.53, n.11, 2023. entification, clinical and epidemiological aspects of an equine infectious anemia outbreak in the Rio Grande do Sul State, Brazil.7 Accessed: Jun. 01, 2016. MAPA. Animal diseases in Brazil. History of the first observations. Boletim de Defesa Sanitária Animal. Brasília: [s.n.], 1988. Available from: <https://www.gov.br/agricultura/pt-br/assuntos/ sanidade-animal-e-vegetal/saude-animal/arquivos-sisa/as-doencas- dos-animais-no-brasil-historico-das-primeiras-observacoes.pdf>. Accessed: Jan. 25, 2019. COSTA, E. et al. Horse-rearing in Rio Grande do Sul. A Hora Veterinária, v. 196, n. 6, p. 35–40, 2013. Available from: <https:// www.agricultura.rs.gov.br/upload/arquivos/201612/02101333- inftec-50-panorama-da-equinocultura-no-rio-grande-do-sul.pdf>. Accessed: Sep. 03, 2018. OAKS, J. L. et al. Equine infectious anemia virus is found in tissue macrophages during subclinical infection. Journal of Virology, v.72, n.9, p.7263–7269, 1998. Available from: <https://doi.org/10.1128/ JVI.72.9.7263-7269.1998>. Accessed: May, 12, 2021. CRUZ, A. P. M. et al. Seroprevalence for equine infectious anaemia in equidae seized in the municipality of Petrópolis, State of Rio de Janeiro, Brazil, 2015/2018. Ciência Rural, v.50, n.4, 2020. Available from: <http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103- 84782020000400452&tlng=en>. Accessed: Jan. 15, 2022. REBELATTO, M. C. et al. Serological diagnosis of equine infectious anaemia virus infection in the central region of the Rio Grande do Sul state. Ciência Rural, v.22, n.2, p.179-196, 1992. Available from: <http://www.scielo.br/scielo.php?script=sci_artt ext&pid=S0103-84781992000200012>. Accessed: Jan. 08, 2016. CURSINO, A. E. et al. Equine infectious anemia virus in naturally infected horses from the Brazilian Pantanal. Archives of Virology, v.163, n.9, p.2385–2394, 2018. Available from: <https://doi. org/10.1007/s00705-018-3877-8>. Accessed: May, 25, 2018. SEAPA. Inquérito soroepidemiológico da anemia infecciosa equina no estado do Rio Grande do Sul. Secretaria da Agricultura, Pecuária e Desenvolvimento Rural, 2014. p.34. DONG, J. B. et al. Development of a nested PCR assay to detect equine infectious anemia proviral DNA from peripheral blood of naturally infected horses. Archives of Virology, 2012. v.157, n.11, p.2105–2111. Available from: <https://doi.org/10.1007/s00705- 012-1406-8>. Accessed: Jan. 15, 2018. SEAPI. Programa Estadual de Sanidade Equina (PESE), 2018. Available from: <https://www.agricultura.rs.gov.br/pese>. Accessed: Nov. 19, 2020. TIGRE, D. M. et al. Characterization of isolates of equine infectious anemia virus in Brazil. Archives of Virology, v.162, n.3, p.873–877, 2017. Available from: <https://doi.org/10.1007/s00705-016-3172-5>. Accessed: Dec. 05, 2016. TIGRE, D. M. et al. Characterization of isolates of equine infectious anemia virus in Brazil. Archives of Virology, v.162, n.3, p.873–877, 2017. Available from: <https://doi.org/10.1007/s00705-016-3172-5>. Accessed: Dec. 05, 2016. IBGE, Instituto Brasileiro de Geografia e Estatística. Censo Agropecuário. [S.l.], 2017. Available from: <https://censos.ibge. Ciência Rural, v.53, n.11, 2023. Ciência Rural, v.53, n.11, 2023.
https://openalex.org/W2122762441
https://europepmc.org/articles/pmc2672059?pdf=render
English
null
High Concentrations of Morphine Sensitize and Activate Mouse Dorsal Root Ganglia via TRPV1 and TRPA1 Receptors
Molecular pain
2,009
cc-by
7,378
BioMed Central BioMed Central Molecular Pain Received: 13 January 2009 Accepted: 16 April 2009 This article is available from: http://www.molecularpain.com/content/5/1/17 © 2009 Forster et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Morphine and its derivatives are key drugs in pain control. Despite its well-known analgesic properties morphine at high concentrations may be proalgesic. Particularly, short-lasting painful sensations have been reported upon dermal application of morphine. To study a possible involvement of TRP receptors in the pro-nociceptive effects of morphine (0.3 – 10 mM), two models of nociception were employed using C57BL/6 mice and genetically related TRPV1 and TRPA1 knockout animals, which were crossed and generated double knockouts. Hindpaw skin flaps were used to investigate the release of calcitonin gene-related peptide indicative of nociceptive activation. Results: Morphine induced release of calcitonin gene-related peptide and sensitized the release evoked by heat or the TRPA1 agonist acrolein. Morphine activated HEK293t cells transfected with TRPV1 or TRPA1. Activation of C57BL/6 mouse dorsal root ganglion neurons in culture was investigated with calcium imaging. Morphine induced a dose-dependent rise in intracellular calcium in neurons from wild-type animals. In neurons from TRPV1 and TRPA1 knockout animals activation by morphine was markedly reduced, in the TRPV1/A1 double knockout animals this morphine effect was abrogated. Naloxone induced an increase in calcium levels similar to morphine. The responses to both morphine and naloxone were sensitized by bradykinin. Conclusion: Nociceptor activation and sensitization by morphine is conveyed by TRPV1 and TRPA1. Research Address: 1Institute of Physiology and Pathophysiology, University of Erlangen-Nürnberg, Universitätsstrasse 17, Erlangen, 91054 Germany and 2Department of Pharmacology, University of Cambridge, Tennis Court Rd, Cambridge, CB2 1PD, UK Email: Alexander B Forster - aforster@physiologie1.uni-erlangen.de; Peter W Reeh - reeh@physiologie1.uni-erlangen.de; Karl Messlinger - messlinger@physiologie1.uni-erlangen.de; Michael JM Fischer* - fischer@physiologie1.uni-erlangen.de * C di h * Corresponding author Published: 16 April 2009 Molecular Pain 2009, 5:17 doi:10.1186/1744-8069-5-17 Received: 13 January 2009 Accepted: 16 April 2009 This article is available from: http://www.molecularpain.com/content/5/1/17 © 2009 Forster et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 13 January 2009 Accepted: 16 April 2009 Open Research High concentrations of morphine sensitize and activate mouse dorsal root ganglia via TRPV1 and TRPA1 receptors Alexander B Forster1, Peter W Reeh1, Karl Messlinger1 and Michael JM Fischer*1,2 Open Access Morphine elicits calcium transients in DRG neurons p A total of 106 dorsal root ganglion (DRG) neurons were exposed to 10 mM morphine for 40 s, 30 μM acrolein for 30 s, 1 μM capsaicin for 5 s and 60 mM potassium for 30 s with an interval of 240 s between applications. Response rates to morphine were 22%, to acrolein 45% and to cap- saicin 58%. Morphine responses in neurons activated by capsaicin but not by acrolein and in neurons activated by acrolein but not capsaicin were larger than in neurons which did not respond to either capsaicin or acrolein (p = 0.024 and p = 0.027, t-test independent samples). Responses to morphine grouped by the sensitivity to cap- saicin and acrolein are presented in Fig. 2a. Morphine (10 mM) was applied three times for 40 s. The second appli- cation of morphine in the presence of BCTC (10 μM) and HC030031 (50 μM) did not elicit a calcium increase (both p < 0.001 compared to the initial and the third mor- phine application, n = 62, Wilcoxon, Fig. 2b). Morphine a cells Figure 1 Morphine activates TRPV1 and TRPA1 expressed in HEK cells. HEK cells transfected with cDNA for hTRPV1 and mTRPA1 were exposed to 10 mM morphine. The upper traces show an inward current in a cell transfected by hTRPV1 and GFP, evoked by 10 mM morphine and by 300 nM capsaicin, both applied for a period of 10 s. The lower traces show an inward current in a cell transfected by mTRPA1 and GFP, evoked by 10 mM morphine and by 30 μM mustard oil, both applied for a period of 40 s. Cells were exposed to 1 μM m-3M3FBS before application of morphine and between applications. The transient receptor potential channel TRPV1 (formerly VR1) receptor is found in small to medium diameter dor- sal root, trigeminal and nodose ganglia primary afferent neurons which sense potentially damaging stimuli such as capsaicin, heat and low pH [8,9]. For TPRV1 more pun- gent activating compounds have been reported than for any other TRPV channel. Another TRP channel, TRPA1 (formerly ANKTM1) equips neurons with a sensitivity for mustard oil and many diverse pungent chemical stimuli [10]. Among these compounds are electrophilic com- pounds such as allyl isothiocyanate, acrolein, formalin, 15-deoxy-Δ12,14-prostaglandin J2, nitric oxide, hydrogen peroxide as well as the inflammatory mediator bradykinin [11]. TRPA1 and TRPV1 are the principal detectors for painful chemical stimuli. The aim of this study was to investigate whether the painful sensations evoked by mor- phine are mediated by these two receptors. Two models of nociceptive activation were employed using mice lacking TRPV1, TRPA1 or both these receptors. Background time morphine and its derivatives, which form the class of opioids, became the main therapeutic option for the treat- ment of moderate to severe pain. The analgesia as well as most side effects are caused by interaction with the opioid receptors, especially the mu-receptors. g After many thousands of years of therapeutic opium use, in 1806 the German pharmacist Sertuerner published the discovery of the main pharmacological compound of opium, which he later called morphine [1]. From this Page 1 of 11 (page number not for citation purposes) Page 1 of 11 (page number not for citation purposes) Molecular Pain 2009, 5:17 Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 Morphine activates TRPV1 and TRPA1 expressed in HEK cells Figure 1 Morphine activates TRPV1 and TRPA1 expressed in HEK cells. HEK cells transfected with cDNA for hTRPV1 and mTRPA1 were exposed to 10 mM morphine. The upper traces show an inward current in a cell transfected by hTRPV1 and GFP, evoked by 10 mM morphine and by 300 nM capsaicin, both applied for a period of 10 s. The lower traces show an inward current in a cell transfected by mTRPA1 and GFP, evoked by 10 mM morphine and by 30 μM mustard oil, both applied for a period of 40 s. Cells were exposed to 1 μM m-3M3FBS before application of morphine and between applications. Opioids have several commonly known side effects including addiction, sedation, constipation and nausea [2]. Besides their well-known antinociceptive actions, opi- oids can cause hyperalgesia by an unknown, opioid recep- tor-independent mechanism [3,4]. This present study was initiated by the clinical observation that in patients suffer- ing from radiodermatitis, which could not be controlled by systemic morphine, high doses of a custom-made opi- oid gel applied topically to the skin can elicit severe burn- ing pain for a few seconds (Clinical research group KFO130, Erlangen, Germany). The isotonic gel prepared by the university's pharmacy contains 2.67 mM (0.1%) morphine and has a neutral pH. Case reports have previ- ously reported hyperalgesia after application of high doses of morphine [5,6]. At lower concentrations mor- phine only causes pruritus, which might be explained by mast cell degranulation [7]. Sensitization by bradykinin and effect of naloxone The inflammatory mediator bradykinin is known to acti- vate nociceptive neurons in higher concentrations but in lower concentrations to sensitize TRPV1 and TRPA1. To determine whether calcium increases evoked by 10 mM morphine are increased by bradykinin at a concentration below activation, we applied the second of four consecu- tive morphine applications in the presence of 5 μM brady- kinin (Fig. 4). Bradykinin was applied 40 seconds before the morphine stimulus and did not elicit a calcium response (n = 111). Among the 22.5% of DRG neurons responding to morphine, bradykinin increased the mor- phine-stimulated calcium influx to 249% (ANOVA, F(3,330) = 5.1, p= 0.002, HSD post-hoc test), the subse- quent two morphine responses were not different com- pared to the first one (p = 0.33 and p = 0.87, HSD post- hoc tests). This protocol was repeated with morphine replaced by naloxone, which is structurally similar but has no intrinsic activity on opioid receptors. The activation by naloxone was also concentration-dependent (n = 10, data not shown). Bradykinin reversibly increased the naloxone-stimulated calcium increase to 165% of the first response (ANOVA, F(3,66) = 5.0, p = 0.025, HSD-post hoc test). Morphine and acrole Figure 2 Morphine activates DRG neurons responsive to capsaicin and acrolein Figure 2 Morphine activates DRG neurons responsive to cap- saicin and acrolein. Upper panel. Morphine, acrolein and capsaicin were applied to 106 wildtype DRG neurons. The average calcium increase evoked by morphine 10 mM is com- pared in neurons grouped by sensitivity to capsaicin and acrolein. Neurons responsive to capsaicin, acrolein or both showed a larger calcium signal response to morphine than neurons not responsive to both capsaicin and acrolein. Lower panel. Wildtype DRG neurons were exposed to three repetitive applications of 10 mM morphine (n = 62). In the presence of 10 μM BCTC and 50 μM HC030031, morphine evoked no significant increase in intracellular calcium. p p p g Morphine activates DRG neurons responsive to cap- saicin and acrolein. Upper panel. Morphine, acrolein and capsaicin were applied to 106 wildtype DRG neurons. The average calcium increase evoked by morphine 10 mM is com- pared in neurons grouped by sensitivity to capsaicin and acrolein. Neurons responsive to capsaicin, acrolein or both showed a larger calcium signal response to morphine than neurons not responsive to both capsaicin and acrolein. Lower panel. Sensitization by bradykinin and effect of naloxone Wildtype DRG neurons were exposed to three repetitive applications of 10 mM morphine (n = 62). In the presence of 10 μM BCTC and 50 μM HC030031, morphine evoked no significant increase in intracellular calcium. Morphine-induced CGRP release from hindpaw skin In order to reappraise these findings in intact innervated tissue, we investigated the stimulated CGRP release from nerve endings in isolated hind paw skin of C57BL/6 mice. In a first series of experiments (n = 8) we assessed the response to application of 10 mM morphine diluted in SIF (Fig. 5). The basal CGRP concentration in the first incubation period (SIF) was 2 ± 1 pg/ml. When the skin flaps were transferred to the test tube containing 10 mM morphine, the CGRP release increased to 20 ± 5 pg/ml (p = 0.012, Wilcoxon). Final exposure to 60 mM KCl resulted in a CGRP release of 54 ± 11 pg/ml. HSD post-hoc tests). The EC50 was 2.6 ± 0.04 mM (Fig. 3). Morphine at 10 mM reached 32% of the 60 mM potas- sium-induced calcium influx. This protocol was repeated in neurons from TRPV1-/- (n = 112), TRPA1-/- (n = 125) as well as from TRPV1-/- TRPA1-/ - animals (n = 135). Compared to neurons from C57BL/6 mice, the increases in calcium levels evoked by 10 mM morphine were smaller in animals lacking TRPV1 or TRPA1 and almost absent in double knockouts (ANOVA, F(9,1485) = 7.0; TRPV1-/-: 51%, p = 0.008; TRPA1-/-: 34%, p = 0.005; TRPV1-/- TRPA1-/-: 2%, p < 0.001; % of AUC in C57BL/6, HSD post-hoc tests). Compared to baseline, the small morphine-induced increases of intracellular cal- cium were still significant in neurons from TRPV1-/- (p < Morphine activates hTRPV1 and TRPA1 expressed in HEK293t In HEK293t cells transfected with hTRPV1 application of 10 mM morphine evoked an inward current, 300 μM cap- saicin was applied as a control thereafter (n = 10, sample recording in Fig. 1). In HEK293t cells transfected with mTRPA1 10 mM morphine evoked inward currents that appeared to rapidly inactivate during continued super- fusion (n = 10). However, upon offset and washout of morphine regularly large tail currents occurred that revealed the sustained activation of TRPA1 and a slow deactivation. Mustard oil (30 μM) was applied as a con- trol thereafter. Increasing concentrations of morphine (0.1 – 10 mM) were consecutively applied for 40 s, separated by 240 s washout intervals. From C57BL/6 animals 134 DRG neu- rons were tested. Morphine at 3 mM and 10 mM evoked a concentration-dependent transient rise in intracellular calcium (ANOVA, F(4,532) = 16.9, p = 0.005 and p < 0.001, Page 2 of 11 (page number not for citation purposes) Page 2 of 11 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/17 Molecular Pain 2009, 5:17 Morphine activates DRG neurons responsive to capsaicin and acrolein Figure 2 Morphine activates DRG neurons responsive to cap- saicin and acrolein. Upper panel. Morphine, acrolein and capsaicin were applied to 106 wildtype DRG neurons. The average calcium increase evoked by morphine 10 mM is com- pared in neurons grouped by sensitivity to capsaicin and acrolein. Neurons responsive to capsaicin, acrolein or both showed a larger calcium signal response to morphine than neurons not responsive to both capsaicin and acrolein. Lower panel. Wildtype DRG neurons were exposed to three repetitive applications of 10 mM morphine (n = 62). In the presence of 10 μM BCTC and 50 μM HC030031, morphine evoked no significant increase in intracellular calcium. 0.001, ANOVA, F(4,444) = 13.0) and TRPA1-/- (p < 0.001, ANOVA, F(4,468) = 24.6), but not so in neurons from TRPV1-/- TRPA1-/- animals (p = 0.10, ANOVA, F(4,536) = 2.0). Due to the limited solubility of morphine at pH 7.4, no higher concentrations than 10 mM could be tested. Neurons activated by morphine were 20.5 ± 0.9 μm in diameter, smaller than the average of 23.0 ± 0.5 μm (p = 0.041, U-test). heat A separate series of experiments was performed to test the hypothesis that morphine concentrations without a direct effect on CGRP release may sensitize cutaneous afferents to noxious stimuli activating TRPV1 or TRPA1. CGRP release in the presence of 310 μM morphine was not dif- ferent from the basal release (Fig. 6). The selective agonist Page 3 of 11 (page number not for citation purposes) Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 activates DRG neurons via TRPA1 and TRPV1 receptors causing increases in intracellular calciu tly activates DRG neurons via TRPA1 and TRPV1 receptors causing increases in phine application periods lasting 40 s are indicated by bars. The upper four panels show the fluo sted genotypes, presented as mean ± SEM of all neurons (number of neurons, C57Bl/6: 134, and TRPV1-/- TRPA1-/-: 135). All neurons responded to 60 mM KCl at the end of the experimen ogram inset in the uppermost panel shows that preferentially small size DRG neurons respond t diameter in μm, y: number of cells). The bottom panel summarizes the dose-dependent activation ce ratio during the application period was normalized to the response elicited by 60 mM KCl. Morphine dose-dependently activates DRG neurons via TRPA1 and TRPV1 receptors causing increases in intracellular calcium Figure 3 Morphine dose-dependently activates DRG neurons via TRPA1 and TRPV1 receptors causing increases in intracellular calcium. Morphine application periods lasting 40 s are indicated by bars. The upper four panels show the fluo- rescence ratios of the four tested genotypes, presented as mean ± SEM of all neurons (number of neurons, C57Bl/6: 134, TRPV1-/-: 112, TRPA1-/-: 125 and TRPV1-/- TRPA1-/-: 135). All neurons responded to 60 mM KCl at the end of the experiment (not shown). The stacked histogram inset in the uppermost panel shows that preferentially small size DRG neurons respond to morphine (black bars, x: cell diameter in μm, y: number of cells). The bottom panel summarizes the dose-dependent activation. The integral of the fluorescence ratio during the application period was normalized to the response elicited by 60 mM KCl. Morphine dose dependently activates DRG neurons via TRPA1 and TRPV1 receptors causing increases in intracellular calcium Figure 3 Morphine dose-dependently activates DRG neurons via TRPA1 and TRPV1 receptors causing increases in intracellular calcium. Morphine application periods lasting 40 s are indicated by bars. heat The upper four panels show the fluo- rescence ratios of the four tested genotypes, presented as mean ± SEM of all neurons (number of neurons, C57Bl/6: 134, TRPV1-/-: 112, TRPA1-/-: 125 and TRPV1-/- TRPA1-/-: 135). All neurons responded to 60 mM KCl at the end of the experiment (not shown). The stacked histogram inset in the uppermost panel shows that preferentially small size DRG neurons respond to morphine (black bars, x: cell diameter in μm, y: number of cells). The bottom panel summarizes the dose-dependent activation. The integral of the fluorescence ratio during the application period was normalized to the response elicited by 60 mM KCl. Page 4 of 11 (page number not for citation purposes) Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 r Pain 2009, 5:17 http://www.molecularpain.com n sensitizes activation of neurons by morphine and naloxone nin sensitizes activation of neurons by morphine and naloxone. Repetitive activation of DRG n pplications of morphine and naloxone. Bradykinin reversibly sensitized calcium influx evoked by both m naloxone (n = 91) but did not evoke a response when applied alone 40 s prior to morphine or naloxon n periods; data are presented as mean ± SEM. The lower panel summarizes the calcium increases nor ponses to 60 mM KCl. *p < 0.05 y y p g Bradykinin sensitizes activation of neurons by morphine and naloxone. Repetitive activation of DRG neurons elicited by 40 s applications of morphine and naloxone. Bradykinin reversibly sensitized calcium influx evoked by both morphine (n = 111) and naloxone (n = 91) but did not evoke a response when applied alone 40 s prior to morphine or naloxone. Bars indicate application periods; data are presented as mean ± SEM. The lower panel summarizes the calcium increases normalized to indi- vidual responses to 60 mM KCl. *p < 0.05 Page 5 of 11 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/17 http://www.molecularpain.com/content/5/1/17 Molecular Pain 2009, 5:17 Morphine elicits release of CGRP Figure 5 Morphine elicits release of CGRP. Morphine (10 mM) applied for 5 minutes reversibly stimulates the release of CGRP from the isolated hindpaw skin of mice (n = 8). Syn- thetic interstitial fluid containing 60 mM KCl was applied for comparison. *p < 0.05 noxious heat stimulation using SIF at 47°C; heat-induced cutaneous CGRP release depends partly on TRPV1 as a transducer. The exposure to 310 μM morphine caused no rise in CGRP release (Fig. Clinical observation The present study was initiated by the observation of pain- ful sensations caused by topical application of a 2.67 mM morphine gel to inflamed skin following radiation ther- apy, as reported in the interdisciplinary pain research group (KFO 130). In these patients, the topical morphine formulation (prepared by the pharmacy unit of the uni- versity) was preferred because systemic opiates could not be applied systemically due to unwanted side effects. The painful sensations were reported to last only for a few sec- onds but exceeded seven on a ten-point rating scale. The time course of the pain was similar to that reported after injection of lidocaine, which has recently been shown to activate nociceptive afferents via TRPV1 and TRPA1 recep- tors before sodium channel blockade leads to local anesthesia [12]. Similarly for morphine, an inhibition of sodium channels has been described [13-15], masking by conduction block the sustained activation. Sodium chan- nel block also occurs with naloxone [16], shedding doubts on alleged naloxone reversal of peripheral opioid effects. acrolein 100 μM was used to activate TRPA1 at the lower end of the concentration-response relationship; in pres- ence of 310 μM morphine acrolein caused markedly greater increases in CGRP release (p = 0.028, n = 6, Wil- coxon). A similar series of experiments was performed for Morphine sensitizes CGRP-release stimulated by acrolein Figure 6 Morphine sensitizes CGRP-release stimulated by acrolein. Release of CGRP from isolated hindpaw skin of mice was stimulated by 100 μM acrolein applied for 5 min (open symbols). The contralateral hindpaw skin of every ani- mal was exposed to acrolein in the presence of 310 μM mor- phine (closed symbols) which sensitized the release of CGRP evoked by acrolein (n = 6). *p < 0.05 The radiation dermatitis involves a tailored therapy; topi- cal application of morphine is not the primary choice but rather an uncommon option. This might explain why sys- tematic studies are not available. Common symptomatic therapy of radiation dermatitis is based on topical wound care predominantly involving moisturizers. There is no uniequivocal evidence for a causal therapy, including the topical application of ascorbic acid, corticosteroids or cal- cineurin inhibitors; opioids have not been investigated so far [17]. Morphine Figure 6 o p e se s t es CG e ease st u ate by ac o e gu e 6 Morphine sensitizes CGRP-release stimulated by acrolein. Clinical observation Release of CGRP from isolated hindpaw skin of mice was stimulated by 100 μM acrolein applied for 5 min (open symbols). The contralateral hindpaw skin of every ani- mal was exposed to acrolein in the presence of 310 μM mor- phine (closed symbols) which sensitized the release of CGRP evoked by acrolein (n = 6). *p < 0.05 heat 7), but slightly sensitized the release evoked by heat stimulation (p = 0.018, n = 7, Wil- coxon, compared to 47°C on the control side). Discussion Morphine and naloxone at high concentrations were found to activate cultured primary afferent neurons in a concentration-dependent manner demonstrated by inward currents and calcium transients. TRPV1 and TRPA1 but not opioid receptors conveyed this activation. The inflammatory mediator bradykinin increased the neuronal sensitivity to morphine and naloxone. Mor- phine concentrations below the activation threshold sen- sitized TRPV1 and TRPA1 in native cutaneous nerve endings releasing CGRP. Morphine Figure 5 p g Morphine elicits release of CGRP. Morphine (10 mM) applied for 5 minutes reversibly stimulates the release of CGRP from the isolated hindpaw skin of mice (n = 8). Syn- thetic interstitial fluid containing 60 mM KCl was applied for comparison. *p < 0.05 Receptors mediating opioid effects There is evidence for peripheral opioid receptor effects [18-20]. The expression profile of opioid receptors on pri- Page 6 of 11 (page number not for citation purposes) Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 Morphine sensitizes CGRP-release stimulated by noxious heat Figure 7 Morphine sensitizes CGRP-release stimulated by noxious heat. Release of CGRP from isolated hindpaw skin of mice stimulated by noxious heat of 47°C applied for 5 min (open symbols). The contralateral hindpaw skin flap of every animal was exposed to 47°C in the presence of 310 μM morphine (closed symbols) which slightly facilitated the release of CGRP stimu- lated by heat (n = 7). The inset shows the results with and without morphine for every animal. *p < 0.05. p y g Morphine sensitizes CGRP-release stimulated by noxious heat. Release of CGRP from isolated hindpaw skin of mice stimulated by noxious heat of 47°C applied for 5 min (open symbols). The contralateral hindpaw skin flap of every animal was exposed to 47°C in the presence of 310 μM morphine (closed symbols) which slightly facilitated the release of CGRP stimu- lated by heat (n = 7). The inset shows the results with and without morphine for every animal. *p < 0.05. not easily detected since the immediate pain of skin punc- ture is closely followed by the nerve conduction block. The only previously reported application of a high con- centration of morphine in a well-controlled setup pro- duced nociceptor activation, as observed in the present study. In this preparation, the superior spermatic nerve of dogs in vitro, morphine applied by superfusion at 1 – 310 μM generated action potentials in slowly conducting affer- ents; the activation was concentration-dependent and the combination of bradykinin and morphine resulted in sen- sitization [27]. In the present study the applied concentra- tions were higher, and 310 μM morphine caused sensitization but no overt activation. A higher expression of TRPA1 and TRPV1 in visceral afferents [28,29] might mary afferent neurons corresponds to the nociceptor pop- ulation [21,22]. Peripheral application of opioid receptor agonists (below 100 μM) or antagonists demonstrated their local efficacy but the relative contribution of periph- eral opioid receptors after systemic opioid administration is still unclear [23]. In animal experiments involving peripheral opioid injec- tions or topical morphine applications, pain-related behavior has not been reported [24-26]. Page 7 of 11 (page number not for citation purposes) Conclusion Morphine, at doses rarely used for pain therapy, is a nox- ious stimulus activating both TRPV1 and TRPA1 receptors. Morphine-induced calcium increase, indicative for nocic- eptive activation, was reduced in DRG neurons from TRPV1 and TRPA1 knockout animals. Moreover, TRPV1/ A1 double knockout mice were used for the first time to demonstrate by the lack of activation that both receptors together fully account for the nociceptive sensitivity to morphine. We suggest that also the sensitivity for several other algogenic compounds that still elicit pain and acti- vate neurons in the single knockouts will be lost in these double knockout animals. The observed morphine-induced increase in calcium lev- els is only a fraction of that elicited by KCl. This might explain why a lower percentage of neurons was activated by morphine in comparison to acrolein and capsaicin. Inhibition of calcium conductance by morphine is a pos- sible explanation. In fact, morphine and the mu-agonist DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) reduce the conduc- tivity of voltage gated calcium channels [30,31], which may be activated secondary to neuronal depolarization via TRPV1 and TRPA1. For TRPA1, both morphine and mustard oil seem to inhibit ion flux through TRPA1, a tail current was observed when the application was stopped. The apparent desensitization is likely due to an additional pore blocking effect that morphine at high concentration may exert on TRPA1. Mustard oil, the index agonist of TRPA1, also induced, yet smaller, tail currents followed by slow deactivation. Together with the very slow activation this may suggest that also mustard oil exerts a double action on TRPA1, activation of the receptor-channel and partial pore block. Receptors mediating opioid effects However, it seems likely that in these studies morphine concentra- tions at the nerve endings were below the activation threshold of nociceptors. A behavioural correlate for the activation of TRPV1 and TRPA1 by morphine injection is Page 7 of 11 (page number not for citation purposes) Page 7 of 11 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/17 Molecular Pain 2009, 5:17 contribute to the lower concentration of morphine required for activation of testicular nociceptors. sensitizes TRPV1 [37] and TRPA1 [38]. Therefore it was not surprising that morphine responses were sensitized by bradykinin. Sensitization to morphine by bradykinin and a reduced barrier function may explain the particular painfulness of the treated skin areas in radiodermatitis patients. For the reported painful sensations in patients or the acti- vation of primary afferents, a G-protein coupled opioid receptor effect seems unlikely in view of the action of the opioid antagonist naloxone. Since TRPV1 and TRPA1 are the principal detectors for painful chemical compounds, they were the primary targets of the investigation. Consid- ering pharmacological results and activation of neurons from knockout animals, both TRP receptors contributed approximately equal to the activation of DRG neurons caused by morphine. Only the knockout of both receptors eliminated the responsiveness. Cell culture Animals were killed in pure CO2 atmosphere. Dorsal root ganglia of all lumbar and the first two thoracic segments of the spinal column were excised and transferred in Dul- becco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, USA) solution containing 50 μg/ml gentamicin (Sigma). The ganglia were treated with 1 mg/ml colla- genase and 0.1 mg/ml protease for 30 minutes (both from Sigma) in TNB 100 solution supplemented by TNB 100 lipid-protein-complex, 100 μg/ml streptomycin and pen- icillin (all from Biochrom, Berlin, Germany) and 200 μg/ ml glutamine (Invitrogen, Carlsbad, USA) for 20 minutes at 37°C. After three wash steps, the suspension was tritu- rated in TNB solution using a fire-polished silicone-coated Pasteur pipette. The cells were plated on poly-D-lysine- coated (Sigma) cover slips and cultured in TNB medium overnight at 37°C in a humidified 5% CO2 atmosphere. Methods Animals Animal care and treatment were in accordance with the guidelines of the International Association for the Study of Pain [39]. Adult C57Bl/6, TRPV1-/- and TRPA1-/- as well as TRPV1-/-TRPA1-/- double knockout mice were used. Breeding pairs of TRPV1 and TRPA1 knockout mice were obtained from Dr. John Davis [40] and Dr. David Corey [41] and backcrossed to C57BL/6. Double knockout ani- mals were generated in our animal facility by cross-mating knockouts of both strains. All animals were genotyped by the previously reported primers. Stimulated CGRP release The neuropeptide calcitonin gene related peptide CGRP is expressed by a large subset of predominantly nociceptive primary afferent neurons and released upon activation of these neurons. The measurement of released CGRP is therefore widely used as an index for the activation of nociceptors. CGRP is expressed in a large proportion of neurons positive for TRPV1 or TRPA1, therefore the release of CGRP is a favorable readout for the activation of these neurons [32,33]. High concentrations of morphine reversibly stimulated the release of CGRP from the iso- lated skin. Furthermore, concentrations below the thresh- old of activation sensitized the CGRP release evoked by noxious heat and acrolein, activators of TRPV1 and TRPA1 receptors, respectively. Calcium imaging ll d Cells were stained by 5 μM fura-2 AM and 0.02% pluronic F-127 (both from Invitrogen) dissolved in the TNB medium for about 30 min in the incubator, followed by a short wash-out period to allow fura-2 AM ester hydrolysis. The cover slips were placed in a custom-made chamber and mounted on a Zeiss Axiovert inverse microscope with a 40× NeoFluar objective. Cells were continuously super- fused throughout the experiment with extracellular fluid (in mM: NaCl 145, KCl 5, CaCl2 1.25, MgCl2 1, Glucose 10, Hepes 10) at a flow rate of approximately 0.3 ml/min at room temperature. Cells were illuminated with a 75W xenon arc lamp and a monochromator alternating between 340 and 380 nm of wavelength (Photon Tech- nology International, New Jersey, USA). Images were acquired at 1 Hz with 200 μs exposure time using a CCD camera, controlled by Image Master software (PTI, Bir- mingham, USA). Fluorescence ratios were computed for regions of interest adapted to the neurons. All experimen- tal protocols were pre-programmed using a custom-made software that controls the valves of a 7-channel gravity- driven common-outlet superfusion system [42]. Enzyme-immuno assay for CGRP Immediately after the incubation, 100 μl of the solution were processed using a commercial CGRP enzyme immune assay kit (SPIbio, France). The details of this method were described previously [44]. The antibodies used are directed against human α/β-CGRP but are 100% cross-reactive against mouse and rat CGRP. The detection level is about 2 pg/ml. Samples of SIF and all stimulation solutions measured on the same enzyme immune assay 96-well plate served as controls. Data analysis The calcium influx elicited by a stimulus was quantified by evaluating the area under the curve (AUC) of the appli- cation period. Absolute increases in calcium concentra- tions were calculated based on Rmin and Rmax [45]. Compared to the foregoing reference period a mean increase of the intracellular calcium concentration of at least 15 nM throughout the application period was con- sidered as activation. At the end of all protocols a 10 s stimulus of KCl 60 mM was applied as control and nor- malization reference. Cell diameter was calculated as the mean of two orthogonal axes. Repeated measurements and independent groups in calcium imaging experiments were compared by ANOVA and HSD post-hoc test. CGRP release experiments were performed using both hindpaw skin flaps of the same animal and compared using the Wilcoxon matched pairs test. Statistical analysis was per- formed using Statistica 7 (Tulsa, OK, USA), sigmoidal dose-response curves were fitted by Origin 7.5 (North- hampton, MA, USA). Data are presented as mean ± SEM; p < 0.05 was considered significant. Patch Clamp Recordings In inflamed skin inflammatory mediators and their recep- tors are frequently overexpressed [34,35], among them particularly bradykinin and its receptors [36]. Bradykinin p g HEK293t cells were transfected with plasmids of hTRPV1 (John Davis) or mTRPA1 (Ardem Patapoutian) along with Page 8 of 11 (page number not for citation purposes) Page 8 of 11 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/17 Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 NaH2PO4 1.7, Na-gluconate 9.6, sucrose 7.6, glucose 5.6, CaCl2 1.5 and MgSO4 0.7 [43] equilibrated with carbogen (pH 7.4). The fixed skin flaps were placed in test tubes and mounted in a shaking bath of 32°C. After washing for 30 minutes, each experiment was composed of four consecu- tive 5 min incubation periods in test tubes filled with 0.8 ml SIF. In the first two periods basal CGRP release was determined, during the third period the preparation was chemically or thermally stimulated, the fourth period was for the post-stimulation control. One separate protocol provided two stimulations in incubation periods two and four. The contralateral hind paw skin of each animal was used as a matched-pairs control without conditioning chemicals. GFP as reporter plasmid using Polyfect (Qiagen, Hilden, Germany) according to the manufacturer's protocol. After incubation for one day, the cells were replated in 35 mm culture dishes and used for experiments within 1–2 days. GFP-expressing cells were identified by fluorescence. Recording electrodes were pulled from borosilicate glass tubes (GB150T-8P, Science Products, Hofheim, Germany) to give a resistance of 3.5 – 5.0 MΩ (P97, Sutter, Novato, CA). The external solution contained (in mM) 140 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 4 glucose, adjusted to pH 7.4. The internal solution contained (in mM) 140 KCl, 1.6 MgCl2, 2 EGTA and 10 HEPES and was adjusted to pH 7.4. Recordings were performed at room tempera- ture and cells were held at -60 mV. Membrane currents were acquired with an Axopatch 200B amplifier and pClamp 10 software (Molecular Devices, Sunnyvale, CA). Solutions were applied with a RSC-100 gravity-driven solution changer (Bio-Logic, Claix, France). All cells were first exposed to morphine, then to capsaicin or mustard oil. TRPA1 was sensitized by PLC activator m-3M3FBS (1 μM, Sigma). CGRP release C57/Bl6 mice of either sex with an average weight of 20 g (range 12–27 g) were used. The skin of both hind paws distal to the knee was subcutaneously excised. The skin flaps with an average weight of 105 mg (range 57–135 mg, n = 36) were fixed to acrylic rods by surgical threads with the corium exposed. During this procedure the skin flaps were constantly immersed in synthetic interstitial fluid (SIF, in mM: NaCl 107.8, KCl 3.5, NaHCO3 26.2, References g p 24. Khasar SG, Wang JF, Taiwo YO, Heller PH, Green PG, Levine JD: Mu- opioid agonist enhancement of prostaglandin-induced hyperalgesia in the rat: a G-protein beta gamma subunit- mediated effect? Neuroscience 1995, 67:189-195. 1. Sertuerner F: Darstellung der reinen Mohnsäure (Opium- säure) nebst einer chemischen Untersuchung des Opiums mit vorzüglicher Hinsicht auf einen darin neu entdeckten Stoff und die dahin gehörigen Bemerkungen. Journal der Phar- macie fuer Aerzte und Apotheker 1806:47-93. 1. Sertuerner F: Darstellung der reinen Mohnsäure (Opium- säure) nebst einer chemischen Untersuchung des Opiums mit vorzüglicher Hinsicht auf einen darin neu entdeckten Stoff und die dahin gehörigen Bemerkungen. Journal der Phar- macie fuer Aerzte und Apotheker 1806:47-93. 25. Yesilyurt O, Dogrul A, Gul H, Seyrek M, Kusmez O, Ozkan Y, Yildiz O: Topical cannabinoid enhances topical morphine antinoci- ception. Pain 2003, 105:303-308. f p 2. Benyamin R, Trescot AM, Datta S, Buenaventura R, Adlaka R, Sehgal N, Glaser SE, Vallejo R: Opioid complications and side effects. Pain Physician 2008, 11:S105-S120. p 26. Kolesnikov YA, Cristea M, Pasternak GW: Analgesic synergy between topical morphine and butamben in mice. Anesth Analg 2003, 97:1103-7. table. y 3. Chu LF, Angst MS, Clark D: Opioid-induced hyperalgesia in humans: molecular mechanisms and clinical considerations. Clin J Pain 2008, 24:479-496. 27. Kumazawa T, Mizumura K, Sato J, Minagawa M: Facilitatory effects of opioids on the discharges of visceral nociceptors. Brain Res 1989, 497:231-238. J 4. Yaksh TL, Harty GJ, Onofrio BM: High dose of spinal morphine produce a nonopiate receptor-mediated hyperesthesia: clin- ical and theoretic implications. Anesthesiology 1986, 64:590-597. 28. Nassenstein C, Kwong K, Taylor-Clark T, Kollarik M, Macglashan DM, Braun A, Undem BJ: Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs. J Physiol 2008, 586:1595-1604. p gy 5. Wilson GR, Reisfield GM: Morphine hyperalgesia: a case report. Am J Hosp Palliat Care 2003, 20:459-461. J p 6. De Conno F, Caraceni A, Martini C, Spoldi E, Salvetti M, Ventafridda V: Hyperalgesia and myoclonus with intrathecal infusion of high-dose morphine. Pain 1991, 47:337-339. 29. Taylor-Clark TE, McAlexander MA, Nassenstein C, Sheardown SA, Wilson S, Thornton J, Carr MJ, Undem BJ: Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal bronchopulmonary C-fibres by the endogenous autacoid 4- oxononenal. J Physiol 2008, 586:3447-3459. g p 7. References Casale TB, Bowman S, Kaliner M: Induction of human cutaneous mast cell degranulation by opiates and endogenous opioid peptides: evidence for opiate and nonopiate receptor partic- ipation. J Allergy Clin Immunol 1984, 73:775-781. J y 30. Schroeder JE, McCleskey EW: Inhibition of Ca2+ currents by a mu-opioid in a defined subset of rat sensory neurons. J Neu- rosci 1993, 13:867-873. p J gy 8. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997, 389:816-824. 31. Yang JC, Shan J, Ng KF, Pang P: Morphine and methadone have different effects on calcium channel currents in neuroblast- oma cells. Brain Res 2000, 870:199-203. p p y 9. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skin- ner K, Raumann BE, Basbaum AI, Julius D: The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998, 21:531-543. 32. Price TJ, Flores CM: Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, tran- sient receptor potential vanilloid subfamily type 1 immuno- reactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse. J Pain 2007, 8:263-272. 10. Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A: ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold tem- peratures. Cell 2003, 112:819-829. 33. Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, Noguchi K: Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/ c-fibers and colocalization with trk receptors. J Comp Neurol 2005, 493:596-606. 11. Garcia-Anoveros J, Nagata K: TRPA1. 2007:347-362. ff 12. Leffler A, Fischer MJ, Rehner D, Kienel S, Kistner K, Sauer SK, Gavva NR, Reeh PW, Nau C: The vanilloid receptor TRPV1 is acti- vated and sensitized by local anesthetics in rodent sensory neurons. J Clin Invest 2008, 118:763-776. 34. Ruzicka T, Simmet T, Peskar BA, Ring J: Skin levels of arachidonic acid-derived inflammatory mediators and histamine in atopic dermatitis and psoriasis. J Invest Dermatol 1986, 86:105-108. J 13. Carratu MR, Dubois JM, Mitolo-Chieppa D: Block of sodium cur- rent in myelinated nerve fibre with enkephalins. Neurophar- macology 1982, 21:619-623. 35. We like to thank Iwona Izydorczyk for excellent technical assistance. We like to thank Iwona Izydorczyk for excellent technical assistance. 23. 23. Stein C, Machelska H, Binder W, Schafer M: Peripheral opioid analgesia. Curr Opin Pharmacol 2001, 1:62-65. The authors declare that they have no competing interests. 19. Coggeshall RE, Zhou S, Carlton SM: Opioid receptors on periph- eral sensory axons. Brain Res 1997, 764:126-132. Authors' contributions y 20. Truong W, Cheng C, Xu QG, Li XQ, Zochodne DW: Mu opioid receptors and analgesia at the site of a peripheral nerve injury. Ann Neurol 2003, 53:366-375. ABF carried out the experiments and performed the statis- tical analysis. KM and PWR helped in experimental design and to draft the manuscript. MJMF designed the study, supervised all aspects and drafted the manuscript. All authors read and approved the final manuscript. 21. Poonyachoti S, Kulkarni-Narla A, Brown DR: Chemical coding of neurons expressing delta- and kappa-opioid receptor and type I vanilloid receptor immunoreactivities in the porcine ileum. Cell Tissue Res 2002, 307:23-33. 22. 22. Minami M, Maekawa K, Yabuuchi K, Satoh M: Double in situ hybridization study on coexistence of mu-, delta- and kappa- opioid receptor mRNAs with preprotachykinin A mRNA in the rat dorsal root ganglia. Brain Res Mol Brain Res 1995, 30:203-210. http://www.molecularpain.com/content/5/1/17 Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 http://www.molecularpain.com/content/5/1/17 Competing interests 18. Fields HL, Emson PC, Leigh BK, Gilbert RF, Iversen LL: Multiple opi- ate receptor sites on primary afferent fibres. Nature 1980, 284:351-353. p g The authors declare that they have no competing interests. Abbreviations CGRP: calcitonin gene-related peptide; DRG: dorsal root ganglion; TRPV1: transient receptor potential cation chan- nel, subfamily V, member 1; TRPA1: transient receptor potential cation channel, subfamily A, member 1; SIF: synthetic interstitial fluid Page 9 of 11 (page number not for citation purposes) Page 9 of 11 (page number not for citation purposes) References Reilly DM, Parslew R, Sharpe GR, Powell S, Green MR: Inflamma- tory mediators in normal, sensitive and diseased skin types. Acta Derm Venereol 2000, 80:171-174. 14. Frazier DT, Ohta M, Narahashi T: Nature of the morphine recep- tor present in the squid axon. Proc Soc Exp Biol Med 1973, 142:1209-1214. 36. Schremmer-Danninger E, Naidoo S, Neuhof C, Valeske K, Snyman C, Sander C, Bhoola KD, Neuhof H: Visualisation of tissue kal- likrein, kininogen and kinin receptors in human skin follow- ing trauma and in dermal diseases. Biol Chem 2004, 385:1069-1076. 15. Sakaue A, Honda M, Tanabe M, Ono H: Antinociceptive effects of sodium channel-blocking agents on acute pain in mice. J Phar- macol Sci 2004, 95:181-188. 16. Carratu' MR, Mitolo-Chieppa D: Inhibition of ionic currents in frog node of Ranvier treated with naloxone. Br J Pharmacol 1982, 77:115-119. 37. Premkumar LS, Ahern GP: Induction of vanilloid receptor chan- nel activity by protein kinase C. Nature 2000, 408:985-990. 17. Hymes SR, Strom EA, Fife C: Radiation dermatitis: clinical pres- entation, pathophysiology, and treatment 2006. J Am Acad Dermatol 2006, 54:28-46. 38. Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Ear- ley TJ, Patapoutian A: Noxious cold ion channel TRPA1 is acti- vated by pungent compounds and bradykinin. Neuron 2004, 41:849-857. Page 10 of 11 (page number not for citation purposes) Page 10 of 11 (page number not for citation purposes) Molecular Pain 2009, 5:17 http://www.molecularpain.com/content/5/1/17 39. Zimmermann M: Ethical guidelines for investigations of exper- imental pain in conscious animals. Pain 1983, 16:109-110. 40. Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL, Rogers DC, Bingham S, Randall A, Shear- down SA: Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000, 405:183-187. p g 41. Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang DS, Woolf CJ, Corey DP: TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 2006, 50:277-289. 42. Dittert I, Vlachova V, Knotkova H, Vitaskova Z, Vyklicky L, Kress M, Reeh PW: A technique for fast application of heated solutions of different composition to cultured neurones. J Neurosci Meth- ods 1998, 82:195-201. 43. Bretag AH: Synthetic interstitial fluid for isolated mammalian tissue. Life Sci 1969, 8:319-329. 44. References Averbeck B, Reeh PW: Interactions of inflammatory mediators stimulating release of calcitonin gene-related peptide, sub- stance P and prostaglandin E(2) from isolated rat skin. Neu- ropharmacology 2001, 40:416-423. p gy 45. Poenie M, Tsien R: Fura-2: a powerful new tool for measuring and imaging [Ca2+]i in single cells. Prog Clin Biol Res 1986, 210:53-56. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 11 of 11 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge
https://openalex.org/W3036035957
https://figshare.com/articles/journal_contribution/Sheep_performance_on_perennial_ryegrass_cultivars_differing_in_concentration_of_water_soluble_carbohydrate/22748297/1/files/40425749.pdf
English
null
Sheep performance on perennial ryegrass cultivars differing in concentration of water-soluble carbohydrate
Journal of New Zealand grasslands
2,015
cc-by
5,978
Abstract value, and conversion to animal product. To date, New Zealand studies on HSGs have focussed on measuring the effects on animal productivity and/or environmental impact at the level of the individual sheep (Proctor et al. 2015) or cow (Cosgrove et al. 2007). While studies conducted in the United Kingdom have measured responses to higher levels of WSC in terms of sheep production per hectare (Munro et al. 1992; Lee et al. 2001; Marley et al. 2007), the authors are not aware of studies in New Zealand conducted on the same basis. High-sugar perennial ryegrass cultivars (HSG) selected for higher concentrations of water-soluble carbohydrate may enhance animal production and reduce emissions of methane and nitrogen. Assessing the effects on economic output and environmental footprint is most robust when related to production per unit of land. Average daily gain (ADG) and liveweight gain per hectare (LWG/ha) of sheep grazing a high-sugar perennial ryegrass cultivar, a diploid perennial ryegrass and a tetraploid perennial ryegrass were compared during measurement periods conducted in spring (84 days duration), autumn (99 days) and late spring- summer (160 days). Continuous variable stocking was used, and stocking rate adjusted to maintain a target sward surface height of 6 cm. Average daily gain was higher (P=0.003) on the HSG than on either control in late spring-summer and higher on the tetraploid control than on the HSG or the diploid control in autumn (P=0.04), but the higher ADGs did not translate to significantly higher LWG/ha. These results can inform farmers on cultivar choice and support analysis of methane and nitrogen emissions on an intensity basis for inventory and regulatory purposes. Recent data from the United Kingdom (DEFRA 2010) and New Zealand (Jonker et al. 2014) suggests that the high-sugar trait may also reduce methane emissions. In order to scale-up the effects of grass cultivar on methane emissions measured per individual animal (g CH4/head/day) to an intensity basis (i.e. g CH4 per hectare of land, or per unit of animal production) for inventory or regulatory purposes, it is necessary to measure all aspects of animal performance in conjunction with measurements of methane emissions. This paper reports a study conducted to measure the effects of grass cultivar on performance per animal and per hectare under New Zealand conditions, and to support the interpretation of associated measurements on methane emissions. 123 123 Sheep performance on perennial ryegrass cultivars differing in concentration of water-soluble carbohydrate G.P. COSGROVE, P.S. TAYLOR and A. JONKER AgResearch Grasslands, PB 11008, Palmerston North 4442 gerald.cosgrove@agresearch.co.nz ISSN 2463-2872 (Print) ISSN 2463-2880 (Online) Abstract The hypothesis tested was that ryegrasses selected for a higher concentration of WSC would increase sheep average daily gain (ADG) and liveweight gain per hectare (LWG/ha). Key words: water-soluble carbohydrate, perennial ryegrasses, high-sugar ryegrass, average daily gain, liveweight gain Measurements S d Pasture: Sward surface heights were recorded by taking 25 readings per plot twice weekly using a Hill Farming Research Organisation sward stick (see Frame (1993) for description of methodology). To collect representative samples of the herbage offered, three (four in late spring-summer 2014/15) grazing exclosure cages (1.0 × 0.5 m) were randomly placed in each plot and after 2 weeks herbage accumulation in spring and 3–4 weeks in autumn (and one interval of 5 weeks in summer 2015) a sample was cut to the mean SSH of that plot recorded when the cage was placed. After each sampling each cage was relocated to a new site within the plot (no pre-trimming). The sample from each cage was composited into a single sample for the plot and immediately frozen in liquid nitrogen and stored frozen. Pasture: Sward surface heights were recorded by taking 25 readings per plot twice weekly using a Hill Farming Research Organisation sward stick (see Frame (1993) for description of methodology). To collect representative samples of the herbage offered, three (four in late spring-summer 2014/15) grazing exclosure cages (1.0 × 0.5 m) were randomly placed in each plot and after 2 weeks herbage accumulation in spring and 3–4 weeks in autumn (and one interval of 5 weeks in summer 2015) a sample was cut to the mean SSH of that plot recorded when the cage was placed. After each sampling each cage was relocated to a new site within the plot (no pre-trimming). The sample from each cage was composited into a single sample for the plot and immediately frozen in liquid nitrogen and stored frozen. Samples were subsequently freeze dried, ground to pass a 1 mm sieve and analysed using near-infrared reflectance spectroscopy (FeedTECH, AgResearch Grasslands, Palmerston North) to predict concentrations of water-soluble carbohydrates (calibrated using the anthrone method (Thomas 1977)), crude protein (CP), neutral detergent fibre (NDF) and organic matter digestibility. These samples were collected six times during spring 2013, five times during autumn 2014, and seven times during late spring-summer 2014/15. Samples from each time point were analysed separately and the season mean concentrations presented. Pastures were fertilised after sowing with 250 kg/ha of Cropmaster 15 (Ravensdown Ltd.; 15% N, 10% P, 10% K, 8% S) in May 2013, and with 300 kg/ha superphosphate (9% P, 11% S) in July 2013, as part of farm fertiliser policy for Aorangi. Site High sugar ryegrasses (HSGs), bred for elevated concentrations of water-soluble carbohydrate (WSC; sugar) can increase sheep liveweight gain (Munro et al. 1992; Lee et al. 2001; Proctor et al. 2015), milk production (Cosgrove et al. 2007; Miller et al. 2001b) and improve the utilisation of dietary protein, thereby reducing nitrogen excretion (Miller et al. 2001a; Pacheco et al. 2007, 2009). However, there are also studies which show no animal response (Cosgrove et al. 2007; Marley et al. 2007; Tas et al. 2006; Taweel et al. 2006 ), and recent reviews highlight the uncertain efficacy of elevated WSC (Edwards et al. 2007; Parsons et al. 2011). An important measure of the efficacy of this trait, especially economic performance, is animal production per unit area of land. This takes into account pasture production, dry matter intake and nutritive This grazing trial with sheep was conducted at the AgResearch Aorangi Research Farm, Manawatu, on Kairanga and Te Arakura recent alluvial soils (Rijkse & Daly 1972). Measurement periods were conducted during spring 2013 (26 September–18 December; 84 days), autumn and early winter 2014 (22 April–30 July; 99 days; henceforth referred to as autumn) and late-spring, summer and early-autumn 2014/15 (25 November–4 May; 160 days; henceforth referred to as late spring-summer). The latter period was interrupted by drought in January (11 mm rainfall compared with the 20-year mean of 65 mm), and all sheep were removed from plots for 2 weeks in February because sward surface height (SSH) declined below the target height of 6 cm (see description under Measurements below). Following ISSN 2463-2872 (Print) ISSN 2463-2880 (Online) Journal of New Zealand Grasslands 77: 124 (2015) 123-130 summer 2014/15, on which measurements of ADG were based. In addition, a variable number of “put- and-take” (P&T) sheep were used. Decisions to add or remove P&T animals were based on changes in SSH from the target of 6 cm. Liveweight gain/ha for the grazing period was calculated from the total number of grazing days (total number of sheep each day × number of days) × ADG of the tester sheep. For late spring- summer 2014/15, 15-month-old heifers (346 ± 13.4 kg LW) were used as P&T grazers in addition to sheep to give greater capacity to control SSH during the period of high pasture accumulation rates when ryegrasses were flowering. Site Each heifer was considered equivalent to 5 lambs for calculating grazing days and LWG per ha, based on the ratio of stock units (s.u.) for each species (heifers 4.5 s.u. and lambs 0.9 s.u.; Woodford & Nicol 2004). Overall, heifers accounted for 15% of total grazing days and lambs 85%. This proportion was similar for each cultivar and replicate, so the assumption of 1 heifer = 5 lambs was considered unlikely to have biased the calculation of LWG/ha. removal, sheep were held for 1 week as a single group on non-trial grass pasture, and then separated again into their respective treatment groups (replicate groups within treatment still combined) and given 1 week on spare plots for readaptation to their assigned cultivar before being returned to treatment plots. Measurements S d A soil test taken in October 2013 indicated pH 5.6 and Olsen-P of 31. Lime was applied at 2.5 t/ha in February 2014 to correct the low pH. Nitrogen fertiliser was applied at 75 kg urea/ha 2–3 times during each grazing period. Between grazing periods, plots were stocked with sheep and occasionally cattle, to maintain the continuously-stocked sward state. Experimental treatments and design Three cultivars of perennial ryegrass, each containing AR1 endophyte, were compared: a high-sugar diploid perennial ryegrass (cv. ‘Abermagic’; flowering date +15 days compared with ‘Nui’), a control diploid perennial ryegrass (cv. ‘Alto’; +14 days) and a control tetraploid perennial ryegrass (cv. ‘Base’, +25 days). The design consisted of a randomised complete block, with four replicate blocks. Across blocks, plots ranged in size from 0.56 to 0.67 ha, but within blocks the plots of each cultivar were identical in size. Each cultivar was sown in monoculture into a cultivated seedbed in autumn 2013. Diploid cultivars were sown at 20 kg seed/ha and the tetraploid at 28 kg seed/ha. Animals For measurements in spring 2013, 120 one-year-old ewes were used (42.0 ± 3.33 kg initial liveweight (LW)); in autumn 2014 120 6-month-old cryptorchid lambs (30.7 ± 1.37 kg LW) and in late spring-summer 2014/15 180 3-month-old newly weaned cryptorchid lambs were used (28.7 ± 1.53 kg LW). For each season sheep were weighed and allocated to 12 groups (n=10 for spring and autumn and n=15 for late spring-summer), with each group having the same mean and range in LW. In each season an additional pool of cohorts from the same flock was identified, from which put-and-take grazers could be drawn (described below). These experiments were conducted under AgResearch Grasslands Animal Ethics Committee approvals #13004 and #13385. y q g Samples were subsequently freeze dried, ground to pass a 1 mm sieve and analysed using near-infrared reflectance spectroscopy (FeedTECH, AgResearch Grasslands, Palmerston North) to predict concentrations of water-soluble carbohydrates (calibrated using the anthrone method (Thomas 1977)), crude protein (CP), neutral detergent fibre (NDF) and organic matter digestibility. These samples were collected six times during spring 2013, five times during autumn 2014, and seven times during late spring-summer 2014/15. Samples from each time point were analysed separately and the season mean concentrations presented. Trial stocking management Animal: After 1 week of adaptation to grazing the assigned cultivar the sheep were weighed again directly off pasture (full liveweight (LW)). At the end of the grazing period full LW was again recorded. Average A continuous, variable stocking method was used. For spring 2013 and autumn 2014 each plot was stocked with 10 “tester” sheep, and 15 sheep for late spring- formance on perennial ryegrass cultivars differing in concentration... (G.P. Cosgrove, P.S. Taylor and A. Jonker) 125 Figure 1 The sward surface height of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar, a diploid control cultivar and a tetraploid control cultivar, under continuous variable stocking by sheep during measurement periods in spring 2013 (a), autumn 2014 (b) and late spring-summer 2014/15 (c). daily gain was calculated from liveweight gain over the period excluding adaptation, and for late spring- summer 2014/15 included the 2-week period when drought forced the removal of lambs from plots. Data analysis h i The experimental unit was the plot and the group of animals grazing on it. Data was analysed for each season separately. Cultivar effects for pasture and animal variables (2 d.f.) were compared using analysis of variance, with the cultivar × block interaction (6 d.f.) used as error term after removing block effects (3 d.f.). Comparison of means is based on Fisher’s protected least significant difference at P<0.05. Discussion Average daily gain was influenced by ryegrass cultivar in two of the three periods of measurement in this study but in one case sheep grazing the tetraploid control had the highest ADG and in the other case sheep grazing the HSG had the highest ADG. These effects did not translate to significantly higher LWG/ha. Some studies conducted in the United Kingdom with sheep similarly continuously stocked on monoculture swards maintained at a SSH of 6–7 cm (Munro et al. 1992; Ryegrass chemical composition In spring 2013 the cultivars did not differ significantly in concentrations of WSC or CP, but the HSG and the tetraploid control were lower in NDF than the diploid control (Table 1). In autumn 2014 the HSG had a significantly higher concentration of WSC (P=0.009) and significantly lower concentration of CP (P=0.02) than either of the control cultivars and had a lower concentration of NDF (P=0.001) compared with the diploid control. In late spring-summer 2014/2015, the HSG had a higher concentration of WSC (P=0.017) and lower concentration of NDF (P=0.0004), than either of the control cultivars. such that LWG/ha did not differ among cultivars (mean 335 kg/ha). In late spring-summer 2014/15, ADG was higher (P=0.003) for sheep grazing the HSG (133 g/ day) than for either of the control cultivars (mean of 119 g/day), but this did not translate to significantly higher LWG/ha. Sward surface height During spring 2013 each cultivar was maintained at a similar SSH, although SSH was marginally below the target of 6 cm during October and increased above the target from early November, and particularly so during December (Figure 1a). In autumn 2014, the tetraploid control had a lower SSH than the other two cultivars during May, but from then on all three cultivars were above the target until the termination in late July (Figure 1b). For late spring-summer 2014/2015, cultivars were similar in SSH until early February, although SSH had declined to 4 cm when lambs were temporarily removed (Figure 1c). Plots recovered to 7 cm SSH during spelling, but the tetraploid control again had lower SSH than the other cultivars during March, yet recovered to be similar to the other cultivars by the end of April. ure 1 The sward surface height of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar, a diploid control cultivar and a tetraploid control cultivar, under continuous variable stocking by sheep during measurement periods in spring 2013 (a), autumn 2014 (b) and late spring-summer 2014/15 (c). The sward surface height of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar, a diploid control cultivar and a tetraploid control cultivar, under continuous variable stocking by sheep during measurement periods in spring 2013 (a), autumn 2014 (b) and late spring-summer 2014/15 (c). Figure 1 Animal performancei There were no significant differences among cultivars in sheep ADG (mean of 164 g/hd/day) or in LWG/ha (427 kg LWG/ha) in spring 2013 (Table 2). In autumn 2014, lambs grazing the tetraploid control had higher ADG (206 g/day) compared with those grazing the HSG or diploid control (179 g/day; P=0.04), but this was offset by lower stocking rate (data not shown), Journal of New Zealand Grasslands 77: 126 123-130 (2015) Lee et al. 2001) have reported higher ADG and LWG/ ha on a high-WSC cultivar compared with a control, but others have recorded no difference (Marley et al. 2007). Munro et al. (1992) reported 19% higher lamb LWG/ha from the high-WSC cultivar which they attributed to higher organic matter digestibility because herbage production was similar for each cultivar (concentrations of WSC were not reported). The study by Lee et al. (2001) recorded ADG of lambs up to 25% higher and LWG/ha up to 35% higher when grazing the experimental line high-WSC ryegrass in two out of three measurement periods, which they attributed to a combination of higher WSC and lower structural fibre in the high-WSC ryegrass. Proctor et al. (2015) also reported higher ADG from a high-WSC compared with a control cultivar when each was growing in mixture with white clover, but did not report the proportions of each species in the mixture. It is possible that differences between the high-WSC and the control pastures in the proportions of ryegrass and white clover may have influenced the cultivar comparison, as was demonstrated in the study by Munro et al. (1992). In the study by Lee et al. (2001) the differences between the high-WSC ryegrass and control cultivar in concentrations of WSC (up to 54 g WSC/kg DM higher in the high-WSC grass) and NDF (up to 76 g NDF/kg DM lower in the high- Table 1 The effects of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar, a diploid control cultivar and a tetraploid control cultivar, on the mean concentrations (g/kg DM) of WSC, crude protein (CP) and neutral detergent fibre (NDF) during measurements periods in spring 2013, autumn 2014 and late spring-summer 2014/15. eans within row followed by different letters differ significantly P<0.05 owed by different letters differ significantly P<0.05 Animal performancei Choosing cultivars selected for high WSC, or tetraploid cultivars, which also achieve higher organic matter digestibility, regardless of whether that results from a greater concentration of WSC or from a lower concentration of NDF, can be expected to result WSC grass) were approximately double the magnitude of differences in the study reported here. In the study by Marley et al. (2007) where no animal response was noted, the difference between cultivars in concentration of WSC was only 11 g WSC/kg DM. While expression of higher WSC in the ryegrass selected for high-WSC largely determines the magnitude of this difference, the choice of control cultivar(s) may also influence the difference and whether or not a significant animal response is observed. The study reported here and those by Lee et al. (2001) and Proctor et al. (2015) each used a different high-WSC cultivar and a different control cultivar. Cultivars used as controls, while not selectively bred on the basis of WSC, can differ among themselves in the concentrations of WSC (Easton et al. 2009). Cultivar choice, as well as environmental effects on WSC concentrations and expression of the high- WSC trait in an HSG (Cosgrove et al. 2014; Parsons et al. 2004), should all be considered in comparisons among studies. Sward physical conditions were controlled as tightly as practicable to minimise limitations to dry matter intake (DMI), with some possible exceptions as discussed below. Of all sward characteristics SSH has a predominant influence on DMI by grazing animals. Maintaining swards at or above 6 cm SSH is considered to be non-limiting to DMI (Penning et al. 1991) and maintaining this target height was the primary measure for ensuring uniformity of feeding conditions across cultivars and across seasons. There were occasions when SSH was lower than the target when adjustments to stocking rate were not fast enough or great enough, and so possibly limiting to DMI. The DMI of sheep grazing the tetraploid control may have been restricted in autumn 2014 when the SSH was below 6 cm for approximately 30 days of the 99 day duration. Even so, sheep are remarkably flexible in their ingestive behaviour (e.g., biting and chewing behaviour) and the time they spend grazing each day, and restrictions in say bite mass because of low SSH can be compensated by increases in the time spent grazing to satisfy their DMI requirement. Animal performancei Cosgrove, P.S. Taylor and A. Jonker) the changes in concentrations of other constituents. For example, if a greater concentration of WSC is offset by a lower concentration of structural carbohydrates this can lead to higher DMI, and so greater ADG (Lee et al. 2001), and if crude protein is low, the greater concentration of WSC can improve microbial protein capture in the rumen and so improve overall protein nutrition (Kingston-Smith & Theodorou 2000). The concentration of WSC was higher in the HSG than in the diploid control by 14–31 g WSC/kg DM, a difference comparable with some New Zealand studies conducted under cutting (Hume et al. 2010) or sheep grazing (Parsons et al. 2004), but lower than the additional 35– 47 g WSC/kg DM in rotationally grazed dairy pastures at four sites differing in latitude (Cosgrove et al. 2014). While cultivars did not differ significantly in WSC (or CP) in spring 2013 (a possible explanation for this is discussed below), the significantly lower concentration of NDF in the high-WSC ryegrass is consistent with the expected difference in chemical composition of that compared with a control, and as the concentration of one constituent increases the concentration of others inevitably decreases (Cosgrove et al. 2009; Rasmussen et al. 2009). Within season there did not appear to be a consistent relationship between a greater concentration of WSC and greater ADG. However, the associated changes in NDF and CP may also have influenced the observed ADG response, as modelling studies have suggested for nitrogen emissions and milk yield (Ellis et al. 2011). In each season the concentration of CP in each of the three cultivars was above the recommended minimum level for growing sheep (National Research Council 2007), and so ADG would be more likely to have been affected by differences in WSC and NDF than by differences in CP. Given the uncertain response to the aggregate effect of an increase in the concentration of WSC and a decrease in NDF, digestibility may be a simpler explanatory variable for assessing overall trends. Across cultivars and seasons, there was a clear, positive relationship between ADG and organic matter digestibility (R2=0.68; Figure 2), although even this does not fully account for the observed ADGs. Overall, ADG increased by 0.5 g/ day for each 1 g/kg DM increase in organic matter digestibility. Animal performancei Season Item Ryegrass cultivar P0.05 LSD0.05 High Diploid Tetraploid WSC control control Spring 2013 WSC 196 182 178 0.71 29.4 CP 216 213 225 0.68 33.3 NDF 434b1 458a 440b 0.02 15.0 Autumn 2014 WSC 186a 155b 162b 0.009 10.5 CP 252b 267a 274a 0.02 13.7 NDF 416b 439a 416b 0.001 14.8 Late spring- WSC 251a 221b 213b 0.017 24.3 summer 2014/15 CP 175 177 189 0.100 14.2 NDF 462c 497a 485b 0.0004 10.1 1 Means within row followed by different letters differ significantly P<0.05 Table 1 The effects of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar, a diploid control cultivar and a tetraploid control cultivar, on the mean concentrations (g/kg DM) of WSC, crude protein (CP) and neutral detergent fibre (NDF) during measurements periods in spring 2013, autumn 2014 and late spring-summer 2014/15. Table 2 The effects of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar, a diploid control cultivar and a tetraploid control cultivar, on average daily gain (ADG; g/day) and liveweight gain per hectare (LWG/ha; kg/ha) of sheep during measurements periods in spring 2013, autumn 2014 and late spring-summer 2014/15. Table 2 The effects of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar, a diploid control cultivar and a tetraploid control cultivar, on average daily gain (ADG; g/day) and liveweight gain per hectare (LWG/ha; kg/ha) of sheep during measurements periods in spring 2013, autumn 2014 and late spring-summer 2014/15. Season Item Ryegrass cultivar P0.05 LSD0.05 High Diploid Tetraploid WSC control control Spring 2013 ADG 170 158 164 0.62 37.3 LWG/ha 478 381 421 0.17 108.3 Autumn 2014 ADG 179b1 179b 206a 0.04 22.8 LWG/ha 329 339 337 0.89 55.2 Late spring- ADG 133a 120b 118b 0.003 6.7 summer 2014/15 LWG/ha 500 434 446 0.28 97.6 1 Means within row followed by different letters differ significantly P<0.05 rformance on perennial ryegrass cultivars differing in concentration... (G.P. Cosgrove, P.S. Taylor and A. Jonker) 127 127 the changes in concentrations of other constituents. For example, if a greater concentration of WSC is offset by a lower concentration of structural carbohydrates this can lead to higher DMI, and so greater ADG (Lee et al. 2001), and if crude protein is low, the greater concentration of WSC can improve microbial protein capture in the rumen and so improve overall protein nutrition (Kingston-Smith & Theodorou 2000). Animal performancei The concentration of WSC was higher in the HSG than in the diploid control by 14–31 g WSC/kg DM, a difference comparable with some New Zealand studies conducted under cutting (Hume et al. 2010) or sheep grazing (Parsons et al. 2004), but lower than the additional 35– 47 g WSC/kg DM in rotationally grazed dairy pastures at four sites differing in latitude (Cosgrove et al. 2014). While cultivars did not differ significantly in WSC (or CP) in spring 2013 (a possible explanation for this is discussed below), the significantly lower concentration of NDF in the high-WSC ryegrass is consistent with the expected difference in chemical composition of that compared with a control, and as the concentration of one constituent increases the concentration of others inevitably decreases (Cosgrove et al. 2009; Rasmussen et al. 2009). Within season there did not appear to be a consistent relationship between a greater concentration of WSC and greater ADG. However, the associated changes in NDF and CP may also have influenced the observed ADG response, as modelling studies have suggested for nitrogen emissions and milk yield (Ellis et al. 2011). In each season the concentration of CP in each of the three cultivars was above the recommended minimum level for growing sheep (National Research Council 2007), and so ADG would be more likely to have been affected by differences in WSC and NDF than by differences in CP. Given the uncertain response to the aggregate effect of an increase in the concentration of WSC and a decrease in NDF, digestibility may be a simpler explanatory variable for assessing overall trends. Across cultivars and seasons, there was a clear, positive relationship between ADG and organic matter digestibility (R2=0.68; Figure 2), although even this does not fully account for the observed ADGs. Overall, ADG increased by 0.5 g/ day for each 1 g/kg DM increase in organic matter digestibility. Choosing cultivars selected for high WSC, or tetraploid cultivars, which also achieve higher organic matter digestibility, regardless of whether that results from a greater concentration of WSC or from a lower concentration of NDF, can be expected to result in higher daily gains. Other factors such as seasonal growing conditions that result in favourable chemical composition and higher organic matter digestibility will also increase daily gains. Higher WSC specifically, may be important when the herbage CP concentration is low because under those conditions the additional entration... (G.P. Animal performancei Exceeding the target SSH is unlikely to affect DMI in the short-term (in the longer-term sward quality would deteriorate), but it indicates stocking rate was lower than the swards could support and so the potential LWG/ha may have been underestimated. In this latter case when SSH exceeded the target all cultivars were similar in height and so unlikely to have biased the comparison among cultivars in LWG/ha. Comparisons among cultivars in nutritive value, expressed through ADG, and herbage accumulation rates, expressed through LWG/ha, were the experimental variables of primary interest in this study. A greater concentration of WSC in herbage may increase nutritive value but this effect is influenced by Journal of New Zealand Grasslands 77: (2015) 128 123-130 (2015) Figure 2 The relationship between organic matter digestibility of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar (circles), a diploid control cultivar (triangles) and a tetraploid control cultivar (squares) and average daily gains of sheep grazing those cultivars during measurement periods in spring 2013 (grey symbols), autumn 2014 (solid symbols) and late spring-summer 2014/15 (open symbols). Figure 2 The relationship between organic matter digestibility of three cultivars of perennial ryegrass, a diploid high water-soluble carbohydrate (WSC) cultivar (circles), a diploid control cultivar (triangles) and a tetraploid control cultivar (squares) and average daily gains of sheep grazing those cultivars during measurement periods in spring 2013 (grey symbols), autumn 2014 (solid symbols) and late spring-summer 2014/15 (open symbols). ryegrasses. More generally, they can be used by farmers to inform cultivar selection in lamb grazing systems. ACKNOWLEDGEMENTS This project was funded from the Sustainable Land Management and Climate Change Fund administered by the Ministry for Primary Industries. Thanks to Steve Lees, Richard Templeton and Derek Birch of the Aorangi Research Farm for managing the stocking rate adjustments and animal measurements, to Fiona Smith and Tara Hancock for assisting with pasture measurements, and to David Pacheco for discussions on data interpretation. REFERENCES Figure 2 Cosgrove, G.P.; Burke, J.L.; Death, A.F.; Hickey, M.J.; Pacheco, D.; Lane, G.A. 2007. Ryegrasses with increased water-soluble carbohydrate: evaluating the potential for grazing dairy cows in New Zealand. Proceedings of the New Zealand Grassland Association 69: 179-185. Cosgrove, G.P.; Koolaard, J.; Luo, D.; Burke, J.L.; Pacheco, D. 2009. The composition of high sugar ryegrasses. Proceedings of the New Zealand Grassland Association 71: 187-193. WSC improves ruminal protein utilisation and so the availability of protein for liveweight gain. Cosgrove, G.P.; Mapp, N.R.; Taylor, P.S.; Harvey, B.M.; Knowler, K.J. 2014. The chemical composition of high-sugar and control ryegrasses in grazed pastures at different latitudes throughout New Zealand. Proceedings of the New Zealand Grassland Association 76: 169-175. For spring 2013, when the difference among cultivars in WSC or CP was not significant, variability among replicate blocks was high, particularly the variability among the six sampling dates underlying the season mean for cultivars within one replicate. There is no clear reason for this, but the replicate in question had been spelled from grazing during August to accumulate herbage of each cultivar tall enough to mechanically harvest in September for a related indoor feeding trial (Jonker et al. 2014). It is possible that this management practice affected herbage chemical composition as the swards recovered from severe defoliation at harvest and progressively readjusted to a short, leafy, continuously stocked state. DEFRA. 2010. Ruminant nutrition regimes to reduce methane and nitrogen emissions. Final Report AC0209, Department for Environment Food & Rural Affairs. 37 pp. Easton, H.S.; Stewart, A.V.; Lyons, T.B.; Parris, M.; Charrier, S. 2009. Soluble carbohydrate content of ryegrass cultivars. Proceedings of the New Zealand Grassland Association 71: 161-166. Edwards, G.R.; Parsons, A.J.; Rasmussen, S. 2007. High sugar ryegrasses for dairy systems. pp. 307-334. In: Meeting the challenges for pasture-based dairying. Proceedings of the 3rd Dairy Science Symposium. Eds. Chapman, D.F.; Clark, D.A.; Macmillan, K.L.; Nation, D.P. 18-20 September 2007, University of Melbourne, Victoria, Australia. Published by the National Dairy Alliance. Conclusions The average daily gain was highest for lambs grazing the HSG in one measurement period, consistent with the hypothesised effect of a greater concentration of WSC. However, the highest ADG for the tetraploid control in another measurement period could not be explained on this basis alone. There was a positive relationship of ADG with organic matter digestibility and factors which increase organic matter digestibility, which includes cultivar attributes such as high WSC and tetraploidy, will enhance ADG. Cultivar effects resulting in higher ADG did not translate to higher LWG/ha. Specifically, these results will inform the assessment of methane emissions on an intensity basis to determine the mitigation potential of high-sugar Ellis, J.L.; Dijkstra, J.; Bannik, A.; Parsons, A.J.; Rasmussen, S.; Edwards, G.R. 2011. The effect of high sugar grass on predicted nitrogen emission and milk yield simulated using a dynamic model. Journal of Dairy Science 94: 3105-3118. Frame, J. 1993. Herbage mass. Chapter 3. In: Sward measurement handbook, 2nd Edition. Eds. Davies, A.; Baker, R.D.; Grant, S. A.; Laidlaw, A.S. British Grassland Society, University of Reading, UK. mance on perennial ryegrass cultivars differing in concentration... (G.P. Cosgrove, P.S. Taylor and A. Jonker) Sheep performance on perennial ryegrass cultivars differing in concentration... (G.P. Cosgrove, P.S. Taylor 129 dairying. Proceedings of the 3rd Dairy Science Symposium. Eds. Chapman, D.F.; Clark, D.A.; Macmillan, K.L.; Nation, D.P. 18-20 September 2007, University of Melbourne, Victoria, Australia. Published by the National Dairy Alliance. Hume, D.E.; Hickey, M.J.; Lyons, T.B.; Baird, D.B. 2010. Agronomic performance and water soluble carbohydrate expression of selected ryegrasses at two locations in New Zealand. New Zealand Journal of Agricultural Research 53: 37-57. Pacheco, D.; Lowe, K.; Burke, J.L.; Cosgrove, G.P. 2009. Urinary nitrogen excretion from cows at different stage of lactation grazing different ryegrass cultivars during spring or autumn. Proceedings of the New Zealand Society of Animal Production 69: 196- 200. Jonker, A.J.; Molano, G.; Sandoval, E.; Taylor, P.S.; Antwi, C.; Cosgrove, G.P. 2014. Methane emissions by sheep offered high sugar or conventional perennial ryegrass at two allowances. Proceedings of the New Zealand Society of Animal Production 74: 145-147. Kingston-Smith, A.H.; Theodorou, M.K. 2000. Post-ingestion metabolism of fresh forage. New Phytologist 148: 37-55. Parsons, A.J.; Rasmussen, S.; Xue, H.; Newman, J.A.; Anderson, C.B.; Cosgrove, G.P. 2004. Some ‘high sugar grasses’ don’t like it hot. Proceedings of the New Zealand Grassland Association 66: 265-271. Conclusions Lee, M.R.F.; Jones, E.L.; Moorby, J.M.; Humphreys, M.O; Theodorou, M.K.; MacRae, J.C.; Scollan, N.D. 2001. Production response from lambs grazed on Lolium perenne selected for an elevated water soluble carbohydrate concentration. Animal Research 50: 441-449. Parsons, A.J.; Rowarth, J.S.; Rasmussen, S. 2011. High-sugar grasses. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 6: 1-12. Penning, P.D. Parsons, A.J.; Orr, R.J.; Treacher, T.T. 1991. Intake and behaviour responses by sheep to changes in sward characteristics under continuous stocking. Grass and Forage Science 46: 15-28. Marley, C.L.; Fraser, M.D.; Fisher, W.J.; Forbes, A.B.; Jones, R.; Moorby, J.M.; MacRae, J.C.; Theodorou, M.K. 2007. Effects of continuous or rotational grazing of two perennial ryegrass varieties on the chemical composition of the herbage and the performance of finishing lambs. Grass and Forage Science 62: 255-264. Proctor, L.E.; Craig, H.J.B.; Mclean, N.J.; Fennessy, P.F.; Chuah, J.; Campbell, A.W. 2015. The effect of grazing high sugar grass on lamb performance. Proceedings of the New Zealand Society of Animal Production 75: 235-238. Miller, L.A.; Baker, D.H.; Theodorou, M.K.; MacRae, J.C.; Humphreys, M.O.; Scollan, N.D.; Moorby, J.M. 2001a. Efficiency of nitrogen use in dairy cows grazing ryegrass with different water soluble carbohydrate concentrations. pp. 377-378. In: Proceedings XIX International Grasslands Congress. Sao Paulo, Brazil. Eds. Gomide, J.A.; Mattos, W.R.M.; da Silva, S.C. Rasmussen, S.; Parsons, A.J.; Xue, H.; Newman, J.A. 2009. High sugar grasses: harnessing the benefit of new cultivars through growth management. Proceedings of the New Zealand Grassland Association 71: 167-175. Rijkse, W.C.; Daly. B.K. 1972. Soils of the Aorangi experimental farm. New Zealand Journal of Agricultural Research 15: 117-136. Miller, L.A.; Moorby, J.M.; Davies, D.R.; Humphreys, M.O.; Scollan, N.D.; MacRae, J.C.; Theodorou, M.K. 2001b. Increased concentration of water-soluble carbohydrate in perennial ryegrass (Lolium perenne L.): milk production from late-lactation dairy cows. Grass and Forage Science 56: 383-394. Tas, B.M.; Taweel, H.Z.; Smit, H.J.; Elgersma, A.; Dijkstra, J.; Tamminga, S. 2006. Effects of perennial ryegrass cultivars on milk yield and nitrogen utilisation in grazing dairy cows. Journal of Dairy Science 89: 3494-3500. Munro, J.M.M.; Davies, D.A.; Evans, W.B.; Scurlock, R.V. 1992. Animal production evaluation of herbage varieties. 1. Comparison of Aurora with Frances, Talbot and Melle perennial ryegrasses when grown alone and with clover. Grass and Forage Science 47: 259-273. Taweel, H.Z.; Tas, B.M.; Smit, H.J.; Elgersma, A.; Dijkstra, J.; Tamminga, S. 2006. Conclusions Grazing behaviour, intake, rumen function and milk production of dairy cows offered Lolium perenne containing different levels of water-soluble carbohydrates. Livestock Science 102: 33-41. National Research Council. 2007. Nutrient requirements of small ruminants: sheep, goats, cervids and new world camelids. Animal Nutrition Series. National Research Council of the National Academies. The National Academies Press, Washington, DC. Thomas, T.A. 1977. An automated procedure for the determination of soluble carbohydrates in herbage. Journal of the Science of Food and Agriculture 28: 639-642. Pacheco, D.; Burke, J.L.; Cosgrove, G.P. 2007. An empirical model to estimate efficiency of nitrogen utilisation in cows grazing fresh forages. pp. 409- 416. In: Meeting the challenges for pasture-based Woodford, K.B.; Nicol, A.M. 2004. A re-assessment of the stock unit system. Ministry of Agriculture Information Paper No. 2005/02. Journal of New Zealand Grasslands 77:    123-130     (2015) Journal of New Zealand Grasslands 77:    123-130 (2015) 130
https://openalex.org/W2962944741
https://www.aclweb.org/anthology/S19-2166.pdf
English
null
Harvey Mudd College at SemEval-2019 Task 4: The D.X. Beaumont Hyperpartisan News Detector
null
2,019
cc-by
2,707
1 Introduction The rest of this paper begins with a descrip- tion of previous work on the related task of fake news detection in Section 2. We then describe our model and features in Section 3, and our results in Section 4. Section 5 discusses some lessons learned with respect to what features are most use- ful in identifying hyperpartisan news, and Sec- tion 6 closes with a brief description of our sys- tem’s namesake, fictional magazine editor D.X. Beaumont. Hyperpartisan news is becoming more mainstream as online sources gain popularity. Hyperpartisan news is news written from an extremely partisan perspective, such that the goal is reinforcing exist- ing belief structures in the party’s ideology rather than conveying facts. Such hyperpartisan writing tends to amplify political divisions and increase animosity between opposing political ideologies. Hyperpartisan news sources also output fake news at startling rates (Silverman et al., 2016). Auto- matic detection of fake news is difficult, but de- tecting hyperpartisan news can help, and it can also expose biases in journalism. This task is challenging to automate because it is even diffi- cult for humans: fake and biased news articles get shared on social media at high rates, and even labels that were hand-generated by professionals have errors (Silverman et al., 2016). We attempt to use various features of political news articles to train a multinomial Naive Bayes classifier to complete this task. We use a set of bag-of-words (BoW) features for words appearing in the title of each article, and for words appearing in the arti- cle text. With these features, we identified a set Abstract We use the 600 hand-labelled articles from Se- mEval Task 4 (Kiesel et al., 2019) to hand- tune a classifier with 3000 features for the Hy- perpartisan News Detection task. Our final system uses features based on bag-of-words (BoW), analysis of the article title, language complexity, and simple sentiment analysis in a naive Bayes classifier. We trained our fi- nal system on the 600,000 articles labelled by publisher. Our final system has an accuracy of 0.653 on the hand-labeled test set. The most effective features are the Automated Readabil- ity Index and the presence of certain words in the title. This suggests that hyperpartisan writ- ing uses a distinct writing style, especially in the title. Proceedings of the 13th International Workshop on Semantic Evaluation (SemEval-2019), pages 967–970 Minneapolis, Minnesota, USA, June 6–7, 2019. ©2019 Association for Computational Linguistics 1 Evan Amason Harvey Mudd College 301 Platt Boulevard Claremont, CA 91711 eamason@hmc.edu Jake Palanker Harvey Mudd College 301 Platt Boulevard Claremont, CA 91711 jpalanker@hmc.edu of words that characterize hyperpartisan writing. We also considered complexity features such as type-to-token ratio and automated readability in- dex. Based on the performance of these features we attempt to answer the question of whether hy- perpartisan writing is more or less complex than non-hyperpartisan writing. A successful classifier could be very useful in today’s society. For exam- ple, it could be used to create a browser plug-in to check online articles for political bias in real time as the user reads. People on social media could use it to verify the legitimacy of a political article before sharing it with their followers. En- couraging people to share factual news rather than inflammatory hyperpartisan articles would hope- fully improve communication between opposing parties and create a more informed population. Harvey Mudd College at SemEval-2019 Task 4: The D.X. Beaumont Hyperpartisan News Detector Mary Clare Shen Harvey Mudd College 301 Platt Boulevard Claremont, CA 91711 mshen@hmc.edu Evan Amason Harvey Mudd College 301 Platt Boulevard Claremont, CA 91711 eamason@hmc.edu Jake Palanker Harvey Mudd College 301 Platt Boulevard Claremont, CA 91711 jpalanker@hmc.edu Julie Medero Harvey Mudd College 301 Platt Boulevard Claremont, CA 91711 jmedero@hmc.edu 2 Previous Work Since the 2016 election, there has been a lot of in- terest in fake news, which is closely related to the hyperpartisan news we focus on. Our approach to the hyperpartisan news task leverages lessons learned in prior work on fake news detection, and explores the extent to which that work is success- ful in a different but related task. Fake news de- tection has been widely studied (e.g., the survey paper by Fuhr et al. (Fuhr et al., 2018)), and we base many of our classifier’s features on previous studies of fake news. The content of fake and real news articles differ 967 substantially. Fake news articles have been found to require a lower reading level than real news arti- cles, to be less technical, and to use more personal pronouns. Further, their titles tend to be longer, use more proper nouns, and use more words that are all capitalized (Horne and Adali, 2017). Our work differs in that we were trying to determine whether an article is hyperpartisan, which is sim- ilar to but not the same as identifying fake news articles. In particular, a hyperpartisan news arti- cle may be factually correct (i.e., not contain any mistruths) but still be written with a hyperparti- san slant. We hypothesize, nonetheless, that the stylistic features that distinguish between real and fake news may be useful in identifying hyperpar- tisan news articles. Potthast, et. al., also showed that there are significant stylistic differences be- tween hyperpartisan and mainstream news articles (Potthast et al., 2017). Consequently, we include reading level and features of each article’s title as features in our model. each vocabulary word occurs in the full arti- cle text. We then drop a fixed number stop words, selected automatically by frequency. We experimented with both 50 and 100 stop words, and the run of our system that was submitted to the SemEval task used 50 stop words. Title Bag of Words: Next, using the same vocab- ulary but without excluding stop words, we add word counts for the title of the article. We also count the number of words in the title that are entirely capitalized, generally a fea- ture of hyperpartisan titles (Horne and Adali, 2017). Sentiment Analyzer: We use two sentiment lex- icons (Hu and Liu, 2004). The first con- tains 2000 words with positive sentiment, and the second contains 4000 words with neg- ative sentiment. 3 Methodology Each article’s content and title was tokenized us- ing spacy’s default English model (AI, 2016–). We use a multinomial naive Bayes classifier from scikit-learn, extracting a large number of features and then using feature selection to re- duce the number of features available to our clas- sifier. 2 Previous Work We count the occurrence of words from each list, hypothesizing that hyperpartisan articles will likely have many more words with polarized sentiment than non-hyperpartisan articles. The success of these features on identifying fake news motivates our decision to focus on arti- cle titles as a differentiating feature, and to include reading level in the set of features available to our model. Complexity Features: Finally, we include fea- tures designed to capture the articles’ com- plexity. This category includes features such as Average Word Length, Type-Token-Ratio, and SMOG Readability Formula. Each of these is designed to capture the complexity of a given text; Average Word Length gives us insights into the vocab choices and uses of ”advanced” words, Type-Token-Ratio mea- sures the amount of ”novel” words in the text, the SMOG Readability Formula is based on the number of polysyllabic words per sen- tence (which is influenced both by vocabu- lary choice, and sentence length). Since prior work shows that hyper-partisan articles are often written at an easier reading level, with more repeating words, and simpler sentence structure, we expect that these complexity features will be useful in identifying hyper- partisan articles. Complexity Features: Finally, we include fea- tures designed to capture the articles’ com- plexity. This category includes features such as Average Word Length, Type-Token-Ratio, and SMOG Readability Formula. Each of these is designed to capture the complexity of a given text; Average Word Length gives us insights into the vocab choices and uses of ”advanced” words, Type-Token-Ratio mea- sures the amount of ”novel” words in the text, the SMOG Readability Formula is based on the number of polysyllabic words per sen- tence (which is influenced both by vocabu- lary choice, and sentence length). Since prior work shows that hyper-partisan articles are often written at an easier reading level, with more repeating words, and simpler sentence structure, we expect that these complexity features will be useful in identifying hyper- partisan articles. Perez-Rosa et al. also examine fake news ar- ticles to create a classifier for them (Prez-Rosas et al., 2018). Their results identify additional fea- tures related to text readability, with fake news articles tending to be written at a lower reading level than real news articles. We incorporate fea- tures from their work, including Average Word Length, Type-Token-Ratio, and SMOG Readabil- ity Formula . 3.2 Feature Selection We make use of features related to the words in the article as a whole, the title of the article, sentiment, and text complexity. The above feature space was very large compared to the number of available articles, so we imple- mented two different methods of feature selection: one using variance, and one using a χ2 test. In each case, we perform statistics on the training set, Bag of Words Features: Using a vocabulary of 30,000 words, we count the number of times 968 2 Feature Title Category χ2 p-value ”trump” Polarity 416 1.77e-92 A.R.I. Complexity 377 3.67e-84 ”*” Title 208 2.95e-47 ”class” Title 179 9.45e-41 ”american” Title 170 7.41e-39 ”most” Title 143 5.04e-33 ”political” Title 137 1.08e-31 ”israel” Title 133 1.16e-30 ”like” Polarity 128.7 7.85e-30 ”these” Title 126 2.92e-29 Table 2: Highest ranked features from our hand labeled data-set. attempting to describe which features are the most distinguishing. Given these statistics, we score each feature, and select a subset of the total feature set using either a threshold score or a target fea- ture count. By experimenting on the smaller hand- labeled data set, we found that reducing to the best 3000 features maximized our performance for 10- fold cross validation. This modification was made after the evaluation, however; our results on the SemEval task represent the performance of our task without feature selection. Table 2: Highest ranked features from our hand labeled data-set. 4.1 Feature Selection As part of additional analysis, we examined the effectiveness of feature selection on the validation set. Table 1 shows that reducing the number of features to 3000 had a negligible effect on both ac- curacy and f-measure. Since the validation set is qualitatively different from the hand-labeled test set used in the official competition, these results are not directly comparable to our final system per- formance. In particular, we note that our system performs slightly better on the validation set than on the test set regardless of the number of fea- tures used, which may indicate that our classifier learned some characteristics of the source-labeled validation set that distinguished it from the hand- labeled test set. The Automated Readability Index feature (a complexity feature measuring word length and sentence length) is the second highest performing, indicating that this way of capturing complexity is worthy of further study. A number of BoW features on the title are also important. The selected words included fall un- der a few categories such as controversial topic (trump, Israel), generalization (most, these), and political terms (political, class). Some, like the presence of ”*” in title, seem like strange outliers that are likely a consequence of a combination of formatting artifacts and the small size of the hand- labeled dataset. Feature Accuracy f1-measure Selection all 0.611 0.675 3000 0.5983 0.667 Table 1: Validation set performance using all of our features or the 3000 most informative features. While an earlier, simpler version of our model achieved 10-fold cross-validation accuracy of .787 on the hand-labeled training set, the submission we submitted performed much more poorly on the final test set. We hypothesize that one source of this difference may have been in the tuning of our hyper-parameter related to feature selection. We tuned this parameter manually using results from 10-fold cross-validation on the hand labeled data- set. Because the hand labeled data was signifi- cantly smaller, it is possible that it took far fewer features to properly classify the space. Improved tuning of this parameter on a larger set could have given us better results. Nonetheless, our work demonstrates that BoW, complexity, and polarity features are all useful in identifying hyperpartisan news articles. Table 1: Validation set performance using all of our features or the 3000 most informative features. Table 1: Validation set performance using all of our features or the 3000 most informative features. 4 Results Our final system achieved an accuracy of 0.653, which ranked 28th out of 42 submissions on the test set hand-labeled by article. Using our χ2 feature selection system, we found the top 10 features over the hand-labeled article set, shown in Table 2. The size of the hand-labeled set is rather small, so the extremely small p-values are likely inflated by this. 5 Discussion Hyperpartisan news has been a concern since the rise of social media, and that concern has only grown since the 2016 election. Giving consumers of social media the knowledge of whether or not what they are reading is hyperpartisan could help to reduce the number of people fooled by fake or misleading facts, and it could help to reduce the partisan divide within the United States. 969 3 Stein. 2018. An information nutritional label for on- line documents. SIGIR Forum, 51(3):46–66. 6 Namesake Our system is named after D.X. Beaumont, a mag- azine editor and publisher on the short-lived TV Series My Sister Eileen that aired on CBS in 1960- 61 (Wikipedia contributors, 2018). The series, based on autobiographical short stories published in The New Yorker by Ruth McKenney (Lippman, 2018). Ruth, who aspired to be a writer, worked for Beaumont (shown in Figure 1 as portrayed by Raymond Bailey). We imagine that the prolifer- ation of hyperpartisan news in modern communi- cation would have caused the orderly Ruth a great deal of frustration, and hope that our contribution to this task will benefit future writers and their publishers. Benjamin D. Horne and Sibel Adali. 2017. This just in: Fake news packs a lot in title, uses simpler, repetitive content in text body, more similar to satire than real news. In The 2nd International Workshop on News and Public Opinion at ICWSM. Cornell University. Minqing Hu and Bing Liu. 2004. Mining and summa- rizing customer reviews. In KDD. Johannes Kiesel, Maria Mestre, Rishabh Shukla, Em- manuel Vincent, Payam Adineh, David Corney, Benno Stein, and Martin Potthast. 2019. SemEval- 2019 Task 4: Hyperpartisan News Detection. In Proceedings of The 13th International Workshop on Semantic Evaluation (SemEval 2019). Association for Computational Linguistics. Laura Lippman. 2018. In praise of ruth mckenney. The New York Times. [Online; accessed 18-February- 2019]. Figure 1: Darren McGavin, who portrayed D.X. Beau- mont in the TV Series My Sister Eileen(NBC Televi- sion, 2017). NBC Television. 2017. Accessed: 2019-02-20 (Public domain). [link]. Martin Potthast, Johannes Kiesel, Kevin Reinartz, Janek Bevendorff, and Benno Stein. 2017. A sty- lometric inquiry into hyperpartisan and fake news. CoRR, abs/1702.05638. Vernica Prez-Rosas, Bennett Kleinberg, Alexandra Lefevre, and Rada Mihalcea. 2018. Automatic de- tection of fake news. In Proceedings of the 27th In- ternational Conference on Computational Linguis- tics. Craig Silverman, Lauren Strapagiel, Hamza Shaban, Ellie Hall, and Jeremy Singer-Vine. 2016. Hyper- partisan facebook pages are publishing false and misleading information at an alarming rate. Figure 1: Darren McGavin, who portrayed D.X. Beau- mont in the TV Series My Sister Eileen(NBC Televi- sion, 2017). Wikipedia contributors. 2018. My sister eileen — Wikipedia, the free encyclopedia. [Online; accessed 18-February-2019]. Acknowledgments We would like to thank our stalwart grutor (a Harvey Mudd portmanteau of grader and tutor!), Jonah Rubin, for his help at all hours on our coursework during the semester that led to this system submission. References Explosion AI. 2016–. spacy: Industrial-strength natu- ral language processing. Norbert Fuhr, Anastasia Giachanou, Gregory Grefen- stette, Iryna Gurevych, Andreas Hanselowski, Kalervo Jarvelin, Rosie Jones, YiquN Liu, Josiane Mothe, Wolfgang Nejdl, Isabella Peters, and Benno Norbert Fuhr, Anastasia Giachanou, Gregory Grefen- stette, Iryna Gurevych, Andreas Hanselowski, Kalervo Jarvelin, Rosie Jones, YiquN Liu, Josiane Mothe, Wolfgang Nejdl, Isabella Peters, and Benno 970 4 4
https://openalex.org/W1973489762
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0106446&type=printable
English
null
Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences?
PloS one
2,014
cc-by
7,986
Abstract Objective: To investigate the association between cigarette use during pregnancy and pregnancy-induced hypertension/ preeclampsia/eclampsia (PIH) by maternal race/ethnicity and age. Methods: This retrospective cohort study was based on the U.S. 2010 natality data. Our study sample included U.S. women who delivered singleton pregnancies between 20 and 44 weeks of gestation without major fetal anomalies in 2010 (n = 3,113,164). Multivariate logistic regression models were fit to estimate crude and adjusted odds ratios and the corresponding 95% confidence intervals. Results: We observed that the association between maternal smoking and PIH varied by maternal race/ethnicity and age. Compared with non-smokers, reduced odds of PIH among pregnant smokers was only evident for non-Hispanic white and non-Hispanic American Indian women aged less than 35 years. Non-Hispanic Asian/Pacific Islander women who smoked during pregnancy had increased odds of PIH regardless of maternal age. Non-Hispanic white and non-Hispanic black women 35 years or older who smoked during pregnancy also had increased odds of PIH. Conclusion: Our study findings suggest important differences by maternal race/ethnicity and age in the association between cigarette use during pregnancy and PIH. More research is needed to establish the biologic and social mechanisms that might explain the variations with maternal age and race/ethnicity that were observed in our study. Citation: Chang JJ, Strauss JF III, Deshazo JP, Rigby FB, Chelmow DP, et al. (2014) Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? PLoS ONE 9(10): e106446. doi:10.1371/journal.pone.0106446 Editor: Ana Claudia Zenclussen, Medical Faculty, Otto-von-Guericke University Magdeburg, Medical Faculty, Germany Received March 16, 2014; Accepted August 5, 2014; Published October 22, 2014 Received March 16, 2014; Accepted August 5, 2014; Published October 22, 2014 ang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. pyright:  2014 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2014 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1*, Jerome F. Strauss III2, Jon P. Deshazo3, Fidelma B. Rigby4, David P. Chelmow4, 5 1 Department of Epidemiology, College for Public Health and Social Justice, Saint Louis University, St. Louis, Missouri, United States of America, 2 VCU Medical Center School of Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America, 3 Department of Health Administration, School of Allied Health Professions, Virginia Commonwealth University, Richmond, Virginia, United States of America, 4 Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, Virginia, United States of America, 5 Department of Obstetrics and Gynecology, Washington University, St. Louis, Missouri, United States of America Abstract Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The primary data utilized in our analyses are deposited in public databases. The 2010 public-use U.S. natality file from Center for Disease Control and Prevention’s National Center for Health Statistics is publicly available at http://www.cdc.gov/nchs/data_access/Vitalstatsonline.htm The U.S. National Inpatient Sample (NIS) database is publicly available at http:// www.hcup-us.ahrq.gov/nisoverview.jsp. Funding: This work was supported by the National Institute of Health (Grant P60 MD002256). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. * Email: jjchang@slu.edu ogen activation [7], apoptosis [8,9], reactive oxygen species formation [10], and sFlt-1, an antiangiogenic factor [11,12]. October 2014 | Volume 9 | Issue 10 | e106446 Materials and Methods This population-based, retrospective cohort study was based on the 2010 public-use U.S. natality file from Center for Disease Control and Prevention’s National Center for Health Statistics. We used data based on the 1989 (unrevised) version of the birth certificate for this analysis because the 2003 release has substantial missing data for some key variables for the present study. The natality file includes data on parental demographics, medical and obstetrical characteristics and complications, and neonatal status at birth. Our study sample consisted of U.S. women who delivered singleton pregnancies between 20 and 44 weeks of gestation in 2010. Pregnancies complicated by major fetal anomalies were excluded. These exclusions resulted in a study sample of 3,113,164 pregnancies. U.S. natality data is a publicly available data set and qualified for exemption from Institutional Review Board approval. The exposure, cigarette use during pregnancy, was indicated with a yes/no binary variable based on maternal self-report on the birth certificate. The outcome was PIH and included all women who had the condition ‘‘pregnancy-associated hypertension (i.e., PIH/preeclampsia)’’ or ‘‘eclampsia’’ checked on the 1989 version of the birth certificate [26,27]. Pregnancy-associated hypertension was defined as pregnancy- induced hypertension after the 20th week of gestation that resulted in an increase in blood pressure of at least 30 mm Hg systolic or 15 mm Hg diastolic on two measurements taken 6 hours apart [27]. Eclampsia, defined as the occurrence of convulsions and/or coma unrelated to other cerebral conditions in women with signs and symptoms of preeclampsia [27], was included in the preeclampsia group due to the small number of women affected within each ethnic group after stratification by maternal race/ethnicity and age. Mother’s race and ethnicity were self-reported and categorized as non- Hispanic white, non-Hispanic black, Hispanic, non-Hispanic Asian/Pacific Islander (non-Hispanic Asian), and non-Hispanic American Indians/Alaskan natives (non-Hispanic American Indi- ans), and Hispanics. This population-based, retrospective cohort study was based on the 2010 public-use U.S. natality file from Center for Disease Control and Prevention’s National Center for Health Statistics. We used data based on the 1989 (unrevised) version of the birth certificate for this analysis because the 2003 release has substantial missing data for some key variables for the present study. The natality file includes data on parental demographics, medical and obstetrical characteristics and complications, and neonatal status at birth. Our study sample consisted of U.S. women who delivered singleton pregnancies between 20 and 44 weeks of gestation in 2010. Materials and Methods Pregnancies complicated by major fetal anomalies were excluded. These exclusions resulted in a study sample of 3,113,164 pregnancies. U.S. natality data is a publicly available data set and qualified for exemption from Institutional Review Board approval. Smoking during Pregnancy and Preeclampsia Smoking during Pregnancy and Preeclampsia chronic hypertension, diabetes, and kidney disease [24]. For instance, it has been shown that white women are more likely to smoke heavily during pregnancy ($20 cigarette daily) than women of other race and ethnicity [23,25]. birth using Diagnosis Related Groups codes (765, 766, 767,768, 774, 775) assigned to inpatient visits from years 2006, 2008, and 2010. NIS data from 2006 and 2008 were included to increase the sample size due to the small number of eclampsia cases available. Multiple births were excluded (as defined by ICD9 codes V31– V37). This resulted in a NIS data sample of 12,326,151 pregnancies. We used ICD-9-CM codes to identify the exposure (i.e. cigarette use during pregnancy) and outcome (PIH) as well as maternal medical conditions as follows: tobacco use (305.1, V. 158.2), preeclampsia and pregnancy-induced hypertension (642.46, 642.56, 642.36), and eclampsia (642.61, 642.62, 642.63, 642.64), diabetes (ICD9 250), and chronic hypertension (642.2, 401). Despite the acknowledged disparity in hypertensive disorders during pregnancy by maternal age and race/ethnicity, few prior studies have examined the impact of their joint interactive effect with other known risk factors. The objective of this study was to examine the association between cigarette use during pregnancy and PIH by maternal race/ethnicity and age. Statistical analysis Mother’s race and ethnicity were self-reported and categorized as non- Hispanic white, non-Hispanic black, Hispanic, non-Hispanic Asian/Pacific Islander (non-Hispanic Asian), and non-Hispanic American Indians/Alaskan natives (non-Hispanic American Indi- ans), and Hispanics. Statistical analysis y Differences in sample characteristics by race/ethnicity were assessed by using the Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. Multivariable binary logistic regression models were used to estimate the crude and adjusted odds ratios (aOR) and the corresponding 95% confidence interval (95% CI). To reduce the bias in the parameter estimation, potential confounders were included in the multivariate analysis, including maternal age, marital status, parity, Kotelchuck prenatal care index, gestational weight gain, and maternal medical risk factors (i.e., diabetes and chronic hypertension). To evaluate if maternal race/ethnicity and age are effect modifiers, the Wald test was used to test if the regression coefficient of the product term of cigarette use during pregnancy, maternal race/ethnicity, and maternal age was statistically different from zero. Advanced maternal age, defined as 35 years or older, has been associated with adverse pregnancy [32]. Therefore, we dichotomized maternal age using a cutoff value of 35. We detected a significant interaction effect (interaction term P- value,0.01) for the product term of cigarette use during pregnancy, maternal race/ethnicity, and maternal age. Therefore, we stratified by race/ethnicity and age in the multivariable analysis. To generate nationally repre- sentative estimates, the analyses based on the NIS sample data were weighted and adjusted for clustering data structure using the survey commands. All tests were 2 tailed and P,0.05 was considered significant. All statistical analyses were performed with STATA (version 10.0, STATA Corp, College Station, TX). q p pp The exposure, cigarette use during pregnancy, was indicated with a yes/no binary variable based on maternal self-report on the birth certificate. The outcome was PIH and included all women who had the condition ‘‘pregnancy-associated hypertension (i.e., PIH/preeclampsia)’’ or ‘‘eclampsia’’ checked on the 1989 version of the birth certificate [26,27]. Pregnancy-associated hypertension was defined as pregnancy- induced hypertension after the 20th week of gestation that resulted in an increase in blood pressure of at least 30 mm Hg systolic or 15 mm Hg diastolic on two measurements taken 6 hours apart [27]. Eclampsia, defined as the occurrence of convulsions and/or coma unrelated to other cerebral conditions in women with signs and symptoms of preeclampsia [27], was included in the preeclampsia group due to the small number of women affected within each ethnic group after stratification by maternal race/ethnicity and age. Introduction Preeclampsia is associated with significant pregnancy-related morbidity and mortality, [1]. The etiology of preeclampsia is still not well understood, but several risk factors have been identified. These include genetic factors [2,3], nulliparity, multifetal gesta- tions, maternal race and age, and pre-existing conditions such as preeclampsia in a prior pregnancy, chronic hypertension, kidney disease, diabetes mellitus, and obesity [1]. Advanced maternal age and race/ethnicity have been well documented to be significant risk factors for a number of adverse pregnancy outcomes. Gregory and Korst observed that older pregnant women experienced increased risk of a number of maternal, fetal, and placental conditions including hypertension [13]. Rates of hypertensive disorders and preeclampsia appear to vary by race and ethnicity, as do the presentation and course of the disease [13–23]. In the U.S., preeclampsia risk is higher in ethnic minority women compared with non-Hispanic white women, with African-American women having the highest rate [18,22,23]. The causal mechanisms explaining the racial and ethnic differences in hypertensive disorders during pregnancy are largely unknown. These disparities might be related to a number of risk factors that are associated with race and ethnicity for preeclampsia, including Numerous studies have documented an inverse association between cigarette smoking during pregnancy and preeclampsia in different populations with a reduction of risk by up to 50% [4]. The underlying mechanism for this association is still not well understood with several hypothesized pathways, including carbon monoxide-mediated inhibition of inflammation [5], enhanced vasodilation [6], suppression of platelet aggregation [7], plasmin- October 2014 | Volume 9 | Issue 10 | e106446 October 2014 | Volume 9 | Issue 10 | e106446 1 PLOS ONE | www.plosone.org Results Characteristic Natality data NIS samples p-value* (n = 3,113,164) (n = 12,326,151) % % Mother’s age Mean (SD) 26.6 (6.1) 27.5 (13.6) ,0.0001 Mother’s age ,20 9.4 10.1 ,0.0001 20–34 76.5 75.6 35+ 14.1 14.3 Tobacco use 9.2 2.1 ,0.0001 PIH/Preeclampsia 4.1 6.7 ,0.0001 Eclampsia 0.27 0.08 ,0.0001 Chronic hypertension 1.3 0.7 ,0.0001 Diabetes 5.0 0.8 ,0.0001 Marital status 40.1 na Not married Parity na 1 59.0 $2 41.0 Prenatal care adequacy Inadequate 14.7 na Intermediate 10.1 Adequate 42.1 Adequate+ 33.1 Weight gain 0–19 lbs 24.8 na 20–39 lbs 51.2 40+ lbs 24.0 Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension. *p value from Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. doi:10.1371/journal.pone.0106446.t001 Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension. *p value from Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. doi:10.1371/journal.pone.0106446.t001 Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension. *p value from Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. doi:10.1371/journal.pone.0106446.t001 Table 4 shows that the inverse relationship between maternal smoking and PIH was age dependent. The odds of PIH increased with age in all ethnic groups after adjusting for potential confounders. The reduced odds of PIH was only evident for non-Hispanic white and non-Hispanic American Indian women young than 35 years old who smoked during pregnancy based on the natality data. This decreased odds of PIH conferred by maternal smoking was similarly observed in non-Hispanic white women younger than 35 years old based on NIS sample, albeit with a borderline significant and weaker strength of association. It is noteworthy that the increased odds of PIH among non-Hispanic Asian women who smoked during pregnancy persisted regardless of maternal age, with stronger association among older women within this ethnic group (Table 4). Furthermore, data from both the natality and the NIS sample indicated increased odds of PIH among non-Hispanic white women 35 years or older who smoked during pregnancy compared with those who did not, with stronger association based on the NIS data (Table 4). Interestingly, non- Hispanic white women also had the lowest aOR in the older age group, suggesting that there may still be a mitigating effect of smoking within this ethnic group. care compared to women of other ethnic origins. Results In this study, data from the birth certificates served as the primary source because they provide more demographic and lifestyle variables than the NIS data as potential confounders for the multivariable analysis. As expected by the differing data sources, there were statistically significant differences in selected maternal characteristics between the natality and the NIS data samples (Table 1). Women in the NIS data were slightly older than those from the natality data. Prevalence of pregnancy-induced hypertension was greater in the NIS sample than in the natality data. The prevalence of cigarette use during pregnancy, eclampsia, chronic hypertension, and diabetes were lower in the NIS data than in the natality data (Table 1). Factors that may be associated with maternal smoking and PIH were evaluated as potential confounders. Data for the following demographic and lifestyle variables were obtained from the birth certificate: maternal age (,20, 20 to 34, and $35 years), marital status (single or married), parity, Kotelchuck prenatal care index [28], gestational weight gain (,20 lbs, 20 to 39 lbs, and $40 lbs), and maternal medical risk factors including diabetes and chronic hypertension. Some studies had suggested potential misclassifications of medical conditions with data from birth certificates [29,30]. To address this issue, we attempted to validate our study findings from the natality data by replicating the same analysis as in the birth certificate data in a nationally representative sample of maternal hospital discharges from the U.S. National Inpatient Sample (NIS) database, the largest all-payer, publicly available inpatient database in the U.S. [31]. We identified in NIS records representing hospital stays for women who were pregnant or gave Table 2 shows the maternal characteristics in different ethnic groups from the natality data. All variables showed significant differences by race/ethnicity. The mean (standard deviation) maternal age of non-Hispanic Asian [30.6 years (5.3)] was higher (P,0.01) than that of other ethnic groups. Non-Hispanic American Indian women had the highest proportions of teenage births (16%), tobacco use during pregnancy (18.6%), PIH/ preeclampsia (5.3%), eclampsia (0.6%), and inadequate prenatal October 2014 | Volume 9 | Issue 10 | e106446 PLOS ONE | www.plosone.org 2 Smoking during Pregnancy and Preeclampsia Table 1. Study Sample Characteristics. October 2014 | Volume 9 | Issue 10 | e106446 Results Relative to women in other ethnic groups, non-Hispanic black women had the greatest proportions of chronic hypertension (2.8%) and being single (72.5%) whereas non-Hispanic Asian had the largest percentage of women with diabetes (8.7%). Using the natality data, we found decreased odds of PIH among non-Hispanic white and non-Hispanic American Indian women who smoked during pregnancy (aOR = 0.90, 95% CI: 0.88, 0.92 and aOR = 0.80, 95% CI: 0.70, 0.91, respectively, Table 3) compared to those who did not smoke during pregnancy, after controlling for confounders that included maternal age, marital status, parity, Kotelchuck prenatal care index, gestational weight gain, chronic hypertension, and diabetes. On the contrary, non- Hispanic black, non-Hispanic Asian, and Hispanic women who smoked during pregnancy had increased likelihood of PIH, after adjusting for confounders (Table 3). The increased odds of PIH for non-Hispanic Asian women who smoked during pregnancy was also observed in the NIS sample (aOR = 1.53, 95% CI: 1.14, 2.04) after controlling for maternal age, chronic hypertension, and diabetes (Table 3). Some covariates including marital status, parity, Kotelchuck prenatal care index, gestational weight gain were not available in NIS dataset and thus could not be included in the multivariate analysis for the NIS sample. There may be potential overlap between the 2010 US natality and the NIS sample, which includes data from year 2006, 2008, and 2010. Therefore, we conducted a subsample analysis for the October 2014 | Volume 9 | Issue 10 | e106446 October 2014 | Volume 9 | Issue 10 | e106446 PLOS ONE | www.plosone.org 3 Smoking during Pregnancy and Preeclampsia Table 2. Maternal Characteristics by Race/Ethnicity, US 2010 Natality file (n = 3,113,164). Results Characteristic NH White NH Black NH American Indian NH Asian/Pacific Islander Hispanic P Value* (n = 1,718,634) (n = 402,472) (n = 34,348) (n = 192,141) (n = 765,569) n(%) n(%) n(%) n(%) n(%) Mother’s age Mean (SD) 28.2 (5.8) 25.9 (6.2) 26.4 (5.8) 30.6 (5.3) 30.2 (5.4) ,0.01 Mother’s age ,0.01 ,20 117,061 (6.8) 62,348 (15.5) 5,503(16.0) 4,155(2.2) 104,003 (13.6) 20–34 1,347,959(78.4) 298,208 (74.1) 26,092 (76.0) 141,816 (73.8) 567,453 (74.1) 35+ 253,614 (14.8) 41,916 (10.4) 2,753(8.0) 46,170 (24.0) 94,113 (12.3) Tobacco use 227,997 (13.3) 33,300 (8.3) 6,389 (18.6) 2,800(1.5) 15,931(2.1) ,0.01 PIH/Preeclampsia 77,812 (4.5) 20,616 (5.1) 1,835 (5.3) 4,558 (2.4) 23,505(3.1) ,0.01 Eclampsia 4,554 (0.3) 1,720 (0.4) 195(0.6) 404(0.2) 1,635(0.2) ,0.01 Chronic hypertension 21,610 (1.3) 11,330 (2.8) 601 (1.8) 1,535 (0.8) 5,363(0.7) ,0.01 Diabetes 79,167 (4.6) 17,915 (4.5) 2,397 (7.0) 16,656(8.7) 40,005 (5.2) ,0.01 Marital status ,0.01 Married 1,224,022(71.2) 110,690 (27.5) 11,519 (33.5) 162,467 (84.6) 356,277 (46.5) Not married 494,612 (28. 8) 291,782 (72.5) 22,829 (66.5) 29,674 (15.4) 409,292 (53.5) Parity ,0.01 1 741,778 (43.2) 164,284 (40.8) 12,524 (36.5) 88,947 (46.3) 269,956 (35.3) $2 976,856 (56.8) 238,188 (59.2) 21,824 (63.5) 103,194 (53.7) 495,613 (64.7) Prenatal care Adequacy ,0.01 Inadequate 184,037 (10.7) 88,795 (22.1) 8,826 (25.7) 23,195 (12.1) 152,607 (19.3) Intermediate 161,565 (9.4) 43,267 (10.8) 4,719 (13.7) 21,244 (11.1) 84,207 (11.0) Adequate 769,893 (44.8) 144,370 (35.9) 12,315 (35.9) 84,611 (44.0) 298,586 (39.0) Adequate+ 603,139 (35.1) 126,040 (31.3) 8,488 (24.7) 63,091 (32.8) 230,169 (30.1) Weight gain ,0.01 0–19 lbs 356,997 (20.8) 128,957 (32.0) 10,390 (30.3) 43,571 (22.7) 232,815 (30.4) 20–39 lbs 899,157 (52.3) 177,754 (44.2) 15,565 (45.3) 113,758 (59.2) 388,071 (50.7) 40+ lbs 462,480 (26.9) 95,761 (23.8) 8,393 (24.4) 34,812 (18.1) 144,683 (18.9) Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension *p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi Square test Table 3. Odds Ratios for the Effect of Smoking on PIH among Ethnic Groups. djusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabete djusted for maternal age, chronic hypertension, and diabetes. ; , p , , g y yp maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi-Square tes p g y yp *p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (contin (categorical variables). Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension bb e at o : SD, sta da d de at o ; , o spa c, , eg a cy duced ype te s o *p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi-Square test Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. + adjusted for maternal age, chronic hypertension, and diabetes. doi:10 1371/journal pone 0106446 t003 reviation: OR, odds ratio, 95% CI, 95% confidence interval. : SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension , ; , p , , g y yp *p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi-Square test (categorical variables) Results * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. +adjusted for maternal age, chronic hypertension, and diabetes. doi:10.1371/journal.pone.0106446.t004 on the natality data and conferred for the older age group by the NIS data. 2010 US natality data set among primiparous women and the NIS sample from only 2006 and 2008, separately. In this subsample analysis, we obtained results that are largely consistent with the analysis of the US natality and the multi-year NIS sample. We observed that the association between maternal cigarette use and hypertensive disorders of pregnancy varied by maternal race/ ethnicity and age (Table S1). Specifically, the decreased odds of PIH among women who smoked during pregnancy was only apparent in non-Hispanic white and American Indian women younger than 35 years old who smoked during pregnancy, based on the natality data and among non-Hispanic white and blacks only based on the NIS sample. We also observed that maternal cigarette use during pregnancy was associated with increased odds of PIH for non-Hispanic Asians younger than 35 years old based on the natality data and the increased odds was also conferred by the NIS data regardless of maternal age (Table S2). In general, we observed an association in the same direction with cigarette use during pregnancy and PIH between the two data sets, albeit with differences in the strength of association with a weaker association from the NIS data for women younger than 35 years of age, but a stronger association for mothers with age greater than or equal to 35 (Table 4). This discrepancy in findings between the two data sets may be explained by the different sources of information between the two data sets. In the NIS data, measures of interest were defined based on ICD-9 discharge diagnosis codes, whereas in the natality data smoking is self- reported by the patient and hypertensive disorders were coded by chart extractors. Hence, compared to the natality data, prevalence of maternal smoking during pregnancy in the NIS data was much lower, whereas, the prevalence of the outcome was higher (Table 1). Geller and colleagues [34] evaluated the accuracy of the ICD-9 revision codes for preeclampsia and eclampsia and observed variation in accuracy of diagnosis with a positive predictive value for severe preeclampsia of 84.8%, 45.3% for mild preeclampsia, and 41.7% for eclampsia. Results The potential misclassification in NIS data for exposure and the outcome were likely non-differential, however, which would bias the point estimate toward the null value and may explain the weaker strength of association observed in the younger women in the NIS data. Results Natality data (n = 3,113,164) NIS data (n = 12,326,151) Crude OR 95 % CI Adjusted OR* 95 % CI Adjusted OR+ 95 % CI NH White 0.89 0.87, 0.91 0.90 0.88, 0.92 0.99 0.94–1.03 NH Black 0.99 0.94, 1.04 1.07 1.02, 1.13 0.95 0.85, 1.06 NH American Indian 0.74 0.65, 0.84 0.80 0.70, 0.91 0.97 0.65, 1.43 NH Asian/Pacific Islander 1.73 1.44, 2.09 1.64 1.36, 1.99 1.53 1.14, 2.04 Hispanic 1.11 1.02, 1.21 1.09 1.01, 1.19 0.98 0.84, 1.14 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. + adjusted for maternal age, chronic hypertension, and diabetes. doi:10 1371/journal pone 0106446 t003 * adjusted for maternal age, marital status, parity, kotelchuck pren + adjusted for maternal age, chronic hypertension, and diabetes. October 2014 | Volume 9 | Issue 10 | e106446 October 2014 | Volume 9 | Issue 10 | e106446 PLOS ONE | www.plosone.org 4 Smoking during Pregnancy and Preeclampsia Table 4. Odds Ratios for the Effect of Smoking on PIH Among Ethnic Groups by Maternal Age. Natality data NIS data (n = 12,326,151) (n = 3,113,164) Women ,35 yrs Adjusted OR* 95 % CI Adjusted OR+ 95 % CI NH White 0.89 0.86, 0.91 0.95 0.91, 1.00 NH Black 1.05 1.00, 1.09 0.91 0.81, 1.03 NH American Indian 0.76 0.66, 0.87 0.96 0.64, 1.45 NH Asian/Pacific Islander 1.66 1.35, 2.05 1.36 0.96, 1.93 Hispanic 1.09 1.00, 1.20 0.99 0.84, 1.17 Natality data NIS data (n = 12,326,151) (n = 3,113,164) Women $35 yrs Adjusted OR* 95 % CI Adjusted OR+ 95 % CI NH White 1.17 1.09, 1.26 1.29 1.13, 1.47 NH Black 1.18 1.01, 1.37 1.30 0.97, 1.62 NH American Indian 1.29 0.88, 1.89 1.07 0.25, 4.63 NH Asian/Pacific Islander 1.71 1.07, 2.72 2.32 1.35, 3.99 Hispanic 1.18 0.91, 1.54 0.91 0.57, 1.46 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. +adjusted for maternal age, chronic hypertension, and diabetes. doi:10.1371/journal.pone.0106446.t004 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. +adjusted for maternal age, chronic hypertension, and diabetes. doi:10.1371/journal.pone.0106446.t004 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. October 2014 | Volume 9 | Issue 10 | e106446 Smoking during Pregnancy and Preeclampsia Nevertheless, this measurement error of exposure was likely non-differential, which we expect would result in the attenuation of our point estimate toward the null value [36],which may explain the weaker association in our findings. In addition, the different rates of maternal smoking and preeclampsia across ethnic groups observed by previous studies may have contributed to this discrepancy. Our observation of the increased odds of PIH among non- Hispanic Asian women was unexpected and of particular interest. In the non-pregnant population, it has been observed that Asian and Indian adults have an increased risk of diabetes, hypertension, and dyslipidemia at lower BMI levels than European adults [37]. Additionally, in a study of ethnic variations in HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome among pregnant women with preexisting hypertension, Williams et al. observed that Caucasian and Chinese women were more likely than East Indians to develop HELLP syndrome [38]. Asian women may also differ from women of other ethnic origins in socioeconomic status, stress, physical activities, diet, and other social or life style factors that could contribute to this differential risk in hypertensive disorders of pregnancy. p p Our study findings are also limited by the self-reported information on tobacco use status during pregnancy. However, the prevalence of cigarette use during pregnancy in natality data from the present study is consistent with the population-based estimate from the 2010 Pregnancy Risk Assessment and Monitor- ing System data from 27 states conducted by the U.S. Centers for Disease Control and Prevention [52]. England et al. reported that 24% of smokers during pregnancy were misclassified as quitters in a large multicenter randomized study of nulliparous women [53]. Llurba and associates observed a 90% concordance between self- reported smoking and cotinine levels among 125 healthy Spanish pregnant women at 24 weeks of gestation in a case control study [54]. To estimate the bias introduced by the potential misclassi- fication of maternal smoking status in our study based on natality data file, we conducted a sensitivity analysis. Assuming a 76% sensitivity and 100% specificity of our exposure measurement, our parameter estimates remained largely the same for results shown in Table 3 and 4 after correcting for the measurement error [55]. Our study is further limited by the lack of information on the timing, intensity, and frequency of maternal smoking, which could potentially vary by race/ethnicity. In addition, our analysis used broad categorizations of ethnicity (e.g. Smoking during Pregnancy and Preeclampsia non-Hispanic Asian and Hispanic), which may obscure an association between maternal smoking and PIH within ethnic subgroups. When analyzing different ethnic and racial groups in the present study based on a U.S. population, it is difficult to ascertain whether the categori- zation of maternal race/ethnicity summarizes genetics or envi- ronment. While racial group implies a specific genetic inheritance, ethnicity reflects culture and is therefore changeable. In this study no attempt has been made to distinguish between the two potential effects in the role of maternal ethnic origin as an effect modifier. p g y In general, race/ethnicity may influence health outcomes due to differences in socio-economic status, life style (i.e., diet and physical activity), access to medical care, and medical conditions among ethnic groups [19]. For instance, blacks have higher prevalence of hypertension [39] and obesity [40] than whites. Cigarette use during pregnancy was less prevalent among Asians and blacks compared to whites [35,41,42]. Knuistet al suggested that risk factors for preeclampsia differed in prevalence among women of different ethnic groups such that the diastolic blood pressure was the strongest predictor for preeclampsia in white women whereas high maternal age was a better predictor for preeclampsia in blacks [23]. Previous studies also suggested that the underlying pathophysiology of hypertensive disorders may be different in blacks than in whites [43,44]. The varying rates of preeclampsia by race/ethnicity could also be attributed to genetic predisposition to developing diseases [45,46]. Prasmusinto et al. demonstrated ethnic differences in the association of factor V Leiden mutation and the C677T methylenetetrahydrofolate reductase gene polymorphism with preeclampsia among white and Indonesian mothers [47]. Variations in the distribution in the risk factors of preeclampsia including maternal smoking among ethnic groups combined with ethnic variation in genetic polymor- phisms predisposing to preeclampsia may explain the observed ethnic differences in the association between maternal smoking and preeclampsia. Further studies are needed to elucidate the biologic mechanisms underlying the ethnic differences in this relationship. The strength of this study rests in its use of a large population- based sample of U.S. women and the availability of information on many potential confounders that may affect the risk of PIH from the natality data. The large sample size provided adequate statistical power to detect significant associations, increased precision in the risk estimates, and the ability to evaluate the potential effect modifying role of maternal race/ethnicity and age. Smoking during Pregnancy and Preeclampsia data [35]. Knuist et al. observed an inverse association between maternal smoking and preeclampsia in both white [adjusted relative risk: 0.8, 95% CI: 0.3, 1.7] and black women (adjusted relative risk: 0.5, 95% CI: 0.1, 4.4), but the results were not statistically significant. Findings from Knuist et al. may be limited by the lack of sufficient statistical power for a stratified analysis by maternal race [23]. discharge data from New York City, Engel et al. found that the reduced risk of preeclampsia among women who smoked during pregnancy was limited to women aged 30 years or younger and that more advanced maternal age may be associated with greater risk of hypertension with preeclampsia [50]. Our findings are in general agreement with this observation. data [35]. Knuist et al. observed an inverse association between maternal smoking and preeclampsia in both white [adjusted relative risk: 0.8, 95% CI: 0.3, 1.7] and black women (adjusted relative risk: 0.5, 95% CI: 0.1, 4.4), but the results were not statistically significant. Findings from Knuist et al. may be limited by the lack of sufficient statistical power for a stratified analysis by maternal race [23]. Some methodological limitations of this study need to be considered in interpreting our study findings. They include the potential for inaccurate reporting, residual confounding by socioeconomic and other unmeasured maternal characteristics (e.g., stress, physical activities, nutrition), the lack of information regarding the diagnosis, timing and severity of preeclampsia, and misclassification of medical and obstetrical conditions. A prior validation study has indicated that the reporting rate of preeclampsia on birth certificates with a check-box format is fairly good, ranging from 85% to 97% when compared with risks based on hospital discharge data [51]. In our study, we used pregnancy induced hypertension to approximate preeclampsia as our outcome which may results in measurement error. It is unclear whether pregnancy induced hypertension and preeclampsia are two distinct disorders that share a similar symptom (i.e., hypertension) or if pregnancy induced hypertension is a precursor of preeclampsia [16]. The weaker association between maternal smoking and PIH from our study may be attributed to potential misclassification of the exposure, maternal smoking, due to recall bias or under- reporting for both natality and the NIS data. Discussion Numerous studies have observed a decreased risk of both preeclampsia and gestational hypertension among women who smoked during pregnancy with an average aOR of 0.7 [4,33]. However, to our knowledge, no prior studies have examined the joint impact of maternal race/ethnicity and age on the relationship between smoking and hypertensive disorders of pregnancy. The present study found that the association between maternal cigarette use and hypertensive disorders of pregnancy varied by maternal race/ethnicity and age. Specifically, the decreased odds of PIH among women who smoked during pregnancy was only apparent in non-Hispanic white and American Indian women younger than 35 years old who smoked during pregnancy, based on the natality data and among non-Hispanic white only based on the NIS sample. Interestingly, we also observed that maternal cigarette use during pregnancy was associated with increased odds of PIH for non-Hispanic Asians regardless of maternal age, based The inverse association between maternal smoking and PIH among non-Hispanic white and American Indian women in our study appears to be weaker compared to findings from prior research [23,33,35]. Misra et al. found that maternal smoking was associated with reduced odds of gestational hypertension in white women (aOR: 0.17, 95% CI: 0.12 to 0.24) but not in black women (aOR: 0.35, 95% CI: 0.09–1.37) [35]. However, results from this study may have been affected by selection bias as more than half of the eligible subjects were excluded from the study due to missing October 2014 | Volume 9 | Issue 10 | e106446 October 2014 | Volume 9 | Issue 10 | e106446 PLOS ONE | www.plosone.org 5 October 2014 | Volume 9 | Issue 10 | e106446 References 20. Janakiraman V, Gantz M, Maynard S, El-Mohandes A (2009) Association of cotinine levels and preeclampsia among African-American women. Nicotine Tob Res 11: 679–684. 1. Ananth CV, Vintzileos AM (2011) Ischemic placental disease: epidemiology and risk factors. Eur J Obstet Gynecol Reprod Biol 159: 77–82. y p 2. Nejatizadeh A, Stobdan T, Malhotra N, Pasha MA (2008) The genetic aspects of pre-eclampsia: achievements and limitations. Biochem Genet 46: 451–479. 21. Anderson NH, Sadler LC, Stewart AW, Fyfe EM, McCowan LM (2012) Ethnicity, body mass index and risk of pre-eclampsia in a multiethnic New Zealand population. Aust N Z J Obstet Gynaecol 52: 552–558. 3. Palei AC, Spradley FT, Warrington JP, George EM, Granger JP (2013) Pathophysiology of hypertension in pre-eclampsia: a lesson in integrative physiology. Acta Physiol (Oxf) 208: 224–233. 22. Caughey AB, Stotland NE, Washington AE, Escobar GJ (2005) Maternal ethnicity, paternal ethnicity, and parental ethnic discordance: predictors of preeclampsia. Obstet Gynecol 106: 156–161. 4. England L, Zhang J (2007) Smoking and risk of preeclampsia: a systematic review. Frontiers in Bioscience 12: 2471–2483. 23. Knuist M, Bonsel GJ, Zondervan HA, Treffers PE (1998) Risk factors for preeclampsia in nulliparous women in distinct ethnic groups: a prospective cohort study. Obstet Gynecol 92: 174–178. 5. Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, et al. (2000) Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 6: 422–428. 24. Roberts J (2009) Maternal-Fetal Medicine: principles and practice. In: Creasy R, Resnik R, Iams JD, editors. Pregnancy-related hypertension. Philadelphia, PA: Saunders Elsevier; . pp. 650–688. p y 6. Zhang F, Kaide JI, Rodriguez-Mulero F, Abraham NG, Nasjletti A (2001) Vasoregulatory function of the heme-heme oxygenase-carbon monoxide system. Am J Hypertens 14: 62S–67S. 25. Perreira KM, Cortes KE (2006) Race/ethnicity and nativity differences in alcohol and tobacco use during pregnancy. Am J Public Health 96: 1629–1636. 7. Fujita T, Toda K, Karimova A, Yan SF, Naka Y, et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat Med 7: 598–604. 26. (1996) ACOG technical bulletin. Hypertension in pregnancy. Number 219– January 1996 (replaces no. 91, February 1986). Committee on Technical Bulletins of the American College of Obstetricians and Gynecologists. Int J Gynaecol Obstet 53: 175–183. 8. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, et al. References (2000) Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 192: 1015–1026. 27. (n.d.) Division of Vital Statistics. Technical appendix from vital statistics of the United States, Natality, March, 2001 Hyattsville, Maryland: National Center for Health Statistics. 9. Liu XM, Chapman GB, Peyton KJ, Schafer AI, Durante W (2002) Carbon monoxide inhibits apoptosis in vascular smooth muscle cells. Cardiovasc Res 55: 396–405. 28. Bloch JR, Dawley K, Suplee PD (2009) Application of the Kessner and Kotelchuck prenatal care adequacy indices in a preterm birth population. Public Health Nurs 26: 449–459. 10. Wang X, Wang Y, Kim HP, Nakahira K, Ryter SW, et al. (2007) Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation. J Biol Chem 282: 1718–1726. 29. Watkins ML, Edmonds L, McClearn A, Mullins L, Mulinare J, et al. (1996) The surveillance of birth defects: the usefulness of the revised US standard birth certificate. Am J Public Health 86: 731–734. 11. Powers RW, Roberts JM, Cooper KM, Gallaher MJ, Frank MP, et al. (2005) Maternal serum soluble fms-like tyrosine kinase 1 concentrations are not increased in early pregnancy and decrease more slowly postpartum in women who develop preeclampsia. Am J Obstet Gynecol 193: 185–191. 30. Allen AM, Dietz PM, Tong VT, England L, Prince CB (2008) Prenatal smoking prevalence ascertained from two population-based data sources: birth certificates and PRAMS questionnaires, 2004. Public Health Rep 123: 586–592. 12. Cudmore M, Ahmad S, Al-Ani B, Fujisawa T, Coxall H, et al. (2007) Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1. Circulation 115: 1789–1797. 31. Health care Cost and Utilization Project (HCUP) (2011) Introduction to the NIS:2009, HCUP NIS related reports. Rockville, MD: Agency for Healthcare Research and Quality. 13. Gregory KD, Korst LM (2003) Age and racial/ethnic differences in maternal, fetal, and placental conditions in laboring patients. Am J Obstet Gynecol 188: 1602–1606; discussion 1606–1608. 32. Waldenstrom U, Aasheim V, Nilsen AB, Rasmussen S, Pettersson HJ, et al. (2014) Adverse pregnancy outcomes related to advanced maternal age compared with smoking and being overweight. Obstet Gynecol 123: 104–112. ; 14. Goodwin AA, Mercer BM (2005) Does maternal race or ethnicity affect the expression of severe preeclampsia? Am J Obstet Gynecol 193: 973–978. 33. Conde-Agudelo A, Althabe F, Belizan JM, Kafury-Goeta AC (1999) Cigarette smoking during pregnancy and risk of preeclampsia: a systematic review. Acknowledgments This work was supported by the National Institute of Health (Grant P60 MD002256). We thank Dr. Emmanuel A. Anum for his contributions in data analysis to the early phases of this work. This work was supported by the National Institute of Health (Grant P60 MD002256). We thank Dr. Emmanuel A. Anum for his contributions in data analysis to the early phases of this work. Smoking during Pregnancy and Preeclampsia In addition, our study used two independent data sources and observed similar findings albeit with variations in the strength of association for the effect of interest. Using the NIS data from Few studies have sufficient sample size to examine the effect modifying role of maternal age in the association between maternal smoking and preeclampsia. Advanced maternal age, especially after age 35, is associated with increased risk of preeclampsia [48] and other adverse pregnancy outcomes [49]. In a large study based on birth records linked with hospital October 2014 | Volume 9 | Issue 10 | e106446 PLOS ONE | www.plosone.org 6 Smoking during Pregnancy and Preeclampsia hospital inpatient records, which is superior to birth certificate check boxes further supports and enhances the validity of our study findings. Author Contributions Conceived and designed the experiments: JS DC JJC GM. Performed the experiments: JD JJC DC. Analyzed the data: JD JJC. Contributed reagents/materials/analysis tools: JJC JS JD FR DC GM. Wrote the paper: JJC JS JD FR DC GM. Supporting Information Table S1 Odds Ratios for the Effect of Smoking on PIH Among Ethnic Groups. (DOCX) Our study findings suggested important differences by maternal race/ethnicity and age in the association between maternal smoking and PIH. How race/ethnicity modifies this relationship is not clearly understood. It may be explained by a combination of social, behavioral, and genetic polymorphisms and disease susceptibility. While this disparity needs to be confirmed in future studies, our study results may help health professional identify specific subgroups of women who are at higher risk for PIH. Although the pathophysiologic pathways of preeclampsia are largely unknown, separation of women into etiologically homoge- neous groups in future studies of preeclampsia may improve our understanding and prediction of the disease. It is plausible that women of different racial and ethnic origins may have different clinical presentations and clinical courses of preeclampsia. More research is needed to establish the biologic and social mechanisms that might explain the variations by maternal age and race/ ethnicity that were observed in our study. Table S2 Odds Ratios for the Effect of Smoking on PIH Among Ethnic Groups by Maternal Age. (DOCX) Smoking during Pregnancy and Preeclampsia 38. Williams KP, Wilson S (1997) Ethnic variation in the incidence of HELLP syndrome in a hypertensive pregnant population. J Perinat Med 25: 498–501. trahydrofolate reductase gene polymorphism with preeclampsia. Eur J Obstet Gynecol Reprod Biol 112: 162–169. 39. Gillum RF (1996) Epidemiology of hypertension in African American women. Am Heart J 131: 385–395. 48. Duckitt K, Harrington D (2005) Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies. BMJ 330: 565. 40. Wattigney WA, Webber LS, Srinivasan SR, Berenson GS (1995) The emergence of clinically abnormal levels of cardiovascular disease risk factor variables among young adults: the Bogalusa Heart Study. Prev Med 24: 617–626. 49. Khalil A, Syngelaki A, Maiz N, Zinevich Y, Nicolaides KH (2013) Maternal age and adverse pregnancy outcomes: a cohort study. Ultrasound Obstet Gynecol. 50. Engel SM, Janevic TM, Stein CR, Savitz DA (2009) Maternal smoking, preeclampsia, and infant health outcomes in New York City, 1995–2003. Am J Epidemiol 169: 33–40. y g g y 41. Tuck SM, Cardozo LD, Studd JW, Gibb DM, Cooper DJ (1983) Obstetric characteristics in different racial groups. Br J Obstet Gynaecol 90: 892–897. 41. Tuck SM, Cardozo LD, Studd JW, Gibb DM, Cooper DJ (19 characteristics in different racial groups. Br J Obstet Gynaecol 90 g p y 42. Perry IJ, Beevers DG, Whincup PH, Bareford D (1995) Predictors of ratio of placental weight to fetal weight in multiethnic community. BMJ 310: 436–439. 42. Perry IJ, Beevers DG, Whincup PH, Bareford D (1995) Predictors of ratio of l t l i ht t f t l i ht i lti th i it BMJ 310 436 439 42. Perry IJ, Beevers DG, Whincup PH, Bareford D (1995) Predict 51. Frost F, Starzyk P, George S, McLaughlin JF (1984) Birth complication reporting: the effect of birth certificate design. Am J Public Health 74: 505–506. placental weight to fetal weight in multiethnic community. BMJ 310: 436–439. 43. Falkner B (1990) Differences in blacks and whites with essential hypertension: biochemistry and endocrine. State of the art lecture. Hypertension 15: 681–686. 43. Falkner B (1990) Differences in blacks and whites with essential hypertension: biochemistry and endocrine. State of the art lecture. Hypertension 15: 681–686. 52. Division of Reproductive Health, U.S. Centers for Disease Control and Prevention. (n.d.) Tobacco use and pregnancy. 44. Frohlich ED (1990) Hemodynamic differences between black patients and white patients with essential hypertension. References Am J Obstet Gynecol 181: 1026–1035. 15. Marshall NE, Guild C, Cheng YW, Caughey AB, Halloran DR (2013) Racial disparities in pregnancy outcomes in obese women. J Matern Fetal Neonatal Med. 34. Geller SE, Ahmed S, Brown ML, Cox SM, Rosenberg D, et al. (2004) International Classification of Diseases-9th revision coding for preeclampsia: how accurate is it? Am J Obstet Gynecol 190: 1629–1633; discussion 1633– 1624. 16. Wolf M, Shah A, Jimenez-Kimble R, Sauk J, Ecker JL, et al. (2004) Differential risk of hypertensive disorders of pregnancy among Hispanic women. J Am Soc Nephrol 15: 1330–1338. 17. Bouthoorn SH, Gaillard R, Steegers EA, Hofman A, Jaddoe VW, et al. (2012) Ethnic differences in blood pressure and hypertensive complications during pregnancy: the Generation R study. Hypertension 60: 198–205. 35. Misra DP, Kiely JL (1995) The effect of smoking on the risk of gestational hypertension. Early Hum Dev 40: 95–107. 36. Dosemeci M, Wacholder S, Lubin JH (1990) Does nondifferential misclassifi- cation of exposure always bias a true effect toward the null value? Am J Epidemiol 132: 746–748. 18. Tanaka M, Jaamaa G, Kaiser M, Hills E, Soim A, et al. (2007) Racial disparity in hypertensive disorders of pregnancy in New York State: a 10-year longitudinal population-based study. Am J Public Health 97: 163–170. 37. (2004) World Health Organization expert consultation. Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet: 157–163. 19. Gong J, Savitz DA, Stein CR, Engel SM (2012) Maternal ethnicity and pre- eclampsia in New York City, 1995–2003. Paediatr Perinat Epidemiol 26: 45–52. October 2014 | Volume 9 | Issue 10 | e106446 October 2014 | Volume 9 | Issue 10 | e106446 7 PLOS ONE | www.plosone.org trahydrofolate reductase gene polymorphism with preeclampsia. Eur J Obstet Gynecol Reprod Biol 112: 162–169. Smoking during Pregnancy and Preeclampsia State of the art lecture. Hypertension 15: 675–680. 53. England LJ, Grauman A, Qian C, Wilkins DG, Schisterman EF, et al. (2007) Misclassification of maternal smoking status and its effects on an epidemiologic study of pregnancy outcomes. Nicotine & Tobacco Research 9: 1005–1013. 45. El Sahly HM, Reich RA, Dou SJ, Musser JM, Graviss EA (2004) The effect of mannose binding lectin gene polymorphisms on susceptibility to tuberculosis in different ethnic groups. Scand J Infect Dis 36: 106–108. 54. Llurba E, Sanchez O, Dominguez C, Soro G, Goya M, et al. (2013) Smoking during pregnancy: changes in mid-gestation angiogenic factors in women at risk of developing preeclampsia according to uterine artery Doppler findings. Hypertens Pregnancy 32: 50–59. g p J 46. Pegoraro RJ, Hira B, Rom L, Moodley J (2003) Plasminogen activator inhibitor type 1 (PAI1) and platelet glycoprotein IIIa (PGIIIa) polymorphisms in Black South Africans with pre-eclampsia. Acta Obstet Gynecol Scand 82: 313–317. 55. Fink AK, Lash TL (2003) A null association between smoking during pregnancy and breast cancer using Massachusetts registry data (United States). Cancer Causes Control 14: 497–503. p p y 47. Prasmusinto D, Skrablin S, Fimmers R, van der Ven K (2004) Ethnic differences in the association of factor V Leiden mutation and the C677T methylenete- October 2014 | Volume 9 | Issue 10 | e106446 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8
https://openalex.org/W4327812527
https://zenodo.org/records/7748030/files/414659911.pdf
English
null
The Effect of Financial Risk, Capital Structure, Banking Liquidity on Profitability: Operational Efficiency as Intervening Variables in Persero Bank and Private Commercial Banks
Zenodo (CERN European Organization for Nuclear Research)
2,023
cc-by
6,489
International Journal of Arts and Social Science ISSN: 2581-7922, Volume 5 Issue 1, January 2022 International Journal of Arts and Social Science ISSN: 2581-7922, Volume 5 Issue 1, January 2022 www.ijassjournal.com The Effect of Financial Risk, Capital Structure, Banking Liquidity on Profitability: Operational Efficiency as Intervening Variables in Persero Bank and Private Commercial Banks Hasmiana1 , Madris2 , Shine Pintor 3 1 Student of Masters Program in Financial Management at the Open University, Indonesia 2 Faculty of Economics, Economics and Business, Hasanuddin University, Indonesia 3 Faculty of Economics, Open University, Indonesia ABSTRACT: This study aims to (1) to partially analyze the effect of financial risk, capital structure, and liquidity on operational efficiency (2) to partially analyze the effect of financial risk, capital structure, liquidity, and operational efficiency on (3) to analyze partially the effect of financial risk, capital structure, and liquidity on profitability through operational efficiency at State-Owned Banks and Private Commercial Banks. The data collection technique used purposive sampling. The type of data used is quantitative data and secondary data sources which are downloaded through the website https://www.idx.co.id/. The results showed (1) financial risk, capital structure, and liquidity partially had a significant effect on operational efficiency (2) financial risk, capital structure, liquidity, and operational efficiency partially had a significant effect on profitability. (3) Financial risk, capital structure and liquidity partially have no significant effect on profitability through operational efficiency. KEYWORDS- Financial Risk, Capital Structure, Liquidity, Operational Efficiency, Profitability I. INTRODUCTION Profitability is one indicator in making it easier for banks to obtain funds from third parties. This is due to public trust in banks to manage their funds in the form of credit. Better credit distribution can affect bank profits or improve profitability . the ratio is getting bigger, it will reflect better financial performance or a high level of profitability . Profitability is one indicator in making it easier for banks to obtain funds from third parties. This is due to public trust in banks to manage their funds in the form of credit. Better credit distribution can affect bank profits or improve profitability . p y Furthermore, there are various aspects that affect Return On Assets which is a measure of profitability according to the findings of several previous researchers, namely Credit Risk, Capital Structure, Operational Efficiency and Liquidity, each of which is proxied by Non Performing Loans (NPL), Capital Adequacy Ratio (CAR). , BOPO and Loan to Deposit Ratio (LDR). ( ) , p ( ) According to the 2016-2019 Indonesian Banking Statistics, a number of measures of banking financial performance show fluctuating values and are shown in the following table: Table 1 NPL average. CAR, LDR, BOPO and ROA at Persero Banks and Private Banks in Indonesia 2016-2019 Rasio Tahun 2016 2017 2018 2019 NPL % 3,22 2,96 2,66 2,73 CAR % 23,19 23,42 22,91 23,54 LDR % 90,77 88,68 94,09 93,96 BOPO % 81,26 78,39 79,13 80,65 ROA % 2,41 2,49 2,50 2,48 Source: Indonesian Banking Statistics. Table 1 NPL average. CAR, LDR, BOPO and ROA at Persero Banks and Table 1.1 shows that the NPL ratio from 2016 to 2019 is already below the BI standard of 5%. This indicates that the ability of banks to manage non-performing loans has been effective, which means that state- owned banks and private banks that were sampled in this study are good enough to carry out monitoring or supervision in providing credit. Meanwhile, seen from the Capital Adequacy Ratio, in 2016-2017, it has a tendency to increase from 23.19% to 23.42%. However, in 2017 and 2018, CAR has decreased. Then seen from the LDR ratio during 2016-2017 , it decreased from 90.77% to 88.68%, as well as in 2018-2019 which also decreased from 94.09% down to 93.96%. I. INTRODUCTION With the LDR ratio which is already above 85%, it can be said that all state-owned and private banks observed in this study are in good category. Furthermore, judging from the BOPO ratio, the BOPO ratio for state-owned banks and private banks observed in this study is classified as good, because the average BOPO ratio is not more than 92% and this is in accordance with Indonesian bank standards. The smaller the BOPO ratio, the more efficient the operational costs incurred by the bank, which means that the bank's financial performance is increasing. On the other hand, the greater the BOPO ratio, the less able the bank to reduce operational costs, which causes the bank to be less efficient in managing the resources available to the bank so that it has a negative effect on bank profits. Some of the results of research conducted by previous researchers related to the relationship between non-performing loans, capital asset ratio, loan deposit ratio , operational costs to operating income (BOPO) on financial performance with indicators of return on assets . Findings from Pinasti & Mustikawati 2018), which found that NPL had a positive and not significant effect on profitability while the research conducted by Fitria (2017), Saerang, et al. (2014) NPL has a negative and insignificant effect on profitability. Research conducted by Harun (2016), Bawono (2018) states that CAR has no effect on ROA. Then research by Kurniasih (2016) found that LDR had no partial effect on ROA. Research by Saerang et al. (2014) that partially LDR has a positive and significant effect on ROA. Meanwhile Kurniasih (2016) found that BOPO had no significant effect on return on assets . In contrast to the results of research by Harun (2016) that BOPO has a negative and significant relationship to return on assets . The increase in NPL creates distrust of banks towards customers. This resulted in a decline in lending to the public and ultimately affected bank profitability . Research results from Nguyen (2020) prove that NPL has a significant effect on profitability. The low BOPO ratio reflects the efficiency in managing bank operational costs. Operational costs have a significant correlation with the bank's ability to manage risky productive assets. The ability of banks to manage their assets properly will ensure capital adequacy (CAR). I. INTRODUCTION A bank is a financial institution that functions as an intermediary between the parties providing funds and those in need of funds. One sector that utilizes the distribution of funds from the bank to trigger economic growth is the real sector. According to Kasmir (2014:4) that managing banking must be done professionally, so that it can obtain continuous profits as the main purpose of the bank was established, therefore in obtaining profit (profitability) in managing the banking business sector it is expected to maintain the continuity of banking business in Indonesia. . One way that is done by a bank is to collect funds for the community and then processed it in the form of credit. To improve the welfare of the community, savings from the community are channeled back to the community in the form of credit. The increasingly fierce competition is reflected in the emergence of relatively new banks. Therefore, one of the factors that are of concern to banks is to maintain the level of bank liquidity. Liquidity is related to the bank's ability to meet obligations that are soon due. Constraints in the provision of liquidity can affect a bank's ability to generate profits or bank profitability . The existence of the ability of banks to earn profits in the banking world has an important role for owners, depositors, government and the public in assessing the financial condition of a Bank. Return on assets (ROA) is one of the ratios to measure a bank's ability to generate profits. Return On Assets focuses on the bank's ability to earn earnings in the company's operations. Meanwhile , Return on Equity (ROE) only measures the return obtained from the company owner's investment in the business. So in this study the emphasis is on ROA as a measure of bank performance. y The reason for choosing Return on Assets (ROA) as a performance measure is because it can be used to measure the company's effectiveness in generating profits through the use of its assets (Sekarwati, 2019). Return On Assets is a ratio in measuring the level of profit obtained based on the management of total assets. If Page 226 Hasmiana International Journal of Arts and Social Science ISSN: 2581-7922, Volume 5 Issue 1, January 2022 www.ijassjournal.com the ratio is getting bigger, it will reflect better financial performance or a high level of profitability . a) Stewardship Theory Stewardship theory describes a situation where customers (stewards) are not motivated by individual goals but are aimed at their primary outcome goals for the benefit of the bank (principals). Stewardship theory views customers as credit recipients as parties who can be trusted to act as well as possible for the benefit of the bank. Customers are expected to pay off their obligations before/at maturity because they can minimize financial risk and increase profitability (Donaldson, et al. 1997). p y Stewarship theory describes a situation where management is not motivated by individual goals but is more focused on their main outcome goals for the benefit of the organization and assumes a strong relationship between satisfaction and organizational success (Yoyo, et al. 2017:60). b) Definition of Financial Risk (Credit Risk) b) Definition of Financial Risk (Credit Risk) Credit risk is a condition where the debtor does not repay the principal and other cash related to investments in accordance with the provisions stipulated in the credit agreement. Credit risk can cause cash flow problems and affect bank liquidity because payments may be delayed or non-existent, Greuning & Bratanovic (2011). Financial Risk is measured by Non Performing Loan (NPL). Non-Performing Loan (NPL) is the ability of banks to manage non-performing loans (Wibisono, 2017:54). I. INTRODUCTION A high CAR can anticipate losses in the event of a decline in asset value and ultimately increase the BOPO ratio and bank profitability . The results of research Page 227 Hasmiana International Journal of Arts and Social Science ISSN: 2581-7922, Volume 5 Issue 1, January 2022 www.ijassjournal.com from Chiaramonte and Casu (2017), Vinh and Thao (2016) which prove that CAR has a significant positive effect on banking efficiency which in turn affects profitability. from Chiaramonte and Casu (2017), Vinh and Thao (2016) which prove that CAR has a significant positive effect on banking efficiency which in turn affects profitability. Based on previous researchers, where in this study a mediation test will be carried out by choosing operational cost efficiency as a mediating variable which aims to examine the effect of Financial Risk, Capital Structure, Banking Liquidity on Profitability mediated by Operational Efficiency in State-Owned Banks and Private Commercial Banks. The reason for choosing operational cost efficiency as a mediating variable is based on the inconsistency of research and statements found by Abbas et al. (2019) that research on the relationship between financial risk, capital structure, and liquidity on banking profitability in Asian countries that are still experiencing economic development is still very rare. Thus, it is necessary to reveal about these relationships, especially in the banking world in Asian countries which are still experiencing economic development, especially after the banking crisis of 2008 (Abbas, et al. 2019). b) Definition of Liquidity Liquidity is a condition related to a certain amount of cash and other assets that are easily converted into cash (Darmawi, 2012: 59). According to Muljono (1999) describes a bank that has a good level of liquidity when: (1) the bank has cash assets as much as needed to be used to ensure liquidity; (2) The bank has other liquid assets; (3) Banks are able to create new assets. Similarly, the measurement of bank liquidity uses ratios, such as: (1) The ratio of the total net liabilities of call money to current activities; (2) The ratio between loans to funds received by the bank. 2. Capital Structure Capital structure is a balance or comparison between foreign capital and own capital. Foreign capital in this case is long-term and short-term debt. While the own capital is divided into retained earnings and the company's ownership participation in which the aim is to increase the value of the company in the market (Brigham & Eharhrdt, 2011:600). 3. Liquidity q y a) Assets and Liabilitiy Management Theory q y a) Assets and Liabilitiy Management Theory Assets and Liabilities Management Theory explains that asset and liability management is aimed at managing liquidity risk, especially cash flow, which aims to maintain an adequate and optimal level of liquidity, such as minimizing idle funds but still taking into account the adequacy of liquidity that will mature (mature). Banks that have a good level of liquidity will have the ability to pay off their obligations, thereby minimizing operational efficiency and increasing profitability. b) Definition of Liquidity 1. Credit Risk a) Stewardship Theory a) Stewardship Theory 4. Profitability Signaling Theory explains that banks with high levels of profitability provide a positive signal for the public to entrust their funds to be managed. Funds sourced from the community will be channeled in the form of credit. Optimal credit management can minimize the occurrence of financial risks that have an impact on operational efficiency and profitability. Page 228 Hasmiana International Journal of Arts and Social Science ISSN: 2581-7922, www.ijassjournal.com Volume 5 Issue 1, January 2022 III. METHOD III. METHOD The population of this research is 40 banks, namely state-owned banks and Indonesian national private commercial banks listed in the Bank Indonesia directory for the period 2016 to 2019. The sampling technique uses purposive sampling. The amount of data that was processed with the help of the SPSS-23 program was 160 data. The type of data used is quantitative data and the data source is secondary data in the form of time series data such as financial reports from the Bank Indonesia directory. The data analysis method used classical assumption testing (normality test, multicollinearity test, heteroscedasticity test, autocorrelation test), multiple/multiple regression , coefficient of determination, and hypothesis testing (t test). The variables used consist of three exogenous variables (financial risk: X1, capital structure: X2, liquidity: X3) and two endogenous variables (operational efficiency: Y1 and profitability: Y2). b) Definition of profitability p y Profitability is a ratio used to measure a company's ability to generate profits from its normal business activities. A company is an organization that operates with the aim of making a profit by selling products (goods and/or services) to its customers. p , p From the results of the descriptive analysis in Table 2, the descriptive statistical analysis can be described as follows: IV. RESULTS AND DISCUSSION Before testing the Data Normality Test and other Statistical Tests, it is first explained about the characteristics of the data, namely NPL, CAR, LDR, BOPO and ROA as follows: Table 2 Descriptive Statistics of Research Variables N Minimum Maximum Mean Std. Deviation NPL 160 .03 9.92 2,10 1,49 CAR 160 9.01 147.44 22,90 12,51 LDR 160 47.54 163.10 87,21 17,87 BOPO 160 8.09 236.20 92,28 23,89 ROA 160 -18,89 4,00 0,64 2,77 Valid N (listwise) 160 Source: SPSS output, processed data Table 2 p p From the results of the descriptive analysis in Table 2, the descriptive statistical analysis can be described as follows: 1. The minimum value for Financial Risk (NPL) is 0.03% obtained from PT Bank National Nobu Tbk. Meanwhile, PT Bank Neo Commerce Tbk has the maximum NPL ratio of 9.92%. This means that the highest NPL among the sample companies is 9.92%. The average value of NPL during 2016-2019 is 2,1003%, meaning that for every Rp. 1, the loan disbursed has the potential for non-payment (loss) of Rp. 0.02. 2. The minimum CAR value at PT. Banten Regional Development Bank TBK 9.01 percent. Meanwhile, PT Bank Jago Indonesia has the highest CAR, which is 147.44%. Then the average NPL value during 2016- 2019 was 22.8986%, meaning that for every Rp. 1, fixed assets at risk can guarantee a capital readiness of Rp. 0.23. 2. The minimum CAR value at PT. Banten Regional Development Bank TBK 9.01 percent. Meanwhile, PT Bank Jago Indonesia has the highest CAR, which is 147.44%. Then the average NPL value during 2016- 2019 was 22.8986%, meaning that for every Rp. 1, fixed assets at risk can guarantee a capital readiness of Rp. 0.23. p 3. The minimum LDR value at PT Bank Jago Indonesia Tbk is 47.54. Meanwhile, PT Bank BTPN, Tbk has the highest LDR, which is 163.10%. The average LDR value during 2016-2019 is 87.2186%, meaning that for every Rp. 1, funds from the public are channeled in the form of loans of Rp. 0.87. p 3. The minimum LDR value at PT Bank Jago Indonesia Tbk is 47.54. Meanwhile, PT Bank BTPN, Tbk has the highest LDR, which is 163.10%. The average LDR value during 2016-2019 is 87.2186%, meaning that for every Rp. 1, funds from the public are channeled in the form of loans of Rp. 0.87. y p p p 4. IV. RESULTS AND DISCUSSION The minimum BOPO ratio is PT Bank Jago Indonesia Tbk at 8.09% . Meanwhile, PT Bank PT Bank Of India Indonesia, Tbk has the highest BOPO , which is 235.20%. The average value of BOPO during 2016- 2019 is 92.2762%, meaning that for every Rp. 1, operating income is used for operational costs of Rp. 0.92. 5. The minimum ROA value is PT Bank Jago Indonesia Tbk which has -18.89%. Meanwhile, PT Bank Mayapada Internasional, Tbk has a maximum ROA of 4.00%. The average ROA value during 2016-2019 is 0 6355% i h R 1 id fi f R 0 006 y p p p 4. The minimum BOPO ratio is PT Bank Jago Indonesia Tbk at 8.09% . Meanwhile, PT Bank PT Bank Of India Indonesia, Tbk has the highest BOPO , which is 235.20%. The average value of BOPO during 2016- 2019 is 92.2762%, meaning that for every Rp. 1, operating income is used for operational costs of Rp. 0.92. g , India Indonesia, Tbk has the highest BOPO , which is 235.20%. The average value of BOPO during 2016- 2019 is 92.2762%, meaning that for every Rp. 1, operating income is used for operational costs of Rp. 0.92. 5. The minimum ROA value is PT Bank Jago Indonesia Tbk which has -18.89%. Meanwhile, PT Bank Mayapada Internasional, Tbk has a maximum ROA of 4.00%. The average ROA value during 2016-2019 is 0.6355%, meaning that every Rp. 1 asset can provide a profit of Rp. 0.006. 2019 is 92.2762%, meaning that for every Rp. 1, operating income is used for operational costs of Rp. 0.92. 5. The minimum ROA value is PT Bank Jago Indonesia Tbk which has -18.89%. Meanwhile, PT Bank Mayapada Internasional, Tbk has a maximum ROA of 4.00%. The average ROA value during 2016-2019 is 0.6355%, meaning that every Rp. 1 asset can provide a profit of Rp. 0.006. Page 229 Hasmiana International Journal of Arts and Social Science www.ijassjournal.com ISSN: 2581-7922, Volume 5 Issue 1, January 2022 Table 3 Residual Data Normality Test Results Uji Kolmogorov-Smirnov Test Unstandardized Coefficient Nilai Kolmogorov smirnov test 0,090 Sign 0,200 Source: SPSS output, processed data From the results of the Kolmogorov-Smirnov test above, the Asymp value is produced. Sig. (2-tailed) of = 0.200 0.05, these results can be concluded that the residual data in this regression model is normally distributed, because the value of Asymp. Sig. IV. RESULTS AND DISCUSSION (2-tailed) is already above 0.005 International Journal of Arts and Social Science ISSN: 2581-7922, www.ijassjournal.com Volume 5 Issue 1, January 2022 Table 3 Residual Data Normality Test Results From the results of the Kolmogorov-Smirnov test above, the Asymp value is produced. Sig. (2-tailed) of = 0.200 0.05, these results can be concluded that the residual data in this regression model is normally distributed, because the value of Asymp. Sig. (2-tailed) is already above 0.005 From the results of the Kolmogorov-Smirnov test above, the Asymp value is produced. Sig. (2-tailed) of = 0.200 0.05, these results can be concluded that the residual data in this regression model is normally distributed, because the value of Asymp. Sig. (2-tailed) is already above 0.005 Table 4 Multicollinearity Test Result Model Collinearity Statistics Conclusion Tolerance VIF NPL 0,774 1,293 There are no symptoms of multicollinearity CAR 0,900 1,111 There are no symptoms of multicollinearity LDR 0,981 1,019 There are no symptoms of multicollinearity BOPO 0,741 1,350 There are no symptoms of multicollinearity Source: SPSS output, processed data Table 4 Multicollinearity Test Result Table 4 Table 4 Multicollinearity Test Result p p Based on the results of the output Table 4, it can be concluded that there is no multicollinearity due to VIF 10 and tolerance ‰¤ 1, meaning that there is no strong correlation or relationship between two or more independent variables in a multiple regression model. Table 5 Heteroscedasticity Test Results Residual Model Independent Variable Sig Description ABRES NPL 0,054 There is no heteroscedasticity because the value of ² value > 0.05 CAR 0,062 LDR 0,059 BOPO 0,057 Source: SPSS output, processed data Table 5 Based on the results of the output Table 5, it can be concluded that the significance value of each variable is 0.05, meaning that there is no variance inequality from the residuals for all observations in the regression model or there is no heteroscedasticity. Table 6 Autocorrelation Test Results Model Mark Description R 0,668 No symptoms of autocorrelation Rsquare 0,473 Nilai du 1,7798 Nilai dl 2,2202 Durbin-Watson 1,929 Source: SPSS output, processed data Based on the Durbin Watson table (Appendix 12), it is known that the value of d (dw) = 1.929 from the upper limit (du) which is 1.7798 and less than (4-du) 4-1.7798 = 2.2202. So it can be concluded that there are no problems / symptoms of Autocorrelation. IV. RESULTS AND DISCUSSION Table 6 Autocorrelation Test Results Model Mark Description R 0,668 No symptoms of autocorrelation Rsquare 0,473 Nilai du 1,7798 Nilai dl 2,2202 Durbin-Watson 1,929 Source: SPSS output, processed data p , p Based on the Durbin Watson table (Appendix 12), it is known that the value of d (dw) = 1.929 from the upper limit (du) which is 1.7798 and less than (4-du) 4-1.7798 = 2.2202. So it can be concluded that there are no problems / symptoms of Autocorrelation. p p Based on the Durbin Watson table (Appendix 12), it is known that the value of d (dw) = 1.929 from the upper limit (du) which is 1.7798 and less than (4-du) 4-1.7798 = 2.2202. So it can be concluded that there are no problems / symptoms of Autocorrelation. p p Based on the Durbin Watson table (Appendix 12), it is known that the value of d (dw) = 1.929 from the upper limit (du) which is 1.7798 and less than (4-du) 4-1.7798 = 2.2202. So it can be concluded that there are no problems / symptoms of Autocorrelation. Page 230 Hasmiana International Journal of Arts and Social Science www.ijassjournal.com ISSN: 2581-7922, Volume 5 Issue 1, January 2022 Table 7 Coefficient of Determination Analysis Model Mark Description R 0,509 The influence is strong enough Rsquare 0,259 Adjusted Rsquare 0,245 Std. Error of the Estimate 25,83 Source: SPSS output, processed data www.ijassjournal.com The effect/contribution simultaneously ( R_1^2 ) between financial risk, capital structure and liquidity on operational efficiency is 46% and the remaining 54% is influenced by other factors not observed in the model. Meanwhile, the effect/contribution simultaneously (R_2^2) between financial risk, capital structure and liquidity on profitability is 24.5% and the remaining 75.5% is influenced by other factors not observed in the model. Simultaneous and Partial Hypothesis Testing Effect of Financial Risk on Operational Efficienc p y The results of statistical hypothesis testing prove that financial risk has a significant effect on operational efficiency. Financial risk is proxied by NPL which is an indication of a problem within the bank, which if not addressed immediately, will have a negative impact on the bank itself. A high NPL indicates the customer's failure or inability to repay the loan amount received along with the interest, according to a predetermined period of time. This results in losses and increases operational efficiency. The results of this study support the opinion of Kasmir (2004), Afkar, (2017). However, the results of research from Sendyvia Candra (2015) show that NPL has no significant effect on the level of efficiency. Simultaneous and Partial Hypothesis Testing (1) The results of simultaneous hypothesis testing show that there is a positive and significant effect (F1 = 19.111; Sig = 0.000 0.05) between financial risk, capital structure and liquidity on operational efficiency. Likewise, there is simultaneously a positive and significant effect (F2 = 36.536; Sig = 0.000 0.05) between financial risk, capital structure, liquidity and operational efficiency on profitability. (1) The results of simultaneous hypothesis testing show that there is a positive and significant effect (F1 = 19.111; Sig = 0.000 0.05) between financial risk, capital structure and liquidity on operational efficiency. Likewise, there is simultaneously a positive and significant effect (F2 = 36.536; Sig = 0.000 0.05) between financial risk, capital structure, liquidity and operational efficiency on profitability. p y (2) The results of partial hypothesis testing show the following: p y (2) The results of partial hypothesis testing show the following: (a) Financial risk (P=0.000 0.05, beta coefficient = 0.419), capital structure (P=0.002 0.05 and beta coefficient = -0.213), and liquidity (P=0.000 0, 05 and beta coefficient = - 0.028 partially significant effect on operational efficiency (P = 0.000 0.05, beta coefficient = 0.419). p y (b) Financial risk (P=0.001‰¤ 0.05 and beta coefficient = -0.242), capital structure (P=0.003 0.05 and beta coefficient = 0.481), liquidity (P=0.000‰¤ 0.05 and beta coefficient = 0.187), and operational efficiency (P = 0.000 0.05 and beta coefficient = -0.435) partially significant effect on profitability. y p y g p y (c) Financial risk (P=0.054 0.05 and beta coefficient = -0.182), capital structure (P=0.061 0.05 and beta coefficient = 0.093), and liquidity (P=0.059 > 0.05 and beta coefficient = 0.012) partially has no significant effect on profitability through operational efficiency. so that the statement of hypothesis five b can be rejected or not supported. In this case, there is no relationship between the influence of financial risk on profitability through operational efficiency. (c) Financial risk (P=0.054 0.05 and beta coefficient = -0.182), capital structure (P=0.061 0.05 and beta coefficient = 0.093), and liquidity (P=0.059 > 0.05 and beta coefficient = 0.012) partially has no significant effect on profitability through operational efficiency. so that the statement of hypothesis five b can be rejected or not supported. In this case, there is no relationship between the influence of financial risk on profitability through operational efficiency. Effect of Capital Structure on Operational Efficiency The results of statistical hypothesis testing prove that the capital structure has a negative and significant effect on operational efficiency. Capital structure is proxied by Capital Adequacy Ratio (CAR). Banks with high CAR have the ability to provide funds as capital for business development and accommodate the risk of losses that may occur. The risk of loss affects operational efficiency. The results of this study are supported by the findings of Jackson and Fethi (2000). However, this is different from the research conducted by Subandi and Ghozali (2014) which proves that CAR has a significant positive relationship to bank efficiency. Effect of Operational Efficiency on Profitability The results of statistical hypothesis testing prove that operational efficiency has a significant effect on profitability. Operational efficiency is proxied by BOPO which is the ratio between operating costs divided by operating income. The BOPO ratio is used to measure the efficiency of a bank's business or to measure the amount of bank costs incurred to obtain income from assets. The results of this study support the findings of Pinasti and Mustikawati (2019), Likewise with Harun's research (2016) the findings that BOPO has a significant effect on ROA. However, the results of this study do not support the findings of Kurniasih, et al. (2016) which shows BOPO has a negative and insignificant effect on ROA. Meanwhile, the findings of Bawono (2018) which show BOPO have a positive and insignificant effect on ROA. Effect of Liquidity on Operational Efficiency The results of statistical hypothesis testing prove that liquidity proxied by Loan to deposit ratio (LDR) has a positive and significant effect on operational efficiency. A high LDR ratio indicates low cash availability and has the potential to pose a liquidity risk. Liquidity risk occurs due to the inability to generate cash flow from Page 231 Hasmiana www.ijassjournal.com Volume 5 Issue 1, January 2022 assets, both from productive assets (payment of installments) or from asset sales and the inability to collect cash flows from fundraising, interbank transactions, and other loans, congestion or delays. cash flow . The results of this study support the findings of Subandi and Ghozali (2014), however, the findings of Purwoko and Sudiyanto (2013) show that LDR has no effect on operational efficiency. The Effect of Financial Risk on Profitability Through Operational Efficiency The results of statistical hypothesis testing prove that operational efficiency has no significant effect on intervening financial risk on profitability. Operational efficiency as proxied by the BOPO ratio is related to the share of operating income used to finance bank operational activities. Banks that have a high level of operational efficiency have a small BOPO ratio. A small BOPO ratio indicates that the bank has the ability to control its operating costs smaller with the assumption that operating income is relatively constant. Likewise, a small BOPO ratio indicates a bank has the ability to increase operating income assuming relatively constant operating costs. Effect of Capital Structure on Profitability through Operational Efficiency Effect of Capital Structure on Profitability through Operational Efficiency The results of statistical hypothesis testing prove that operational efficiency has no significant effect on intervening capital structure on profitability. operational efficiency which is proxied by the BOPO ratio in relation to the share of operating income used to finance the bank's operational activities. Banks that have a high level of operational efficiency have a small BOPO ratio. A small BOPO ratio indicates that the bank has the ability to control its operational costs. Likewise, a small BOPO ratio indicates the bank has the ability to increase its operating income. The results of this study do not support the findings of Chiaramonte and Casu (2017), Vinh and Thao (2016) which prove that capital structure has a significant effect on banking efficiency which in turn affects profitability. Effect of Financial Risk on Profitability The results of statistical hypothesis testing prove that financial risk proxied by Non Performing Loans (NPL) has a significant effect on profitability. NPLs are related to the existence of credit problems in the bank, which if not addressed immediately, will have a bad impact on the bank itself. A high NPL indicates the customer's failure or inability to repay the loan amount received along with the interest, according to the specified time period. This causes losses and reduces the bank 's profitability . The results of this study support the opinions of several experts, as stated by Kasmir (2004), Kurniasih (2016), however, the findings of Anton Bawono, Usman Harun (2016) show that NPL has a negative and insignificant effect on ROA. Effect of Capital Structure on Profitability The results of statistical hypothesis testing prove that capital structure has a positive and significant effect on profitability. Capital structure is proxied by Capital Adequacy Ratio (CAR). Banks with high CAR have the ability to provide funds as capital for business development and accommodate the risk of losses that may occur. The risk of loss affects the profitability of the bank. To increase CAR, banks need to optimize the management of risky assets. This study is supported by research conducted by Kurniasih (2016) which found that capital structure as measured by CAR has a partial effect on ROA. However, the results of this study do not support the findings of Harun (2016); Bawono and Falakh (2018) which show that CAR has a positive and insignificant effect on ROA. While the findings from Pinasti and Mustikawati (2018) show that CAR has a negative and significant effect on ROA. V. CONCLUSION Based on the results of research conducted which aims to determine the effect of credit risk, NPL, CAR, and LDR on BOPO and ROA processed using path analysis, conclusions can be drawn from the overall results of this study, namely: 1. The results of the analysis carried out can be concluded that credit risk has a positive and significant e on operational cost efficiency at state-owned banks and private banks. 2. The effect of capital structure (CAR) on operational cost efficiency, it can be concluded that ca structure (CAR) is negative and significant on operational cost efficiency 3. The effect of operational efficiency (BOPO) on profitability, where from the results of the analysis it can be concluded that operational cost efficiency has a negative and significant effect on return on assets (ROA). ( ) 4. The effect of financial risk (NPL) on profitability (ROA) it can be concluded that NPL has a negativ significant effect on return on assets (ROA). 5. Based on the results of the path test, it shows that operational cost efficiency (BOPO) cannot mediate the effect of financial risk (NPL) on profitability (ROA). 6. Based on the path test results, it can be concluded that operational cost efficiency cannot mediate the effect of capital structure (CAR) on profitability (ROA). 7. From the path test results, it can be concluded that operational cost efficiency can mediate the effect of liquidity (LDR) on profitability (ROA). The Effect of Liquidity on Profitability Through Operational Efficiency The results of statistical hypothesis testing prove that operational efficiency has no significant effect in intervening Liquidity on profitability. Operational efficiency as proxied by the BOPO ratio is related to the share of operating income used to finance the bank's operational activities. Banks that have a high level of operational efficiency have a small BOPO ratio. A small BOPO ratio indicates that the bank has the ability to control its operating costs smaller with the assumption that operating income is relatively constant. Likewise, a small BOPO ratio indicates a bank has the ability to increase operating income assuming relatively constant operating costs. Effect of Liquidity on Profitability. q y y The results of statistical hypothesis testing prove that liquidity has a positive and significant effect on profitability. Liquidity is proxied by Loan to deposit ratio (LDR). A high LDR ratio indicates high cash availability and has the potential not to pose a liquidity risk. Liquidity risk occurs due to the inability to generate Page 232 Hasmiana www.ijassjournal.com cash flow from assets, both from productive assets (payment of repayments/installments) or from asset sales and the inability to collect cash flows from fundraising, interbank transactions, and other loans, congestion or the existence of cash flow delays . The results of this study are supported by Wibisono (2017:48), Usman Harun (2016); Saerang, et al. (2014). However, the results of this study do not support the findings of Erma Kurniasih (2016) which shows that LDR has a positive and insignificant effect on ROA. Volume 5 Issue 1, January 2022 [8.] Donaldson, L. Davis, J. H., dan Schoorman, F. D. (1997). Toward A Stewardship Theory of Management. Academy of Management Review Vol. 22, No.1, 49-64 [9.] Fitria, L. N. (2017). Analisis Pengaruh Rasio Keuangan dan Dana Pihak Ketiga terhadap Profitabilitas melalui Financing to Deposit Ratio sebagai variabel Intervening pada Perbankan Syariah: Studi pada Bank Umum Syariah periode 2011–2015. [10.] Greuning, H. V., & Bratanovic, S. B. (2011). Analisis Risiko Perbankan. Edisi Ketiga. Jakarta: Salemba. [11.] Harun, U. (2016). Pengaruh Ratio-ratio Keuangan CAR, LDR, NIM, BOPO, NPL terhadap ROA. Jurnal Riset Bisnis dan Manajemen, Vol.4, No.2, 67-82. [12.] Jackson, P. M., & Fethi, M. D. 2000. Evaluating the efficiency of Turkish commercial banks: An application of DEA and Tobit Analysis. Diakses dari https://pdfs.semanticscholar.org/bb67/ deb5d904234e5cca 3bfeddb d1ceb122bf68e.pdf. Pada tanggal 2 Agustus 2021 15.34 WIB [13.] Kasmir (2014). Manajemen Perbankan. Edisi Revisi. Cetakan ke 12, Jakarta: Rajawali Pers. [14.] Kurniasih, T.,& Sari, Ratna Sari, M.M. (2016). Pengaruh Return on Assets, Leverage, Corporate Governance, Ukuran Perusahaan, dan Kompensasi Laba Fiskal Pada Tax Avoidance. Buletin Studi Ekonomi, Vol.18, No.1, 58-66. [15.] Muljono, T.P. (1999). Analisis Laporan Keuangan Untuk Perbankan. Edisi revisi, Cetakan 6. Yogyakarta: BPFE. . [16.] Nguyen, T. H. (2020). Impact of bank capital adequacy on bank profitabilityunder basel II accord: evidence from vietnam. Journal of Economic Development, Vol.45, No.1, 31–46. [17.] Pinasti, W. F. & Mustikawati, R. I. (2018). Pengaruh CAR, BOPO, NPL, NIM Dan LDR Terhadap Profitabilitas Bank Umum Periode 2011-2015. Nominal: Barometer Riset Akuntansi dan Manajemen, Vol. 7, No. 1, 126-142. Purwoko, D., & Sudiyatno, B. (2013). Faktor-faktor yang memengaruhi kinerja bank (Studi empirik pada indust perbankan di Bursa Efek Indonesia). Jurnal Bisnis dan Ekonomi, Vol.20, No. 1, 24-192. [19.] Saerang, I., Tommy, P., & Christiano, M. (2014). Analisis Terhadap Rasio-rasio Keuangan Untuk Mengukur Profitabilitas Pada Bank-bank Swasta Yang Go Public Di Bursa Efek Indonesia. Jurnal Riset Ekonomi, Manajemen, Bisnis Dan Akuntansi, Vol.2, No.4, 817–830. [20.] Sekarwati, A. (2019). Pengaruh CAR, DPK, BOPO, dan NPF Terhadap Profitabilitas dengan FDR sebagai Variabel Intervening pada Perbankan Syariah di Indonesia. Thesis IAIN Salatiga 21.] Sendyvia, C. dan Agung Y. (2015), Analisis Rasio Keuangan terhadap Tingkat Efisiensi Bank Umum Syariah Stage SFA),Accounting Analysis JournalVol.4, No. 2,2015,http://journal.unnes.ac.id/sju/index.php/aaj. 22.] Subandi, S., & Ghozali, I. (2014). Determinan efisiensi dan kinerja terhadap kinerja profitabilitas industri perb di Indonesia. Jurnal Keuangan dan Perbankan Vol.17, No.1,15-20 [23.] Vinh, NTH. (2016). REFERENCE [1.] Abbas, F., Iqbal, S., & Aziz, B. (2019). The impact of bank capital, bank liquidity and credit risk on profitability in postcrisis period:‎ A comparative study of US and Asia. Cogent Economics and Finance, 7(1). https://doi.org/ 10.1080/23322039.2019.1605683 [2.] Afkar, T. (2017). Analisis pengaruh kredit macet dan kecukupan likuiditas terhadap pengelolaan biaya operasional bank umum Syariah di Indonesia. Jurnal Inovasi dan Kewirausahaan Asia , 2 (02), 177-192. [3.] Bawono, A. 2018. Multivariate Analysis dengan SPSS. Salatiga: STAIN. Salatiga Press. [4.] Bawono, A, & Falakh, F. (2018). Analisis Pengaruh Rasio Keuangan dengan Likuiditas dan Profitabilitas sebagai Intervening terhadap Kinerja Perbankan Syariah pada PT Indonesia Tahun 2015-2018. Human Falah Jurnal Ekonomi Dan Bisnis Islam, Vol. 6, No. 2, 265-277. [5.] Brigham, E. F., & Ehrhardt. (2011). Financial Management Theory And Practice (Eleventh ed.). Ohio: South Western Cengage Learning. [6.] Chiaramonte, L., & Casu, B. (2017). Capital and liquidity ratios and financial distress. Evidence from the European banking industry. The British Accounting Review, 49, 138-161. [7.] Darmawi, H. (2012). Manajemen Perbankan. Cetakan Kedua. Jakarta: Bumi Aksara. Page 233 Hasmiana Page 233 International Journal of Arts and Social Science ISSN: 2581-7922, www.ijassjournal.com Volume 5 Issue 1, January 2022 The Impact of Non-performing Loans on Bank Profitability and Lending Behaviour; Evidence from Vietnam. Journal of Economics and Development.DOI:10.24311/JED/2017.24.3.06 Corpus ID: 16889 9420. https://www.semanticscholar.org/paper/The-impact-of-non-performing-loans-on-bank-and-from-Vinh [24.] Wibisono, M.Y. & Salamah, W. (2017). Pengaruh CAR, NPF, BOPO, FDR, terhadap ROA yang Dimediasi Oleh NOM. Jurnal Bisnis & Manajemen: Vol. 17, No. 1,41-62. [25.] Yoyo, S. Devyanthi, S. Sofiati, Nunung, A. (2017). Keuangan di Era Otonomi Daerah. Yogyakarta: Andi. Page 234 Page 234 Hasmiana
https://openalex.org/W2770150710
https://europepmc.org/articles/pmc5702312?pdf=render
English
null
Identification and Description of Novel Mood Profile Clusters
Frontiers in psychology
2,017
cc-by
12,055
ORIGINAL RESEARCH published: 21 November 2017 doi: 10.3389/fpsyg.2017.01958 Edited by: Martin S. Hagger, Curtin University, Australia Keywords: affect, BRUMS, emotion, mood profiling, online assessment Identification and Description of Novel Mood Profile Clusters Renée L. Parsons-Smith*, Peter C. Terry and M. Anthony Machin School of Psychology and Counselling, University of Southern Queensland, Toowoomba, QLD, Australia Mood profiling has been a popular assessment strategy since the 1970s, although little evidence exists of distinct mood profiles beyond the realm of sport and exercise. In the present study, we investigated clusters of mood profiles derived from the six subscales of the Brunel Mood Scale using the In The Mood website. Mood responses in three samples (n = 2,364, n = 2,303, n = 1,865) were analyzed using agglomerative, hierarchical cluster analysis, which distinguished six distinct and theoretically meaningful profiles. K-means clustering further refined the final parameter solution. Mood profiles identified were termed the iceberg, inverse iceberg, inverse Everest, shark fin, surface, and submerged profiles. Simultaneous multiple discriminant function analysis showed that cluster membership was correctly classified with a high degree of accuracy. Chi-squared tests indicated that the six mood profiles were unequally distributed according to the gender, age, and education of participants. Future research should investigate the antecedents, correlates and consequences of these six mood profile clusters. Keywords: affect, BRUMS, emotion, mood profiling, online assessment INTRODUCTION Reviewed by: Carla Lynn Kuesten, Amway (United States), United States Aleksandra Luszczynska, University of Colorado Colorado Springs, United States Mood has been described as “a set of feelings, ephemeral in nature, varying in intensity and duration, and usually involving more than one emotion” (Lane and Terry, 2000, p. 17). Mood measurement has typically occurred using self-report scales to assess transient emotions. The individualized and subjective nature of moods and emotions mean that responses elicited from self-report measures are considered to provide valid and useful information (Paulhus and Vazire, 2007). The 65-item Profile of Mood States (POMS; McNair et al., 1971, 1992) and, more recently, the abbreviated 24-item Brunel Mood Scale (BRUMS; Terry et al., 1999, 2003) have been used extensively to assess mood responses across a range of diverse contexts. *Correspondence: Renée L. Parsons-Smith rparsons-smith@bigpond.com Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology The most popular applications of mood profiling have been in the sport and exercise domains. More specifically, the role of mood in predicting sport performance has generated a considerable body of knowledge (see Beedie et al., 2000). The mental health model (Morgan, 1985) predicts that psychological health, as reflected by positive mood, associates with athletic success, whereas psychopathology associates with an increased incidence of failure (Rowley et al., 1995). The graphical representation of mood responses, proposed by Morgan (1980) to be typical of successful athletes approximates the shape of an iceberg, where the mean scores of the normative group represent the water line beneath which most scores fall. Parsimoniously termed the iceberg profile, this pattern of mood responses combines high vigor with low tension, depression, anger, fatigue, and confusion scores. The iceberg profile has subsequently been found to be the typical profile reported among athletes, successful or otherwise, and therefore is less indicative of athletic success than previously claimed (Renger, 1993; Terry and Lane, 2000). Received: 14 August 2017 Accepted: 24 October 2017 Published: 21 November 2017 Citation: Parsons-Smith RL, Terry PC and Machin MA (2017) Identification and Description of Novel Mood Profile Clusters. Front. Psychol. 8:1958. doi: 10.3389/fpsyg.2017.01958 November 2017 | Volume 8 | Article 1958 1 Frontiers in Psychology | www.frontiersin.org Parsons-Smith et al. Mood Profile Clusters Two additional mood profiles have previously been identified in the literature. The first profile, referred to as the Everest profile (Terry, 1995), is a more pronounced iceberg profile, characterized by higher vigor scores (>60%) and lower tension, depression, anger, fatigue, and confusion scores (<40%), and associates with superior performance. The second profile, referred to as the inverse iceberg profile, is characterized by below average scores for vigor and above average scores for tension, depression, anger, fatigue, and confusion, and typically debilitates performance efforts (Terry, 1995). Research now suggests that athletic performance is closely related to mood for some individuals but relatively independent of mood for others (Totterdell, 1999; Lane and Chappell, 2001). between psychologist and client, which may facilitate both early identification of problems and timely resolution (Terry, 1995). An internet-based mood profiling system based on the BRUMS, referred to as the In The Mood website (http://www. moodprofiling.com; Lim, 2011; Terry et al., 2013) has been developed. Transcending the barriers of distance and access to expertise, the In The Mood website facilitates mood profiling in populations not previously considered. In summary, several previous studies have demonstrated that mood profiles of athletes often differ from the general population, and explicated how mood responses relate to sports performance (Beedie et al., 2000; Prapavessis, 2000). Three stereotypical mood profiles have previously been identified, termed the iceberg profile (Morgan, 1980), the Everest profile (Terry, 1995), and the inverse iceberg profile (Terry, 1995). Despite several hundred published studies on mood profiling, it remains unknown if distinct mood profile clusters are identifiable beyond the realm of sport and exercise. Hence, the primary objective of the present study was to investigate if relatively consistent mood patterning was evident among the general population using a web-based delivery method to assess mood. From an applied practitioner perspective, mood profiling has utility for predicting the performance of athletes by means of individualized assessment of idiosyncratic mood-performance relationships (Terry, 1995). Citation: Mood profiling has been used to monitor adaptations to rigorous training schedules (Prapavessis et al., 1992; Raglin and Morgan, 1994), assess risk of athlete staleness or burnout caused by overtraining (Morgan et al., 1987), and monitor recovery from overtraining syndrome (Terry, 2004), defined as an acute or chronic state of incompetence causing decreases or plateaux in performance ability (Lemyre et al., 2007). The inverse iceberg mood profile may provide an important diagnostic indicator for overtraining syndrome (Budgett, 1998) and may possibly be indicative of a range of mental health disorders. Frontiers in Psychology | www.frontiersin.org Participants The total number of participants involved in the study was 6,532 individuals (male = 3,659, female = 2,873) spread over three samples. Sample A included 2,364 participants (male = 1,219, female = 1,145), Sample B included 2,303 participants (male = 1,288, female = 1,015), and Sample C included 1,865 participants (male = 1,152, female = 713). Participants in each sample were aged from 18 to 65 years or older, and reported a range of educational levels and ethnicities. The sociodemographic composition of each sample is detailed in Table 1. The three samples differed significantly by gender, age, education and ethnicity. Additional applications within the sport context include monitoring psychological responses to travel fatigue and jetlag (Terry and Lane, 2011), assessing the effectiveness of injury rehabilitation programs (Pearson and Jones, 1992) and quantifying the mood benefits of music (Terry et al., 2012). Mood profiling can also help to discriminate athletes at risk of eating disorders, with BRUMS scores successfully screening out athletes not at risk of pathogenic behaviors with 91% efficiency (Terry and Galambos, 2004). Beyond the realm of sport, mood profiling has been used as a screening tool for post-traumatic stress risk in military populations. For example, van Wijk et al. (2013) found that a BRUMS cut-offscore of ≥24 for total mood disturbance (i.e., sum of scores for tension, depression, anger, fatigue and confusion minus vigor score) at demobilization gave a sensitivity of 100% and specificity of 79% for subsequent post-traumatic symptoms. In a similar vein, mood profiling was used to investigate effects of stress during basic army combat training (Lieberman et al., 2016). Other applications of mood profiling include monitoring the psychological well- being of cardiac rehabilitation patients (Sties et al., 2014), post- operative adjustment following prostate surgery (Braslis et al., 1995), post-menopausal symptomology (Wyrwich and Yu, 2011), adolescent suicide risk (Gould et al., 2005), and subjective effects of pharmaceuticals (Salzman et al., 1995) and illicit drugs (Weddington et al., 1990). Mood profiling has also played a valuable role in workplace assessment (Morfeld et al., 2007). Brunel Mood Scale (BRUMS) Mood responses were assessed using the BRUMS (Terry et al., 1999, 2003), a scale of 24 mood descriptors using a standard response timeframe of “How do you feel right now?” Participants rated their mood responses on a 5-point Likert scale of 0 = not at all, 1 = a little, 2 = moderately, 3 = quite a bit, and 4 = extremely. The BRUMS has six subscales (i.e., anger, confusion, depression, fatigue, tension, and vigor), with four items each. Total subscale scores range from 0 to 16. The BRUMS measurement model was validated using multi-sample confirmatory factor analysis (Terry et al., 2003) across samples of adult students (n = 656), adult athletes (n = 1,984), young athletes (n = 676), and schoolchildren (n = 596). Subscales have demonstrated adequate internal consistency, with Cronbach alpha coefficients reported as: tension = 0.74, depression = 0.85, anger = 0.82, vigor = 0.85, fatigue = 0.90, and confusion = 0.83 (Terry et al., 1999). Test- retest reliability coefficients have ranged from 0.26 to 0.53 over a 1-week period (Terry et al., 1999, 2003), which is appropriate for a measure of transient feeling states. The psychometric robustness Collectively, research points to the utility of mood profiling in both clinical and non-clinical settings. More generally, mood profiles provide a valuable catalyst for discussion November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 2 Mood Profile Clusters Parsons-Smith et al. TABLE 1 | Sociodemographic characteristics of participants. Brunel Mood Scale (BRUMS) Sample A Sample B Sample C Source n % n % n % χ2 Total 2,364 100.0 2,303 100.0 1,865 100.0 Gender 44.07† Male 1,219 51.6 1,288 55.9 1,152 61.8 Female 1,145 48.4 1,015 44.1 713 38.2 Age Group (years) 541.47† 18–24 1,416 59.9 1,491 64.7 767 41.1 25–35 356 15.1 420 18.2 277 14.9 36–45 353 14.9 201 8.7 306 16.4 46–55 138 5.8 120 5.2 352 18.9 56–65 87 3.7 54 2.3 163 8.7 >65 14 0.6 17 0.7 0 0.0 Education 818.14† < High School 41 1.7 204 8.9 57 3.1 High School 1,221 51.6 745 32.3 654 35.1 Trade 0 0.0 0 0.0 96 5.2 TAFE 0 0.0 0 0.0 107 5.7 University 709 30.0 896 38.9 571 30.6 Postgraduate 393 16.6 458 19.8 380 20.4 Ethnicity 1, 144.61† African 123 5.2 137 5.9 159 8.5 Asian 136 5.8 0 0.0 0 0.0 Caucasian 962 40.7 1,628 70.7 1,513 81.1 Indigenous 39 1.6 19 0.8 12 0.6 Middle Eastern 81 3.4 68 3.0 23 1.2 Other 1,023 43.3 451 19.6 158 8.5 TAFE, Technical and Further Education (e.g., hospitality, tourism); Trade, qualified tradesperson (e.g., plumber, electrician). TAFE and Trade categories not used in Sample A and Sample B. †p < 0.001. TABLE 1 | Sociodemographic characteristics of participants. Alternatively, users could navigate away from the informed consent webpage, or withdraw from the study by clicking “I do not wish to participate, take me away.” Closing the browser window also exited the In The Mood website without data collection. Using a snowballing technique, data were collected over a 3-year period, with data downloaded periodically into three separate datasets. The respective data collection periods for Sample A, Sample B, and Sample C were March 2011 to October 2011, November 2011 to October 2013, and November 2013 to May 2014. Lim (2011) showed that mood responses derived from the In The Mood website did not differ significantly from data collected using the hardcopy version of the BRUMS. The research was granted ethical approval by the Human Research Ethics Committee at the University of Southern Queensland (H13REA169). Data Screening C Data Screening Cases were screened for implausible responses and deleted where identified. Given that all BRUMS questions required a response before data were transferred to the secure database, no missing values were detected. Although significant univariate non-normality was evident for some subscales (e.g., depression), it is typical that the distribution of negative mood scores show large numbers at the lower end of the scale, and smaller numbers at the upper end (Terry et al., 1999). Following visual inspection of the frequency distributions for skewness and kurtosis, it was concluded that deviations from normal distribution were unlikely to make a substantive difference to the analyses, and no trimming of the dataset occurred. A total of 217 multivariate outliers, based on Mahalanobis distance statistics at p < 0.001, were identified but scrutiny of individual cases suggested that they were all plausible response patterns. Further, in population studies, scores approaching the extremes of scale ranges are of particular interest when they reflect unusual although legitimate mood responses (Tabachnick and Fidell, 1996). Hence, multivariate outliers were retained in the dataset. The full range of scores from 0 to 16 was evident for each of the BRUMS subscales. Mean T-scores, standard deviations, and In The Mood Website Development of the In The Mood website (Lim, 2011; Terry et al., 2013) was guided by the conceptual framework of Lane and Terry (2000). Once respondents complete the BRUMS, an automated report is generated that interprets scores for the six mood dimensions with reference to normative scores, and a brief summary of the potential influence of obtained mood scores on performance. Raw and standard scores plus a graphical representation of the individual mood profile are presented to respondents, and where appropriate, a series of evidence- based mood regulation strategies corresponding with each mood dimension is provided. Data Analysis Cluster Analysis y Agglomerative, hierarchical cluster analysis was used to distinguish cluster metrics, and k-means clustering with random seeds was used to refine the final parameter solution. Cluster analysis is an exploratory technique designed to delineate natural groups that are undefined a priori. Given that hierarchical and partitioning computations will group even random unrelated data (Mooi and Sarstedt, 2011), theoretical considerations are especially salient. Using an iterative procedure, cluster membership is re-evaluated and proximity metrics re-calculated to minimize within-group variance and maximize between- group variance (Everett, 1993). Ward’s method was used to determine cluster numbers, followed by the k-means method to fine tune cluster boundaries, as recommended by Clatworthy et al. (2007). All analyses were conducted using the Statistical Package for the Social Sciences, version 23. TAFE, Technical and Further Education (e.g., hospitality, tourism); Trade, qualified tradesperson (e.g., plumber, electrician). TAFE and Trade categories not used in Sample A and Sample B. †p < 0.001. of the BRUMS makes it an appropriate measure in performance environments and its brevity lends itself well to web-based mood profiling. Sample A p Data were analyzed using agglomerative, hierarchical cluster analysis. Ward’s method was the chosen clustering algorithm, given theoretical considerations that both the shape and magnitude of the mood profiles would be relevant. Squared euclidean distance was used as the proximity measure to maximize differences between heterogeneous groups. Three checks were conducted to verify the cluster solution (Blashfield, 1980). First, visual examination of the scree plot showed a clear change in trajectory at a 6-cluster solution. Second, review of the final 25 cases of the agglomeration schedule showed a change in distance coefficients at case 2,358. Third, each cluster solution was reviewed, including the member contributions for each possible solution. Five distinct clusters were traced back from step six: H2 (n = 109), H3 (n = 474), H4 (n = 455), H5 (n = 302), and H6 (n = 630). H1 (n = 394) was not as stable as the other five clusters, in that H1 (n = 284) and H7 (n = 110) combined immediately before the sixth step. However, given that the scree plot showed a distinct elbow, and distance coefficients increased from case 2,358, a 6-cluster solution was judged to provide the best fit to the data. Cluster 1 was previously identified in the literature as the inverse iceberg profile (Terry, 1995), characterized by low vigor, together with high tension, depression, anger, fatigue, and confusion. Cluster 2, a novel mood profile, was termed the inverse Everest profile, characterized by low vigor, together with high tension and fatigue, and very high depression, anger, and confusion. Cluster 3, a second novel mood profile, was termed the surface profile, characterized by slightly above average levels of tension, depression, anger, vigor, fatigue, and confusion. Cluster 4 was the classic iceberg profile (Morgan, 1980), characterized by high vigor, together with low tension, depression, anger, fatigue, TABLE 3 | Inter-correlation matrix of the hierarchical and K-means clusters (n = 2,364). Hierarchical K-means H1 H2 H3 H4 H5 H6 K1 0.97 0.18 0.89 0.27 0.38 0.15 K2 −0.03 1.00 −0.06 −0.89 −0.80 −0.93 K3 0.57 −0.83 0.50 0.99 0.93 0.94 K4 0.37 −0.94 0.41 0.97 0.96 1.00 K5 0.90 0.02 0.99 0.33 0.55 0.31 K6 0.67 −0.76 0.70 0.93 0.99 0.93 H1, H2, … H6 denotes hierarchical clusters identified in Sample A. K1, K2, … K6 denotes k-means clusters identified in Sample A. Procedure Adult participants (≥18 years) were recruited from the general population via the In The Mood website (Lim, 2011; Terry et al., 2013). Respondents provided informed consent by clicking on the “I agree” checkbox, which initiated a link to the BRUMS. November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 3 Mood Profile Clusters Parsons-Smith et al. 95% confidence intervals for each mood dimension within each sample are provided in Table 2. Following the initial identification of the six clusters, a partitioning method was used to validate the findings, and further refine the final parameter solution. K-means clustering was conducted using random aggregation centers with a prescribed 6-cluster solution. The hierarchical and k-means techniques both produced clusters that pooled a large proportion of shared variance (see Table 3). Additionally, the inter-correlations between the prescribed k-means solution yielded a similar result to the inter-correlations from the hierarchical cluster analysis. Large negative correlations were found between K2 and K3, K4, and K6. A positive relationship was found between K1 and K5 with 82.8% common variance, while clusters K3, K4, and K6 were also related to one another sharing 88.4% to 90.3% common variance. Taken together, these findings provided strong evidence that the cluster structures were both independent and stable. All scores are T-scores. Sample A Function Eigenvalue % of Variance Cumulative % Canonical correlation SAMPLE A 1 5.678 71.3 71.3 0.922 2 1.693 21.3 92.5 0.793 3 0.498 6.2 98.8 0.576 4 0.094 1.2 99.9 0.293 5 0.004 0.1 100.0 0.067 SAMPLE B 1 6.607 76.3 76.3 0.932 2 1.558 18.0 94.3 0.780 3 0.393 4.5 98.8 0.531 4 0.099 1.1 99.9 0.300 5 0.004 0.1 100.0 0.065 SAMPLE C 1 6.739 77.4 77.4 0.933 2 1.475 16.9 94.3 0.772 3 0.438 5.0 99.4 0.552 4 0.051 0.6 99.9 0.220 5 0.005 0.1 100.0 0.073 TABLE 5 | Discriminant functions for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). In line with the cut-offcriterion, only predictor variables with loadings of ± 0.30 were interpreted. Based on the structure matrix (see Table 6), mood dimensions that associated with DF1A included high levels of confusion, fatigue, tension, depression, and anger. Variables associated with DF2A included high levels of vigor, and low levels of fatigue, whereas those associated with DF3A included high levels of vigor and fatigue. Variables associated with DF4A included low levels of tension and high levels of depression, and those associated with DF5A included low levels of confusion and depression, and a high level of anger. DFA showed cluster membership to be classified correctly with a high degree of accuracy for all clusters (see Table 7). Prior probabilities were 10.3, 2.7, 14.8, 29.4, 17.3, and 25.5% for the inverse iceberg profile, inverse Everest profile, surface profile, iceberg profile, shark fin profile, and submerged profile, respectively. The proportional by chance accuracy rate was computed by squaring and summing the proportion of cases TABLE 4 | Descriptive statistics of the 6-cluster solution in Sample A (n = 2,364). Sample A H1 (n = 394), H2 (n = 109), H3 (n = 474), H4 (n = 455), H5 (n = 302), H6 (n = 630). K1 (n = 244), K2 (n = 64), K3 (n = 349), K4 (n = 695), K5 (n = 409), K6 (n = 603). Inter-correlations among the six clusters were examined to evaluate the extent to which clusters were mutually exclusive. Given the large sample size, even small correlations reached statistical significance and hence a criterion of < 0.70 was applied to signify that inter-correlations represented less than half of the covariance and were therefore indicative of substantial independence. Large negative correlations between H2 and H4, H5, and H6 represented reverse cluster patterning rather than denoting similarity. A strong positive relationship was found between cluster H1 and H3, with 81.0% shared variance. Additionally, H4, H5, and H6 were also found to be closely related, sharing 84.6–92.2% common variance. These inter- relationships suggested homogeneous clusters. Despite some clusters sharing a similar shape, mean scores for the six mood dimensions were sufficiently different to satisfy the criterion of heterogeneous groups according to a Ward’s analysis. TABLE 3 | Inter-correlation matrix of the hierarchical and K-means clusters (n = 2,364). TABLE 2 | Descriptive statistics for BRUMS subscales. Sample A (n = 2,364) Sample B (n = 2,303) Sample C (n = 1,865) Subscale M SD 95% CI M SD 95% CI M SD 95% CI Tension 46.65 7.81 [46.33, 46.96] 47.24 8.52 [46.89, 47.59] 45.89 8.00 [45.53, 46.25] Depression 49.95 10.26 [49.54, 50.36] 51.85 11.83 [51.37, 52.33] 51.12 10.82 [50.63, 51.61] Anger 49.80 8.29 [49.46, 50.13] 52.15 10.28 [51.73, 52.57] 50.89 9.35 [40.47, 51.32] Vigor 48.59 9.12 [48.22, 48.95] 49.44 9.26 [49.06, 49.82] 49.58 8.92 [49.18, 49.99] Fatigue 52.26 9.55 [51.88, 52.65] 52.64 9.42 [52.26, 53.03] 52.17 9.38 [51.74, 52.59] Confusion 49.81 9.53 [49.43, 50.20] 51.72 10.66 [51.29, 52.16] 49.48 9.54 [49.04, 49.91] All scores are T-scores. November 2017 | Volume 8 | Article 1958 4 Frontiers in Psychology | www.frontiersin.org Mood Profile Clusters Parsons-Smith et al. in each group from the table of prior probabilities for groups (i.e., 0.1032 + 0.0272 + 0.1482 + 0.2942 + 0.1732 + 0.2552 = 0.215). Additionally, when the discriminant functions were used to predict group membership, the hit ratio was very high. A total of 95.2% of cases were correctly reclassified back into the original categories. November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org Sample A This percentage was notably higher than the minimum classification accuracy rate of 46.5% (i.e., the proportional by chance accuracy rate + 25%), suggesting that the overlap of the distributions was small, and the function was a good discriminator between groups. and confusion. Cluster 5, a third novel mood profile, was termed the shark fin profile, characterized by low tension, depression, anger, vigor, and confusion together with high fatigue. Finally, cluster 6, a fourth novel mood profile, was termed the submerged profile, characterized by low scores for tension, depression, anger, vigor, fatigue, and confusion. Table 4 includes descriptive statistics for the 6-cluster solution in Sample A. and confusion. Cluster 5, a third novel mood profile, was termed the shark fin profile, characterized by low tension, depression, anger, vigor, and confusion together with high fatigue. Finally, cluster 6, a fourth novel mood profile, was termed the submerged profile, characterized by low scores for tension, depression, anger, vigor, fatigue, and confusion. Table 4 includes descriptive statistics for the 6-cluster solution in Sample A. A post-hoc discriminant function analysis (DFA) was used to calculate the extent to which cases could be correctly classified into clusters. DFA is a two-step statistical procedure that involves significance testing of discriminant functions followed by calculation of correctly classified cases. The ratio of cases to independent variables was 394 to 1, which far exceeded the requirement of ≥20 to 1. The number of cases in the smallest cluster was 64, which exceeded the preferred number of cases (i.e., ≥20) per group. Five discriminant functions collectively accounted for 100% of the variance, and each function predicted significant variance (see Table 5). TABLE 5 | Discriminant functions for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). Sample A Iceberg profile (n = 695) Inverse Everest profile (n = 64) Inverse iceberg profile (n = 244) Mood dimension M SD 95% CI M SD 95% CI M SD 95% CI Tension 42.84 3.59 [42.58, 43.11] 67.70 8.64 [65.54, 69.86] 56.65 7.64 [55.69, 57.61] Depression 44.98 2.58 [44.79, 45.17] 87.17 11.95 [84.19, 90.16] 63.86 9.95 [62.61, 65.11] Anger 46.26 2.69 [46.06, 46.47] 79.05 10.81 [76.35, 81.75] 59.82 9.20 [58.66, 60.98] Vigor 57.33 5.32 [56.93, 57.73] 42.50 10.64 [39.84, 45.16] 45.73 7.54 [44.77, 46.68] Fatigue 45.72 4.69 [45.37, 46.07] 68.80 7.23 [67.02, 70.58] 60.80 8.38 [59.74, 61.85] Confusion 44.80 3.38 [44.55, 45.05] 80.39 11.22 [77.59, 83.19] 63.20 8.23 [62.16, 64.24] Shark fin profile (n = 409) Submerged profile (n = 603) Surface profile (n = 349) Tension 44.42 5.23 [43.91, 44.92] 43.23 4.18 [42.89, 43.56] 51.90 6.10 [51.26, 52.54] Depression 48.97 6.67 [48.32, 49.62] 46.34 4.75 [45.96, 46.72] 50.68 7.14 [49.93, 51.43] Anger 48.00 4.58 [47.55, 48.45] 46.50 3.14 [46.25, 46.75] 52.26 7.02 [51.52, 53.00] Vigor 41.12 6.58 [40.48, 41.76] 42.52 4.67 [42.15, 42.89] 53.51 6.34 [52.85, 54.18] Fatigue 64.16 6.22 [63.55, 64.76] 46.99 4.51 [46.63, 47.35] 51.46 5.85 [50.84, 52.07] Confusion 47.47 5.59 [46.93, 48.02] 45.99 4.65 [45.61, 46.36] 54.20 7.16 [53.44, 54.95] TABLE 4 | Descriptive statistics of the 6-cluster solution in Sample A (n = 2,364). 5 Mood Profile Clusters Parsons-Smith et al. TABLE 6 | Structure matrix and unstandardized canonical coefficients for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). Sample A Predicted group membership Cluster 1 2 3 4 5 6 N % SAMPLE A Iceberg 695 0 0 0 0 0 695 100.0 Inverse Everest 0 63 1 0 0 0 64 98.4 Inverse Iceberg 0 0 225 7 0 12 244 92.2 Shark Fin 4 0 1 386 17 1 409 94.4 Submerged 0 0 2 0 593 8 603 98.3 Surface 35 0 10 5 10 289 349 82.8 SAMPLE B Iceberg 684 0 0 1 1 0 686 99.7 Inverse Everest 0 76 7 0 0 0 83 91.6 Inverse Iceberg 0 1 273 1 0 9 284 96.1 Shark Fin 0 0 1 289 26 2 318 90.9 Submerged 15 0 0 0 565 6 586 96.4 Surface 34 0 4 2 12 294 346 85.0 SAMPLE C Iceberg 519 0 0 0 3 1 523 99.2 Inverse Everest 0 41 3 0 0 0 44 93.2 Inverse Iceberg 0 0 171 1 0 2 174 98.3 Shark Fin 1 0 0 286 18 2 307 93.2 Submerged 10 0 0 0 531 0 541 98.2 Surface 18 0 4 9 17 228 276 82.6 1, Iceberg Profile; 2, Inverse Everest Profile; 3, Inverse Iceberg Profile; 4, Shark Fin Profile; 5, Submerged Profile; 6, Surface Profile. The same six mood profile clusters identified in Sample A (i.e., iceberg, inverse Everest, inverse iceberg, shark fin, submerged, and surface profiles) were also evident in the other two samples. Descriptive statistics and cluster sizes for Sample B and Sample C are shown in Tables 9, 10, respectively. The smallest cluster had 83 cases in Sample B and 44 cases in Sample C, exceeding the minimum threshold of 20. The five discriminant functions extracted accounted for 100% of the variance in both samples, and each function predicted significant variance (see Table 5). p g Based on the structure matrix for Sample B, the mood dimensions strongly associated with DF1B included high levels of depression, confusion, tension, anger, and fatigue. The predictor variables strongly associated with DF2B included a high level of vigor and low fatigue. The predictor variables strongly associated with DF3B included a high level of vigor and fatigue, together with a low level of depression. Sample A Structure matrix Mood dimension DF1A DF2A DF3A DF4A DF5A DF1B DF2B DF3B DF4B DF5B DF1C DF2C DF3C DF4C DF5C Tension 0.445 0.268 −0.063 −0.691* −0.126 0.500 0.270 0.032 −0.647* 0.485 0.478 0.257 −0.075 0.604* 0.432 Depression 0.560 0.149 −0.169 0.673* −0.303 0.630* 0.169 −0.354 0.593 0.298 0.570 0.145 −0.130 −0.727* 0.119 Anger 0.494 0.234 −0.114 0.259 0.781* 0.487 0.225 −0.112 0.220 −0.521* 0.520* 0.254 −0.228 −0.225 0.247 Vigor −0.176 0.728* 0.663 0.012 0.015 −0.139 0.700* 0.671 0.187 −0.052 −0.141 0.756* 0.612 −0.025 −0.032 Fatigue 0.444 −0.545 0.706* −0.082 0.015 0.449 −0.590 0.659* 0.110 0.052 0.438 −0.531 0.715* 0.029 0.001 Confusion 0.546* 0.250 −0.155 −0.255 −0.404 0.560* 0.211 −0.039 −0.255 −0.412 0.564 0.220 −0.152 0.281 −0.712* Unstandardised canonical coefficients Tension 0.178 0.108 −0.059 −0.341 0.007 0.194 0.128 0.012 −0.353 0.285 0.187 0.115 −0.008 0.316 0.355 Depression 0.255 0.089 −0.026 0.451 −0.304 0.252 0.121 −0.164 0.467 0.352 0.222 0.100 0.023 −0.520 0.082 Anger 0.254 0.118 −0.078 0.044 0.571 0.178 0.044 −0.069 −0.011 −0.341 0.237 0.070 −0.187 0.019 0.134 Vigor −0.064 0.301 0.278 0.049 −0.026 −0.047 0.274 0.258 0.107 0.030 −0.055 0.312 0.265 −0.036 −0.020 Fatigue 0.182 −0.239 0.311 −0.013 0.005 0.180 −0.273 0.306 0.060 0.023 0.189 −0.236 0.321 0.022 0.005 Confusion 0.234 0.122 −0.075 −0.112 −0.228 0.210 0.089 0.003 −0.145 −0.329 0.267 0.105 −0.090 0.153 −0.594 *Largest absolute correlation between each variable and any discriminant function. DF1, DF2, … DF5 denotes discriminant functions. ABC denotes Sample A, Sample B, and Sample C, respectively. TABLE 6 | Structure matrix and unstandardized canonical coefficients for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). TABLE 6 | Structure matrix and unstandardized canonical coefficients for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). for each mood dimension in each sample are presented in Table 8. TABLE 7 | Classifications for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). TABLE 7 | Classifications for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). Sample A The predictor variables strongly associated with DF4B included low tension and high depression, while the predictor variables strongly associated with DF5B included low levels of anger and confusion, and a high level of tension. Based on the structure matrix for Sample C, the mood dimensions strongly associated with DF1C included high levels of anger, fatigue, depression, tension, and confusion. The predictor variables strongly associated with DF2C included a high level of vigor and low fatigue. The predictor variables strongly associated with DF3C included high levels of fatigue and vigor. The predictor variables strongly associated with DF4C included low levels of depression and high tension, while the predictor variables strongly associated with DF5C included high tension and low levels of confusion. The structure matrix and unstandardized canonical coefficients for each sample are shown in Table 6. 1, Iceberg Profile; 2, Inverse Everest Profile; 3, Inverse Iceberg Profile; 4, Shark Fin Profile; 5, Submerged Profile; 6, Surface Profile. Sociodemographic Distribution of Mood Profiles Chi-squared tests of goodness-of-fit were used to assess the distribution of mood profiles according to the gender, age and level of education of participants. The distribution of inverse Everest and surface profiles was independent of gender in all three samples (see Table 12). Females were significantly over- represented for the inverse iceberg profile in all three samples and for the shark fin profile in two samples. Conversely, males were significantly over-represented for the iceberg profile in all three samples. No clear trend was evident for the submerged profile. For age groupings, distribution of the submerged profile was independent of age in all three samples. Similarly, in Sample B and Sample C, the surface profile was independent of age grouping. Participants aged 18–24 were significantly over- represented for the shark fin profile and significantly under- represented for the inverse Everest profile in both Sample A and Sample B. Those aged 25–35 were significantly over-represented for the inverse Everest profile in Sample A and Sample B, significantly over-represented for the inverse iceberg in Sample B and Sample C, and significantly under-represented for the iceberg profile in Sample B and Sample C. Those aged 56–65 were significantly over-represented for the iceberg profile in Sample A and Sample B. The DFA showed that cluster membership was correctly classified with a high degree of accuracy for both Sample B and Sample C. Prior probabilities for Sample B and Sample C respectively were 12.3, 3.6, 15.0, 29.8, 13.8, 25.4% and 9.3, 2.4, 14.8, 28.0, 16.5, 29.0%, for the inverse iceberg profile, inverse Everest profile, surface profile, iceberg profile, shark fin profile, and submerged profile. The proportional by chance accuracy rates were also computed (i.e., 0.1232 + 0.0362 + 0.1502 + 0.2982 + 0.1382 + 0.2542 = 0.211 and 0.0932 + 0.0242 + 0.1482 + 0.2802 + 0.1652 + 0.2902 = 0.221, respectively). For level of education, distribution of the surface profile was independent in all three samples. Similarly, the submerged profile was independent of education level in Sample B and Sample C. High school certificate participants were significantly over-represented for the iceberg profile and significantly under- represented for the inverse iceberg profile in both Sample B and Sample C. Postgraduate participants were significantly under-represented for the shark fin profile in Sample A and Sample B. Replication of Mood Profile Clusters—Sample B and Sample C Clusters—Sample B and Sample C K-means clustering using random aggregation centers and a prescribed 6-cluster solution was used to replicate the findings derived from Sample A. Mean T-scores of the cluster centroids November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 6 Mood Profile Clusters Parsons-Smith et al. TABLE 8 | Cluster centroids for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). Cluster Mood dimension 1 2 3 4 5 6 SAMPLE A Tension 42.84 67.70 56.65 44.42 43.23 51.90 Depression 44.98 87.17 63.86 48.97 46.34 50.68 Anger 46.26 79.05 59.82 48.00 46.50 52.26 Vigor 57.33 42.50 45.73 41.12 42.52 53.51 Fatigue 45.72 68.80 60.80 64.16 46.99 51.46 Confusion 44.80 80.39 63.20 47.47 45.99 54.20 SAMPLE B Tension 42.33 66.76 59.16 45.22 42.91 51.72 Depression 45.22 89.53 67.97 50.09 47.26 52.10 Anger 47.24 81.71 64.87 50.15 47.26 54.49 Vigor 57.13 42.71 47.25 42.59 42.51 55.66 Fatigue 45.11 67.59 61.45 65.11 49.21 51.11 Confusion 45.27 80.67 66.09 50.39 46.55 55.77 SAMPLE C Tension 42.25 70.45 57.82 44.97 41.67 50.64 Depression 45.65 87.43 69.87 52.00 45.95 53.03 Anger 46.64 81.86 66.69 49.48 46.66 53.92 Vigor 58.84 45.39 46.90 42.35 44.58 52.26 Fatigue 45.69 70.00 60.91 64.10 47.02 52.92 Confusion 44.42 78.73 66.05 48.98 44.62 54.00 1, Iceberg Profile; 2, Inverse Everest Profile; 3, Inverse Iceberg Profile; 4, Shark Fin Profile; 5, Submerged Profile; 6, Surface Profile. TABLE 8 | Cluster centroids for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). narrow bounds (see Table 8). Additionally, the percentage of participants in each cluster was very similar across all three samples: inverse iceberg ∼10.6% (range = 9.3–12.3%), inverse Everest ∼2.9% (range = 2.4–3.6%), surface ∼14.9% (range = 14.8–15.0%), iceberg ∼29.1% (range = 28.0–29.8%), shark fin ∼15.9% (range = 13.8–17.3%), and submerged ∼26.6% (range = 25.4–29.0%) profile. Finally, the percentage of correct classification of cluster membership also showed little variation: inverse iceberg (range = 92.2–98.3%), inverse Everest (range = 91.6–98.4%), surface (range = 82.6–85.0%), iceberg (range = 99.2–100%), shark fin (range = 90.9–94.4%), and submerged (range = 96.4–98.3%) profile. Frontiers in Psychology | www.frontiersin.org Sociodemographic Distribution of Mood Profiles Those with a TAFE certificate were significantly over-represented for the inverse Everest and inverse iceberg profiles and significantly under-represented for the iceberg profile in Sample C. Finally, those with a trade qualification were significantly over-represented for the inverse iceberg profile in Sample C. p y Additionally, when the discriminant functions were used to predict group membership, the hit ratio was very high for both samples. A total of 94.7 and 95.2% of the cases were correctly reclassified back into the original categories for Sample B and Sample C, respectively. These percentages were notably higher than the minimum classification accuracy rate of 46.1% for Sample B, and 47.1% for Sample C. These findings indicate that overlap of distributions was small, and functions within each sample were good discriminators between groups. Table 11 lists the classification function coefficients for each sample. Overall, the k-means cluster analyses for Sample B and Sample C produced cluster structures that were very similar to those identified in Sample A. A visual summary of the 6-cluster solution across samples is provided in Figure 1. For ethnicity, African participants were significantly over- represented for the iceberg profile and significantly under- represented for the surface profile in both Sample A and Sample C. Asian participants were significantly over-represented for the Cluster scores were consistent across the three samples, with mean values for the various profiles constrained within relatively November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 7 Mood Profile Clusters Parsons-Smith et al. TABLE 9 | Descriptive statistics of the 6-cluster solution in Sample B (n = 2,303). Sociodemographic Distribution of Mood Profiles Iceberg profile (n = 686) Inverse Everest profile (n = 83) Inverse iceberg profile (n = 284) Mood dimension M SD 95% CI M SD 95% CI M SD 95% CI Tension 42.33 3.28 [42.08, 42.57] 66.76 9.75 [64.63, 68.89] 59.16 7.01 [58.34, 59.98] Depression 45.22 2.85 [45.01, 45.44] 89.53 10.30 [87.28, 91.78] 67.97 9.57 [66.85, 69.09] Anger 47.24 4.68 [46.89, 47.59] 81.71 11.18 [79.27, 84.15] 64.87 8.83 [63.84, 65.90] Vigor 57.13 5.41 [56.72, 57.53] 42.71 10.52 [40.41, 45.01] 47.25 8.01 [46.31, 48.18] Fatigue 45.11 4.66 [44.76, 45.46] 67.59 7.54 [65.94, 69.24] 61.45 7.15 [60.62, 62.29] Confusion 45.27 3.61 [45.00, 45.54] 80.67 10.00 [78.49, 82.86] 66.09 7.97 [65.16, 67.02] Shark fin profile (n = 318) Submerged profile (n = 586) Surface profile (n = 346) Tension 45.22 5.06 [44.67, 45.78] 42.91 3.71 [42.61, 43.21] 51.72 6.44 [51.04, 52.40] Depression 50.09 6.57 [49.37, 50.82] 47.26 5.41 [46.82, 47.70] 52.10 6.84 [51.37, 52.82] Anger 50.15 6.77 [49.40, 50.90] 47.26 4.02 [46.93, 47.58] 54.49 7.59 [53.68, 55.29] Vigor 42.59 7.44 [41.77, 43.41] 42.51 5.17 [42.09, 42.93] 55.66 6.59 [54.97, 56.36] Fatigue 65.11 5.88 [64.46, 65.76] 49.21 4.79 [48.82, 49.60] 51.11 5.12 [50.57, 51.65] Confusion 50.39 6.92 [49.62, 51.15] 46.55 4.89 [46.16, 46.95] 55.77 7.37 [54.99, 56.55] TABLE 10 | Descriptive statistics of the 6-cluster solution in Sample C (n = 1,865). Sociodemographic Distribution of Mood Profiles Iceberg profile (n = 523) Inverse Everest profile (n = 44) Inverse iceberg profile (n = 174) Mood dimension M SD 95% CI M SD 95% CI M SD 95% CI Tension 42.25 3.20 [41.97, 42.52] 70.45 7.32 [68.23, 72.68] 57.82 6.81 [56.80, 58.84] Depression 45.65 3.69 [45.33, 45.97] 87.43 11.91 [83.81, 91.05] 69.87 9.03 [68.52, 71.22] Anger 46.64 3.27 [46.36, 46.92] 81.86 9.86 [78.87, 84.86] 66.69 9.40 [65.28, 68.10] Vigor 58.84 4.94 [58.42, 59.27] 45.39 8.67 [42.75, 48.02] 46.90 7.95 [45.71, 48.09] Fatigue 45.69 4.72 [45.28, 46.09] 70.00 6.59 [68.00, 72.00] 60.91 7.21 [59.83, 61.99] Confusion 44.42 2.73 [44.19, 44.66] 78.73 9.26 [75.91, 81.54] 66.05 8.61 [64.76, 67.34] Shark fin profile (n = 307) Submerged profile (n = 541) Surface profile (n = 276) Tension 44.97 5.67 [44.33, 45.61] 41.67 2.84 [41.43, 41.91] 50.64 7.31 [49.77, 51.51] Depression 52.00 7.68 [51.14, 52.87] 45.95 3.95 [45.61, 46.28] 53.03 7.13 [52.18, 53.87] Anger 49.48 5.33 [48.88, 50.07] 46.66 3.19 [46.39, 46.93] 53.92 7.57 [53.03, 54.82] Vigor 42.35 6.37 [41.63, 43.06] 44.58 5.96 [44.07, 45.08] 52.26 6.38 [51.50, 53.02] Fatigue 64.10 6.43 [63.38, 64.82] 47.02 4.68 [46.62, 47.41] 52.92 5.83 [52.23, 53.61] Confusion 48.98 6.35 [48.26, 49.69] 44.62 3.25 [44.35, 44.90] 54.00 7.16 [53.15, 54.84] TABLE 10 | Descriptive statistics of the 6-cluster solution in Sample C (n = 1,865). ptive statistics of the 6-cluster solution in Sample C (n = 1,865). Sociodemographic Distribution of Mood Profiles Iceberg profile (n = 686) Inverse Everest profile (n = 83) Inverse iceberg profile (n = 284) Mood dimension M SD 95% CI M SD 95% CI M SD 95% CI Tension 42.33 3.28 [42.08, 42.57] 66.76 9.75 [64.63, 68.89] 59.16 7.01 [58.34, 59.98] Depression 45.22 2.85 [45.01, 45.44] 89.53 10.30 [87.28, 91.78] 67.97 9.57 [66.85, 69.09] Anger 47.24 4.68 [46.89, 47.59] 81.71 11.18 [79.27, 84.15] 64.87 8.83 [63.84, 65.90] Vigor 57.13 5.41 [56.72, 57.53] 42.71 10.52 [40.41, 45.01] 47.25 8.01 [46.31, 48.18] Fatigue 45.11 4.66 [44.76, 45.46] 67.59 7.54 [65.94, 69.24] 61.45 7.15 [60.62, 62.29] Confusion 45.27 3.61 [45.00, 45.54] 80.67 10.00 [78.49, 82.86] 66.09 7.97 [65.16, 67.02] Shark fin profile (n = 318) Submerged profile (n = 586) Surface profile (n = 346) Tension 45.22 5.06 [44.67, 45.78] 42.91 3.71 [42.61, 43.21] 51.72 6.44 [51.04, 52.40] Depression 50.09 6.57 [49.37, 50.82] 47.26 5.41 [46.82, 47.70] 52.10 6.84 [51.37, 52.82] Anger 50.15 6.77 [49.40, 50.90] 47.26 4.02 [46.93, 47.58] 54.49 7.59 [53.68, 55.29] Vigor 42.59 7.44 [41.77, 43.41] 42.51 5.17 [42.09, 42.93] 55.66 6.59 [54.97, 56.36] Fatigue 65.11 5.88 [64.46, 65.76] 49.21 4.79 [48.82, 49.60] 51.11 5.12 [50.57, 51.65] Confusion 50.39 6.92 [49.62, 51.15] 46.55 4.89 [46.16, 46.95] 55.77 7.37 [54.99, 56.55] TABLE 9 | Descriptive statistics of the 6-cluster solution in Sample B (n = 2,303). TABLE 10 | Descriptive statistics of the 6-cluster solution in Sample C (n = 1,865). Sociodemographic Distribution of Mood Profiles Iceberg profile (n = 523) Inverse Everest profile (n = 44) Inverse iceberg profile (n = 174) Mood dimension M SD 95% CI M SD 95% CI M SD 95% CI Tension 42.25 3.20 [41.97, 42.52] 70.45 7.32 [68.23, 72.68] 57.82 6.81 [56.80, 58.84] Depression 45.65 3.69 [45.33, 45.97] 87.43 11.91 [83.81, 91.05] 69.87 9.03 [68.52, 71.22] Anger 46.64 3.27 [46.36, 46.92] 81.86 9.86 [78.87, 84.86] 66.69 9.40 [65.28, 68.10] Vigor 58.84 4.94 [58.42, 59.27] 45.39 8.67 [42.75, 48.02] 46.90 7.95 [45.71, 48.09] Fatigue 45.69 4.72 [45.28, 46.09] 70.00 6.59 [68.00, 72.00] 60.91 7.21 [59.83, 61.99] Confusion 44.42 2.73 [44.19, 44.66] 78.73 9.26 [75.91, 81.54] 66.05 8.61 [64.76, 67.34] Shark fin profile (n = 307) Submerged profile (n = 541) Surface profile (n = 276) Tension 44.97 5.67 [44.33, 45.61] 41.67 2.84 [41.43, 41.91] 50.64 7.31 [49.77, 51.51] Depression 52.00 7.68 [51.14, 52.87] 45.95 3.95 [45.61, 46.28] 53.03 7.13 [52.18, 53.87] Anger 49.48 5.33 [48.88, 50.07] 46.66 3.19 [46.39, 46.93] 53.92 7.57 [53.03, 54.82] Vigor 42.35 6.37 [41.63, 43.06] 44.58 5.96 [44.07, 45.08] 52.26 6.38 [51.50, 53.02] Fatigue 64.10 6.43 [63.38, 64.82] 47.02 4.68 [46.62, 47.41] 52.92 5.83 [52.23, 53.61] Confusion 48.98 6.35 [48.26, 49.69] 44.62 3.25 [44.35, 44.90] 54.00 7.16 [53.15, 54.84] inverse Everest and inverse iceberg profiles and significantly under-represented for the shark fin profile in Sample A DISCUSSION TABLE 9 | Descriptive statistics of the 6-cluster solution in Sample B (n = 2,303). DISCUSSION inverse Everest and inverse iceberg profiles and significantly under-represented for the shark fin profile in Sample A. Indigenous participants were significantly over-represented for the iceberg profile in Sample A and significantly over-represented for the inverse iceberg profile in Sample B and Sample C. Middle Eastern participants were significantly over-represented for the iceberg profile in Sample A and Sample C. Caucasian participants, who formed the largest proportion of the total sample, showed several significant deviations from the expected distribution across mood profiles (Table 12) but no clear tends were evident. The sociodemographic trends reported above should be treated with caution due to violations of the underlying assumption of minimum cell counts for some categories of participants. Three distinct mood profiles (iceberg, inverse iceberg, and Everest profiles) were previously identified within athletic samples (Morgan, 1980; Terry, 1995). We investigated whether relatively consistent mood patterns were evident within the general population using a web-based delivery method. Three datasets gathered via the In The Mood website were interrogated using cluster analytic methodology. More specifically, the mood responses of Sample A (n = 2,364) were analyzed using a two- step clustering procedure. Six mood profiles were identified, including two established profiles (i.e., iceberg, inverse iceberg profiles) and four novel profiles (i.e., inverse Everest, shark fin, submerged, and surface profiles). Cluster membership was November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 8 Mood Profile Clusters Parsons-Smith et al. TABLE 11 | Classification function coefficients for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). DISCUSSION Cluster Mood dimension 1 2 3 4 5 6 SAMPLE A Tension 0.102 2.023 1.201 0.227 0.198 0.878 Depression 0.378 3.575 1.734 0.584 0.328 0.651 Anger 0.006 3.186 1.351 0.206 0.085 0.698 Vigor 1.852 0.945 1.094 0.753 0.829 1.571 Fatigue 0.458 2.116 1.551 1.859 0.540 0.854 Confusion 0.158 2.838 1.550 0.306 0.237 0.875 SAMPLE B Tension 0.161 1.938 1.484 0.324 0.203 0.964 Depression 0.441 3.442 1.830 0.509 0.393 0.677 Anger −0.212 1.793 0.859 0.091 −0.064 0.290 Vigor 1.642 0.854 1.079 0.769 0.750 1.539 Fatigue 0.447 2.083 1.673 2.086 0.773 0.885 Confusion 0.169 2.358 1.461 0.488 0.235 0.890 SAMPLE C Tension 0.285 2.548 1.468 0.472 0.189 0.994 Depression 0.506 3.223 1.997 0.769 0.314 0.771 Anger −0.283 2.715 1.480 0.095 −0.065 0.476 Vigor 1.942 1.060 1.145 0.835 0.953 1.484 Fatigue 0.473 2.373 1.668 1.957 0.577 1.055 Confusion 0.091 3.071 2.076 0.496 0.133 0.970 1, Iceberg Profile; 2, Inverse Everest Profile; 3, Inverse Iceberg Profile; 4, Shark Fin Profile; 5, Submerged Profile; 6, Surface Profile. TABLE 11 | Classification function coefficients for Sample A (n = 2,364), Sample B (n = 2,303), and Sample C (n = 1,865). et al., 2013). Our finding that ∼11% of the general population reported an inverse iceberg profile suggests that its prevalence is sufficient to warrant further investigations of associated risks and consequences in a range of environments beyond sport, exemplified by van Wijk’s et al. (2013) use of mood profiling to screen for risk of post-traumatic stress in military populations. By extension, the inverse Everest profile, a novel mood profile reported by about 3% of our combined sample and representing the most negative of the six mood profiles, may be indicative of clinical psychopathology. High scores for tension and fatigue, combined with very high scores for depression, anger and confusion, represents a profile that shares many of the symptoms of clinically diagnosable mental health conditions. Mood disorder severity occurs along a continuum, with increased symptomology corresponding with greater cognitive deficits, such as distorted thinking, reduced concentration, distractibility, slower reaction time, memory loss, and indecision (Sarapas et al., 2012). The inverse Everest profile would therefore likely associate with a broader range of negative cognitive and behavioral outcomes than previously demonstrated for the inverse iceberg profile, including debilitated performance. Confirming such associations empirically is a clear line of enquiry for future investigations. DISCUSSION The influence on human functioning of the shark fin profile, the second of the novel mood profiles, is also unknown. Notably, the shark fin profile lacks some markers of negative mood, such as high levels of tension, depression, anger, and confusion. It is reasonable to speculate, however, that a profile with the lowest vigor scores of all the profiles, combined with higher fatigue scores than any profile except the inverse Everest (see Figure 1) would have deleterious effects on functioning, particularly in environments requiring energy and alertness. The combination of high fatigue and low vigor is a well-established concern for patient safety in clinical health environments (Gaba and Howard, 2002), for road safety (Summala and Mikkola, 1994), and the safety of pilots and passengers in the aviation industry (Bourgeois-Bougrine et al., 2003; Jackson and Earl, 2006). Therefore, future investigations are needed to assess whether the shark fin profile is a potential contributor to accidents caused by impaired functioning in high-risk environments. correctly classified with a high degree of accuracy. Results were replicated in Sample B (n = 2,303) and Sample C (n = 1,865). Chi-squared tests of goodness-of-fit indicated that the distributions of sociodemographic variables (i.e., gender, age, education level, and ethnicity) across the six mood profiles were significantly different from expected cell counts in all three samples. These findings raise many research questions worthy of future investigation relating to the antecedents, correlates and consequences of the four novel mood profiles, as well as the profiles previously identified in the literature. The iceberg and Everest profiles have long been associated with healthy cognitive functioning and high to superior levels of physical performance (Morgan, 1980, 1985; Terry, 1995). Despite the longstanding line of investigation into the effects of mood in sporting and to a lesser extent educational contexts, far less is known about how mood affects human functioning in other domains. Hence, there is much scope for assessing the influence of mood profiles generally, and the potentially beneficial effects of the iceberg and Everest profiles in particular, in other intense performance environments, such as in medical, military, business, construction, and mining contexts. A third novel mood profile, the submerged profile, shares many characteristics of the iceberg profile, with below average scores for tension, depression, anger, fatigue, and confusion. Frontiers in Psychology | www.frontiersin.org DISCUSSION The sole difference between the submerged and iceberg profiles is the stark contrast in vigor scores, which sit about 15 percentile points (1.5 standard deviations) apart. Hence, the submerged profile is characterized by being relatively devoid of emotion, including vigor, which may well be described as feeling flat. Such a mood profile may impede goal-directed behavior in a variety of contexts, although the veracity of this suggestion is unknown, and consequently in need of investigation. The fourth novel mood profile, referred to as the surface profile, is characterized by scores on all mood dimensions being within the 50–56% range. As such, the surface profile approximates the baseline or waterline test norms originally identified by Morgan (1985), suggesting that this profile would be associated with normal functioning across a range of tasks and environments. The inverse iceberg profile has frequently been associated with debilitating conditions among athletes, including overtraining syndrome (Budgett, 1998), risk of eating disorders (Terry and Galambos, 2004) and reduced physical performance (Lahart November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 9 Parsons-Smith et al. Mood Profile Clusters FIGURE 1 | Visual summary of the 6-cluster solutions in three samples. FIGURE 1 | Visual summary of the 6-cluster solutions in three samples. Our findings and those derived from subsequent investigations of the six mood profiles identified in the present study may serve to extend and/or refine existing theoretical frameworks related to the mood construct. For example, Lane and Terry’s (2000) conceptual model of relationships between mood and performance emphasized the key role played by depressed mood in moderating the effects of anger and tension on performance. A recent study of more than 73,000 participants in an online experiment suggested several ways in which the conceptual model could be extended, for example, by accounting for the effects of mood regulation, suppression and re-appraisal strategies, use of psychological skills, and greater effort (Lane et al., 2017). Our identification of novel mood profiles will help to inform future iterations of the conceptual model. the inverse Everest and inverse iceberg profile. This trend is consistent with the age-of-onset distribution reported by Kessler et al. (2005), who identified 30 as the median age for mood disorders to emerge, and the lifetime incidence of mood disorders to be 20.8%. DISCUSSION Given that the inverse Everest profile was reported by ∼3% of the total number of participants in the present study, and the inverse iceberg profile by another 11% of participants, these two negative mood profiles may be indicative of risk of clinical mood disorders and hence could have utility for mental health screening purposes (e.g., van Wijk et al., 2013). g p p g j No clear trends emerged for the incidence of specific mood profiles according to level of education. This is consistent with recent overviews of the literature around the influence of education and socioeconomic status on mood disorders, notably bipolar disorder (Schoeyen et al., 2011; Eid et al., 2013). Further investigation of the relationship between education status and mood responses is worthwhile, although other sociodemographic variables appear more likely to yield meaningful insights. The trends for incidence of specific mood profiles by ethnicity were complex and should be treated with caution. There is strong evidence of differences in health status according to ethnicity, including variations in the incidence of mood disorders (Johnson-Lawrence et al., 2013). Given that examination of the link between mood profiles and ethnic background was not the central focus of the present study, we advise against drawing conclusions about the link from our data and recommend that further investigations be conducted in this area. The finding of sociodemographic differences in the incidence of specific mood profiles clearly warrants further investigation. Males were more likely to report the iceberg profile, whereas females were more likely to report the inverse iceberg and shark fin profiles. Given that the lifetime prevalence of clinical mood disorders in women has been shown to be approximately twice that of men (Steiner et al., 2003), the finding that females were significantly over-represented for the more negative mood profiles is not surprising, and offers support for the predictive validity of the profiles. Sub-clinical negative moods are also more prevalent among females than males (e.g., Butler and Nolen-Hoeksema, 1994; Monteagudo et al., 2013), and gender differences in hormonal activity (Soares, 2013) and use of mood regulation strategies such as rumination (Nolen-Hoeksema and Jackson, 2001) have been proposed as mechanisms to explain the greater prevalence of negative mood profiles among women. Frontiers in Psychology | www.frontiersin.org DISCUSSION From an applied practitioner perspective, identification of the six mood profile clusters may assist the interpretation of BRUMS test scores and may extend the utility of the measure in quantifying mood responses by providing a point of reference for attaching meaning to the mood profile. Moreover, the inverse Everest profile may function as an indicator of potential psychopathology among non-clinical samples. Determining the Findings about age-related effects on the incidence of specific mood profiles showed the 25–35 age group to be under- represented for the iceberg profile and over-represented for November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 10 Mood Profile Clusters Parsons-Smith et al. TABLE 12 | Continued Cluster Source 1 2 3 4 5 6 CaucasianB 515§+ 52 179§− 214 443§+ 225*− IndigenousB 1*− 3§+ 6§+ 1 3 5 Middle EasternB 21 7§+ 12 4 7§− 17*+ OtherB 112§− 14 73§+ 73 98*− 81 AfricanC 60§+ 3 12 14§− 59*+ 11§− CaucasianC 403§− 38 140 273†+ 427 232 IndigenousC 4 0 4§+ 1 2 1 Middle EasternC 12§+ 2*+ 1 0*− 6 2 OtherC 44 1 17 19 47 30 1, Iceberg Profile; 2, Inverse Everest Profile; 3, Inverse Iceberg Profile; 4, Shark Fin Profile; 5, Submerged Profile; 6, Surface Profile. TAFE, Technical and Further Education (e.g., hospitality, tourism); Trade, qualified tradesperson (e.g., plumber, electrician). ASample A (n = 2,364). BSample B (n = 2,303). CSample C (n = 1,865). +, over-represented; -, under-represented. *p < 0.05, §p < 0.01, †p < 0.001. TABLE 12 | Distribution of mood profile clusters by gender, age, education, and ethnicity. TABLE 12 | Continued TABLE 12 | Distribution of mood profile clusters by gender, age, education, and ethnicity. DISCUSSION Cluster Source 1 2 3 4 5 6 GENDER MaleA 406†+ 33 107*− 196 288*− 189 FemaleA 289†− 31 137*+ 213 315*+ 160 MaleB 431†+ 51 139*− 150†− 318 199 FemaleB 255†− 32 145*+ 168†+ 268 147 MaleC 381†+ 24 92*− 137†− 357*+ 161 FemaleC 142†− 20 82*+ 170†+ 184*− 115 AGE GROUP (YR.) 18–24A 358†− 29*− 151 274§+ 374 230*+ 25–35A 110 22†+ 33 54 89 48 36–45A 138†+ 7 35 55 79 39*− 46–55A 46 3 19 15*− 35 20 56–65A 38§+ 1 5 10 24 9 > 65A 5 2§+ 1 1 2 3 18–24B 448 38†− 173 226*+ 374 232 25–35B 94†− 24§+ 68§+ 59 112 63 36–45B 67 10 15*− 22 61 26 46–55B 45 6 17 10 27 15 56–65B 23*+ 2 10 1§− 8 10 > 65B 9*+ 3§+ 1 0 4 0 18–24C 223 15 55§− 131 225 118 25–35C 52†− 9 40§+ 63§+ 72 41 36–45C 87 8 26 44 99 42 46–55C 112 8 31 50 104 47 56–65C 49 4 22 19 41 28 EDUCATION < High SchoolA 4§− 1 8*+ 5 20†+ 3 High SchoolA 337*− 25*− 124 235§+ 317 183 UniversityA 233*+ 22 64 116 178 96 PostgraduateA 121 16 48 53*− 88 67 < High SchoolB 52 8 32 23 55 34 High SchoolB 255§+ 21 76*− 101 184 108 UniversityB 243*− 31 102 149§+ 234 137 PostgraduateB 136 23 74§+ 45§− 113 67 < High SchoolC 19 0 5 10 18 5 High SchoolC 202*+ 15 42§− 110 187 98 TAFEC 20*− 6*+ 23†+ 17 26 15 TradeC 28 1 15*+ 16 26 10 UniversityC 165 12 49 89 170 86 PostgraduateC 89*− 10 40 65 114 62 ETHNICITY AfricanA 56†+ 0 7 19 32 9*− AsianA 33 9§+ 22*+ 9†− 43 20 CaucasianA 263 32 117*+ 169 216§− 165§+ IndigenousA 20§+ 0 2 6 9 2 Middle EasternA 41†+ 2 8 11 14 5*− OtherA 282 21 88*− 195*+ 289§+ 148 AfricanB 37 7 14 26 35 18 (Continued) therapeutic meaningfulness and predictive effectiveness of mood profiles appear to be logical directions for future research. Further, the empirical examination of potential links between specific mood profiles and dimensions of personality according to the five-factor model (i.e., extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience; Costa and McCrae, 1992) may also yield beneficial findings, from both theoretical and practical standpoints. November 2017 | Volume 8 | Article 1958 REFERENCES Jackson, C. A., and Earl, L. (2006). Prevalence of fatigue among commercial pilots. Occupat. Med. 56, 263–268. doi: 10.1093/occmed/kql021 Beedie, C. J., Terry, P. C., and Lane, A. M. (2000). The Profile of Mood States and athletic performance: two meta-analyses. J. Appl. Sport Psychol. 12, 49–68. doi: 10.1080/10413200008404213 Johnson-Lawrence, V., Griffith, D. M., and Watkins, D. C. (2013). The effects of race, ethnicity, and mood/anxiety disorders on the chronic physical health conditions of men from a national sample. Am. J. Men’s Health 7, 58S−67S. doi: 10.1177/1557988313484960 Blashfield, R. K. (1980). Propositions regarding the use of cluster analysis in clinical research. J. Consult. Clin. Psychol. 48, 456–459. doi: 10.1037/0022-006X.48.4.456 Kessler, R. C., Berglund, P., Demler, O., Jin, R., Merikangas, K. R., and Walters, E. E. (2005). Lifetime prevalence and age-of-onset distributions of DSM- IV disorders in the National Comorbidity Survey Replication. Arch. Gen. Psychiatry 62, 593–602. doi: 10.1001/archpsyc.62.6.593 Bourgeois-Bougrine, S., Carbon, P., Gounelle, C., Mollard, R., and Coblentz, A. (2003). Perceived fatigue for short- and long-haul flights: a survey of 739 airline pilots. Aviat. Space Environ. Med. 74, 1072–1077. Available online at: http:// www.ingentaconnect.com/content/asma/asem/2003/00000074/00000010/ art00010 Lahart, I. M., Lane, A. M., Hulton, A., Williams, K., Godfrey, R., Pedlar, C., et al. (2013). Challenges in maintaining emotion regulation in a sleep and energy deprived state induced by the 4800 km ultra-endurance bicycle race: the Race Across AMerica (RAAM). J. Sports Sci. Med. 12, 481–488. Available online at: https://doaj.org/article/2a2f3c6e2e5e4160bad6b7817a89117e Braslis, K. G., SantaCruz, C., Brickman, A. L., and Soloway, M. S. (1995). Quality of life 12 months after radical prostatectomy. BJU Int. 75, 48–53. doi: 10.1111/j.1464-410X.1995.tb07231.x https://doaj.org/article/2a2f3c6e2e5e4160bad6b7817a89117e Lane, A. M., and Chappell, R. C. (2001). Mood and performance relationships among players at the World Student Games basketball competition. J. Sport Behav. 24, 182–196. Available online at: http://www.biomedsearch.com/article/ Mood-performance-relationships-among-players/75179370.html Budgett, R. (1998). Fatigue and underperformance in athletes: the overtraining syndrome. Br. J. Sports Med. 32, 107–110. doi: 10.1136/bjsm.32.2.107 ood-performance-relationships-among-players/75179370.html Butler, L. D., and Nolen-Hoeksema, S. (1994). Gender differences in responses to depressed mood in a college sample. Sex Roles 30, 331–346. doi: 10.1007/BF01420597 Lane, A. M., and Terry, P. C. (2000). The nature of mood: development of a conceptual model with a focus on depression. J. Appl. Sport Psychol. 12, 16–33. doi: 10.1080/10413200008404211 Clatworthy, J., Hankins, M., Buick, D., Weinman, J., and Horne, R. (2007). Cluster analysis in illness perception research: a Monte Carlo study to identify the most appropriate method. Psychol. ETHICS STATEMENT This study was carried out in accordance with the protocol approved by the University of Southern Queensland’s Office of Research and Higher Degrees, Human Research Ethics Committee (approval number: H13REA169). All subjects gave informed consent in accordance with the National Statement on Ethical Conduct in Human Research (2007). ACKNOWLEDGMENTS RP-S—Conception and design of study, acquisition of data, performed analysis on all samples, interpreted data, drafting the manuscript and acted as corresponding author. Final approval of the version to be published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the We thank Associate Professor Graeme Senior from the University of Southern Queensland for his assistance with the hierarchical and k-means cluster analytic techniques. Strengths and Limitations The primary strength of the current research lies in the fact that the same six mood profile clusters were identified in three large samples that were sociodemographically heterogeneous. The agglomerative, hierarchical cluster analysis followed by the k-means iterative technique produced similar multivariate structures, signaling a robust method of allocation of cases. Although the web-based delivery method and snowballing technique for data collection produced three large and heterogeneous samples, the convenience sampling method may have introduced an element of bias, given that access to the Internet was required for participation. However, replication of the 6-cluster solution in each of the three independent samples represents substantive evidence to support the consistency of the cluster structures. Limitations are evident regarding the sociodemographic analyses. The small number of participants in the less than high school certificate category (range = 1.7–8.9%) and over 65 age group (range = 0–0.7%) raises the question of whether they adequately represent the underlying populations of interest. Additionally, small cell sizes made results for some analyses uninterpretable and others questionable due to violation of underlying assumptions. Analyses involving the inverse Everest mood profile were most affected given the modest number of participants reporting that profile. Finally, the ecological validity November 2017 | Volume 8 | Article 1958 11 Frontiers in Psychology | www.frontiersin.org Mood Profile Clusters Parsons-Smith et al. accuracy or integrity of any part of the work are appropriately investigated and resolved. PT—Conception and design of study, acquisition of data, supervised development of work, helped in data interpretation, drafting and revising the manuscript critically for important intellectual content, final approval of the version to be published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. MM—Conception and design of study, co-supervised development of work, revising the manuscript critically for important intellectual content, final approval of the version to be published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. of the mood profiles is still to be determined. However, further investigation of relationships between various aspects of human functioning and the distinct mood profiles identified in the present study seems likely to yield new insights. REFERENCES Health 22, 123–142. doi: 10.1080/14768320600774496 Lane, A. M., Terry, P. C., Devonport, T. J., Friesen, A. P., and Totterdell, P. A. (2017). A test and extension of Lane and Terry’s (2000) conceptual model of mood-performance relationships using a large internet sample. Front. Psychol. 8:470. doi: 10.3389/fpsyg.2017.00470 Costa, P. T. Jr., and McCrae, R. R. (1992). Revised NEO Personality Inventory (NEO-PI-R) and NEO Five-Factor (NEO-FFI) Inventory Professional Manual. Odessa, FL: Psychological Assessment Resources. Lemyre, P. N., Roberts, G. C., and Stray-Gundersen, J. (2007). Motivation, overtraining, and burnout: can self-determined motivation predict overtraining and burnout in elite athletes? Eur. J. Sport Sci. 7, 115–126. doi: 10.1080/17461390701302607 Eid, L., Heim, K., Doucette, S., McCloskey, S., Duffy, A., and Grof, P. (2013). Bipolar disorder and socioeconomic status: what is the nature of this relationship? Int. J. Bipolar Disord. 1:9. doi: 10.1186/2194-7511-1-9 Lieberman, H. R., Karl, J. P., McClung, J. P., Williams, K. W., and Cable, S. (2016). Improved mood state and absence of sex differences in response to the stress of Army Basic Combat Training. Appl. Psychol. Health Well Being 8, 351–363. doi: 10.1111/aphw.12075 p p Everett, B. S. (1993). Cluster Analysis, 3rd Edn. New York, NY: Wiley. Gaba, D. M., and Howard, S. K. (2002). Fatigue among clinicians and the safety of patients. New Engl. J. Med. 347, 1249–1255. doi: 10.1056/NEJMsa020846 Gould, M. S., Marrocco, F. A., Kleinman, M., Thomas, J. G., Mostkoff, K., Cote, J., et al. (2005). Evaluating iatrogenic risk of youth suicide screening programs: a randomized controlled trial. J. Am. Med. Assoc. 293, 1635–1643. doi: 10.1001/jama.293.13.1635 Lim, J. (2011). The Development and Subsequent Evaluation of a Website to Assess and to Provide Mood Regulation Strategies Based on the Pattern of Responses to the Brunel Mood Scale (BRUMS). Unpublished doctoral thesis, University of Southern Queensland, Australia. November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 12 Parsons-Smith et al. Mood Profile Clusters McNair, D. M., Lorr, M., and Droppelman, L. F. (1971). Manual for the Profile of Mood States. San Diego, CA: Educational and Industrial Testing Services. Steiner, M., Dunn, E., and Born, L. (2003). Hormones and mood: from menarche to menopause and beyond. J. Affect. Disord. 74, 67–83. doi: 10.1016/S0165-0327(02)00432-9 McNair, D. M., Lorr, M., and Droppelman, L. F. (1992). Revised Manual for the Profile of Mood States. San Diego, CA: Educational and Industrial Testing Services. Sties, S. W., Gonzales, A. I., Netto, A. REFERENCES doi: 10.1080/10413200008404215 Nolen-Hoeksema, S., and Jackson, B. (2001). Mediators of the gender difference in rumination. Psychol. Women Q. 25, 37–47. doi: 10.1111/1471-6402.00005 Terry, P. C., and Lane, A.M. (2011). “Moods and emotions,” in The New Sport and Exercise Psychology Companion, eds T. Morris and P. C. Terry (Morgantown, WV: Fitness Information Technology), 63–87. Paulhus, D. L., and Vazire, S. (2007). “The self-report method,” in Handbook of Research Methods in Personality Psychology, eds R. W. Robins, R. C. Fraley, and R. F. Krueger (New York, NY: Guilford), 224–239. Pearson, L., and Jones, G. (1992). Emotional effects of sports injuries: implications for physiotherapists. Physiotherapy 78, 762–770. doi: 10.1016/S0031-9406(10)61642-2 Terry, P. C., Lane, A. M., and Fogarty, G. J. (2003). Construct validity of the Profile of Mood States-Adolescents for use with adults. Psychol. Sport Exerc. 4, 125–139. doi: 10.1016/S1469-0292(01)00035-8 Terry, P. C., Lane, A. M., Lane, H. J., and Keohane, L. (1999). Development and validation of a mood measure for adolescents. J. Sports Sci. 17, 861–872. doi: 10.1080/026404199365425 Prapavessis, H. (2000). The POMS and sports performance: a review. J. Appl. Sport Psychol. 12, 34–48. doi: 10.1080/10413200008404212 Prapavessis, H., Berger, B., and Grove, J. R. (1992). The relationship of training and pre-competition mood states to swimming performance: an exploratory investigation. Aust. J. Sci. Med. Sport 24, 12–17. Terry, P. C., Lim, J., and Parsons-Smith, R. L. (2013). In The Mood. Available online at: http://www.moodprofiling.com Raglin, J. S., and Morgan, W. P. (1994). Development of a scale for use in monitoring training induced distress in athletes. Int. J. Sports Med. 15, 84–88. doi: 10.1055/s-2007-1021025 Totterdell, P. (1999). Mood scores: mood and performance in professional cricketers. Br. J. Psychol. 90, 317–332. doi: 10.1348/000712699161422 van Wijk, C. H., Martin, J. H., and Hans-Arendse, C. (2013). Clinical utility of the Brunel Mood Scale in screening for post-traumatic stress risk in a Military population. Military Med. 178, 372–376. doi: 10.7205/MILMED-D-12-00422 Renger, R. (1993). A review of the Profile of Mood States (POMS) in the prediction of athletic success. J. Appl. Sport Psychol. 5, 78–84. doi: 10.1080/10413209308411306 Weddington, W. W., Brown, B. S., Haertzen, C. A., Cone, E. J., Dax, E. M., Herning, R. I., , et al. (1990). Changes in mood, craving, and sleep during short-term abstinence reported by male cocaine addicts. Arch. Gen. Psychiatry 47, 861–868. doi: 10.1001/archpsyc.1990.01810210069010 Rowley, A. J., Landers, D. M., Kyllo, L. B., and Etnier, J. L. (1995). REFERENCES S., Wittkopf, P. G., Lima, D. P., and de Carvalho, T. (2014). Validation of the Brunel Mood Scale for cardiac rehabilitation program. Revista Bras. Med. Esporte 20, 281–284. doi: 10.1590/1517-86922014200401999 Monteagudo, M., Rodriguez-Blanco, T., Pueyo, M. J., Zabaleta-del-Olmo, E., Mercader, M., García, J., et al. (2013). Gender differences in negative mood states in secondary school students: health survey in Catalonia (Spain). Gaceta Sanitaria 27, 32–39. doi: 10.1016/j.gaceta.2012.01.009 Summala, H., and Mikkola, T. (1994). Fatal accidents among car and truck drivers: effects of fatigue, age, and alcohol consumption. Hum. Factors 36, 315–326. doi: 10.1177/001872089403600211 Mooi, E. A., and Sarstedt, M. (2011). A Concise Guide to Market Research: The Process, Data, and Methods Using IBM SPSS Statistics. Berlin: Springer Science and Business media. Tabachnick, B. G., and Fidell, L. S. (1996). Using Multivariate Statistics, 3rd Edn. New York, NY: Harper Collins. Terry, P. C. (1995). The efficacy of mood state profiling among elite performers: a review and synthesis. Sport Psychol. 9, 309–324. doi: 10.1123/tsp.9.3.309 Morfeld, M., Petersen, C., Krüger-Bödeker, A., von Mackensen, S., and Bullinger, M. (2007). The assessment of mood at workplace-psychometric analyses of the revised Profile of Mood States (POMS) questionnaire. GMS Psycho Soc. Med. 4. Available online at: https://www.scienceopen.com/document?id=cdc50f14- 960c-426b-926a-b8258f320680 Terry, P. C. (2004). “Mood and emotion in sport,” in Sport Psychology: Theory, Application, and Issues, eds T. Morris and S. J. Summers (Brisbane, QLD: Wiley), 48–73. Morgan, W. P. (1980). Test of champions: the iceberg profile. Psychol. Today 14, 92–99. Terry, P. C., and Galambos, S. (2004). “Utility of mood profiles in identifying risk of eating disorders among adolescent rowers,” in Proceedings of the 39th Australian Psychological Society Annual Conference (Sydney, NSW). 269–273. Morgan, W. P. (1985). “Selected psychological factors limiting performance: a mental health model,” in Limits of Human Performance, eds D. H. Clarke and H. M. Eckert (Champaign, IL: Human Kinetics), 70–80. Terry, P. C., Karageorghis, C. I., Saha, A. M., and, D’Auria, S. (2012). Effects of synchronous music on treadmill running among elite triathletes. J. Sci. Med. Sport 15, 52–57. doi:10.1016/j.jsams.2011.06.003 Morgan, W. P., Brown, D. R., Raglin, J. S., O’Connor, P. J., and Ellickson, K. A. (1987). Psychological monitoring of overtraining and staleness. Br. J. Sports Med. 21, 107–114. Terry, P. C., and Lane, A. M. (2000). Development of normative data for the Profile of Mood States for use with athletic samples. J. Appl. Sport Psychol. 12, 69–85. REFERENCES Does the iceberg profile discriminate between successful and less successful athletes? A meta-analysis. J. Sport Exer. Psychol. 17, 185–199. doi: 10.1123/jsep.17.2.185 Wyrwich, K. W., and Yu, H. (2011). Validation of POMS questionnaire in postmenopausal women. Q. Life Res. 20, 1111–1121. doi: 10.1007/s11136-011-9846-2 Salzman, C., Wolfson, A. N., Schatzberg, A., Looper, J., Henke, R., Albanese, M., et al. (1995). Effect of fluoxetine on anger in symptomatic volunteers with borderline personality disorder. J. Clin. Psychopharmacol. 15, 23–29. doi: 10.1097/00004714-199502000-00005 Sarapas, C., Shankman, S. A., Harrow, M., and Goldberg, J. F. (2012). Parsing trait and state effects of depression severity on neurocognition: evidence from a 26- year longitudinal study. J. Abnor. Psychol. 121, 830–837. doi: 10.1037/a0028141 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Schoeyen, H. K., Birkenaes, A. B., Vaaler, A. E., Auestad, B. H., Malt, U. F., Andreassen, O. A., et al. (2011). Bipolar disorder patients have similar levels of education but lower socio-economic status than the general population. J. Affect. Disord. 129, 68–74. doi: 10.1016/j.jad.2010.08.012 Copyright © 2017 Parsons-Smith, Terry and Machin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2017 Parsons-Smith, Terry and Machin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Soares, C. N. (2013). Depression in peri- and postmenopausal women: prevalence, pathophysiology and pharmacological management. Drugs Aging 30, 677–685. doi: 10.1007/s40266-013-0100-1 November 2017 | Volume 8 | Article 1958 Frontiers in Psychology | www.frontiersin.org 13
https://openalex.org/W1978315188
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0051339&type=printable
English
null
Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome
PloS one
2,012
cc-by
7,715
Abstract This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Tai Shan Scholar Foundation, National Natural Science Foundation of China (No. 81070407, No. 81170515, No. 81070396, No. 81270578), 973 Program (No. 2009CB521904, No. 2011CB503906), State Program of National Natural Science Foundation of China for Innovative Research Group 2011–2013 (No. 81021001), National Public Health Grand Research Foundation (No. 201202017), Key Clinical Research Project of Public Health Ministry of China 2010–2012, National Key Vocational School About Clinical Speciality for Blood Disorders, Clinical Medicine Center Foundation of Shandong Province, Leading Medical Professionals Foundation of Shandong Province, and Outstanding Young Scientist Research Award Foundation of Shandong Province (No. BS2009SW014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: daoxinma@sdu.edu.cn Recently, a separate CD4+ T-cell subset was identified on the basis of cytokine interleukin(IL)-17 [4]. This subset was subse- quently named T-helper cell type17 (Th17), expressing the lineage-specific transcription factor retinoic acid receptor-related orphan receptor C (RORC) [5]. Since then, Th17 cells have entered the limelight because of their comprehensive involvement in inflammations [6]. IL-17A, a representative Th17 cytokine, has been described in various models of immune-mediated tissue injury, including rheumatoid arthritis, lupus, myeloma as well as others likely to be defined. Though Kordasti and colleagues recently showed the increased number of peripheral Th17 cells in Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome Lin-lin Shao1, Lei Zhang2, Yu Hou3, Shuang Yu1, Xin-guang Liu1, Xiao-yang Huang4, Yuan-xin Sun1, Tian Tian1, Na He1, Dao-xin Ma1*, Jun Peng1, Ming Hou1 1 Department of Hematology, Qilu Hospital, Shandong University, Jinan, China, 2 Department of Orthopedics, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, China, 3 Medical College, Shandong University, Jinan, China, 4 Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, China Abstract Background: Immunological mechanisms are increasingly recognized in the progression of myelodysplastic syndrome (MDS). Early-stage MDS (E-MDS) is characterized by autoimmune-mediated myelosuppression whereas late-stage MDS (L- MDS) involves immune evasion, giving dysplastic cells growth potential to progress into acute myeloid leukemia. T-helper (Th) 22 is involved in the pathogenesis of inflammatory autoimmunity and tumorigenesis. The roles of Th22 cells in the pathophysiology of E-MDS and L-MDS remain unsettled. Design and Methods: We studied 37 MDS patients (E-MDS, n = 17; L-MDS, n = 20) and 20 healthy controls to characterize their peripheral blood (PB), as well as 25 MDS patients and 10 healthy controls to characterize their bone marrow(BM). The expression of Interleukin-22 (IL-22), IL-17 or interferon gamma (IFN-c) was examined in E-MDS, L-MDS patients and controls by flow cytometry. The mRNA expression levels of RAR-related orphan receptor C (RORC), IL-6, tumor necrosis factor alpha (TNF-a) and IL-23 in peripheral blood mononuclear cells (PBMCs) were determined by real-time quantitative polymerase chain reaction. The levels of IL-22 and IL-17 both in PB and BM plasma were examined by enzyme-linked immunosorbent assay. Results: In E-MDS, peripheral Th17 cells were significantly elevated and correlated with peripheral Th22 cells compared with healthy controls and L-MDS. Significantly higher levels of peripheral Th22 expansion, mRNA expression of IL-6, TNF-a and lower level of RORC mRNA expression were observed in L-MDS compared with E-MDS. No statistical difference was found in IL-23 mRNA expression or plasma IL-22, IL-17 levels among E-MDS, L-MDS and controls. Conclusions: Our data demonstrated that L-MDS cohort had increased frequencies of peripheral Th22 cells and higher mRNA expression levels of IL-6 and TNF-a, indicating that Th22 cells along with Th17 cells or not are involved in the dynamic immune responses of MDS. Citation: Shao L-l, Zhang L, Hou Y, Yu S, Liu X-g, et al. (2012) Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome. PLoS ONE 7(12): e51339. doi:10.1371/journal.pone.0051339 Editor: Daniel T. Starczynowski, Cincinnati Children’s Hospital Medical Center, United States of America Received August 3, 2012; Accepted November 7, 2012; Published December 7, 2 ao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright:  2012 Shao et al. December 2012 | Volume 7 | Issue 12 | e51339 Introduction Primary myelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem-cell disorders, characterized by ineffective hematopoiesis and an increased probability of developing acute leukemia. Autoimmune-mediated myelosuppression and immune evasion of malignant clone are increasingly recognized in the process of MDS. Clinical responses to immunoregulatory therapy and findings of expanded clonal T lymphocytes have led to the speculation that both CD4+ helper and CD8+ cytotoxic T cells are involved in the immunological pathophysiology of MDS [1,2,3]. December 2012 | Volume 7 | Issue 12 | e51339 December 2012 | Volume 7 | Issue 12 | e51339 PLOS ONE | www.plosone.org 1 Th22 and Th17 Cells in Different Stages of MDS low risk MDS [7],the mechanism of cellular immune abnormal- ities remains unclear. low risk MDS [7],the mechanism of cellular immune abnormal- ities remains unclear. FISH was performed to record cytogenetic karyotype regarding 5q31, 5q33, CEP7, 7q31, CEP8, 20q and CEPY. The patients were further divided into two subgroups based on International Prognostic Scoring System (IPSS) score [19], early-stage MDS (E- MDS, low/intermediate-1 risk, n = 29) and late-stage MDS (L- MDS, intermediate-2/high risk, n = 25). 5% BM blasts was chosen as the cut-off value delimiting fifty-six percent of MDS patients ,5% and forty-four percen t $5%. The demographic and key clinical features of MDS patients are listed in Table 1. Th22, capable of secreting IL-22 and tumor necrosis factor alpha (TNF-a), is a new subset of T cells clearly separated from other known Th cells [8]. This subset expresses none of interferon (IFN)-c, IL-17 cytokine, or respectively associated transcription factors T-bet and RORC [9]. Promoted by IL-6 and TNF-a, aryl hydrocarbon receptor (AHR) activation participates in priming human naive CD4+ T cells to Th22 subset [8,10]. Th22 cells have been found within the epidermal layer in prominence and regulating epidermal responses in inflammatory skin diseases [8]. In addition, Zhuang et al. have described a preferential expansion of Th22 cells which contribute to gastric cancer progression [11]. These data support the contention that Th22 cells are involved in the pathophysiology of autoimmunity and tumorigenesis. Preparation of Peripheral Blood Mononuclear Cells, Blood and Bone Marrow Plasma Peripheral whole blood was collected from 37 patients (E-MDS, n = 17; L-MDS, n = 20) while bone marrow were drawn from 25 cases. Plasma was obtained by centrifugation of heparinized peripheral blood and stored at 280uC for cytokine analysis. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA anticoagulated blood by gradient centrifugation (400 g, 20 minutes) using Ficoll-Paque (Pharmacia Diagnostics) and stored at 280uC for RNA isolation. IL-22 belongs to the IL-10 family of cytokines and is primarily secreted by activated Th22 cells [12]. The expression of IL-22 in cancers and autoimmune disorders is various, with IL-17 as siblings but not twins regarding their biological characteristics. IL- 22 was up-regulated in skin pathology and anaplastic lymphoma kinase positive anaplastic large cell lymphoma, providing signaling directionality from the immune system to targeted tissue-resident cells [12,13,14]. Meanwhile, it was down-regulated in systemic lupus erythematosus [15]. However, within disorders such as inflammatory bowel disease (IBD), IL-22 played either protective or pathogenic role in discrepant induction by naive and memory/ effector cells [16,17]. Ethics Statement Our research has been approved by the Institutional Review Boards of Qilu Hospital of Shandong University. A written informed consent document has been obtained from each participant. The informed consent stated that the excess of peripheral blood for Flow Cytometry - Clinical Diagnostics or unused portion of bone marrow for Fluorescence in situ hybridization (FISH) - Clinical Diagnostics was the sample source of our research. The peripheral blood drawn from healthy subjects and bone marrow drawn from hematologically normal individuals undergoing orthopedic femoral surgery for this research was voluntary. Flow Cytometric Analysis Intracellular cytokines were studied by flow cytometry to reflex the cytokine-producing cells. Briefly, heparinized peripheral whole blood (400 ml) with an equal volume of Roswell Park Memorial Table 1. Demographic and clinical characteristics of MDS patients. Characteristics value No. of patients 54 Age(y) 52.6615.4 Sex(male/female) 39/15 IPSS risk group, n(%) E-MDS: Low+Intermediate-1 29 (53.7%) L-MDS: Intermediate-2+ High 25 (46.3%) WHO MDS category, n(%) Unknown 15(27.7%) RCUD 13(24.1%) RARS 1(0.2%) RCMD 2(3.7%) RAEB-1 6(11.1%) RAEB-2 13(24.1%) MDS/MPD 2(3.7%) CMML-1 1(0.2%) CMML-2 1(0.2%) BM blasts, n(%) ,5 30(55.6%) $5 24(44.4%) IPSS karyotype, n(%) Favorable 33(61.1%) Intermediate 12(22.2%) Unfavorable 9(16.7%) Abbreviations: BM, bone marrow; n, number; RCUD, refractory cytopenia with unilineage dysplasia; RARS, refractory anemia with ring sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess blasts; CMML, chronic myelo-monocytic leukemia. doi:10.1371/journal.pone.0051339.t001 Table 1. Demographic and clinical characteristics of MDS patients. Up to now, no data exist with regard to Th22 cells and their association with Th17 or Th1 in MDS patients. To investigate possible roles of the above in the pathophysiology of MDS, we measured the percentages of peripheral Th22, Th17, Th1, mRNA expression levels of RORC, IL-6, TNF-a and IL-23 in peripheral blood mononuclear cells (PBMCs) as well as cytokine level of IL-22 or IL-17 in peripheral blood (PB) and bone marrow (BM), and evaluated their relevance. Patients and Controls A total of 54 patients (15 females and 39 males; mean age 52.6615.4 years) with MDS according to the World Health Organization (WHO) classification [18] were recruited in this study. All patients were treatment-naive or had no medical interventions for at least 3 months when sampling. Twenty age- matched healthy PB donors (6 females and 14 males; mean age 51.0615.9 years) were also included in the study. Ten hemato- logically normal age-matched individuals (3 females, 7 males; mean age 53.1611.8 years) were used as BM controls. Enrollment took place between March 2011 and May 2012 in the Department of Hematology of Qilu Hospital, Shandong University, China. Abbreviations: BM, bone marrow; n, number; RCUD, refractory cytopenia with unilineage dysplasia; RARS, refractory anemia with ring sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess blasts; CMML, chronic myelo-monocytic leukemia. doi:10.1371/journal.pone.0051339.t001 Abbreviations: BM, bone marrow; n, number; RCUD, refractory cytopenia with unilineage dysplasia; RARS, refractory anemia with ring sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess blasts; CMML, chronic myelo-monocytic leukemia. doi:10.1371/journal.pone.0051339.t001 December 2012 | Volume 7 | Issue 12 | e51339 PLOS ONE | www.plosone.org 2 Th22 and Th17 Cells in Different Stages of MDS Institute 1640 medium was incubated for 4h at 37uC, 5% CO2 in the presence of 25 ng/ml of phorbol myristate acetate (PMA), 1 mg/ml of ionomycin, and 1.7 mg/ml Golgiplug (monensin; all from Alexis Biochemicals, San Diego, CA, USA). PMA and ionomycin are pharmacological T-cell-activating agents that mimic signals generated by the T-cell receptor (TCR) complex and have the advantage of stimulating T cells of any antigen specificity. Monensin was used to block the intracellular transport mechanisms, thereby leading to an accumulation of cytokines in the cells. After incubation, cells were stained with PE-Cy5- conjugated anti-CD4 monoclonal antibody at room temperature in the dark for 20 min. The cells were next stained with FITC- conjugated anti-IFN-c monoclonal antibody, PE-conjugated anti- IL-17 monoclonal antibody, and APC-conjugated anti-IL-22 monoclonal antibody after fixation and permeabilization. All antibodies above were from eBioscience (SanDiego, CA, USA). Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACS Calibur cytometer equipped with CellQuest software (BD Bioscience PharMingen). t-test or the Wilcoxon rank-sum test to compare parametric and non-parametric data respectively. IL-22 and IL-17 Enzyme-Linked Immunosorbent Assay (ELISA) For peripheral Th1 cells, no significant difference was observed between MDS patients and HC (median, 9.65%; range, 6.16– 21.08% vs. median, 9.06%; range, 6.01–14.02%) in this study (Fig. 1 E, F, G and Fig. 2B). For peripheral Th1 cells, no significant difference was observed between MDS patients and HC (median, 9.65%; range, 6.16– 21.08% vs. median, 9.06%; range, 6.01–14.02%) in this study (Fig. 1 E, F, G and Fig. 2B). PB and BM were collected into heparin-anticoagulant vacutai- ner tubes. Plasma was obtained by centrifugation and stored at 280uC for determination of cytokines. IL-22 and IL-17 levels were determined with a quantitative sandwich enzyme immuno- assay technique in accordance with the manufacturer’s recom- mendations (lower detection limit 9 pg/ml; eBioscience). mRNA Expression Levels of RORC, IL-6, TNF-a and IL-23 in E-MDS, L-MDS Cohort and Controls RORC, the key transcription factor directing Th17 lineage commitment, was determined by real-time PCR. Our result demonstrated that RORC transcript was notably higher in PBMCs of E-MDS patients, consistent with the flow cytometry mRNA Expression Levels of RORC, IL-6, TNF-a and IL-23 in E-MDS, L-MDS Cohort and Controls Elevated Circulating Th17 Cells in E-MDS Patients Elevated Circulating Th17 Cells in E-MDS Patients The frequency of Th17 cells (CD4+IL-17+) was profoundly increased in MDS patients compared to healthy controls (1.6461.04% vs. 0.9760.29%, P = 0.002). Further on, a signifi- cant increase of peripheral Th17 cells was seen in E-MDS(median, 1.90%; range, 0.58–6.01%)compared with L-MDS(median, 1.16%; range, 0.15–1.86%)(P = 0.002) (Fig. 1 B, C, D and Fig. 2A). When compared to HC, E-MDS patients but not L- MDS ones, had a distinctly greater median percentage of committed Th17 cells among peripheral CD4+ cells (P = 0.002 vs. P = 0.34) (Fig. 2A). However, there was no marked difference regarding peripheral IL-17 between MDS patients (median, 25.1 pg/ml; range, 16.9–50.1) and HC (median, 22.4 pg/ml; range, 17.9–37.0; P = 0.74) (Fig. 3B). No significant difference of IL-17 level was shown between BM and matched PB (P.0.05) (Fig. 3D). No correlation was identified between peripheral Th17 frequency and circulating IL-17 level. mRNA Expression Levels of RORC, IL-6, TNF-a and IL-23 in E-MDS, L-MDS Cohort and Controls RORC, the key transcription factor directing Th17 lineage commitment, was determined by real-time PCR. Our result demonstrated that RORC transcript was notably higher in PBMCs of E-MDS patients, consistent with the flow cytometry Results Elevated Circulating Th22 Cells in MDS Patients We analyzed the frequency of peripheral Th22 based on cytokine patterns after in vitro stimulation by PMA plus ionomycin in short-term cultures. The expression of a typical dot plot of circulating Th22 (CD4+IL-22+IL-172IFNc2) cells in representative MDS patients and healthy controls (HC) is shown in Fig. 1 H, I, J. Compared with HC, the percentage of peripheral Th22 cells was significantly increased in MDS patients (0.7160.17% vs. 1.5560.74%, P,0.0001). The percentage of peripheral Th22 cells in E-MDS is higher than that in controls(1.2760.50% vs. 0.7160.17%, P = 0.002). Also a signifi- cant increase was shown in L-MDS compared with E-MDS patients (1.7760.84% vs. 1.2760.50%, P = 0.03) (Fig. 2C). The levels of IL-22 in PB and BM were measured by ELISA. No significant difference of PB IL-22 level between MDS patients (median, 22.64 pg/ml; range, 16.02–54.66) and normal controls (median, 23.86 pg/ml; range, 14.05–36.49) was observed, consis- tent with BM findings (Fig. 3 A, C). No correlation was found between peripheral Th22 cells and circulating IL-22 in MDS patients. Meanwhile, peripheral Th1 and Th17 cells failed to show a statistical correlation with circulating level of IL-22 in our present research. Patients and Controls Statistical significance among three groups was determined by ANOVA, and difference between any two groups was determined by Newman–Keuls multiple comparison test (q test) unless the data were not normally distributed, in which case Kruskal–Wallis test (H test) and Nemenyi test were used. The Pearson or Spearman correlation test was used for correlation analysis depending on data distribution. All tests were performed by SAS 9.1 system. P value less than 0.05 was considered statistically significant. Determination of the Expression of RORC, IL-6, TNF-a, IL- 23 mRNA Determination of the Expression of RORC, IL-6, TNF-a, IL- 23 mRNA TRIzol reagent (Invitrogen) was used to isolate total RNA of PBMCs. RNA was converted into cDNA using the PrimeScript RT reagent kit (Perfect Real Time; Takara) according to the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was performed for RORC, IL-6, TNF-a, IL-23 and the endogenous control (b-actin) on an ABI 7500 Real-time PCR System (Applied Biosystems) using SYBR Green (Toyobo) as a double-strand DNA-specific binding dye. The primers for all mRNA arrays were intron spanned. The PCR reactions were cycled 40 times after initial denaturation (95uC, 5 minutes) with the following parameters: denaturation at 95uC for 15 seconds, annealing at 65uC (RORC, b-actin)/62uC(IL-6, TNF-a, IL-23, b- actin)for 15 seconds, extension at 72uC for 45 seconds. The primers are shown as follows: RORC forward: TTT TCC GAG GAT GAG ATT GC; reverse: CTT TCC ACA TGC TGG CTA CA; IL-6 forward: TTC TCC ACA AGC GCC TTC GGT CCA, reverse: AGG GCT GAG ATG CCG TCG AGG ATG TA; TNF-a forward: CGA GTG ACA AGC CTG TAG C, reverse: GGT GTG GGT GAG GAG CAC AT; IL-23 forward: GCA GAT TCC AAG CCT CAG TC, reverse: TTC AAC ATA TGC AGG TCC CA; b-actin forward: CCT TCC TGG GCA TGG AGT CCT G, reverse: GGA GCA ATG ATC TTG ATC TTC. The amplification efficiency of the PCR products was calculated using Applied Biosystems System software. Target gene concen- trations were expressed relative to the number of b-actin transcripts used as the reference control. All experiments were conducted in triplicate. Statistical Analysis Results were expressed as mean 6 SD or median (range). Comparisons between two groups were assessed by the non-paired PLOS ONE | www.plosone.org December 2012 | Volume 7 | Issue 12 | e51339 3 Th22 and Th17 Cells in Different Stages of MDS Figure 1. Circulating percentages of Th17, Th1 and Th22 cells in representative healthy controls, E-MDS and L-MDS patients. Heparinized peripheral whole blood from 37 MDS(E-MDS, n = 17; L-MDS, n = 20)patients and 20 healthy PB donors were stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, and then stained with labeled antibodies for FACS analysis. (A) Lymphocytes were gated by flow cytometry. (B, C, D) Representative FACS dot plots of circulating Th17 (CD4+IL-17+) cells from healthy controls, E-MDS and L-MDS patients. (E, F, G) Representative FACS dot plots of circulating Th1 (CD4+IFNc+) cells from healthy controls, E-MDS and L-MDS patients. (H, I, J) Representative FACS dot plots of circulating Th22 (CD4+IL-22+IL-172IFNc2 ) cells from healthy controls, E-MDS and L-MDS patients. Numbers in plots indicate relative percentages per quadrant. doi:10.1371/journal.pone.0051339.g001 Figure 1. Circulating percentages of Th17, Th1 and Th22 cells in representative healthy controls, E-MDS and L-MDS patients. Heparinized peripheral whole blood from 37 MDS(E-MDS, n = 17; L-MDS, n = 20)patients and 20 healthy PB donors were stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, and then stained with labeled antibodies for FACS analysis. (A) Lymphocytes were gated by flow cytometry. (B, C, D) Representative FACS dot plots of circulating Th17 (CD4+IL-17+) cells from healthy controls, E-MDS and L-MDS patients. (E, F, G) Representative FACS dot plots of circulating Th1 (CD4+IFNc+) cells from healthy controls, E-MDS and L-MDS patients. (H, I, J) Representative FACS dot plots of circulating Th22 (CD4+IL-22+IL-172IFNc2 ) cells from healthy controls, E-MDS and L-MDS patients. Numbers in plots indicate relative percentages per quadrant. doi:10.1371/journal.pone.0051339.g001 data. The relative amount of RORC mRNA in E-MDS patients was increased 4.7- and 3.3-fold compared with healthy controls (HC) and L-MDS patients (P = 0.0007; P = 0.002, respectively) (Fig. 4A). levels of regulatory factors IL-6, TNF-a and IL-23 in MDS patients and controls. The relative amount of mRNA of IL-6 in L-MDS patients was 2.4-(P,0.05) and 5.3-fold (P,0.001) of that in E- MDS patients and healthy controls, respectively (Fig. 4B). Statistical Analysis TNF-a mRNA level was also present at higher levels in L-MDS compared In view of increased frequencies of Th22 cells in L-MDS and Th17 cells in E-MDS, we also determined the mRNA expression December 2012 | Volume 7 | Issue 12 | e51339 PLOS ONE | www.plosone.org 4 Th22 and Th17 Cells in Different Stages of MDS with E-MDS and controls (3.5-fold, P,0.05 and 10.6-fold, P,0.005, respectively) (Fig. 4C). Correlation between Th22, Th17, and Th1 Cells in MDS PATIENTS In E-MDS patients, a significantly positive correlation was found between peripheral Th22 cells and Th17 cells (r = 0.675, P = 0.004, Pearson correlation analysis) while no statistical correlation was obtained in L-MDS patients (r = 0.138, P = 0.610, Spearman correlation analysis). Peripheral Th22 cells showed no significant correlation with peripheral Th1 (P = 0.053). Discussion The presence of immune reactions against hematopoietic cells in MDS patients has crystallized as specific cell subsets and cytokines turmoil have been found as indispensable components of MDS pathophysiology. Th17, a new CD4+ T cell lineage that Figure 2. Circulating percentages of Th17 cells, Th1 cells, and Th22 cells in MDS. (A) Circulating percentages of Th17 (CD4+ IL-17+) cells from healthy controls, E-MDS and L-MDS. Significantly increased percentage of Th17 cells was found in E-MDS patients (median, 1.90%; range, 0.58– 6.01%) compared to L-MDS (median, 1.16%; range, 0.15–1.86%) (*P = 0.002) or healthy controls (median, 1.01%; range, 0.55–1.69%) (*P = 0.002). (B) Circulating percentages of Th1 (CD4+IFNc+) cells from healthy controls, E-MDS and L-MDS. There was no significant difference between E-MDS (median, 9.65%; range, 6.16–21.08%) patients and L-MDS (median, 8.41%; range, 2.59–15.23%) or healthy controls (median, 9.06%; range, 6.01– 14.02%). (C) Circulating percentages of Th22 (CD4+ IL-22+ IL-172IFNc2 ) cells from healthy controls, E-MDS and L-MDS. Significantly elevated percentage of Th22 cells was found in L-MDS patients (1.7760.84%) compared to E-MDS (1.2760.50%) (*P = 0.03) and healthy controls (0.7160.17%) (*P,0.0001). Obviously increased percentage of Th22 cells was shown between E-MDS and healthy controls (*P = 0.002). (D) Circulating percentages of Th17 cells from healthy controls, bone marrow (BM) blasts ,5% and blasts $5% patients. The circulating percentages of Th17 cells remained significantly higher in blasts ,5% compared with healthy donors. (*P = 0.003). doi:10.1371/journal.pone.0051339.g002 Figure 2. Circulating percentages of Th17 cells, Th1 cells, and Th22 cells in MDS. (A) Circulating percentages of Th17 (CD4+ IL-17+) cells from healthy controls, E-MDS and L-MDS. Significantly increased percentage of Th17 cells was found in E-MDS patients (median, 1.90%; range, 0.58– 6.01%) compared to L-MDS (median, 1.16%; range, 0.15–1.86%) (*P = 0.002) or healthy controls (median, 1.01%; range, 0.55–1.69%) (*P = 0.002). (B) Circulating percentages of Th1 (CD4+IFNc+) cells from healthy controls, E-MDS and L-MDS. There was no significant difference between E-MDS (median, 9.65%; range, 6.16–21.08%) patients and L-MDS (median, 8.41%; range, 2.59–15.23%) or healthy controls (median, 9.06%; range, 6.01– 14.02%). (C) Circulating percentages of Th22 (CD4+ IL-22+ IL-172IFNc2 ) cells from healthy controls, E-MDS and L-MDS. Significantly elevated percentage of Th22 cells was found in L-MDS patients (1.7760.84%) compared to E-MDS (1.2760.50%) (*P = 0.03) and healthy controls (0.7160.17%) (*P,0.0001). Obviously increased percentage of Th22 cells was shown between E-MDS and healthy controls (*P = 0.002). Clinical Relevance of Peripheral Th22, Th17 and Circulating IL-22 in MDS Patients Clinical Relevance of Peripheral Th22, Th17 and Circulating IL-22 in MDS Patients IL-23 is a heterodimeric cytokine consisting of two subunits: p40, which is also a component of IL-12, and p19, a unique subunit of IL-23. So we examined IL-23p19 mRNA expression as representative of IL-23. Quantitative real-time PCR analysis showed that there was no significant difference in IL-23p19 mRNA expression levels among E-MDS, L-MDS and healthy controls (P.0.05) (Fig. 4D). Peripheral Th17 percentage significantly increased in BM blasts ,5% patients compared with healthy donors (1.7660.80% vs. 0.9860.30%, P = 0.003), while no significant difference was seen between BM blasts $5% patients and healthy donors (1.3860.80% vs. 0.9860.30%, P.0.05) or between blasts $5% and ,5% patients (1.3860.80% vs. 1.7660.80%, P.0.05) (Fig. 2D). As for the correlations between peripheral Th22 frequency and the levels of three kinds of blood cells in MDS patients, no significant differences were found. Correlation between Th22, Th17, and Th1 Cells in MDS PATIENTS Discussion There was no significant difference between E-MDS (median, 40.34 pg/ml; range, 11.08–101.33 pg/ml, P.0.05) or L-MDS patients (median, 30.68 pg/ml; range, 23.86–49.43 pg/ml, P.0.05) and healthy controls (median, 33.59 pg/ml; range, 20.93–61.74 pg/ml, P.0.05). (D) Concentrations of IL-17 in BM plasm from healthy controls, E-MDS and L-MDS patients. No significant difference was found between E-MDS (median, 25.68 pg/ml; range, 20.96–83.13 pg/ml, P.0.05) or L-MDS patients (median, 23.48 pg/ml; range, 20.49–31.80 pg/ml, P.0.05) and healthy controls (median, 31.99 pg/ml; range, 25.11–66.37 pg/ml, P.0.05). doi:10.1371/journal.pone.0051339.g003 preferentially produces IL-17, has been described to be involved in various autoimmune diseases. Currently, there are two reports addressing discrepant ideas that Th17 cells operate to regress or enhance leukemic progression of MDS [7,20]. Compared with the well-known Th1, Th2 and Th17 subset,another distinct CD4+ T cell lineage, capable of secreting IL-22 but not IL-17 or IFN-c, has been denoted as Th22 subset [9,12]. Although previous studies have indicated that IL-22 secreted by Th22 participates in certain tumorigenicity and autoimmunity [14,21], it is not clear whether they are involved in MDS yet. infection, inflammation, autoimmunity and cancer suggest that Th22 may play a biphasic role varying on the focal microenvi- ronment [8,11]. With respect to disordered immune function in preleukemic states, E-MDS and L-MDS can be considered as two separate entities. The former is characterized by excessive apoptotic activity with autoimmune assault in the bone marrow whereas the latter involves decreased apoptotic indices and dramatic suppression of host anti-tumor responses, giving dysplastic cells the growth potential to progress into acute myeloid leukemia [22,23]. In our present study, increased Th17 cells have been advocated in E-MDS in a pattern reminiscent of autoim- munity, backed up by an analogous result from Mufti’s group [7]. Different from previous report [20], we found elevated RORC mRNA expression level in peripheral blood of E-MDS patients compared with normal controls and L-MDS patients, suggesting that the differentiation of Th17 cells takes part in E-MDS pathophysiology specifically. In our present study, no significant difference of IL-17 concentration whether in the BM or PB among E-MDS patients, L-MDS patients, or healthy controls was found. To study whether Th22 subset is compromised in the process of MDS, the percentages of peripheral Th22 cells in MDS patients and healthy controls were determined. Discussion (D) Circulating percentages of Th17 cells from healthy controls, bone marrow (BM) blasts ,5% and blasts $5% patients. The circulating percentages of Th17 cells remained significantly higher in blasts ,5% compared with healthy donors. (*P = 0.003). doi:10.1371/journal.pone.0051339.g002 December 2012 | Volume 7 | Issue 12 | e51339 PLOS ONE | www.plosone.org 5 Th22 and Th17 Cells in Different Stages of MDS Figure 3. Concentrations of IL-22 and IL-17 in PB and BM from healthy controls and MDS patients. (A) Concentrations of IL-22 in P plasma from healthy controls, E-MDS and L-MDS patients. There was no significant difference between E-MDS (median, 21.66 pg/ml; range, 16.02 36.00 pg/ml, P.0.05) or L-MDS patients (median, 23.37 pg/ml; range, 17.00–54.66 pg/ml, P.0.05) and healthy controls (median, 23.86 pg/ml; range 14.04–36.49 pg/ml, P.0.05). (B) Concentrations of IL-17 in PB plasma from healthy controls, E-MDS and L-MDS patients. No significant difference wa found between E-MDS (median, 22.32 pg/ml; range, 19.13–50.11 pg/ml, P.0.05) or L-MDS (median, 25.39 pg/ml; range, 17.86–46.31 pg/ml, P.0.05 patients and healthy controls (median, 25.11 pg/ml; range, 16.87–37.00 pg/ml, P.0.05).(C)Concentrations of IL-22 in BM plasm from healthy control E-MDS and L-MDS patients. There was no significant difference between E-MDS (median, 40.34 pg/ml; range, 11.08–101.33 pg/ml, P.0.05) or L-MD patients (median, 30.68 pg/ml; range, 23.86–49.43 pg/ml, P.0.05) and healthy controls (median, 33.59 pg/ml; range, 20.93–61.74 pg/ml, P.0.05). (D Concentrations of IL-17 in BM plasm from healthy controls, E-MDS and L-MDS patients. No significant difference was found between E-MDS (median 25.68 pg/ml; range, 20.96–83.13 pg/ml, P.0.05) or L-MDS patients (median, 23.48 pg/ml; range, 20.49–31.80 pg/ml, P.0.05) and healthy contro (median, 31.99 pg/ml; range, 25.11–66.37 pg/ml, P.0.05). doi:10.1371/journal.pone.0051339.g003 g Figure 3. Concentrations of IL-22 and IL-17 in PB and BM from healthy controls and MDS patients. (A) Concentrations of IL-22 in PB plasma from healthy controls, E-MDS and L-MDS patients. There was no significant difference between E-MDS (median, 21.66 pg/ml; range, 16.02– 36.00 pg/ml, P.0.05) or L-MDS patients (median, 23.37 pg/ml; range, 17.00–54.66 pg/ml, P.0.05) and healthy controls (median, 23.86 pg/ml; range, 14.04–36.49 pg/ml, P.0.05). (B) Concentrations of IL-17 in PB plasma from healthy controls, E-MDS and L-MDS patients. No significant difference was found between E-MDS (median, 22.32 pg/ml; range, 19.13–50.11 pg/ml, P.0.05) or L-MDS (median, 25.39 pg/ml; range, 17.86–46.31 pg/ml, P.0.05) patients and healthy controls (median, 25.11 pg/ml; range, 16.87–37.00 pg/ml, P.0.05).(C)Concentrations of IL-22 in BM plasm from healthy controls, E-MDS and L-MDS patients. Discussion Our results demonstrated that the percentage of peripheral Th22 subset (defined as CD4+IL- 22+IL-172IFNc2) was markedly elevated in MDS patients compared with healthy donors, and notably higher in L-MDS than in E-MDS. These results indicated that Th22 might be more involved in the immune evasion of MDS contributing to disease progression. Facts provided by advanced studies of Th22 cells in December 2012 | Volume 7 | Issue 12 | e51339 PLOS ONE | www.plosone.org 6 Th22 and Th17 Cells in Different Stages of MDS Figure 4. The ratio of RORC, IL-6, TNF-a, IL-23 mRNA in healthy controls and MDS patients. (A) The ratio of RORC mRNA in E-MDS patients compared with that of healthy controls or L-MDS was 4.7 (*P = 0.0007) or 3.3 (*P = 0.002), respectively. (B) The ratio of IL-6 mRNA in L-MDS patients compared with that of healthy controls or E-MDS was 5.3 (*P = 0.0001) or 2.4 (*P = 0.037), respectively. (C) The ratio of TNF-a mRNA in L-MDS patients compared with that of healthy controls or E-MDS was 10.6 (*P = 0.002) or 3.5 (*P = 0.049), respectively. (D) IL-23p19 mRNA expression level among E- MDS, L-MDS and healthy controls was comparable (P.0.05). Bars represent SD. doi:10.1371/journal.pone.0051339.g004 Figure 4. The ratio of RORC, IL-6, TNF-a, IL-23 mRNA in healthy controls and MDS patients. (A) The ratio of RORC mRNA in E-MDS patients compared with that of healthy controls or L-MDS was 4.7 (*P = 0.0007) or 3.3 (*P = 0.002), respectively. (B) The ratio of IL-6 mRNA in L-MDS patients compared with that of healthy controls or E-MDS was 5.3 (*P = 0.0001) or 2.4 (*P = 0.037), respectively. (C) The ratio of TNF-a mRNA in L-MDS patients compared with that of healthy controls or E-MDS was 10.6 (*P = 0.002) or 3.5 (*P = 0.049), respectively. (D) IL-23p19 mRNA expression level among E- MDS L MDS and healthy controls was comparable (P.0 05) Bars represent SD Figure 4. The ratio of RORC, IL-6, TNF-a, IL-23 mRNA in healthy controls and MDS patients. (A) The ratio of RORC mRNA in E-MDS patients compared with that of healthy controls or L-MDS was 4.7 (*P = 0.0007) or 3.3 (*P = 0.002), respectively. (B) The ratio of IL-6 mRNA in L-MDS patients compared with that of healthy controls or E-MDS was 5.3 (*P = 0.0001) or 2.4 (*P = 0.037), respectively. Th22 and Th17 Cells in Different Stages of MDS Th22 and Th17 Cells in Different Stages of MDS anaplastic large cell lymphoma (ALK+ ALCL) cell lines [14]. Several reports have identified that IL-22 secreted by Th22 cells interacts with CD4+ T cells, monocyte-derived human macro- phages and bone marrow-derived dendritic cells (DCs) [33,34,35]. To some instances, we hypothesize that Th22 cells participate in the immune regulation of hematopoiesis probably through interaction with DCs or other Th subsets. Despite these appearant correlations, at this period we cannot confirm the direct effect of Th22 on the impaired immune surveillance of L-MDS. cells are reduced and cytokine secretion are decreased [40]. The data above unambiguously indicate that the unilateral increased frequency of Th22 cells contributes little to the IL-22 production capacity. On the other hand, mounting evidences support that IL- 6 possesses a developmental relationship to the IL-22 production [24]. Inversely, TGF-b down-regulates the IL-22 production [41]. Compelling evidence has been achieved from several groups that levels of IL-6 as well as TGF-b are increased in MDS patients [42,43]. So the unaltered extracellular level of IL-22 can be partially attributed to the regulating homeostasis between IL-6 stimulative and TGF-b inhibitory effect. p Numerous evidences suggest Th1 cells have been linked to the development of autoimmune inflammatory processes. To further investigate the role of Th1 cells in the pathogenesis of MDS, we also examined the percentages of periphery Th1 cells in MDS patients and healthy donors. In contrast to the results of periphery Th22 and Th17 cells, no statistical difference was shown in Th1 frequencies between patients with MDS and healthy donors. Although previous studies showed that the conventional Th1- prototypical cytokines IFN-c and TNF-a were predominant in MDS, it was later confirmed that these cytokines were derived from macrophage lineage cells [36]. Meanwhile, statistical correlations between Th1 cells and Th22 cells or Th17 cells were not found in this study. In summary, for the first time we showed a clear difference between E- and L-MDS in terms of Th22-cell related immuno- logical environment. Circulating Th22 expansion occurs much more frequently in late stage, which may favor the escape of the preleukemic clone. By contrast, in early stage, circulating Th17 expansion tends to be predominant, thereby underpinning the inflammatory autoimmune assault and eventually the apoptosis of bone marrow. Th22 and Th17 Cells in Different Stages of MDS The numerical alterations of Th22 subset in early and late disease stage would suggest that shifty in the dynamics of Th22 could be a parameter affecting disease progression, exerting antithetical effects in the regulation of immune homeostasis and tumor immunity. Blockade of Th22 cells might be of clinical profit in both E-MDS and L-MDS patients. Further studies on more patients are needed to substantiate whether this is indeed the case, and it is necessary to clarify the situation of Th22 cells in MDS bone marrow. IL-22 has been involved in the pathophysiology of some malignant diseases, such as multiple myeloma [37]. As to MDS, the expression level of IL-22 has not been investigated until today. Our results demonstrated for the first time that the level of IL-22 either in BM or in PB of MDS patients was comparable with that of healthy controls, and no correlation with peripheral Th22 cells was revealed. IL-22 is not exclusively produced by Th22 cells, but rather appears to be produced by other T cells such as CD4+IL- 17+IL-22+ cells and CD4+IFNc+IL-22+ cells, as well as circulating NK cells [38,39]. The frequency of Th22 cells among total IL-22- producing T cells ranges from 37% to 63% [9]. When taking IL- 22-producing NK cells into account, that proportion will become smaller. In MDS, groups have reported that function of MDS-NK References Th22 cells correlate with gastric cancer progression and predict poor patient survival. Cancer Immunol Immunother. 1. Molldrem JJ, Jiang YZ, Stetler-Stevenson M, Mavroudis D, Hensel N, et al. (1998) Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte-mediated inhibition of CFU-GM and alterations in T-cell receptor Vbeta profiles. Br J Haematol 102: 1314–1322. 12. Trifari S, Kaplan CD, Tran EH, Crellin NK, Spits H (2009) Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol 10: 864–871. 2. Kochenderfer JN, Kobayashi S, Wieder ED, Su C, Molldrem JJ (2002) Loss of T-lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppression. Blood 100: 3639–3645. 13. Zheng Y, Danilenko DM, Valdez P, Kasman I, Eastham-Anderson J, et al. (2007) Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 445: 648–651. 3. Fozza C, Contini S, Galleu A, Simula MP, Virdis P, et al. (2009) Patients with myelodysplastic syndromes display several T-cell expansions, which are mostly polyclonal in the CD4(+) subset and oligoclonal in the CD8(+) subset. Exp Hematol 37: 947–955. 14. Bard JD, Gelebart P, Anand M, Amin HM, Lai R (2008) Aberrant expression of IL-22 receptor 1 and autocrine IL-22 stimulation contribute to tumorigenicity in ALK+ anaplastic large cell lymphoma. Leukemia 22: 1595–1603. 15. Cheng F, Guo Z, Xu H, Yan D, Li Q (2009) Decreased plasma IL22 levels, but not increased IL17 and IL23 levels, correlate with disease activity in patients with systemic lupus erythematosus. Ann Rheum Dis 68: 604–606. 4. Steinman L (2007) A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med 13: 139– 145. 16. Andoh A, Zhang Z, Inatomi O, Fujino S, Deguchi Y, et al. (2005) Interleukin- 22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. Gastroenterology 129: 969–984. 5. Ivanov, II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, et al. (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: 1121–1133. 17. Sugimoto K, Ogawa A, Mizoguchi E, Shimomura Y, Andoh A, et al. (2008) IL- 22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. J Clin Invest 118: 534–544. 6. Author Contributions Conceived and designed the experiments: DxM LlS LZ JP MH. Performed the experiments: LlS LZ SY XyH YxS TT. Analyzed the data: LlS LZ XgL. Contributed reagents/materials/analysis tools: DxM LZ NH. Wrote the paper: LlS LZ YH DxM. Discussion (C) The ratio of TNF-a mRNA in L-MDS patients compared with that of healthy controls or E-MDS was 10.6 (*P = 0.002) or 3.5 (*P = 0.049), respectively. (D) IL-23p19 mRNA expression level among E- MDS, L-MDS and healthy controls was comparable (P.0.05). Bars represent SD. doi:10.1371/journal.pone.0051339.g004 widely recognized features [25,26,27]. On the other hand, L- MDS, a disease stage progressing towards AML with additional genetic lesions, is characterized by increased numbers of Tregs [28], dysfunctional NK cells [29] and higher immunoinhibitory molecule B7-H1 expression on MDS blasts [22], resulting in immune evasion of the malignant clone. Stimulation of IL-6 plus TNF-a could promote Th22-cell differentiation from CD4+ T cells [30]. Both TNF-a and IL-6 have been detected excessively expressed in the sera of patients with high risk MDS [31]. Our data demonstrated that L-MDS patients had increased IL-6 and TNF-a mRNA expression compared with E-MDS patients or normal controls. Thus, it can be concluded that a cell-cytokine milieu within the tumor microenvironment of L-MDS may be suitable for the polarization of Th22 cells. IL-22R1, the critical member of IL-22 receptor complex, was not detected in normal immune cells [32]. Notwithstanding, recent study found aberrant expression of IL-22R1 on anaplastic lymphoma kinase positive Although IL-23 signaling is dispensable for Th17 commitment, it induces IL-17 production as one of the essential cofactors [24]. Our present study regarding IL-23 mRNA expression level did not show the difference between E-MDS, L-MDS and controls. Along with previous studies regarding the serum level of IL-23 [7], we speculate that the unaltered IL-17 level vs. elevated Th17 cells in E-MDS attributes to IL-23 insufficiency. In our study, there existed a positive correlation between peripheral Th22 and Th17 subset in E-MDS patients, implying that differentiation of Th22 and Th17 cells may be induced in an influential manner in E-MDS. Co-existence of Th22 and Th17 cells along with pro-inflammatory cytokines leads to a dramatic increase in the overexuberant inflammatory immune reaction [8]. Such functional synergism may happen in the persistent low-level inflammatory state of E-MDS in which elevated levels of pro- apoptotic cytokines, low regulatory T-cells (Tregs) number and increased natural killer (NK) cytotoxicity against hematopoiesis are December 2012 | Volume 7 | Issue 12 | e51339 7 PLOS ONE | www.plosone.org Th22 and Th17 Cells in Different Stages of MDS B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes. Blood 116: 1124–1131. 33. Xie MH, Aggarwal S, Ho WH, Foster J, Zhang Z, et al. (2000) Interleukin (IL)- 22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2–4 and IL-22R. J Biol Chem 275: 31335–31339. , y g g proteins CRF2–4 and IL-22R. J Biol Chem 275: 31335–31339. y 23. Aggarwal S, van de Loosdrecht AA, Alhan C, Ossenkoppele GJ, Westers TM, et al. (2011) Role of immune responses in the pathogenesis of low-risk MDS and high-risk MDS: implications for immunotherapy. Br J Haematol 153: 568–581. p J 34. Dhiman R, Indramohan M, Barnes PF, Nayak RC, Paidipally P, et al. (2009) IL- 22 produced by human NK cells inhibits growth of Mycobacterium tuberculosis by enhancing phagolysosomal fusion. J Immunol 183: 6639–6645. 24. Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 Cells. Annu Rev Immunol 27: 485–517. 35. Schnyder B, Lima C, Schnyder-Candrian S (2010) Interleukin-22 is a negative regulator of the allergic response. Cytokine 50: 220–227. 25. Parker JE, Mufti GJ, Rasool F, Mijovic A, Devereux S, et al. (2000) The role of apoptosis, proliferation, and the Bcl-2-related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood 96: 3932– 3938. 36. Kitagawa M, Saito I, Kuwata T, Yoshida S, Yamaguchi S, et al. (1997) Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)- gamma by bone marrow cells from patients with myelodysplastic syndromes. Leukemia 11: 2049–2054. 26. Kotsianidis I, Bouchliou I, Nakou E, Spanoudakis E, Margaritis D, et al. (2009) Kinetics, function and bone marrow trafficking of CD4+CD25+FOXP3+ regulatory T cells in myelodysplastic syndromes (MDS). Leukemia 23: 510–518. 37. Prabhala RH, Pelluru D, Fulciniti M, Prabhala HK, Nanjappa P, et al. (2010) Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma. Blood 115: 5385–5392. 27. Chamuleau MED, Westers TM, van Dreunen L, Groenland J, Zevenbergen A, et al. (2009) Immune mediated autologous cytotoxicity against hematopoietic precursor cells in patients with myelodysplastic syndrome. Haematologica-the Hematology Journal 94: 496–506. 38. Sabat R, Wolk K, Kunz S, Asadullah K (2002) Cutting edge: Immune cells as sources and targets of the IL-10 family members? Journal of Immunology 168: 5397–5402. 39. Wolk K, Witte E, Wallace E, Docke WD, Kunz S, et al. References McGeachy MJ, Cua DJ (2008) Th17 cell differentiation: the long and winding road. Immunity 28: 445–453. 7. Kordasti SY, Afzali B, Lim Z, Ingram W, Hayden J, et al. (2009) IL-17- producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome. Br J Haematol 145: 64–72. 18. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, et al. (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 114: 937–951. 8. Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, et al. (2009) Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest 119: 3573–3585. 19. Cheson BD, Bennett JM, Kantarjian H, Pinto A, Schiffer CA, et al. (2000) Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood 96: 3671–3674. g 9. Duhen T, Geiger R, Jarrossay D, Lanzavecchia A, Sallusto F (2009) Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells. Nat Immunol 10: 857–863. 20. Bouchliou I, Miltiades P, Nakou E, Spanoudakis E, Goutzouvelidis A, et al. (2011) Th17 and Foxp3(+) T regulatory cell dynamics and distribution in myelodysplastic syndromes. Clin Immunol 139: 350–359. 10. Chizzolini C, Ramirez JM, Brembilla NC, Sorg O, Chicheportiche R, et al. (2010) Activation of the aryl hydrocarbon receptor reveals distinct requirements for IL-22 and IL-17 production by human T helper cells. European Journal of Immunology 40: 2450–2459. 21. Zhang L, Li JM, Liu XG, Ma DX, Hu NW, et al. (2011 ) Elevated Th22 Cells Correlated with Th17 Cells in Patients with Rheumatoid Arthritis. J Clin Immunol 31: 606–614. 11. Zhuang Y, Peng LS, Zhao YL, Shi Y, Mao XH, et al. (2012 Apr 13. [Epub ahead of print]) Increased intratumoral IL-22-producing CD4(+) T cells and 22. Kondo A, Yamashita T, Tamura H, Zhao W, Tsuji T, et al. (2010) Interferon- gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, PLOS ONE | www.plosone.org December 2012 | Volume 7 | Issue 12 | e51339 8 Th22 and Th17 Cells in Different Stages of MDS Th22 and Th17 Cells in Different Stages of MDS Th22 and Th17 Cells in Different Stages of MDS (2006) IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis. Eur J Immunol 36: 1309–1323. 28. Kordasti SY, Ingram W, Hayden J, Darling D, Barber L, et al. (2007) CD4+CD25high Foxp3+ regulatory T cells in myelodysplastic syndrome (MDS). Blood 110: 847–850. 29. Epling-Burnette PK, Bai F, Painter JS, Rollison DE, Salih HR, et al. (2007) Reduced natural killer (NK) function associated with high-risk myelodysplastic syndrome (MDS) and reduced expression of activating NK receptors. Blood 109: 4816–4824. 40. Kiladjian JJ, Bourgeois E, Lobe I, Braun T, Visentin G, et al. (2006) Cytolytic function and survival of natural killer cells are severely altered in myelodysplastic syndromes. Leukemia 20: 463–470. 41. Volpe E, Servant N, Zollinger R, Bogiatzi SI, Hupe P, et al. (2008) A critical function for transforming growth factor-beta, interleukin 23 and proinflamma- tory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 9: 650–657. 30. Ye ZJ, Zhou Q, Yuan ML, Du RH, Yang WB, et al. (2012) Differentiation and Recruitment of IL-22-Producing Helper T Cells Stimulated by Pleural Mesothelial Cells in Tuberculous Pleurisy. Am J Respir Crit Care Med 185: 660–669. 42. Pardanani A, Finke C, Lasho TL, Al-Kali A, Begna KH, et al. (2012 ) IPSS- independent prognostic value of plasma CXCL10, IL-7 and IL-6 levels in myelodysplastic syndromes. Leukemia 26: 693–699. 31. Alexandrakis M, Coulocheri S, Xylouri I, Ganotakis E, Eliakis P, et al. (1998) Elevated serum TNF-alpha concentrations are predictive of shortened survival in patients with high-risk myelodysplastic syndromes. Haematologia (Budap) 29: 13–24. 43. Zhou L, McMahon C, Bhagat T, Alencar C, Yu Y, et al. (2011) Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase. Cancer Res 71: 955–963. 32. Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, et al. (2004) IL-22 increases the innate immunity of tissues. Immunity 21: 241–254. December 2012 | Volume 7 | Issue 12 | e51339 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9
https://openalex.org/W4390144979
https://journals.uco.es/hikma/article/download/16331/15048
es
¿Conocen y usan los estudiantes de traducción suficientes recursos lexicográficos y terminográficos?
Hikma
2,023
cc-by
8,187
ISSN: 1579-9794 ¿Conocen y usan los estudiantes de Traducción suficientes recursos lexicográficos y terminográficos? Resultados de un estudio empírico Do students of Translation know and use enough lexicographic and terminographic resources? Results of an empirical study MIRIAM BUENDÍA CASTRO mbuendia@ugr.es Universidad de Granada Fecha de recepción: 24/08/2023 Fecha de aceptación: 01/12/2023 Resumen: Los avances tecnológicos del siglo XXI y, en especial la inteligencia artificial, están cambiando la forma de documentarnos y obtener la información. La traducción no ha permanecido ajena a estos cambios. No obstante, ahora más que nunca, conviene destacar el papel fundamental de los diccionarios en el proceso traductor, ya que estos proporcionan información conceptual y lingüística de calidad que difícilmente podrá sustituirse por muchos avances que se produzcan. En este sentido, resulta esencial determinar el conocimiento que los estudiantes de Traducción poseen acerca de los recursos lexicográficos y terminográficos existentes, e investigar sobre el uso que hacen de estos recursos. Así pues, el presente artículo estudia el conocimiento y el uso de los recursos lexicográficos y terminográficos por parte de 165 estudiantes del Grado en Traducción e Interpretación y del Máster en Traducción Profesional, de la Universidad de Granada, a través de un cuestionario. Los resultados revelan diferencias significativas entre los distintos cursos y, en general, el desconocimiento y el empleo poco frecuente de los recursos terminográficos, incluso por parte de los estudiantes de máster. Así pues, este trabajo destaca la necesidad de incluir más formación en torno al uso y tipología de diccionarios en los estudios de Traducción. Palabras clave: Recurso lexicográfico, Recurso terminográfico, Estudiante de Traducción, Diccionario, Traducción Abstract: Technological advances in the 21st century, especially artificial intelligence, are changing the way we document and obtain information. Translation has also been affected by these changes. However, now more Hikma 22(2) (2023), 309 - 330 310 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? than ever, it is worth highlighting the fundamental role of dictionaries in the translation process, as they provide quality conceptual and linguistic information that is difficult to be replaced. In this sense, it is essential to determine Translation students' knowledge of existing lexicographic and terminographic resources, and to investigate the use they make of them. Thus, this article studied the knowledge and utilisation of lexicographic and terminographic resources by 165 students of the four-year Degree in Translation and Interpreting and the Master's Degree in Professional Translation, at the University of Granada, by means of a questionnaire. Results reveal significant differences between the different academic years and, in general, a lack of knowledge and little use of terminographic resources, even by master’s students. Thus, this work highlights the need to include more training in the utilisation and typology of dictionaries in Translation studies. Keywords: Lexicographic resource, Terminographic resource, Student of Translation, Dictionary, Translation INTRODUCCIÓN La consulta de diccionarios constituye una parte esencial en el proceso traductor. Los diccionarios pueden ayudar a los profesionales de la traducción tanto en situaciones cognitivas como comunicativas. En cuanto a las situaciones cognitivas, estas están motivadas por la necesidad de adquirir conocimiento nuevo o verificar el ya existente sobre un determinado tema (Bergenholtz y Tarp, 2010). Por su parte, las funciones comunicativas vienen determinadas por la necesidad de obtener ayuda del diccionario cuando el usuario está realizando alguna actividad textual, como leer o revisar un texto, traducir un texto de una lengua origen a una lengua meta, o escribir un texto en la lengua materna o en una lengua extranjera (L'Homme y Leroyer, 2009, p. 270). Así pues, si queremos que los futuros traductores y traductoras conozcan y utilicen los recursos lexicográficos y terminográficos disponibles, resulta necesario investigar sobre el conocimiento y el uso que los estudiantes de Traducción tienen acerca de estos recursos. Hasta ahora, los estudios que evalúan el uso de los diccionarios por parte de los estudiantes de Traducción son escasos y, más concretamente, dentro del panorama nacional. Algunas excepciones son Sánchez-Ramos (2005), Kodura (2016), Altuwairesh (2021, 2022), Karami y Riassati (2023), o Svike (2022). Así pues, el presente artículo estudia el conocimiento y el uso de los recursos lexicográficos y terminográficos por parte de 143 estudiantes del Grado en Traducción e Interpretación (lengua B inglés) y 22 estudiantes del Máster en Traducción Profesional (con el inglés en su combinación lingüística) de la Universidad de Granada. Los datos se recabaron a través Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 311 de un cuestionario. Concretamente, este se fundamenta en las siguientes preguntas de investigación: - ¿Qué conocimiento tienen los estudiantes del Grado en Traducción e Interpretación, con lengua B inglés, y los estudiantes del Máster en Traducción Profesional, con el inglés en su combinación lingüística, acerca de los recursos lexicográficos y terminográficos existentes? - ¿Con qué frecuencia usan los distintos recursos lexicográficos y terminográficos? - ¿Existen diferencias significativas entre los estudiantes en función del curso? Este artículo se estructura como se detalla a continuación. La primera sección presenta la revisión bibliográfica sobre los recursos lexicográficos y terminográficos y los estudios sobre el uso de diccionarios con estudiantes de traducción. La segunda describe la metodología de recogida y análisis de datos. La tercera se centra en el análisis de los datos y los resultados obtenidos mediante métodos estadísticos. La cuarta analiza las principales inferencias extraídas de nuestro estudio y las relaciona con otros estudios. Por último, la sección final recoge las limitaciones del estudio y las conclusiones. 1. ESTADO DE LA CUESTIÓN 1.1. Recursos lexicográficos y terminográficos Para distinguir entre terminología y lexicología, la mayoría de los investigadores empiezan estableciendo una correspondencia entre, por un lado, la lexicología y la lexicografía, y por otro, la terminología y la terminografía. Por ejemplo, Pérez (2002, cap. 3.3) afirma que la lexicología es la disciplina que estudia y describe el léxico de una lengua, mientras que la lexicografía constituye la lexicología aplicada a la compilación de diccionarios de la lengua general. De la misma forma, la terminología se ocupa de la descripción teórica y metodológica del lenguaje especializado, mientras que la terminografía consiste en la terminología aplicada a la elaboración de diccionarios especializados. No obstante, la frontera entre la terminografía y la lexicografía no siempre está clara. Dado que los diccionarios especializados son recursos que se centran en ámbitos especializados (por ejemplo, negocios, química o derecho) (Bowker, 2010, p. 156), muchos autores se refieren a la terminografía como lexicografía especializada (Bergenholtz y Tarp, 2010). Así pues, se puede concluir que la lexicografía especializada y la terminografía se refieren básicamente al mismo tipo de actividad. En esta investigación, se Hikma 22(2) (2023), 309 - 330 312 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? ha utilizado la denominación «recursos lexicográficos» para hacer referencia a los diccionarios de la lengua general, y la denominación «recursos terminográficos» para aludir a los recursos especializados. La tipología de diccionarios que incluimos en el cuestionario, y por la que interrogamos a los estudiantes (véase el subapartado 2.3.), se deriva de Hausmann (1989), quien distingue entre diccionarios especializados y generales, y tiene en cuenta el número de lenguas implicadas (monolingües y bilingües. Sin embargo, dado que en aquella época la tipología de diccionarios se refería exclusivamente a los impresos, también hemos distinguido entre diccionarios en papel y electrónicos, en línea con Lew y De Schryver (2014). 1.1.1 Estudios sobre el uso de diccionarios con estudiantes de Traducción Existe un gran número de estudios sobre el uso de diccionarios que se centran en el público general o en estudiantes de segundas lenguas (Hamouda, 2013; Gromann y Schnitzer, 2015; Kosem et al., 2019; Márkus, Fajt y Dringó-Horwáth, 2023, inter alia). No obstante, los trabajos que tienen como perfil de usuario al estudiante de Traducción son mucho más limitados y los que hay disponibles suelen ofrecer tendencias generales, sin distinguir entre los distintos cursos de grado o máster (Sánchez-Ramos, 2005; Kodura, 2016; Altuwairesh, 2021, 2022; Karami y Riassati, 2023; Svike, 2022). Sánchez-Ramos (2005) llevó a cabo un estudio con 98 estudiantes de Traducción de la Universidad Jaume I, en el que destacaba la necesidad de que los estudiantes se familiarizaran más con los diccionarios electrónicos. Kodura (2016) realizó una encuesta con 63 estudiantes del Grado en Filología Inglesa de la Universidad Pedagógica de Cracovia. Los resultados destacaron el cambio de tendencia hacia el uso de los recursos electrónicos. Karami y Riasati (2023) distribuyeron un cuestionario entre 64 estudiantes de Traducción de inglés de la Universidad Azad de Rasht (Irán). Estos autores llegaron a la conclusión de que el 97 % de los encuestados preferían los diccionarios electrónicos a los de papel. Svike (2022), por su parte, realizó una encuesta a 78 estudiantes de Traducción de la Universidad de Ciencias Aplicadas de Ventspils (Letonia). Los resultados pusieron de relieve que los diccionarios bilingües eran los más utilizados (87,2 %), seguidos de los monolingües letones (80 %) y los monolingües en lengua inglesa (55 %). Altuwairesh (2021, 2022) encuestó a 95 alumnas de la Facultad de Lenguas y Traducción de la Universidad Rey Saud de Riad. Los resultados confirmaron que las estudiantes prefieren los diccionarios electrónicos a los de papel, y los recursos bilingües a los monolingües. Los resultados obtenidos con los estudiantes de Traducción coinciden en gran medida con los mostrados en otros estudios sobre el uso de Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 313 diccionarios. Parece que hoy en día los estudiantes prefieren los diccionarios electrónicos (Lew y Szarowska, 2017). En cuanto al número de lenguas de un diccionario, muchos trabajos han destacado la preferencia de los diccionarios bilingües sobre los monolingües (por ejemplo, Gromann y Schnitzer, 2015). Sin embargo, no son muchos los estudios que analizan el uso de los recursos especializados por parte de usuarios semiespecializados. Algunas excepciones son Gromann y Schnitzer (2015), quienes llevaron a cabo una investigación con 430 estudiantes de lenguas y concluyeron que menos del 10 % consultaba recursos especializados. 2. METODOLOGÍA A continuación, se especifica el contexto en el que se enmarca esta investigación: el Grado en Traducción e Interpretación (lengua B inglés) y el Máster en Traducción Profesional (con los estudiantes que tienen el inglés en su combinación lingüística) (2.1). Asimismo, se proporciona información detallada de los sujetos que participaron en este estudio (2.2) y del cuestionario que se utilizó para recabar los datos (2.3). 2.1. Contextualización de los estudios de grado y máster El plan de estudios del Grado en Traducción e Interpretación de la Universidad de Granada se encuentra estructurado en cuatro cursos académicos de 60 créditos ECTS cada uno. Cada curso académico está dividido en dos semestres y, en cada uno, los estudiantes pueden cursar 30 créditos ECTS. Todos los estudiantes cuentan con una lengua B (primera lengua extranjera), que es la lengua de acceso a la titulación, y una lengua C (segunda lengua extranjera). Las lenguas B ofertadas son cuatro, a saber, árabe, alemán, francés e inglés, siendo los estudiantes de lengua B inglés los más numerosos y los estudiantes de lengua B árabe los menos. Las lenguas C son nueve: árabe, alemán, chino, francés, inglés, italiano, griego moderno, portugués y ruso, aunque muy pronto se va a incluir la lengua de signos como lengua C. Por su parte, el Máster Universitario en Traducción Profesional de la Universidad de Granada es un programa de posgrado de 60 ECTS que comprende un curso académico. Este máster ha sido incluido en la red de European Master’s in Translation, que aúna los másteres de mayor calidad en el campo de la traducción en Europa. Las lenguas utilizadas en la impartición del título son, además del español, las cuatro lenguas B reseñadas anteriormente. Hikma 22(2) (2023), 309 - 330 314 2.2. ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? Muestra La muestra del estudio se compone de 165 estudiantes: 143 del Grado en Traducción e Interpretación y 22 del Máster en Traducción Profesional de la Universidad de Granada. De los 165 estudiantes, el 83 % (n = 137) eran mujeres, el 15,8 % (n = 26) eran hombres y el 1,2 % (n = 2) no especificó el sexo. La edad media de los encuestados es de 20,07 años. El encuestado más joven tenía 18 años y, el más mayor, 42 años. De los 165 encuestados, la distribución por año de estudios es la siguiente: 30,9 % (n = 51) estudiantes de primer curso, 27,9 % (n = 46) de segundo, 14,5 % (n = 24) de tercero, 13,3 % (n = 22) de cuarto, y 13,3 % (n = 22) de máster. Cabe recordar que todos los estudiantes son nativos de español y tienen como lengua B inglés. 2.3. Cuestionario Para la recogida de datos se elaboró un cuestionario en papel. Las preguntas se eligieron a partir de ejemplos recurrentes en la literatura sobre investigación de usuarios de diccionarios (véase 1.1), y se adaptaron a nuestras preguntas y objetivos de investigación. El cuestionario se distribuyó en aquellas clases cuyo profesorado aceptó participar en el estudio, con el objetivo de contar con una muestra representativa de cada curso. Se explicó a los estudiantes que la participación era voluntaria y anónima. Por lo tanto, aquellos que decidieran no participar no se verían perjudicados académicamente. El cuestionario se facilitó en español, ya que estaba dirigido únicamente a estudiantes de la Universidad de Granada que tuvieran el español como lengua materna. Dicho cuestionario se compone de cinco secciones. La primera corresponde a la introducción y contiene los objetivos de la investigación y las instrucciones. Se garantizó el anonimato de los sujetos y se solicitó consentimiento informado para recabar los datos. La segunda sección incluye cinco preguntas demográficas: edad, sexo, lengua materna, lengua B y curso académico. Estas cuestiones permitieron descartar los estudiantes que no fueran nativos españoles y no tuvieran el inglés como lengua B. Asimismo, fueron de extrema utilidad para posteriormente realizar los análisis comparativos por subgrupos, especialmente por curso académico. La tercera sección contiene dos preguntas sobre tendencias generales en el uso de recursos lexicográficos y terminográficos. Más concretamente, se les preguntó por la frecuencia de uso de los diccionarios, mediante una pregunta de escala Likert de cuatro puntos (pregunta 6), y acerca de qué formato de diccionario suelen utilizar, mediante una pregunta de opción múltiple en la que los encuestados debían elegir entre un conjunto de opciones predefinidas (pregunta 7). Conviene recordar que las preguntas eran de selección única y los estudiantes tan solo podían elegir una opción. La cuarta sección se centra Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 315 en las tendencias específicas en el uso de recursos lexicográficos y terminográficos (diez preguntas relativas a la frecuencia de uso de cada tipo de recurso; por ejemplo, diccionario especializado bilingüe electrónico, etc.). Las respuestas de los participantes se basaron exclusivamente en una escala Likert de cuatro puntos («nunca», «casi nunca», «a veces», «con frecuencia») para evitar la tendencia de los participantes en escalas con un número impar de puntos a marcar la posición central o neutra. Por último, la sección 5 se ocupa de los recursos lexicográficos y terminográficos específicos utilizados (diez preguntas para conocer los diccionarios específicos más empleados para cada categoría de la sección 4; según las instrucciones, únicamente se podía especificar un diccionario para cada categoría). Las preguntas de la sección 5 eran preguntas abiertas en las que los estudiantes disponían de un texto libre para proporcionar los diccionarios específicos que utilizan. Una vez finalizado el primer borrador, se invitó a un experto en cuestionarios a revisarlo. El análisis sirvió para conocer el tiempo necesario para completarlo (en torno a 10 minutos), y detectar posibles mejoras en la redacción y el contenido de las preguntas. Las preguntas y sus ítems se ajustaron y retocaron en función de los comentarios recibidos. El cuestionario se administró en el primer semestre, entre noviembre y diciembre, del curso académico 2021-2022. Los datos se recogieron en varias clases de distintas asignaturas de los diferentes cursos de grado y máster, elegidas en función de la disposición del profesorado a participar en el estudio y con la idea de contar con una muestra equilibrada de cada curso académico. Como afirma Camacho (2013), el estudio de la lexicografía y terminografía se encuentra, sobre todo, en la asignatura de Terminología, aunque también está presente en la asignatura de Lengua Española, Lingüística o Documentación. Así pues, conviene destacar que, en el momento de la administración del cuestionario, los estudiantes de primer curso no habían cursado ninguna asignatura de traducción ni ninguna asignatura específica sobre Documentación aplicada a la Traducción o Terminología, pero sí estaban cursando la asignatura de Lengua A1 Español. En el caso de los estudiantes de segundo curso, ya habían completado la asignatura de Documentación aplicada a la Traducción y Lingüística; los de tercer curso, además, ya tenían experiencia con asignaturas de traducción y estaban terminando de cursar la asignatura de Terminología; y los de cuarto curso habían completado una amplia variedad de módulos de traducción, tanto general como especializada, en diversas combinaciones lingüísticas. En lo que se refiere a los estudiantes de máster, la gran mayoría contaba con una formación previa en Traducción. Hikma 22(2) (2023), 309 - 330 316 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? 3. RESULTADOS Para examinar los datos se utilizó el programa de análisis estadístico SPSS Versión 25.0. Dado que el cuestionario se administró en papel, los datos se tuvieron que introducir manualmente en el programa informático. Se utilizaron estadísticas descriptivas para responder a las preguntas de la investigación. Más concretamente, se empleó la prueba de chi-cuadrado para determinar si existían asociaciones significativas entre las variables, dado que es la prueba más común para determinar si hay una relación significativa entre dos variables categóricas, como es el caso de este estudio. Existen diferencias significativas cuando el valor de p es menor que 0,05 (p < 0,05). En primer lugar, investigamos sobre las tendencias generales de uso de los recursos lexicográficos y terminográficos, es decir, la frecuencia con la que los estudiantes utilizan los diccionarios (pregunta 6), y a qué formato de diccionario recurren (pregunta 7), que son las preguntas contenidas en la sección 3 del cuestionario. En respuesta a la pregunta 6 («¿con qué frecuencia usas los diccionarios?»), el 63 % (n = 104) de los estudiantes afirma utilizar los diccionarios con frecuencia; el 25,5 % (n = 42), a veces; y el 11,5 %, casi nunca. Sin embargo, los resultados varían significativamente entre los distintos cursos de grado y máster (p = 0,000) (Figura 1). Como se puede apreciar en la Figura 1, la frecuencia de uso parece aumentar conforme aumenta el curso. De esta forma, mientras que los estudiantes de segundo, tercer, cuarto curso y máster consultan los diccionarios con frecuencia (65,2 %, n = 30; 87,5 %, n = 21; 86,4 %, n = 19; y 81,8 %, n = 18, respectivamente), el porcentaje disminuye drásticamente para el caso de los estudiantes de primer curso (31,4 %, n = 16). Asimismo, mientras que el 27,5 % (n = 14) de los estudiantes de primer curso manifiesta que casi nunca se vale de diccionarios, el porcentaje de estudiantes que consultan los diccionarios con muy poca frecuencia es mucho menor para el resto de los cursos de grado y máster, situándose en torno al 4 %. Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 317 Figura 1. ¿Con qué frecuencia usas los diccionarios? Respuestas en función del curso Fuente. Elaboración propia La pregunta 7 («¿qué formato de diccionario usas con más frecuencia?») pretendía indagar acerca del tipo de formato de diccionario que los estudiantes utilizaban con más asiduidad. Solo el 1,8 % (n = 3) de los encuestados respondió que usa diccionarios en papel, siendo los ordenadores la opción preferida para consultar los diccionarios (64,8 %, n = 107). El porcentaje de estudiantes que utiliza principalmente los teléfonos inteligentes para acceder a los diccionarios es del 27,9 % (n = 46) y las tabletas representan el 5,5 % (n = 9). Como se muestra en la Figura 2, existen diferencias estadísticamente significativas (p = 0,011) entre los estudiantes de los distintos cursos de grado y máster en relación con el formato que suelen utilizar con más frecuencia para acceder a los diccionarios. En otras palabras, aunque la mayoría de los estudiantes utiliza el ordenador para consultar los diccionarios en todos los cursos académicos, el porcentaje de alumnado que emplea los diccionarios a través del ordenador es mucho mayor a partir del segundo curso (60,9 %, n = 28, para los alumnos de segundo curso; 87,5 %, n = 21, para los de tercero; 86,4 %, n = 19, para los de cuarto; y 86,4 %, n = 19, para los estudiantes de máster; frente al 39,2 %, n = 20, de los estudiantes de primer curso). Además, también cabe destacar que hay un número notablemente mayor de estudiantes de primer y segundo curso que acceden a los Hikma 22(2) (2023), 309 - 330 318 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? diccionarios a través de sus teléfonos inteligentes (51 %, n = 26; y 32,6 %, n = 15, respectivamente) en comparación con el resto de alumnado de otros cursos, que oscila entre el 0,0 % para los estudiantes de tercero, el 9,1 % (n = 2) para los de máster, y el 13,6 % (n = 3) para los de cuarto curso. Figura 2. ¿Qué formato de diccionario usas más? Respuestas en función del curso Fuente. Elaboración propia Una vez analizados los patrones generales de uso de los diccionarios (sección 3), la sección 4 incluía diez preguntas que pretendían estudiar las tendencias específicas de consulta de los distintos tipos de recursos lexicográficos y terminográficos. Como se ha mencionado anteriormente, las respuestas de los participantes se basaron exclusivamente en una escala Likert de cuatro puntos («nunca», «casi nunca», «a veces», «con frecuencia») para evitar la tendencia de los participantes en escalas con un número impar de puntos a marcar la posición central o neutra. Los diccionarios se clasificaron ateniendo al nivel de especialización (general o especializado) y, posteriormente, en cada nivel de especialización, en función del formato de representación (papel o electrónico) y del número de lenguas (monolingües español o inglés, o bilingües inglés/español). De esta forma, se preguntó a los estudiantes con qué frecuencia consultaban los siguientes tipos de recursos: (i) diccionario general en español (en papel), (ii) diccionario general en español (electrónico), Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 319 (iii) diccionario general en inglés (en papel), (iv) diccionario general en inglés (electrónico), (v) diccionario general bilingüe inglés/español (en papel), (vi) diccionario general bilingüe inglés/español (electrónico), (vii) diccionario especializado en inglés o en español (en papel), (viii) diccionario especializado en inglés o en español (electrónico), (ix) diccionario especializado bilingüe inglés/español (en papel), y (x) diccionario especializado bilingüe inglés/español (electrónico). La Tabla 1 recogida más abajo resume la frecuencia de uso de los distintos recursos indicada por los 165 encuestados, mas sin ninguna distinción entre los diferentes cursos académicos. La primera columna de la izquierda contiene las diversas categorías de diccionarios establecidas, distinguiendo entre recursos de la lengua general y recursos especializados; y las columnas de la derecha ofrecen la asiduidad de consulta («nunca», «casi nunca», «a veces», «con frecuencia») en términos de frecuencias y porcentajes. Como se puede observar en la Tabla 1, los recursos electrónicos fueron, con diferencia, más utilizados que los de papel, siendo el porcentaje de consulta de los recursos especializados en papel aún menor. En este sentido, de los 165 encuestados, 145 (87,9 %) respondieron que nunca consultan diccionarios especializados monolingües en español o en inglés en papel; y, 137 (83 %), que nunca acceden a diccionarios especializados bilingües inglés-español en formato impreso. Asimismo, los datos demuestran que los recursos de la lengua general (recursos lexicográficos) se utilizaron más que los especializados (recursos terminográficos). A este respecto, mientras que solo en torno a un 10 % de los estudiantes afirma utilizar con frecuencia los diccionarios monolingües o bilingües especializados en formato electrónico, el porcentaje de uso frecuente de los diccionarios bilingües inglés-español en formato electrónico asciende a un 68,5 %; el uso de diccionarios generales en inglés en soporte digital, a un 63 %; y el de diccionarios generales en español en formato electrónico, a un 43 %. De hecho, estos tres recursos lexicográficos resultaron los más empleados por los estudiantes. Nunca Diccionario general en español (papel) en español (electrónico) en inglés (papel) en inglés (electrónico) A veces N (%) Casi nunca N (%) N (%) Con frecuencia N (%) 86 (52,1) 2 (1,2) 98 (59,4) 3 (1,8) 66 (40) 42 (25,5) 50 (30,3) 24 (14,5) 10 (6,1) 50 (30,3) 14 (8,5) 34 (20,6) 3 (1,8) 71 (43) 3 (1,8) 104 (63) Hikma 22(2) (2023), 309 - 330 320 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? bilingüe inglés/español 104 (63) 45 (27,3) 10 (6,1) 6 (3,6) (papel) bilingüe inglés/español 7 (4,2) 19 (11,5) 26 (15,8) 113 (68,5) (electrónico) Diccionario especializado en español o en inglés 145 (87,9) 12 (7,3) 6 (3,6) 2 (1,2) (papel) en español o en inglés 83 (50,3) 39 (23,6) 27 (16,4) 16 (9,7) (electrónico) bilingüe inglés/español 137 (83) 16 (9,7) 9 (5,5) 3 (1,8) (papel) bilingüe inglés/español 97 (58,8) 38 (23) 13 (7,9) 17 (10,3) (electrónico) Tabla 1. Frecuencia de uso de los distintos tipos de recursos lexicográficos y terminográficos (preguntas 8-18). Los datos se expresan en frecuencias y porcentajes Fuente. Elaboración propia A continuación, se ofrece un desglose completo de la frecuencia de uso de cada tipo de recurso por curso académico con el objetivo de estudiar si existen diferencias significativas entre los estudiantes de los cuatro cursos de grado y máster. En aquellos casos en los que se dan estas, se ofrece un diagrama de barras con los resultados divididos por curso académico. Además, se incluyen también los diccionarios específicos reseñados por los estudiantes para cada categoría general de diccionarios. Estos datos proceden de las respuestas recogidas en la última sección del cuestionario (sección 5, preguntas 19-29). Como ya se ha mencionado anteriormente, se trataba de preguntas abiertas que permitían respuestas de texto libre. Pasamos, en primer lugar, a describir la frecuencia de uso del diccionario general en español en papel. Para esta categoría, no hubo diferencias significativas entre los cursos (p = 0,314). En general, se observó que la mayoría de los estudiantes de los cuatro cursos de grado y la titulación de máster nunca o casi nunca utilizaban tales diccionarios. Los recursos específicos más consultados por el alumnado dentro de esta categoría (pregunta 19) fueron los siguientes: el Diccionario de la lengua española, reseñado por el 16,4 % (n = 27); el Diccionario de uso del español, de María Moliner, por el 3 % (n = 5); el Diccionario Clave (Diccionario de uso del español actual), por el 1,8 % (n = 3); el Diccionario Santillana del español, por el 1,2 % (n = 2); y el Diccionario VOX esencial de la lengua española, por el 1,2 % (n = 2). Conviene destacar que el 76,4 % (n = 126) de los estudiantes no especificó ningún recurso en esta categoría y dejó la respuesta en blanco. La frecuencia de uso del diccionario general en español en formato electrónico sí arrojó diferencias significativas entre los distintos cursos Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 321 (p = 0,010). Como se aprecia en la Figura 3, la frecuencia de consulta de este tipo de recurso se acrecentó, en general, a medida que aumenta el curso académico. En este sentido, mientras que el porcentaje de alumnado que recurre con frecuencia a este tipo de recurso se sitúa en el 43,5 % (n = 20) para los estudiantes de segundo curso, el 45,8 % (n = 211) para los de tercero, el 63,6 % (n = 14) para los de cuarto y el 68,2 % (n = 15) para los de máster, el porcentaje baja hasta el 21,6 % (n = 11) para los estudiantes de primer curso. Figura 3. Frecuencia de uso de diccionarios generales en español (electrónicos). Respuestas en función del curso Fuente. Elaboración propia En consonancia con los recursos lexicográficos más populares dentro de la categoría de diccionario general en español en papel, el homólogo en formato electrónico más reseñado fue el Diccionario de la lengua española (67,9 %, n = 112), seguido del Diccionario Clave (Diccionario de uso del español actual) (4,2 %, n = 7) y del Diccionario Panhispánico de dudas (3 %, n = 5). El 24,8 % (n = 41) no citó ningún recurso dentro de esta categoría. En la frecuencia de uso del diccionario general en inglés en papel, no se detectaron diferencias relevantes entre los cursos (p = 0,054). En general, los estudiantes consultaron muy poco estos recursos y solo un 1,8 % (n = 3) afirmó utilizarlos con frecuencia (véase la Tabla 4). El Oxford English dictionary fue el recurso más consultado (13,9 %, n = 23); seguido del Collins English dictionary (4,2 %, n = 7), el Cambridge English dictionary (3,6 %, Hikma 22(2) (2023), 309 - 330 322 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? n = 6) y el Longman dictionary of contemporary English (2,4 %, n = 4). El 75,8 % (n = 125) de los estudiantes no citó ningún recurso dentro de esta categoría de diccionarios. Tampoco se apreciaron disparidades significativas entre los cursos en el uso de los diccionarios generales en inglés en formato electrónico (p = 0,701). En este caso, el Cambridge English dictionary fue, con diferencia, el recurso más consultado (71 estudiantes, 43 %), seguido del Oxford English dictionary (10,3 %, n = 17), el WordReference (English definition) (8,5 %, n = 14), el Collins English dictionary (6,7 %, n = 11), el Merriam Webster dictionary (3,6 %, n = 6), el Urban dictionary (1,8 %, n = 3), el Longman dictionary of contemporary English y el Macmillan English dictionary (0,6 %, n = 1). El 24,8 % (n = 41) dejó la respuesta en blanco. En lo que se refiere a la frecuencia de uso de los diccionarios generales bilingües en papel, como era de esperar como con los recursos en papel, se utilizaron poco y no se encontraron diferencias sustanciales (p = 0,221). El recurso más consultado fue el Gran diccionario Oxford español-inglés, inglésespañol (16,4 %, n = 27); seguido del Diccionario Cambridge Compact English-Spanish, español-inglés y el Collins pocket plus English-Spanish, español-inglés, (2,4 %, n = 4) cada uno; el Diccionario manual VOX inglésespañol, español-inglés (1,2 %, n = 2) y el Longman diccionario pocket inglésespañol, español-inglés (0,6 %, n = 1). De los 165 estudiantes, el 77 % (n = 127) no detalló ningún diccionario para esta categoría. Como se ha mencionado anteriormente, el diccionario general bilingüe en formato electrónico fue el recurso más consultado por los estudiantes de todos los cursos de grado y máster. Para esta categoría, no hubo variaciones significativas entre los cursos académicos (p = 0,258). El WordReference constituyó el recurso más utilizado con un 41,8 % (n = 69), seguido del Cambridge dictionary. Diccionario inglés-español (15,2 %, n = 25). El resto de las fuentes reseñadas, aunque con una representación menor, fueron Reverso diccionario inglés-español (3,6 %, n = 6), Oxford bilingüe y Collins Spanish dictionary (3 % cada uno, n = 5), Diccionario en línea inglés-español de PONS (1,2 %, n = 2) y Longman English-Spanish dictionary (0,6 %, n = 1). El 31,5 % (n = 52) no indicó ningún recurso en este apartado. Pasamos ahora a describir los resultados referidos al uso de los recursos terminográficos (sección 4, preguntas 14-17). Como se adelantó previamente, más del 90 % de los encuestados afirmó utilizar nunca o casi nunca los diccionarios especializados en papel, ni monolingües (en inglés o español) ni bilingües. No se identificaron diferencias significativas entre los cuatro cursos de grado o el máster para los recursos en papel (diccionario especializado en inglés o en español en papel: p = 0,366; y diccionario Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 323 especializado bilingüe inglés/español en papel: p = 0,080). El 95,2 % de los encuestados, tanto para el caso de los diccionarios especializados monolingües (en inglés o en español), como para los diccionarios especializados bilingües inglés/español en papel, dejó la respuesta en blanco y no especificó ningún recurso concreto. No obstante, los que sí detallaron los recursos que utilizaban con más asiduidad dentro de los diccionarios especializados en papel, en inglés o en español, indicaron el Black's law dictionary (4,2 %, n = 7), y el Diccionario de términos jurídicos de la editorial Comares (0,6 %, n = 1). En el caso de los bilingües, el 3,6 % (n = 6) incluyó el Diccionario de términos jurídicos (inglés-español, español-inglés) de Alcaraz Varó y, el 1,2 % (n = 2), el Libro rojo: diccionario de dudas y dificultades de traducción del inglés médico de Fernando Navarro. El uso de los recursos terminográficos en formato electrónico se situó en torno al 10 %. Más concretamente, el 9,7 % (n = 16) afirmó emplean con frecuencia los diccionarios especializados monolingües (en inglés o español) en formato electrónico; y, el 10,3 % (n = 17), los recursos especializados bilingües en el mismo formato. No obstante, al contrario de lo que ocurría con sus homólogos en papel, en el caso de los recursos especializados en formato electrónico sí se detectaron diferencias significativas entre los distintos cursos (p = 0,000), tanto para los diccionarios especializados monolingües en formato electrónico (véase Figura 4), como para los bilingües (véase Figura 5). La Figura 4 muestra el uso de los diccionarios especializados en inglés o en español en formato electrónico. Como se puede observar, el porcentaje de los estudiantes que declaran no consultar nunca un recurso de estas características es muy alto en el primer curso (76,5 %, n = 39), continúa siendo alto para los estudiantes de segundo curso (60,9 %, n = 28) y baja considerablemente para el resto de cursos (25 %, n = 6; 22,7 %, n = 5; y 22,7 %, n = 5 para tercero, cuarto y máster respectivamente). De forma similar, se aprecia un aumento importante de uso frecuente de estos recursos en los estudiantes de cuarto curso y máster en comparación con el resto de cursos. De esta forma, mientras que ningún estudiante de primero afirma utilizar con frecuencia los diccionarios especializados monolingües en inglés o en español en formato electrónico, este porcentaje se sitúa en el 4,3 % (n = 2) para los estudiantes de segundo y en el 4,2 % (n = 1) para los estudiantes de tercero; y sube hasta el 31,8 % (n = 7) para los estudiantes de cuarto de grado y 27,3 % (n = 6) para los de máster. En cuanto a los recursos específicos señalados por los estudiantes, conviene destacar que el 77 % (n = 127) no especificó nada. El diccionario más citado fue el Diccionario panhispánico del español jurídico, por el 20 % Hikma 22(2) (2023), 309 - 330 324 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? de los estudiantes (n = 33); el Black’s law dictionary, por el 1,8 % (n = 3); y el Oxford concise medical dictionary, por el 1,2 % (n = 2). Figura 4. Frecuencia de uso de diccionarios especializados en inglés o en español (electrónicos). Respuestas en función del curso Fuente. Elaboración propia La Figura 5 recoge la frecuencia de uso de los diccionarios especializados bilingües (inglés/español) en formato electrónico en función del curso. Como se puede observar, dicha frecuencia parece incrementarse a medida que aumenta el curso. Así, mientras que menos del 10 % del alumnado de los tres primeros cursos consulta este recurso (más concretamente, ningún estudiante de primero; el 8,7 %, n = 4, de los estudiantes de segundo; y el 8,7 %, n = 2, de los de tercero), el porcentaje aumenta hasta el 22,7 % (n = 5) y el 27,3 % (n = 6) para los estudiantes de cuarto y máster respectivamente. En contraposición, mientras que el porcentaje de estudiantes que afirma no recurrir nunca a un diccionario especializado bilingüe en formato electrónico resulta alto en los tres primeros cursos de grado (84,3 %, n = 43, primer curso; 52,3 %, n = 24, segundo curso; y 66,7 %, n = 16, tercer curso), dicho porcentaje es significativamente menor en los estudiantes de cuarto (36,4 %, n = 8) y los de máster (27,3 %, n = 6). Más del 92,7 % (n = 153) no citaron ningún recurso específico; el 2,4 % (n = 4) nombró la base de datos de la Unión Europea denominada IATE; y, el 2,4 % (n = 4), el Libro rojo. Por último, la UNTERM (base de datos de las Naciones Unidas), el English-Spanish business dictionary, el Vademecum y la Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 325 EcoLexicon (base de conocimiento sobre el medio ambiente) fueron enumeradas por un 0,6 % (n = 1) de los estudiantes. Figura 5. Frecuencia de uso de diccionarios especializados bilingües (electrónicos). Respuestas en función del curso Fuente. Elaboración propia 4. DISCUSIÓN La sección de resultados se ha centrado en el análisis y presentación de los datos estadísticos. Así pues, en el presente apartado, se abordarán las inferencias más importantes que pueden derivarse del estudio realizado. Esta investigación ha corroborado que los recursos electrónicos se utilizan mucho más que los de papel (98,2 % frente a 1,8 %). Tales resultados coinciden con el resto de estudios recientes acerca del uso de diccionarios pro parte de los estudiantes de Traducción (Kodura, 2016; Altuwairesh, 2021, 2022; Karami y Riasati, 2023). Los resultados confirmaron que el ordenador constituye el dispositivo más empleado por la mayoría de los encuestados de los cuatro cursos de grado y máster para la consulta de diccionarios, seguido de los teléfonos inteligentes y las tabletas. Esto coincide con los resultados del cuestionario que evaluó el uso de diccionarios monolingües en toda Europa, con más de 9000 participantes (Kosem et al., 2019). Sin embargo, en nuestro estudio se encontraron diferencias significativas entre los cursos de grado y el máster. Hikma 22(2) (2023), 309 - 330 326 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? Por lo tanto, aunque la mayoría de los estudiantes se valen de los ordenadores para recurrir a los diccionarios, el porcentaje parece incrementarse a medida que aumenta el curso. En cambio, el uso de los teléfonos inteligentes parece disminuir a medida que los estudiantes avanzan en sus estudios y, por ende, se registró un número sustancialmente mayor de estudiantes de primer año que consultan los diccionarios a través de un teléfono inteligente en comparación con el resto de cursos. A diferencia de otras investigaciones con estudiantes de segundas lenguas (como aquella de Gromann y Schnitzer, 2015), en las que la utilización de recursos bilingües supera a la de los monolingües, nuestros resultados muestran un uso similar de los diccionarios monolingües y bilingües, tanto de los recursos lexicográficos como terminográficos. El tipo de fuente más empleada entre los estudiantes de Traducción es el diccionario general bilingüe inglés/español en formato electrónico (68,5 %, n = 113), seguido del diccionario general en inglés en formato electrónico (63 %, n = 104) y del diccionario general en español en formato electrónico (43 %, n = 71). Los resultados son similares a los obtenidos con estudiantes de Traducción de otras universidades (Spike, 2022). No sorprende que el diccionario bilingüe de la lengua general sea el más utilizado porque, tal y como advierte Sycz-Opón (2019, p. 167), la mayoría de las búsquedas de los estudiantes de Traducción comienzan con la consulta de un diccionario bilingüe (78,63 %). En general, hubo diferencias significativas entre los cursos académicos y parece que el uso de los diccionarios monolingües se acrecienta a medida que aumenta el curso académico. Este hecho se halla en consonancia con la postura de Gavriilidou (2014, p. 44), quien establece que los diccionarios monolingües suelen ser más útiles para los estudiantes con un nivel de competencia lingüística superior. El diccionario general más consultado en español, tanto en papel como en formato electrónico, fue el Diccionario de la lengua española de la Real Academia Española; y en inglés, el Oxford English dictionary, en papel, y el Cambridge English Dictionary, en formato electrónico. En lo que se refiere a los diccionarios generales bilingües, el Oxford español-inglés, inglés-español constituyó el recurso más usado en papel; y, el WordReference, el más popular en formato electrónico, reseñado por casi la mitad de los estudiantes (41,8 %, n = 69). En este artículo ha quedado de manifiesto el escaso uso de los recursos terminográficos y el desconocimiento de diccionarios especializados por parte de los estudiantes de Traducción, incluso en aquellos de cursos superiores y de máster. Más del 80 % del alumnado declaró no utilizar nunca un recurso especializado en español o en inglés en papel, y el porcentaje de estudiantes que consultaba con frecuencia un diccionario electrónico en Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 327 inglés o en español o bilingüe inglés/español se situaba en el 16 % y 17 % respectivamente. Estas cifras son ligeramente superiores a las señaladas por Gromann y Schnitzer (2015), quienes ubicaban en torno al 10 % el uso de los recursos especializados por los estudiantes de segundas lenguas. Sin embargo, el hecho de que nuestro trabajo se centre en estudiantes de Traducción que cuentan con varias asignaturas de traducción especializada (tanto en el grado como en el máster) y necesitan documentarse para cada encargo de traducción, nos hizo pensar que los recursos terminográficos iban a ser más populares entre dichos estudiantes. Sin embargo, llama la atención que sí se aprecian diferencias significativas entre los distintos cursos, y que parece que la consulta de los recursos especializados aumenta especialmente en el último curso de grado y en el máster, situándose en torno al 30 %. Con la excepción de la categoría de diccionarios especializados en inglés o en español en formato electrónico, para el resto de recursos especializados, más del 90 % de los estudiantes no especificó ningún diccionario en particular. Además, la mayoría de los recursos enumerados correspondían al ámbito jurídico. Esto es algo previsible, ya que las asignaturas de traducción especializada incluyen la traducción jurídica. No obstante, la traducción científica, médica o económica también se integran dentro del contenido de las asignaturas de traducción especializada, por lo que sorprende que casi nadie las haya mencionado. Dentro de la categoría de recursos especializados en español en papel, el Diccionario de términos jurídicos, de la editorial Comares (solo indicado por un estudiante), y el Diccionario panhispánico del español jurídico (formato electrónico) fueron los más utilizados. En cuanto al inglés, el Black's law dictionary fue el más citado, tanto en formato papel como electrónico. Con relación a los recursos especializados bilingües en papel, el Diccionario de términos jurídicos: a dictionary of legal terms. inglés-español/ Spanish-English de Alcaraz-Varó fue el más reseñado (1,2 %) y, para la categoría de recursos electrónicos bilingües o multilingües, la base de datos IATE (2,4 %). CONCLUSIONES El objetivo de este artículo era identificar las tendencias de uso de los recursos lexicográficos y terminográficos por parte de los estudiantes del Grado en Traducción e Interpretación con lengua B inglés y del Máster en Traducción Profesional, con inglés entre su combinación lingüística, de la Universidad de Granada. Asimismo, se pretendía investigar sobre la posible existencia de diferencias significativas entre los estudiantes en función del curso y averiguar si estos conocen la variedad de recursos existentes. Hikma 22(2) (2023), 309 - 330 328 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? Algunas de las principales aportaciones del presente estudio a la investigación sobre usuarios de diccionarios son el hecho de que se centra en un usuario menos analizado, es decir, el estudiante de Traducción; que la muestra es relativamente alta (concretamente de 165 sujetos), teniendo en cuenta que se refiere a un perfil de usuario muy específico; que se compone de una muestra representativa de los cuatro cursos de grado y el curso de máster; y que es uno de los pocos estudios que compara las diferencias significativas entre los cuatro cursos de grado y el curso de máster. Sin embargo, este trabajo tiene algunas limitaciones que conviene destacar. Dado que la muestra está formada en su totalidad por estudiantes de Traducción de la misma universidad (la Universidad de Granada), nuestras conclusiones podrían no ser generalizables al resto de estudiantes de Traducción. Así pues, resultaría pertinente llevar a cabo la misma investigación en otras Facultades de Traducción en España, o en el extranjero, para poder confirmar las tendencias puestas de manifiesto en este estudio. En conclusión, nuestros resultados revelan que los estudiantes de Traducción utilizan los diccionarios con menos frecuencia de la que se esperaría, y que conocen, en general, pocos recursos. Esto es especialmente cierto en el caso de los recursos especializados. Tal hecho podría deberse a que las nuevas generaciones están cambiando sus hábitos de consulta de forma drástica a favor de plataformas virtuales, como Linguee, y de traductores automáticos, por la necesidad de encontrar respuestas rápidas a las búsquedas que realizan (Maldonado y Liébana, 2021, p. 208). Asimismo, el presente estudio confirma que los recursos impresos han quedado relegados a un segundo plano. No obstante, hay que tener en cuenta que muchos diccionarios especializados siguen estando disponibles únicamente en formato papel, por lo que los recursos terminográficos en papel no pueden abandonarse. A raíz de los resultados, esta investigación pone de manifiesto que se necesita más formación acerca de los recursos lexicográficos y terminográficos entre los estudiantes de Traducción para mejorar su competencia instrumental. REFERENCIAS BIBLIOGRAFICAS Altuwairesh, N. (2021). Translation trainee’s uses of and attitudes towards different types of dictionaries: a case study. International Journal of Applied Linguistics and English Literature, 10(2), 73-80. https://journals.aiac.org.au/index.php/IJALEL/article/view/6604 Altuwairesh, N. (2022). Successful translation student’s use of dictionaries. International Journal of English Linguistics, 12(2), 25-35. https://doi.org/10.5539/ijel.v12n2p25 Hikma 22(2) (2023), 309 - 330 Miriam Buendía Castro 329 Bergenholtz, H. y Tarp, S. (2010). LSP lexicography or terminography? The lexicographer’s point of view. En P. A. Fuertes-Olivera (Ed.), Specialized dictionaries for learners (pp. 27-37). De Gruyter. Bowker, L. (2010). The contribution of corpus linguistics to the development of specialised dictionaries for learners. En P. A. Fuertes-Olivera (Ed.), Specialized dictionaries for learners (pp. 155-168). De Gruyter. Camacho Niño, J. (2013). La lexicografía en los planes de estudio de traducción e interpretación en España. Hikma, 12, 47-63. https://doi.org/10.21071/hikma.v12i.5235 Gavriilidou, Z. (2014). Users’ abilities and performance in dictionary look up. En N. Lavidas, T. Alexiou y A. M. Sougari (Eds.), Major trends in theoretical and applied linguistics (pp. 41-52). De Gruyter. https://doi.org/10.2478/9788376560885.p13 Gromann, D. y Schnitzer, J. (2015). Where do business students turn for help? An empirical study on dictionary use inn foreign-language learning. International Journal of Lexicography, 29(1), 55-99. https://doi.org/10.1093/ijl/ecv027 Hamouda, A. (2013). A study of dictionary use by Saudi EFL students at Qassim University. Studies in English Language Teaching, 1(1), 227-257. https://doi.org/10.22158/selt.v1n1p227 Hausmann, F. J. (1989). Wörterbuchtypologie. En F. J. Hausmann, O. Reichmann, H. E. Wiegand y L. Zgusta (Eds.), Dictionaries. An international encyclopedia of lexicography 1 (pp. 968-981). De Gruyter. Karami, S. y Riasati, M. J. (2023). A study of electronic dictionary use and attitudes among Iranian students of translation during Covid-19 pandemic. Education Research International, 1-9. https://doi.org/10.1155/2023/7616256 Kodura, M. (2016). Dictionary-using skills of translation students. The Central European Journal of Social Sciences and Humanities, 4, 235-242. Kosem, I. et al. (2019). The image of the monolingual dictionary across Europe. Results of the European Survey of Dictionary use and Culture, International Journal of Lexicography, 32(1), 92-114. https://doi.org/10.1093/ijl/ecy022 L’Homme, M. C. y Leroyer, P. (2009). Combining the semantics of collocations with situation-driven search paths in specialized dictionaries. Terminology, 15(2), 258-283. https://doi.org/10.1075/term.15.2.05lho Hikma 22(2) (2023), 309 - 330 330 ¿Conocen y usan los estudiantes de Traducción suficientes recursos […]? Lew, R. y De Schryver, G. M. (2014). Dictionary users in the digital revolution. International Journal of. Lexicography, 27(4), 341-359. https://doi.org/10.1093/ijl/ecu011 Lew, R. y Szarowska, A. (2017). Evaluating online bilingual dictionaries: The case of popular free English-Polish dictionaries. ReCALL, 29(2), 138-159. https://doi.org/10.1017/S0958344016000252 Maldonado González, M. C. y Liébana González, M. (2021). Los motores de traducción automática y su uso como herramienta lexicográfica en la traducción de unidades léxicas aisladas. Círculo de Lingüística Aplicada a la Comunicación, 88, 189-211. https://dx.doi.org/10.5209/clac.77002 Márkus, K. P., Fajt, B. y Dringó-Horváth, I. (2023). Dictionary skills in teaching English and German as a foreign language in Hungary: a questionnaire study. International Journal of Lexicography, 36(2), 173-194. https://doi.org/10.1093/ijl/ecad004 Pérez Hernández, M. C. (2002). Explotación de los córpora textuales informatizados para la creación de bases de datos terminológicas basadas en el conocimiento [Tesis doctoral]. Universidad de Málaga. http://elies.rediris.es/elies18 Sánchez-Ramos, M. M. (2005). Research on dictionary use by trainee translators. Translator Journal 9(2). https://translationjournal.net/journal/32dictuse.htm Svike, S. (2022). Survey analysis of dictionary-using skills and habits among translation students. En A. Klosa-Kückelhaus, S. Engelberg, C. Möhrs y P. Storjohann (Eds.), Dictionaries and Society. Proceedings of the XX EURALEX International Congress (pp. 494-508). IDS-Verlag. https://euralex.org/wpcontent/uploads/2022/09/EURALEX2022_Proceedings.pdf Sycz-Opoń, J. (2019) Information-seeking behaviour of translation students at the University of Silesia during legal translation – an empirical investigation, The Interpreter and Translator Trainer, 13(2), 152-176. https://doi.org/10.1080/1750399X.2019.1565076 Hikma 22(2) (2023), 309 - 330
https://openalex.org/W4210485366
https://eumj.med.sumdu.edu.ua/index.php/journal/article/download/209/155
Kirghiz, Kyrgyz
null
THE RESULTS OF SURGICAL TREATMENT IN PATIENTS WITH VERTEBRAL METASTASES
Shìdnoukraïnsʹkij medičnij žurnal
2,021
cc-by
3,973
Abstract Yevgeniy I. Slynko1, Olexandr O. Potapov2, Yuriy V. Derkach1, 1Department of Spinal Neuro- surgery, Romodanov Neurosurgery Institute, Kyiv, Ukraine; 2Department of Neurosurgery and Neurology, Medical Institute, Sumy State University, Sumy, Ukraine Yevgeniy I. Slynko1, Olexandr O. Potapov2, Yuriy V. Derkach1, 1Department of Spinal Neuro- surgery, Romodanov Neurosurgery Institute, Kyiv, Ukraine; 2Department of Neurosurgery and Neurology, Medical Institute, Sumy State University, Sumy, Ukraine DOI: https://doi.org/10.21272/eumj.2021;9(4):310-317 DOI: https://doi.org/10.21272/eumj.2021;9(4):310-317 Corresponding author: Yuriy V. Derkach, Department of Spinal Neurosurgery, Romodanov Neurosurgery Institute, Kyiv, Ukraine e-mail: 290986@ukr.net DOI: https://doi.org/10.21272/eumj.2021;9(4):310-317 THE RESULTS OF SURGICAL TREATMENT IN PATIENTS WITH VERTEBRAL METASTASES. Ромоданова НАМН України, м. Київ, Україна; 2Kафедрa нейрохірургії та неврології медичного інституту Сумського державного універ- ситету, м. Суми, Україна Євгеній І. Слинько1, Олександр О. Потапов2, Юрій В. Деркач1, The results of surgical treatment in patients with vertebral metastases. EUMJ. 2021;9(4):310-317 DOI: https://doi.org/10.21272/eumj. 2021;9(4):310-317 РЕЗУЛЬТАТИ ХІРУРГІЧНОГО ЛІКУВАННЯ ХВОРИХ ЗI ВТОРИННИМИ УРАЖЕННЯМИ ХРЕБЦІВ. Резюме Євгеній І. Слинько1, Олександр О. Потапов2, Юрій В. Деркач1, 1Відділення патології спинного мозку, Інститут нейрохірургії ім. акад. А. П. Ромоданова НАМН України, м. Київ, Україна; 2Kафедрa нейрохірургії та неврології медичного інституту Сумського державного універ- ситету, м. Суми, Україна Євгеній І. Слинько1, Олександр О. Потапов2, Юрій В Деркач1 Євгеній І. Слинько1, Олександр О. Потапов2, Юрій В. Деркач1, Матеріали та методи. В дослідження включено 108 пацієнтів із метастазами в хребті, які отримували хірургічне лікування в Інсти- туті нейрохірургії ім. акад. А. П. Ромоданова НАМН України в пе- ріод з 2015 року по 2019 роки. Результати. Вибір хірургічного доступу залежав від таких фак- торів: розташування пухлини відносно твердої мозкової оболонки і нервових структур, кісток та був такий: задній доступ використову- вали для видалення пухлин, які займають задній та задньо-боковий простір по відношенню до мозку; боковий доступ ми використову- вали для видалення пухлин розташованих латерально від мозку; передній доступ ми використовували для видалення пухлин, розта- шованих спереду від спинного мозку. В І групі спостережень ми використовували задні доступи в 49 спостереженнях, передні доступи – в 19 спостереженнях, бокові доступи – в 5 спостереженнях. В ІІ групі спостережень використо- вували лише задні групи доступів. Обговорення. Вибір адекватного хірургічного доступу до пух- лини хребців, який мінімізує травму нервових структур під час ви- далення пухлини, значно покращує результати хірургічного ліку- вання. Використання передніх та бокових доступів при вентраль- них, вентролатеральних локалізація пухлин дає змогу тотально ви- далити пухлину, зменшити тракцію нервових структур та отримати достатній візуальний контроль операційного поля під час видалення пухлини, що в свою чергу позитивно впливає на регрес больового синдрому та провідникових порушень. Висновки. Диференційний підхід до вибору доступу для вида- лення пухлини зменшує неврологічний дефіцит в післяопераційно- му періоді, дає змогу максимально радикально видалити пухлину, що дозволяє у віддаленому періоді значно зменшити кількість ре- цидивів пухлин. Ключові слова: видалення пухлин хребців; особливості хірур- гічного пухлин хребців; вторинне ураження хребців. Автор, відповідальний за листування: Юрій В. Деркач, відділення патології спинного мозку, Інститут нейрохірургії ім. акад. А. П. Ромоданова, м. Київ, Україна e-mail: 290986@ukr.net How to сite/ Як цитувати статтю: Slynko YeI, Potapov OO, Derkach YuV. The results of surgical treatment in patients with vertebral metastases. EUMJ. 2021;9(4):310-317 DOI: https://doi.org/10.21272/eumj. 2021;9(4):310-317 Автор, відповідальний за листування: Юрій В. Деркач, відділення патології спинного мозку, Інститут нейрохірургії ім. акад. А. П. Ромоданова, м. Київ, Україна e-mail: 290986@ukr net Автор, відповідальний за листування: e-mail: 290986@ukr.net How to сite/ Як цитувати статтю: Slynko YeI, Potapov OO, Derkach YuV. The results of surgical treatment in patients with vertebral metastases. EUMJ. 2021;9(4):310-317 DOI: https://doi.org/10.21272/eumj. 2021;9(4):310-317 How to сite/ Як цитувати статтю: Slynko YeI, Potapov OO, Derkach YuV. The results of surgical treatment in patients with vertebral metastases. EUMJ. 2021;9(4):310-317 DOI: https://doi.org/10.21272/eumj. 2021;9(4):310-317 How to сite/ Як цитувати статтю: Slynko YeI, Potapov OO, Derkach YuV. THE RESULTS OF SURGICAL TREATMENT IN PATIENTS WITH VERTEBRAL METASTASES. Study Objectives. The study was conducted to analyze and compare different types of surgical access in the treatment of patients with metastatic vertebral lesions to improve the outcome of surgery. Materials and Methods. The study included 108 patients with vertebral metastases who were operated on at the Romodanov Neurosurgery Institute of the National Academy of Medical Sciences of Ukraine in 2015–2019. Results. The choice of surgical access depended on a few factors such as tumor location relative to the dura mater, bones, and nerve structures and was as follows: posterior access was used to resect tumors located posteriorly and posterolaterally to the brain; lateral access was used for tumors located laterally to the brain; anterior access was used to resect tumors located in front of the spinal cord. In Group I (73 patients), posterior access was used in 49 cases (67%), anterior access – in 19 cases (26%), and lateral access – in 5 cases (7%). In Group II (35 patients), only posterior access was used. Discussion. Selection of adequate surgical access for vertebral tumor resection in order to minimize nerve structure injury significantly improved the results of surgical treatment. Anterior and lateral access for ventral and ventrolateral tumors operation made it possible to completely resect the tumor, reduce the traction of nerve structures, and obtain sufficient visual control of the operating field during the surgery, which in turn had a positive effect on regression of pain and conduction disorders. Conclusions. A differential approach to the choice of surgical access reduces the neurological deficit in the postoperative period and allows radical resection of the tumor, which in turn helps to significantly reduce the number of tumor recurrences in the long- term period. Keywords: vertebral tumor resection, features of surgical treatment of vertebral tumors, secondary vertebral lesions.– This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ 310 Slynko YeI, Potapov OO, Derkach YuV EUMJ, 2021;9(4):310-317 Резюме Євгеній І. Слинько1, Олександр О. Потапов2, Юрій В. Деркач1, 1Відділення патології спинного мозку, Інститут нейрохірургії ім. акад. А. П. Ромоданова НАМН України, м. Київ, Україна; 2Kафедрa нейрохірургії та неврології медичного інституту Сумського державного універ- ситету, м. Суми, Україна Резюме Євгеній І. Слинько1, Олександр О. Потапов2, Юрій В. Деркач1, 1Відділення патології спинного мозку, Інститут нейрохірургії ім. акад. А. П. Introduction/Вступ In order to relieve pain and improve spinal cord function and the quality of life of patients, surgery is increasingly performed, including minimally invasive surgery, palliative surgery, or radical surgery. In turn, most studies report a significant clinical effect with carefully selected surgical methods in patients with vertebral metastases [2]. Flavio Tancioni et al. used minimally invasive surgery, resulting in clinical remission of pain (in Vertebral metastases are the most common type of secondary involvement of the vertebral spine with a prevalence of 30–70% in cancer patients; in 5–10% of cases, metastases cause epidural compression of the spinal cord, leading to conduction disorders, pain, and reduced quality of life [1]. 311 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ Slynko YeI, Potapov OO, Derkach YuV EUMJ, 2021;9(4):310-317 96% of cases) and improvement of neurological deficit after 2 weeks (in 88% of cases) [3]. Masuda et al. evaluated the surgical outcomes of 44 patients who underwent spinal cord decompression and spine stabilization and reported that all patients presented with improved scores by the Frankel Scale and ECOG Scale of Performance Status after surgery [4]. the most severe compression site. This classification includes 4 stages: stage 1 – no compression of the spinal cord; stage 2 falls into: 2a – involvement of the epidural space without deformation of the dural sac, 2b – involvement of the epidural space with deformation of the dural sac and no signs of spinal cord compression, 2c – deformation of the dural sac with signs of spinal cord compression; stage 3 – spinal cord compression with intact extra-axial fluid spaces; stage 4 – spinal cord compression with affected extra-axial fluid spaces. However, there are still some issues in the treatment of spinal metastases. Complications such as perioperative bleeding and spinal cord injury should be considered after surgery. Along with the rapid development of immunotherapy, endocrine therapy, radiation therapy, and chemotherapy (especially targeted therapy), multidisciplinary combination treatment in patients with malignant vertebral tumors has become a trend [5]. Therefore, the indications and contraindications for the surgical treatment of spinal metastases should be clearly understood. In order to resect the tumor completely, patients underwent radical surgery, including total or partial vertebrectomy, with subsequent stabilization of the spine, if necessary. All observations were divided into two groups. Introduction/Вступ In Group I (73 observations), a differential approach to the choice of surgical access was used depending on the direction of vertebral tumor growth and epidural component. In group II (35 observations), the posterior access was exclusively used to resect vertebral tumors. Accordingly, this study was conducted to analyze and compare different types of surgical access in the treatment of patients with metastatic vertebral lesions to improve the outcome of surgery. The minimum post-surgery follow-up duration was 2 weeks, the maximum – 72 months. The duration of the post-surgery follow-up period averaged 24.4 ± 1.2 months. Materials and methods The study included 108 patients with malignant vertebral tumors who were operated on at the Romodanov Neurosurgery Institute of the National Academy of Medical Sciences of Ukraine in the period from 2015 till 2019. The inclusion criteria were as follows: patients diagnosed with spinal metastases using clinical and instrumental examinations (CT, MRI, or PET-CT); patients with hematologic malignancies of the spine, including lymphoma and myeloma; patients with spinal cord disorders, such as primary spinal tumors, spinal tuberculosis or degenerative spine conditions. Results Results To objectify the degree of spinal cord compression, the epidural spinal cord compression scale (ESCC, 2011) was used, which was based on the assessment of axial T2-weighted MRI images of 312 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under EUMJ, 2021;9(4):310-317 Slynko YeI, Potapov OO, Derkach YuV Figure 1 – Distribution of cases with regard to the involved part of the spine 17 21 19 16 5 5 2 9 9 10 7 2 2 0 5 10 15 20 25 Cervical spine Thoracic spine Lumbar spine Sacral spine > 1 part involved One vertebral body involved, Group I More than one vertebral body involved, Group I One vertebral body involved, Group II More than one vertebral body involved, Group II Figure 1 – Distribution of cases with regard to the involved part of the spine The primary tumor sites in Group II were: renal cancer – 12 cases (34.2%), prostate cancer – 9 cases (25.7%), lymphoma and multiple myeloma – 5 cases (14.3%), lung cancer – 5 cases (14.3%), metastases of mesenchymal sarcoma – 2 cases (5.7%), thyroid cancer – 1 case (2.9%), unknown primary site – 1 case (2.9%) (Fig. 2). In all observations, the epidural spinal cord compression scale (ESCC, 2011) was used (Fig. 3). Figure 2 – Distribution of cases with regard to the primary tumor site 24 19 10 10 5 4 2 2 2 9 7 5 5 2 1 1 0 5 10 15 20 25 Renal cancer Prostate cancer Lymphoma and multiple myeloma Lung cancer Metastases of mesenchymal sarcoma Thyroid cancer Liver cancer Gastrointestinal cancer Unknown primary site Primary tumor site, Group II Primary tumor site, Group I Figure 2 – Distribution of cases with regard to the primary tumor site Group I showed the average score of 2.12 ± 0.21 in the preoperative period and 1.13 ± 0.14 – in the postoperative period (p < 0.05). Motor segmental disorders (MD) in Group I amounted to 3.13 ± 0.31 before surgery and 1.9 ± 0.23 after surgery, respectively (p < 0.05). Sensitive segmental disorders (SD) amounted to 3.23 ± 0.33 before surgery and 2.12 ± 0.24 in the postoperative period (p < 0.05). Results In Group I, thoracic spine was most commonly involved – 22 observations (30%); lumbar spine was involved in 20 cases (27.5%), cervical spine – in 18 cases (24.7%), and sacral spine – in 13 cases (17.8%). Tumors affecting more than two parts of the spine were observed in 6 cases. At the same time, 14 patients with only one affected part of the spine had more than one vertebra involved (Fig. 1). In Group II, cases with regard to involved part of the spine were distributed as follows: cervical spine – 11 observations (31%), thoracic spine – 12 observations (33.9%), lumbar spine – 7 observations (20%). In 5 cases (15.1%), vertebral tumors affected two or more parts of the spine (Fig. 1). Among the patients included in this study, Group I comprised 42 men (55.4%) and 36 women (44.6%). Group II included 16 men (53.3%) and 14 women (46.6%). Neurological deficits were evaluated using parameters of pain syndrome and conduction disorders before and after surgery. The primary tumor sites in Group I were: renal cancer – 21 cases (28.8%), prostate cancer – 17 cases (23.3%), lymphoma and multiple myeloma – 10 cases (13.7%), lung cancer – 10 cases (13.7%), metastases of mesenchymal sarcoma – 5 cases (6.9%), thyroid cancer – 4 cases (5.5%), liver cancer – 2 cases (2.7%), gastrointestinal cancer – 2 cases (2.7%), unknown primary site – 2 cases (2.7%) (Fig. 2). Pain syndrome was assessed according to a 5- point scale (0 – no pain, 5 – the maximum level of pain). Motor and sensory functions were assessed using the Frankel scale. Results Pain syndrome (PS) in Group I before surgery scored 4.2 ± 0.3 and after surgery it was 2.4 ± 0.4 (p < 0.05) (Fig. 4). PS in Group II equaled 4.3 ± 0.3 before surgery and 3.1 ± 0.2 after surgery (p < 0.05) (Fig. 4). The frequency of conduction disorders (motor and sensory segmental disorders), which were assessed using the Frankel scale before surgery and after surgery in Groups I and II, is presented in Fig. 5. Comparison of conduction disorders in 313 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ EUMJ, 2021;9(4):310-317 Slynko YeI, Potapov OO, Derkach YuV Figure 3 – Distribution of cases by the epidural spinal cord compression scale (ESCC, 2011) 16 27 21 7 4 3 Number of cases 1 2а 2b 2c 3 4 16 27 21 7 4 3 Number of cases 1 2а 2b 2c 3 4 amounted to 3.55 ± 0.36 before surgery and 3.22 ± 0.24 in the postoperative period (p > 0.05) (Fig. 5). Number of cases Number of cases 16 27 21 7 4 3 1 2а 2b 2c 3 4 The choice of surgical access depended on a few factors such as tumor location relative to the dura mater, bones and nerve structures and was as follows: posterior access was used to resect tumors located posteriorly and posterolaterally to the brain; lateral access was used for tumors located laterally to the brain; anterior access was used to resect tumors located in front of the spinal cord. In Group I, posterior access was used in 49 cases (67%), anterior access – in 19 cases (26%), and lateral access – in 5 cases (7%). In Group II, only posterior access was used (Fig. 6). The analysis of quality of life indicators showed that patients in Group I significantly more often (p = 0.035) had a satisfactory condition in the early and long-term postoperative period vs. patients in Group II: 33 cases (45.4%) in Group I and 7 cases (18.7%) in Group II in the early period and 35 cases (48%) in Group I and 12 cases (33.3%) in Group II in the long-term period. Results Figure 3 – Distribution of cases by the epidural spinal cord compression scale (ESCC, 2011) In Group II, the values were as follows: 2.34 ± 0.23 before surgery and 1.67 ± 0.19 in the postoperative period (p < 0.05). MD equaled 3.23 ± 0.32 before surgery and 2.45 ± 0.26 after surgery. SD Figure 4 – Pain syndrome before and after surgery 1 1 16 20 21 6 0 1 6 25 23 10 0 1 3 6 8 8 4 1 4 5 7 8 5 0 5 10 15 20 25 30 0 1 2 3 4 5 Before surgery, Group I After surgery, Group I Before surgery, Group II After surgery, Group II Figure 4 – Pain syndrome before and after surgery Discussion improving the results of combined treatment in patients with vertebral metastases. In contrast to primary tumors of the spine, spinal metastases were operated in order to improve the general condition of patients and maintain a good quality of life. That is, surgeons must take into account the somatic state of health of patients, instrumental study results, and the existing neurological deficit. In all cases, patients were first consulted in oncology centers and referred for further neurosurgical treatment. The degree of epidural compression according to the epidural spinal cord compression scale (ESCC, 2011) has a direct correlation with neurological disorders. Significant regression of pain and conduction disorders was observed in patients with stages 1, 2a, and 2b. Possible regression of pathological symptoms was reported in patients with stages 2b and 3. There was almost no regression of conduction disorders in patients with stage 4. The multifactorial approach to the differentiated selection of patients eligible for surgery allows 314 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ Slynko YeI, Potapov OO, Derkach YuV EUMJ, 2021;9(4):310-317 Figure 5 – Changes in neurological deficit by the Frankel scale 12 12 18 22 14 10 7 16 26 19 5 4 7 9 5 4 5 6 7 8 0 5 10 15 20 25 30 Frankel A Frankel B Frankel C Frankel D Frankel E Before surgery, Group I After surgery, Group I Before surgery, Group II After surgery, Group II After surgery, Group I After surgery, Group II Figure 5 – Changes in neurological deficit by the Frankel scale Selection of adequate surgical access for vertebral tumor resection in order to minimize nerve structure injury significantly improved the results of surgical treatment. Anterior and lateral access for ventral and ventrolateral tumors operation (Fig. 7, 8) made it possible to completely resect the tumor, reduce the traction of nerve structures, and obtain sufficient visual control of the operating field during the surgery, which in turn had a positive effect on regression of pain and conduction disorders. Conclusions/Висновки Anterior and lateral access for ventral and ventrolateral tumors operation made it possible to completely resect the tumor, reduce the traction of nerve structures, and obtain sufficient visual control of the operating field during the surgery. A differential approach to the choice of surgical access depending on tumor location in patients with vertebral tumors led to a reduction (38.7 ± 1.1 months in Group II; 18.5 ± 1.3 months in Group I) of neurological deficit and pain regression in the postoperative period. Discussion Figure 6 – Surgical accesses chosen for surgical treatment of patients 9 14 12 14 8 2 7 2 5 0 2 4 6 8 10 12 14 16 Cervical spine Thoracic spine Lumbar spine Sacral spine Posterior Anterior Lateral Figure 6 – Surgical accesses chosen for surgical treatment of patients the differentiated choice of surgical access to remove metastatic vertebral tumors provided a better quality of life in patients of Group I vs. Group II. Thus, evaluation of positive changes in performance status of patients with account of regression of neurological deficit in the early and long-term postoperative period demonstrated that This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ 315 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ Slynko YeI, Potapov OO, Derkach YuV EUMJ, 2021;9(4):310-317 a b c Figure 7 – Recection of L3 vertebral tumor using anterior access a b a Figure 7 – Recection of L3 vertebral tumor using anterior access a. MRI frontal image (red arrow indicates tumor location); b. Intraoperative photo of tumor resection; c. Intraoperative photo after tumor resection and spine stabilization b. Intraoperative photo of tumor resection; c. Intraoperative photo after tumor resection and spine stabilization a b c Figure 8 – Resection of Tn5–Tn7 vertebral tumors using anterior-lateral transthoracic access c a b b c a a Figure 8 – Resection of Tn5–Tn7 vertebral tumors using anterior-lateral transthoracic access a. MRI sagittal view image of a patient with a tumor of Tn5–Tn7 vertebrae; a. MRI sagittal view image of a patient with a tumor of Tn5–Tn7 vertebrae; b. MRI axial view image of a patient with a tumor of Tn5–Tn7 vertebrae; c. Intraoperative photo of tumor resection; c. Intraoperative photo after tumor resection and spine stabilization b. MRI axial view image of a patient with a tumor of Tn5–Tn7 vertebrae; c. Intraoperative photo of tumor resection; c. Intraoperative photo after tumor resection and spine stabi g f p f c. Intraoperative photo of tumor resection; c. Intraoperative photo after tumor resection and spine stabilization The authors declare no conflict of interest. The authors declare no conflict of interest. The authors declare no conflict of interest. 10.1016/B978-0-12-811161-1.00016-5. PMID: 29307356. 10.1016/B978-0-12-811161-1.00016-5. PMID: 29307356. 10.1016/B978-0-12-811161-1.00016-5. PMID: 29307356. multidisciplinary approaches in the management of metastatic epidural spinal cord compression. Future Oncol. 2018 Jul;14(17):1665-1668. doi: 10.2217/fon- 2018-0133. Epub 2018 Jun 25. PMID: 29939082. 4. Mossa-Basha M, Gerszten PC, Myrehaug S, Mayr NA, Yuh WT, Jabehdar Maralani P, Sahgal A, Lo SS. Spinal metastasis: diagnosis, management and follow-up. Br J Radiol. 2019 Nov;92(1103):20190211. doi: 10.1259/bjr.20190211. Epub 2019 Jul 25. PMID: 31322920; PMCID: PMC6849675 4. Mossa-Basha M, Gerszten PC, Myrehaug S, Mayr NA, Yuh WT, Jabehdar Maralani P, Sahgal A, Lo SS. Spinal metastasis: diagnosis, management and follow-up. Br J Radiol. 2019 Nov;92(1103):20190211. doi: 10.1259/bjr.20190211. Epub 2019 Jul 25. PMID: 31322920; PMCID: PMC6849675 (received 04.07.2021, published online 29.12.2021) (одержано 04.07.2021, опубліковано 29.12.2021) 5. Vellayappan BA, Kumar N, Chang EL, Sahgal A, Sloan AE, Lo SS. Novel Conflict of interest/Конфлікт інтересів The authors declare no conflict of interest. References/Список літератури 1. Guo Y, Ngo-Huang AT, Fu JB. Perspectives on Spinal Precautions in Patients Who Have Cancer and Spinal Metastasis. Phys Ther. 2020 Mar 10;100(3):554-563. doi: 10.1093/ptj/pzz178. PMID: 32043130. 2. Patnaik S, Turner J, Inaparthy P, Kieffer WK. Metastatic spinal cord compression. Br J Hosp Med (Lond). 2020 Apr 2;81(4):1-10. doi: 10.12968/hmed.2019.0399. PMID: 32339020. 3. Nater A, Sahgal A, Fehlings M. Management - spinal metastases. Handb Clin Neurol. 2018;149:239-255. doi: J Hosp Med (Lond). 2020 Apr 2;81(4):1-10. doi: 10.12968/hmed.2019.0399. PMID: 32339020. 1. Guo Y, Ngo-Huang AT, Fu JB. Perspectives on Spinal Precautions in Patients Who Have Cancer and Spinal Metastasis. Phys Ther. 2020 Mar 10;100(3):554-563. doi: 10.1093/ptj/pzz178. PMID: 32043130. 3. Nater A, Sahgal A, Fehlings M. Management - spinal metastases. Handb Clin Neurol. 2018;149:239-255. doi: 3. Nater A, Sahgal A, Fehlings M. Management - spinal metastases. Handb Clin Neurol. 2018;149:239-255. doi: 2. Patnaik S, Turner J, Inaparthy P, Kieffer WK. Metastatic spinal cord compression. Br This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ 316 Slynko YeI, Potapov OO, Derkach YuV EUMJ, 2021;9(4):310-317 Іnformation about the authors/Відомості про авторів Слинько Євгеній Ігорович – завідувач відділення патології спинного мозку, Інститут нейрохірургії ім. акад. А.П. Ромоданова НАМН України, м. Київ, Україна; Потапов Олександр Олександрович – завідувач кафедри нейрохірургії та неврології медичного ін- ституту Сумського державного університету, м.Суми, Україна; Деркач Юрій Володимирович – лікар-нейрохірург відділення патології спинного мозку, Інститут нейрохірургії ім. акад. А.П. Ромоданова НАМН України, м. Київ, Україна 317 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/
https://openalex.org/W2134625693
https://europepmc.org/articles/pmc3118331?pdf=render
English
null
Supplementation of a western diet with golden kiwifruits (Actinidia chinensis var.'Hort 16A':) effects on biomarkers of oxidation damage and antioxidant protection
Nutrition journal
2,011
cc-by
7,647
* Correspondence: asgeir.brevik@fhi.no 1Department of Chemical Toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O.Box 4404 Nydalen, N-0403 OSLO, Norway Full list of author information is available at the end of the article Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Abstract Background: The health positive effects of diets high in fruits and vegetables are generally not replicated in supplementation trials with isolated antioxidants and vitamins, and as a consequence the emphasis of chronic disease prevention has shifted to whole foods and whole food products. Methods: We carried out a human intervention trial with the golden kiwifruit, Actinidia chinensis, measuring markers of antioxidant status, DNA stability, plasma lipids, and platelet aggregation. Our hypothesis was that supplementation of a normal diet with kiwifruits would have an effect on biomarkers of oxidative status. Healthy volunteers supplemented a normal diet with either one or two golden kiwifruits per day in a cross-over study lasting 2 × 4 weeks. Plasma levels of vitamin C, and carotenoids, and the ferric reducing activity of plasma (FRAP) were measured. Malondialdehyde was assessed as a biomarker of lipid oxidation. Effects on DNA damage in circulating lymphocytes were estimated using the comet assay with enzyme modification to measure specific lesions; another modification allowed estimation of DNA repair. Results: Plasma vitamin C increased after supplementation as did resistance towards H2O2-induced DNA damage. Purine oxidation in lymphocyte DNA decreased significantly after one kiwifruit per day, pyrimidine oxidation decreased after two fruits per day. Neither DNA base excision nor nucleotide excision repair was influenced by kiwifruit consumption. Malondialdehyde was not affected, but plasma triglycerides decreased. Whole blood platelet aggregation was decreased by kiwifruit supplementation. Conclusion: Golden kiwifruit consumption strengthens resistance towards endogenous oxidative damage. © 2011 Brevik et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Supplementation of a western diet with golden kiwifruits (Actinidia chinensis var.’Hort 16A’:) effects on biomarkers of oxidation damage and antioxidant protection Supplementation of a western diet with golden kiwifruits (Actinidia chinensis var.’Hort 16A’:) effects on biomarkers of oxidation damage and antioxidant protection Asgeir Brevik1*, Isabel Gaivão2, Tirill Medin3, Aud Jørgenesen3, Anita Piasek4, Johanna Elilasson5, Anette Karlsen3, Rune Blomhoff3, Turid Veggan3, Asim K Duttaroy3 and Andrew R Collins3 Introduction attributed to their high content of antioxidants [4,5]. However, attempts to boost human resistance to cardio- vascular disease and cancer through supplementation trials with isolated antioxidants and vitamins have proved disappointing [6-8], and there is no reason to believe that negative effects of unhealthy diets and life- style can be remedied through the use of antioxidant supplements. Hence, the emphasis of chronic disease prevention policy has shifted to whole foods and whole food products. In addition to recognised antioxidants such as vitamins C and E, carotenoids and flavonoids, fruits and vegetables contain innumerable other phyto- chemicals, with known or (mostly) unknown effects on Diets rich in fruits and vegetables offer protection against the development of cardiovascular diseases (CVD), diabetes and cancer [1-3]. A common factor in the aetiology of these diseases seems to be damage to biomolecules caused by reactive oxygen species. Power- ful endogenous antioxidant defences are thought to be augmented by dietary antioxidants, and so much of the protective effect of fruits and vegetables has been Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Page 2 of 9 human metabolism. Antioxidant activity is clearly not the whole story. Subjects received two different ‘doses’ of kiwifruit in two 4-week supplementation periods separated by a 4- week washout period. The cross-over design of the study minimises so-called seasonal effects. Subjects were randomized into two groups taking the kiwifruit doses in different order; either one kiwifruit per day in the first period, two in the second, or two per day in the first period, one in the second. Subjects were not given specific times at which to consume the fruit, but were asked to add the fruit to their normal diet. They were specifically instructed to avoid increasing their habitual low to moderate intake of fruits and vegetables. y Kiwifruit is particularly rich in vitamin C (ascorbic acid), but also contains a wide range of other phyto- chemicals. The common green kiwifruit, Actinidia deli- ciosa, has been used as a ‘model’ fruit in several trials to examine effects on biomarkers relevant to both cancer and CVD. Typically green kiwifruit contains approxi- mately 85 mg vitamin C per 100 mg fresh weight [9]. Blood sampling F bl p g Four venous blood samples were taken from overnight- fasted subjects: 1) pre-intervention, first period; 2) post- intervention, first period; 3) pre-intervention, second period; 4) post-intervention, second period. A pre-study run-in sample was taken to practice sampling routines. A post-study sample was taken to check for return to baseline levels 4 weeks after the end of supplementation. Blood was collected in Cell Preparation Tubes (Beck- ton Dickinson) and centrifuged (700 × g, 10 min at 4°C) to separate plasma, red cells and mononuclear cells. Plasma aliquots were snap-frozen in liquid nitrogen and stored at -80°C; those intended for vitamin C analysis were acidified with an equal volume of 10% meta-phos- phoric acid (MPA). The MPA solution was freshly pre- pared (within 14 days) and stored at 4°C. Mononuclear cells were analysed for DNA damage on the day of iso- lation. Mononuclear cells suspended in 45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, pH7.8 at 5 × 107 cells per ml were snap-frozen in liquid nitrogen and stored at -80°C for use in the in vitro BER and NER assays. Four venous blood samples were taken from overnight- fasted subjects: 1) pre-intervention, first period; 2) post- intervention, first period; 3) pre-intervention, second period; 4) post-intervention, second period. A pre-study run-in sample was taken to practice sampling routines. A post-study sample was taken to check for return to baseline levels 4 weeks after the end of supplementation. Blood was collected in Cell Preparation Tubes (Beck- ton Dickinson) and centrifuged (700 × g, 10 min at 4°C) to separate plasma, red cells and mononuclear cells. Plasma aliquots were snap-frozen in liquid nitrogen and stored at -80°C; those intended for vitamin C analysis were acidified with an equal volume of 10% meta-phos- phoric acid (MPA). The MPA solution was freshly pre- pared (within 14 days) and stored at 4°C. Mononuclear cells were analysed for DNA damage on the day of iso- lation. Mononuclear cells suspended in 45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, pH7.8 at 5 × 107 cells per ml were snap-frozen in liquid nitrogen and stored at -80°C for use in the in vitro BER and NER assays. Introduction A kiwifruit extract has powerful antioxidant activity in vitro [10], and in humans, regular consumption of this fruit inhibits platelet aggregation [11], decreases endo- genous oxidation of lymphocyte DNA, protects lympho- cyte DNA from oxidation in vitro, and enhances the capacity of lymphocytes to repair DNA oxidation damage [10,12-15]. The more recently available ‘golden’ kiwifruit Actinidia chinensis var. Hort 16A, differs signif- icantly in phytochemical make-up (with 20% higher vita- min C content [9]), demonstrating higher FRAP values [16] than the green kiwifruit. Based on these properties the golden kiwifruit would be expected to show stronger protection against effects of oxidative damage in-vivo. To test this hypothesis, we conducted a human dietary intervention trial with golden kiwifruit, examining potential effects on platelet function, plasma antioxidant status, DNA oxidation, and base excision repair (BER), as well as nucleotide excision repair (NER) activity. Plasma malondialdehyde (MDA) was measured by HPLC. As a product of lipid peroxidation, it acts as a marker for overall oxidative stress. Measurement of plasma vitamin C Measurement of plasma vitamin C The frozen, acidified samples were thawed, and centri- fuged (3500 × g) at +4°C for 10 min. Following the sen- trifugation of the acidified heparin plasma, 100 μL of the clear supernatant was diluted with 400 μL of the mobile phase (2% acetonitrile in 2.5 mM NaH2PO4, 2.5 mM dodecyltrimethyl ammonium chloride and 1.25 mM Na2EDTA in Milli-Q water) for direct determina- tion of ascorbic acid (AA). For the determination of total ascorbic acid (TAA), 100 uL of the clear superna- tant was subjected to reduction for 7 min with the addi- tion of 50 μL 2.3 mmol/L TCEP in 800 mmol/L trizmabuffer (pH 9.0). Subsequently, 350 μL of the mobile phase was added. For separation of ascorbic acid from interfering plasma constituents, a Chromolith Per- formance RP18-e, 4.6 mm × 100 mm column was used, with a Chromolith Performance RP18-e, 4.6 mm × 10 mm guard column. The injection volume used was 5 μL Blood sampling F bl Our results indicate that golden kiwifruit strengthens our resistance towards endogenous oxidative damage, but our results do not support the view that the golden kiwifruit provides noticeably stronger protection against oxidative damage than the green variety. Study design and participants Twenty-four men and women (20-57 years, BMI 20-30 kg/ m²) were recruited from the university and neighbouring companies through poster advertising and email cam- paigns. After a screening interview, subjects eating modest amounts of fruits and vegetables were selected. Exclusion criteria were: use of contraceptive pills, medicines or sup- plements; diets aimed at weight correction; diagnosed dia- betes, cancer or cardiovascular disease; habitual consumption of >30 units of alcohol/week (15 glasses of wine); habitual undertaking of >6h vigorous exercise/week (assessed by exercise questionnaires completed at screen- ing session); abnormal menstrual cycle/hormone replace- ment therapy; or a high intake of fruits and vegetables (> 450 grams per day). The study was approved by the regio- nal committee of ethics in medical science. Written con- sent was obtained from all participants. Page 3 of 9 Page 3 of 9 Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 photometric techniques (Modular P Roche; Roche Diag- nostics). Plasma LDL-cholesterol was calculated using the Friedwald formula [19]. Plasma fibrinopeptide A (FPA) concentration was measured by competitive ELISA and plasma C-reactive protein (CRP) concentra- tion using a semi-quantitative latex agglutination assay, as described [20,21]. and the flow rate was 6.0 mL/min. A variable wave- length UV detector was used at 264 nm. A detailed pro- tocol has been published [17]. Measurement of malondialdehyde in plasma Plasma samples were diluted ten-fold in a final reaction mix containing 0.8% SDS, 10% acetic acid, and 0.17% thiobarbituric acid, incubated for 1 h at 100°C, centri- fuged, and then injected (50 μL) onto a Chromolith Per- formance RP 18e 10 cm (internal diameter: 3 cm) column (Merck) connected to a Brownlee pre-column (RP-18, 5u, OD-GU, 30 × 4,6 mm); the mobile phase was a 6:4 mixture of 50 mM sodium phosphate buffer, pH 6.8, and methanol. Detection was by fluorescence (excitation 532 nm, emission 553 nm) with a Hewlett Packard 1046A programmable detector. FRAP assay d Ferric Reducing Activity of Plasma (FRAP) was deter- mined as described [18]. The modified assay measures FRAP after removal of uric acid and proteins from the plasma and more accurately indicates the contribution of dietary antioxidants. Briefly, 30 μl uricase (0.1 unit/10 μl in Tris buffer, pH 8.5) was added to 60 μl plasma at room temperature. The samples were incubated for 6 min. 120 μl absolute ethanol was added and the samples were vortex-mixed. After incubation for 5 min at 4°C to allow complete protein precipitation, the samples were centrifuged at 13,000 × g for 5 min. The supernatant was used for FRAP analysis as described [18]. In addition to frank DNA strand breaks, oxidised bases were measured by incubating, after lysis, with endonuclease III (to detect oxidised pyrimidines) or for- mamidopyrimidine DNA glycosylase (FPG, recognising 8-oxoGua and other oxidised purines) in 40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin (pH adjusted to 8.0) for 30 min at 37°C. Net enzyme-sensitive sites were calculated by subtract- ing the comet score after incubation with buffer alone from the score with enzyme. Due to a comet assay cali- bration problem in the first intervention period with respect to assessment of endonuclease III sensitive sites, we choose to base all our endonuclease III calculations on samples from the second intervention period. This reduced the effective sample size of endonuclease III sensitive sites from 24 to 12. DNA damage measured in mononuclear cells DNA damage measured in mononuclear cells The comet assay (single cell gel electrophoresis) [22] was used to measure DNA strand breaks. Mononuclear cells were embedded in low melting point agarose (1%, 75 μL) on microscope slides. Gels were allowed to set at 4°C. Cells were lysed for 1h in 2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Tris-HCl, pH 10, 1% Triton X-100 at 4°C to remove membranes, cytoplasm and most nuclear proteins, leaving DNA as nucleoids. To measure strand breaks, the slides were then immersed in elctrophoresis solution (0.3 M NaOH, 1 mM Na2EDTA) for 40 min at 4°C and then electrophoresed at 0.8 V/cm for 30 min at an ambient temperature of 4°C. DNA loops containing breaks extend under electrophoresis to form ‘comet tails’, and the relative intensity of DNA in the tail indi- cates the DNA break frequency. After neutralisation, gels were stained with 4`,6-diamidine-2`-phenylindole dihydrocloride (DAPI) and viewed by fluorescence microscopy. Tail intensity was assessed by visual scoring; 100 comets selected at random were graded according to degree of damage into five classes (0-4) to give an overall score for each gel of between 0 and 400 arbitrary units [22]). Measurement of plasma carotenoids The carotenoids lutein, zeaxanthin, b-kryptoxanthin, a- carotene, b-carotene and lycopene were determined in plasma by HPLC. Proteins were precipitated and removed by the addition of a 450 μL isopropanol to 100 μL plasma, followed by centrifugation at 3.000 g at 4°C for 15 min. The internal standard astaxanthin was added with the isopropanol. Twenty five μL of the clear supernatant were used for analysis. The mobile phases consisted of A: 20% water and 24% acetone in ethanol and B: acetone. The gradient conditions were as follows: From 2 to 100% mobile phase B within 20 min, followed by 100% mobile phase B for 15 min. Detection was per- formed at 453 nm using a variable wavelength detector. Plasma calibrators quantified against the NIST 968c SRM were used as standards. In vitro assay for DNA repair capacity In this assay, a mononuclear cell extract is incubated with DNA substrate containing specific damage; either 8-oxoGua (to measure BER), or cyclobutane pyrimidine dimers (to measure NER) [22]. HeLa cells were used to prepare substrate for the BER assay by incubating them The blood samples were sent to a certified medical laboratory (Fürst Medical Laboratory, Oslo, Norway) for lipid and glucose analyses. Serum blood samples were analyzed for total-, HDL- and LDL-cholesterol, triacyl- glycerols (TAG), and glucose using automated Page 4 of 9 Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 with 1 μM Ro 19-8022 (gift from F. Hoffmann-La Roche) and irradiating on ice with visible light (5 min at 30 cm from a 500 W tungsten halogen source). As sub- strate for the NER assay, HeLa cells were irradiated on ice with 1 Jm2 ultraviolet light (UVC). Substrate cells were embedded in agarose and lysed as in the standard comet assay (above). To a thawed aliquot of snap-frozen lymphocytes, 1% Triton X-100 was added to a final con- centration of 0.2%, the lysate was centrifuged (15,000 × g, 5 min, 4°C) and the supernatant mixed with 4 volumes of the endonuclease III/FPG incubation buffer (see above) - with addition of 1.6 mM MgCl2 in the case of the NER assay [23]. Forty five μL of extract was added to the agarose gel containing substrate nucleoids, incubated for 10 min (BER) or 30 min (NER) and then processed as in the standard comet assay. The produc- tion of DNA breaks during the incubation indicates the ability of the extract to carry out the the initial stage of NER. One repair extract was destroyed accidentally in group 2, reducing the effective size of DNA repair sam- ples to 23. repair and related processes. The number of paired sam- ples available for gene expression studies was limited to seven. Gene expression analysis The blood was collected in Tempus™blood RNA tubes (4342792, Applied Biosystems). Tempus™spin RNA isolation kit (4380204, Applied Biosystems) was used with the optional DNase treatment wash buffer (4305545, Applied Biosystems). The extracted RNA was quantified spectophotometrically (NanoDrop) and stored at -80°C. The RNA integrity was assessed on Experion (Bio-Rad) and all samples included in the analysis had RQI values higher than 8. The RNA concentrations were normalized before reverse transcription with the Transcriptor First Strand Synthesis Kit (04896866001, Roche Diagnostics). 5 ng cDNA was added to each PCR reaction and the qPCR was performed using assays from DNA Damage Response Gene Panel (G101, TATAA Biocenter AB) with primer concentrations of 300 nM. The PCR program consisted of 95°C for 3 min, 40 cycles of 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec followed by a melt curve with a predenaturation step at 95°C and a melting range of 65-95°C. The PCR was per- formed on LightCycler® 480 (Roche Diagnostics) using PerfeCTa® SYBR® green SuperMix, Low Rox™(95056- 02K, Quanta Biosciences) and VisiBlue™qPCR mix col- orant (K101, TATAA Biocenter AB). Assays for 12 can- didate reference genes from the Human endogenous control gene panel (A101, TATAA Biocenter AB) were analyzed for all samples. The two most stable (beta- actin and beta-glucuronidase) were selected using GenEx software (MultiD Analyses AB) and used for nor- malization. Matched samples of RNA from blood col- lected before and after supplementation were analysed by quantitative RT-PCR using probes to genes for DNA Statistical analysis l Results are presented as the mean ± SEM. Student’s t tests were used to test for differences before and after treatment. Results from intervention groups were com- bined so that data for all subjects receiving one kiwifruit per day, whether in the first or second period, were ana- lysed together, and similarly all data for two per day. Values were considered to be significantly different when p < 0.05. Simple regression analysis was per- formed to evaluate correlation between plasma vitamin C and strand breaks. Whole blood platelet aggregation A Chronolog whole blood aggregometer was used to measure the electrical resistance (impedance) to the pas- sage of small electric current across two platinum elec- trodes immersed in the sample of blood. Platelet aggregation increases impedance. For whole blood aggregation, blood (5 ml) mixed with sodium citrate (final concentration 13 mM) was kept at room tempera- ture for 15 min. Typically, 0.5 mL of blood and 0.5 mL of PBS were placed in a plastic cuvette at 37°C. ADP and collagen at various concentrations were used to initiate the aggregation response [16,24,25]. Due to a technical problem with the aggrometer electrode results from whole blood aggregation measurements are based exclusively on the second intervention period. Results Both reduced ascorbic acid and total (reduced plus dehydro-) ascorbic acid increased after supplementation with kiwifruit (Table 1), reduced ascorbic acid being borderline significant (p = 0.054) and total ascorbic acid approaching statistical significance (p = 0.068) at 2 kiwi- fruits per day. No differences were seen between pre- and post-intervention plasma levels of the carotenoids or FRAP values (Table 1). There were no changes in plasma MDA as a result of kiwifruit supplementation (Table 1). Table 2 shows plasma concentrations of total choles- terol, HDL, LDL, triglycerides and glucose. Mean glucose, cholesterol, LDL, and HDL cholesterol values were unchanged over the 4-week intervention periods, whereas triglyceride concentrations were significantly lower (by approximately 13%) after supplementation with 1 or 2 kiwifruits per day. Plasma concentrations of FPA over 6 ng/ml were assumed to be the result of unsatisfactory venepuncture and excluded from data analysis; this applied to 4 out of 180 samples. CRP has been shown to induce cytokine imbalance which affects many aspects of Brevik et al. Results Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Page 5 of 9 Page 5 of 9 Table 1 Plasma antioxidants, FRAP and MDA measured before and after kiwifruit intervention (n = 24) Fruits Before After Diff Change p Mean SEM Mean SEM Mean SEM (%) (μmol/L) (μmol/L) (μmol/L) Total ascorbic acid 1 57.11 15.6 59.58 10.9 2.5 19.0 4 0.53 2 52.83 13.3 60.27 14.3 7.4 19.5 14 0.07 Ascorbic acid, reduced 1 52.79 14.7 55.66 11.2 2.9 18.5 5 0.45 2 47.97 13 55.43 13.1 7.5 18.5 16 0.05 Lutein, μmol/L 1 0.22 0.1 0.22 0.1 0.0 0.1 0 0.95 2 0.22 0.1 0.23 0.1 0.0 0.1 5 0.70 b-Cryptoxanthin, μmol/L 1 0.32 0.3 0.31 0.3 0.0 0.4 -3 0.92 2 0.31 0.3 0.31 0.3 0.0 0.4 0 0.99 a-Carotene, μmol/L 1 0.14 0.1 0.14 0.1 0.0 0.1 0 0.91 2 0.14 0.1 0.14 0.1 0.0 0.1 0 0.98 b-Carotene, μmol/L 1 0.55 0.3 0.56 0.4 0.0 0.5 2 0.89 2 0.55 0.3 0.56 0.3 0.0 0.4 2 0.97 Zeaxanthin, μmol/L 1 0.05 0 0.04 0 0.0 0.0 -20 0.91 2 0.04 0 0.04 0 0.0 0.0 0 0.87 Lycopene, μmol/L 1 0.57 0.1 0.57 0.1 0.0 0.1 0 0.99 2 0.57 0.2 0.56 0.1 0.0 0.2 -2 0.97 FRAP1, μmol/L 1 1203.17 208 1201.13 228.5 -2.0 309.0 0 0.97 2 1189.38 243.9 1260.46 164.4 71.1 294.1 6 0.24 MDA2, μmol/L 1 0.88 0.4 0.88 0.3 0.0 0.5 0 0.97 2 0.89 0.5 0.88 0.5 0.0 0.7 -1 0.53 SEM: standard error of the mean. 1Ferric reducing activity of plasma. 2Malondialdehyde. FRAP and MDA measured before and after kiwifruit intervention (n = 24) Table 1 Plasma antioxidants, FRAP and MDA measured before and after kiwifruit intervention (n = 24) (Table 3). Following 4 weeks of golden kiwifruit inter- vention, a decrease in FPG-sensitive sites in lymphocyte DNA was observed (Table 3), though the decrease was statistically significant only after one kiwifruit per day. Decreases in endonuclease III-sensitive sites were signif- icant after 2 kiwifruits per day but not after one per day (Table 3). In the in vitro DNA repair assays, extract pre- pared from lymphocytes is incubated with specifically damaged DNA containing 8-oxoGua for assessing BER, and cyclobutane pyrimidine dimers for NER. No change in either BER or NER activity was observed following supplementation (Table 3). Results At two fruits per day there was a borderline significant inverse correlation between plasma levels of reduced vitamin C and lymphocyte strandbreaks (Pearson’s r = -0.55, p = 0.066). platelet function. Base levels of hsCRP (CRP measured with a high sensitivity assay) were 2.34 ± 0.05 mg/L, and were not affected by consumption of kiwifruit. Kiwifruit supplementation, both one and two per day, significantly decreased H2O2-induced DNA damage Kiwifruit supplementation, both one and two per day, significantly decreased H2O2-induced DNA damage Table 2 Effects of kiwifruit consumption on fasting plasma levels of lipids and glucose (n = 24) Before After Diff Change p Mean SEM Mean SEM Mean SEM (%) (mM) (mM) (mM) One kwifruit per day Glucose 4.79 0.1 4.69 0.1 -0.10 0.1 -2.1 0.41 HDL 1.53 0.1 1.52 0.1 -0.01 0.1 -6.5 0.71 LDL 2.32 0.1 2.20 0.1 -0.12 0.1 -3.4 0.68 Total cholesterol 4.05 0.1 3.83 0.1 -0.22 0.2 -4.4 0.26 Triglycerides 0.88 0.1 0.76 0.1 -0.12 0.1 -13.3 0.05 Two kwifruits per day Glucose 4.68 0.1 4.80 0.1 0.12 0.1 2.6 0.20 HDL 1.50 0.1 1.43 0.1 -0.07 0.1 -7.1 0.26 LDL 2.39 0.1 2.51 0.1 0.12 0.1 3.3 0.57 Total cholesterols 4.05 0.1 4.10 0.1 0.05 0.2 4.7 0.51 Triglycerides 0.86 0.1 0.76 0.0 -0.10 0.1 -7.4 0.05 SEM: standard error of the mean Table 2 Effects of kiwifruit consumption on fasting plasma levels of lipids and glucose (n = 24) No differences in the expression of any of the genes studied were observed before and after kiwifruit con- sumption (Table 4). Table 5 shows the whole blood platelet aggregation response to different concentrations of ADP and col- lagen before and after daily consumption of kiwifruit. Whole blood platelet aggregation induced by ADP was reduced at both concentrations after consuming 1 kiwi- fruit per day; however significance was attained only in the case of 5.0 μM ADP. We observed after 1 kiwifruit per day a significant reduction in whole blood platelet aggregation in response to collagen at 2 μg/ml. Brevik et al. Results Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Page 6 of 9 Page 6 of 9 Table 3 Endogenous DNA damage and DNA repair activity in lymphocytes, before and after kiwifruit intervention; Arbitrary units (n = 24) Fruits Before After Diff Differ (%) p Mean SEM Mean SEM Mean SEM FPG-sensitive sites 1 196.38 73.7 130.33 85.3 -66.05 112.7 -34 0.01 2 188.58 61.5 148.26 106.7 -40.32 123.2 -21 0.12 Endonuclease III-sensitive sites1 1 139.42 71.1 104.00 59.8 -35.42 92.9 -25 0.20 2 138.17 41.2 92.25 62.1 -45.92 74.5 -33 0.04 Resistance towards H2O2 oxidation 1 283.08 43.9 189.25 117.2 -93.83 125.2 -33 0.00 2 278.00 51.6 191.52 124.5 -86.48 134.8 -31 0.00 Base exision repair (BER) 1 48.43 16.3 46.61 14.5 -1.82 21.8 -4 0.69 2 52.54 13.8 49.11 15.1 -3.43 20.5 -7 0.43 Nucleotide excision repair (NER)2 1 39.38 21.3 38.13 23.9 -1.25 32.0 -3 0.85 2 37.71 18.8 35.50 23.3 -2.21 29.9 -6 0.72 SEM: standard error of the mean. 1Based on samples exclusively from the second intervention period (n = 12). 2n = 23. Table 3 Endogenous DNA damage and DNA repair activity in lymphocytes, before and after kiwifruit intervention; Arbitrary units (n = 24) DNA damage and DNA repair activity in lymphocytes, before and after kiwifruit intervention; 4) ymphocytes, before and after kiwifruit interventio Consuming 2 kiwifruits per day had no significant effect on whole blood platelet aggregation. Previously, we reported increases in mean plasma vita- min C concentrations of up to 26% after suplementation with 3 green kiwifruits per day for 3 weeks [26]. There were no restrictions in either study as to when during the day the fruit should be eaten, and in practice the last fruit was consumed 10 h or more prior to blood sampling. Peak plasma vitamin C concentrations are reached a few hours after supplementation and then decline [27], and so the 14-16% increase in vitamin C seen here is all the more impressive. Measured a short time after kiwifruit consumption, the concentration would presumably have been much higher. This increase in plasma concentration of reduced and total ascorbic acid indicates that (golden) kiwifruit may be an effective booster of antioxidant status, especially since vitamin C activity is likely to be enhanced by acting in concert with other phytochemicals from fruit. In vitro whole blood platelet aggregation was mea- sured in the presence of undiluted green or golden kiwi- fruit extract. Results At similar wet weight the green fruit extract was almost twice as effective as golden kiwifruit extract at inhibiting ADP-induced whole blood platelet aggregation. In contrast, golden kiwifruit extract was sig- nificantly more effective than green kiwifruit extract at inhibiting collagen-induced platelet aggregation (data not shown). SEM: standard error of the mean. Discussion Here we report increases in plasma vitamin C and increases in total antioxidant capacity (as indicated by H2O2-induced DNA damage) as well as a trend towards decrease in oxidised pyrimidines and oxidised purines. Table 4 Expression of essential DNA repair genes in seven randomly selected subjects during kiwifruit consumption and after wash-out Before After Diff Change p Mean SEM Mean SEM Mean SEM (%) Cyclin-dependent kinase inhibitor 1A (p21) 5.32 0.1 5.28 0.2 -0.04 0.2 -0.8 0.80 Replication protein A1 (RPA1) 6.15 0.1 6.31 0.1 0.16 0.2 2.6 0.28 Xeroderma pigmentosum, complementation group A (XPA) 5.56 0.2 5.38 0.2 -0.18 0.2 -3.2 0.30 Excision reapir cross-complementing protein 1 (ERCC1) 3.55 0.1 3.48 0.2 -0.07 0.2 -2.0 0.67 Excision reapir cross-complementing protein 2 (ERCC2) 4.87 0.1 4.79 0.3 -0.08 0.3 -1.6 0.77 8-oxoguanine DNA glycosylas (OGG1) 4.83 0.1 4.83 0.1 0 0.2 0.0 1.00 Proliferating cell nuclear antigen (PCNA) 5.06 0.2 5.01 0.2 -0.05 0.2 -1.0 0.75 X-ray repair cross-complementing protein (XRCC1) 4.07 0.2 3.92 0.1 -0.15 0.2 -3.7 0.41 NAD(P)H dehydrogenase, quinone 1 (NQO1) 7.40 0.1 7.32 0.2 -0.08 0.2 -1.1 0.54 Nuclease (multifunctional DNA repair enzyme) 1 (APEX1) 3.08 0.1 3.00 0.2 -0.08 0.2 -2.6 0.52 Homolog recombination protein (RecA homolog, E. coli)(RAD51) 11.68 0.3 11.58 0.2 -0.1 0.3 -0.9 0.81 Xeroderma pigmentosum, complementation group C (XPC) 4.49 0.1 4.59 0.2 0.1 0.2 2.2 0.58 SEM: standard error of the mean. essential DNA repair genes in seven randomly selected subjects during kiwifruit consumption Table 4 Expression of essential DNA repair genes in seven randomly selected subjects during kiw and after wash-out Brevik et al. Discussion Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Page 7 of 9 Page 7 of 9 Table 5 Effects of daily consumption of one and two golden kiwifruits per day on whole blood aggregation (n = 24) Aggregating agent Before After Diff Change p Mean SEM Mean SEM Mean SEM (%) (Resistance, Ohm) (Resistance, Ohm) (Resistance, Ohm) One kiwifruit per day ADP (5.0 μM) 13.00 2.7 11.40 1.95 -1.6 3.3 -12.3 0.03 ADP (7.5 μM) 13.04 2.0 11.88 2.78 -1.2 3.4 -8.9 0.08 Collagen (1 μg/ml) 16.00 2.1 15.60 2.46 -0.4 3.3 -2.5 0.53 Collagen (2 μg/ml) 18.75 2.2 16.18 1.93 -2.6 2.9 -13.7 0.00 Two kiwifruits per day ADP (5.0 μM) 13.04 3.4 12.17 3.34 -0.9 4.7 -6.7 0.32 ADP (7.5 μM) 12.42 2.7 11.50 2.29 -0.9 3.5 -7.4 0.07 Collagen (1 μg/ml) 15.77 2.4 15.15 1.59 -0.6 2.8 -3.9 0.35 Collagen (2 μg/ml) 17.77 3.4 17.70 2.15 -0.1 4.0 -0.4 0.93 SEM: standard error of the mean. Table 5 Effects of daily consumption of one and two golden kiwifruits per day on whole blood aggregation (n = 24) umption of one and two golden kiwifruits per day on whole blood aggregation (n = 24) unexpected, since a stimulation of BER was the most pronounced effect in the previous kiwifruit study [10]. Significant effects of green kiwifruit, and of a high fruit and vegetable diet, on both BER and NER, have been seen in another recent intervention trial [28], and so we can hypothesise that golden kiwifruit may lack specific ingredient(s) that can modulate DNA repair. The lack of significant effects of golden kiwifruit supplementation on the expression of key DNA repair genes is consistent with the lack of effect on repair enzyme activities. Lack of effect of antioxidant rich whole food on repair enzyme activities have been reported previously. Despite reduced DNA damage and increased resistance towards H2O2 induced strand breaks, Rizo et al. reported no effect of broccoli consumption on the expression of DNA repair enzymes [29]. After both one and two fruits per day resistance towards H2O2-oxidation increased by ~30%. The reduced level of strandbreaks may in part be caused by the increased intake of vitamin C. At two fruits per day there was a borderline significant inverse correlation between plasma levels of reduced vitamin C and lym- phocyte strandbreaks, but this association was not pre- sent at one fruit per day. Discussion Resistance towards H2O2- oxidation seemed to increase somewhat more following consumption of yellow kiwifuit than after consumption of green kiwifruit [26], even if a direct comparison can- not be performed between the two studies. Supplementation with one kiwifruit per day for four weeks protected lymphocytes against DNA base oxida- tion, as indicated by a decrease of more than 20% in the number of FPG-sensitive sites measured with the comet assay. This effect was not seen after supplementation with two kiwifruits per day. The lack of a clear dose- response effect is not unprecedented; in our previous study with green kiwifruit [10], protective effects against DNA oxidation did not increase with higher daily doses of fruits. This may represent a saturation effect, if the active ingredient in one kiwifruit is sufficient to exert a maximal protective effect. We presently have no idea of the identity of the active ingredient. There is increasing evidence that some natural com- ponents can reduce levels of specific CVD risk factors by modulating platelet function [11,30]. In recent years, research groups including ours have reported inhibitory effects of different fruits on platelet aggregation [11]. Potent anti-platelet-aggregation factor(s) are present in tomatoes and green kiwifruits [11,24,25]. Extracts of these fruits inhibit platelet aggregation in response to ADP, collagen, and thrombin both in vitro and in vivo [11,21,25]. Endonuclease III-sensitive sites were also reduced after supplementation by about one quarter. The reduction was significant after two kiwifruits per day but not after one. There was no significant correlation between endo- nuclease III-sensitive sites and H2O2-induced DNA damage or plasma vitamin C, indicating that the reduc- tion in endonuclease III-sensitive sites may be caused by specific modulating compounds of the kiwifruits rather than by its antioxidant content per se. In the present study, consuming 1 golden kiwifruit per day for 4 weeks reduced whole blood platelet aggrega- tion. This effect disappeared during the washout period (data not shown). Consuming 2 golden kiwifruits gener- ally did not inhibit whole blood platelet aggregation. References 1. Jenkins DJ, Kendall CW, Marchie A, Jenkins AL, Augustin LS, Ludwig DS, Barnard ND, Anderson JW: Type 2 diabetes and the vegetarian diet. American Journal of Clinical Nutrition 2003, 78:610S-616S. 1. Jenkins DJ, Kendall CW, Marchie A, Jenkins AL, Augustin LS, Ludwig DS, Barnard ND, Anderson JW: Type 2 diabetes and the vegetarian diet. American Journal of Clinical Nutrition 2003, 78:610S-616S. A possible limitation of the present study was the use of healthy subjects. The protective effect of antioxidant supplementation may be more pronounced in subjects exposed to increased levels of oxidative stress, such as e. g. endurance athletes, diseased or old people [31]. Mod- erate sample size may be another limitation. Since this was a cross-over intervention any possible weak seasonal variation would cancel out in the final analysis, but the inclusion of a control group could have improved our knowledge of seasonal variations in the measured biomarkers. 2. Ness AR, Powles JW: Fruit and vegetables, and cardiovascular disease: a review. International Journal of Epidemiology 1997, 26:1-13. 3. Steinmetz KA, Potter JD: Vegetables, fruit, and cancer prevention: a 2. Ness AR, Powles JW: Fruit and vegetables, and cardiovascular disease: a review. International Journal of Epidemiology 1997, 26:1-13. 3. Steinmetz KA, Potter JD: Vegetables, fruit, and cancer prevention: a review. Journal of the American Dietetic Association 1996, 96:1027-39. 3. Steinmetz KA, Potter JD: Vegetables, fruit, and cancer prevention: a review. Journal of the American Dietetic Association 1996, 96:1027-39. 4. Gordon M: Dietary antioxidants in disease prevention. Natural Product Reports 1996, 13:265-73. 4. Gordon M: Dietary antioxidants in disease prevention. Natural Product Reports 1996, 13:265-73. 5. Rock CL, Jacob RA, Bowen PE: Update on the biological characteristics of the antioxidant micronutrients: vitamin C, vitamin E, and the carotenoids.[see comment]. Journal of the American Dietetic Association 1996, 96:693-702. 5. Rock CL, Jacob RA, Bowen PE: Update on the biological characteristics of the antioxidant micronutrients: vitamin C, vitamin E, and the carotenoids.[see comment]. Journal of the American Dietetic Association 1996, 96:693-702. 6. Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C: Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. JAMA 2007, 297:842-857. 6. Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C: Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. JAMA 2007, 297:842-857. References To summarise, this intervention trial, with biomarkers relevant to both cancer and CVD indicates that one kiwifruit per day is as effective as two per day, when considering both effects on DNA oxidation, and platelet aggregation. The concentration of plasma triglycerides is decreased by golden kiwifruit, with no parallel effect on cholesterol. 7. Huang HY, Caballero B, Chang S, Alberg AJ, Semba RD, Schneyer CR, Wilson RF, Cheng TY, Vassy J, Prokopowicz G, Barnes GJ, Bass EB: The efficacy and safety of multivitamin and mineral supplement use to prevent cancer and chronic disease in adults: a systematic review for a National Institutes of Health state-of-the-science conference. Annals of Internal Medicine 2006, 145:372-85. 7. Huang HY, Caballero B, Chang S, Alberg AJ, Semba RD, Schneyer CR, Wilson RF, Cheng TY, Vassy J, Prokopowicz G, Barnes GJ, Bass EB: The efficacy and safety of multivitamin and mineral supplement use to prevent cancer and chronic disease in adults: a systematic review for a National Institutes of Health state-of-the-science conference. Annals of Internal Medicine 2006, 145:372-85. 8. Miller ER III, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E: Meta-analysis: high-dosage vitamin E supplementation may increase all- cause mortality. Ann Intern Med 2005, 142:37-46. 9. Ferguson AR, Fillion L: Are kiwifruit really good for you? Acta Horticulturae 2003, 610:131-135. Discussion (Only with 7.5 μM ADP was there a marginally signifi- cant decrease.) There is no obvious explanation for this lack of effect, but it corresponds to the lack of dose response in relation to markers of DNA oxidation, dis- cussed above - though there is no reason to assume the The lack of any effect of kiwifruit supplementation on lymphocyte DNA repair - either BER or NER - is Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Page 8 of 9 Page 8 of 9 would also like to thank Anne Randi Alvestad for drawing blood virtually every morning during the course of the study. There were no conflicts of interest for any of the authors in the study. operation of a common molecular mechanism. The lack of effects of 2 golden kiwifruits per day could be caused by unknown confounders. Author details 1 f Our in vitro platelet aggregation experiments suggest that green and golden kiwifruit extracts inhibit both ADP and collagen-induced whole blood platelet aggrega- tion (to different degrees). The fruit extract may contain a wide variety of different types of compounds that have anti-platelet activity in vitro and that affect different mechanisms of activation and aggregation. We have ear- lier shown that anti-platelet effects of fruits including green kiwifruits and tomatoes are independent of their antioxidant activity [11,16,25]. 1Department of Chemical Toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O.Box 4404 Nydalen, N-0403 OSLO, Norway. 2Genetic and Biotechnology Department, University of Trás-os- Montes and Alto Douro, Quinta de Prados, 5001 Vila Real Codex, Portugal. 3Department of Nutrition, Faculty of Medicine, University of Oslo, PB 1046 Blindern, 0316 Oslo, Norway. 4Gdansk University of Technology, Gdansk, Poland. 5TATAA Biocenter AB, Odinsgatan 28, 411 03 Göteborg, Sweden. Authors’ contributions AB carried out the intervention study and wrote the initial draft. IG did the NER measurements together with AP. TM and AJ did the blood aggregation measurements. AK measured carotenoids and FRAP in association with RB. JE did the gene expression analysis. AKD and ARC planned the intervention and wrote the manuscript together with AB. All authors read and approved the final manuscript. Consumption of golden kiwifruit reduced plasma tri- glyceride levels without affecting cholesterol levels: origi- nal levels were restored after the washout period (data not shown). Lowering of plasma triglycerides by kiwifruit was observed despite the fact that the volunteers main- tained their regular diet during the supplementation per- iod. Such effects of fruits and vegetables have been reported before [31]. Our data imply that both green and golden kiwifruits might have cardio-protective effects. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 10 December 2010 Accepted: 18 May 2011 Published: 18 May 2011 Received: 10 December 2010 Accepted: 18 May 2011 Published: 18 May 2011 Conclusion Regular intake of golden kiwifruit can protect against lym- phocyte DNA oxidation as well as increase total antioxi- dant activity and specifically plasma vitamin C. Regular consumption can also reduce platelet aggregation, and lower plasma triglycerides. Consumption of these fruits could therefore benefit public health by countering oxida- tive stress factors, and by reducing the risk of thrombotic events mediated by platelet activation. 10. Collins BH, Horska A, Hotten PM, Riddoch C, Collins AR: Kiwifruit protects against oxidative DNA damage in human cells and in vitro. Nutr Cancer 2001, 39:148-153. 10. Collins BH, Horska A, Hotten PM, Riddoch C, Collins AR: Kiwifruit protects against oxidative DNA damage in human cells and in vitro. Nutr Cancer 2001, 39:148-153. 11. Dutta-Roy AK: Dietary components and human platelet activity. Platelets 2002, 13:67-75. 11. Dutta-Roy AK: Dietary components and human platelet activity. Platelets 2002, 13:67-75. 12. Fillion L, Collins A, Southon S: Beta-carotene enhances the recovery of lymphocytes from oxidative DNA damage. Acta Biochim Pol 1998, 45:183-190. 12. Fillion L, Collins A, Southon S: Beta-carotene enhances the recovery of lymphocytes from oxidative DNA damage. Acta Biochim Pol 1998, 45:183-190. 13. Guarnieri S, Loft S, Riso P, Porrini M, Risom L, Poulsen HE, Dragsted LO, Moller P: DNA repair phenotype and dietary antioxidant supplementation. Br J Nutr 2008, 99:1018-1024. 13. Guarnieri S, Loft S, Riso P, Porrini M, Risom L, Poulsen HE, Dragsted LO, Moller P: DNA repair phenotype and dietary antioxidant supplementation. Br J Nutr 2008, 99:1018-1024. supplementation. Br J Nutr 2008, 99:1018-1024. 14. Spormann TM, Albert FW, Rath T, Dietrich H, Will F, Stockis JP, Eisenbrand G, Janzowski C: Anthocyanin/polyphenolic-rich fruit juice reduces oxidative cell damage in an intervention study with patients on hemodialysis. Cancer Epidemiol Biomarkers Prev 2008, 17:3372-3380. 14. Spormann TM, Albert FW, Rath T, Dietrich H, Will F, Stockis JP, Eisenbrand G, Janzowski C: Anthocyanin/polyphenolic-rich fruit juice reduces oxidative cell damage in an intervention study with patients on hemodialysis. Cancer Epidemiol Biomarkers Prev 2008, 17:3372-3380. g This trial was supported by Zespri International Ltd. and the Throne Holst Fund. We are grateful to all the volunteers for taking part in this study. We Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 Acknowledgements Thi i l This trial was supported by Zespri International Ltd. and the Throne Holst Fund. We are grateful to all the volunteers for taking part in this study. We Page 9 of 9 Page 9 of 9 Brevik et al. Nutrition Journal 2011, 10:54 http://www.nutritionj.com/content/10/1/54 15. Stoner GD, Wang LS, Casto BC: Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries. Carcinogenesis 2008, 29:1665-1674. 15. Stoner GD, Wang LS, Casto BC: Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries. Carcinogenesis 2008, 29:1665-1674. 16. Duttaroy AK, Jorgensen A: Effects of kiwi fruit consumption on platelet aggregation and plasma lipids in healthy human volunteers. Platelets 2004, 15:287-292. 17. Karlsen A, Blomhoff R, Gundersen TE: High-throughput analysis of vitamin C in human plasma with the use of HPLC with monolithic column and UV-detection. Journal of Chromatography B: Analytical Technologies in the Biomedical & Life Sciences 2005, 824:132-8. 18. Benzie IF, Strain JJ: Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 1999, 299:15-27. 19. Friedewald WT, Levy RI, Fredrickson DS: Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972, 18:499-502. 20. O’Kennedy N, Crosbie L, van Lieshout M, Broom JI, Webb DJ, Duttaroy AK: Effects of antiplatelet components of tomato extract on platelet function in vitro and ex vivo: a time-course cannulation study in healthy humans. Am J Clin Nutr 2006, 84:570-579. 21. O’Kennedy N, Crosbie L, Whelan S, Luther V, Horgan G, Broom JI, Webb DJ, Duttaroy AK: Effects of tomato extract on platelet function: a double- blinded crossover study in healthy humans. Am J Clin Nutr 2006, 84:561-569. 22. Collins AR: The comet assay for DNA damage and repair: principles, applications, and limitations. Molecular Biotechnology 2004, 26:249-61. 23. Gaivão I, Piasek A, Brevik A, Collins AR: Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation. Cell Biology and Toxicology 2009, 25(1):45-52. gy gy 24. Dutta-Roy AK, Gordon MJ, Kelly C, Hunter K, Crosbie L, Knight-Carpentar T, Williams BC: Inhibitory effect of Ginkgo biloba extract on human platelet aggregation. Platelets 1999, 10:298-305. 25. Dutta-Roy AK, Crosbie L, Gordon MJ: Effects of tomato extract on human platelet aggregation in vitro. Platelets 2001, 12:218-227. p gg g 26. Acknowledgements Thi i l Collins AR, Harrington V, Drew J, Melvin R: Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis 2003, 24:511-5. 27. Jacobson JS, Begg MD, Wang LW, Wang Q, Agarwal M, Norkus E, Singh VN, Young TL, Yang D, Santella RM: Effects of a 6-month vitamin intervention on DNA damage in heavy smokers. Cancer Epidemiol Biomarkers Prev 2000, 9:1303-1311. 28. Brevik A, Karlsen A, Azqueta A, Tirado AE, Blomhoff R, Collins A: Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors. Cell Biochem Funct 2011, 29:36-42. 29. Riso P, Martini D, Moller P, Loft S, Bonacina G, Moro M, Porrini M: DNA damage and repair activity after broccoli intake in young healthy smokers. Mutagenesis 2010, 25:595-602. 30. Beretz A, Cazenave JP: Old and new natural products as the source of modern antithrombotic drugs. Planta Med 1991, 57:S68-S72. 31. Moller P, Loft S: Dietary antioxidants and beneficial effect on oxidatively damaged DNA. Free Radical Biology & Medicine 2006, 41:388-415. doi:10.1186/1475-2891-10-54 Cite this article as: Brevik et al.: Supplementation of a western diet with golden kiwifruits (Actinidia chinensis var.’Hort 16A’:) effects on biomarkers of oxidation damage and antioxidant protection. Nutrition Journal 2011 10:54. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of:
https://openalex.org/W3199395440
https://www.frontiersin.org/articles/10.3389/fneur.2021.695085/pdf
English
null
Efficacy of a Direct Aspiration First-Pass Technique (ADAPT) for Endovascular Treatment in Different Etiologies of Large Vessel Occlusion: Embolism vs. Intracranial Atherosclerotic Stenosis
Frontiers in neurology
2,021
cc-by
6,150
ORIGINAL RESEARCH published: 09 September 2021 doi: 10.3389/fneur.2021.695085 Keywords: etiology, endovascular treatment, mechanical thrombectomy, embolism, intracranial atherosclerotic stenosis, stroke Reviewed by: Reviewed by: Alhamza R. Al-Bayati, Emory University, United States Nabil Kitchener, General Organization for Teaching Hospitals and Institutes, Egypt Alberto Maud, Texas Tech University Health Sciences Center El Paso, United States Qazi Zeeshan, University at Buffalo, United States Background and Aims: Aspiration thrombectomy is an effective method of recanalizing large vessel occlusion (LVO). However, the efficacy of a direct aspiration first-pass technique (ADAPT) for recanalization of LVO of different etiologies is not properly understood. Methods: The prospectively collected database on ADAPT was reviewed retrospectively. We defined two groups of enrolled patients: the embolism-related occlusions (EMB-O) group and the intracranial atherosclerotic stenosis (ICAS)-related occlusion (ICAS-O) group. Baseline characteristics, procedural variables, and post- procedural variables were collected. Multivariate logistic regression analysis was used to identify first-pass recanalization predictors. Results: Of 114 registered patients, 94 were eligible for this study (51 patients in the EMB-O group and 43 patients in the ICAS-O group). Achieving successful reperfusion immediately after direct aspiration was more frequent in the EMB-O group than in the ICAS-O group (64.71 vs. 27.91%, respectively, p = 0.006), with fewer additional rescue treatments needed (35.29 vs. 70.09%, respectively, p = 0.001). The EMB-O group also showed a higher final successful reperfusion rate (96.8 vs. 74.41%, p = 0.006). However, the 90-day good functional outcomes were not affected by the groups. Independent predictors of first-pass success of aspiration included the isolated middle cerebral artery site of occlusion, embolic etiology, and use of larger bore catheters. Specialty section: This article was submitted to Endovascular and Interventional Neurology, a section of the journal Frontiers in Neurology Specialty section: This article was submitted to Endovascular and Interventional Neurology, a section of the journal Frontiers in Neurology Received: 14 April 2021 Accepted: 14 July 2021 Published: 09 September 2021 Received: 14 April 2021 Accepted: 14 July 2021 Published: 09 September 2021 Geng Liao 1*, Zhenyu Zhang 1, Guangzhi Zhang 1, Weijie Du 1, Chaomao Li 1 and Hanxiang Liang 2 Geng Liao 1*, Zhenyu Zhang 1, Guangzhi Zhang 1, Weijie Du 1, Chaomao Li 1 and Hanxiang Liang 2 Edited by: Osama O. Zaidat, Northeast Ohio Medical University, United States 1 Department of Neurology, Maoming People’s Hospital, Maoming, China, 2 Department of Magnetic Resonance Imaging, Maoming People’s Hospital, Maoming, China INTRODUCTION (mTICI) grade (3). Successful reperfusion was defined as mTICI grade 2b or higher. First attempt recanalization (FAR) was defined as successful recanalization at the first attempt (4). All patients had a computed tomography (CT) at the end of the procedure and a CT or magnetic resonance image (MRI) scan 24 h after treatment onset to assess hemorrhagic complications. Intracerebral hemorrhages were classified in accordance with the European Cooperative Acute Stroke Study criteria (5). Subarachnoid hemorrhage (SAH) was classified using the modified Fisher scale (6). Symptomatic intracranial hemorrhage was defined as parenchymal hematoma type 2 using the ECASS III grading (European Cooperative Acute Stroke Study) according to imaging at 24 h, associated with an increase of at least four NIHSS points within 24 h, or resulting in death (7). New embolism in other vessels was defined as an occlusion of a previously unaffected non-downstream vascular territory observed on the angiogram after clot removal. A direct aspiration first-pass technique (ADAPT) as first- line therapy for stroke thrombectomy has shown non-inferior functional outcome at 90 days compared with stent retriever first-line thrombectomy (1). However, the efficacy of aspiration thrombectomy as a first-line approach for the recanalization of large vessel occlusions (LVOs) of different etiologies is not properly understood. There have been only a few studies about the endovascular treatment (EVT) of intracranial atherosclerotic stenosis (ICAS)-related LVO (ICAS-O) (2). They revealed that mechanical thrombectomy (MT) with a stent retriever or contact aspiration was less effective and more time-consuming in ICAS-O than in embolic LVO (EMB-O). In this study, we aimed to evaluate the efficacy of using ADAPT in LVO of different etiologies. Exclusion criteria encompassed the following: Exclusion criteria encompassed the following: Exclusion criteria encompassed the following: • the etiology of LVO was classified as dissection, chronic total occlusion, moyamoya disease, vasculitis, or undetermined; and Citation: Liao G, Zhang Z, Zhang G, Du W, Li C and Liang H (2021) Efficacy of a Direct Aspiration First-Pass Technique (ADAPT) for Endovascular Treatment in Different Etiologies of Large Vessel Occlusion: Embolism vs. Intracranial Atherosclerotic Stenosis. Front. Neurol. 12:695085. doi: 10.3389/fneur.2021.695085 Conclusions: The efficacy of ADAPT recanalization approach was better in EMB-O than in ICAS-O. In case of embolic etiology and the isolated MCA site of occlusion, using a larger aspiration catheter for direct aspiration thrombectomy may be reasonable. September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org Aspiration for Different Etiologies of Occlusion Liao et al. Frontiers in Neurology | www.frontiersin.org Study Populations In the current study, we divided patients into two groups based on the etiology of occlusion: the EMB-O group and ICAS-O group. ADAPT was used as the first-line approach for MT in both groups. In brief, a large-bore guide catheter such as a MPA1 0.088 inches (Cordis, USA) or 8-9F balloon guide catheter (Stryker, CA, USA) was advanced as far safely as possible into the internal carotid artery for anterior circulation thrombi and to the largest caliber vertebral artery in the posterior circulation thrombi. Then, the aspiration catheter was advanced and inserted into the internal thrombosis, usually over a microcatheter and micro guidewire. Dual aspiration was applied, and the clot was either ingested within the catheter or it was affixed at the catheter tip and then withdrawn under continuous vacuum exerted by a 50 or 60-ml syringe. If aspiration alone failed three times, access was maintained through the aspiration catheter, and then the second- line option was considered with the use of a stent retriever. Rescue treatments were allowed, including angioplasty, intra- arterial infusion of antithrombotics, and intracranial stenting. A control angiogram was performed to confirm reperfusion. Devices were selected at the discretion of neurointerventionalists based on the consensus of the stroke team. We performed a retrospective analysis of our prospectively gathered EVT database of patients with acute ischemic stroke (AIS). The intention was to identify patient cohorts in whom aspiration had been used as a first-line EVT approach in the anterior and posterior circulation (internal carotid artery, middle cerebral artery M1 and M2 segments, basilar artery, and vertebral artery in the V4 segment) and in whom the etiology of occlusion was identified. The database included all patients who presented with AIS due to LVO and who were treated with EVT between January 1, 2018, and June 30, 2020. The inclusion criteria were as follows: • patients with intracranial large artery occlusions; • patients with intracranial large artery occlusions; • underlying etiology classified as ICAS or embolism; and • the onset time was defined as time from symptom onset to puncture ≤24 h. The onset time was defined as the last time when patient was still well. Etiologic Classification of Target Occlusive Lesions • patients referred for pre-onset modified Rankin Scale (mRS) score > 2. The etiology of target LVO was determined by an independent core laboratory imaging analysis group based on medical history, angiography, and magnetic resonance imaging. Two experienced neurointerventionalists who were blinded to patient identification independently reviewed the clinical and image data of all the patients to determine the etiology of target LVO, and an experienced neurologist solved disparities. Interrater agreement for etiology of LVOs was assessed using Cohen’s kappa coefficient (Cohen κ). Vasospasm caused by catheter and uncommon cerebral arterial diseases such as chronic total occlusion, dissection, and moyamoya disease were excluded. Etiological identification referred to TOAST criteria (Trial of Org 10172 in Acute Stroke Treatment) (8): if the occluded vessel was Patient demographics, comorbidities, premorbid functional status, conventional vascular risk factors, and laboratory findings assessed during admission, National Institutes of Health Stroke Scale (NIHSS), arterial occlusion site and lateralization, time from onset to puncture, Alberta Stroke Program Early CT Score (ASPECTS), intravenous thrombolysis before MT, angiographic, the number of passes with the aspiration device, time from puncture to reperfusion, and clinical data were collected. The location of initial occlusion site was determined using baseline computed tomography angiography or digital-subtraction angiography (DSA). Reperfusion performance was evaluated using the modified Thrombolysis in Cerebral Infarction grade September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org 2 Aspiration for Different Etiologies of Occlusion Liao et al. completely recanalized after primary thrombectomy, the etiology was classified as embolic occlusion, and a remnant stenosis >50% was classified as ICAS-O. In addition, the etiology of LVO in some patients was further evaluated by high-resolution magnetic resonance vascular wall analysis following MT during admission. Consequently, the EMB-O and ICAS-O groups were included in the analyses. were expressed as means ± standard deviations, medians (interquartile ranges), or numbers (percentages). Variables were compared using a non-parametric alternative for t-test (Mann- Whitney U-test) for non-continuous or non-normally distributed variables, Student’s t-test for continuous variables, and χ2 test for categorical variables. Multivariate logistic regression was used to identify first-pass effect predictors in overall patients. p-values were two-tailed, and variables were considered significant at the < 0.05 level. Analysis was performed using PASWstat 18.0 (IBM Corporation, New York, USA). RESULTS From January 2018 to June 2020, a total of 429 patients were screened for EVT of LVO ischemic stroke. Of these, 114 patients were treated with aspiration thrombectomy as first- line approach, and they constituted the study sample. Among them, 94 patients were included in the final analysis. The patient inclusion flowchart is shown in Figure 1. There was a good agreement on identification of the etiology of target LVO (Cohen κ: 0.871) between two independent neurointerventionalists. Overall, FAR and successful recanalization rates after simple contact aspiration were achieved in 44.68% (42/94) and 47.87% (45/94) of patients, respectively. The final successful recanalization rate was 86.17% (81/94). Outcome Measures The primary outcome was the rate of immediate and final successful reperfusion (mTICI score of 2b or 3) after the use of aspiration thrombectomy as the first-line approach; secondary outcomes included safety issues (procedural complications), procedural times (onset and femoral puncture to reperfusion), and 90-day all-cause mortality. Favorable outcome was defined by an modified Rankin Scale score (mRS) of 0 to 2 as evaluated by an independent senior vascular neurologist during face-to- face interviews or via telephone conversations at the 90th day of follow-up. Otherwise, since the clinical outcome may differ between the anterior and posterior circulation occlusions, the occlusion site as a subgroup was also included in the analysis of clinical outcomes. Effects of ADAPT for MT Intracerebral hemorrhagic transformation of any type and parenchymal hematoma type 2 occurred at similar rates in the two groups. The occurrence of new embolism in other vessels and iatrogenic dissection or rupture were similar between the two groups. Table 2 summarizes comparative results regarding the treatment. Successful reperfusion after first pass of aspiration was more common in the EMB-O group than in the ICAS-O group (56.86 vs. 30.23%, p = 0.017). Immediate successful reperfusion TABLE 1 | Comparison of baseline characteristics in 94 patients with aspiration thrombectomy as first-line approach. TABLE 1 | Comparison of baseline characteristics in 94 patients with aspiration thrombectomy as first-line approach. Variable Overall Embolism-related occlusions ICAS-related occlusions p-values* No. of patients 94 51 43 Age, years; mean ± SD 68.71 ± 13.33 68.82 ± 13.36 67.28 ± 11.20 0.307 Men 56 (59.6) 27 (52.9) 29 (67.4) 0.224 Hypertension 51 (54.3) 19 (37.3) 32 (74.4) 0.001 Diabetes mellitus 19 (20.2) 5 (9.8) 14 (32.6) 0.013 Dyslipidemia 12 (12.8) 3 (5.9) 9 (20.9) 0.062 Atrial fibrillation 39 (41.5) 37 (72.5) 2 (4.7) 0.000 Cardiovascular disease 18 (19.1) 10 (19.6) 8 (18.6) 1.000 Current smoking 8 (8.50) 4 (7.80) 4 (9.30) 1.000 Site of occlusion ICA siphon 28 (29.79) 18 (35.29) 10 (23.26) NA ICA siphon and MCA tandem 16 (17.02) 10 (19.61) 6 (13.95) NA Middle cerebral artery, M1 20 (21.28) 9 (17.65) 11 (25.58) NA Middle cerebral artery, M2 4 (4.26) 3 (5.88) 1 (2.33) NA Basilar artery 18 (19.15) 10 (19.61) 8 (18.60) NA Vertebral artery 8 (8.51) 0 8 (18.60) NA Initial NIHSS score, median (IQR) 14.95 (10–25) 15.73 (10–21) 14.30 (10–19) 0.345 ASPECTS, median (IQR) 8.44 (7–10) 8.0 (5–10) 8.91 (8–10) 0.078 Previous use of IV thrombolysis 15 (16.0) 10 (19.6) 5 (11.6) 0.441 Onset-to-door time, min, median (IQR) 256 (120–360) 224 (120–300) 293 (180–390) 0.040 Onset-to-puncture time, min, median (IQR) 353 (201–481) 320 (217–444) 394 (259–513) 0.029 The number of passes with the aspiration device (mean ± SD) 1.11 ± 0.373 1.10 ± 0.300 1.12 ± 0.448 0.815 Use of BGC 11 9 2 NA Use of larger bore catheters (≥060) 29 23 6 NA SOFIA PLUS 070 19 16 3 NA Catalyst 060 8 6 2 NA NAVIEN 072 2 1 1 NA HR-MR following MT 19 2 17 NA Values expressed as n (%) unless otherwise indicated. Statistical Analysis Baseline characteristics and outcome measures were compared using univariate comparison and descriptive statistics. Variables FIGURE 1 | Patient inclusion flowchart. AIS, acute ischemic stroke; LVO, large vessel occlusion; ACA, anterior cerebral artery. FIGURE 1 | Patient inclusion flowchart. AIS, acute ischemic stroke; LVO, large vessel occlusion; ACA, anterior cerebral artery. September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org Aspiration for Different Etiologies of Occlusion Liao et al. Values expressed as n (%) unless otherwise indicated. NA, not applicable; ICAS, intracranial atherosclerotic stenosis; ASPECTS, Alberta Stroke Program Early CT Score; ICA, internal carotid artery; IQR, interquartile range; IV, intravenous; MCA, middle cerebral artery; NIHSS, National Institutes of Health Stroke Scale; HR-MR, high-resolution magnetic resonance vascular wall analysis; BGC, balloon guide catheter; MT, mechanical thrombectomy. *p-values calculated using Student’s t-test or Mann–Whitney U-test or χ2 test, as appropriate. Baseline Characteristics after aspiration was achieved more frequently in the EMB- O group (64.71%) than in the ICAS-O group (27.91%; p = 0.006), as well as the complete reperfusion (mTICI 2C-3) rate (51.94 vs. 23.26%, p = 0.003). The rate of total switching to another thrombectomy device was more frequent in the ICAS- O group than in the EMB-O group (70.09 vs. 35.29%, p = 0.001). As rescue treatments, balloon angioplasty and permanent stenting were performed more frequently in the ICAS-O group. Tirofiban/eptifibatide infusion was performed in both groups with no significant difference. characteristics of study sample are described in Tab Demographics and comorbidities showed that the risk factors for atherosclerosis, such as hypertension (74.4 vs. 37.3%; p = 0.001) and diabetes (32.6 vs. 9.8%; p = 0.013), were higher in the ICAS-O group than in the EMB-O group. The risk factors for atrial fibrillation in the EMB-O group were much more common than in the ICAS-O group (72.5 vs. 4.7%, respectively; p < 0.001). Time from onset to main hospital arrival and puncture time were shorter in the EMB-O group. Other demographic variables and comorbidities revealed no significant differences between the two groups. The final successful reperfusion rate was higher in the EMB- O group than in the ICAS-O group (EMB-O, 96.8% vs. ICAS- O, 74.41%; p = 0.006). The time from puncture to final revascularization was longer in the ICAS-O group. Effects of ADAPT for MT Effects of ADAPT for MT NA, not applicable; ICAS, intracranial atherosclerotic stenosis; ASPECTS, Alberta Stroke Program Early CT Score; ICA, internal carotid artery; IQR, interquartile range; IV, intravenous; MCA, middle cerebral artery; NIHSS, National Institutes of Health Stroke Scale; HR-MR, high-resolution magnetic resonance vascular wall analysis; BGC, balloon guide catheter; MT, mechanical thrombectomy. *p-values calculated using Student’s t-test or Mann–Whitney U-test or χ2 test, as appropriate. Values expressed as n (%) unless otherwise indicated. NA, not applicable; ICAS, intracranial atherosclerotic stenosis; ASPECTS, Alberta Stroke Program Early CT Score; ICA, internal carotid artery; IQR, interquartile range; IV, intravenous; MCA, middle cerebral artery; NIHSS, National Institutes of Health Stroke Scale; HR-MR, high-resolution magnetic resonance vascular wall analysis; BGC, balloon guide catheter; MT, mechanical thrombectomy. *p-values calculated using Student’s t-test or Mann–Whitney U-test or χ2 test, as appropriate. September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org 4 Aspiration for Different Etiologies of Occlusion Liao et al. TABLE 2 | Procedure details. Variable Embolism-related occlusions (n = 51) ICAS-related occlusions (n = 43) p-values Immediate effects following first-line thrombectomy of direct aspiration mTICI 2b or greater on first pass of aspiration 29 (56.86) 13 (30.23)* 0.017 mTICI 2c-3 on first pass of aspiration 27 (51.94) 10 (23.25) 0.003 mTICI 2b or greater after aspiration 33 (64.71) 12 (27.91) 0.006 Time from groin puncture to first revascularization, min, median (IQR) 27 (20–30) 42 (30–60) 0.000 Rescue treatments after first-line thrombectomy of direct aspiration 18 (35.29) 31 (70.09) 0.001 Switching to stent retriever device 18 (35.29) 11 (25.58) 0.429 Balloon angioplasty 1 (1.96) 18 (41.86) 0.000 Permanent stenting 2 (3.92) 10 (23.26) 0.013 Tirofiban/Eptifibatide infusion 18 (35.29) 25 (58.14) 0.344 Final endovascular treatment results Final mTICI flow [n (%)] 2b−3 49 (96.8) 32 (74.41) 0.006 0–2a 2 (3.92) 11 (25.58) 0.000 Time from groin puncture to final revascularization, min, median (IQR) 42 (29–50) 83 (60–103) 0.000 Complications Hemorrhage, all types 6 (11.76) 3 (6.98) 0.876 Hemorrhage, PH2 3 (5.88) 2 (4.65) 0.791 SAH, grade 3 or 4 1 (1.96) 1 (2.33) 0.903 New embolism in other vessels 6 (11.76) 2 (4.65) 0.546 Iatrogenic dissection or rupture 0 1 (2.33) 0.932 Spasm 3 (5.88) 2 (4.65) 0.791 Data presented as n (%, 95% confidence interval), median (95% confidence interval), n (%), or mean (SD). Predictors of FAR Using ADAPT Predictors of FAR Using ADAPT To investigate predictors of FAR, multivariate logistic regression analysis was applied in all cases of the two groups. Embolic etiology, isolated MCA occlusion, and the use of larger aspiration catheters were predictors of FAR (Table 4). Multivariate logistic regression analysis was applied in all cases of the two groups. DISCUSSION Our study demonstrates that ADAPT is more successful to achieve full recanalization in embolic LVO compared to intracranial atherosclerotic stenosis LVO. Additionally, we also found that independent predictors of FAR of aspiration included the isolated middle cerebral artery site of occlusion, embolic etiology, and use of larger bore catheters. Subgroup analysis showed that trends for the mortality of ICAS-O patients were higher than those of EMB-O in the posterior circulation. g ICAS is one of the main causes of acute stroke in Asian, Hispanic, and African populations (9). Furthermore, some studies have documented that ICAS-O is responsible for ∼12–34% of all causes of LVO in east Asia (2, 10) and 1.9%−5.5% in Western countries (11, 12). It has been shown that the pathomechanism of ICAS-O is likely due to in situ thromboocclusion (13). Some studies have shown that stent retriever thrombectomy for obtaining initial recanalization is equally effective in ICAS-O and EMB-O, although reocclusion is frequent after an initial recanalization in ICAS-O (2). Unlike stent retriever thrombectomy, aspiration first seemed less effective for the recanalization of ICAS-O (14). Effects of ADAPT for MT mTICI, modified thrombolysis in cerebral infarction; NIHSS, National Institutes of Health stroke scale; IQR, interquartile range; PH2, parenchymal hematoma type 2; SAH, subarachnoid hemorrhage. *One patient was treated successfully by aspiration, but due to re-occlusion after 10 min, another round of rescue therapy was required. Clinical Outcomes After EVT There was no significant difference in favorable clinical outcome at 3 months (Table 3). To investigate the clinical outcome, we divided patients into two subgroups based on the location of the occlusion (anterior circulation vs. posterior circulation). The subgroup analysis showed that the location of the occlusion TABLE 3 | Clinical outcomes at 90 days after endovascular treatment. Embolism- related occlusions (n = 51) ICAS-related occlusions (n = 43) OR (95% CI) p-values mRS 0–2 at 90 days 24 (47.06) 15 (34.88) 1.659 (0.721–3.821) 0.233 Anterior circulation 18 (35.29) 11 (25.58) 1.587 (0.649–3.879) 0.310 Posterior circulation 6 (11.76) 4 (9.30) 1.064 (0.281–4.022) 0.928 Mortality at 90 days 10 (19.61) 13 (30.23) 0.563 (0.218–1.455) 0.233 Anterior circulation 9 (17.65) 6 (13.95) 1.321 (0.43–4.064) 0.626 Posterior circulation 1 (1.96) 7 (16.28) 0.101 (0.012–0.856) 0.012 Data presented as n (%) or median (IQR). OR, odds ratio; mRS, modified Rankin Scale; CI, confidence interval; ICAS, intracranial atherosclerotic stenosis. TABLE 3 | Clinical outcomes at 90 days after endovascular treatment. ects following first-line thrombectomy of direct aspiration Data presented as n (%) or median (IQR). OR, odds ratio; mRS, modified Rankin Scale; CI, confidence interval; ICAS, intracranial atherosclerotic stenosis. in the EMB-O group (16.28 vs. 1.96%, p = 0.012; Table 3, Figure 2B). Clinical Outcomes After EVT There was no significant difference in favorable clinical outcome at 3 months (Table 3). To investigate the clinical outcome, we divided patients into two subgroups based on the location of the occlusion (anterior circulation vs. posterior circulation). The subgroup analysis showed that the location of the occlusion did not affect good clinical outcome at 3 months (Table 3, Figure 2A). However, 90-day mortality in patients with occlusion in the posterior circulation was higher in the ICAS-O than In this cohort, baseline characteristics of the two groups were different in many risk factors for stroke, such as September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org Liao et al. Liao et al. Aspiration for Different Etiologies of Occlusion FIGURE 2 | Comparison of clinical outcome at 90 days after a direct aspiration first-pass technique (ADAPT) for the endovascular treatment of stroke in embolism-related occlusions and intracranial atherosclerotic stenosis-related occlusions in the anterior circulation (A) and in the posterior circulation (B). FIGURE 2 | Comparison of clinical outcome at 90 days after a direct aspiration first-pass technique (ADAPT) for the endovascular treatment of stroke in mbolism-related occlusions and intracranial atherosclerotic stenosis-related occlusions in the anterior circulation (A) and in the posterior circulation (B). arison of clinical outcome at 90 days after a direct aspiration first-pass technique (ADAPT) for the endovascular treatment of stroke i cclusions and intracranial atherosclerotic stenosis-related occlusions in the anterior circulation (A) and in the posterior circulation (B) FIGURE 2 | Comparison of clinical outcome at 90 days after a direct aspiration first-pass technique (ADAPT) for the endovascular treatment of stroke in embolism-related occlusions and intracranial atherosclerotic stenosis-related occlusions in the anterior circulation (A) and in the posterior circulation (B). hypertension, diabetes mellitus, and atrial fibrillation, which stems from the fact that the grouping was based on etiology. It is noteworthy that the onset-to-door time in the ICAS-O group was much higher than in the EMB-O group. Indeed, neurologic deficit at onset was mild in some ICAS-O cases, which, along with insufficient health education, resulted in delayed visit to hospital. Underlying ICAS was identified in approximately a third of patients, which is consistent with the previous reports in Asia (2, 10). The primary outcomes of this study, immediate (56.86%) and final reperfusion (96.8%) performance, were better in the EMB-O group. Frontiers in Neurology | www.frontiersin.org Clinical Outcomes After EVT In addition, time from groin puncture to final revascularization in the EMB- O group (42 min) was significantly lower than in the ICAS- O group (83 min). Interestingly, the higher rescue treatment (70.09%) involving the use of other devices in the ICAS- O group matches those from the other reports (40–59.7%) (2, 14, 15). It is suggested that ICAS-O may require a different first-line treatment strategy compared with a direct aspiration thrombectomy. MT may cause vessel damage, which has been confirmed by clinical and animal studies (16, 17). In the current study, there were no inter-group differences in all and severe complications. September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org 6 Aspiration for Different Etiologies of Occlusion Liao et al. TABLE 4 | Multivariate regression analysis for predictors of first attempt recanalization of ADAPT. Variable* OR 95% CI p-values Age 0.997 0.956–1.039 0.871 Male gender 0.585 0.196–1.743 0.336 Diabetes 1.870 0.487–7.181 0.362 Hypertension 1.201 0.330–4.263 0.777 Hyperlipidemia 0.970 0.207–4.556 0.969 Pre-stroke mRS score 0.753 0.038–14.802 0.753 Baseline NIHSS score 0.939 0.865–1.019 0.133 IV tPA 1.764 0.397–7.846 0.456 Onset-to-puncture time 1.001 0.998–1.004 0.364 Site of occlusion Isolated MCA 4.506 1.066–19.045 0.041 Isolated VBA 3.5 0.887–13.807 0.074 Isolated ICA 0.560 0.194–0.615 0.284 Tandem occlusions 0.313 0.057–1.721 0.182 Etiology: embolism 3.505 1.031–11.917 0.045 Use of larger bore catheters (≥060) 9.167 2.6–32.322 0.001 *Variables showing statistical significance (p < 0.05) are shown in bold font. ADAPT, a direct aspiration first-pass technique; IV tPA, intravenous tissue plasminogen activator; mRS, modified Rankin Scale; NIHSS, National Institutes of Health Stroke Scale; MCA, middle cerebral artery; VBA, vertebral basilar artery; ICA, internal carotid artery. TABLE 4 | Multivariate regression analysis for predictors of first attempt recanalization of ADAPT. large-artery atherosclerosis, and erythrocyte components were positively related to successful reperfusion (21–23). In our study, the presence of embolic etiology, isolated MCA occlusion, and the use of larger aspiration catheters were independent predictors of FAR with aspiration first-line approach. In contrast, underlying atherosclerosis has been shown to be a predictor of unsuccessful recanalization. Given that ACE catheters have not yet been allowed for application in our stroke center, we mainly use the Sofia catheter as a primary aspiration catheter. Sofia catheter is a distal aspiration catheter with a specific hybrid design. Clinical Outcomes After EVT Its braid and coil construction combines different softness segments with a distal inner lumen of 0.055–0.070 inches in 5F and 6F Plus versions, respectively (24). The safety and efficacy of the Sofia aspiration catheter in a large population with first-line use has been reported (25). Our experience shows that a large inner lumen aspiration catheter such as Sofia 6F PLUS (0.070 inches) works better than a small one. Besides we found that the embolic etiology of LVO was a positive FAR predictor with direct aspiration thrombectomy, which may be a useful point for clinical reference. The limitations of this study were as follows. First, since this was a single-center study, the sample size and representativeness are insufficient. Second, it is difficult to determine whether ICAS was really the cause of LVO. Although we had collected the clots in this study, we are not yet providing histopathological analysis due to technical limitations, which requires further research. Thus, the diagnosis of underlying ICAS after thrombectomy mainly depended on the imaging characteristics of the local lesion during the procedure. Third, we used an unblinded approach for the evaluation of NIHSS and mRS. At present, due to time constraints, it is difficult to design a randomized controlled study based on the etiology of LVO stroke before intervention. Future prospective and multicentric studies with blinded endpoint registration are warranted. However, new embolism to previously uninvolved territory (8.5%) was more frequent in this study than in previous reports (1, 14, 15). This may be attributed to the lesser application of the balloon guide catheter (11.7%), and associated with histological clot composition. Despite many disadvantages in the ICAS-O compared with the EMB-O group, 3-month favorable outcomes and mortality did not differ between the groups. The enhanced lateral circulation of atherosclerotic stenosis occlusion and ischemic preconditioning compared to embolic occlusion may play crucial roles, as the ASPECTS in the ICAS-O group was higher than that of EMB-O (8.9 vs. 8.0), indicating the better collaterals in the ICAS group, though it may not be statistically significant. Of note, this study included the patients with posterior circulation LVO. It is not clear whether revascularization of posterior circulation occlusion can benefit from EVT (18, 19). Therefore, the clinical outcome was further analyzed in two subgroups separately (anterior and posterior circulation groups). CONCLUSIONS The evaluation of the efficacy of ADAPT showed higher immediate and final reperfusion in EMB-O than in ICAS-O. However, there were no significant differences in the 90-day outcomes between the two etiological groups of LVO. In case of embolic etiology and isolated MCA site of occlusion, using a larger aspiration catheter pretreatment for direct aspiration thrombectomy may be reasonable. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. Successful recanalization using ADAPT depends on multiple factors such as location of the occlusion, vascular anatomy, clot characteristics, device characteristic, and underlying etiology (4, 20). Previous studies have shown that clots from cardioembolism had a significantly higher proportion of erythrocyte and a lower proportion of fibrin compared with those from Clinical Outcomes After EVT To our surprise, although there was no difference in good outcomes between the two subgroups, we revealed that mortality was significantly higher in the posterior circulation subgroup of ICAS-O than EMB-O. Although the exact mechanism has not been illuminated, it is likely that shorter recanalization time plays a role here. Frontiers in Neurology | www.frontiersin.org REFERENCES and review of the literature. Cardiovasc Intervent Radiol. (2019) 42:863– 72. doi: 10.1007/s00270-019-02187-9 1. Turk AS, Siddiqui A, FifiJT, De Leacy RA, Fiorella DJ, Gu E, et al. Aspiration thrombectomy versus stent retriever thrombectomy as first-line approach for large vessel occlusion (COMPASS): a multicentre, randomised, open label, blinded outcome, non-inferiority trial. Lancet. (2019) 393:998– 1008. doi: 10.1016/S0140-6736(19)30297-1 13. Baek JH, Kim BM, Kim DJ, Heo JH, Nam HS, Song D, et al. Importance of truncal-type occlusion in stentriever-based thrombectomy for acute stroke. Neurology. (2016) 87:1542–50. doi: 10.1212/WNL.00000000000 03202 14. Yoo J, Lee SJ, Hong JH, Kim YW, Hong JM, Kim CH, et al. Immediate effects of first-line thrombectomy devices for intracranial atherosclerosis- related occlusion: stent retriever versus contact aspiration. BMC Neurol. (2020) 20:283–92. 2. Park H, Baek JH, Kim BM. Endovascular treatment of acute stroke due to intracranial atherosclerotic stenosis–related large vessel occlusion. Front Neurol. (2019) 10:308. doi: 10.3389/fneur.2019.00308 15. Lapergue B, Blanc R, Guedin P, Decroix JP, Labreuche J, Preda C, et al. A direct aspiration, first pass technique (ADAPT) versus stent retrievers for acute stroke therapy: an observational comparative study. AJNR Am J Neuroradiol. (2016) 37:1860–5. doi: 10.3174/ajnr.A4840 3. Tomsick T, Broderick J, Carrozella J, Khatri P, Hill M, Palesch Y, et al. Revascularization results in the interventional Management of Stroke II trial. AJNR Am J Neuroradiol. (2008) 29:582–7. doi: 10.3174/ajn r.A0843 16. Liao G, Zhang Z, Che X, Liang H. Mechanical thrombectomy using a stent retriever with an intermediate catheter for partially occluded middle cerebral artery fenestration. World Neurosurg. (2020) 138:355– 9. doi: 10.1016/j.wneu.2020.03.096 4. Anadani M, Alawieh A, Vargas J, Chatterjee AR, Turk A, Spiotta A. First attempt recanalization with ADAPT: rate, predictors, and outcome. J Neurointerv Surg. (2018) 2018:1–6. doi: 10.1136/neurintsurg-2018-0 14294 17. Koge J, Kato S, Hashimoto T, Nakamura Y, Kawajiri M, Yamada T. Vessel wall injury after stent retriever thrombectomy for internal carotid artery occlusion with duplicated middle cerebral artery. World Neurosurg. (2019) 123:54–8. doi: 10.1016/j.wneu.2018.11.223 5. Fiorelli M, Bastianello S, von Kummer R, del Zoppo GJ, Larrue V, Lesaffre E, et al. Hemorrhagic transformation within 36 hours of a cerebral infarct: relationships with early clinical deterioration and 3-month outcome in the European cooperative acute stroke study I (ECASS I) cohort. Stroke. (1999) 30:2280–4. 18. Liu X, Dai Q, Ye R, Zi W, Liu Y, Wang H, et al. REFERENCES Endovascular treatment versus standard medical treatment for vertebrobasilar artery occlusion (BEST): an open-label, randomised controlled trial. Lancet Neurol. (2020) 19:115– 22. doi: 10.1016/S1474-4422(19)30395-3 6. Frontera JA, Claassen J, Schmidt JM, Wartenberg KE, Temes R, Connolly ES, et al. Prediction of symptomatic vasospasm after subarachnoid hemorrhage: the modified fisher scale. Neurosurgery. (2006) 59:21–7. doi: 10.1227/01.Neu.0000218821.34014.1b 19. Siebert E, Bohner G, Zweynert S, Maus V, Mpotsaris A, Liebig T, et al. Revascularization techniques for acute basilar artery occlusion: technical considerations and outcome in the setting of severe posterior circulation steno-occlusive disease. Clin Neuroradiol. (2019) 29:435–43. doi: 10.1007/s00062-018-0683-3 7. Wahlgren N, Ahmed N, Dávalos A, Ford GA, Grond M, Hacke W, et al. SITS-MOST Investigators. Thrombolysis with alteplase for acute ischaemic stroke in the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST): an observational study. Lancet. (2007) 369:275–82. doi: 10.1016/S0140-6736(07)60149-4 20. Blanc R, Redjem H, Ciccio G, Smajda S, Desilles JP, Orng E, et al. Predictors of the aspiration component success of a direct aspiration first pass technique (ADAPT) for the endovascular treatment of stroke reperfusion strategy in anterior circulation acute stroke. Stroke. (2017) 48:16149. doi: 10.1161/STROKEAHA.116.016149 8. Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in acute stroke treatment. Stroke. (1993) 24:34–41. doi: 10.1161/01.str.24.1.35 21. Kamalian S, Morais LT, Pomerantz SR, Aceves M, Sit SP, Bose A, et al. Clot length distribution and predictors in anterior circulation stroke: implications for intra-arterial therapy. Stroke. (2013) 44:3553– 6. doi: 10.1161/STROKEAHA.113.003079 9. Wong LK. Global burden of intracranial atherosclerosis. Int J Stroke. (2006) 1:158–9. doi: 10.1111/j.1747-4949.2006.00045.x 10. Jia B, Feng L, Liebeskind DS, Huo X, Gao F, Ma N, et al. Mechanical thrombectomy and rescue therapy for intracranial large artery occlusion with underlying atherosclerosis. J NeuroIntervent Surg. (2017) 2017:1– 6. doi: 10.1136/neurintsurg−2017-013489 22. Kim SK, Yoon W, Kim TS, Kim HS, Heo TW, Park MS. Histologic analysis of retrieved clots in acute ischemic stroke: correlation with stroke etiology and gradient-echo MRI. AJNR Am J Neuroradiol. (2015) 36:1756– 62. doi: 10.3174/ajnr.A4402 11. Gascou G, Lobotesis K, Machi P, Maldonado I, Vendrell JF, Riquelme C, et al. Stent retrievers in acute ischemic stroke: complications and failures during the perioperative period. AJNR Am J Neuroradiol. (2014) 35:734– 40. doi: 10.3174/ajnr.A3746 23. FUNDING People’s Hospital. They granted approval for our work (2019027). The patients/participants provided their written informed consent to participate in this study. This work was supported by grants from the High-level Hospital Construction Research Project of Maoming People’s Hospital (Reference No. Yueweihan 2018413) and the Special Fund of Science and Technology of Guangdong Province, China (Reference No. 2020S00050). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur. 2021.695085/full#supplementary-material AUTHOR CONTRIBUTIONS GL designed the report, completed the statistical analysis, wrote the protocol, wrote the first draft of the manuscript, and took overall responsibility. GL, ZZ, WD, CL, and GZ contributed to the acquisition of data. GL, ZZ, and HL managed the literature searches and analyses. All authors have provided a substantial contribution to the study. ETHICS STATEMENT The studies involving human participants were reviewed and approved by The Institutional Ethics Committee of Maoming September 2021 | Volume 12 | Article 695085 7 Aspiration for Different Etiologies of Occlusion Liao et al. 24. Wong JHY, Do HM, Telischak NA, MoraffAM, Dodd RL, Marks MP, et al. Initial experience with SOFIA as an intermediate catheter in mechanical thrombectomy for acute ischemic stroke. J Neurointerv Surg. (2017) 9:1103– 6. doi: 10.1136/neurintsurg-2016-012750 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 25. Marnat G, Barreau X, Detraz L, Bourcier R, Gory B, Sgreccia A, et al. First-line sofia aspiration thrombectomy approach within the endovascular treatment of ischemic stroke multicentric registry: efficacy, safety, and predictive factors of success. Am J Neuroradiol. (2019) 40:1006–12. doi: 10.3174/ajn r.A6074 Frontiers in Neurology | www.frontiersin.org September 2021 | Volume 12 | Article 695085 REFERENCES Hashimoto T, Hayakawa M, Funatsu N, Yamagami H, Satow T, Takahashi JC, et al. Histopathologic analysis of retrieved thrombi associated with successful reperfusion after acute stroke thrombectomy. Stroke. (2016) 47:3035–7. doi: 10.1161/STROKEAHA.116.01 5228 12. Dobrocky T, Kaesmacher J, Bellwald S, Piechowiak E, Mosimann PJ, Zibold F, et al. Stent-retriever thrombectomy and rescue treatment of M1 occlusions due to underlying intracranial atherosclerotic stenosis: cohort analysis September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org 8 Aspiration for Different Etiologies of Occlusion Liao et al. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 25. Marnat G, Barreau X, Detraz L, Bourcier R, Gory B, Sgreccia A, et al. First-line sofia aspiration thrombectomy approach within the endovascular treatment of ischemic stroke multicentric registry: efficacy, safety, and predictive factors of success. Am J Neuroradiol. (2019) 40:1006–12. doi: 10.3174/ajn r.A6074 Copyright © 2021 Liao, Zhang, Zhang, Du, Li and Liang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. September 2021 | Volume 12 | Article 695085 Frontiers in Neurology | www.frontiersin.org 9
https://openalex.org/W4225589037
https://www.frontiersin.org/articles/10.3389/fpsyg.2022.826121/pdf
English
null
Effect and Mechanisms of State Boredom on Consumers’ Livestreaming Addiction
Frontiers in psychology
2,022
cc-by
10,125
Effect and Mechanisms of State Boredom on Consumers’ Livestreaming Addiction 1 School of Economics and Management, Beijing Jiaotong University, Beijing, China, 2 School of Management, Minzu University of China, Beijing, China With the rapid development of livestreaming marketing in China, consumers spend an increasing amount of time watching and purchasing on the platform, which shows a trend of livestreaming addiction. In the early stage of the COVID-19 pandemic, the addiction exacerbated by a surge of boredom caused by home quarantine. Based on the observation of this phenomenon, this research focused on whether state boredom could facilitate consumers’ livestreaming addiction and explored the associated mechanisms of this relationship. Based on three studies, this research found that state boredom had a positive effect on consumers’ livestreaming addiction, and this relationship worked through the mediating effect of consumers’ sensation seeking. We further verified a moderated mediation effect of consumers’ life meaning perception, where the indirect effect of state boredom on consumers’ livestreaming addiction via consumers’ sensation seeking existed for high and low levels of life meaning perception, but in opposite directions. The conclusions provided theoretical and practical implications of livestreaming marketing and healthy leisure consumption. Reviewed by: Reviewed by: Kun-Shan Wu, Tamkang University, Taiwan Marco Roccetti, University of Bologna, Italy Shuailei Lian, Yangtze University, China *Correspondence: Jian Li lijian@muc.edu.cn Reviewed by: Kun-Shan Wu, Tamkang University, Taiwan Marco Roccetti, University of Bologna, Italy Shuailei Lian, Yangtze University, China ORIGINAL RESEARCH published: 05 April 2022 doi: 10.3389/fpsyg.2022.826121 ORIGINAL RESEARCH published: 05 April 2022 doi: 10.3389/fpsyg.2022.826121 *Correspondence: Jian Li Specialty section: This article was submitted to Health Psychology, a section of the journal Frontiers in Psychology With the rapid development of the livestreaming industry, individuals become addicted to livestreaming platforms and apps. Livestreaming addiction is defined as the consumers watching for a long time and purchasing frequently on the livestreaming platform. This phenomenon has become more serious during the COVID-19 pandemic. The COVID-19 pandemic restricted people’s activities, and related leisure consumption suffered, such as traveling, eating at restaurant, outdoor activities, etc. The perceived severity of COVID-19 led to an increase in boredom and sensation-seeking expression, which significantly promoted consumers’ post-pandemic consumption willingness (Deng et al., 2020). According to IIMedia Research, China’s livestreaming e-commerce industry has grown rapidly when consumers were isolated at Received: 01 December 2021 Accepted: 22 February 2022 Published: 05 April 2022 Keywords: state boredom, livestreaming addiction, life meaning perception, sensation seeking, China consumers Edited by: Edited by: Zongkui Zhou, Central China Normal University, China Central China Normal University, China INTRODUCTION The livestreaming industry is growing rapidly worldwide. Livestreaming platforms are recognized as a unique form of social media, which extend the forms of computer-mediated communications from text and image to audio and video. As a special combination of multiple media forms, livestreaming allows individuals to publicly broadcast live video streams, chat with broadcasters and other users, purchase products, and send gifts at the same time (Hamilton et al., 2014). The livestreaming platform includes all kinds of live streams ranging from eating, gaming, singing, shopping, to traveling anytime and anywhere. It offers a new approach for individuals to relax and communicate by an exciting and more interactive social media. *Correspondence: Jian Li lijian@muc.edu.cn Citation: Zhang N and Li J (2022) Effect and Mechanisms of State Boredom on Consumers’ Livestreaming Addiction. Front. Psychol. 13:826121. doi: 10.3389/fpsyg.2022.826121 April 2022 | Volume 13 | Article 826121 1 Frontiers in Psychology | www.frontiersin.org Boredom and Live Streaming Addiction Zhang and Li home because of the COVID-19 pandemic. For example, in the first quarter of 2020, 41.7% of users of livestreaming e-commerce shopped 5–8 times per week on average, 37.5% of users shopped 1–4 times, 6.7% of users shopped 9–12 times, 3.3% of users shopped 13 or 16 times, and 0.8% of users shopped more than 16 times per week on average. concentration, and motivation (Huang et al., 2010). Additionally, the self-regulatory process helps bored people to seek sensation (Reisenzein, 2017), and sensation seeking is positively related to Internet addiction (Müller et al., 2016). Therefore, we proposed the mediating effect of sensation seeking on the relationship between state boredom and livestreaming addiction. Life meaning is defined as a generally stable sense of purpose in life and an accompanying sense of fulfillment (Baumeister, 1991), and a contributor to psychological health (Brassai et al., 2011). Faced with a disaster context, individuals with high-perceived life meaning could cope well and be satisfied with life (Drescher et al., 2012). However, the role of life meaning in boredom has rarely been studied. In addition, life meaning could prevent individuals’ unhealthy behaviors, such as Internet addiction (Zhang et al., 2015). Therefore, life meaning may modify the positive effect of state boredom on livestreaming addiction. Researchers have paid sufficient attention to users’ addiction or continuance intention on other types of social media, such as gaming disorders (Jeon et al., 2019), computer addiction (Cho and Kim, 2010), Internet addiction (Kuss and Lopez-Fernandez, 2016), social media addiction (Brand et al., 2019), and addiction to heavy viewing (Horvath, 2004). However, extant literature still lacks a comprehensive explanation for livestreaming addiction. Citation: Limited studies have tried to figure out this question from two aspects: on the one hand, the factors of individual traits, such as escape from loneliness (Chen and Chang, 2019), information seeking (Hilvert-Bruce et al., 2018), flow experience (Chen and Lin, 2018), perceived value (Singh et al., 2021), and Big Five personality traits (Cheng et al., 2019); on the other hand, a social relationship between broadcasters and audiences, such as the parasocial interaction between broadcasters and audiences (Lim et al., 2020), the social identification of the co-viewers, and the sense of community within the streaming room (Lim et al., 2012; Hu et al., 2017). Specifically, this research intends to address the following research questions. Could state boredom lead to livestreaming addiction, just like proneness boredom? How does state boredom promote livestreaming addiction? In addition, under what condition, the positive effect of state boredom on livestreaming addiction would be weakened? Based on the theoretical analysis and practical observation, this study proposed that state boredom has a positive effect on livestreaming addiction, and this effect works through the mediating effect of sensation seeking. The boundary effect of the above relationship is the perception of life meaning. Although some factors for livestreaming addiction have been recognized, few researchers have examined the relationship between state boredom and livestreaming addiction. Boredom refers to a negative experience of desiring but being unable to engage with the environment or in satisfying activities (Biolcati et al., 2018). According to the stability across time and situations, boredom can be categorized into proneness boredom and state boredom. Proneness boredom refers to the stable tendency of boredom generated by individuals in various situations. It is a part of personality traits, related to self-adjusting ability, intrinsic motivation, and values (Musharbash, 2007). On the contrary, state boredom is a temporary boring experience generated by an individual in a specific situation. It is a subjective feeling that can be triggered by monotonous and repetitive external stimuli. In recent years, scholars realize that proneness boredom is not enough to understand boredom itself, but a deeper understanding of boredom and its influence on individual decisions and behaviors could be possible by exploring state boredom (Bench and Lench, 2019). Citation: However, previous studies mainly discussed proneness boredom and its effects on some addictive behaviors, such as mobile phone addiction (Chou et al., 2018; Wang et al., 2020), Internet addiction (Lin et al., 2009), and Facebook addiction (Donati et al., 2022), and less is talked about state boredom. Therefore, paying attention to the effect of state boredom on addiction is a supplement to boredom and addiction theoretical research. The structure of this research proceeds as follows: Section Theoretical Background and Research Hypotheses reviewed the theoretical background of state boredom and livestreaming addiction and proposed three hypotheses. Section Materials and Methods described the research method and showed the results to test the three hypotheses. Section Conclusion and Implications discussed the conclusion, theoretical and practical implications, research limitations, and future research directions. Frontiers in Psychology | www.frontiersin.org State Boredom and Livestreaming Addiction In clinical psychology, boredom is often defined as an emotional state characterized by unpleasant feelings, a lack of stimulation, and low physical arousal (Harris, 2000). Individuals describe boredom as stress, anxiety, exhaustion, pain, and suffering (Mann and Robinson, 2009). Other symptoms include feeling that time is passing so slowly, escaping out of boredom through physical and mental relief (e.g., daydreaming), and talking in a slow and monotonous way (Biolcati et al., 2018). Therefore, scholars believe that boredom is a state of under-stimulation, under- arousal, and a lack of psychological participation associated with dissatisfaction and that individuals try to cope with it by seeking extra stimulation (Brissett and Snow, 1993). The mechanisms of state boredom on livestreaming addition may be related to sensation seeking and life-meaning perception. Sensation seeking is defined as “a trait by the seeking of varied, novel, complex, and intense sensations and experiences and the willingness to take physical, social, legal, and financial risks for the sake of such experience” (Zuckerman, 1996). Scholars believe that boredom is characterized by a lack of interest, To get rid of boredom, individuals indulge themselves in behaviors full of stimulants, such as overeating, gambling, April 2022 | Volume 13 | Article 826121 2 Boredom and Live Streaming Addiction Zhang and Li television, and Internet addiction (Musharbash, 2007). Previous studies have shown that boredom is positively correlated with addictive behaviors such as alcoholism, drug abuse, Internet addiction, and gambling (Pekrun et al., 2010). In terms of food consumption, boredom highly correlated with consumers’ overeating (Wilson, 1986), significantly increased the frequency of eating (Robin, 1975), led to eating disorders (Stickney and Miltenberger, 1999), and enabled obese people to eat more food than normal-weight people faced with boring tasks (Abramson and Stinson, 1977). For social media addiction, Whelan et al. (2020) suggested a strong association between proneness boredom and both information and communication overload, which in turn increased social media fatigue. group chose more exciting television programs than those in the state stress group. Moynihan et al. (2015) found that state boredom increased a person’s preference for stimulus-inducing food (such as candy) but not for non-stimulus-inducing food (such as saltines). At the same time, sometimes people choose negative stimulation to relieve state boredom. Wilson et al. (2014) left the participants alone in a room for 15 min to induce their state boredom. State Boredom and Livestreaming Addiction They were then told that they were free to choose whether or not to receive an electric shock during the solitude. It was found that individuals would rather receive a small negative electric shock than keep waiting. Sensation seeking leads to addictive behaviors. Numerous studies have been conducted to explain why consumers use social media addictively. Some studies found that seeking entertainment and killing time are the strong predictors of social media overload (Quan-Haase and Young, 2010; Ku et al., 2013). Meanwhile, sensation seeking has proved to trigger addictive behaviors in previous research, such as Internet addiction (Müller et al., 2016). Livestreaming addiction could be seen as one kind of Internet addiction. Besides, as one of the most effective ways to relax and entertain, livestreaming can make boring people feel new, exciting, and fun. Therefore, when consumers are bored, they may frequently watch livestreaming and buy products to seek stimulation. Based on this, we proposed the second hypothesis, the mechanisms, and the mediating effect of sensation seeking: Boredom is recognized as one of the common causes of addiction, such as Internet addiction (Chou et al., 2018), Facebook use (Donati et al., 2022), gambling behavior (Mercer and Eastwood, 2010), and smartphone addiction (Wang et al., 2020). Boredom is a negative state of under-arousal, and people usually try to escape from boredom and achieve physiological arousal through media, which leads to media dependence (Khang et al., 2013). Livestreaming is a combination of novel and exciting activities, such as chatting, gambling, viewing videos, shopping, and traveling. Thus, these activities make live streaming a good tool for people with state boredom to easily arouse their psychology, which in turn reinforces livestreaming addiction. State boredom is a temporary boring experience generated by an individual in a specific situation. Previous studies on boredom focused more on proneness boredom, but in fact, state boredom is more common in our daily life, such as college students’ sense of meaninglessness and boredom in study and life (Nett et al., 2011) and white-collar workers’ state boredom in the bottleneck period of work (Loukidou et al., 2009). When individuals are under daily state boredom, they may indulge in the popular livestreaming platforms. As a new form of social media, the characteristics of livestreaming provide individuals with the opportunities of seeking entertainment and killing time. State Boredom and Livestreaming Addiction In a relaxing atmosphere, individuals can interact with the broadcasters and participate in a lucky draw, which has resulted in livestreaming addiction, especially when individuals are boring. Therefore, we suggested the first hypothesis: H2: Sensation seeking mediated the positive influence of state boredom on consumers’ livestreaming addiction. Moderated Mediation Effect of Life Meaning Perception State boredom is closely related to life meaning. A central feature of boredom is a lack of perceived meaning (Fahlman et al., 2009). Boredom indicates that the current situation is purposeless, which is a meaningful threat (Van Tilburg and Igou, 2012). Researchers suggest that the loss or failure of developing meaning in life is a critical factor for boredom (Van Tilburg et al., 2018). Life meaning could decrease the negative results of boredom. For example, studies proved that life meaning prevents Internet addiction (Zhang et al., 2015). This is because life meaning is associated with feelings of control (Martela and Steger, 2016), and those with self-control or high self-esteem demonstrated lower tendency to be addicted (Leung, 2007). Therefore, life meaning might affect how people cope with boredom, and whether they would stave offboredom through livestreaming addiction. H1: State boredom promotes individuals’ livestreaming addiction. April 2022 | Volume 13 | Article 826121 Procedures and Measures Study 1 was a survey, to test the main effect of state boredom on consumers’ livestreaming addition. To guarantee the reliability and validity of the questionnaires, we choose the mature scales that have been verified with high reliability and validity. The measurement was originally in English and was subsequently translated into Chinese, following the back-translation process (Brislin, 1970). H3: Life-meaning perception has a mediating moderation effect on the relationship between state boredom and livestreaming addiction. When the perception of life meaning is low, state boredom will increase livestreaming addiction through sensation seeking; when the perception of life meaning is high, state boredom will reduce livestreaming addiction through sensation seeking. After obtaining informed consent, participants were asked to answer the scales of state boredom, their daily behaviors on the livestreaming platform, livestreaming addition, current affects, and finally the demographics. After the data were qualified, the reward was paid. First, state boredom was measured using a 29-item state boredom scale adapted from Fahlman et al. (2011), which had good reliability (α = 0.965). Participants were asked to indicate the degree of boredom on a 7-point Likert scale (1 = strongly disagree and 7 = strongly agree). The sample items included “I am stuck in a situation that I feel is irrelevant,” “Everything seems repetitive and routine to me,” “I feel empty,” and so on. Second, livestreaming addiction was measured, adapted from the Bergen Facebook Addiction Scale (Andreassen et al., 2012), with 18 items (α = 0.933). Participants were asked how often have they “Spent a lot of time thinking about livestreaming or planned use of livestreaming?” “Thought about how you could free more time to spend on livestreaming?” and so on. Each item was scored on a five-point scale (1 = very rarely, 2 = rarely, 3 = sometimes, 4 = often, and 5 = very often). Third, the positive and negative affects were tested, because the research showed that the boredom would have an effect on person affects (Alda et al., 2015). We chose five related affects from the Positive and Negative Affect Schedule (PANAS; Watson et al., 1988), namely, excited, irritated, bored, impatient, and happy, with a 5-point Likert scale (1 = very slightly, 5 = very strongly). Fourth, demographic information was collected, such as gender, age, education, household income, and perceived socioeconomic status. Procedures and Measures Based on the above hypotheses, we explored the effect of state boredom on livestreaming addiction and the mediating role of sensation seeking and the moderating role that life meaning played on the mediating relationship. The overall research framework is shown in Figure 1. Mediating Effect of Sensation Seeking Mediating Effect of Sensation Seeking State boredom is positively related to sensation seeking. Boredom is caused by monotony, which means that there are no external stimuli or a single stimulus (Hill and Perkins, 1985). People feel bored because they lack interest or stimulation caused by the surrounding environment (Sansone et al., 1992). The self- regulatory process triggered by state boredom can influence specific behaviors by sensation seeking (Van Tilburg and Igou, 2012), which is designed to make the boring situation more interesting or challenging (Jessi et al., 2009). Individuals who suffer from boredom would actively seek out more and stronger complex external stimuli (Reisenzein, 2017). Bryant and Zillmann (1984) found that subjects in the state boredom Recent research indicated that boring people attempted to escape from the meaninglessness associated with boredom by engaging in simulating activities (Moynihan et al., 2017). The sensations involved in these simulating acts and addiction may help to distract people from meaninglessness (Moynihan et al., 2015). Therefore, when boring people try to seek sensation, if they have high life-meaning perception, the meaninglessness threat signaled by state boredom is low and then may weaken the positive effect of state boredom on sensation seeking and addiction; and if they have low life-meaning perception, the meaninglessness threat signaled by state boredom is high, they Frontiers in Psychology | www.frontiersin.org April 2022 | Volume 13 | Article 826121 3 Boredom and Live Streaming Addiction Zhang and Li have a strong motivation for sensation seeking and addiction, and the positive effect of state boredom on sensation seeking and addiction is enhanced. In a word, life meaning moderated the relationship of state boredom on consumers’ sensation seeking and addiction. have a strong motivation for sensation seeking and addiction, and the positive effect of state boredom on sensation seeking and addiction is enhanced. In a word, life meaning moderated the relationship of state boredom on consumers’ sensation seeking and addiction. 1https://www.credamo.com/#/ Study 1 Participants A number of 100 participants were recruited using the sample database on the Credamo. Among them, 34% are men, 69% aged 21–30 years, and 31% aged 31–40 years (refer to Table 1 for more information of demographics). The participants have rich experience with livestreaming. Totally, 65% of participants indicated that livestreaming shopping is the most popular type of livestreaming. On average, they spent 1,165.8 Yuan [standard deviation (SD) = 1,053.785] in the last month on the livestreaming platform and watched livestreaming for 355.09 min (SD = 255.916) per week. In general, individuals suffering from state boredom tend to engage in activities that help restore a sense of meaning (Barbalet, 1999). Life meaning is proved to modify the association of state boredom on media use, which is served as a risk factor for the negative psychological outcomes when individuals experienced boredom during the COVID-19 outbreak in China (Miao et al., 2020). Therefore, when individuals have a high sense of life meaning, they will regulate their behaviors to maintain a high sense of life meaning, to reduce sensation seeking and addictive behavior in a state of boredom. However, when the sense of life meaning is low, they will have lower limits on sensation seeking and addictive behaviors in a state of boredom, thus promoting consumers to engage in more addictive behaviors. Based on the above analysis, hypothesis 3 is proposed. MATERIALS AND METHODS Totally, three studies were conducted to test the above hypotheses. Study 1 was a self-reported survey to test the main effect of state boredom and livestreaming addiction. Study 2 was a survey-embedded randomized experiment to test the mediation effects of sensation seeking on the relationship of state boredom and livestreaming addiction, and Study 3 was a self-reported survey to test the moderated mediation effect of life meaning perception on the relationship among state boredom, sensation seeking, and livestreaming addiction. Participants were recruited from the online survey platform Credamo.1 At the beginning of the survey and experiment, all participants signed informed consent online. The participants were guaranteed anonymity and allowed to discontinue the survey at any time. They were told that the survey was a sociological study that consists of several unrelated sub-surveys. The survey included an attention check test, which needs to be answered carefully, and the corresponding reward can only be obtained after passing the researcher’s review. Each participant can get 10 Yuan as a reward. Results Given the nature of the single-shot cross-sectional survey, we checked whether there is a common method bias before the formal data analysis. Harman’s one-factor analysis was conducted following the recommendations of Podsakoffand Organ (1986). An exploratory factor analysis using a maximum likelihood solution was conducted on all of the items of key variables in this April 2022 | Volume 13 | Article 826121 Frontiers in Psychology | www.frontiersin.org 4 Zhang and Li Boredom and Live Streaming Addiction FIGURE 1 | Research framework. FIGURE 1 | Research framework. was the inclusion of sensation seeking to test whether sensation seeking played a mediating role in the relationship between state boredom and livestreaming addiction. study. A number of seven factors emerged with eigenvalues larger than 1.00, which suggests that more than one factor underlies the data. Moreover, the first factor accounted for only 42.596% of the total variance, which suggests that the common method variance may not be a serious concern in this study (Eby and Dobbins, 1997). study. A number of seven factors emerged with eigenvalues larger than 1.00, which suggests that more than one factor underlies the data. Moreover, the first factor accounted for only 42.596% of the total variance, which suggests that the common method variance may not be a serious concern in this study (Eby and Dobbins, 1997). Main Effect of State Boredom on Livestreaming Addiction Main Effect of State Boredom on Livestreaming Addiction Using SPSS 26.0, we regressed consumers’ state boredom on their livestreaming addiction, respectively, in three models to test H1. In model 1, state boredom was the only predictor of livestreaming addiction. In model 2, demographic variables were included, and in model 3, five affects were analyzed as the control variables. As expected, state boredom had a significantly positive effect on consumers’ livestreaming addiction (β = 0.576, t = 6.981, p < 0.000). The result was consistently significant when controlling for the demographics in model 2 (β = 0.636, t = 7.031, p < 0.000) and for the PANAS in model 3 (β = 0.592, t = 5.101, p < 0.000), and H1 was approved. Detailed information is shown in Table 3. Participants A total of 100 participants were recruited from the sample database on the Credamo platform, who have not joined in Study 1. They were divided into two groups: the boring group (N = 50) and the non-boring group (N = 50). Among them, 36 are men (36%), and their average age was 28.51 (SD = 5.76, min = 19, Description of State Boredom and Livestreaming Addiction Description of State Boredom and Livestreaming Addiction Factor analysis was conducted for state boredom and livestreaming addiction. State boredom was proved to combine as one factor (KMO = 0.934, p < 0.000) and also for livestreaming addiction (KMO = 0.899, p < 0.000). Besides, we conducted a confirmatory factor analysis (CFA) for livestreaming addiction, and the resulting 18-item scale showed an excellent fit to the data (NFI = 0.923, CFI = 0.987, RMSEA = 0.043), averaged the items, and got the mean-centered score of state boredom and livestreaming addiction. Means, SD, and correlation are shown in Table 2. TABLE 1 | Description of participants’ demographics in Study 1. Education N Percentage Secondary education 1 1% Associate’s degree 12 12% Bachelor’s degree 74 74% Master’s degree 13 13% Occupation Students 7 7% State-owned enterprises 23 23% Private enterprises 56 56% Foreign-invested enterprises 4 4% Public institutions 9 9% Civil servant 1 1% Household income monthly (RMB) <5,000 2 2% 5,000–9,999 14 14% 10,000–14,999 24 24% 15,000–19,999 22 22% 20,000–24,999 16 16% 25,000–29,999 10 10% ≥30000 12 12% Perceived SES Between the bottom and the middle of SES 23 23% The middle of SES 63 63% Between the middle and the upper of SES 14 14% N = 100. TABLE 1 | Description of participants’ demographics in Study 1. Study 2 In Study 1, we tested the main effect of state boredom on livestreaming addiction based on the participants’ self-reported survey. In Study 2, two more things were conducted. One was the manipulation of state boredom. State boredom was primed with a typical psychological experiment, a one-factor between- subject design, namely, state boredom (yes vs. no). The other Frontiers in Psychology | www.frontiersin.org April 2022 | Volume 13 | Article 826121 5 Boredom and Live Streaming Addiction Zhang and Li TABLE 4 | Description of participants demographics in Study 2. Education N Percentage Secondary education 1 0.8% Associate’s degree 16 13.3% Bachelor’s degree 88 73.3% Master’s degree 15 12.5% Occupation Student 8 6.7% State-owned enterprises 27 22.5% Private enterprises 69 57.5% Foreign-invested enterprises 5 4.2% Public institutions 9 7.5% Civil servant 2 1.7% Household income monthly (RMB) <5,000 2 1.7% 5,000–9,999 17 14.2% 10,000–14,999 25 20.8% 15000–19,999 26 21.7% 20,000–24,999 22 18.3% 25,000–29,999 12 10% ≥30,000 16 13.3% Perceived SES Between the bottom and the middle of SES 26 21.7% The middle of SES 78 65% Between the middle and the upper of SES 16 13.3% N = 100. max = 56). All the participants had experience in watching or purchasing in livestreaming (refer to Table 4 for information on other demographic variables). Procedures and Measures Before the formal experiment, participants were asked to give informed consent online, just like Study 1. The whole procedures of the formal experiment included four steps. When the data were reviewed and qualified, the reward was paid. First was the manipulation of state boredom. Participants were randomly assigned into two conditions: the boring condition and the non- boring condition. They were asked to recall a time in their life when they felt bored or non-bored and to describe it in as much detail as possible. Second was the manipulation check. Participants answered two questions. “How boring do you think it is today?” (1 = not boring at all, 7 = extremely boring) and “Do you often feel bored in the last week?” (1 = very rarely, 7 = very often). Third was the measurement of the addiction scale (Andreassen et al., 2012), which had good reliability (α = 0.916). Fourth was the measurement of the Impulsive Sensation Seeking [ImpSS; Zuckerman et al. (1993)] scale. This scale included 19 questions (α = 0.862): seven questions were about impulsivity and 12 questions were about sensation seeking. Every question was scored for 1 (yes) or 0 (no), and the final score of the impulsivity and sensation seeking is the sum of all questions. A higher score indicated a higher level of impulsivity and sensation seeking. TABLE 2 | Means, standard deviations (SDs), and correlations of variables in Study 1. M SD Correlation 1 State boredom 2.39 1.05 2 Livestreaming addiction 2.69 0.75 0.576** **p < 0.01. TABLE 3 | The effect of state boredom on livestreaming addiction. Model 1 Model 2 Model 3 Main effect State boredom 0.576*** 0.636*** 0.592*** Controls Gender −0.110 −0.088 Age −0.016 0.016 Education 0.066 0.028 Work 0.191 0.089 Household income monthly 0.133 0.136 Perceived SES −0.140 −0.209 Excited 0.250 Irritated 0.076 Bored 0.189 Impatient 0.060 Happy 0.166 R2 0.332 0.391 0.490 1R2 0.325 0.345 0.419 N = 100; ***p < 0.001. TABLE 2 | Means, standard deviations (SDs), and correlations of variables in Study 1. Finally, demographic information, such as gender, age, education, work, and household income, was collected. Finally, demographic information, such as gender, age, education, work, and household income, was collected. Manipulation Check of State Boredom The results showed that there is a significant difference in the perception of boredom between the boring group and the non- boring group, and people in the boring group perceived more state boredom (M boring = 4.02, SD boring = 1.68) than those in the non-boring group [(Mnotboring = 2.62, SDnotboring = 1.31), F(1,99) = 21.557, p < 0.000]. There is also a significant difference in the frequency of boredom between the boring group and the non-boring group, [F(1,99) = 17.490, p < 0.000, Mboring = 4.08, SDboring = 1.76, Mnotboring = 2.68, SDnotboring = 1.58]. The result showed that the manipulation of state boredom is effective. Procedures and Measures Before the formal survey, participants were asked to provide informed consent online, just like Studies 1 and 2. The whole procedures of the formal survey included five steps. When the data were reviewed and qualified, the reward was paid. First was the measurement of state boredom with the same scale as used in Study 1 (Fahlman et al., 2011), which has good reliability (α = 0.965). Second was the measurement of the livestreaming addiction scale (Andreassen et al., 2012) with good reliability (α = 0.931). Third was the measurement of consumers’ sensation seeking (Steinberg et al., 2008) with six items (α = 0.847), which includes “I like to have new and exciting experiences and sensations even if they are a little frightening,” “I like doing things just for the thrill of it,” “I sometimes like to do things that are a little frightening,” “I’ll try anything once,” “I sometimes do ‘crazy’ things just for fun,” and “I like wild and uninhibited parties.” We adopted seven-point Likert scale, with one representing “strongly disagree” and seven representing “strongly agree.” Fourth was the measurement of life-meaning perception [Meaning in Life Questionnaire, MLQ; Steger et al. (2006)] with 10 items (α = 0.808). The typical items included “I understand my life’s meaning,” “My life has a clear sense of purpose,” and participants chose their agreement on a seven- point Likert scale (1 = absolutely untrue, 7 = absolutely true). Finally, demographic information was collected. Studies 1 and 2 measured and primed state boredom with different methods. We found that state boredom significantly promoted consumers’ livestreaming addictive behaviors, which means that consumers are more likely to indulge in livestreaming and purchasing behavior under the condition of state boredom. At the same time, we found that consumers’ sensation seeking played a mediating role in the above relationship. The boundary of mediating effect of sensation seeking is discussed in Study 3. In other words, under what circumstances TABLE 5 | Mediating effects of sensation seeking by bootstrapping analysis. Impulsivity and sensation seeking Effect BootSE BootLLCI BootULCI Indirect effect −0.1059 0.0577 −0.2488 −0.0207 Direct effect −0.1686 0.1370 −0.4405 0.1034 Total effect −0.2744 0.1325 −0.5373 −0.0116 TABLE 6 | Mediating effects of sensation seeking by regression analysis. Mediating Effect of Sensation Seeking Mediating Effect of Sensation Seeking Factor analysis was conducted on the ImpSS scale of the livestreaming platform and was combined into one factor, KMO = 0.743, p < 0.000, it averaged the 19 items and got the mean-centered score of livestreaming addiction, M = 0.33, SD = 0.22. Following model 4 of the PROCESS Macro (Hayes, 2012), we performed a 5,000-resampling bootstrapping- moderated mediation analysis with state boredom as the independent variable (0 = boring condition, 1 = non-boring condition), sensation seeking as the mediator, consumer’s livestreaming addiction as the dependent variable, and demographics as the control variables. The result showed that impulsive sensation seeking played a complete mediating role and that state boredom promotes consumers’ livestreaming addiction, β = −0.1059, 95% CI (−0.2488, −0.0207) (refer to Table 5 for more information). Besides, we conducted a regression analysis according to the mediation analysis of Baron and Kenny (1986), and the results are shown in Table 6. Therefore, H2 was supported. Procedures and Measures Model 1 Model 2 Model 3 Variables Livestreaming Livestreaming Sensation addiction addiction seeking β p β p β p State boredom −0.205 0.041 −0.126 0.222 −0.327 0.001 Sensation- seeking 0.242 0.020 F 4.292 5.035 11.745 R2 0.042 0.094 0.107 1R2 0.032 0.075 0.098 TABLE 5 | Mediating effects of sensation seeking by bootstrapping analysis. Mediating effects of sensation seeking by bootstrapping analysis. Participants A number of 120 subjects were recruited from the sample database on the Credamo platform, which excluded people who participated in Studies 1 and 2. Among them, 38 are men (31.7%), 81 aged between 21 and 30 years (67.5%), 38 aged between 32 and 40 years (31.7%), and 1 aged between 41 and 50 years (0.8%). Using behavioral experiment in Study 2, we manipulated state boredom in different conditions and the result verified H1, that is, state boredom would increase consumers’ livestreaming addictive behaviors. H2 was proved, that is, consumers’ sensation seeking motivation played a mediating role in the relationship between state boredom and consumers’ livestreaming addiction. Study 3 Study 3 aimed to examine the moderating effect of life meaning on the mediating effect of sensation seeking on the relationship of state boredom and consumers’ livestreaming addiction. In Study 3, measurement of life meaning was added, and a new and simple sensation seeking scale was used. In Study 2, the ImpSS scale included seven items for impulsivity, which were not the key variable we cared about. In view of our interest in distinguishing between impulsivity and sensation seeking, we changed to use only the six Zuckerman items that clearly index thrill or novelty seeking (Steinberg et al., 2008). Frontiers in Psychology | www.frontiersin.org Effect of State Boredom on Livestreaming Addiction Factor analysis was conducted on the livestreaming addiction scale and was combined into one factor, KMO = 0.883, p < 0.000, and it averaged the 18 items and got the mean-centered score of livestreaming addiction, M = 2.92, SD = 0.67. Using SPSS 26.0, ANOVA was conducted to test the main effect of consumers’ state boredom on their livestreaming addiction. Results showed a significant difference in livestreaming addiction between the two conditions [F(1,99) = 4.292, p = 0.041]. The addictive behavior of participants in the boring condition (M boring = 3.05, SD boring = 0.70) was significantly higher than that in the non- boring group (Mnotboring = 2.78, SDnotboring = 0.62). Thus, H1 was approved again. April 2022 | Volume 13 | Article 826121 Frontiers in Psychology | www.frontiersin.org 6 Boredom and Live Streaming Addiction Zhang and Li will state boredom be restricted to improve consumers’ livestreaming addictive behavior through sensation seeking will be discussed in that study. Seeking Following model 8 of the PROCESS Macro (Hayes, 2012), we performed a 5,000-resampling bootstrapping-moderated mediation analysis with state boredom as the independent variable, life meaning as the moderator, sensation seeking as the mediator, and live-streaming addiction as the dependent variable. The result showed a moderated mediation effect: life meaning perception moderated the mediation effect of sensation seeking between state boredom and livestreaming addiction [indirect effect = −0.0653, 95% CI = (−0.1329, −0.0229)]. In particular, when the life meaning was low, the indirect effect of state boredom on livestreaming addiction through sensation seeking was significantly positive [0.0315, 95% CI = (0.0038, 0.0802)]. In contrast, when the life meaning was high, the indirect effect of state boredom on livestreaming addiction through sensation seeking was significantly negative [−0.0797, 95% CI = (−0.1709, −0.0234)]. The finding of state boredom and sensation seeking has been proved in the previous research. For example, Deng et al. (2020) showed that boredom from limited activities during the COVID- 19 pandemic has positive effect on individuals’ sensation seeking expressions. Bench and Lench (2019) manipulated boredom in high and low conditions and found that boredom is a seeking state and boredom prompts the pursuit of novel (even negative) experiences. The boredom in both of the above studies is actually state boredom, and the results mean that state boredom promotes sensation seeking. The positive effects of state boredom and sensation seeking on individuals’ addiction is consistent with previous research. Wang et al. (2020) found that both proneness boredom and sensation seeking could promote smartphone addiction. However, they did not investigate the relationship between boredom and sensation seeking, in which sensation seeking was an independent variable, rather than a mediator of the relationship between boredom and addition. The results indicated that the effect of state boredom on consumers livestreaming addiction via sensation seeking existed in both high and low levels of life meaning perception, but in opposite directions. When life meaning perception was low, state boredom would increase livestreaming addiction This research argued that life meaning perception could moderate the mediating effect of sensation seeking on the positive effect of state boredom on livestreaming addiction. If ones’ life meaning perception is high, he could control the sensation seeking expression and then decrease the desire of livestreaming addiction. Description of Key Variables Factor analysis was conducted for state boredom, livestreaming addiction, sensation seeking, and life meaning. State boredom was proved to combine as one factor (KMO = 0.945, p < 0.000) and also for livestreaming addiction (KMO = 0.912, p < 0.000), sensation seeking (KMO = 0.847, p < 0.000), and life meaning perception (KMO = 0.879, p < 0.000). Factor analysis averaged the items and got the mean-centered score of the four key variables. Means, SDs, and correlation are shown in Table 7. Results A common method bias was conducted first, just like that in Study 1. An exploratory factor analysis using a maximum likelihood solution was conducted on all of the items of key variables in this study. Totally, eighteen factors emerged with eigenvalues larger than 1.00, which suggests that more than one factor underlies the data. Moreover, the first factor accounted for only 33.231% of the total variance, which suggests that common April 2022 | Volume 13 | Article 826121 7 Boredom and Live Streaming Addiction Zhang and Li method variance may not be a serious concern in this study (Eby and Dobbins, 1997). through sensation seeking; however, when the life meaning perception was high, state boredom would decrease livestreaming addiction through sensation seeking. Therefore, H1, H2, and H3 were approved. Frontiers in Psychology | www.frontiersin.org Main Effect of State Boredom on Livestreaming Addiction ff f g Using SPSS 26.0, we tested the main effect of consumers’ state boredom on their livestreaming addiction by ANOVA. The results showed a significant difference in the livestreaming addictive behavior between the two conditions, F(1,118) = 13.158, p < 0.000. The addiction of participants in the boring condition (M boring = 2.88, SD boring = 0.70) was significantly higher than that in the non-boring condition (Mnotboring = 2.42, SDnotboring = 0.70). Thus, H1 was approved again. Moderated Mediation Effect of Life Meaning and Sensation Seeking General Discussion This article investigated the relationship between state boredom and livestreaming addiction and the mechanisms of the moderated mediation of sensation seeking and life meaning perception. Three studies were conducted to test the main effect of state boredom on consumers’ live-streaming addiction, the mediating effect of consumers’ sensation seeking, and the moderated mediation effect of life meaning perception. The results indicated that state boredom, one common kind of boredom, could lead to livestreaming addiction, and when individuals are under state boredom, they are motived by sensation seeking to have addictive behaviors on the livestreaming platforms. When consumers possess high life meaning perception, the state boredom will reduce sensation seeking and further reduce the livestreaming addictive behavior. However, when the perception of life meaning is low, bored consumers can hardly control their own behavior, which will strengthen the promotion effect of sensation seeking on livestreaming addiction. Main Effect of State Boredom on Livestreaming Addiction multidimensional state boredom scale (MSBS). Health Qual. Life Outcomes 13:59. doi: 10.1186/s12955-015-0252-2 Andreassen, C. S., Torsheim, T., Brunborg, G. S., and Pallesen, S. (2012). Development of a facebook. addiction scale. Psychol. Rep 110, 501–517. doi: 10.2466/02.09.18.PR0.110.2.501-517 Barbalet, J. M. (1999). Boredom and social meaning. Br. J. Sociol. 50, 631–646. doi: 10.1111/j.1468-4446.1999.00631.x Seeking In the previous literature, meaning threat involves a strong self-regulatory process and helps individuals to change the state of boredom (Leary et al., 1986; Van Tilburg and Igou, 2011). For example, boredom increases people’s evaluation of inner group and also demeaning of external group, so as to establish their sense of meaning (Van Tilburg and Igou, 2011). Similarly, people sometimes get nostalgic and indulge in reveries to offset TABLE 7 | Means, SDs, and correlations of variables in Study 3. M SD Correlation 1 2 3 1. State boredom 2.57 1.04 2. Live-streaming addiction 2.65 0.73 0.574** 3. Sensation seeking 4.44 1.00 −0.069 0.316** 4. Life meaning 5.46 0.85 −0.391** 0.077 0.348** **p < 0.01. Frontiers in Psychology | www.frontiersin.org 8 TABLE 7 | Means, SDs, and correlations of variables in Study 3. April 2022 | Volume 13 | Article 826121 8 Boredom and Live Streaming Addiction Zhang and Li the lack of meaning caused by boredom (Van Tilburg et al., 2013). However, this research paid attention to life meaning perception, rather than the meaning threat and meaning seeking, to explain the mechanism of state boredom on livestreaming addiction. to analyze the livestreaming addiction behaviors: company- shared data of individuals’ real behaviors on the livestreaming platform or some instruments can be used to record individuals’ livestreaming behaviors at their leisure boredom time. Second, considering the limitation of data collected online, we adopted the situation recall method to deal with the priming of state boredom. Although manipulation was verified successfully, the method was relatively simple. In future, classical initiation methods of state boredom can be adopted and manipulated in the laboratory, which include repetitive action tasks (Markey et al., 2014), cognitive tasks (Van Tilburg and Igou, 2011), and video tasks (Merrifield and Danckert, 2014). Besides, we could provide some real livestreaming video materials to test the participants’ addiction intention and real watching time. The video materials could include different kinds of products, such as cultural product and food, and entertainment products (e.g., singing, dancing, and traveling). ETHICS STATEMENT Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Electronic written informed consent for participation was required for this study in accordance with the national legislation and the institutional requirements. Contributions and Implications This research had both theoretical contributions and practical implications. Theoretically speaking, first, this research extended the literature of boredom and consumer’s behaviors. Previous research focused on proneness boredom and its effects on consumer’s behaviors [e.g., Chou et al. (2018) and Wang et al. (2020)]. However, this research figured out that state boredom, which could be stimulated by environment and happen on everyone, could also influence consumers’ behaviors. We explored how state boredom leads to livestreaming addiction and thus further expands research on the mechanism of addictive behaviors in the field of consumer’s behavior. Second, this research supplied the existing literature about social media addiction. Social media addiction has been studied about Facebook addiction (Ryan et al., 2014), addiction to social network sites (Griffiths et al., 2014), Twitter addiction (Saaid et al., 2014), and microblogging dependence (Wang et al., 2015). We paid attention to the topic of livestreaming addiction, a new kind of social media addiction, and explored the mechanisms of it. FUNDING We acknowledge the financial supported from the Fundamental Research Funds for the Central Universities (2020RCW006) and National Natural Science Foundation of China (72102012 and 71832015). We acknowledge the financial supported from the Fundamental Research Funds for the Central Universities (2020RCW006) and National Natural Science Foundation of China (72102012 and 71832015). Limitation and Future Research There are two deficiencies in this research, which can be made up in future. First, individuals’ livestreaming addiction was measured through self-report questionnaire and individuals’ viewing data were lacking. Considering the reality that individuals tend to watch livestreaming at night after work, this real situation is difficult to be designed and present in the laboratory. In the future, two sources of data could be used multidimensional state boredom scale (MSBS). Health Qual. Life Outcomes 13:59. doi: 10.1186/s12955-015-0252-2 Andreassen, C. S., Torsheim, T., Brunborg, G. S., and Pallesen, S. (2012). Development of a facebook. addiction scale. Psychol. Rep 110, 501–517. doi: 10.2466/02.09.18.PR0.110.2.501-517 Barbalet, J. M. (1999). Boredom and social meaning. Br. J. Sociol. 50, 631–646. doi: 10.1111/j.1468-4446.1999.00631.x DATA AVAILABILITY STATEMENT The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. In practice, this research can help to guide consumers to correctly deal with the negative impact of state boredom, can reduce the consumption caused by livestreaming addiction, and can promote health livestreaming time consumption. Specifically, on the one hand, marketers or broadcasters can appropriately enhance the interest and attraction of the livestream room and thus increase individuals’ watching time and shopping amount in the livestreaming platform by stimulating consumers’ sensation seeking. On the other hand, for consumers, high perception of life meaning can be used to inhibit the promoting effect of state boredom on livestreaming addiction. Therefore, individuals could think about their life meaning when they are watching live streaming to help allocate their leisure time rationally and form healthy livestreaming consumption habits. AUTHOR CONTRIBUTIONS NZ developed the theoretical framework and worked on data analysis and manuscript writing. JL worked on literature review and manuscript writing. Both authors contributed to the article and approved the submitted version. REFERENCES H., and Tseng, C. H. (2013). Gratifications for using CMC technologies: a comparison among SNS, IM, and e-mail. Comput. Hum. Behav. 29, 226–234. Cho, E. J., and Kim, J. H. (2010). Preschoolers’ usage of internet-game in a family context and their level of internet-game addiction. Korean J. Hum. Ecol. 19, 967–980. doi: 10.5934/kjhe.2010.19.6.967 Chou, W. J., Chang, Y. P., and Yen, C. F. (2018). Boredom proneness and its correlation with Internet addiction and Internet activities in adolescents with attention-deficit/hyperactivity disorder. Kaohsiung J. Med. Sci. 34, 467–474. doi: 10.1016/j.kjms.2018.01.016 Kuss, D. J., and Lopez-Fernandez, O. (2016). Internet addiction and problematic internet use: a systematic review of clinical research. World J. Psychiatry 6:143. doi: 10.5498/wjp.v6.i1.143 Leary, M. R., Rogers, P. A., Canfield, R. W., and Coe, C. (1986). Boredom and inter- personal encounters: antecedents and social implications. J. Pers. Soc. Psychol. 51, 968–975. doi: 10.1037/0022-3514.51.5.968 Deng, S., Wang, W., Xie, P., Chao, Y., and Zhu, J. (2020). Perceived severity of COVID-19 and post-pandemic consumption willingness: the roles of boredom and sensation-seeking. Front. Psychol. 11:567784. doi: 10.3389/fpsyg.2020. 567784 Leung, L. (2007). Leisure boredom, sensation seeking, self-esteem, addiction symptoms and patterns of mobile phone use. Mediated Interpers. Commun. 1, 359–381. Donati, M. A., Beccari, C., and Primi, C. (2022). Boredom and problematic Facebook use in adolescents: what is the relationship considering trait or state boredom? Addict. Behav. 125:107132. doi: 10.1016/j.addbeh.2021.107132 Lim, J. S., Choe, M. J., Zhang, J., and Noh, G. Y. (2020). The role of wishful identification, emotional engagement, and parasocial relationships in repeated viewing of live-streaming games: a social cognitive theory perspective. Comput. Hum. Behav. 108:106327. doi: 10.1016/j.chb.2020.10 6327 Drescher, C. F., Baczwaski, B. J., Walters, A. B., Aiena, B. J., Schulenberg, S. E., and Johnson, L. R. (2012). Coping with an ecological disaster: the role of perceived meaning in life and self-efficacy following the Gulf oil spill. Ecopsychology 4, 56–63. doi: 10.1089/eco.2012.0009 Lim, S., Cha, S. Y., Park, C., Lee, I., and Kim, J. (2012). Getting closer and experiencing together: Antecedents and consequences of psychological distance in social media-enhanced real-time streaming video. Comput. Hum. Behav. 28, 1365–1378. doi: 10.1016/j.chb.2012.02.022 Eby, L. T., and Dobbins, G. H. (1997). Collectivistic orientation in teams: an individual and group-level analysis. J. Org. Behav. 18, 275–295. doi: 10.1002/ (sici)1099-1379(199705)18:3<275::aid-job796>3.0.co;2-c Fahlman, S. A., Mercer, K. B., Gaskovski, P., Eastwood, A. E., and Eastwood, J. D. (2009). Does a lack of life meaning cause boredom? REFERENCES Abramson, E. E., and Stinson, S. G. (1977). Boredom and eating in obese and non-obese individuals. Addict. Behav. 2, 181–185. doi: 10.1016/0306-4603(77) 90015-6 Alda, M., Minguez, J., Montero-Marin, J., Gili, M., Puebla-Guedea, M., Herrera- Mercadal, P., et al. (2015). Validation of the spanish version of the Frontiers in Psychology | www.frontiersin.org April 2022 | Volume 13 | Article 826121 9 Zhang and Li Boredom and Live Streaming Addiction Baron, R. M., and Kenny, D. A. (1986). The moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations. J. Pers. Soc. Psychol. 51, 1173–1182. doi: 10.1037/0022-3514.51. 6.1173 Criteria, Evidence, And Treatment, eds K. P. Rosenberg and L. Curtiss Feder (New York, NY: Elsevier), 119–141. ( ) Hamilton, W. A., Garretson, O., and Kerne, A. (2014). “Streaming on twitch: Fostering participatory communities of play within live mixed media,” in Proceedings of the SIGCHI Conference On Human Factors In Computing Systems, (Toronto, ON: ACM), 1315–1324. Fostering participatory communities of play within live mixed media,” in Proceedings of the SIGCHI Conference On Human Factors In Computing Baumeister, R. F. (1991). Meanings Of Life. New York, NY: Guilford Press. Proceedings of the SIGCHI Conference On Human Factors In Computing Systems, (Toronto, ON: ACM), 1315–1324. Bench, S. W., and Lench, H. C. (2019). Boredom as a seeking state: boredom prompts the pursuit of novel (even negative) experiences. Emotion 19, 242–254. doi: 10.1037/emo0000433 Harris, M. B. (2000). Correlates and characteristics of boredom proneness and boredom. J. Appl. Soc. Psychol. 30, 576–598. doi: 10.1111/j.1559-1816.2000. tb02497.x Biolcati, R., Mancini, G., and Trombini, E. (2018). Proneness to boredom and risk behaviors during adolescents’ free time. Psychol. Rep. 121, 303–323. doi: 10.1177/0033294117724447 Hayes, A. F. (2012). PROCESS: A Versatile Computational Tool for Observed Variable Mediation, Moderation, and Conditional Process Modeling. Available onine at: http://www.afhayes.com/public/process2012.pdf (accessed October 1, 2021). Brand, M., Wegmann, E., Stark, R., Muller, A., Wolfling, K., Robbins, T. W., et al. (2019). The interaction of Person-Affect-Cognition-Execution (IPACE) model for addictive behaviors: update, generalization to addictive behaviors beyond internet-use disorders, and specification of the process character of addictive behaviors. Neurosci. Biobehav. Rev. 104, 1–10. doi: 10.1016/j.neubiorev.2019.06. 032 Hill, A. B., and Perkins, R. E. (1985). Towards a model of boredom. Br. J. Psychol. 76, 235–240. doi: 10.1111/j.2044-8295.1985.tb01947.x Hilvert-Bruce, Z., Neill, J. T., and Sjöblom, M. (2018). Game mechanics on social live streaming websites. Comput. Hum. Behav. 84, 58–67. live streaming websites. Comput. Hum. Behav. 84, 58–67. REFERENCES Brassai, L., Piko, B. F., and Steger, M. F. (2011). Meaning in life: Is it a protective factor for adolescents’ psychological health? Int. J. Behav. Med. 18, 44–51. doi: 10.1007/s12529-010-9089-6 Horvath, C. W. (2004). Measuring television addiction. J. Broadcast. Electron. Media 48, 378–398. doi: 10.1207/s15506878jobem4803_3 Hu, M., Zhang, M., and Wang, Y. (2017). Why do audiences choose to keep watching on live video streaming platforms? An explanation of dual identification framework. Comput. Hum. Behav. 75, 594–606. doi: 10.1016/j. chb.2017.06.006 Brislin, R. W. (1970). Back-translation for cross-cultural research. J. Cross Cult. Psychol. 1, 185–216. doi: 10.1037/a0021453 Brissett, D., and Snow, R. P. (1993). Boredom: where the future isn’t. Symbolic Interact. 16, 237–256. doi: 10.1525/si.1993.16.3.237 Huang, S., Li, D., Zhang, W., Li, D., Zhong, H., and Huang, Z. (2010). The development of boredom proneness questionnaire for college students. Psychol. Dev. Educ. 26, 308–314. doi: 10.2147/PRBM.S31 5879 Bryant, J., and Zillmann, D. (1984). Using television to alleviate boredom and stress: selective exposure as a function of induced excitational states. J. Broadcasting Electronic Media 28, 1–20. doi: 10.1080/08838158409386511 Jeon, Y. A., Son, H., Chung, A. D., and Drumwright, M. E. (2019). Temporal certainty and skippable in-stream commercials: effects of ad length, timer, and skip-ad button on irritation and skipping behavior. J. Interact. Market. 47, 144–158. doi: 10.1016/j.intmar.2019.02.005 Chen, C. C., and Lin, Y. C. (2018). What drives live-stream usage intention? The perspectives of flow, entertainment, social interaction, and endorsement. Telematics Informatics 35, 293–303. doi: 10.1016/j.tele.2017.12.003 Chen, C. Y., and Chang, S. L. (2019). Moderating effects of information- oriented versus escapism-oriented motivations on the relationship between psychological well-being and problematic use of video game live-streaming services. J. Behav. Addict. 8, 564–573. doi: 10.1556/2006.8.2019.34 Jessi, L. S., Wagaman, J., and Handley, I. M. (2009). Keeping it dull or making it fun: Task variation as a function of promotion versus prevention focus. Motiv. Emot. 33, 150–160. Khang, H., Kim, J. K., and Kim, Y. (2013). Self-traits and motivations as antecedents of digital media flow and addiction: the internet, mobile phones, and video games. Comput. Hum. Behav. 29, 2416–2424. doi: 10.1016/j.chb.2013. 05.027 Cheng, S. S., Chang, S. L., and Chen, C. Y. (2019). “Problematic use of live video streaming services: Impact of personality traits, psychological factors, and motivations,” in Proceedings of the 2019 8th International Conference on Software and Computer Applications, Penang, Malaysia, 487–490. oftware and Computer Applications, Penang, Malaysia, 487–490. Ku, Y. C., Chu, T. REFERENCES doi: 10.1037/a0019243 Wilson, T. D., Reinhard, D. A., Westgate, E. C., Gilbert, D. T., Ellerbeck, N., Hahn, C., et al. (2014). Just think: the challenges of the disengaged mind. Science 345, 75–77. Zhang, Y., Mei, S., Li, L., Chai, J., Li, J., and Du, H. (2015). The relationship between impulsivity and internet addiction in Chinese college students: a moderated mediation analysis of meaning in life and self-esteem. PLoS One 10:e0131597. doi: 10.1371/journal.pone.0131597 Podsakoff, P. M., and Organ, D. W. (1986). Self-reports in organizational research: problems and prospects. J. Manag. 12, 531–544. doi: 10.1177/ 014920638601200408 Zuckerman, M. (1996). The psychobiological model for impulsive unsocialized sensation seeking: a comparative approach. Neuropsychobiology 34, 125–129. Quan-Haase, A., and Young, A. L. (2010). Uses and gratifications of social media: a comparison of facebook and instant messaging. Bull. Sci. Technol. Soc. 30, 350–361. doi: 10.1177/0270467610380009 Zuckerman, M., Kuhlman, D. M., and Joireman, J. (1993). A comparison of three structural models for personality: the big three, the big five, and the alternative five. J. Pers. Soc. Psychol. 65, 757–768. Reisenzein, R. (2017). Varieties of cognition-arousal theory. Emot. Rev. 9, 17–26. doi: 10.1177/1754073916639665 Robin, J. (1975). Causes and consequences of time perception differences in overweight and normal weight people. J. Pers. Soc. Psychol. 31, 898–904. doi: 10.1037/h0076866 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ryan, T., Chester, A., Reece, J., and Xenos, S. (2014). The uses and abuses of Facebook: a review of Facebook addiction. J. Behav. Addict. 3, 133–148. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Saaid, S. A., Al-Rashid, N. A. A., and Abdullah, Z. (2014). “The impact of addiction to Twitter among university students,” in Future Information Technology, eds F. Xhafa, I. Stojmenovic, J. J. Park, and M. Cho (Berlin: Springer), 231–236. Sansone, C., Weir, C., Harpster, L., and Morgan, C. (1992). Once a boring task always a boring task? Interest as a self-regulatory strategy. J. Pers. Soc. Psychol. 63, 379–390. REFERENCES Results from psychometric, longitudinal, and experimental analyses. J. Soc. Clin. Psychol. 28, 307–340. doi: 10.1521/jscp.2009.28.3.307 Lin, C.-H., Lin, S.-L., and Wu, C.-P. (2009). The effects of parental monitoring and leisure boredom on adolescents’ Internet addiction. Adolescence 44, 993–1004. Loukidou, L., Loan-Clarke, J., and Daniels, K. (2009). Boredom in the workplace: more than monotonous tasks. Int. J. Manag. Rev. 11, 381–405. doi: 10.1111/j. 1468-2370.2009.00267.x Fahlman, S. A., Mercer-Lynn, K. B., Flora, D. B., and Eastwood, J. D. (2011). Development and validation of the multidimensional state boredom scale. Assessment 20, 68–85. doi: 10.1177/1073191111421303 Mann, S., and Robinson, A. (2009). Boredom in the lecture theatre: an investigation into the contributors, moderators and outcomes of boredom amongst university students. Br. Educ. Res. J. 35, 243–258. doi: 10.1080/ 01411920802042911 Griffiths, M. D., Kuss, D. J., and Demetrovics, Z. (2014). “Social networking addiction: an overview of preliminary findings,” in Behavioral Addictions. April 2022 | Volume 13 | Article 826121 Frontiers in Psychology | www.frontiersin.org 10 Zhang and Li Boredom and Live Streaming Addiction Markey, A., Chin, A., Vanepps, E. M., and Loewenstein, G. (2014). Identifying a reliable boredom induction. Percept. Motor Skills 119, 237–253. doi: 10.2466/ 27.PMS.119c18z6 Steinberg, L., Albert, D., Cauffman, E., Banich, M., Graham, S., and Woolard, E. (2008). Age differences in sensation seeking and impulsivity as indexed by behavior and self-report: evidence for a dual systems model. Dev. Psychol. 44, 1764–1778. Martela, F., and Steger, M. F. (2016). The three meanings of meaning in life: distinguishing coherence, purpose, and significance. J. Positive Psychol. 11, 531–545. doi: 10.1080/17439760.2015.1137623 Stickney, M. L., and Miltenberger, R. G. (1999). Evaluating direct and indirect measures for the functional assessment of binge eating. Int. J. Eating Disord. 26, 195–204. Mercer, K. B., and Eastwood, J. D. (2010). Is boredom associated with problem gambling behaviour? It depends on what you mean by ‘boredom’. Int. Gambling Stud. 10, 91–104. doi: 10.1080/14459791003754414 Van Tilburg, W. A. P., and Igou, E. R. (2011). On boredom and social identity: a pragmatic meaning-regulation approach. Pers. Soc. Psychol. Bull. 37, 1679– 1691. Merrifield, C., and Danckert, J. (2014). Characterizing the psychophysiological signature of boredom. Exp. Brain Res. 232, 481–491. doi: 10.1007/s00221-013- 3755-2 Van Tilburg, W. A. P., and Igou, E. R. (2012). On boredom: lack of challenge and meaning as distinct boredom experiences. Motiv. Emot. 36, 181–194. Van Tilburg, W. A. P., Igou, E. R., and Sedikides, C. (2013). REFERENCES In search of meaningfulness: using nostalgia as an antidote to boredom. Emotion 13, 450– 461. Miao, C., Chen, X., Liu, T., Yang, H., and Hall, B. J. (2020). Psychological distress and state boredom during the COVID-19 outbreak in China: the role of meaning in life and media use. Eur. J. Psychotraumatol. 11:1769379. doi: 10. 1080/20008198.2020.1769379 Van Tilburg, W. A. P., Igou, E. R., Maher, P. J., Moynihan, A. B., and Martin, D. (2018). Bored like hell: Religiosity prevents boredom and tempers the quest for meaning. Emotion 19:255. Moynihan, A. B., Igou, E. R., and Van Tilburg, W. A. P. (2017). Boredom increases impulsiveness: A meaning-regulation perspective. Soc. Psychol. 48, 293–309. doi: 10.1027/1864-9335/a000317 g Wang, C., Lee, M. K., and Hua, Z. (2015). A theory of social media dependence: evidence from microblog users. Decis. Support Syst. 69, 40–49. Moynihan, A. B., Van Tilburg, W. A. P., Igou, E. R., Wisman, A., Donnelly, A. E., and Mulcaire, J. B. (2015). Eaten up by boredom: consuming food to escape awareness of the bored self. Front. Psychol. 6:369. doi: 10.3389/fpsyg.2015.00369 Wang, Z., Yang, X., and Zhang, X. (2020). Relationships among boredom proneness, sensation seeking and smartphone addiction among Chinese college students: mediating roles of pastime, flow experience and self-regulation. Technol. Soc. 62:101319. Müller, K. W., Dreier, M., Beutel, M. E., and Wölfling, K. (2016). Is Sensation Seeking a correlate of excessive behaviors and behavioral addictions? A detailed examination of patients with Gambling disorder and internet addiction. Psychiatry Res. 242, 319–325. doi: 10.1016/j.psychres.2016.06.004 Watson, D., Clark, L. A., and Tellegen, A. (1988). Development and validation of brief measures of positive and negative affect: the PANAS scales. J. Pers. Soc. Psychol. 54, 1063–1070. Musharbash, Y. (2007). Boredom, time, and modernity: an example from aboriginal Australia. Am. Anthropol. 109, 307–317. doi: 10.1525/aa.2007.109. 2.307 Whelan, E., Najmul Islam, A. K. M., and Brooks, S. (2020). Is boredom proneness related to social media overload and fatigue? A stress–strain– outcome approach. Internet Res. 30, 869–887. Nett, U. E., Goetz, T., and Hall, N. C. (2011). Coping with boredom in school: an experience sampling perspective. Contem. Educ. Psychol. 36, 49–59. doi: 10.1016/j.cedpsych.2010.10.003 Wilson, G. D. (1986). Eating style, obesity, and health. Pers. Individ. Dif. 7, 215–224. Pekrun, R., Goetz, T., Daniels, L. M., Stupnisky, R. H., and Perry, R. P. (2010). Boredom in achievement settings: exploring control–value antecedents and performance outcomes of a neglected emotion. J. Educ. Psychol. 102:531. REFERENCES Singh, S., Singh, N., Kalini´c, Z., and Liébana-Cabanillas, F. J. (2021). Assessing determinants influencing continued use of live streaming services: an extended perceived value theory of streaming addiction. Expert Syst. Appl. 168:114241. Copyright © 2022 Zhang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Steger, M. F., Frazier, P., Oishi, S., and Kaler, M. (2006). The meaning in life questionnaire: assessing the presence of and search for meaning in life. J. Counseling Psychol. 53:80. April 2022 | Volume 13 | Article 826121 Frontiers in Psychology | www.frontiersin.org 11
https://openalex.org/W1831989259
https://bmcmusculoskeletdisord.biomedcentral.com/track/pdf/10.1186/s12891-015-0775-z
English
null
The mechanism of action for hyaluronic acid treatment in the osteoarthritic knee: a systematic review
BMC musculoskeletal disorders
2,015
cc-by
9,611
* Correspondence: journals@royaltman.com 1Division of Rheumatology and Immunology, David Geffen School of Medicine, University of California at Los Angeles, 1000 Veterans Ave, 90024 Los Angeles, CA, USA Full list of author information is available at the end of the article © 2015 Altman et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 DOI 10.1186/s12891-015-0775-z Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 DOI 10.1186/s12891-015-0775-z Open Access Abstract Background: Knee osteoarthritis (OA) is one of the leading causes of disability within the adult population. Current treatment options for OA of the knee include intra-articular (IA) hyaluronic acid (HA), a molecule found intrinsically within the knee joint that provides viscoelastic properties to the synovial fluid. A variety of mechanisms in which HA is thought to combat knee OA are reported in the current basic literature. Methods: We conducted a comprehensive literature search to identify currently available primary non-clinical basic science articles focussing on the mechanism of action of IA-HA treatment. Included articles were assessed and categorized based on the mechanism of action described within them. The key findings and conclusions from each included article were obtained and analyzed in aggregate with studies of the same categorical assignment. Results: Chondroprotection was the most frequent mechanism reported within the included articles, followed by proteoglycan and glycosaminoglycan synthesis, anti-inflammatory, mechanical, subchondral, and analgesic actions. HA-cluster of differentiation 44 (CD44) receptor binding was the most frequently reported biological cause of the mechanisms presented. High molecular weight HA was seen to be superior to lower molecular weight HA products. HA derived through a biological fermentation process is also described as having favorable safety outcomes over avian-derived HA products. Conclusions: The non-clinical basic science literature provides evidence for numerous mechanisms in which HA acts on joint structures and function. These actions provide support for the purported clinical benefit of IA-HA in OA of the knee. Future research should not only focus on the pain relief provided by IA-HA treatment, but the disease modification properties that this treatment modality possesses as well. words: Osteoarthritis, Hyaluronic acid, Therapy, Intra-articular therapy, Systematic review, Mechanis The mechanism of action for hyaluronic acid treatment in the osteoarthritic knee: a systematic review RD Altman1*, A. Manjoo2, A. Fierlinger3, F. Niazi3 and M. Nicholls4 Background 1 or 2 or 3 or 4 6. Osteoarthrit*.mp 6. Osteoarthrit* 7. Arthrit*.mp 7. Arthrit* 8. Joint pain.mp 8. Joint pain 9. 6 or 7 or 8 9. 6 or 7 or 8 10. Effect*.mp 10. Effect* 11. Mechanism*.mp 11. Mechanism* 12. 10 or 11 12. 10 or 11 13. 5 and 9 and 12 13. 5 and 9 and 12 *denotes the use of the truncation function within the database diabetic patients [8]. IA injection of hyaluronic acid (HA) is another treatment option for knee OA pain. HA is nearly ubiquitous in the body, and is a molecule found in- trinsically within the knee joint where it provides visco- elastic properties to synovial fluid [9]. As OA progresses, natural HA concentration and the distribution of HA within the joint shifts towards lower ranges of HA mo- lecular weight, leading to a degradation of the mechan- ical/viscoelastic properties of the endogenous synovial fluid [2, 10, 11]. Lower ranges of molecular weight distri- butions have also been shown to be strongly correlated to pain [11]. IA-HA administration has aimed to restore this decline in HA concentration and the average molecular weight distribution within the OA knee [9]. g IA-HA has been proposed to have many therapeutic mechanisms of action in the OA knee, including shock absorption, joint lubrication, anti-inflammatory effects, chondroprotection, proteoglycan synthesis, and cartilage matrix alterations [2]. The correlation between these various effects has created a better understanding of how IA-HA treatment could provide therapeutic effects for patients with knee OA [12]. There is also evidence suggesting distinct mechanism of action differences be- tween HA products of varying molecular weight. That is, higher molecular weight (HMW) HA has been re- ported to provide greater anti-inflammatory and pro- teoglycan synthesis effects, as well as joint lubrication and viscoelasticity maintenance [9, 13]. There also ap- pears to be evidence of variable safety profiles be- tween HA derived through a biological fermentation process (Bio-HA) and avian-derived HA (AD-HA), as AD-HA has the potential for local IA reactions [14, 15]. *denotes the use of the truncation function within the database by the same author, only the most recent study was accepted for inclusion. by the same author, only the most recent study was accepted for inclusion. Literature search We conducted a comprehensive literature search using the MEDLINE, EMBASE, and PubMed databases (Table 1). The search was conducted on May 4th, 2014. The inclusion criteria followed throughout the screening process were as follows: 1) Articles describing the mech- anism of HA treatment for OA, 2) Articles focused on OA of the knee, and 3) Primary non-clinical basic science articles focussing on “viscosupplementation”/HA treat- ment. Articles that were published before 1990 or were not published in English were excluded from the study. If multiple studies outlining similar results were published Data abstraction Included articles were assessed and categorized based on the mechanism of action described within them. Single articles were included in multiple categories if they ana- lyzed more than one mechanism of HA action within their study design. The mechanism categories utilized in the data abstraction process were: chondroprotection, al- terations in proteoglycan and/or glycosaminoglycan (GAG) synthesis, anti-inflammatory effects, mechanical modifications, alterations in subchondral bone, and analgeisic effects. We aim to summarize the mechanisms of action for IA-HA treatment of knee OA described in the current literature in order to determine the validity of the above mechanisms of action. We will systematic- ally assess and outline the defined mechanisms in which HA may provide a therapeutic benefit, while analyzing re- ported distinction between product characteristic- dependent effects of IA-HA treatment. Data analysis Th k fi d The key findings and conclusions from each included article were obtained and analyzed in aggregate with studies of the same assigned category. The results pre- sented have been derived through interpretation of com- mon and consistent mechanism of action presentations within each aforementioned category. Background disability often leads to impaired work performance and early retirement [4]. Although osteoarthritis (OA) of the knee is most often a slowly progressive joint disorder, it is one of the leading causes of disability of the adult population [1]. Knee OA, a disease of the entire joint, is characterized by joint pain, cartilage degeneration, and an increase in dis- ability [2]. The progressive nature of OA leads to de- creased knee function, affecting an individual’s ability to perform daily activities [3]. Knee OA also nega- tively impacts socioeconomic factors, as the associated Since there is no established disease modifying agent for OA, there are many options for the treatment of knee OA. Among the pharmacologic therapies, non-steroidal anti-inflammatory drugs (NSAIDs) and intra-articular (IA) corticosteroid injections are most commonly pre- scribed [5]. These options have inherent limitations, as NSAIDs have potentially serious adverse events associated with their use [6], and IA corticosteroid injections often provide a relatively short period of effective relief [7]. Al- though corticosteroid injection generally has a positive safety profile, it has been shown to cause a transient in- crease in blood glucose, which may be a concern for * Correspondence: journals@royaltman.com 1Division of Rheumatology and Immunology, David Geffen School of Medicine, University of California at Los Angeles, 1000 Veterans Ave, 90024 Los Angeles, CA, USA Full list of author information is available at the end of the article Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 Page 2 of 10 Table 1 Search strategy MEDLINE and EMBASE – 1470 articles PubMed – 1395 articles 1. Hyaluronic acid[title] 1. Hyaluronic acid[title] 2. Hylan[title] 2. Hylan[title] 3. Hyaluronan[title] 3. Hyaluronan[title] 4. Viscosupplementation[title] 4. Viscosupplementation[title] 5. 1 or 2 or 3 or 4 5. 1 or 2 or 3 or 4 6. Osteoarthrit*.mp 6. Osteoarthrit* 7. Arthrit*.mp 7. Arthrit* 8. Joint pain.mp 8. Joint pain 9. 6 or 7 or 8 9. 6 or 7 or 8 10. Effect*.mp 10. Effect* 11. Mechanism*.mp 11. Mechanism* 12. 10 or 11 12. 10 or 11 13. 5 and 9 and 12 13. 5 and 9 and 12 *denotes the use of the truncation function within the database Table 1 Search strategy MEDLINE and EMBASE – 1470 articles PubMed – 1395 articles 1. Hyaluronic acid[title] 1. Hyaluronic acid[title] 2. Hylan[title] 2. Hylan[title] 3. Hyaluronan[title] 3. Hyaluronan[title] 4. Viscosupplementation[title] 4. Viscosupplementation[title] 5. 1 or 2 or 3 or 4 5. Search strategy The literature search identified 2,782 potential articles and, of these, 91 articles met the inclusion criteria (Fig. 1). Thirteen additional articles were retrieved by a content expert’s hand search of the literature. This re- sulted in a total of 104 articles included. The conclusions and key findings of each article were identified within the aforementioned mechanism categories (Table 2). Page 3 of 10 Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 Page 3 of 10 Fig. 1 Article screening process receptors. HA binding to CD44 inhibits interleukin (IL)- 1β expression, leading to a decline in matrix metallopro- teinase (MMP) -1, 2, 3, 9, and 13 productions [32, 34]. This binding to CD44 has been shown to be of greater ef- fect for higher MW HA products [21]. HA also binds to the receptor for hyaluronan mediated motility (RHAMM), which is thought to aid in chondroprotection in addition to CD44 binding [34]. The inhibition of IL-1β expression through CD44 binding is carried out through induction of mitogen-activated protein kinase phosphatase (MKP) -1: a negative regulator of IL-1β [43]. This inhibition of various MMPs impedes catabolic enzyme activity within the joint cartilage [59]. HMW HA was shown to have a greater ef- fect in the inhibitory action of MMP production [21], al- though these results are unclear, as another study has demonstrated the favourable MMP inhibitory effect of lower molecular weight (LMW) products [72]. Chondrocyte apoptotic events are further decreased by HA-CD44 binding through the reduction of a disinte- grin and metalloproteinase with thrombospondin mo- tifs (ADAMTS) expression [38]. These peptidases are involved in the cleavage of important synovial com- ponents, including aggrecan, versican, and brevican [81, 82]. Various ADAMTS expression has been shown to decrease as a result of HA-CD44 binding, providing an additional mode of chondroprotection for IA-HA treatment [36, 38, 43]. The production of reactive oxygen species (ROS), such as nitric oxide (NO), results in degeneration of cartilage through in- creased chondrocyte apoptosis [33]. IA-HA treatment demonstrated a reduction in IL-1β-induced oxidative stress, through inhibition of NO production within the synovium [48, 57]. Additional results of CD44- HA binding resulting in chondroprotective effects men- tioned in the current literature include reduction of pros- taglandin E2 (PGE2) synthesis [42, 46, 61], and increased heat shock protein 70 (Hsp70) overexpression [25, 70]. These effects similarly provide therapeutic benefit through reduction of chondrocyte apoptosis. Search strategy It is important to note that HMW HA products demonstrated greater in- hibition of PGE2 expression than LMW comparators in a comparative study, resulting in greater chondroprotective effects [61]. Proteoglycan and glycosaminoglycan synthesis demonstrated to suppress numerous inflammatory me- diators through TLR 2 and 4 binding, including TNF-α, IL-1-β, IL-17, MMP-13 and inducible nitrogen oxide synthase (iNOS) [106]. A direct correlation between mo- lecular weight and anti-inflammatory effects has demon- strated larger effects of PGE2 and IL-6 inhibition for HMW HA treatment [13]. IL-6 is a pro-inflammatory cytokine, regulated by nuclear factor kappa-light-chain- enhancer of activated B cells (Nf-кB). HA binding to ICAM-1 down regulates Nf-kB, which in turn decreases the production of IL-6 [21, 104]. HMW HA down regula- tion of TNFα, IL-1B and IL-8 is an additional contributory factor to the anti-inflammatory effects provided by HMW HA [72, 97]. Proteoglycan and glycosaminoglycan synthesis Twenty-two of the identified studies reported on the en- hanced proteoglycan and glycosaminoglycan synthesis re- lated to IA-HA treatment [27, 42, 44, 48, 62, 69, 83–98]. As OA progresses, intrinsic proteoglycan and GAG con- centrations decline within the cartilage. Results demon- strated that IA-HA treatment stimulated proteoglycan synthesis, delaying the progression of OA [69, 88]. Aggre- can is the primary proteoglycan within articular cartilage, and IA-HA treatment is shown to both suppress aggrecan degradation, as well as promote intrinsic aggrecan devel- opment [62, 93]. IA-HA treatment is shown to mobilize newly synthesized proteoglycan to the outer chondrocyte matrix, potentially providing protection from degradation. Extrinsic HA promoted movement of newly synthesized proteoglycan from the cell-associated matrix to the further-removed matrix in an alginate gel model, which suggests that IA-HA could provide therapeutic relief of OA by strengthening the interterritorial cartilage matrix [92]. A marker of proteoglycan synthesis, sulphate (35SO4 ), is seen to be increasingly incorporated within chondro- cytes after HA introduction [87]. The biological pathway in which HA alters aggrecan levels is shown to be through CD44 and intercellular adhesion molecule (ICAM)-1 binding effects [62]. HMW HA was shown by one study to provide a greater effect of proteoglycan synthesis than LMW HA through stimulation of the insulin-like growth factor (IGF)-1 pathway [89]. IA-HA treatment is also shown to increase endogenous GAG production. IA-HA treatment not only supplemented the synovium with HA, it promoted intrinsic production of HA [27]. Mechanical Ten of the included studies described mechanical effects of HA in the treatment of knee OA [41, 49, 86, 108–114]. The viscous nature of HA treatment is shown to lubricate the joint capsule, preventing degeneration through de- creased friction [86]. HA further protects the joint capsule through beneficial shock absorption effects. HA provides cushioning to absorb pressure and vibration within the joint that otherwise may lead to chondrocyte degradation [41]. Osteoarthritic knees are reported to have higher fric- tion within the joint space than healthy knees, which is counteracted by the joint lubrication capabilities that HA possesses [110]. HMW HA has been demonstrated to pro- vide a greater effect of friction reduction due to its viscous properties. The reduction of friction within the joint can provide therapeutic effects, as the cartilage is protected from mechanical degradation [111]. Chondroprotection Sixty-seven of the included articles described chondro- protective effects of IA-HA treatment [9, 12, 16–80]. IA- HA has been shown to reduce chondrocyte apoptosis, while increasing chondrocyte proliferation [19, 20]. There are multiple observed effects of IA-HA treatment that produce chondroprotection, many of which are results of HA binding to cluster of differentiation 44 (CD44) Table 2 Key mechanisms of action reported by the included articles Mechanism of action Number of articles % of included articles References Chondroprotective 67 64.42 % [9, 12, 16–80] Proteoglycan/Glycosaminoglycan synthesis 22 21.15 % [27, 42, 44, 48, 62, 69, 83–98] Anti-inflammatory 21 20.19 % [13, 20, 21, 25, 27, 52, 54, 55, 68, 72, 96, 97, 99–107] Mechanical 10 9.61 % [41, 49, 86, 108–114] Subchondral bone 8 7.69 % [23, 31, 32, 45, 115–118] Analgesic 6 5.76 % [76, 80, 119–122] Table 2 Key mechanisms of action reported by the included articles Page 4 of 10 Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 Subchondral bone E h f h l Twenty-one of the identified studies reported on the anti-inflammatory effects of IA-HA treatment [13, 20, 21, 25, 27, 52, 54, 55, 68, 72, 96, 97, 99–107]. IL-1β is known to demonstrate pro-inflammatory effects, and the aforementioned suppression of IL-1β expression by HA provides anti-inflammatory effects [52]. IL-1β is a key mediator in the anti-inflammatory effects of HA, and is regulated through HA-CD44 binding [59, 104]. IL-1β suppression results in a down-regulation of MMPs as previously mentioned, which also aids in the anti- inflammatory effect of HA [52]. Further suppression of pro-inflammatory mediators IL-8, IL-6, PGE2, and tumor necrosis factor (TNFα) provides anti-inflammatory ef- fects of IA-HA treatment [13, 21]. The relation between Toll-Like Receptors (TLR) and an inflammatory re- sponse is demonstrated as HA degradation products in- duced an inflammatory response through CD44 and TLR interaction. This pro-inflammatory response from HA degradation product binding to TLR and CD44 re- ceptors results in increased Nf-kB, IL-1β, TNFα, IL-6, and IL-33 production [105]. HMW HA has been Eight of the included studies outlined the effects that IA-HA has on the subchondral bone [23, 31, 32, 45, 115–118]. It previously has been shown that interaction between subchondral bone osteoblasts and articular car- tilage chondrocytes in osteoarthritic joints alters ADAMTS-4/5 and MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13 expression and regulation, medi- ated by mitogen-activated protein kinase (MAPK) and extracellular signal regulated kinase 1 and 2 (ERK 1/2) signalling pathways [118]. IA-HA also affects the subchondral bone through suppression of MMP-13 and IL-6 via CD44 binding, which potentially prevents abnormal osseous tissue metabolism [116]. The suppres- sion of MMP-13 expression by IA-HA has been sug- gested to be a critical factor in the effect on OA subchondral bone [117]. The effect that IA-HA has on MMPs, specifically MMP-13, through CD44 binding has been suggested to inhibit the effects of OA within the subchondral bone in numerous non-clinical basic sci- ence investigations [32, 45, 116–118]. HA effectively Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 Page 5 of 10 normalization of HA distribution within the synovial fluid [124]. A large number of reports describe CD44 binding as a primary mode in which HA provides action against knee OA in non-clinical basic science studies. CD44-mediated effects of IA-HA are shown to contrib- ute to the potential chondroprotection, proteoglycan/ glycosaminoglycan synthesis, anti-inflammatory, and subchondral mechanisms. Subchondral bone E h f h l This binding is shown to have a variety of effects on numerous signalling pathways, all of which demonstrate some sort of intervention in the progression of OA [13, 21, 32, 36, 38, 42, 43, 46, 59, 61, 104, 116]. The suppression of IL-1β and IL-6 and subse- quent effects of this suppression have been suggested to be a key factor in the therapeutic mechanism provided by HA-CD44 binding [123]. It is evident that HA-CD44 binding is a major component in the mechanism in which HA provides therapeutic effect; however, there are additional mechanisms that provide alternate pathways for the effectiveness of HA treatment in OA knees, in- cluding ICAM binding, mechanical improvements at- tributed to shock absorption and lubrication, an increase in cartilage/bone interface type II collagen turnover, as well as analgesic effects through interaction with nerve endings and joint nociceptors [31, 41, 86, 97, 121, 122]. HA binding to the RHAMM receptor promotes wound repair, activates pro-migration and invasion functions, regulates cellular responses to growth factors, and plays a role in fibroblast migration and motility [125–127]. These results of HA-RHAMM binding are potential fac- tors involved in the disease modification effects of HA treatment for OA. changes subchondral bone density and thickness through trabecular structure alterations, resulting in greater sub- chondral bone compliancy. This ultimately is shown to re- duce the stress put on cartilage during impact loading [23]. An indication of IA-HA stimulation of cartilage/bone interface type II collagen turnover is the increase of urine carboxy-terminal collagen crosslinks (CTX)-II levels ob- served following IA-HA treatment, which demonstrated improvement in the osteoarthritic knee [31]. Ultimately, HA appeared to limit subchondral bone changes that are characteristic of early OA [115]. Analgesic Six of the included articles described the analgesic ef- fects of IA-HA treatment [76, 80, 119–122]. A single in- jection of HA demonstrated a significant decrease in pain-associated behaviour within a murine model [119]. One study suggested that HA did not directly bind to bradykinin receptors, but provided analgesic effects through interaction with HA receptors and/or free nerve endings within the joint tissue [121]. HA analgesic ef- fects have been shown to occur at mechanosensitive stretch-activated ion channels, where channel activity is significantly decreased upon HA binding [122]. HMW HA was shown to decrease mechanical sensitivity of stretch-activated ion channels, which effectively blocked the pain response. LMW HA was seen to be less effective in blocking this response [122]. HA reduces the action of joint nociceptors, which provides pain reduction within the joint. Sensitized nociceptive terminals within the joint tissue are affected by HA concentration, reducing the pain response exhibited by these terminals [120]. There is evidence which demonstrates that certain in- trinsic properties of particular IA-HA products may pro- vide beneficial results in comparison to other IA-HA products. The most recognized of these intrinsic proper- ties is molecular weight. Contrary to a previous basic science review by Ghosh et al., which suggested a poten- tial benefit of LMW HA in providing rheological prop- erty restoration over HMW HAs [128], the evidence within the current review has demonstrated advanta- geous results for HMW HA treatments. The current re- view supports the view that HMW HA provides superior chondroprotective, proteoglycan and glycosami- noglycan synthesis, anti-inflammatory, mechanical, and analgesic mechanisms of action [61, 89, 97, 111, 122]. A study by Huang et al. demonstrated superior anti- inflammatory effects of HMW HA but superior chon- droprotective effects of LMW HA; however, these results regarding chondroprotection are unclear due to lack of additional evidence within the knee OA basic science lit- erature [72]. An increased production of inflammatory cytokines and chemokines, recruitment of inflammatory mediators, and blood vessel formation have been shown to be a response to LMW HA below 500 kDa. While the Discussion Our review of the existing literature provides a general consensus that IA-HA for knee OA has beneficial effects through several mechanisms of action; however, the pre- dominant mechanism in which therapeutic effect is pro- vided is not clearly understood [2]. In perspective, it is not clear which of these mechanisms are clinically rele- vant, as it is appreciated that beneficial mechanisms of action are not necessarily transferrable to benefit in the clinical setting. At this time, it is presumed that the clin- ical benefit of IA-HA in knee OA is due to several con- current mechanisms of action, instead of any one single specific mechanism of action. The majority of exogenous HA remains in the joint for a few days; however, the clinical therapeutic effects of HA treatment may be seen for up to 6 months, or more. This may suggest that IA-HA contains disease modifying properties, and does not act solely by restor- ing viscoelastic properties to the synovial fluid [123]. HA injections may stimulate endogenous production of add- itional HA by human synoviocytes, aiding in the Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 Page 6 of 10 average MW of available HA products on the market vary greatly, it should be noted that, to our knowledge, all currently available products worldwide have a mo- lecular weight >500 kDa [129, 130]. An analysis of HA- CD44 interaction demonstrated that HA size has direct impact on the affinity in which HA binds to the CD44 receptor [131]. These results demonstrate the capacity of HMW HA to treat the progression of knee OA through CD44 binding of HA. These basic science find- ings are consistent with systematic reviews of clinical tri- als and comparative studies which have demonstrated that HMW HA provides greater therapeutic benefit than LMW HA in the treatment of knee OA [6, 132], al- though the current literature does not provide consensus regarding the clinical efficacy difference between low and high molecular weight HA [133]. but were not classified into the corresponding category because the mentioned alternate mechanism was not a key result or conclusion of the study. Future research should analyze the relationship between the various mech- anisms presented in this report, and clarify the way in which these mechanisms overlap and may work together to alleviate symptoms of knee OA. Conclusions The non-clinical basic science literature provides evi- dence for numerous mechanisms in which IA-HA may provide clinical benefit in knee OA. Chondroprotection is the most frequently reported mechanism, with HA- CD44 binding being the most frequently reported source of these effects. IA-HA is also reported to provide proteoglycan and glycosaminoglycan synthesis, anti- inflammatory, mechanical, subchondral, and analgesic effects. There is evidence of favorable results for HMW HA treatments in comparison to LMW HA. Bio HA is also demonstrated to provide an advantageous safety profile over AD-HA, as reports demonstrate the associ- ation between injection site flare ups and avian-derived proteins. There are a variety of reported mechanisms in which IA-HA is demonstrated to treat knee OA, as well as numerous product characteristics that impact the re- sults of IA-HA treatment. A thorough understanding of the variety of mechanisms in which IA-HA provides beneficial effects within the OA knee, as well as the characteristic-specific effects of various IA-HA products, is required to recognize the applicability and appropri- ateness of IA-HA treatment for knee OA. Future re- search should not only focus on the pain relief provided by IA-HA treatment, but the disease modification prop- erties that this treatment modality may possess as well. Traditionally, HA products had been derived from avian sources; however, some available products are pro- duced by biological fermentation. This process avoids the presence of avian-derived molecules which are sug- gested to be a potential cause of adverse local reactions [134]. There is a lack of thorough reporting regarding the potential of Bio-HA over AD-HA. One study has suggested AD-HA injection sites may be the cause of synovitis in their patient group, yet the exact patho- logical agent is unknown [135]. Results from a second study also outline the potential for hylan AD-HA to cause a foreign body giant cell type granulomatous reac- tion [136]. Research has demonstrated that the flare-ups associated with hylan injection may be correlated to the accumulation of hylan or its breakdown products, as in- jection site flare ups typically do not occur upon first in- jection [14]. Avian derived proteins have been shown to be the cause of injection site flare up, as antibodies to chicken serum protein were present in patients who demonstrated injection site adverse reaction after being treated with AD-HA [15]. Abbreviations ADAMTS: A disintegrin and metalloproteinase with thrombospondin motifs; AD-HA: Avian-derived hyaluronic acid; Bio-HA: Biologically-derived hyaluronic acid; CD: Cluster of differentiation; CTX: Carboxy-terminal collagen crosslinks; ERK 1/2: Extracellular signal regulated kinase 1 and 2; GAG: Glycosaminoglycan; HA: Hyaluronic acid; HMW: Higher molecular weight; Hsp70: Heat shock protein 70; IA: Intra-articular; ICAM: Intercellular adhesion molecule; IGF: Insulin-like growth factor; IL: Interleukin; iNOS: Inducible nitrogen oxideLMW, lower molecular weight; MAPK: Mitogen-activated protein kinase; MKP: Protein kinase phosphate; MMP: Metalloproteinase; Nf-кB: Nuclear factor kappa-light-chain- enhancer of activated B cells; NO: Nitric oxide; NSAIDs: Nonsteroidal anti-inflammatory drugs; OA: Osteoarthritis; PGE2: Prostaglandin E2; RHAMM: Receptor for hyaluronan mediated motility; ROS: Reactive oxygen species; TLR: Toll-like receptor. This review has methodological strength in its system- atic and thorough search of available basic science evi- dence within multiple databases. The current report also demonstrates rigor in its presentation of multiple mecha- nisms in which HA acts, providing evidence on all mecha- nisms whether comprehensively or infrequently reported within the current literature. Limitations of the current study arise due to the subjective classification of included article mechanism of action key conclusions. Included ar- ticles may briefly mention alternate mechanisms of action, Discussion Future research should also aim to recognize differences between mechanisms ex- hibited by high and lower molecular weight products, as well as analyze the safety profile differences between Bio- HA and AD-HA. Conclusions There is some high-quality clinical evidence that Bio-HA has a significantly smaller incidence of injection site adverse events than AD-HA [134]; however, this is not thoroughly investigated within the current literature. More comprehensive investigation of the difference in incidence of injection site adverse events between Bio-HA and AD-HA from both a basic science and clinical perspective is needed. Received: 12 August 2015 Accepted: 15 October 2015 22. Creamer P, Sharif M, George E, Meadows K, Cushnaghan J, Shinmei M, et al. Intra-articular hyaluronic acid in osteoarthritis of the knee: an investigation into mechanisms of action. Osteoarthritis Cartilage. 1994;2(2):133–40. Competing interests Roy D. Altman: Consultant: Cytori, DuPuy (Bioventus Global), Ferring, Flexion, Iroko, McNeil, Novartis, Oletec, Pfizer, QMed, Rotta, Strategic Science and Technologies, Teva. Speaker: Ferring, Iroko. Page 7 of 10 Page 7 of 10 Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 Ajay Manjoo: No conflicts of interest Ajay Manjoo: No conflicts of interest Anke Fierlinger: Paid employee of Ferring Pharmaceuticals Inc. Faizan Niazi: Paid employee of Ferring Pharmaceuticals Inc. Mathew Nicholls: Serves on advisory boards for Ferring. 14. Pullman-Mooar S, Mooar P, Sieck M, Clayburne G, Schumacher HR. Are there distinctive inflammatory flares after hylan g-f 20 intraarticular injections? J Rheumatol Dec. 2002;29(12):2611–4. 15. Goldberg VM, Coutts RD. Pseudoseptic reactions to hylan 15. Goldberg VM, Coutts RD. Pseudoseptic reactions to hylan viscosupplementation: diagnosis and treatment. Clin Orthop Relat Res Feb. 2004;419:130–7. Authors’ contributions d d 16. Ando A, Hagiwara Y, Chimoto E, Hatori K, Onoda Y, Itoi E. Intra-articular injection of hyaluronan diminishes loss of chondrocytes in a rat immobilized-knee model. Tohoku J Exp Med. 2008;215(4):321–31. AF and FN participated in the design and development of the study. RDA provided interpretation of the data, and significant writing contributions to the manuscript. All authors provided critical review and revisions to the manuscript. All authors read and approved the final manuscript. 17. Ariyoshi W, Okinaga T, Knudson CB, Knudson W, Nishihara T. High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-kappaB ligand through Rho kinase. Osteoarthritis Cartilage. 2014;22(1):111–20. Acknowledgements 18. Asari A, Miyauchi S, Matsuzaka S, Itoh T, Uchiyama Y. Hyaluronate on heat shock protein and synovial cells in a canine model of osteoarthritis. Osteoarthritis Cartilage. 1996;4(3):213–5. This review is funded by Ferring Pharmaceuticals Inc. The authors would like to thank Mark Phillips and Global Research Solutions Inc. for their assistance in preparing the manuscript. 19. Brun P, Panfilo S, Daga Gordini D, Cortivo R, Abatangelo G. The effect of hyaluronan on CD44-mediated survival of normal and hydroxyl radical- damaged chondrocytes. Osteoarthritis Cartilage. 2003;11(3):208–16. References 23. Ding M, Christian Danielsen C, Hvid I. Effects of hyaluronan on three- dimensional microarchitecture of subchondral bone tissues in guinea pig primary osteoarthrosis. Bone. 2005;36(3):489–501. 1. Cheng OT, Souzdalnitski D, Vrooman B, Cheng J. Evidence-based knee injections for the management of arthritis. Pain Med. 2012;13(6):740–53. 1. Cheng OT, Souzdalnitski D, Vrooman B, Cheng J. Evidence-based knee injections for the management of arthritis. Pain Med. 2012;13(6):740–53. 2. Moreland LW. Intra-articular hyaluronan (hyaluronic acid) and hylans for the treatment of osteoarthritis: mechanisms of action. Arthritis Res Therapy. 2003;5(2):54. 24. Ehlers EM, Behrens P, Wunsch L, Kuhnel W, Russlies M. Effects of hyaluronic acid on the morphology and proliferation of human chondrocytes in primary cell culture. Ann Anat. 2001;183(1):13–7. 3. Bellamy N, Campbell J, Robinson V, Gee T, Bourne R, Wells G. Viscosupplementation for the treatment of osteoarthritis of the knee. Cochrane Database Syst Rev. 2006;2, CD005321. 3. Bellamy N, Campbell J, Robinson V, Gee T, Bourne R, Wells G. Viscosupplementation for the treatment of osteoarthritis of the knee. Cochrane Database Syst Rev. 2006;2, CD005321. 25. Galois L, Etienne S, Henrionnet C, Scala-Bertola J, Grossin L, Mainard D, et al. Ambivalent properties of hyaluronate and hylan during post-traumatic OA in the rat knee. Biomed Mater Eng. 2012;22(4):235–42. 4. Trigkilidas D, Anand A. The effectiveness of hyaluronic acid intra-articular injections in managing osteoarthritic knee pain. Ann R Coll Surg Engl. 2013;95(8):545–51. 4. Trigkilidas D, Anand A. The effectiveness of hyaluronic acid intra-articular injections in managing osteoarthritic knee pain. Ann R Coll Surg Engl. 2013;95(8):545–51. 26. Gonzalez-Fuentes AM, Green DM, Rossen RD, Ng B. Intra-articular hyaluronic acid increases cartilage breakdown biomarker in patients with knee osteoarthritis. Clin Rheumatol. 2010;29(6):619–24. 5. Colen S, van den Bekerom MP, Mulier M, Haverkamp D. Hyaluronic acid in the treatment of knee osteoarthritis: a systematic review and meta-analysis with emphasis on the efficacy of different products. BioDrugs. 2012;26(4):257–68. 27. Greenberg DD, Stoker A, Kane S, Cockrell M, Cook JL. Biochemical effects of two different hyaluronic acid products in a co-culture model of osteoarthritis. Osteoarthritis Cartilage. 2006;14(8):814–22. 28. Grishko V, Xu M, Ho R, Mates A, Watson S, Kim JT, et al. Effects of hyaluronic acid on mitochondrial function and mitochondria-driven apoptosis following oxidative stress in human chondrocytes. J Biol Chem. 2009;284(14):9132–9. 6. Rutjes A, Juni P, da Costa B, Trelle S, Nuesch E, Reichenbach S. Viscosupplementation for osteoarthritis of the knee: a systematic review and meta-analysis. Ann Intern Med. Author details 1D f Rh 1Division of Rheumatology and Immunology, David Geffen School of Medicine, University of California at Los Angeles, 1000 Veterans Ave, 90024 Los Angeles, CA, USA. 2Division of Orthopaedics, McMaster University, Hamilton, ON, Canada. 3Ferring Pharmaceuticals Inc., Parsippany, NJ, USA. 4Kentucky Orthopaedic and Hand Surgeons, A division of Ortho Kentucky, Lexington, KY, USA. 1Division of Rheumatology and Immunology, David Geffen School of Medicine, University of California at Los Angeles, 1000 Veterans Ave, 90024 Los Angeles, CA, USA. 2Division of Orthopaedics, McMaster University, 20. Brun P, Zavan B, Vindigni V, Schiavinato A, Pozzuoli A, Iacobellis C, et al. In vitro response of osteoarthritic chondrocytes and fibroblast-like synoviocytes to a 500-730 kDa hyaluronan amide derivative. J Biomed Mater Res B Appl Biomater. 2012;100(8):2073–81. Hamilton, ON, Canada. Ferring Pharmaceuticals Inc., Parsippany, NJ, USA. 4Kentucky Orthopaedic and Hand Surgeons, A division of Ortho Kentucky, Lexington, KY, USA. 21. Chang CC, Hsieh MS, Liao ST, Chen YH, Cheng CW, Huang PT, et al. Hyaluronan regulates PPARgamma and inflammatory responses in IL-1beta- stimulated human chondrosarcoma cells, a model for osteoarthritis. Carbohydr Polym. 2012;90(2):1168–75. Received: 12 August 2015 Accepted: 15 October 2015 References 2012;157:180–91. 7. Bannuru RR, Natov NS, Obadan IE, Price LL, Schmid CH, McAlindon TE. Therapeutic trajectory of hyaluronic acid versus corticosteroids in the treatment of knee osteoarthritis: a systematic review and meta-analysis. Arthritis Rheum. 2009;61(12):1704–11. 29. Hashizume M, Mihara M. Desirable effect of combination therapy with high molecular weight hyaluronate and NSAIDs on MMP production. Osteoarthritis Cartilage. 2009;17(11):1513–8. , , , , , Therapeutic trajectory of hyaluronic acid versus corticosteroids in the treatment of knee osteoarthritis: a systematic review and meta-analysis. Arthritis Rheum. 2009;61(12):1704–11. 8. Habib GS. Systemic effects of intra-articular corticosteroids. Clin Rheumatol. 2009;28(7):749–56. 30. Homandberg GA, Hui F, Wen C, Kuettner KE, Williams JM. Hyaluronic acid suppresses fibronectin fragment mediated cartilage chondrolysis: I. In vitro. Osteoarthritis Cartilage. 1997;5(5):309–19. 8. Habib GS. Systemic effects of intra-articular corticosteroids. Clin Rheumatol. 2009;28(7):749–56. 8. Habib GS. Systemic effects of intra-articular corticosteroids. Clin Rheumatol. 2009;28(7):749–56. 9. Elmorsy S, Funakoshi T, Sasazawa F, Todoh M, Tadano S, Iwasaki N. Chondroprotective effects of high-molecular-weight cross-linked hyaluronic acid in a rabbit knee osteoarthritis model. Osteoarthritis Cartilage. 2014;22(1):121–7. 31. Ishijima M, Nakamura T, Shimizu K, Hayashi K, Kikuchi H, Soen S, et al. Different changes in the biomarker CTX-II following intra-articular injection of high molecular weight hyaluronic acid and oral non-steroidal anti- inflammatory drugs for patients with knee osteoarthritis: a multi-center randomized controlled study. Osteoarthr Cartil. 2013;21:S292. 10. Kosinska MK, Ludwig TE, Liebisch G, Zhang R, Siebert HC, Wilhelm J, et al. Articular joint lubricants during osteoarthritis and rheumatoid arthritis display altered levels and molecular species. PLoS One. 2015;10(5), e0125192. 32. Julovi SM, Yasuda T, Shimizu M, Hiramitsu T, Nakamura T. Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage. Arthritis Rheum. 2004;50(2):516–25. 11. Band PA, Heeter J, Wisniewski HG, Liublinska V, Pattanayak CW, Karia RJ, et al. Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression. Osteoarthritis Cartilage. 2015;23(1):70–6. 11. Band PA, Heeter J, Wisniewski HG, Liublinska V, Pattanayak CW, Karia RJ, et al. Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression. Osteoarthritis Cartilage. 2015;23(1):70–6. 12. Diaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment. J Orthop Res. 2005;23(6):1370–6. 13. Lajeunesse D, Delalandre A, Martel-Pelletier J, Pelletier J-P. Hyaluronic acid reverses the abnormal synthetic activity of human osteoarthritic subchondral bone osteoblasts. Bone. References 2003;33(4):703–10. 33. Kalaci A, Yilmaz HR, Aslan B, Sogut S, Yanat AN, Uz E. Effects of hyaluronan on nitric oxide levels and superoxide dismutase activities in synovial fluid in knee osteoarthritis. Clin Rheumatol. 2007;26(8):1306–11. p g g 12. Diaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment. J Orthop Res. 2005;23(6):1370–6. 34. Karna E, Miltyk W, Surazynski A, Palka JA. Protective effect of hyaluronic acid on interleukin-1-induced deregulation of beta1-integrin and insulin-like growth factor-I receptor signaling and collagen biosynthesis in cultured human chondrocytes. Mol Cell Biochem. 2008;308(1-2):57–64. y 13. Lajeunesse D, Delalandre A, Martel-Pelletier J, Pelletier J-P. Hyaluronic acid reverses the abnormal synthetic activity of human osteoarthritic subchondral bone osteoblasts. Bone. 2003;33(4):703–10. Page 8 of 10 Page 8 of 10 Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 35. Kobayashi K, Amiel M, Harwood FL, Healey RM, Sonoda M, Moriya H, et al. The long-term effects of hyaluronan during development of osteoarthritis following partial meniscectomy in a rabbit model. Osteoarthritis Cartilage. 2000;8(5):359–65. 55. Smith MM, Russell AK, Schiavinato A, Little CB. A hexadecylamide derivative of hyaluronan (HYMOVIS(R)) has superior beneficial effects on human osteoarthritic chondrocytes and synoviocytes than unmodified hyaluronan. J Inflamm (Lond). 2013;10:26. 36. Koga H. Effects of hyaluronic acid on arthritic articular cartilage. Connect Tissue Res. 2012;53(1):48–93. 56. Takahashi K, Goomer RS, Harwood F, Kubo T, Hirasawa Y, Amiel D. The effects of hyaluronan on matrix metalloproteinase-3 (MMP-3), interleukin- 1beta(IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression during the development of osteoarthritis. Osteoarthritis Cartilage 1999;7(2):182–90. 37. Lazaro J, Granado P, del Sol G, Medina A, Gallego L, Sandoval D, et al. The role of different hyaluronic acids in the articular cartilage of rabbit. Open Orthopaedics J. 2010;4:44–7. 38. Li J, Gorski DJ, Anemaet W, Velasco J, Takeuchi J, Sandy JD, et al. Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism. Arthritis Res Ther. 2012;14(3):R151. 57. Takahashi K, Hashimoto S, Kubo T, Hirasawa Y, Lotz M, Amiel D. Hyaluronan suppressed nitric oxide production in the meniscus and synovium of rabbit osteoarthritis model. J Orthop Res. 2001;19(3):500–3. 58. Tanaka M, Masuko-Hongo K, Kato T, Nishioka K, Nakamura H. Suppressive effects of hyaluronan on MMP-1 and RANTES production from chondrocytes. Rheumatol Int. 2006;26(3):185–90. 39. References Li P, Raitcheva D, Hawes M, Moran N, Yu X, Wang F, et al. Hylan G-F 20 maintains cartilage integrity and decreases osteophyte formation in osteoarthritis through both anabolic and anti-catabolic mechanisms. Osteoarthritis Cartilage. 2012;20(11):1336–46. 59. Waddell DD, Kolomytkin OV, Dunn S, Marino AA. Hyaluronan suppresses IL- 1beta-induced metalloproteinase activity from synovial tissue. Clin Orthop Relat Res. 2007;465:241–8. 60. Yasuda T. Nuclear factor-kappaB activation by type II collagen peptide in articular chondrocytes: its inhibition by hyaluronan via the receptors. Mod Rheumatol. 2013;23(6):1116–23. 40. Lisignoli G, Grassi F, Zini N, Toneguzzi S, Piacentini A, Guidolin D, et al. Anti- Fas-induced apoptosis in chondrocytes reduced by hyaluronan: evidence for CD44 and CD54 (intercellular adhesion molecule 1) invovement. Arthritis Rheum. 2001;44(8):1800–7. 61. Yasui T, Akatsuka M, Tobetto K, Hayaishi M, Ando T. The effect of hyaluronan on interleukin-1 alpha-induced prostaglandin E2 production in human osteoarthritic synovial cells. Agents Actions. 1992;37(1-2):155–6. 41. Lu HT, Sheu MT, Lin YF, Lan J, Chin YP, Hsieh MS, et al. Injectable hyaluronic-acid-doxycycline hydrogel therapy in experimental rabbit osteoarthritis. BMC Vet Res. 2013;9:68. 62. Yatabe T, Mochizuki S, Takizawa M, Chijiiwa M, Okada A, Kimura T, et al. Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes. Ann Rheum Dis. 2009;68(6):1051–8. 42. Maneiro E, de Andres MC, Fernandez-Sueiro JL, Galdo F, Blanco FJ. The biological action of hyaluronan on human osteoartritic articular chondrocytes: the importance of molecular weight. Clin Exp Rheumatol. 2004;22(3):307–12. 63. Yoshimi T, Kikuchi T, Obara T, Yamaguchi T, Sakakibara Y, Itoh H, et al. Effects of high-molecular-weight sodium hyaluronate on experimental osteoarthrosis induced by the resection of rabbit anterior cruciate ligament. Clin Orthop Relat Res. 1994;298:296–304. 43. Mihara M, Hashizume M. The effect of high molecular hyaluronic acid on the induction of matrix degradation enzymes By IL-6, IL-1β and TNF-α. Osteoarthr Cartil. 2012;20:S134–5. 64. Yoshioka M, Shimizu C, Harwood FL, Coutts RD, Amiel D. The effects of hyaluronan during the development of osteoarthritis. Osteoarthritis Cartilage. 1997;5(4):251–60. 44. Miki Y, Teramura T, Tomiyama T, Onodera Y, Matsuoka T, Fukuda K, et al. Hyaluronan reversed proteoglycan synthesis inhibited by mechanical stress: possible involvement of antioxidant effect. Inflamm Res. 2010;59(6):471–7. 65. Yu CJ, Ko CJ, Hsieh CH, Chien CT, Huang LH, Lee CW, et al. Proteomic analysis of osteoarthritic chondrocyte reveals the hyaluronic acid-regulated proteins involved in chondroprotective effect under oxidative stress. J Proteomics. 2014;99:40–53. 45. Mladenovic Z, Saurel AS, Berenbaum F, Jacques C. References Potential role of hyaluronic acid on bone in osteoarthritis: matrix metalloproteinases, aggrecanases, and RANKL expression are partially prevented by hyaluronic acid in interleukin 1-stimulated osteoblasts. J Rheumatol. 2014;41(5):945–54. 66. Zhang FJ, Gao SG, Cheng L, Tian J, Xu WS, Luo W, et al. The effect of hyaluronic acid on osteopontin and CD44 mRNA of fibroblast-like synoviocytes in patients with osteoarthritis of the knee. Rheumatol Int. 2013;33(1):79–83. 46. Mongkhon JM, Thach M, Shi Q, Fernandes JC, Fahmi H, Benderdour M. Sorbitol-modified hyaluronic acid reduces oxidative stress, apoptosis and mediators of inflammation and catabolism in human osteoarthritic chondrocytes. Inflamm Res. 2014;63(8):691–701. 67. Zhou JL, Liu SQ, Qiu B, Hu QJ, Ming JH, Peng H. The protective effect of sodium hyaluronate on the cartilage of rabbit osteoarthritis by inhibiting peroxisome proliferator-activated receptor-gamma messenger RNA expression. Yonsei Med J. 2009;50(6):832–7. 47. Ohno S, Im HJ, Knudson CB, Knudson W. Hyaluronan oligosaccharide- induced activation of transcription factors in bovine articular chondrocytes. Arthritis Rheum. 2005;52(3):800–9. 68. Zhou PH, Liu SQ, Peng H. The effect of hyaluronic acid on IL-1beta-induced chondrocyte apoptosis in a rat model of osteoarthritis. J Orthop Res. 2008;26(12):1643–8. 48. Peng H, Zhou JL, Liu SQ, Hu QJ, Ming JH, Qiu B. Hyaluronic acid inhibits nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes in vitro. Inflamm Res. 2010;59(7):519–30. 69. Williams J. The effects of hyaluronic acid on fibronectin fragment mediated cartilage chondrolysis in skeletally mature rabbits. Osteoarthr Cartil. 2003;11(1):44–9. 49. Plaas A, Li J, Riesco J, Das R, Sandy JD, Harrison A. Intraarticular injection of hyaluronan prevents cartilage erosion, periarticular fibrosis and mechanical allodynia and normalizes stance time in murine knee osteoarthritis. Arthritis Res Ther. 2011;13(2):R46. 70. Xu H, Ito T, Tawada A, Maeda H, Yamanokuchi H, Isahara K, et al. Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72. J Biol Chem. 2002;277(19):17308–14. 50. Qiu B, Liu SQ, Peng H. Influence of sodium hyaluronate on iNOS expression in synovium and NO content in synovial fluid of rabbits with traumatic osteoarthritis. Chin J Traumatol. 2008;11(5):293–6. 71. Zhou J-L, Liu S-Q, Qiu B, Hu Q-J, Ming J-H, Peng H. Effects of hyaluronan on vascular endothelial growth factor and receptor-2 expression in a rabbit osteoarthritis model. J Orthop Sci. 2009;14(3):313–9. 51. Sakakibara Y, Miura T, Iwata H, Kikuchi T, Yamaguchi T, Yoshimi T, et al. Effect of high-molecular-weight sodium hyaluronate on immobilized rabbit knee. Clin Orthop Relat Res. 1994;299:282–92. 72. References Huang TL, Hsu HC, Yang KC, Yao CH, Lin FH. Effect of different molecular weight hyaluronans on osteoarthritis-related protein production in fibroblast-like synoviocytes from patients with tibia plateau fracture. J Trauma. 2010;68(1):146–52. 52. Sasaki A, Sasaki K, Konttinen YT, Santavirta S, Takahara M, Takei H, et al. Hyaluronate inhibits the interleukin-1beta-induced expression of matrix metalloproteinase (MMP)-1 and MMP-3 in human synovial cells. Tohoku J Exp Med. 2004;204(2):99–107. 73. Amiel D, Toyoguchi T, Kobayashi K, Bowden K, Amiel ME, Healey RM. Long- term effect of sodium hyaluronate (Hyalgan) on osteoarthritis progression in a rabbit model. Osteoarthritis and cartilage / OARS, Osteoarthritis Res Soc. 2003;11(9):636–43. 53. Shimizu C, Yoshioka M, Coutts RD, Harwood FL, Kubo T, Hirasawa Y, et al. Long-term effects of hyaluronan on experimental osteoarthritis in the rabbit knee. Osteoarthritis Cartilage. 1998;6(1):1–9. 74. Kim NH, Han CD, Lee HM, Yang IH. Effect of sodium hyaluronate on prevention of osteoarthritis. Yonsei Med J. 1991;32(2):139–46. 54. Smith MM, Cake MA, Ghosh P, Schiavinato A, Read RA, Little CB. Significant synovial pathology in a meniscectomy model of osteoarthritis: modification by intra-articular hyaluronan therapy. Rheumatology (Oxford). 2008;47(8):1172–8. 75. Liu J, Song W, Yuan T, Xu Z, Jia W, Zhang C. A comparison between platelet-rich plasma (PRP) and hyaluronate acid on the healing of cartilage defects. PLoS One. 2014;9(5), e97293. Page 9 of 10 Page 9 of 10 Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 76. Mihara M, Higo S, Uchiyama Y, Tanabe K, Saito K. Different effects of high molecular weight sodium hyaluronate and NSAID on the progression of the cartilage degeneration in rabbit OA model. Osteoarthritis Cartilage / OARS, Osteoarthritis Res Soc. 2007;15(5):543–9. 99. Asari A, Mizuno S, Tanaka I, Sunose A, Kuriyama S, Miyazaki K, et al. Suppression of hyaluronan and prostaglandin E2 production in traumatic arthritic synovial cells by sodium hyaluronate. Connective Tissue. 1997;29:1–5. 100. Lisignoli G, Grassi F, Piacentini A, Cocchini B, Remiddi G, Bevilacqua C, et al. Hyaluronan does not affect cytokine and chemokine expression in osteoarthritic chondrocytes and synoviocytes. Osteoarthritis Cartilage. 2001;9(2):161–8. 77. Qiu B, Liu SQ, Peng H, Wang HB. The effects of sodium hyaluronate on mRNA expressions of matrix metalloproteinase-1, -3 and tissue inhibitor of metalloproteinase-1 in cartilage and synovium of traumatic osteoarthritis model. Chin J Traumatol. 2005;8(1):8–12. 101. Oliviero F, Scanu A, Ramonda R, Frallonardo P, Sfriso P, Dayer J, et al. References Mechanisms involved in inhibition of inflammation in THP-1 cells by the hexadecylamide derivative of hyaluronic acid. Osteoarthr Cartil. 2014;22:S292–3. 78. Ozkan FU, Ozkan K, Ramadan S, Guven Z. Chondroprotective effect of N- acetylglucosamine and hyaluronate in early stages of osteoarthritis–an experimental study in rabbits. Bull NYU Hosp Jt Dis. 2009;67(4):352–7. 102. Sezgin M, Demirel AÇ, Karaca C, Ortancıl Ö, Ülkar GB, Kanık A, et al. Does hyaluronan affect inflammatory cytokines in knee osteoarthritis? Rheumatol Int. 2004;25(4):264–9. 79. Yang L, Zhang J, Wang G. The effect of sodium hyaluronate treating knee osteoarthritis on synovial fluid interleukin -1beta and clinical treatment mechanism. Pak J Pharm Sci. 2015;28(1 Suppl):407–10. 103. Sheehan KM, Delott LB, Day SM, Deheer DH. Hyalgan® has a dose- dependent differential effect on macrophage proliferation and cell death. J Orthop Res. 2003;21(4):744–51. 80. Yoshioka K, Yasuda Y, Kisukeda T, Nodera R, Tanaka Y, Miyamoto K. Pharmacological effects of novel cross-linked hyaluronate, Gel-200, in experimental animal models of osteoarthritis and human cell lines. Osteoarthritis Cartilage. 2014;22(6):879–87. 104. Yasuda T. Hyaluronan inhibits Akt, leading to nuclear factor-kappaB down- regulation in lipopolysaccharide-stimulated U937 macrophages. J Pharmacol Sci. 2011;115(4):509–15. 81. Hui AY, McCarty WJ, Masuda K, Firestein GS, Sah RL. A systems biology approach to synovial joint lubrication in health, injury, and disease. Wiley Interdiscip Rev Syst Biol Med. 2012;4(1):15–37. 105. Campo GM, Avenoso A, D'Ascola A, Prestipino V, Scuruchi M, Nastasi G, et al. Inhibition of hyaluronan synthesis reduced inflammatory response in mouse synovial fibroblasts subjected to collagen-induced arthritis. Arch Biochem Biophys. 2012;518(1):42–52. 82. Lin EA, Liu CJ. The role of ADAMTSs in arthritis. Protein Cell. 2010;1(1):33–47. 83. Asari A, Miyauchi S, Matsuzaka S, Ito T, Kominami E, Uchiyama Y. Molecular weight-dependent effects of hyaluronate on the arthritic synovium. Arch Histol Cytol. 1998;61(2):125–35. 106. Campo GM, Avenoso A, Nastasi G, Micali A, Prestipino V, Vaccaro M, et al. Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression. Biochim Biophys Acta. 2011;1812(9):1170–81. 84. Bagga H, Burkhardt D, Sambrook P, March L. Longterm effects of intraarticular hyaluronan on synovial fluid in osteoarthritis of the knee. J Rheumatol. 2006;33(5):946–50. 107. Schumacher HR, Paul C, Hitchon CA, El-Gabalawy H, Zonay L, Clayburne G, et al. Hyaluronate effects on synovium and synovial fluid. A prospective blinded study in patients with osteoarthritis of the knee. Osteoarthritis Cartilage. 2006;14(5):501–3. 85. Bauer C, Baumgartner R, Hornof M, Halbwirth F, Niculescu-Morzsa E, Zwickl H, et al. References Cross-linked hyaluronic acid scaffolds: a potential usage in cartilage regeneration? Osteoarthr Cartil. 2013;21:S312. 108. Bell CJ, Ingham E, Fisher J. Influence of hyaluronic acid on the time- dependent friction response of articular cartilage under different conditions Proc Inst Mech Eng H. 2006;220(1):23–31. 86. Forsey R, Fisher J, Thompson J, Stone M, Bell C, Ingham E. The effect of hyaluronic acid and phospholipid based lubricants on friction within a human cartilage damage model. Biomaterials. 2006;27(26):4581–90. 109. Ghosh P, Read R, Numata Y, Smith S, Armstrong S, Wilson D. The effects of intraarticular administration of hyaluronan in a model of early osteoarthritis in sheep. II. Cartilage composition and proteoglycan metabolism. Semin Arthritis Rheum. 1993;22(6 Suppl 1):31–42. 87. Frean S, Abraham L, Lees P. In vitro stimulation of equine articular cartilage proteoglycan synthesis by hyaluronan and carprofen. Res Vet Sci. 1999;67:181–8. 88. Han F, Ishiguro N, Ito T, Sakai T, Iwata H. Effects of sodium hyaluronate on experimental osteoarthritis in rabbit knee joints. Med Sci. 1999;62. 110. Obara T, Mabuchi K, Iso T, Yamaguchi T. Increased friction of animal joints by experimental degeneration and recovery by addition of hyaluronic acid. Clin Biomech (Bristol, Avon). 1997;12(4):246–52. 89. Homandberg GA, Ummadi V, Kang H. The role of insulin-like growth factor-I in hyaluronan mediated repair of cultured cartilage explants. Inflamm Res. 2004;53:8. 111. Waller KA, Zhang LX, Fleming BC, Jay GD. Preventing friction-induced chondrocyte apoptosis: comparison of human synovial fluid and Hylan G-F 20. J Rheumatol. 2012;39(7):1473–80. 90. Hulmes D. Intra-articular hyaluronate in experimental rabbit osteoarthritis can prevent changes in cartilage proteoglycan content. Osteoarthr Cartil. 2004;12(3):232–8. 112. Yu L-P, Yang H, Voschin E, Skrabut E. Viscoelastic properties and molecular weight of hylan G-F 20 compared with other commercial hyaluronan based viscosupplements. Osteoarthr Cartil. 2011;19(S1):S235. 91. Kang Y, Eger W, Koepp H, Williams JM, Kuettner KE, Homandberg GA. Hyaluronan suppresses fibronectin fragment-mediated damage to human cartilage explant cultures by enhancing proteoglycan synthesis. J Orthop Res. 1999;17(6):858–69. 113. Mori S, Naito M, Moriyama S. Highly viscous sodium hyaluronate and joint lubrication. Int Orthop. 2002;26(2):116–21. 92. Kikuchi T, Yamada H, Fujikawa K. Effects of high molecular weight hyaluronan on the distribution and movement of proteoglycan around chondrocytes cultured in alginate beads. Osteoarthr Cartil. 2001;9(4):351–6. 114. Tang SF, Chen CP, Chen MJ, Pei YC, Lau YC, Leong CP. Changes in sagittal ground reaction forces after intra-articular hyaluronate injections for knee osteoarthritis. Arch Phys Med Rehabil. 2004;85(6):951–5. 93. References Kobayashi K, Matsuzaka S, Yoshida Y, Miyauchi S, Wada Y, Moriya H. The effects of intraarticularly injected sodium hyaluronate on levels of intact aggrecan and nitric oxide in the joint fluid of patients with knee osteoarthritis. Osteoarthr Cartil. 2004;12(7):536–42. 115. Armstrong S, Read R, Ghosh P. The effects of intraarticular hyaluronan on cartilage and subchondral bone changes in an ovine model of early osteoarthritis. J Rheumatol. 1994;21(4):680–8. 94. Nishida Y, Knudson CB, Knudson W. Extracellular matrix recovery by human articular chondrocytes after treatment with hyaluronan hexasaccharides or Streptomyces hyaluronidase. Mod Rheumatol. 2003;13(1):62–8. 116. Hiraoka N, Takahashi Y, Arai K, Honjo S, Nakawaga S, Tsuchida S, et al. Hyaluronan and intermittent hydrostatic pressure synergistically suppressed MMP-13 and Il-6 expressions in osteoblasts from OA subchondral bone. Osteoarthr Cartil. 2009;17(1):S97. 95. Smith Jr GN, Mickler EA, Myers SL, Brandt KD. Effect of intraarticular hyaluronan injection on synovial fluid hyaluronan in the early stage of canine post-traumatic osteoarthritis. J Rheumatol. 2001;28(6):1341–6. 117. Hiraoka N, Takahashi KA, Arai Y, Sakao K, Mazda O, Kishida T, et al. Intra- articular injection of hyaluronan restores the aberrant expression of matrix metalloproteinase-13 in osteoarthritic subchondral bone. J Orthop Res. 2011;29(3):354–60. 96. Stove J, Gerlach C, Huch K, Gunther KP, Puhl W, Scharf HP. Effects of hyaluronan on proteoglycan content of osteoarthritic chondrocytes in vitro. J Orthop Res. 2002;20(3):551–5. 118. Prasadam I, Crawford R, Xiao Y. Aggravation of ADAMTS and matrix metalloproteinase production and role of ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes – possible pathogenic role in osteoarthritis. J Rheumatol. 2012;39(3):621–34. 97. Wang C, Lin Y, Chiang B, Hou S. High molecular weight hyaluronic acid down-regulates the gene expression of osteoarthritis-associated cytokines and enzymes in fibroblast-like synoviocytes from patients with early osteoarthritis. Osteoarthr Cartil. 2006;14(12):1237–47. 98. Abatangelo G, Botti P, Del Bue M, Gei G, Samson JC, Cortivo R, et al. Intraarticular sodium hyaluronate injections in the Pond-Nuki experimental model of osteoarthritis in dogs. I. Biochemical results. Clin Orthop Relat Res. 1989;241:278–85. 119. Boettger MK, Kummel D, Harrison A, Schaible HG. Evaluation of long-term antinociceptive properties of stabilized hyaluronic acid preparation (NASHA) in an animal model of repetitive joint pain. Arthritis Res Ther. 2011;13(4):R110. Page 10 of 10 Page 10 of 10 Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 120. Gomis A, Miralles A, Schmidt RF, Belmonte C. Altman et al. BMC Musculoskeletal Disorders (2015) 16:321 References Am J Respir Cell Mol Biol. 2000;23(4):431–3. 131. Mizrahy S, Raz SR, Hasgaard M, Liu H, Soffer-Tsur N, Cohen K, et al. Hyaluronan-coated nanoparticles: the influence of the molecular weight on CD44-hyaluronan interactions and on the immune response. J Control Release. 2011;156(2):231–8. 132. Jevsevar DS, Jones DL, Matzkin EG, Manner PA, Mooar P, Schousboe JT, et al. American academy of orthopaedic surgeons. Treatment of osteoarthritis of the knee: evidence based guideline 2nd Edition. JBJS. 2013;95(20):1885–6. Full document at: http://www.aaos.org/Research/guidelines/ TreatmentofOsteoarthritisoftheKneeGuideline.pdf. 133. Lee PB, Kim YC, Lim YJ, Lee CJ, Sim WS, Ha CW, et al. Comparison between high and low molecular weight hyaluronates in knee osteoarthritis patients: open-label, randomized, multicentre clinical trial. J Int Med Res. 2006;34(1):77–87. 134. Kirchner M, Marshall D. A double-blind randomized controlled trial comparing alternate forms of high molecular weight hyaluronan for the treatment of osteoarthritis of the knee. Osteoarthr Cartil. 2006;14(2):154–62. 135. Chen AL, Desai P, Adler EM, Di Cesare PE. Granulomatous inflammation after Hylan G-F 20 viscosupplementation of the knee : a report of six cases. J Bone Joint Surg Am. 2002;84-A(7):1142–7. 135. Chen AL, Desai P, Adler EM, Di Cesare PE. Granulomatous inflammation after Hylan G-F 20 viscosupplementation of the knee : a report of six cases. J Bone Joint Surg Am. 2002;84-A(7):1142–7. 136. Zardawi IM, Chan I. Synvisc perisynovitis. Pathology. 2001;33(4):519–20. 136. Zardawi IM, Chan I. Synvisc perisynovitis. Pathology. 2001;33(4):519–20. References Intra-articular injections of hyaluronan solutions of different elastoviscosity reduce nociceptive nerve activity in a model of osteoarthritic knee joint of the guinea pig. Osteoarthr Cartil. 2009;17(6):798–804. 121. Gotoh S, Onaya J, Abe M, Miyazaki K, Hamai A, Horie K, et al. Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats. Ann Rheum Dis. 1993;52(11):817–22. 122. Pena Ede L, Sala S, Rovira JC, Schmidt RF, Belmonte C. Elastoviscous substances with analgesic effects on joint pain reduce stretch-activated ion channel activity in vitro. Pain. 2002;99(3):501–8. 122. Pena Ede L, Sala S, Rovira JC, Schmidt RF, Belmonte C. Elastoviscous substances with analgesic effects on joint pain reduce stretch-activated ion channel activity in vitro. Pain. 2002;99(3):501–8. y 123. Dougados M. Sodium hyaluronate therapy in osteoarthritis: arguments for a potential beneficial structural effect. Semin Arthritis Rheum. 2000;30(2 Suppl 1):19–25. 123. Dougados M. Sodium hyaluronate therapy in osteoarthritis: arguments for a potential beneficial structural effect. Semin Arthritis Rheum. 2000;30(2 Suppl 1):19–25. 124. Balazs EA, Denlinger JL. Viscosupplementation: a new concept in the treatment of osteoarthritis. J Rheumatol Suppl. 1993;39:3–9. 124. Balazs EA, Denlinger JL. Viscosupplementation: a new concept in the treatment of osteoarthritis. J Rheumatol Suppl. 1993;39:3–9. 125. Karbownik MS, Nowak JZ. Hyaluronan: towards novel anti-cancer therapeutics. Pharmacol Rep. 2013;65(5):1056–74. 125. Karbownik MS, Nowak JZ. Hyaluronan: towards novel anti-ca 125. Karbownik MS, Nowak JZ. Hyaluronan: towards novel anti-cancer therapeutics. Pharmacol Rep. 2013;65(5):1056–74. therapeutics. Pharmacol Rep. 2013;65(5):1056–74. 126. Fakhari A, Berkland C. Applications and emerging trends of hyaluronic acid in tissue engineering, as a dermal filler and in osteoarthritis treatment. Acta Biomater. 2013;9(7):7081–92. 126. Fakhari A, Berkland C. Applications and emerging trends of hyaluronic acid in tissue engineering, as a dermal filler and in osteoarthritis treatment. Acta Biomater. 2013;9(7):7081–92. 127. Harris E, Weigel PH. Functional Aspects of the Hyaluronan and Chondroitin Sulfate Receptors. In: Raton B, ed: CRC Press; 2009:171-192. 128. Ghosh P, Guidolin D. Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent? Semin Arthritis Rheum. 2002;32(1):10–37. 129. Jiang D, Liang J, Noble PW. Hyaluronan as an immune regulator in human diseases. Physiol Rev. 2011;91(1):221–64. 129. Jiang D, Liang J, Noble PW. Hyaluronan as an immune regulator in human diseases. Physiol Rev. 2011;91(1):221–64. 130. Camenisch TD, McDonald JA. Hyaluronan: is bigger better? Am J Respir Cell Mol Biol. 2000;23(4):431–3. 130. Camenisch TD, McDonald JA. Hyaluronan: is bigger better? References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review
https://openalex.org/W4313901786
https://ems.press/content/serial-article-files/26037
English
null
Comparison of spectral sequences involving bifunctors
Documenta mathematica
2,008
cc-by
30,877
677 677 Documenta Math. Documenta Math. Comparison of Spectral Sequences Involving Bifunctors Matthias K¨unzer Received: October 15, 2006 Revised: October 29, 2008 Communicated by Max Karoubi Abstract. Suppose given functors A × A′ F−→B G −→C between abelian categories, an object X in A and an object X′ in A′ such that F(X, −), F(−, X′) and G are left exact, and such that further conditions hold. We show that, E1-terms exempt, the Grothendieck spectral sequence of the composition of F(X, −) and G evaluated at X′ is isomorphic to the Grothendieck spectral sequence of the com- position of F(−, X′) and G evaluated at X. The respective E2-terms are a priori seen to be isomorphic. But instead of trying to com- pare the differentials and to proceed by induction on the pages, we rather compare the double complexes that give rise to these spectral sequences. 2000 Mathematics Subject Classification: 18G40 2000 Mathematics Subject Classification: 18G40 Keywords and Phrases: Grothendieck spectral sequence, Lyndon- Hochschild-Serre spectral sequence. Keywords and Phrases: Grothendieck spectral sequence, Lyndon- Hochschild-Serre spectral sequence. Keywords and Phrases: Grothendieck spectral sequence, Lyndon- Hochschild-Serre spectral sequence. Contents 0 Introduction 679 0.1 Language . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679 0.2 First comparison . . . . . . . . . . . . . . . . . . . . . . . . . . 679 0.3 Second comparison . . . . . . . . . . . . . . . . . . . . . . . . . 680 0.4 Results of Beyl and Barnes . . . . . . . . . . . . . . . . . . . . 681 0.5 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . 681 Documenta Mathematica 13 (2008) 677–737 678 Matthias K¨unzer 1 Double and triple complexes 683 1.1 Double complexes . . . . . . . . . . . . . . . . . . . . . . . . . . 683 1.1.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . 683 1.1.2 Applying H in different directions . . . . . . . . . . Communicated by Max Karoubi . . 684 1.1.3 Concentrated double complexes . . . . . . . . . . . . . . 684 1.1.4 Row- and columnwise notions . . . . . . . . . . . . . . . 684 1.1.5 Horizontally and vertically split acyclic double complexes 685 1.1.6 Total complex . . . . . . . . . . . . . . . . . . . . . . . 687 1.1.7 The homotopy category, first quadrant . . . . . . . . . . 687 1.2 Triple complexes . . . . . . . . . . . . . . . . . . . . . . . . . . 688 1.2.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . 688 1.2.2 Planewise total complex . . . . . . . . . . . . . . . . . . 688 2 Cartan-Eilenberg resolutions 689 2.1 A remark . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689 2.2 Exact categories . . . . . . . . . . . . . . . . . . . . . . . . . . 689 2.3 An exact category structure on C(A) . . . . . . . . . . . . . . . 691 2.4 An exact category structure on C[0(A) . . . . . . . . . . . . . . 694 2.5 The Cartan-Eilenberg resolution of a quasiisomorphism . . . . 694 3 Formalism of spectral sequences 696 3.1 Pointwise split and pointwise finitely filtered complexes . . . . 697 3.2 Spectral objects . . . . . . . . . . . . . . . . . . . . . . . . . . . 697 3.3 Spectral sequences . . . . . . . . . . . . . . . . . . . . . . . . . 698 3.4 A short exact sequence . . . . . . . . . . . . . 0.1 Language In §3, we give a brief introduction to the Deligne-Verdier spectral sequence language; cf. [17, II.§4], [6, App.]; or, on a more basic level, cf. [11, Kap. 4]. This language amounts to considering a diagram E(X) containing all the images between the homology groups of the subquotients of a given filtered complex X, instead of, as is classical, only selected ones. This helps to gain some elbow room in practice : to govern the objects of the diagram E(X) we can make use of a certain short exact sequence; cf. §3.4. Dropping the E1-terms and similar ones, we obtain the proper spectral sequence ˙E(X) of our filtered complex X. Amongst others, it contains all Ek-terms for k ≥2 in the classical language; cf. §§ 3.6, 3.5. 0 Introduction To calculate Ext∗(X, Y ), one can either resolve X projectively or Y injectively; the result is, up to isomorphism, the same. To show this, one uses the double complex arising when one resolves both X and Y ; cf. [5, Chap. V, Th. 8.1]. Two problems in this spirit occur in the context of Grothendieck spectral se- quences; cf. §§ 0.2, 0.3. To calculate Ext∗(X, Y ), one can either resolve X projectively or Y injectively; the result is, up to isomorphism, the same. To show this, one uses the double complex arising when one resolves both X and Y ; cf. [5, Chap. V, Th. 8.1]. Two problems in this spirit occur in the context of Grothendieck spectral se- quences; cf. §§ 0.2, 0.3. Comparison of Spectral Sequences Involving Bifunctors 679 7 Acyclic CE-resolutions 719 7.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 719 7.2 A theorem of Beyl . . . . . . . . . . . . . . . . . . . . . . . . . 720 8 Applications 721 8.1 A Hopf algebra lemma . . . . . . . . . . . . . . . . . . . . . . . 721 8.1.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . 721 8.1.2 Some basic properties . . . . . . . . . . . . . . . . . . . 722 8.1.3 Normality . . . . . . . . . . . . . . . . . . . . . . . . . . 724 8.1.4 Some remarks and a lemma . . . . . . . . . . . . . . . . 725 8.2 Comparing Hochschild-Serre-Hopf with Grothendieck . . . . . . 730 8.3 Comparing Lyndon-Hochschild-Serre with Grothendieck . . . . 732 8.4 Comparing Hochschild-Serre with Grothendieck . . . . . . . . . 733 8.5 Comparing two spectral sequences for a change of rings . . . . 734 8.6 Comparing two spectral sequences for Ext and ⊗. . . . . . . . 734 8.7 Comparing two spectral sequences for Ext of sheaves . . . . . . 735 Documenta Mathematica 13 (2008) 677–737 Communicated by Max Karoubi . . . . . . . . . . 699 3.5 Classical indexing . . . . . . . . . . . . . . . . . . . . . . . . . . 699 3.6 Comparing proper spectral sequences . . . . . . . . . . . . . . . 700 3.7 The first spectral sequence of a double complex . . . . . . . . . 701 4 Grothendieck spectral sequences 703 4.1 Certain quasiisomorphisms are preserved by a left exact functor 703 4.2 Definition of the Grothendieck spectral sequence functor . . . . 704 4.3 Haas transformations . . . . . . . . . . . . . . . . . . . . . . . . 710 4.3.1 Situation . . . . . . . . . . . . . . . . . . . . . . . . . . 710 4.3.2 Construction of the first Haas transformation . . . . . . 710 4.3.3 Construction of the second Haas transformation . . . . 712 5 The first comparison 712 5.1 The first comparison isomorphism . . . . . . . . . . . . . . . . 712 5.2 Naturality of the first comparison isomorphism . . . . . . . . . 713 6 The second comparison 715 6.1 The second comparison isomorphism . . . . . . . . . . . . . . . 715 6.2 Naturality of the second comparison isomorphism . . . . . . . . 717 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 679 0.2 First comparison Suppose given abelian categories A, A′ and B with enough injectives and an abelian category C. Suppose given objects X ∈Ob A and X′ ∈Ob A′. Let A × A′ - F B be a biadditive functor such that F(X, −) and F(−, X′) are left exact. Let B - G C be a left exact functor. Suppose further conditions to hold; see §5.1. Documenta Mathematica 13 (2008) 677–737 680 Matthias K¨unzer We have a Grothendieck spectral sequence for the composition G ◦F(X, −) and a Grothendieck spectral sequence for the composition G ◦F(−, X′). We evaluate the former at X′ and the latter at X. We have a Grothendieck spectral sequence for the composition G ◦F(X, −) and a Grothendieck spectral sequence for the composition G ◦F(−, X′). We evaluate the former at X′ and the latter at X. In both cases, the E2-terms are (RiG)(RjF)(X, X′). Moreover, they both converge to Ri+j(G ◦F)  (X, X′). So the following assertion is well-motivated. Theorem 31. The proper Grothendieck spectral sequences just described are isomorphic; i.e. ˙EGr F (X,−),G(X′) ≃˙EGr F (−,X′),G(X) . Theorem 31. The proper Grothendieck spectral sequences just described are isomorphic; i.e. ˙EGr F (X,−),G(X′) ≃˙EGr F (−,X′),G(X) . So instead of “resolving X′ twice”, we may just as well “resolve X twice”. So instead of “resolving X′ twice”, we may just as well “resolve X twice”. In fact, the underlying double complexes are connected by a chain of dou- ble homotopisms, i.e. isomorphisms in the homotopy category as defined in double roww [5, IV.§4], and rowwise homotopisms (the proof uses a chain •  double •  roww. • roww. double ) Th hi th i d i hi th i - roww. • - double •). These morphisms then induce isomorphisms on the associ- ated proper first spectral sequences. Documenta Mathematica 13 (2008) 677–737 0.5 Acknowledgements Results of Beyl and Haas are included for sake of documentation that they work within our framework; cf. Theorem 40 and §4. No originality from my part is claimed. I thank B. Keller for directing me to [12, XII.§11]. I thank the referee for helping to considerably improve the presentation, and for suggesting Lemma 47 and §8.2. I thank G. Carnovale and G. Hiß for help with Hopf algebras. and §8.2. I thank G. Carnovale and G. Hiß for help with Hopf algebras. 0.4 Results of Beyl and Barnes Let R be a commutative ring. Let G be a group. Let N ⊴G be a normal subgroup. Let M be an RG-module. Let R be a commutative ring. Let G be a group. Let N ⊴G be a normal subgroup. Let M be an RG-module. Beyl generalises Grothendieck’s setup, allowing for a variant of a Cartan- Eilenberg resolution that consists of acyclic, but no longer necessarily injective objects [4, Th. 3.4]. We have documented Beyl’s Theorem as Theorem 40 in our framework, without claiming originality. Beyl generalises Grothendieck’s setup, allowing for a variant of a Cartan- Eilenberg resolution that consists of acyclic, but no longer necessarily injective objects [4, Th. 3.4]. We have documented Beyl’s Theorem as Theorem 40 in our framework, without claiming originality. spectral sequence for RG -Mod - ( ) RN -Mod - ( ) R -Mod at M is iso- morphic to the Lyndon-Hochschild-Serre spectral sequence, i.e. the spectral sequence associated to the double complex RG(BarG/N;R ⊗R BarG;R , M); cf. [4, Th. 3.5], [3, §3.5]. This is now also a consequence of Theorems 31 and 34, as explained in §§ 8.2, 8.3. spectral sequence for RG -Mod - ( ) RN -Mod - ( ) R -Mod at M is iso- morphic to the Lyndon-Hochschild-Serre spectral sequence, i.e. the spectral sequence associated to the double complex RG(BarG/N;R ⊗R BarG;R , M); cf. [4, Th. 3.5], [3, §3.5]. This is now also a consequence of Theorems 31 and 34, as explained in §§ 8.2, 8.3. Barnes works in a slightly different setup. He supposes given a commutative ring R, abelian categories A, B and C of R-modules, and left exact functors F : A - B and G : B - C, where F is supposed to have an exact left adjoint J : B - A that satisfies F ◦J = 1B. Moreover, he assumes A to have ample injectives and C to have enough injectives. In this setup, he obtains a general comparison theorem. See [2, Sec. X.5, Def. X.2.5, Th. X.5.4]. [ ] Beyl [4] and Barnes [2] also consider cup products; in this article, we do not. Beyl [4] and Barnes [2] also consider cup products; in this article, we do not. 0.3 Second comparison Suppose given abelian categories A and B′ with enough injectives and abelian categories B and C. Suppose given objects X ∈Ob A and Y ∈Ob B. Let A - F B′ be a left exact functor. Let B × B′ - G C be a biadditive functor such that G(Y, −) is left exact. A - F B′ be a left exact functor. Let B × B′ - G C be a biadditive functor such that G(Y, −) is left exact. Let B ∈Ob C[0(B) be a resolution of Y , i.e. a complex B admitting a quasiiso- morphism Conc Y - B. Suppose that G(Bk, −) is exact for all k ≥0. Let A ∈Ob C[0(A) be, say, an injective resolution of X. Suppose further conditions to hold; see §6.1. We have a Grothendieck spectral sequence for the composition G(Y, −) ◦F, which we evaluate at X. On the other hand, we can consider the double complex G(B, FA), where the indices of B count rows and the indices of A count columns. To the first filtration of its total complex, we can associate the proper spectral sequence ˙EI G(B, FA)  .  If B has enough injectives and B is an injective resolution of Y , then in both cases the E2-terms are a priori seen to be (RiG) Y, (RjF)(X)  . So also the following assertion is well-motivated. Theorem 34. We have ˙EGr F,G(Y,−)(X) ≃˙EI G(B, FA)  . Theorem 34. We have ˙EGr F,G(Y,−)(X) ≃˙EI G(B, FA)  . So instead of “resolving X twice”, we may just as well “resolve X once and Y once”. The left hand side spectral sequence converges to Ri+j(G(Y, −) ◦F)  (X). By this theorem, so does the right hand side one. The underlying double complexes are connected by two morphisms of double complexes (in the directions • - •  •) that induce isomorphisms on the associated proper spectral sequences. Of course, Theorems 31 and 34 have dual counterparts. Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Conventions We have a full embedding A - Conc C[0(A), where, given X ∈Ob A, the complex Conc X has entry X at position 0 and zero elsewhere. • Denote by C(A) the category of complexes X = (· · · - d Xi−1 - d Xi - d Xi+1 - d · · · ) X = (· · · - Xi−1 - Xi - Xi+1 - · · · ) with values in A. Denote by C[0(A) the full subcategory of C(A) consisting of com- plexes X with Xi = 0 for i < 0. We have a full embedding A - Conc C[0(A), where, given X ∈Ob A, the complex Conc X has entry X at position 0 and zero elsewhere. with values in A. Denote by C[0(A) the full subcategory of C(A) consisting of com- Conc [ plexes X with Xi = 0 for i < 0. We have a full embedding A - Conc C[0(A), where, given X ∈Ob A, the complex Conc X has entry X at position 0 and zero elsewhere. • Given a complex X ∈Ob C(A) and k ∈Z, we denote by X•+k the complex that has differential Xi+k - (−1)kd Xi+1+k between positions i and i + 1. We also write X•−1 := X•+(−1) etc. • Given a complex X ∈Ob C(A) and k ∈Z, we denote by X•+k the complex that has differential Xi+k - (−1)kd Xi+1+k between positions i and i + 1. We also write X•−1 := X•+(−1) etc. • Suppose given a full additive subcategory M ⊆A. Then A/M denotes the quotient of A by M, which has the same objects as A, and which has as morphisms residue classes of morphisms of A, where two morphisms are in the same residue class if their difference factors over an object of M. • Suppose given a full additive subcategory M ⊆A. Then A/M denotes the quotient of A by M, which has the same objects as A, and which has as morphisms residue classes of morphisms of A, where two morphisms are in the same residue class if their difference factors over an object of M. • A morphism in A is split if it isomorphic, as a diagram on • - •, to a morphism of the form X ⊕Y - “ 1 0 0 0 ” X ⊕Z. Conventions Throughout these conventions, let C and D be categories, let A be an additive category, let B and B′ be abelian categories, and let E be an exact category in which all idempotents split. • For a, b ∈Z, we write [a, b] := {c ∈Z : a ≤c ≤b}, [a, b[ := {c ∈Z : a ≤c < b}, etc. • For a, b ∈Z, we write [a, b] := {c ∈Z : a ≤c ≤b}, [a, b[ := {c ∈Z : a ≤c < b}, etc. • For a, b ∈Z, we write [a, b] := {c ∈Z : a ≤c ≤b}, [a, b[ := {c ∈Z : a ≤c < b etc. • Given I ⊆Z and i ∈Z, we write I≥i := {j ∈I : j ≥i} and I<i := {j ∈I : j < i} • The disjoint union of sets A and B is denoted by A ⊔B. b • Composition of morphisms is written on the right, i.e. - a - b = - ab . • Functors act on the left. Composition of functors is written on the left, i.e. - F - G = - G◦F • Given objects X, Y in C, we denote the set of morphisms from X to Y by C(X, Y ). • The category of functors from C to D and transformations between them is denoted by C, D . Documenta Mathematica 13 (2008) 677–737 682 Matthias K¨unzer • Denote by C(A) the category of complexes X = (· · · - d Xi−1 - d Xi - d Xi+1 - d · · · ) with values in A. Denote by C[0(A) the full subcategory of C(A) consisting of com- plexes X with Xi = 0 for i < 0. We have a full embedding A - Conc C[0(A), where, given X ∈Ob A, the complex Conc X has entry X at position 0 and zero elsewhere. • Denote by C(A) the category of complexes X = (· · · - d Xi−1 - d Xi - d Xi+1 - d · · · ) with values in A. Denote by C[0(A) the full subcategory of C(A) consisting of com- plexes X with Xi = 0 for i < 0. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 683 • A morphism X - Y in C(E) between complexes X and Y with pure differentials is a quasiisomorphism if Hk applied to it yields an isomorphism for all k ∈Z. A complex X with pure differentials is acyclic if HkX ≃0 for all k ≥0. Such a complex is also called a purely acyclic complex. • Suppose that B has enough injectives. Given a left exact functor B - F B′, an object X ∈Ob B is F -acyclic if RiF X ≃0 for all i ≥1. In other words, X is F -acyclic if for an injective resolution I ∈C[0(Inj B) of X (and then for all such injective resolutions), we have HiF I ≃0 for all i ≥1. • By a module, we understand a left module, unless stated otherwise. If A is a ring, we abbreviate A(−, =) := A -Mod(−, =) = HomA(−, =). Conventions A complex X ∈Ob C(A) is split if all of its differentials are split. • An elementary split acyclic complex in C(A) is a complex of the form where the entry T is at positions k and k + 1 for some k ∈Z. A split acyclic complex is a complex isomorphic to a direct sum of elementary split acyclic complexes, i.e. a complex isomorphic to a complex of the form p p p · · · - “ 0 0 1 0 ” T i ⊕T i+1 - “ 0 0 1 0 ” T i+1 ⊕T i+2 - “ 0 0 1 0 ” T i+2 ⊕T i+3 - “ 0 0 1 0 ” · · Let Csp ac(A) ⊆C(A) denote the full additive subcategory of split acyclic complexes. Let K(A) := C(A)/Csp ac(A) denote the homotopy category of complexes with values in A. Let K[0(A) denote the image of C[0(A) in K(A). A morphism in C(A) is a homotopism if its image in K(A) is an isomorphism. • We denote by Inj B ⊆B the full subcategory of injective objects. We denote by Inj B ⊆B the full subcategory of injective objects. • Concerning exact categories, introduced by Quillen [14, p. 15], we use the conventions of [10, Sec. A.2]. In particular, a commutative quadrangle in E being a pullback is indicated by • Concerning exact categories, introduced by Quillen [14, p. 15], we use the conventions of [10, Sec. A.2]. In particular, a commutative quadrangle in E being a pullback is indicated by A /  B  C / D , A /  B  C / D , a commutative quadrangle being a pushout by A /  B  C / D . A /  B  C / D . • Given X ∈Ob C(E) with pure differentials, and given k ∈Z, we denote by ZkX the kernel of the differential Xk - Xk+1, by Z′kX the cokernel of the differ- ential Xk−1 - Xk, and by BkX the image of the differential Xk−1 - Xk. Furthermore, we have pure short exact sequences BkX - r ZkX - HkX and HkX - r Z′kX - Bk+1X. Documenta Mathematica 13 (2008) 677–737 1 Double and triple complexes We fix some notations and sign conventions. We fix some notations and sign conventions. Let A and B be additive categories. Let C(A) - H B be an additive functor. 1.1.2 Applying H in different directions Given X ∈Ob CC(A), we let H(X∗,−) ∈Ob C(A) denote the complex that has H(X∗,j) at position j ∈Z, and as differential H(X∗,j) - H(X∗,j+1) the image of the morphism X∗,j - X∗,j+1 of complexes under H. Similarly, H(X−,∗) ∈Ob C(A) has H(Xj,∗) at position j ∈Z. In other words, a “∗” denotes the index direction to which H is applied, a “−” denotes the surviving index direction. For short, “∗” before “−”. 1.1.3 Concentrated double complexes Given a complex U ∈Ob C[0(A), we denote by Conc2 U ∈Ob CC⌞(A) the double complex whose 0th row is given by U, and whose other rows are zero; i.e. given j ∈Z, then (Conc2 U)i,j equals U j if i = 0, and 0 otherwise, the differentials taken accordingly. Similarly, Conc1 U ∈Ob CC⌞(B) denotes the double complex whose 0th column is given by U, and whose other columns are zero. 1.1.1 Definition Similarly, given j ∈Z, X∗,j ∈Ob C(A) denotes the jth column of X. 1.1.1 Definition A double complex with entries in A is a diagram A double complex with entries in A is a diagram ... ... ... · · · d / Xi+2,j d / ∂ O Xi+2,j+1 d / ∂ O Xi+2,j+2 d / ∂ O · · · X = · · · d / Xi+1,j d / ∂ O Xi+1,j+1 d / ∂ O Xi+1,j+2 d / ∂ O · · · · · · d / Xi,j d / ∂ O Xi,j+1 d / ∂ O Xi,j+2 d / ∂ O · · · ... ∂ O ... ∂ O ... ∂ O in A such that dd = 0, ∂∂= 0 and d∂= ∂d everywhere. As morphisms between double complexes, we take all diagram morphisms. Let CC(A) denote the category of double complexes. We may identify CC(A) = C(C(A)). The double complexes considered in this §1.1 are stipulated to have entries in A. Let CC⌞(A) := C[0(C[0(A)) be the category of first quadrant double complexes, consisting of double complexes X such that Xi,j = 0 whenever i < 0 or j < 0. X = in A such that dd = 0, ∂∂= 0 and d∂= ∂d everywhere. As morphisms between double complexes, we take all diagram morphisms. Let CC(A) denote the category of double complexes. We may identify CC(A) = C(C(A)). The double complexes considered in this §1.1 are stipulated to have entries in A. Let CC⌞(A) := C[0(C[0(A)) be the category of first quadrant double complexes, consisting of double complexes X such that Xi,j = 0 whenever i < 0 or j < 0. in A such that dd = 0, ∂∂= 0 and d∂= ∂d everywhere. As morphisms between double complexes, we take all diagram morphisms. Let CC(A) denote the category of double complexes. We may identify CC(A) = C(C(A)). The double complexes considered in this §1.1 are stipulated to have entries in Let CC⌞(A) := C[0(C[0(A)) be the category of first quadrant double complexes, consisting of double complexes X such that Xi,j = 0 whenever i < 0 or j < 0. Documenta Mathematica 13 (2008) 677–737 684 Matthias K¨unzer Given a double complex X and i ∈Z, we let Xi,∗∈Ob C(A) denote the com- plex that has entry Xi,j at position j ∈Z, the differentials taken accordingly; Xi,∗is called the ith row of X. 1.1.4 Row- and columnwise notions A morphism X - f Y of double complexes is called a rowwise homotopism if Xi,∗ - f i,∗ Y i,∗is a homotopism for all i ∈Z. Provided A is abelian, it is called a rowwise quasiisomorphism if Xi,∗ - f i,∗ Y i,∗is a quasiisomorphism for all i ∈Z. A morphism X - f Y of double complexes is called a columnwise homotopism if X∗,j - f ∗,j Y ∗,j is a homotopism for all j ∈Z. Provided A is abelian, it is called a columnwise quasiisomorphism if X∗,j - f ∗,j Y ∗,j is a quasiisomorphism for all j ∈Z. A morphism X - f Y of double complexes is called a columnwise homotopism if X∗,j - f ∗,j Y ∗,j is a homotopism for all j ∈Z. Provided A is abelian, it is called a columnwise quasiisomorphism if X∗,j - f ∗,j Y ∗,j is a quasiisomorphism for all j ∈Z. Provided A is abelian, a double complex X is called rowwise split if Xi,∗is split for all i ∈Z; a short exact sequence X′ - X - X′′ of double complexes is called rowwise split short exact if X′i,∗ - Xi,∗ - X′′i,∗is split short exact for all i ∈Z. A double complex X is called rowwise split acyclic if Xi,∗is a split acyclic complex for all i ∈Z. It is called columnwise split acyclic if X∗,j is a split acyclic complex for all j ∈Z. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 685 1.1.5 Horizontally and vertically split acyclic double complexes Comparison of Spectral Sequences Involving Bifunctors 685 1.1.5 Horizontally and vertically split acyclic double complexes 1.1.5 Horizontally and vertically split acyclic double complexes An elementary horizontally split acyclic double complex is a double complex of the form ... ... ... ... · · · / 0 O / T i+1 O T i+1 / O 0 O / · · · · · · / 0 O / T i ∂ O T i / ∂ O 0 O / · · · ... O ... O ... O ... O . A horizontally split acyclic double complex is a double complex isomorphic to a direct sum of elementary horizontally split acyclic double complexes, i.e. 1.1.4 Row- and columnwise notions to one of the form A horizontally split acyclic double complex is a double complex isomorphic to a direct sum of elementary horizontally split acyclic double complexes, i.e. to one of the form A horizontally split acyclic double complex is a double complex isomorphic to a direct sum of elementary horizontally split acyclic double complexes, i.e. to one of the form ... ... · · · / T i+1,j⊕T i+1,j+1 “ 0 0 1 0 ” / O T i+1,j+1⊕T i+1,j+2 / O · · · · · · / T i,j⊕T i,j+1 “ 0 0 1 0 ” / “∂0 0 ∂ ” O T i,j+1⊕T i,j+2 / “∂0 0 ∂ ” O · · · ... O ... O . Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 686 Matthias K¨unzer An elementary vertically split acyclic double complex is a double complex of the form An elementary vertically split acyclic double complex is a double complex of the form ... ... · · · / 0 / O 0 / O · · · · · · / T i d / O T i+1 / O · · · · · · / T i d / T i+1 / · · · · · · / 0 / O 0 / O · · · ... O ... O . A vertically split acyclic double complex is a double complex isomorphic to a direct sum of elementary vertically split acyclic double complexes, i.e. to one of the form A vertically split acyclic double complex is a double complex isomorphic to a direct sum of elementary vertically split acyclic double complexes, i.e. to one of the form ... ... · · · / T i+1,j⊕T i+2,j “ d 0 0 d ” / O T i+1,j+1⊕T i+2,j+1 / O · · · · · · / T i,j⊕T i+1,j “ d 0 0 d ” / “0 0 1 0 ” O T i,j+1⊕T i+1,j+1 / “ 0 0 1 0 ” O · · · ... O ... O . A horizontally split acyclic double complex is in particular rowwise split acyclic. A vertically split acyclic double complex is in particular columnwise split Documenta Mathematica 13 (2008) 677–737 ... ... 1.1.6 Total complex Let KK⌞(A) be the full image of CC⌞(A) in KK(A). Th t t l l tX f d bl l X ∈Ob CC⌞(A) i gi b th Let KK⌞(A) be the full image of CC⌞(A) in KK(A). ( ) g ( ) ( ) The total complex tX of a double complex X ∈Ob CC⌞(A) is given by the complex tX =  X0,0 - ( d ∂) X0,1 ⊕X1,0 - “d ∂ 0 0 −d −∂ ” X0,2 ⊕X1,1 ⊕X2,0 - d ∂ 0 0 0 −d −∂0 0 0 d ∂ ! X0,3 ⊕X1,2 ⊕X2,1 ⊕X3,0 - · · ·  in Ob C[0(A). Using the induced morphisms, we obtain a total complex func- tor CC⌞(A) - t C[0(A). Since t maps elementary horizontally or vertically split acyclic double complexes to split acyclic complexes, it induces a functor KK⌞(A) - t K[0(A). If, in addition, A is abelian, the total complex func- tor maps rowwise quasiisomorphisms and columnwise quasiisomorphisms to quasiisomorphisms, as one sees using the long exact homology sequence and induction on a suitable filtration. in Ob C[0(A). Using the induced morphisms, we obtain a total complex func- tor CC⌞(A) - t C[0(A). Since t maps elementary horizontally or vertically split acyclic double complexes to split acyclic complexes, it induces a functor KK⌞(A) - t K[0(A). If, in addition, A is abelian, the total complex func- tor maps rowwise quasiisomorphisms and columnwise quasiisomorphisms to quasiisomorphisms, as one sees using the long exact homology sequence and induction on a suitable filtration. Note that we have an isomorphism U - ∼ t Conc1 U, natural in U ∈Ob C[0(A), having entries 1U0, 1U1, −1U2, −1U3, 1U4, etc. Moreover, U = t Conc2 U, natural in U ∈Ob C[0(A). KK(A) := CC(A)/CCsp ac(A) ; cf. [5, IV.§4]. A morphism in CC(A) that is mapped to an isomorphism in KK(A) is called a double homotopism. A speculative aside. The category K(A) is Heller triangulated; cf. [10, Def. 1.5.(i), Th. 4.6]. Such a Heller triangulation hinges on two in- duced shift functors, one of them induced by the shift functor on K(A). Now KK(A) carries two shift functors, and so there might be more isomorphisms between induced shift functors one can fix. How can the formal structure of KK(A) be described? 1.1.4 Row- and columnwise notions · · · / T i+1,j⊕T i+2,j “ d 0 0 d ” / O T i+1,j+1⊕T i+2,j+1 / O · · · · · · / T i,j⊕T i+1,j “ d 0 0 d ” / “0 0 1 0 ” O T i,j+1⊕T i+1,j+1 / “ 0 0 1 0 ” O · · · ... O ... O . A horizontally split acyclic double complex is in particular rowwise split acyclic. A vertically split acyclic double complex is in particular columnwise split Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 687 acyclic. y A double complex is called split acyclic if it is isomorphic to the direct sum of a horizontally and a vertically split acyclic double complex. Let CCsp ac(A) denote the full additive subcategory of split acyclic double complexes. Let KK(A) := CC(A)/CCsp ac(A) ; Documenta Mathematica 13 (2008) 677–737 1.2.1 Definition Let CCC(A) := C(C(C(A))) be the category of triple complexes. A triple complex Y has entries Y k,ℓ,m for k, ℓ, m ∈Z. Let CCC(A) := C(C(C(A))) be the category of triple complexes. A triple complex Y has entries Y k,ℓ,m for k, ℓ, m ∈Z. We denote the differentials in the three directions by Y k,ℓ,m - d1 Y k+1,ℓ,m, Y k,ℓ,m - d2 Y k,ℓ+1,m and Y k,ℓ,m - d3 Y k,ℓ,m+1, respectively. Let k, ℓ, m ∈Z. We shall use the notation Y −,ℓ,= for the double complex hav- ing at position (k, m) the entry Y k,ℓ,m, differentials taken accordingly. Similarly the complex Y k,ℓ,∗etc. Given a triple complex Y ∈Ob CCC(A), we write HY −,=,∗∈Ob CC(A) for the double complex having at position (k, ℓ) the entry H(Y k,ℓ,∗), differentials taken accordingly. Denote by CCC (A) ⊆CCC(A) the full subcategory of first octant triple com- plexes; i.e. triple complexes Y having Y k,ℓ,m = 0 whenever k < 0 or ℓ< 0 or m < 0. 1.1.7 The homotopy category of first quadrant double com- plexes as a quotient Lemma 1 The residue class functor CC(A) - KK(A), restricted to CC⌞(A) - KK⌞(A), induces an equivalence CC⌞(A)/ CCsp ac(A) ∩CC⌞(A)  - ∼ KK⌞(A) . CC⌞(A)/ CCsp ac(A) ∩CC⌞(A)  - ∼ KK⌞(A) . Documenta Mathematica 13 (2008) 677–737 688 Matthias K¨unzer Proof. We have to show faithfulness; i.e. that if a morphism X - Y in CC⌞(A) factors over a split acyclic double complex, then it factors over a split acyclic double complex that lies in Ob CC⌞(A). By symmetry and additivity, it suffices to show that if a morphism X - Y in CC⌞(A) factors over a hor- izontally split acyclic double complex, then it factors over a horizontally split acyclic double complex that lies in Ob CC⌞(A). Furthermore, we may assume X - Y to factor over an elementary horizontally split acyclic double complex S concentrated in the columns k and k + 1 for some k ∈Z. We may assume that Si,j = 0 for i < 0 and j ∈Z. If k < 0, and in particular, if k = −1, then X - Y is zero because S - Y is zero, so that in this case we may assume S = 0. On the other hand, if k ≥0, then S ∈Ob CC⌞(A). Cf. also the similar Remark 2. Cf. also the similar Remark 2. Cf. also the similar Remark 2. 2 Cartan-Eilenberg resolutions We shall use Quillen’s language of exact categories [14, p. 15] to deal with Cartan-Eilenberg resolutions [5, XVII.§1], as it has been done by Mac Lane already before this language was available; cf. [12, XII.§11]. The assertions in this section are for the most part wellknown. Remark 2 Let A be an additive category. Then C[0(A)/ C[0(A) ∩Csp ac(A)  - K[0(A) C[0(A)/ C[0(A) ∩Csp ac(A)  - K[0(A) is an equivalence. Proof. Faithfulness is to be shown. A morphism X - Y in C[0(A) that factors over an elementary split acyclic complex of the form (· · · - 0 - T T - 0 - · · · ) with T in positions k and k + 1 is zero, provided k < 0. ( is zero, provided k < 0. By means of induced morphisms, this furnishes a functor CCC (A) - t1,2 CC⌞(A) Y - t1,2Y . CCC (A) - t1,2 CC⌞(A) Y - t1,2Y . 1.2.2 Planewise total complex For Y ∈Ob CCC (A) we denote by t1,2Y ∈Ob CC⌞(A) the planewise total complex of Y , defined for m ∈Z as (t1,2Y )∗,m := t(Y −,=,m) , with the differentials of t1,2Y in the horizontal direction being induced by the differentials in the third index direction of Y , and with the differentials of t1,2Y in the vertical direction being given by the total complex differentials. Explicitly, given k, ℓ≥0, we have with the differentials of t1,2Y in the horizontal direction being induced by the differentials in the third index direction of Y , and with the differentials of t1,2Y in the vertical direction being given by the total complex differentials. Explicitly, given k, ℓ≥0, we have (t1,2Y )k,ℓ= M i, j ≥0, i+j = k Y i,j,ℓ. Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 2.2 Exact categories Concerning the terminology of exact categories, introduced by Quillen [14, p. 15], we refer to [10, Sec. A.2]. Concerning the terminology of exact categories, introduced by Quillen [14, p. 15], we refer to [10, Sec. A.2]. Let E be an exact category in which all idempotents split. An object I ∈Ob E is called relatively injective, or a relative injective (relative to the set of pure short exact sequences, that is), if E(−, I) maps pure short exact sequences of E to short exact sequences. We say that E has enough relative injectives, if for all X ∈Ob E, there exists a relative injective I and a pure monomorphism X - r I. In case E is an abelian category, with all short exact sequences stipulated to be pure, then we omit “relative” and speak of “injectives” etc. Definition 3 Suppose given a complex X ∈Ob C[0(E) with pure differentials. A relatively injective complex resolution of X is a complex I ∈Ob C[0(E), together with a quasiisomorphism X - I, such that the following properties are satisfied. (1) The object entries of I are relatively injective. (2) The differentials of I are pure. (3) The quasiisomorphism X - I consists of pure monomorphisms. Documenta Mathematica 13 (2008) 677–737 690 Matthias K¨unzer We often refer to such a relatively injective complex resolution just by I. A relatively injective object resolution, or just a relatively injective resolution, of an object Y ∈Ob E is a relatively injective complex resolution of Conc Y . A relatively injective resolution is the complex of a relatively injective object resolution of some object in E. We often refer to such a relatively injective complex resolution just by I. A relatively injective object resolution, or just a relatively injective resolution, of an object Y ∈Ob E is a relatively injective complex resolution of Conc Y . A relatively injective resolution is the complex of a relatively injective object resolution of some object in E. of an object Y ∈Ob E is a relatively injective complex resolution of Conc Y . A relatively injective resolution is the complex of a relatively injective object resolution of some object in E. Remark 4 Suppose that E has enough relative injectives. Every complex X ∈ Ob C[0(E) with pure differentials has a relatively injective complex resolution I ∈Ob C[0(E). 2.2 Exact categories Embed them into a morphism from the pure short exact sequence X′ - r X - X′′ X′ - r X - X′′ to the split short exact sequence I′ - (1 0) I′ ⊕I′′ - “0 1 ” I′′ . Insert the pushout T of X′ - r X along X′ - r I′0 and the pullback of I′0 ⊕I′′0 - I′′0 along X′′ - r I′′0 to see that X - I′0 ⊕I′′0 is purely monomorphic. So we can take the cokernel B1I′ - B1I - B1I′′ of this morphism of pure short exact sequences. Considering the cokernels on the commutative triangle (X, T, I′0 ⊕I′′0) of pure monomorphisms, we obtain a bicartesian square (T, I′0 ⊕I′′0, B1I′, B1I) and conclude that the sequence of cokernels is itself purely short exact. So we can iterate. Insert the pushout T of X′ - r X along X′ - r I′0 and the pullback of I′0 ⊕I′′0 - I′′0 along X′′ - r I′′0 to see that X - I′0 ⊕I′′0 is purely monomorphic. So we can take the cokernel B1I′ - B1I - B1I′′ of this morphism of pure short exact sequences. Considering the cokernels on the commutative triangle (X, T, I′0 ⊕I′′0) of pure monomorphisms, we obtain a bicartesian square (T, I′0 ⊕I′′0, B1I′, B1I) and conclude that the sequence of cokernels is itself purely short exact. So we can iterate. 2.2 Exact categories In particular, every object Y ∈Ob E has a relatively injective resolution J ∈Ob C[0(E). Proof. Let X0 - r I0 be a pure monomorphism into a relatively injective object I0. Forming a pushout along X0 - r I0, we obtain a pointwise purely monomorphic morphism of complexes X - X′ with X′0 = I0 and X′k = Xk for k ≥2. By considering its cokernel, we see that it is a quasiisomorphism. So we may assume X0 to be relatively injective. Let X1 - r I1 be a pure monomorphism into a relatively injective object I1. Form a pushout along X1 - r I1 etc. Remark 5 Suppose given X ∈Ob C[0(E) with pure differentials such that HkX ≃0 for k ≥1. Suppose given I ∈Ob C[0(E) such that Ik is purely injective for k ≥0, and such that the differential I0 - d I1 has a kernel in E. Then the map K[0(E)(X, I) - E Kern(X0 - d X1), Kern(I0 - d I1)  that sends a representing morphism of complexes to the morphism induced on the mentioned kernels, is bijective. Suppose E to have enough relative injectives. Let I ⊆E denote the full subcat- egory of relative injectives. Let C[0, res(I) denote the full subcategory of C[0(I) consisting of complexes X with pure differentials such that HkX ≃0 for k ≥1. Let K[0, res(I) denote the image of C[0, res(I) in K(E). Remark 6 The functor C[0, res(I) - E, X - H0(X), induces an equiva- lence K[0, res(I) - ∼ E . K[0, res(I) - ∼ E . Proof. This functor is dense by Remark 4, and full and faithful by Remark 5. Remark 7 (exact Horseshoe Lemma) Remark 7 (exact Horseshoe Lemma) Given a pure short exact sequence X′ - X - X′′ and relatively injective resolutions I′ of X′ and I′′ of X′′, there exists a relatively injective resolution I of X and a pointwise split short exact sequence I′ - I - I′′ that maps under H0 to X′ - X - X′′. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 691 Comparison of Spectral Sequences Involving Bifunctors 691 Proof. Choose pure monomorphisms X′ - r I′0 and X′′ - r I′′0 into relative injectives I′0 and I′′0. 2.3 An exact category structure on C(A) Let A be an abelian category with enough injectives. Let A be an abelian category with enough injectives. Remark 8 The following conditions on a short exact sequence X′ - X - X′′ in C(A) are equivalent. (1) All connectors in its long exact homology sequence are equal to zero. (1) All connectors in its long exact homology sequence are equal to zero (2) The sequence BkX′ - BkX - BkX′′ is short exact for all k ∈Z. (2) The sequence BkX′ - BkX - BkX′′ is short exact for all k ∈Z. (3) The morphism ZkX - ZkX′′ is epimorphic for all k ∈Z. (3′) The morphism Z′kX′ - Z′kX is monomorphic for all k ∈Z. (4) The diagram BkX′ /  ZkX′ /  HkX′  BkX /  ZkX /  HkX  BkX′′ / ZkX′′ / HkX′′ has short exact rows and short exact columns for all k ∈Z. has short exact rows and short exact columns for all k ∈Z. Proof. We consider the diagram in (4) as a (horizontal) short exact sequence of (vertical) complexes and regard its long exact homology sequence. Taking into account that all assertions are supposed to hold for all k ∈Z, we can employ the long exact homology sequence on X′ - X - X′′ to prove the equivalence of (1), (2), (3) and (4). ( ) ( ) ( ) ( ) Now the assertion (1) ⇐⇒(3) is dual to the assertion (1) ⇐⇒(3′). Documenta Mathematica 13 (2008) 677–737 692 Matthias K¨unzer Remark 9 The category C(A), equipped with the set of short exact sequences that have zero connectors on homology as pure short exact sequences, is an exact category with enough relatively injective objects in which all idempotents split. With respect to this exact category structure on C(A), a complex is relatively injective if and only if it is split and has injective object entries. Cf. [12, XII.§11], where pure short exact sequences are called proper. A rela- tively injective object in C(A) is also referred to as an injectively split complex. To a relatively injective resolution of a complex X ∈Ob C(A), we also refer as a Cartan-Eilenberg-resolution, or, for short, as a CE-resolution of X; cf. [5, XVII.§1]. A CE-resolution is a CE-resolution of some complex. Considered as a double complex, it is in particular rowwise split and has injective object entries. Cf. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 693 Comparison of Spectral Sequences Involving Bifunctors 693 we may complete to we may complete to A •@ @ @ @ @ @ @ @ / B Y @ @@@ @ @@ @ >~ ~~~ >~ ~~~ X •~ ~ ~ ~ >~ ~ ~~ • / Z in C(A) with (X, Y, B) and (A, Y, Z) pure short exact sequences. Applying Z′k to this diagram, we conclude that Z′k(A - B) is a monomorphism for k ∈Z, whence A - B is a pure monomorphism. This proves the claim. h d C(A) l C(A) l b l A •@ @ @ @ @ @ @ @ / B Y @ @@@ @ @@ @ >~ ~~~ >~ ~~~ X •~ ~ ~ ~ >~ ~ ~~ • / Z in C(A) with (X, Y, B) and (A, Y, Z) pure short exact sequences. Applying Z′k to this diagram, we conclude that Z′k(A - B) is a monomorphism for k ∈Z, whence A - B is a pure monomorphism. This proves the claim. in C(A) with (X, Y, B) and (A, Y, Z) pure short exact sequences. Applying Z′k to this diagram, we conclude that Z′k(A - B) is a monomorphism for k ∈Z, whence A - B is a pure monomorphism. This proves the claim. Note that idempotents in C(A) are split since C(A) is also an abelian category. We claim relative injectivity of complexes with split differentials and injec- tive object entries. By a direct sum decomposition, and using the fact that any monomorphism from an elementary split acyclic complex with injective entries to an arbitrary complex is split, we are reduced to showing that a pure monomorphism from a complex with a single nonzero injective entry, at posi- tion 0, say, to an arbitrary complex is split. So suppose given I ∈Ob Inj A, X ∈Ob C(A) and a pure monomorphism Conc I - r X. Using Remark 8.(3′), we may choose a retraction to the composite (I - X0 - Z′0X). This yields a retraction to I - X0 that composes to 0 with X−1 - X0, which can be employed for the sought retraction X - Conc I. This proves the claim. Let X ∈Ob C(A). We claim that there exists a pure monomorphism from X to a relatively injective complex. 2.3 An exact category structure on C(A) [12, XII.§11], where pure short exact sequences are called proper. A rela- tively injective object in C(A) is also referred to as an injectively split complex. To a relatively injective resolution of a complex X ∈Ob C(A), we also refer as a Cartan-Eilenberg-resolution, or, for short, as a CE-resolution of X; cf. [5, XVII.§1]. A CE-resolution is a CE-resolution of some complex. Considered as a double complex, it is in particular rowwise split and has injective object entries. Given a morphism X - f X′ in C(A), CE-resolutions J of X and J′ of X′, a morphism J - ˆ f J′ in CC(A) such that (Ji,j - ˆf i,j J′i,j) = (0 - 0) for i < 0 and such that H0(J∗,− - ˆ f ∗,− J′∗,−) = (X - f X′) is called a CE-resolution of X - f X′. By Remarks 9 and 6, each morphism in C(A) has a CE-resolution. Proof of Remark 9. We claim that C(A), equipped with the said set of short exact sequences, is an exact category. We verify the conditions (Ex 1, 2, 3) listed in [10, Sec. A.2]. The conditions (Ex 1◦, 2◦, 3◦) then follow by duality. Note that by Remark 8.(3′), a monomorphism X - Y in C(A) is pure if and only if Z′k(X - Y ) is monomorphic in A for all k ∈Z. Ad (Ex 1). To see that a split monomorphism is pure, we may use additivity of the functor Z′k for k ∈Z. Ad (Ex 2). To see that the composition of two pure monomorphisms is pure, we may use Z′k being a functor for k ∈Z. Ad (Ex 3). Suppose given a commutative triangle Ad (Ex 3). Suppose given a commutative triangle Y @ @ @ @ @@@@ X ?~ ~ ~ ~ ~ ~ ~ ~ • / Z , in C(A). Applying the functor Z′k to it, for k ∈Z, we conclude that Z′k(X - Y ) is monomorphic, whence X - Y is purely monomorphic. So Comparison of Spectral Sequences Involving Bifunctors 693 Comparison of Spectral Sequences Involving Bifunctors 693 Since A has enough injectives, by a direct sum decomposition we are reduced to finding a pure monomorphism from X to a split complex. Consider the following morphism φk of complexes for k ∈Z, · · · / 0 / Xk (1 0) / Xk ⊕Z′kX / 0 / · · · · · · / Xk−2 d / O Xk−1 d / d O Xk d / (1 p) O Xk+1 / O · · · , · · · / 0 / Xk (1 0) / Xk ⊕Z′kX / 0 / · · · · · · / Xk−2 d / O Xk−1 d / d O Xk d / (1 p) O Xk+1 / O · · · , where Xk - p Z′kX is taken from X. The functor Z′k maps it to the identity. We take the direct sum of the upper complexes over k ∈Z and let the mor- phisms φk be the components of a morphism φ from X to this direct sum. At position k, this morphism φ is monomorphic because φk is. Moreover, Z′k(φ) is a monomorphism because Z′k(φk) is. Hence φ is purely monomorphic by condition (3′) of Remark 8. This proves the claim. where Xk - p Z′kX is taken from X. The functor Z′k maps it to the identity. We take the direct sum of the upper complexes over k ∈Z and let the mor- phisms φk be the components of a morphism φ from X to this direct sum. At position k, this morphism φ is monomorphic because φk is. Moreover, Z′k(φ) is a monomorphism because Z′k(φk) is. Hence φ is purely monomorphic by condition (3′) of Remark 8. This proves the claim. y Remark 10 Write E := C(A). Given ℓ≥0, we have a homology functor E - Hℓ A, which induces a functor C(E) - C(Hℓ) C(A). Suppose given a purely acyclic complex X ∈Ob C(E). Then C(Hℓ)X ∈Ob C(A) is acyclic. Documenta Mathematica 13 (2008) 677–737 694 Matthias K¨unzer Proof. This follows using the definition of pure short exact sequences, i.e. Remark 8.(1). 2.4 An exact category structure on C[0(A) Write CC⌞, CE(Inj A) for the full subcategory of CC⌞(A) whose objects are CE-resolutions. Write KK⌞, CE(Inj A) for the full subcategory of KK⌞(A) whose objects are CE-resolutions. Write CC⌞, CE(Inj A) for the full subcategory of CC⌞(A) whose objects are CE-resolutions. Write KK⌞, CE(Inj A) for the full subcategory of KK⌞(A) whose objects are CE-resolutions. Remark 11 The category C[0(A), equipped with the short exact sequences that lie in C[0(A) and that are pure in C(A) in the sense of Remark 9 as pure short exact sequences, is an exact category wherein idempotents are split. It has enough relative injectives, viz. injectively split complexes that lie in C[0(A). Proof. To show that it has enough relative injectives, we replace φ0 in the proof of Remark 9 by X - φ′ 0 Conc X0, defined by X0 - 1X0 X0 at position 0. 2.5 The Cartan-Eilenberg resolution of a quasiisomorphism 2.5 The Cartan-Eilenberg resolution of a quasiisomorphism Abbreviate E := C(A), which is an exact category as in Remark 9. Consider CC⌞(A) ⊆C[0(E), where the second index of X ∈Ob CC⌞(A) counts the positions in E = C(A); i.e. when X is viewed as a complex with values in E, its entry at position k is given by Xk,∗∈E = C(A). Remark 12 Suppose given a split acyclic complex X ∈Ob C[0(A). There exists a horizontally split acyclic CE-resolution J ∈Ob CC⌞, CE(Inj A) of X. Proof. This holds for an elementary split acyclic complex, and thus also in the general case by taking a direct sum. Lemma 13 Suppose given X ∈Ob CC⌞(A) with pure differentials when con- sidered as an object of C[0(E), and with HkX∗,− ≃0 in C[0(A) for k ≥1. Suppose given J ∈Ob CC⌞(Inj A) with split rows Jk,∗for k ≥1. In other words, J is supposed to consist of relative injective object entries when consid- ered as an object of C[0(E). Then the map (∗) KK⌞(A)(X, J) - H0((−)∗,−) K[0(A) H0X∗,− , H0J∗,− is bijective. Proof. First, we observe that by Remark 5, we have (∗∗) K[0(E)(X, J) - H0((−)∗,−) ∼ E H0X∗,− , H0J∗,− . (∗∗) Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 695 Comparison of Spectral Sequences Involving Bifunctors 695 So it remains to show that (∗) is injective. Let X - f J be a morphism that vanishes under (∗). Then H0X∗,− - H0J∗,− factors over a split acyclic complex S ∈Ob C[0(A); cf. Remark 2. Let K be a horizontally split acyclic CE-resolution of S; cf. Remark 12. By Remark 5, we obtain a mor- phism X - K that lifts H0X∗,− - S and a morphism K - J that lifts S - H0J∗,− . The composite X - K - J vanishes in KK⌞(A). The difference (X - f J) −(X - K - J) lifts H0X∗,− - 0 H0J∗,− . Hence by (∗∗), it vanishes in K[0(E) and so a fortiori in KK⌞(A). Altogether, X - f J vanishes in KK⌞(A). lifts H0X∗,− - 0 H0J∗,− . Hence by (∗∗), it vanishes in K[0(E) and so a fortiori in KK⌞(A). Altogether, X - f J vanishes in KK⌞(A). Proposition 14 The functor CC⌞, CE(Inj A) - H0((−)∗,−) C[0(A) induces an equivalence KK⌞, CE(Inj A) - H0((−)∗,−) ∼ K[0(A) . Proof. Documenta Mathematica 13 (2008) 677–737 “ 1 0 ” Since ¯J is rowwise split acyclic and since the sequence J - ˜J′ - ¯J is row- wise split short exact, J - ˜J′ is a rowwise homotopism. Since J - ˜J′ is a f CE-resolution of X - f X′, this proves the proposition. 2.5 The Cartan-Eilenberg resolution of a quasiisomorphism By Lemma 13, this functor is full and faithful. By Remark 4, it is dense. Corollary 15 Suppose given X, X′ ∈Ob C[0(A). Let J be a CE-resolution of X. Let J′ be a CE-resolution of X′. If X and X′ are isomorphic in K[0(A), then J and J′ are isomorphic in KK⌞(A). Corollary 15 Suppose given X, X′ ∈Ob C[0(A). Let J be a CE-resolution of X. Let J′ be a CE-resolution of X′. If X and X′ are isomorphic in K[0(A), then J and J′ are isomorphic in KK⌞(A). Corollary 15 Suppose given X, X′ ∈Ob C[0(A). Let J be a CE-resolution of X. Let J′ be a CE-resolution of X′. If X and X′ are isomorphic in K[0(A), then J and J′ are isomorphic in KK⌞(A). The following lemma is to be compared to Remark 12. Lemma 16 Suppose given an acyclic complex X ∈Ob C[0(A). There exists a rowwise split acyclic CE-resolution J of X. Each CE-resolution of X is isomorphic to J in KK⌞(A). Proof. By Corollary 15, it suffices to show that there exists a rowwise split acyclic CE-resolution of X. Recall that a CE-resolution of an arbitrary com- plex Y ∈Ob C[0(A) can be constructed by a choice of injective resolutions of HkY and BkY for k ∈Z, followed by an application of the abelian Horseshoe Lemma to the short exact sequences BkY - ZkY - HkY for k ∈Z and then to ZkY - Y k - Bk+1Y for k ∈Z; cf. [5, Chap. XVII, Prop. 1.2]. Since HkX = 0 for k ∈Z, we may choose the zero resolution for it. Applying this construction, we obtain a rowwise split acyclic CE-resolution. ˆ Given X - f X′ in C[0(A), a morphism J - f J′ in CC⌞(A) is called a CE-resolution of X - f X′ if H0( ˆf ∗,−) ≃f, as diagrams of the form • - •. By Remark 5, given CE-resolutions J of X and J′ of X′, there exists a CE-resolution J - ˆ f J′ of X - f X′. Documenta Mathematica 13 (2008) 677–737 696 Matthias K¨unzer Proposition 17 Let X - f X′ be a quasiisomorphism in C[0(A). Let J - ˆ f J′ be a CE-resolution of X - f X′. Let A be an abelian category. Documenta Mathematica 13 (2008) 677–737 “ 1 0 ” Since X′⊕A - “ 0 ” X′ is a homotopism, J′⊕B - “ 0 ” J is a double homotopism; cf. Corollary 15. Hence ˆf is a composite of a rowwise homotopism and a double homotopism if and only if this holds for ( ˆf b). So we may assume that f is pointwise split monomorphic, so in particular, monomorphic. By Proposition 14, we may replace the given CE-resolution ˆf by an arbitrary CE-resolution of f between J and an arbitrarily chosen CE-resolution of X′ without changing the property of being a composite of a rowwise homotopism and a double homotopism for this newly chosen CE-resolution of f. Let X - f X′ - ¯X be a short exact sequence in C[0(A). Since f is a quasi- isomorphism, ¯X ∈Ob C[0(A) is acyclic. Let ¯J be a rowwise split acyclic CE-resolution of ¯X; cf. Lemma 16. The short exact sequence X - f X′ - ¯X ¯ ( ) Let X - f X′ - ¯X be a short exact sequence in C[0(A). Since f is a quasi- isomorphism, ¯X ∈Ob C[0(A) is acyclic. Let ¯J be a rowwise split acyclic f Let X - f X′ - ¯X be a short exact sequence in C[0(A). Since f is a quasi- isomorphism, ¯X ∈Ob C[0(A) is acyclic. Let ¯J be a rowwise split acyclic CE-resolution of ¯X; cf. Lemma 16. The short exact sequence X - f X′ - ¯X is pure by acyclicity of ¯X; cf. Remark 8.(1). Hence by the exact Horseshoe Lemma, there exists a rowwise split short exact sequence J - ˜J′ - ¯J of CE-resolutions that maps to X - f X′ - ¯X under H0(−)∗,− ; cf. Remark 7. Since ¯J is rowwise split acyclic and since the sequence J - ˜J′ - ¯J is row- wise split short exact, J - ˜J′ is a rowwise homotopism. Since J - ˜J′ is a f CE-resolution of ¯X; cf. Lemma 16. The short exact sequence X - f X′ - ¯X is pure by acyclicity of ¯X; cf. Remark 8.(1). Hence by the exact Horseshoe Lemma, there exists a rowwise split short exact sequence J - ˜J′ - ¯J of CE-resolutions that maps to X - f X′ - ¯X under H0(−)∗,− ; cf. Remark 7. 2.5 The Cartan-Eilenberg resolution of a quasiisomorphism Then ˆf can be written as a com- posite in CC⌞, CE(Inj A) of a rowwise homotopism, followed by a double homo- topism. Proof. Choose a pointwise split monomorphism X - a A into a split acyclic complex X. We can factor (X - f X′) =  X - (f a ) X′ ⊕A - “1 0 ” X′  , so that (f a) is a pointwise split monomorphism. Let B be a CE-resolution of A. Choosing a CE-resolution b of a, we obtain the factorisation (J - ˆ f J′) =  J - ( ˆ f b) J′ ⊕B - “ 1 0 ” J′  3.1 Pointwise split and pointwise finitely filtered complexes Let Z∞:= {−∞} ⊔Z ⊔{∞}, considered as a linearly ordered set, and thus as a category. Write ]α, β] := {σ ∈Z∞: α < σ ≤β} for α, β ∈Z∞such that α ≤β; etc. Given X ∈Ob Z∞, C(A) , the morphism of X on α ≤β in Z∞shall be denoted by X(α) - x X(β). An object X ∈Ob Z∞, C(A) is called a pointwise split and pointwise finitely filtered complex (with values in A), provided (SFF 1, 2, 3) hold. (SFF 1) We have X(−∞) = 0. i (SFF 1) We have X(−∞) = 0. (SFF 2) The morphism X(α)i - xi X(β)i is split monomorphic for all i ∈Z and all α ≤β in Z∞. i (SFF 3) For all i ∈Z, there exist β0, α0 ∈Z such that X(α)i - xi X(β)i is an identity whenever α ≤β ≤β0 or α0 ≤α ≤β in Z∞. The pointwise split and pointwise finitely filtered complexes with values in A The pointwise split and pointwise finitely filtered complexes with values in A form a full subcategory SFFC(A) ⊆ Z∞, C(A) . Suppose given a pointwise split and pointwise finitely filtered complex X with values in A for the rest of the present §3. form a full subcategory SFFC(A) ⊆ Z∞, C(A) . Suppose given a pointwise split and pointwise finitely filtered complex X with values in A for the rest of the present §3. Let α ∈Z∞. Write ¯X(α) := Cokern X(α −1) - X(α)  for α ∈Z. Given i ∈Z, we obtain X(α)i ≃L σ∈]−∞,α] ¯X(σ)i, which is a finite direct sum. We identify along this isomorphism. In particular, we get as a matrix representa- tion for the differential X(α)i - d X(α)i+1 =  L σ∈]−∞,α] ¯X(σ)i - (di σ,τ)σ,τ L τ∈]−∞,α] ¯X(τ)i+1  , X(α)i - d X(α)i+1 =  L σ∈]−∞,α] ¯X(σ)i - (di σ,τ)σ,τ L τ∈]−∞,α] ¯X(τ)i+1  , where di σ,τ = 0 whenever σ < τ; a kind of lower triangular matrix. Documenta Mathematica 13 (2008) 677–737 3 Formalism of spectral sequences We follow essentially Verdier [17, II.4]; cf. [6, App.]; on a more basic level, cf. [11, Kap. 4]. We follow essentially Verdier [17, II.4]; cf. [6, App.]; on a more basic level, cf. [11, Kap. 4]. We follow essentially Verdier [17, II.4]; cf. [6, App.]; on a more basic level, cf. [11, Kap. 4]. Comparison of Spectral Sequences Involving Bifunctors 697 3.1 Pointwise split and pointwise finitely filtered complexes 3.2 Spectral objects Write Di β/α, β′/α′ := (di σ,τ)σ∈]α,β], τ∈]α′,β′] : X(β/α)i - X(β′/α′)i+1 for i ∈Z and β/α, β′/α′ ∈Z# ∞. Given −∞≤α ≤β ≤γ ≤∞and i ∈Z, we let Di β/α, β′/α′ := (di σ,τ)σ∈]α,β], τ∈]α′,β′] : X(β/α)i - X(β′/α′)i+1 for i ∈Z and β/α, β′/α′ ∈Z# ∞. Given −∞≤α ≤β ≤γ ≤∞and i ∈Z, we let for i ∈Z and β/α, β′/α′ ∈Z# ∞. for i ∈Z and β/α, β′/α′ ∈Z# ∞. Given −∞≤α ≤β ≤γ ≤∞and i ∈Z, we let  X(β/α)i - xi X(γ/α)i  :=  X(β/α)i - (1 0) X(β/α)i ⊕X(γ/β)i   X(γ/α)i - xi X(γ/β)i  :=  X(β/α)i ⊕X(γ/β)i - “ 0 1 ” X(γ/β)i   X(γ/β)i - xi X(α+1/β)i  :=  X(γ/β)i - Di γ/β, β/α X(β/α)i+1  . By periodicity up to shift of complexes, this defines Sp(X). The construction is functorial in X ∈Ob SFFC(A). By periodicity up to shift of complexes, this defines Sp(X). The construction is functorial in X ∈Ob SFFC(A). 3.2 Spectral objects Let ¯Z∞:= Z∞× Z. Write α+k := (α, k), where α ∈Z∞and k ∈Z. Let α+k ≤β+ℓin ¯Z∞if k < ℓor (k = ℓand α ≤β), i.e. let ¯Z∞be linearly ordered via a lexicographical ordering. We have an automorphism α+k - α+k+1 of the poset ¯Z∞, to which we refer as shift. Note that −∞+k = (−∞)+k. We have an order preserving injection Z∞ - ¯Z∞, α - α+0. We use this injection as an identification of Z∞with its image in ¯Z∞, i.e. we sometimes write α := α+0 by abuse of notation. Let ¯Z∞:= Z∞× Z. Write α+k := (α, k), where α ∈Z∞and k ∈Z. Let α+k ≤β+ℓin ¯Z∞if k < ℓor (k = ℓand α ≤β), i.e. let ¯Z∞be linearly ordered via a lexicographical ordering. We have an automorphism α+k - α+k+1 of the poset ¯Z∞, to which we refer as shift. Note that −∞+k = (−∞)+k. p ∞, f ( ) We have an order preserving injection Z∞ - ¯Z∞, α - α+0. We use this injection as an identification of Z∞with its image in ¯Z∞, i.e. we sometimes write α := α+0 by abuse of notation. Let ¯Z# ∞:= {(α, β) ∈¯Z∞× ¯Z∞ : β−1 ≤α ≤β ≤α+1}. We usually write β/α := (α, β) ∈¯Z# ∞; reminiscent of a quotient. The set ¯Z# ∞is partially ordered by β/α ≤β′/α′ :⇐⇒(β ≤β′ and α ≤α′). We have an automorphism β/α - (β/α)+1 := α+1/β of the poset ¯Z# ∞, to which, again, we refer as shift. Documenta Mathematica 13 (2008) 677–737 698 Matthias K¨unzer We write Z# ∞:= {β/α ∈¯Z# ∞: −∞≤α ≤β ≤∞}. Note that any element of ¯Z# ∞can uniquely be written as (β/α)+k for some β/α ∈Z# ∞and some k ∈Z. We shall construct the spectral object Sp(X) ∈Ob ¯Z# ∞, K(A) . The morphism of Sp(X) on β/α ≤β′/α′ in ¯Z# ∞shall be denoted by X(β/α) - x X(β′/α′). We require that  X (β/α)+k - x X (β′/α′)+k =  X(β/α) - x X(β′/α′) •+k for β/α ≤β′/α′ in ¯Z# ∞; i.e., roughly put, that Sp(X) be periodic up to shift of complexes. D fi X β/α  := Cokern X(α) - x X(β)  for β/α ∈Z# ∞. By periodicity, we conclude that X α/α  = 0 and X α+1/α  = 0 for all α ∈¯Z∞. 3.3 Spectral sequences Let ¯Z## ∞ := {(γ/α, δ/β) ∈¯Z# ∞× ¯Z# ∞: δ−1 ≤α ≤β ≤γ ≤δ ≤α+1}. Given (γ/α, δ/β) ∈¯Z## ∞, we usually write δ/β//γ/α := (γ/α, δ/β). The set ¯Z## ∞ is partially ordered by Let ¯Z## ∞ := {(γ/α, δ/β) ∈¯Z# ∞× ¯Z# ∞: δ−1 ≤α ≤β ≤γ ≤δ ≤α+1}. Given (γ/α, δ/β) ∈¯Z## ∞, we usually write δ/β//γ/α := (γ/α, δ/β). The set ¯Z## ∞ is partially ordered by δ/β//γ/α ≤δ′/β′//γ′/α′ :⇐⇒ (γ/α ≤γ′/α′ and δ/β ≤δ′/β′) . δ/β//γ/α ≤δ′/β′//γ′/α′ :⇐⇒ (γ/α ≤γ′/α′ and δ/β ≤δ′/β′) . Define the spectral sequence E(X) ∈Ob ¯Z## ∞, A of X by letting its value on δ/β//γ/α ≤δ′/β′//γ′/α′ Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 699 Comparison of Spectral Sequences Involving Bifunctors 699 in ¯Z## ∞ be the morphism that appears in the middle column of the diagram in ¯Z## ∞ be the morphism that appears in the middle column of the diagram H0X(γ/α)   / H0(x)  E(δ/β//γ/α)(X) • / e  H0X(δ/β)  H0(x)  H0X(γ′/α′)   / E(δ′/β′//γ′/α′)(X) • / H0X(δ′/β′)  . Given δ/β//γ/α ∈¯Z## ∞ and k ∈Z, we also write E(δ/β//γ/α)+k(X) := E (δ/β)+k//(γ/α)+k (X) . Given δ/β//γ/α ∈¯Z## ∞ and k ∈Z, we also write E(δ/β//γ/α)+k(X) := E (δ/β)+k//(γ/α)+k (X) . Altogether, Z∞, C(A) ⊇ SFFC(A) - ¯Z# ∞, K(A) - ¯Z## ∞, A X - Sp(X) - E(X) . E(ε/γ//δ/α)(X) - r e E(ε/γ//δ/β)(X) - e E(α+1/γ//δ/β)(X) . Proof. Apply the functor induced by β/α - α+1/β to Sp(X). Then apply [10, Lem. 3.9]. Proof. Apply the functor induced by β/α - α+1/β to Sp(X). Then apply [10, Lem. 3.9]. [ , ] The short exact sequence in Lemma 18 is called a fundamental short exact sequence (in first notation), the short exact sequence in Lemma 19 is called a fundamental short exact sequence (in second notation). They will be used without further comment. E(ε/β//γ/α)(X) - r e E(ε/β//δ/α)(X) - e E(ε/γ//δ/α)(X) . E(ε/β//γ/α)(X) - r e E(ε/β//δ/α)(X) - e E(ε/γ//δ/α)(X) . Proof. See [10, Lem. 3.9]. Lemma 19 Given ε−1 ≤α ≤β ≤γ ≤δ ≤ε ≤α+1 in ¯Z∞, we have a short exact sequence Lemma 19 Given ε−1 ≤α ≤β ≤γ ≤δ ≤ε ≤α+1 in ¯Z∞, we have a short exact sequence 3.4 A short exact sequence Lemma 18 Given ε−1 ≤α ≤β ≤γ ≤δ ≤ε ≤α+1 in ¯Z∞, we have a short exact sequence 3.5 Classical indexing Let 1 ≤r ≤∞and let p, q ∈Z. Denote Ep,q r = Ep,q r (X) := E(−p −1 + r/−p −1//−p/−p −r)+p+q(X) , where i + ∞:= ∞and i −∞:= −∞for all i ∈Z. where i + ∞:= ∞and i −∞:= −∞for all i ∈Z. where i + ∞:= ∞and i −∞:= −∞for all i ∈Z. Documenta Mathematica 13 (2008) 677–737 700 Matthias K¨unzer Example 20 The short exact sequences in Lemmata 18, 19 allow to derive the exact couples of Massey. Write Di,j r = Di,j r (X) := E(−i/−∞//−i−r+1/−∞)+i+j(X) Di,j r = Di,j r (X) := E(−i/−∞//−i−r+1/−∞)+i+j(X) for i, j ∈Z and r ≥1. We obtain an exact sequence for i, j ∈Z and r ≥1. We obtain an exact sequence Di, j r - e Di−1, j+1 r - e Ei+r−2, j−r+2 r - e Di+r−1, j−r+2 r - e Di+r−2, j−r+3 r by Lemmata 18, 19. We record the following wellknown lemma in the language we use here. E(∞/β//γ/δ−1)+k - r e E(∞/β//γ/α)+k - e E(δ/β//γ/α)+k . Claim 7. The morphism ˙E(f) is an isomorphism. Suppose given α, β, γ, δ ∈¯Z∞such that δ−1 ≤α ˙< β ≤γ ˙< δ ≤α+1. Via a shift, we may assume that we are in the situation of Claim 5 or of Claim 6. Suppose given α, β, γ, δ ∈¯Z∞such that δ−1 ≤α ˙< β ≤γ ˙< δ ≤α+1. Via a shift, we may assume that we are in the situation of Claim 5 or of Claim 6. E(δ/β//γ −1/α)+k - r e E(δ/β//γ/α)+k - e E(δ/γ −1//γ/α)+k . E(δ/β//γ −1/α)+k - r e E(δ/β//γ/α)+k - e E(δ/γ −1//γ/α)+k . Claim 4. We have an isomorphism E(δ/β//γ/α)+k(f) for all k ∈Z and all α, β, γ, δ ∈Z∞such that α < β ≤γ < δ. Claim 4. We have an isomorphism E(δ/β//γ/α)+k(f) for all k ∈Z and all α, β, γ, δ ∈Z∞such that α < β ≤γ < δ. In view of Claim 3, it suffices to choose ˜α ∈Z small enough such that E(δ/β// /˜)+k(f) E(δ/β// / )+k(f) t In view of Claim 3, it suffices to choose ˜α ∈Z small enough such that E(δ/β//γ/˜α)+k(f) = E(δ/β//γ/−∞)+k(f); etc. Claim 5. We have an isomorphism E(δ/β//γ/α)+k(f) for all k ∈Z and all α, β, γ, δ ∈Z∞such that α ˙< β ≤γ ˙< δ. In view of Claim 4, it suffices to choose ˜β ∈Z small enough such that E(δ/ ˜β//γ/−∞)+k(f) = E(δ/−∞//γ/−∞)+k(f); etc. ( / //γ/ ) ( ) ( / //γ/ ) ( ) Claim 6. We have an isomorphism E(δ/β//γ/α)+k(f) for all k ∈Z and all α, β, γ, δ ∈¯Z∞such that −∞≤δ−1 ≤α ˙< β ≤γ ≤∞< −∞+1 ≤δ ≤α+1. In view of Claim 5, it suffices to consider the short exact sequence 3.6 Comparing proper spectral sequences Let X - f Y be a morphism in SFFC(A), i.e. a morphism of pointwise split and pointwise finitely filtered complexes with values in A. Write E(X) - E(f) E(Y ) for the induced morphism on the spectral sequences. For α, β ∈¯Z∞, we write α ˙< β if For α, β ∈¯Z∞, we write α ˙< β if α < β  or α = β and α ∈{∞+k : k ∈Z} ∪{−∞+k : k ∈Z}  . We write ˙¯Z## ∞ := {δ/β//γ/α ∈¯Z## ∞ : δ−1 ≤α ˙< β ≤γ ˙< δ ≤α+1} . We write ˙E = ˙E(X) := E(X)| ˙¯Z## ∞ ∈Ob ˙¯Z## ∞, A ˙E = ˙E(X) := E(X)| ˙¯Z## ∞ ∈Ob ˙¯Z## ∞, A for the proper spectral sequence of X; analogously for the morphisms. Lemma 21 If E(α + 1/α −1//α/α −2)+k(f) is an isomorphism for all α ∈Z and all k ∈Z, then ˙E(f) is an isomorphism. Proof. Claim 1. We have an isomorphism E(γ/β −1//β/β −2)+k(f) for all k ∈Z, all β ∈Z and all γ ∈Z such that γ > β. We have an isomorphism E(β + 1/β −1//β/α −1)+k(f) for all k ∈Z, all β ∈Z and all α ∈Z such that α < β. The assertions follow by induction using the exact sequences E(γ + 2/γ//γ + 1/β)+k−1 - e E(γ/β −1//β/β −2)+k - e E(γ + 1/β −1//β/β −2)+k - 0 and and 0 - E(β + 1/β −1//β/α −2)+k - e E(β + 1/β −1//β/α −1)+k - e E(β −1/α −2//α −1/α −3)+k+1 . Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 701 Comparison of Spectral Sequences Involving Bifunctors 701 Comparison of Spectral Sequences Involving Bifunctors 701 Claim 2. We have an isomorphism E(γ/β −1//β/α −1)+k(f) for all k ∈Z and all α, β, γ ∈Z such that α < β < γ. We proceed by induction on γ −α. By Claim 1, we may assume that α < β −1 < β + 1 < γ. Consider the image diagram E(γ−1/β−1//β/α−1)+k - e E(γ/β−1//β/α−1)+k - r e E(γ/β−1//β/α)+k . Claim 3. We have an isomorphism E(δ/β//γ/α)+k(f) for all k ∈Z and all α, β, γ, δ ∈Z such that α < β ≤γ < δ. We may assume that γ −β ≥1, for E(δ/β//β/α)+k = 0. We proceed by induction on γ −β. By Claim 2, we may assume that γ −β ≥2. Consider the short exact sequence Claim 3. We have an isomorphism E(δ/β//γ/α)+k(f) for all k ∈Z and all α, β, γ, δ ∈Z such that α < β ≤γ < δ. We may assume that γ −β ≥1, for E(δ/β//β/α)+k = 0. We proceed by induction on γ −β. By Claim 2, we may assume that γ −β ≥2. Consider the short exact sequence 3.7 The first spectral sequence of a double complex Let A be an abelian category. Let X ∈Ob CC⌞(A). Given n ∈Z∞, we write X[n,∗for the double complex arising from X by replacing Xi,j by 0 for all i ∈ [0, n[. We define a pointwise split and pointwise finitely filtered complex tIX, called the first filtration of tX, by letting tIX(α) := tX[−α,∗for α ∈Z∞; and by letting tIX(α) - tIX(β) be the pointwise split inclusion tX[−α,∗ - tX[−β,∗ for α, β ∈Z∞such that α ≤β. Let EI = EI(X) := E(tIX). This construction is functorial in X ∈Ob CC⌞(A). Note that tIX(α) = X−α,k+α. Documenta Mathematica 13 (2008) 677–737 702 Matthias K¨unzer Lemma 22 Let α ∈]−∞, 0]. Let k ∈Z such that k ≥−α. We have Lemma 22 Let α ∈]−∞, 0]. Let k ∈Z such that k ≥−α. We have EI(α/α −1//α/α −1)+k(X) = Hk+α(X−α,∗) EI(α + 1/α −1//α/α −2)+k(X) = H−αHk+α(X−,∗)  , naturally in X ∈Ob CC⌞(A). naturally in X ∈Ob CC⌞(A). naturally in X ∈Ob CC⌞(A). Proof. The first equality follows by EI(α/α−1//α/α−1)+k = HktIX(α/α−1) = Hk+α(X−α,∗). The morphism tIX(α/α−1) - tIX (α−2)+1/α−1  = tIX α−1/α−2 •+1 from Sp(tIX) is at position k ≥0 given by tIX(α)k = X−α,k+α - (−1)α ∂ X−α+1,k+α = tIX(α −1)k+1 ; cf. §1.1.6. In particular, the morphisms EI(α + 1/α//α + 1/α)+k−1 - e EI(α/α −1//α/α −1)+k - e EI(α −1/α −2//α −1/α −2)+k+1 are given by Hk+α(X−α−1,∗) - (−1)α+1Hk+α(∂) Hk+α(X−α,∗) - (−1)αHk+α(∂) Hk+α(X−α+1,∗) . Now the second equality follows by the diagram EI(α+1/α−1//α/α−2)+k •U U U U U U U U e *U U U U U U U U EI(α/α−1//α/α−2)+k •T T T T T T T T e )T T T T T T T T *j j j j j j j j e 5j j j j j j j j EI(α+1/α−1//α/α−1)+k EI(α+1/α//α+1/α)+k−1 e / EI(α/α−1//α/α−1)+k e / )i i i i i i i i e 4i i i i i i i i EI(α−1/α−2//α−1/α−2)+k+1 . Remark 23 Let X - f Y be a rowwise quasiisomorphism in CC⌞(A). Then EI(δ/β//γ/α)+k(f) is an isomorphism for δ−1 ≤α ≤β ≤γ ≤δ ≤α+1 in ¯Z∞ and k ∈Z. Proof. It suffices to show that the morphism Sp(tIf) in ¯Z# ∞, K(A) is point- wise a quasiisomorphism. To have this, it suffices to show that tf [k,∗is a quasi- isomorphism for k ≥0. HktIU(γ/α)  - Hk+1tIU(β/δ−1)  . The double complex U [−β,∗/U [−(δ−1),∗is columnwise acyclic except possibly if −(δ−1) = i + 1 or if −β = i + 1. The double complex U [−γ,∗/U [−α,∗is columnwise acyclic except possibly if −γ = i+1 or if −α = i+1. Both remaining combinations of these exceptional cases are excluded by (∗∗), however. Hence EI(δ/β//γ/α)+k(U) = 0. This proves the claim. Both claims taken together show that ˙EI annihilates U. Comparison of Spectral Sequences Involving Bifunctors 703 Lemma 24 The functor CC⌞(A) - ˙EI ˙¯Z## ∞, A factors over KK⌞(A) - ˙EI ˙¯Z## ∞, A . Proof. By Lemma 1, we have to show that ˙EI annihilates all elementary horizon- tally split acyclic double complexes in Ob CC⌞(A) and all elementary vertically split acyclic double complexes in Ob CC⌞(A). Let U ∈Ob CC⌞(A) be an elementary vertically split acyclic double complex concentrated in rows i and i + 1, where i ≥0. Let V ∈Ob CC⌞(A) be an elementary horizontally split acyclic double complex concentrated in columns j and j + 1, where j ≥0. Since V is rowwise acyclic, EI annihilates V by Remark 23, whence so does ˙EI. Suppose given (∗) −∞≤α ˙< β ≤γ ˙< δ ≤∞ −∞≤α ˙< β ≤γ ˙< δ ≤∞ (∗) in ¯Z∞and k ∈Z. We claim that the functor EI(δ/β//γ/α)+k annihilates U. We may assume that β < γ. Note that EI(δ/β//γ/α)+k(U) is the image of HktIU(γ/α)  - HktIU(δ/β)  . The double complex U [−δ,∗/U [−β,∗is columnwise acyclic except possibly if −β = i + 1 or if −δ = i + 1. The double complex U [−γ,∗/U [−α,∗is columnwise acyclic except possibly if −α = i + 1 or if −γ = i + 1. All three remaining combinations of these exceptional cases are excluded by (∗), however. Hence EI(δ/β//γ/α)+k(U) = 0. This proves the claim. S i (∗∗) δ−1 ≤α ˙< β ≤γ ≤∞≤−∞+1 ≤δ ≤α+1 . in ¯Z∞and k ∈Z. We claim that the functor EI(δ/β//γ/α)+k annihilates U. We may assume that β < γ and that δ−1 < α. Note that EI(δ/β//γ/α)+k(U) is the image of HktIU(γ/α)  - Hk+1tIU(β/δ−1)  . 3.7 The first spectral sequence of a double complex But f [k,∗is a rowwise quasiisomorphism for k ≥0; cf. §1.1.6. Docum n a Ma h ma ica 13 (2008) 677 737 naturally in X ∈Ob CC⌞(A). Proof. The first equality follows by EI(α/α−1//α/α−1)+k = HktIX(α/α−1) = Hk+α(X−α,∗). The morphism tIX(α/α−1) - tIX (α−2)+1/α−1  = tIX α−1/α−2 •+1 Proof. The first equality follows by EI(α/α−1//α/α−1)+k = HktIX(α/α−1) = Hk+α(X−α,∗). The morphism tIX(α/α−1) - tIX (α−2)+1/α−1  = tIX α−1/α−2 •+1 from Sp(tIX) is at position k ≥0 given by Proof. The first equality follows by EI(α/α−1//α/α−1)+k = HktIX(α/α−1) = Hk+α(X−α,∗). The morphism tIX(α/α−1) - tIX (α−2)+1/α−1  = tIX α−1/α−2 •+1 from Sp(tIX) is at position k ≥0 given by H (X ). The morphism tIX(α/α−1) - tIX (α−2)+1/α−1  = tIX α−1/α−2 •+1 from Sp(tIX) is at position k ≥0 given by tIX(α)k = X−α,k+α - (−1)α ∂ X−α+1,k+α = tIX(α −1)k+1 ; tIX(α)k = X−α,k+α - (−1)α ∂ X−α+1,k+α = tIX(α −1)k+1 ; cf. §1.1.6. In particular, the morphisms cf. §1.1.6. In particular, the morphisms EI(α + 1/α//α + 1/α)+k−1 - e EI(α/α −1//α/α −1)+k - e EI(α −1/α −2//α −1/α −2)+k+1 are given by are given by Hk+α(X−α−1,∗) - (−1)α+1Hk+α(∂) Hk+α(X−α,∗) (−1)αH Hk+α(X−α−1,∗) - (−1)α+1Hk+α(∂) Hk+α(X−α,∗) Hk+α(X−α−1,∗) - (−1)α+1Hk+α(∂) Hk+α(X−α,∗) - (−1)αHk+α(∂) Hk+α(X−α+1,∗) . Now the second equality follows by the diagram Remark 23 Let X - f Y be a rowwise quasiisomorphism in CC⌞(A). Then EI(δ/β//γ/α)+k(f) is an isomorphism for δ−1 ≤α ≤β ≤γ ≤δ ≤α+1 in ¯Z∞ and k ∈Z. Proof. It suffices to show that the morphism Sp(tIf) in ¯Z# ∞, K(A) is point- wise a quasiisomorphism. To have this, it suffices to show that tf [k,∗is a quasi- isomorphism for k ≥0. But f [k,∗is a rowwise quasiisomorphism for k ≥0; cf. §1.1.6. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 703 4.1 Certain quasiisomorphisms are preserved by a left exact functor Let U - I′ be a quasiisomorphism to an injective complex resolution I′ that is mapped to a quasiisomorphism by F; cf. Remark 26. Since U - I′ is a quasiisomorphism, the induced map K(A)(U, I)  K(A)(I′, I) is surjec- tive, so that there exists a morphism I′ - I such that (U - I′ - I) = Proof. Let U - I′ be a quasiisomorphism to an injective complex resolution I′ that is mapped to a quasiisomorphism by F; cf. Remark 26. Since U - I′ is a quasiisomorphism, the induced map K(A)(U, I)  K(A)(I′, I) is surjec- tive, so that there exists a morphism I′ - I such that (U - I′ - I) = Proof. Let U - I′ be a quasiisomorphism to an injective complex resolution I′ that is mapped to a quasiisomorphism by F; cf. Remark 26. Since U - I′ is a quasiisomorphism, the induced map K(A)(U, I)  K(A)(I′, I) is surjec- tive, so that there exists a morphism I′ - I such that (U - I′ - I) = (U - f I) in K(A). Since, moreover, U - f I is a quasiisomorphism, I′ - I is a homotopism. Since FU - FI′ is a quasiisomorphism and FI′ - FI is a homotopism, we conclude that FU - FI is a quasiisomorphism. (U - f I) in K(A). Since, moreover, U - f I is a quasiisomorphism, I′ - I is a homotopism. Since FU - FI′ is a quasiisomorphism and FI′ - FI is a homotopism, we conclude that FU - FI is a quasiisomorphism. (U - f I) in K(A). Since, moreover, U - f I is a quasiisomorphism, I′ - I is a homotopism. Since FU - FI′ is a quasiisomorphism and FI′ - FI is a homotopism, we conclude that FU - FI is a quasiisomorphism. 4.1 Certain quasiisomorphisms are preserved by a left exact functor Suppose given abelian categories A, B, and suppose that A has enough injec- tives. Let A - F B be a left exact functor. Documenta Mathematica 13 (2008) 677–737 704 Matthias K¨unzer Remark 25 Suppose given an F-acyclic object X ∈Ob A and an injective res- olution I ∈Ob C[0(Inj A) of X. Let Conc X - f I be its quasiisomorphism. Then Conc FX - F f FI is a quasiisomorphism. Proof. This follows since F is left exact and since Hi(FI) ≃(RiF)X ≃0 for i ≥1. Remark 26 Suppose given a complex U ∈Ob C[0(A) consisting of F-acyclic objects. There exists an injective complex resolution I ∈Ob C[0(Inj A) of U such that its quasiisomorphism U - f I maps to a quasiisomorphism FU - F f FI. Proof. Let J ∈Ob CC⌞, CE(Inj A) be a CE-resolution of U; cf. Remark 9. Since the morphism of double complexes Conc2 U - J is a columnwise quasiiso- morphism consisting of monomorphisms, taking the total complex, we obtain a quasiisomorphism U - tJ consisting of monomorphisms. By F-acyclicity of the entries of U, the image Conc2 FU - FJ under F is a columnwise quasiisomorphism, too; cf. Remark 25. Hence F maps the quasiisomorphism U - tJ to the quasiisomorphism FU - FtJ. So we may take I := tJ. Proof. Let J ∈Ob CC⌞, CE(Inj A) be a CE-resolution of U; cf. Remark 9. Since the morphism of double complexes Conc2 U - J is a columnwise quasiiso- morphism consisting of monomorphisms, taking the total complex, we obtain a quasiisomorphism U - tJ consisting of monomorphisms. By F-acyclicity of the entries of U, the image Conc2 FU - FJ under F is a columnwise quasiisomorphism, too; cf. Remark 25. Hence F maps the quasiisomorphism U - tJ to the quasiisomorphism FU - FtJ. So we may take I := tJ. Lemma 27 Suppose given a complex U ∈Ob C[0(A) consisting of F-acyclic objects and an injective complex resolution I ∈Ob C[0(Inj A) of U. Let U - f I be its quasiisomorphism. Then FU - F f FI is a quasiisomorphism. Lemma 27 Suppose given a complex U ∈Ob C[0(A) consisting of F-acyclic objects and an injective complex resolution I ∈Ob C[0(Inj A) of U. Let U - f I be its quasiisomorphism. Then FU - F f FI is a quasiisomorphism. Proof. Comparison of Spectral Sequences Involving Bifunctors 705 Comparison of Spectral Sequences Involving Bifunctors 70 705 Comparison of Spectral Sequences Involving Bifunctors An object X ∈Ob A that possesses an (F, G)-acyclic resolution is called (F, G)-acyclicly resolvable. The full subcategory of (F, G)-acyclicly resolvable objects in A is denoted by A(F,G). An object X ∈Ob A that possesses an (F, G)-acyclic resolution is called (F, G)-acyclicly resolvable. The full subcategory of (F, G)-acyclicly resolvable objects in A is denoted by A(F,G). ( , ) A complex A ∈Ob C[0(A), together with a quasiisomorphism Conc X - A, is called an F-acyclic resolution of X ∈Ob A if (A 2) holds. Remark 28 If F carries injective objects to G-acyclic objects, then (A 1) and (A 3) imply (A 2). Proof. Given i ≥0, we let I be an injective resolution of Ai, and ˜I the acyclic complex obtained by appending Ai to I in position −1. Since Ai is F-acyclic, the complex F ˜I is acyclic; cf. Remark 25. Note that FB0 ˜I ≃FAi is G-acyclic by assumption. Since Proof. Given i ≥0, we let I be an injective resolution of Ai, and ˜I the acyclic complex obtained by appending Ai to I in position −1. Since Ai is F-acyclic, the complex F ˜I is acyclic; cf. Remark 25. Note that FB0 ˜I ≃FAi is G-acyclic by assumption. Since (RkG)F ˜Ij - (RkG)FBj+1 ˜I - (Rk+1G)FBj ˜I is exact in the middle for j ≥0 and k ≥1, we may conclude by induction on j and by G-acyclicity assumption on F ˜Ij that FBj ˜I is G-acyclic for j ≥0. In particular, we have (R1G)(FBj ˜I) ≃0 for j ≥0, whence GFBj ˜I - GF ˜Ij - GFBj+1 ˜I is exact in the middle for j ≥0 and k ≥1, we may conclude by induction on j and by G-acyclicity assumption on F ˜Ij that FBj ˜I is G-acyclic for j ≥0. In particular, we have (R1G)(FBj ˜I) ≃0 for j ≥0, whence is exact in the middle for j ≥0 and k ≥1, we may conclude by induction on j and by G-acyclicity assumption on F ˜Ij that FBj ˜I is G-acyclic for j ≥0. In particular, we have (R1G)(FBj ˜I) ≃0 for j ≥0, whence GFBj ˜I - GF ˜Ij - GFBj+1 ˜I GFBj ˜I - GF ˜Ij - GFBj+1 ˜I is short exact for j ≥0. We conclude that (G ◦F)˜I is acyclic. 4.2 Definition of the Grothendieck spectral sequence functor Suppose given abelian categories A, B and C, and suppose that A and B have enough injectives. Let A - F B and B - G C be left exact functors. A (F, G)-acyclic resolution of X ∈Ob A is a complex A ∈Ob C[0(A), together with a quasiisomorphism Conc X - A, such that the following hold. enough injectives. Let A - F B and B - G C be left exact functors. A (F, G)-acyclic resolution of X ∈Ob A is a complex A ∈Ob C[0(A), together with a quasiisomorphism Conc X - A, such that the following hold. (A 1) The object Ai is F-acyclic for i ≥0. (A 2) The object Ai is (G ◦F)-acyclic for i ≥0. (A 2) The object Ai is (G ◦F)-acyclic for i ≥0. (A 3) The object FAi is G-acyclic for i ≥0. (A 3) The object FAi is G-acyclic for i ≥0. Comparison of Spectral Sequences Involving Bifunctors 705 Hence Ai is (G ◦F)-acyclic. To see Remark 28, one could also use a Grothendieck spectral sequence, once established. To see Remark 28, one could also use a Grothendieck spectral sequence, once established. Remark 29 Suppose given X ∈Ob A, an injective resolution I of X and an F-acyclic resolution A of X. Then there exists a quasiisomorphism A - I that is mapped to 1X by H0. Moreover, any morphism A - u I that is mapped to 1X by H0 is a quasiisomorphism and is mapped to a quasiisomorphism FA - F u FI by F. F acyclic resolution A of X. Then there exists a quasiisomorphism A - I that is mapped to 1X by H0. Moreover, any morphism A - u I that is mapped to 1X by H0 is a quasiisomorphism and is mapped to a quasiisomorphism FA - F u FI by F. Proof. Let I′ be an injective complex resolution of A such that its quasiisomor- phism A - I′ is mapped to a quasiisomorphism by F; cf. Remark 26. We use the composite quasiisomorphism Conc X - A - I′ to resolve X by I′. To prove the first assertion, note that there is a homotopism I′ - I resolving 1X; whence the composite (A - I′ - I) is a quasiisomorphism resolving 1X. To prove the second assertion, note that the induced map K(A)(A, I)  K(A)(I′, I) is surjective, whence there is a factorisation (A - I′ - I) = (A - u I) in K(A) for some morphism I′ - I, which, since resolving 1X as well, is a homotopism. In particular, A - u I is a quasi- isomorphism. Finally, since FI′ - FI is a homotopism, also FA - F u FI is a quasiisomorphism. Documenta Mathematica 13 (2008) 677–737 706 Matthias K¨unzer Alternatively, in the last step of the preceding proof we could have invoked Lemma 27. The following construction originates in [5, XVII.§7] and [7, Th. 2.4.1]. In its present form, it has been carried out by Haas in the classical framework [8]. We do not claim any originality. I do not know whether the use of injectives in A in the following construction can be avoided; in any case, it would be desirable to do so. We set out to define the proper Grothendieck spectral sequence functor A(F,G) - ˙EGr F,G ˙¯Z## ∞, C . We define ˙EGr F,G on objects. Suppose given X ∈Ob A(F,G). Choose an (F, G)-acyclic resolution AX ∈Ob C[0(A) of X. Choose a CE-resolution JX ∈Ob CC⌞(Inj B) of FAX. Let EGr F,G(X) := EI(GJX) = E(tIGJX) ∈ Ob ¯Z## ∞, C be the Grothendieck spectral sequence of X with respect to F and G. Accordingly, let ˙EGr F,G(X f−→Y ) := ˙EGr F,G(X) := ˙EI(GJX) = ˙E(tIGJX) ∈Ob ˙¯Z## ∞, C ˙EGr F,G(X) := ˙EI(GJX) = ˙E(tIGJX) ∈Ob ˙¯Z## ∞, C be the proper Grothendieck spectral sequence of X with respect to F and G. We define ˙EGr F,G on morphisms. Suppose given X ∈Ob A(F,G), and let AX and JX be as above. Choose an injective resolution IX ∈Ob C[0(Inj A) of X. Choose a quasiisomorphism AX - pX IX that is mapped to 1X by H0 and to a quasiisomorphism by F; cf. Remark 29. Choose a CE-resolution KX ∈ Ob CC⌞(Inj B) of FIX. Choose a morphism JX - qX KX in CC⌞(Inj B) that is mapped to FpX by H0(−)∗,− ; cf. Remark 6. Note that JX - qX KX can be written as a composite in CC⌞, CE(Inj B) of a rowwise homotopism, followed by a double homotopism; cf. Proposition 17. Hence, so can GJX GqX −−−→GKX. Thus ˙EI(GJX) ˙EI(GqX) −−−−−→˙EI(GKX) is an isomorphism; cf. Remark 23, Lemma 24. Suppose given X - f Y in A(F,G). Choose a morphism IX - f ′ IY in C[0(A) that is mapped to f by H0. Choose a morphism KX - f ′′ KY in CC⌞(Inj B) that is mapped to Ff ′ by H0(−)∗,− ; cf. Remark 6. Let ˙EGr F,G(X f−→Y ) :=  ˙EI(GJX) ˙EI(GqX) −−−−−→ ∼ ˙EI(GKX) ˙EI(Gf ′′) −−−−−→˙EI(GKY ) ˙EI(GqY ) ←−−−−− ∼ ˙EI(GJY )  . ˙EGr F,G(X f−→Y ) := Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 707 Comparison of Spectral Sequences Involving Bifunctors 707 Comparison of Spectral Sequences Involving Bifunctors 707 The procedure can be adumbrated as follows. The procedure can be adumbrated as follows. The procedure can be adumbrated as follows. The procedure can be adumbrated as follows. AX  pX IX - f′ AY  pY IY JX  qX KX - f′′ JY  qY KY X - f Y We show that this defines a functor ˙EGr F,G : A(F,G) - ˙¯Z## ∞, C . We need to show independence of the construction from the choices of f ′ and f ′′, for then functoriality follows by appropriate choices. Let IX - ˜f ′ IY and KX - ˜ f ′′ KY be alternative choices. The residue classes of f ′ and ˜f ′ in K[0(A) coincide, whence so do the residue classes of Ff ′ and F ˜f ′ in K[0(B). Therefore, the residue classes of f ′′ and ˜f ′′ in KK⌞(B) coincide; cf. Proposition 14. Hence, so do the residue classes of Gf ′′ and G ˜f ′′ in KK⌞(C). Thus ˙EI(Gf ′′) = ˙EI(G ˜f ′′); cf. Lemma 24. ( ) ( ) We show that alternative choices of AX, IX and pX, and of JX, KX and qX, yield isomorphic proper Grothendieck spectral sequence functors. ˜ ˜ We show that alternative choices of AX, IX and pX, and of JX, KX and qX, yield isomorphic proper Grothendieck spectral sequence functors. ˜ ˜ We show that alternative choices of AX, IX and pX, and of JX, KX and qX, yield isomorphic proper Grothendieck spectral sequence functors. Let ˜AX - ˜pX ˜IX and ˜JX - ˜qX ˜KX be alternative choices, where X runs through Ob A(F,G). Let ˜AX - ˜pX ˜IX and ˜JX - ˜qX ˜KX be alternative choices, where X runs through Ob A(F,G). Let ˜AX - ˜pX ˜IX and ˜JX - ˜qX ˜KX be alternative choices, where X runs through Ob A(F,G). Suppose given X - f Y in A(F,G). We resolve the commutative quadrangle X f / Y X f / Y X f / Y X f / Y X f / Y X f / Y A to a commutative quadrangle IX f ′ / uX  IY uY  ˜IX ˜f ′ / ˜IY Documenta Mathematica 13 (2008) 677–737 in A to a commutative quadrangle in A to a commutative quadrangle IX f ′ / uX  IY uY  ˜IX ˜f ′ / ˜IY Documenta Mathematica 13 (2008) 677–737 708 Matthias K¨unzer in K[0(A), in which uX and uY are homotopisms; cf. Remark 6. Documenta Mathematica 13 (2008) 677–737 X - f Y Abbreviate E := ˙E(−k + 1/−k −1//−k/−k −2)+k+ℓ. E(tIGJX) E(tIGqX ) ∼ / ≀  E(tIGKX) E(tIGf′′) / ≀  E(tIGKY ) ≀  E(tIGJY ) E(tIGqY ) ∼ o ≀  HkHℓGJ−,∗ X HkHℓGq−,∗ X ∼ / ≀  HkHℓGK−,∗ X HkHℓGf′′−,∗/ ≀  HkHℓGK−,∗ Y ≀  HkHℓGJ−,∗ Y HkHℓGq−,∗ Y ∼ o ≀  HkGHℓJ−,∗ X HkGHℓq−,∗ X ∼ / ≀  HkGHℓK−,∗ X HkGHℓf′′−,∗/ ≀  HkGHℓK−,∗ Y ≀  HkGHℓJ−,∗ Y HkGHℓq−,∗ Y ∼ o ≀  (RkG)HℓF AX (RkG)HℓF pX ∼ / (RkG)HℓF IX (RkG)HℓF f′/ ≀  (RkG)HℓF IY ≀  (RkG)HℓF AY (RkG)HℓF pY ∼ o (RkG)(RℓF )(X) (RkG)(RℓF )(f)/ (RkG)(RℓF )(Y ) We shall prove the second isomorphism. By Lemma 27, the quasiisomorphism FAX - tJX maps to a quasiisomorphism GFAX - tGJX ≃GtJX. By Lemma 27, the quasiisomorphism AX - pX IX maps to a quasiisomorphism GFAX - GF pX GFIX. So ˙EGr F,G(∞/−∞//∞/−∞)+k+ℓ(X) ≃Hk+ℓ(tGJX) ≃Hk+ℓ(GtJX) ≃Hk+ℓ(GFAX) ≃Hk+ℓ(GFIX) E(tIGJX) E(tIGqX ) ∼ / ≀  E(tIGKX) E(tIGf′′) / ≀  E(tIGKY ) ≀  E(tIGJY ) E(tIGqY ) ∼ o ≀  HkHℓGJ−,∗ X HkHℓGq−,∗ X ∼ / ≀  HkHℓGK−,∗ X HkHℓGf′′−,∗/ ≀  HkHℓGK−,∗ Y ≀  HkHℓGJ−,∗ Y HkHℓGq−,∗ Y ∼ o ≀  HkGHℓJ−,∗ X HkGHℓq−,∗ X ∼ / ≀  HkGHℓK−,∗ X HkGHℓf′′−,∗/ ≀  HkGHℓK−,∗ Y ≀  HkGHℓJ−,∗ Y HkGHℓq−,∗ Y ∼ o ≀  (RkG)HℓF AX (RkG)HℓF pX ∼ / (RkG)HℓF IX (RkG)HℓF f′/ ≀  (RkG)HℓF IY ≀  (RkG)HℓF AY (RkG)HℓF pY ∼ o (RkG)(RℓF )(X) (RkG)(RℓF )(f)/ (RkG)(RℓF )(Y ) We shall prove the second isomorphism. By Lemma 27, the quasiisomorphism FAX - tJX maps to a quasiisomorphism GFAX - tGJX ≃GtJX. By Lemma 27, the quasiisomorphism AX - pX IX maps to a quasiisomorphism GFAX - GF pX GFIX. So ˙EGr F,G(∞/−∞//∞/−∞)+k+ℓ(X) ≃Hk+ℓ(tGJX) ≃Hk+ℓ(GtJX) ≃Hk+ℓ(GFAX) ≃Hk+ℓ(GFIX) ≃ Rk+ℓ(G ◦F)  (X) . We shall prove naturality of the second isomorphism. Consider the following diagram. Abbreviate ˜E := ˙EGr F,G(∞/−∞//∞/−∞)+k+ℓ. X - f Y Then we resolve the commutative quadrangle FIX F f ′ / F uX  FIY F uY  F ˜IX F ˜ f ′ / F ˜IY FIX F f ′ / F uX  FIY F uY  F ˜IX F ˜ f ′ / F ˜IY in K[0(B) to a commutative quadrangle in K[0(B) to a commutative quadrangle KX f ′′ / vX  KY vY  ˜KX ˜ f ′′ / ˜KY KX f ′′ / vX  KY vY  ˜KX ˜ f ′′ / ˜KY in KK⌞(B); cf. Proposition 14. Therein, vX and vY are each composed of a rowwise homotopism, followed by a double homotopism; cf. Proposition 17. So are GvX and GvY . An application of ˙EI G(−)  yields the sought isotransfor- mation, viz.  ˙EI(GJX) ˙EI(GqX) −−−−−→ ∼ ˙EI(GKX) ˙EI(GvX) −−−−−→ ∼ ˙EI(G ˜KX) ˙EI(G˜qX) ←−−−−− ∼ ˙EI(G ˜JX)  at X ∈Ob A(F,G); cf. Remark 23, Lemma 24. at X ∈Ob A(F,G); cf. Remark 23, Lemma 24. Finally, we recall the starting point of the whole enterprise. Remark 30 ([5, XVII.§7], [7, Th. 2.4.1]) Suppose given X ∈Ob A(F,G) and k, ℓ∈Z≥0. We have ˙EGr F,G(−k + 1/−k −1//−k/−k −2)+k+ℓ(X) ≃ (RkG)(RℓF)(X) ˙EGr F,G(∞/−∞//∞/−∞)+k+ℓ(X) ≃ Rk+ℓ(G ◦F)  (X) , naturally in X. Proof. Keep the notation of the definition of ˙EGr F,G . We shall prove the first isomorphism. By Lemma 22, we have Proof. Keep the notation of the definition of ˙EGr F,G . Proof. Keep the notation of the definition of EGr F,G . We shall prove the first isomorphism. By Lemma 22, we have f p F,G We shall prove the first isomorphism. By Lemma 22, we have ˙EGr F,G(−k + 1/−k −1//−k/−k −2)+k+ℓ(X) ≃Hk(Hℓ(GJ−,∗ X )) . Since JX is rowwise split, we have Hℓ(GJ−,∗ X ) ≃G(HℓJ−,∗ X ). Note that HℓJ−,∗ X is an injective resolution of HℓFAX; cf. Remark 8.(1). By Remark 29, HℓFAX - HℓF pX ∼ HℓFIX ≃(RℓF)(X). So Hk(Hℓ(GJ−,∗ X )) ≃Hk(G(HℓJ−,∗ X )) ≃(RkG)(HℓFAX) ≃(RkG)(RℓF)(X) . Hk(Hℓ(GJ−,∗ X )) ≃Hk(G(HℓJ−,∗ X )) ≃(RkG)(HℓFAX) ≃(RkG)(RℓF)(X) . Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 709 Comparison of Spectral Sequences Involving Bifunctors 709 Comparison of Spectral Sequences Involving Bifunctors 709 We shall prove naturality of the first isomorphism. Suppose given X - f Y in A(F,G). Consider the following commutative diagram. 4.3.1 Situation Consider the following diagram of abelian categories, left exact functors and transformations, A F / U  B G / V  C W  A′ F ′ / B′ G′ / C′ , µ 8@yy y y y y ν 8@y y y y y y i.e. F ′ ◦U - µ V ◦F and G′ ◦V - ν W ◦G. Suppose that the conditions (1, 2, 3) hold. (1) The categories A, B, A′ and B′ have enough injectives. (1) The categories A, B, A′ and B′ have enough injectives. (2) The functors U and V carry injectives to injectives. (2) The functors U and V carry injectives to injectives. (3) The functor F carries injective to G-acyclic objects. The functor F ′ carries injective to G′-acyclic objects. (3) The functor F carries injective to G-acyclic objects. The functor F ′ carries injective to G′-acyclic objects. We have A(F,G) = A since an injective resolution is an (F, G)-acyclic resolution. Likewise, we have A′ (F ′,G′) = A′. We have A(F,G) = A since an injective resolution is an (F, G)-acyclic resolution. Likewise, we have A′ (F ′,G′) = A′. (F ,G ) Note in particular the case U = 1A , V = 1B and W = 1C . (F ,G ) Note in particular the case U = 1A , V = 1B and W = 1C . We set out to define the Haas transformations ˙EGr F ′,G′ U(−)  - hI µ ˙EGr F,G′◦V −  - hII ν ˙EGr F,W◦G −  , where hI µ depends on F, F ′, G′, U, V and µ, and where hII ν depends on F, G, G′, V , W and ν. where hI µ depends on F, F ′, G′, U, V and µ, and where hII ν depends on F, G, G′, V , W and ν. 4.3 Haas transformations The following transformations have been constructed in the classical framework by Haas [8]. We do not claim any originality. X - f Y ˜ E(tIGJX) ˜ E(tIGqX ) ∼ / ≀  ˜ E(tIGKX) ˜ E(tIGf′′) / ≀  ˜ E(tIGKY ) ≀  ˜ E(tIGJY ) ˜ E(tIGqY ) ∼ o ≀  Hk+ℓtGJX Hk+ℓtGqX ∼ / ≀  Hk+ℓtGKX Hk+ℓtGf′′/ ≀  Hk+ℓtGKY ≀  Hk+ℓtGJY Hk+ℓtGqY ∼ o ≀  Hk+ℓGtJX Hk+ℓGtqX ∼ / Hk+ℓGtKX Hk+ℓGtf′′/ Hk+ℓGtKY Hk+ℓGtJY Hk+ℓGtqY ∼ o Hk+ℓGF AX Hk+ℓGF pX ∼ / ≀ O Hk+ℓGF IX Hk+ℓGF f′/ ≀  O Hk+ℓGF IY ≀  O Hk+ℓGF AY Hk+ℓGF pY ∼ o ≀ O ` Rk+ℓ(G ◦F ) ´ (X) (Rk+ℓ(G◦F ))(f)/ ` Rk+ℓ(G ◦F ) ´ (Y ) Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 710 Documenta Mathematica 13 (2008) 677–737 4.3.2 Construction of the first Haas transformation Given T ∈Ob A, we let ˙EGr F,G(T ) be defined via an injective resolution IT of T and via a CE-resolution JT of FIT ; cf. §4.2. Given T ∈Ob A, we let ˙EGr F,G(T ) be defined via an injective resolution IT of T and via a CE-resolution JT of FIT ; cf. §4.2. ; § Given T ′ ∈Ob A′, we let ˙EGr F ′,G′(T ′) be defined via an injective resolution I′ T ′ of T ′ and via a CE-resolution J′ T ′ of F ′I′ T ′; cf. §4.2. We define hI µ. Let X ∈Ob A. By Remark 5, there is a unique morphism I′ UX - h′X UIX in K[0(A′) that maps to 1UX under H0. Let J′ UX - h′′X V JX Given T ′ ∈Ob A′, we let ˙EGr F ′,G′(T ′) be defined via an injective resolution I′ T ′ of T ′ and via a CE-resolution J′ T ′ of F ′I′ T ′; cf. §4.2. Given T ′ ∈Ob A′, we let ˙EGr F ′,G′(T ′) be defined via an injective resolution I′ T ′ of T ′ and via a CE-resolution J′ T ′ of F ′I′ T ′; cf. §4.2. T T We define hI µ. Let X ∈Ob A. By Remark 5, there is a unique morphism ′ h′X [0( ′) 0 ′ h′′X We define hI µ. Let X ∈Ob A. By Remark 5, there is a unique morphism I′ h′X UI i K[0(A′) h 1 d H0 L J′ h′′X V J I′ UX - h X UIX in K[0(A′) that maps to 1UX under H0. Let J′ UX - h X V JX I′ UX - h X UIX in K[0(A′) that maps to 1UX under H0. Let J′ UX - h X V JX Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 711 Comparison of Spectral Sequences Involving Bifunctors 711 Comparison of Spectral Sequences Involving Bifunctors 711 be the unique morphism in KK⌞(B′) that maps to the composite mor- phism  F ′I′ UX - F ′h′X F ′UIX - µ V FIX  in K[0(B′) under H0(−)∗,− ; cf. Lemma 13. Let the first Haas transformation be defined by  ˙EGr F ′,G′ UX  - hI µX ˙EGr F,G′◦V X  :=  EI(G′J′ UX) - EI(G′h′′X) EI(G′V JX)  . 4.3.3 Construction of the second Haas transformation We maintain the notation of §4.3.2. Given X ∈Ob A, we let the second Haas transformation be defined by  ˙EGr F,G◦V X  - hII ν X ˙EGr F,W◦G X  :=  ˙EI(G′V JX) - ˙EI(ν) ˙EI(WGJX)  . It is a transformation since ν is. It is a transformation since ν is. in ˙¯Z## ∞, C′ . in ˙¯Z## ∞, C′ . 4.3.2 Construction of the first Haas transformation  ˙EGr F ′,G′ UX  - hI µX ˙EGr F,G′◦V X  Gr F,G′◦V X  :=  EI(G′J′ UX) - EI(G′h′′X) EI(G′V JX)  .  :=  EI(G′J′ UX) - EI(G′h′′X) EI(G′V JX)  . :=  EI(G′J′ UX) We show that hI µ is a transformation. Let X - f Y be a morphism in A. Let IX - f ′ IY resolve X - f Y . Let JX - f ′′ JY resolve FIX - f ′ FIY . Let I′ UX - ˜ f ′ I′ UY resolve UX - Uf UY . Let J′ UX - ˜ f ′′ JUY resolve F ′IUX - F ′ ˜ f ′ F ′IUY . The quadrangle UX Uf  UX Uf  UY UY UX Uf  UX Uf  UY UY commutes in A′. Hence, by Remark 5, applied to I′ UX and UIY , the resolved quadrangle I′ UX h′X / ˜ f ′  UIX Uf ′  I′ UY h′Y / UIY I′ UX h′X / ˜ f ′  UIX Uf ′  I′ UY h′Y / UIY commutes in K[0(A′). Hence both quadrangles in commutes in K[0(A′). Hence both quadrangles in F ′I′ UX F ′h′X / F ′ ˜f ′  F ′UIX F ′Uf ′  µ / V FIX V F f ′  F ′I′ UY F ′h′Y / F ′UIY µ / V FIY commute in K[0(B′). By Lemma 13, applied to J′ UX and V JY , the outer quad- rangle in the latter diagram can be resolved to the commutative quadrangle J′ UX h′′X / ˜f ′′  V JX V f ′′  J′ UY h′′Y / V JY Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 712 Matthias K¨unzer in KK⌞(B′). Applying EI G′(−)  and employing the definitions of ˙EGr F ′,G′ , ˙EGr F,G′◦V and hI µ , we obtain the sought commutative diagram ˙EGr F ′,G′(UX) hI µX / ˙EGr F ′,G′(Uf)  ˙EGr F,G′◦V (X) ˙EGr F,G′◦V (f)  ˙EGr F ′,G′(UY ) hI µY / ˙EGr F,G′◦V (Y ) 5.1 The first comparison isomorphism ˙EGr F (X,−),G(X′) ≃˙EGr F (−,X′),G(X) 5.1 The first comparison isomorphism Suppose given abelian categories A, A′ and B with enough injectives and an abelian category C. Let A×A′ - F B be a biadditive functor. Let B - G C be an additive functor. Suppose given objects X ∈Ob A and X′ ∈Ob A′. Suppose the following properties to hold. (a) The functor F(−, X′) : A - B is left exact. (a) The functor F(−, X′) : A - B is left exact. (a) The functor F(−, X′) : A - B is left exact. (a′) The functor F(X, −) : A′ - B is left exact. (a′) The functor F(X, −) : A′ - B is left exact. (a′) The functor F(X, −) : A′ - B is left exact. (b) The functor G is left exact. (b) The functor G is left exact. (c) The object X possesses a F(−, X′), G  -acyclic resolution A ∈ Ob C[0(A). (c) The object X possesses a F(−, X′), G  -acyclic resolution A ∈ Ob C[0(A). (c) The object X possesses a F(−, X′), G  -acyclic resolution A ∈ Ob C[0(A). (c′) The object X′ possesses a F(X, −), G  -acyclic resolution A′ ∈ Ob C[0(A′). (c′) The object X′ possesses a F(X, −), G  -acyclic resolution A′ ∈ Ob C[0(A′). Moreover, the resolutions appearing in (c) and (c′) are stipulated to have the following properties. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 713 Comparison of Spectral Sequences Involving Bifunctors 713 (d) For all k ≥0, the quasiisomorphism Conc X - A is mapped to a quasi- isomorphism Conc F(X, A′k) - F(A, A′k) under F(−, A′k). (d′) For all k ≥0, the quasiisomorphism Conc X′ - A′ is mapped to a quasiisomorphism Conc F(Ak, X′) - F(Ak, A′) under F(Ak, −). The conditions (d, d′) are e.g. satisfied if F(−, A′k) and F(Ak, −) are exact for all k ≥0. Theorem 31 (first comparison) The proper Grothendieck spectral se- quence for the functors F(X, −) and G, evaluated at X′, is isomorphic to the proper Grothendieck spectral sequence for the functors F(−, X′) and G, evaluated at X; i.e. Theorem 31 (first comparison) The proper Grothendieck spectral se- quence for the functors F(X, −) and G, evaluated at X′, is isomorphic to the proper Grothendieck spectral sequence for the functors F(−, X′) and G, evaluated at X; i.e. in ˙¯Z## ∞, C . Proof. Let JA , JA′ , JA,A′ ∈Ob CC⌞(Inj B) be CE-resolutions of the com- plexes F(A, X′), F(X, A′), tF(A, A′) ∈Ob C[0(B), respectively. The quasiisomorphism Conc X - A is mapped to the morphism F(Conc X, A′) - F(A, A′), yielding F(X, A′) - tF(A, A′), which is a quasiisomorphism since Conc F(X, A′k) - F(A, A′k) is a quasiisomorphism for all k ≥0 by (d). Choose a CE-resolution JA′ - JA,A′ of F(X, A′) - tF(A, A′); cf. Re- mark 6. Since the morphism F(X, A′) - tF(A, A′) is a quasiisomorphism, JA′ - JA,A′ is a composite in CC⌞, CE(Inj B) of a rowwise homotopism and a double homotopism; cf. Proposition 17. So is GJA′ - GJA,A′. Hence, by Remark 23 and by Lemma 24, we obtain an isomorphism of the proper spectral sequences of the first filtrations of the total complexes, ˙EGr F (X,−),G(X′) = ˙EI(GJA′) - ∼ ˙EI(GJA,A′) . Likewise, we have an isomorphism ˙EGr F (−,X′),G(X) = ˙EI(GJA) - ∼ ˙EI(GJA,A′) . We compose to an isomorphism ˙EGr F (X,−),G(X′) - ∼ ˙EGr F (−,X′),G(X) as sought. 5.2 Naturality of the first comparison isomorphism We narrow down the assumptions just as we have done for the introduction of the Haas transformations in §4.3.1 in order to be able to express, in this narrower case, a naturality of the first comparison isomorphism from Theorem 31. 5.2 Naturality of the first comparison isomorphism 5.2 Naturality of the first comparison isomorphism We narrow down the assumptions just as we have done for the introduction of the Haas transformations in §4.3.1 in order to be able to express, in this narrower case, a naturality of the first comparison isomorphism from Theorem 31. Suppose given abelian categories A, A′ and B with enough injectives and an abelian category C. Let A×A′ - F B be a biadditive functor. Let B - G C be an additive functor. Suppose that the following properties hold. Documenta Mathematica 13 (2008) 677–737 Matthias K¨unzer 714 (a) The functor F(−, X′) : A - B is left exact for all X′ ∈Ob A′. (a) The functor F(−, X′) : A - B is left exact for all X′ ∈Ob A′. (a′) The functor F(X, −) : A′ - B is left exact for all X ∈Ob A. (a′) The functor F(X, −) : A′ - B is left exact for all X ∈Ob A. (b) The functor G is left exact. (b) The functor G is left exact. (b) The functor G is left exact. (b) The functor G is left exact. (b) The functor G is left exact. (c) For all X′ ∈Ob A′, the functor F(−, X′) carries injective objects to G-acyclic objects. (c) For all X′ ∈Ob A′, the functor F(−, X′) carries injective objects to G-acyclic objects. (c) For all X′ ∈Ob A′, the functor F(−, X′) carries injective objects to G-acyclic objects. (c′) For all X ∈Ob A, the functor F(X, −) carries injective objects to G-acyclic objects. (c′) For all X ∈Ob A, the functor F(X, −) carries injective objects to G-acyclic objects. (d) The functor F(I, −) is exact for all I ∈Ob Inj A. (d′) The functor F(−, I′) is exact for all I′ ∈Ob Inj A′. (d) The functor F(I, −) is exact for all I ∈Ob Inj A. (d′) The functor F(−, I′) is exact for all I′ ∈Ob Inj A′. (d′) The functor F(−, I′) is exact for all I′ ∈Ob Inj A′. Proposition 32 Suppose given X - x ˜X in A and X′ ∈Ob A′. Note that we have a transformation F(x, −) : F(X, −) - F( ˜X, −). The following quadrangle, whose vertical isomorphisms are given by the construction in the proof of Theorem 31, commutes. 5.2 Naturality of the first comparison isomorphism We refrain from investigating naturality of the first comparison isomorphism in G. We refrain from investigating naturality of the first comparison isomorphism in G. 5.2 Naturality of the first comparison isomorphism ˙EGr F (X,−),G(X′) hI F (x,−)X′ / ≀  ˙EGr F ( ˜ X,−),G(X′) ≀  ˙EGr F (−,X′),G(X) ˙EGr F(−,X′),G(x) / ˙EGr F (−,X′),G( ˜X) For the definition of the first Haas transformation hI F (x,−), see §4.3.2. For the definition of the first Haas transformation hF (x,−), see §4.3.2. An analogous assertion holds with interchanged roles of A and A′. Proof of Proposition 32. Let I resp. ˜I be an injective resolution of X resp. ˜X in A. Let I - ˆx ˜I be a resolution of X - x ˜X. Let I′ be an injective resolution of X′ in A′. Let J(X) I′ resp. J( ˜ X) I′ be a CE-resolution of F(X, I′) resp. F( ˜X, I′). Let JI,I′ resp. J˜I,I′ be a CE-resolution of tF(I, I′) resp. tF(˜I, I′). Let JI resp. J˜I be a CE-resolution of F(I, X′) resp. F(˜I, X′). We have a commutative diagram ( , ) An analogous assertion holds with interchanged roles of A and A′. Proof of Proposition 32. Let I resp. ˜I be an injective resolution of X resp. ˜X in A. Let I - ˆx ˜I be a resolution of X - x ˜X. Let I′ be an injective resolution of X′ in A′. (X) ( ˜ X) ˜ Let J(X) I′ resp. J(X) I′ be a CE-resolution of F(X, I′) resp. F( ˜X, I′). Let JI,I′ resp. J˜I,I′ be a CE-resolution of tF(I, I′) resp. tF(˜I, I′). Let JI resp. J˜I be a CE-resolution of F(I, X′) resp. F(˜I, X′). We have a commutative diagram F(X, I′) F (x,I′) /  F( ˜X, I′)  tF(I, I′) tF (ˆx,I′) / tF(˜I, I′) F(I, X′) F (ˆx,X′) / O F(˜I, X′) O Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 715 in C[0(B), hence in K[0(B). By Proposition 14, it can be resolved to a commu- tative diagram ˜ J(X) I′ /  J( ˜ X) I′  JI,I′ / J˜I,I′ JI / O J˜I O J(X) I′ /  J( ˜ X) I′  JI,I′ / J˜I,I′ JI / O J˜I O J(X) I′ /  J( ˜ X) I′  JI,I′ / J˜I,I′ JI / O J˜I O in KK⌞(B). Application of ˙EI G(−)  yields the result; cf. Lemma 24. in KK⌞(B). Application of ˙EI G(−)  yields the result; cf. Lemma 24. Documenta Mathematica 13 (2008) 677–737 6.1 The second comparison isomorphism Theorem 34 (second comparison) The proper Grothendieck spectral se- quence for the functors F and G(Y, −), evaluated at X, is isomorphic to ˙EI(G(B, FA)); i.e. ˙EGr F,G(Y,−)(X) ≃˙EI(G(B, FA)) in ˙¯Z## ∞, C . Proof. Let J′ ∈Ob CC⌞(Inj B′) be a CE-resolution of FA. By definition, ˙EGr F,G(Y,−)(X) = ˙EI(G(Y, J′)). By Remark 33, it suffices to find D ∈Ob CC⌞(C) and two morphisms of double complexes 6.1 The second comparison isomorphism Suppose given abelian categories A and B′ with enough injectives, and abelian categories B and C. Let A - F B′ be an additive functor. Let B ×B′ - G C be a biadditive functor. Suppose given objects X ∈Ob A and Y ∈Ob B. Let B ∈Ob C[0(B) be a resolution of Y , i.e. suppose a quasiisomorphism Conc Y - B to exist. Suppose the following properties to hold. Let A - F B′ be an additive functor. Let B ×B′ - G C be a biadditive functor. Suppose given objects X ∈Ob A and Y ∈Ob B. Let B ∈Ob C[0(B) be a resolution of Y , i.e. suppose a quasiisomorphism Conc Y - B to exist. Suppose the following properties to hold. (a) The functor F is left exact. (b) The functor G(Y, −) is left exact. (c) The object X possesses an (F, G(Y, −))-acyclic resolution A ∈Ob C[0(A). (d) The functor G(Bk, −) is exact for all k ≥0. (d) The functor G(Bk, −) is exact for all k ≥0. (e) The functor G(−, I′) is exact for all I′ ∈Ob Inj B′. (e) The functor G(−, I′) is exact for all I′ ∈Ob Inj B′. Remark 33 Suppose given a morphism D - f D′ in CC⌞(C). If Hℓ(f −,∗) is a quasiisomorphism for all ℓ≥0, then f induces an isomorphism Remark 33 Suppose given a morphism D - f D′ in CC⌞(C). If Hℓ(f −,∗) is a quasiisomorphism for all ℓ≥0, then f induces an isomorphism Remark 33 Suppose given a morphism D - f D′ in CC⌞(C). If Hℓ(f −,∗) is a quasiisomorphism for all ℓ≥0, then f induces an isomorphism ˙EI(D) - ˙EI(f) ˙EI(D′) of proper spectral sequences. of proper spectral sequences. Proof. By Lemma 21, it suffices to show that EI(α + 1/α −1//α/α −2)+k(f) is an isomorphism for all α ∈Z and all k ∈Z. By Lemma 22, this amounts to isomorphisms HkHℓ(f −,∗) for all k, ℓ≥0, i.e. to quasiisomorphisms Hℓ(f −,∗) for all ℓ≥0. Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 716 Matthias K¨unzer Consider the double complex G(B, FA) ∈Ob CC⌞(C), where the indices of B count rows and the indices of A count columns. To the first filtration of its total complex, we can associate the proper spectral sequence ˙EI(G(B, FA)) ∈ Ob ˙¯Z## ∞, C . G(B, FA) - u D v G(Y, J′) such that Hℓ(u−,∗) and Hℓ(v−,∗) are quasiisomorphisms for all ℓ≥0. Given a complex U ∈Ob C[0(B), recall that we denote by Conc2U ∈Ob CC⌞(B) the double complex whose row number 0 is given by U, and whose other rows are zero. such that Hℓ(u−,∗) and Hℓ(v−,∗) are quasiisomorphisms for all ℓ≥0. Given a complex U ∈Ob C[0(B), recall that we denote by Conc2U ∈Ob CC⌞(B) the double complex whose row number 0 is given by U, and whose other rows are zero. We have a diagram We have a diagram G(B, Conc2 FA) - G(B, J′)  G(Conc Y, J′) in CCC (C). Let ℓ≥0. Application of Hℓ(−)−,=,∗ yields a diagram (∗) Hℓ`G(B, Conc2 FA)−,=,∗´ - Hℓ`G(B, J′)−,=,∗´  Hℓ`G(Conc Y, J′)−,=,∗´ in CC⌞(C). We have in CCC (C). Let ℓ≥0. Application of Hℓ(−)−,=,∗ yields a diagram (∗) Hℓ`G(B, Conc2 FA)−,=,∗´ - Hℓ`G(B, J′)−,=,∗´  Hℓ`G(Conc Y, J′)−,=,∗´ in CCC (C). Let ℓ≥0. Application of Hℓ(−)−,=,∗ yields a diagram (∗) Hℓ`G(B, Conc2 FA)−,=,∗´ - Hℓ`G(B, J′)−,=,∗´  Hℓ`G(Conc Y, J′)−,=,∗´ i CC⌞(C) W h in CC⌞(C). We have HℓG(B, Conc2 FA)−,=,∗ ≃ G  B , Hℓ(Conc2 FA)−,∗ = G B, Conc Hℓ(FA)  and and and HℓG(B, J′)−,=,∗ ≃ G B, Hℓ(J′−,∗)  , since the functor G(Bk, −) is exact for all k ≥0 by (d), or, since the CE-resolution J is rowwise split. Since the CE-resolution J′ is rowwise split, we moreover have HℓG(Conc Y, J′)−,=,∗ ≃ G Conc Y, Hℓ(J′−,∗)  . So the diagram (∗) is isomorphic to the diagram (∗∗) G B, Conc Hℓ(FA)  - G B, Hℓ(J′−,∗)   G Conc Y, Hℓ(J′−,∗)  , Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 This diagram in turn, is isomorphic to Hℓ G(B, FA)−,∗ - Hℓ (t1,2G(B, J′))−,∗  HℓG(Y, J′) −,∗ , where the left hand side morphism is obtained by precomposition with the isomorphism G(B, FAk) - ∼ t Conc1 G(B, FAk) = (t1,2G(B, Conc2 FA))−,k, where k ≥0; cf. §1.1.6. H k where the left hand side morphism is obtained by precomposition with the isomorphism G(B, FAk) - ∼ t Conc1 G(B, FAk) = (t1,2G(B, Conc2 FA))−,k, where k ≥0; cf. §1.1.6. Hence we may take G(B, FA) - u D v G(B, J′)  := G(B, FA) - t1,2G(B, J′)  G(Y, J′)  . Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 71 whose left hand side morphism is induced by the quasiisomorphism Conc Hℓ(FA) - Hℓ(J′−,∗), and whose right hand side morphism is induced by the quasiisomorphism Conc Y - B. By exactness of G(Bk, −) for k ≥0, the left hand side morphism of (∗∗) is a row- wise quasiisomorphism. Since Hℓ(J′k,∗) is injective, the functor G(−, Hℓ(J′k,∗)) is exact by (e), and therefore the right hand side morphism of (∗∗) is a column- wise quasiisomorphism. Thus an application of t to (∗∗) yields two quasi- isomorphisms; cf. §1.1.6. Hence, also an application of t to (∗) yields two quasiisomorphisms in the diagram whose left hand side morphism is induced by the quasiisomorphism Conc Hℓ(FA) - Hℓ(J′−,∗), and whose right hand side morphism is induced by the quasiisomorphism Conc Y - B. y q p By exactness of G(Bk, −) for k ≥0, the left hand side morphism of (∗∗) is a row- wise quasiisomorphism. Since Hℓ(J′k,∗) is injective, the functor G(−, Hℓ(J′k,∗)) is exact by (e), and therefore the right hand side morphism of (∗∗) is a column- wise quasiisomorphism. Thus an application of t to (∗∗) yields two quasi- isomorphisms; cf. §1.1.6. Hence, also an application of t to (∗) yields two quasiisomorphisms in the diagram o an application of t to (∗ B, J′)−,=,∗´  tHℓ`G(Conc Y −,∗ ◦t1,2, where t1,2 denot ond index of a triple comple G(B, J′))−,∗´  Hℓ`(t1,2G(C This diagram in turn, is iso B, J′))−,∗  HℓG(Y tHℓ`G(B, Conc2 FA)−,=,∗´ - tHℓ`G(B, J′)−,=,∗´  tHℓ`G(Conc Y, J′)−,=,∗´. tHℓ`G(B, Conc2 FA)−,=,∗´ - tHℓ`G(B, J′)−,=,∗´  tHℓ`G(Conc Y, J′)−,=,∗´. Note that t ◦Hℓ(−)−,=,∗ = Hℓ(−)−,∗ ◦t1,2, where t1,2 denotes taking the total complex in the first and the second index of a triple complex; cf. §1.2.2. Hence we have a diagram Hℓ`(t1,2G(B, Conc2 FA))−,∗´ - Hℓ`(t1,2G(B, J′))−,∗´  Hℓ`(t1,2G(Conc Y, J′))−,∗´ consisting of two quasiisomorphisms. This diagram in turn, is isomorphic to (−)−,=,∗ = Hℓ(−)−,∗ ◦t1,2, where t1,2 denotes taking the first and the second index of a triple complex; cf. §1 diagram )) ∗´ ℓ`( ( ′)) ∗´ ℓ`( ( ′)) Note that t ◦Hℓ(−)−,=,∗ = Hℓ(−)−,∗ ◦t1,2, where t1,2 denotes taking the total complex in the first and the second index of a triple complex; cf. §1.2.2. Hence we have a diagram Hℓ`(t1,2G(B, Conc2 FA))−,∗´ - Hℓ`(t1,2G(B, J′))−,∗´  Hℓ`(t1,2G(Conc Y, J′))−,∗´ consisting of two quasiisomorphisms. 6.2 Naturality of the second comparison isomorphism 6.2 Naturality of the second comparison isomorphism Again, we narrow down the assumptions just as we have done for the introduc- tion of the Haas transformations in §4.3.1 to express a naturality of the second comparison isomorphism from Theorem 34. Suppose given abelian categories A and B′ with enough injectives, and abelian categories B and C. Suppose given additive functors A - - F ˜ F B′ and a transfor- mation F - φ ˜F. Let B × B′ - G C be a biadditive functor. Suppose given a morphism X - x ˜X in A and an object Y ∈Ob B. Let B ∈ Ob C[0(B) be a resolution of Y , i.e. suppose a quasiisomorphism Conc Y - B to exist. Suppose the following properties to hold. (a) The functors F and ˜F are left exact and carry injective to G(Y, −)-acyclic objects. (b) The functor G(Y, −) is left exact. (b) The functor G(Y, −) is left exact. (b) The functor G(Y, −) is left exact. (b) The functor G(Y, −) is left exact. Documenta Mathematica 13 (2008) 677–737 718 Matthias K¨unzer (c) The functor G(Bk, −) is exact for all k ≥0. (d) The functor G(−, I′) is exact for all I′ ∈Ob Inj B′. Let A - a ˜A in C[0(Inj A) be an injective resolution of X - x ˜X in A. Note that we have a commutative quadrangle G(B, FA) G(B,φA) / G(B,F a)  G(B, ˜FA) G(B, ˜ F a)  G(B, F ˜A) G(B,φ ˜ A) / G(B, ˜F ˜A) in CC⌞(C). ( ) Note that once chosen injective resolutions A of X and ˜A of ˜X, the image of G(B, Fa) in KK⌞(C) does not depend on the choice of the resolution A - a ˜A of X - x ˜X, for C[0(A) - G(B,F (−)) CC⌞(C) maps an elementary split acyclic complex to an elementary horizontally split acyclic complex. Lemma 35 The quadrangle ˙EGr F,G(Y,−)(X) ˙EGr F,G(Y,−)(x) / ≀  ˙EGr F,G(Y,−)( ˜X) ≀  ˙EI(G(B, FA)) ˙EI(G(B,F a)) / ˙EI(G(B, F ˜A)) commutes, where the vertical isomorphisms are those constructed in the proof of Theorem 34. Proof. Let J′ - ˆa ˜J′ be a CE-resolution of FA - F a F ˜A. Consider the following commutative diagram in CC⌞(C). Comparison of Spectral Sequences Involving Bifunctors 719 Lemma 36 The quadrangle Lemma 36 The quadrangle ˙EGr F,G(Y,−)(X) hI φX / ≀  ˙EGr ˜ F,G(Y,−)(X) ≀  ˙EI(G(B, FA)) ˙EI(G(B,φA)) / ˙EI(G(B, ˜F A)) commutes, where the vertical morphisms are those constructed in the proof of Theorem 34. For the definition of the first Haas transformation hI F (x,−), see §4.3.2. ˆ For the definition of the first Haas transformation hI F (x,−), see §4.3.2. Proof. Let J′ - ˆφ ˘J′ be a CE-resolution of FA - F φ ˜FA. Consider the following commutative diagram in CC⌞(C). ( , ) Proof. Let J′ - ˆφ ˘J′ be a CE-resolution of FA - F φ ˜FA. Consider the following commutative diagram in CC⌞(C). G(Y, J′) G(Y, ˆφ) /  G(Y, ˘J′)  t1,2G(B, J′) t1,2G(B, ˆφ) / t1,2G(B ˘J′) G(B, FA) G(B,φA) / O G(B, ˜F A) O G(Y, J′) G(Y, ˆφ) /  G(Y, ˘J′)  t1,2G(B, J′) t1,2G(B, ˆφ) / t1,2G(B ˘J′) G(B, FA) G(B,φA) / O G(B, ˜F A) O An application of ˙EI yields the result. An application of ˙EI yields the result. An application of ˙EI yields the result. An application of ˙EI yields the result. We refrain from investigating naturality of the second comparison isomorphism in Y . We refrain from investigating naturality of the second comparison isomorphism in Y . 7 Acyclic CE-resolutions We record Beyl’s Theorem [4, Th. 3.4] (here Theorem 40) in order to document that it fits in our context. The argumentation is entirely due to Beyl [4, Sec. 3], so we do not claim any originality. We record Beyl’s Theorem [4, Th. 3.4] (here Theorem 40) in order to document that it fits in our context. The argumentation is entirely due to Beyl [4, Sec. 3], so we do not claim any originality. Let A, B and C be abelian categories. Suppose A and B to have enough injectives. Let A - F B - G C be left exact functors. Let A, B and C be abelian categories. Suppose A and B to have enough injectives. Let A - F B - G C be left exact functors. Let A, B and C be abelian categories. Suppose A and B to have enough injectives. Let A - F B - G C be left exact functors. Documenta Mathematica 13 (2008) 677–737 6.2 Naturality of the second comparison isomorphism G(Y, J′) G(Y,ˆa) /  G(Y, ˜J′)  t1,2G(B, J′) t1,2G(B,ˆa)/ t1,2G(B, ˜J′) G(B, FA) G(B,F a) / O G(B, F ˜A) O G(Y, J′) G(Y,ˆa) /  G(Y, ˜J′)  t1,2G(B, J′) t1,2G(B,ˆa)/ t1,2G(B, ˜J′) G(B, FA) G(B,F a) / O G(B, F ˜A) O An application of ˙EI yields the result. An application of ˙EI yields the result. An application of ˙EI yields the result. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 719 7.1 Definition Let T ∈Ob C[0(B). In this §7, a CE-resolution of T will synonymously (and not quite correctly) be called an injective CE-resolution, to emphasise the fact that its object entries are injective. We regard C[0(B) as an exact category as in Remarks 9 and 11. 720 Matthias K¨unzer Definition 37 A double complex B ∈ CC⌞(B) is called a G-acyclic CE-resolution of T if the following conditions are satisfied. (1) We have H0(B∗,−) ≃T and Hk(B∗,−) ≃0 for all k ≥1. (1) We have H0(B∗,−) ≃T and Hk(B∗,−) ≃0 for all k ≥1. (2) The morphism of complexes Bk,∗ - Bk+1,∗, consisting of vertical dif- ferentials of B, is a pure morphism for all k ≥0. (3) The object Bℓ(Bk,∗) is G-acyclic for all k, ℓ≥0 . (3) The object Bℓ(Bk,∗) is G-acyclic for all k, ℓ≥0 . (4) The object Zℓ(Bk,∗) is G-acyclic for all k, ℓ≥0 . (4) The object Zℓ(Bk,∗) is G-acyclic for all k, ℓ≥0 . A G-acyclic CE-resolution is a G-acyclic CE-resolution of some T ∈Ob C[0(B). A G-acyclic CE-resolution is a G-acyclic CE-resolution of some T ∈Ob C[0(B). From (3, 4) and the short exact sequence Zℓ(Bk,∗) - Bk,ℓ - Bℓ+1(Bk,∗), we conclude that Bk,ℓis G-acyclic for all k, ℓ≥0 . From (3, 4) and the short exact sequence Bℓ(Bk,∗) - Zℓ(Bk,∗) - Hℓ(Bk,∗), we conclude that Hℓ(Bk,∗) is G-acyclic for all k, ℓ≥0 . Example 38 An injective CE-resolution of T is in particular a G-acyclic CE-resolution of T . Note that given Y ∈Ob C(B) and ℓ∈Z, we have ZℓGY ≃GZℓY , whence the universal property of the cokernel HℓGY of GY ℓ−1 - ZℓGY induces a morphism HℓGY - GHℓY . This furnishes a transformation Hℓ(GXk,∗) - θX GHℓ(Xk,∗), natural in X ∈Ob CC⌞(B). Remark 39 If B is a G-acyclic CE-resolution, then the morphism Hℓ(GB−,∗) - θB GHℓ(B−,∗) is an isomorphism for all ℓ≥0. Proof. The sequences GBℓ(Bk,∗) - GZℓ(Bk,∗) - GHℓ(Bk,∗) GZℓ−1(Bk,∗) - GBk,ℓ−1 - GBℓ(Bk,∗) are short exact for k, ℓ≥0 by G-acyclicity of Bℓ(Bk,∗) resp. of Zℓ−1(Bk,∗). In particular, the cokernel of GBk,ℓ−1 - GZℓ(Bk,∗) is given by GHℓ(Bk,∗). 7.2 A theorem of Beyl 7.2 A theorem of Beyl 7.2 A theorem of Beyl Let X ∈Ob A(F,G). Let A ∈C[0(A) be a (F, G)-acyclic resolution of X. Let B ∈CC⌞(B) be a G-acyclic CE-resolution of FA. Theorem 40 (Beyl, [4, Th. 3.4]) We have an isomorphism of proper spec- tral sequences ˙EGr F,G(X) ≃˙EI(GB) in ˙¯Z## ∞, C . Comparison of Spectral Sequences Involving Bifunctors 721 Proof. Since the proper Grothendieck spectral sequence is, up to isomorphism, independent of the choice of an injective CE-resolution, as pointed out in §4.2, our assertion is equivalent to the existence of an injective CE-resolution J of FA such that ˙EI(GJ) ≃ ˙EI(GB). So by Remark 33, it suffices to show that there exists an injective CE-resolution J of FA and a morphism B - J that induces a quasiisomorphism Hℓ(GB−,∗) - Hℓ(GJ−,∗) for all ℓ≥0. By Remark 39 and Example 38, it suffices to show that GHℓ(B−,∗) - GHℓ(J−,∗) is a quasiisomorphism for all ℓ≥0. By the conditions (1, 2) on B and by G-acyclicity of Hℓ(Bk,∗) for k, ℓ≥0, the complex Hℓ(B−,∗) is a G-acyclic resolution of Hℓ(FA); cf. Remark 10. By Remark 4, there exists J ∈Ob CC⌞(Inj B) with vertical pure morphisms and split rows, and a morphism B - J consisting rowwise of pure monomor- phisms such that Hk(B∗,−) - Hk(J∗,−) is an isomorphism of complexes for all k ≥0. In particular, the composite (Conc2 FA - B - J) turns J into an injective CE-resolution of FA. j Let ℓ≥0. Since B is a G-acyclic and J an injective CE-resolution of FA, both Conc Hℓ(FA) - Hℓ(B−,∗) and Conc Hℓ(FA) - Hℓ(J−,∗) are quasiiso- morphisms. Hence Hℓ(B−,∗) - Hℓ(J−,∗) is a quasiisomorphism, too. Now Lemma 27 shows that GHℓ(B−,∗) - GHℓ(J−,∗) is a quasiisomorphism as well. 8.1 A Hopf algebra lemma We will establish Lemma 47 in §8.1.4, needed to prove an acyclicity that enters the proof of the comparison result Theorem 52 in §8.2 for Hopf algebra coho- mology, which in turn allows to derive comparison results for group cohomology and Lie algebra cohomology; cf. §§ 8.3, 8.4. 8 Applications We will apply Theorems 31 and 34 in various algebraic situations. In particular, we will re-prove a theorem of Beyl; viz. Theorem 53 in §8.3. In several instances below, we will make tacit use of the fact that a left exact functor between abelian categories respects injectivity of objects provided it has an exact left adjoint. 8.1.1 Definition Let R be a commutative ring. Write ⊗:= ⊗R . A Hopf algebra over R is an R-algebra H together with R-algebra morphisms H - ε R (counit) and H - ∆ H ⊗H (comultiplication), and an R-linear map H - S H (antipode) such that the following conditions (i–iv) hold. ( ) Write x∆= P i xui ⊗xvi for x ∈H, where ui and vi are chosen maps from H to H, and where i runs over a suitable indexing set. Note that P i (r · x + s · y)ui ⊗(r · x + s · y)vi = r · (P i xui ⊗xvi) + s · (P i yui ⊗yvi) Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 in ˙¯Z## ∞, C . Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 721 Documenta Mathematica 13 (2008) 677–737 722 Matthias K¨unzer for x, y ∈H and r, s ∈R, whereas ui and vi are not necessarily R-linear maps. The elegant Sweedler notation [15, §1.2] for the images under ∆(∆⊗1) etc. led the author, being new to Hopf algebras, to confusion in a certain case. So we will express them in these more naive terms. Write H ⊗H - ∇ H, x ⊗y - x · y and R - η H, r - r · 1H. Write H ⊗H - τ H ⊗H, x ⊗y - y ⊗x. (i) We have ∆(ε ⊗idH) = (x - 1R ⊗x), i.e. P i xuiε · xvi = x for x ∈H. (i′) We have ∆(idH ⊗ε) = (x - x ⊗1R), i.e. P i xui · xviε = x for x ∈H. (ii) We have ∆(idH ⊗∆) = ∆(∆⊗idH), i.e. P i,j xui ⊗xviuj ⊗xvivj = P i,j xuiuj ⊗xuivj ⊗xvi for x ∈H. (ii) We have ∆(idH ⊗∆) = ∆(∆⊗idH), i.e. P i,j xui ⊗xviuj ⊗xvivj = P i,j xuiuj ⊗xuivj ⊗xvi for x ∈H. (iii) We have ∆(S ⊗idH)∇= εη, i.e. P i xuiS · xvi = xε · 1H for x ∈H. (iii′) We have ∆(idH ⊗S)∇= εη, i.e. P i xui · xviS = xε · 1H for x ∈H. (iv) We have S2 = idH. In particular, imposing (iv), we stipulate a Hopf algebra to have an involutive antipode. 8.1.2 Some basic properties In an attempt to be reasonably self-contained, we recall some basic facts on Hopf algebras needed for Lemma 47 below; cf. [15, Ch. IV], [1, §2], [13, §§1-3]. In doing so, we shall use direct arguments. Suppose given a Hopf algebra H over R. Documenta Mathematica 13 (2008) 677–737 Suppose given a Hopf algebra H over R. Remark 41 ([15, Prop. 4.0.1], [1, Th. 2.1.4], [13, 3.4.2]) The following hold. Remark 41 ([15, Prop. 4.0.1], [1, Th. 2.1.4], [13, 3.4.2]) The following hold. (1) We have P i(x·y)ui⊗(x·y)vi = P i,j(xui·yuj)⊗(xvi·yvj) for x, y ∈H. (1) We have P i(x·y)ui⊗(x·y)vi = P i,j(xui·yuj)⊗(xvi·yvj) for x, y ∈H. (2) We have 1HS = 1H. (2) We have 1HS = 1H. (3) We have (x · y)S = y · x for x, y ∈H. (3) We have (x · y)S = y · x for x, y ∈H. (4) We have Sε = ε. (4) We have Sε = ε. (4) We have Sε = ε. (5) We have ∆(S ⊗S)τ = S∆, i.e. P i xuiS ⊗xviS = P i xSvi ⊗xSui for x ∈H. (5) We have ∆(S ⊗S)τ = S∆, i.e. P i xuiS ⊗xviS = P i xSvi ⊗xSui for x ∈H. (6) We have x · y = P i P j(xui)uj · y · (xui)vjS  · xvi for x, y ∈H.   (6) We have x · y = P i P j(xui)uj · y · (xui)vjS  · xvi for x, y ∈H.   (6) We have x · y = P i P j(xui)uj · y · (xui)vjS  · xvi for x, y ∈H. (6′) We have y · x = P i xui · P j(xvi)ujS · y · (xvi)vj  for x, y ∈H. (6′) We have y · x = P i xui · P j(xvi)ujS · y · (xvi)vj  for x, y ∈H. (6′) We have y · x = P i xui · P j(xvi)ujS · y · (xvi)vj  for x, y ∈H. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 723 Comparison of Spectral Sequences Involving Bifunctors 723 (7) We have P i xvi · xuiS = xε · 1H for x ∈H. (7′) We have P i xviS · xui = xε · 1H for x ∈H. Proof. Ad (1). Given x, y ∈H, we obtain Proof. Ad (1). Given x, y ∈H, we obtain P i (xy)ui ⊗(xy)vi = (xy)∆= x∆· y∆= P i,j (xui · yuj) ⊗(xvi · yvj) . Ad (2). Remarking that 1H∆= 1H ⊗1H, we obtain 1HS = 1H∆(S ⊗idH)∇ (iii) = 1Hε · 1H = 1H . Ad (3). Given x, y ∈H, we obtain (x · y)S 2 × (i′) = P i,k(xui · xviε · yuk · yvkε)S (iii′) = P i,j,k(xui · yuk · yvkε)S · xviuj · xvivjS (iii′) = P i,j,k,ℓ(xui · yuk)S · xviuj · yvkuℓ· yvkvℓS · xvivjS 2 × (ii) = P i,j,k,ℓ(xuiuj · yukuℓ)S · xuivj · yukvℓ· yvkS · xviS (1) = P i,j,k(xui · yuk)ujS · (xui · yuk)vj · yvkS · xviS (iii) = P i,k(xui · yuk)ε · yvkS · xviS = P i,k(yukε · yvk)S · (xuiε · xvi)S 2 × (i) = yS · xS . 2 × (i) = yS · xS . Ad (4). Note that (yε · z)ε = yε · zε = (y · z)ε for y, z ∈H. Given x ∈H, we obtain xSε (i) = (P i xuiε · xvi)Sε = (P i xuiε · xviS)ε = (P i xui · xviS)ε (iii′) Documenta Mathematica 13 (2008) 677–737 Ad (5). Given x ∈H, we obtain d (5). Given x ∈H, we obtain x∆(S ⊗S)τ (i) = P i(xuiε · xvi)∆(S ⊗S)τ = P i(xuiε · 1H)∆· xvi∆(S ⊗S)τ (iii) = P i,j(xuiujS · xuivj)∆· xvi∆(S ⊗S)τ = P i,j xuiujS∆· xuivj∆· xvi∆(S ⊗S)τ (ii) = P i,j xuiS∆· xviuj∆· xvivj∆(S ⊗S)τ = P i,j,k,ℓxuiS∆· (xviujuk ⊗xviujvk) · (xvivjvℓS ⊗xvivjuℓS) = P i,j,k,ℓxuiS∆· (xviujuk · xvivjvℓS ⊗xviujvk · xvivjuℓS) (ii) = P i,j,k,ℓxuiS∆· (xviuj · xvivjvkvℓS ⊗xvivjuk · xvivjvkuℓS) (ii) = P i,j,k,ℓxuiS∆· (xviuj · xvivjvkS ⊗xvivjukuℓ· xvivjukvℓS) (iii′) = P i,j,k xuiS∆· (xviuj · xvivjvkS ⊗xvivjukε · 1H) = P i,j,k xuiS∆· (xviuj · (xvivjvk · xvivjukε)S ⊗1H) (i) = P i j xuiS∆· (xviuj · xvivjS ⊗1H) (i) = P i,j xuiS∆· (xviuj · xvivjS ⊗1H) Documenta Mathematica 13 (2008) 677–737 724 Matthias K¨unzer (iii′) = P i xuiS∆· (xviε · 1H ⊗1H) = P i(xui · xviε)S∆ (i′) = xS∆. Ad (6). Given x, y ∈H, we obtain x·y (i′) = P i xui·y·xviε (iii) = P i,j xui·y·xviujS·xvivj (ii) = P i,j xuiuj·y·xuivjS·xvi. Ad (6′). Given x ∈H, we obtain x·y (i′) = P i xui·y·xviε (iii) = P i,j xui·y·xviujS·xvivj (ii) = P i,j xuiuj·y·xuivjS·xvi. Ad (6′). Given x ∈H, we obtain x·y (i′) = P i xui·y·xviε (iii) = P i,j xui·y·xviujS·xvivj (ii) = P i,j xuiuj·y·xuivjS·xvi. Ad (6′). Given x ∈H, we obtain y·x (i) = P i xuiε·y·xvi (iii′) = P i,j xuiuj·xuivjS·y·xvi (ii) = P i,j xui·xviujS·y·xvivj. Ad (7). Given x ∈H, we have P i xvi · xuiS (iv) = P i xS2vi · xS2uiS (5) = P i xSuiS · xSviS2 (iv) = P i xSuiS · xSvi (iii) = xSε · 1H (4) = xε · Ad (7′). Given x ∈H, we have P i xviS · xui (iv) = P i xS2viS · xS2ui (5) = P i xSuiS2 · xSviS (iv) = P i xSui · xSviS (iii′) = xSε · P i xviS · xui (iv) = P i xS2viS · xS2ui (5) = P i xSuiS2 · xSviS (iv) = P i xSui · xSviS (iii′) = xSε · 1H (4) = xε · 1H . In the present §8.1, we shall refer to the assertions Remark 41.(1–7′) just by (1–7′). 8.1.3 Normality Suppose given a Hopf algebra H over R, and an R-subalgebra K ⊆H. Suppose H and K to be flat as modules over R. Suppose given a Hopf algebra H over R, and an R-subalgebra K ⊆H. Suppose H and K to be flat as modules over R. Note that K ⊗K - H ⊗H is injective. We will identify K ⊗K with its image. Note that K ⊗K - H ⊗H is injective. We will identify K ⊗K with its image. The R-subalgebra K ⊆H is called a Hopf-subalgebra if K∆⊆K ⊗K and KS ⊆K. In this case, we may and will suppose the maps ui and vi to restrict to maps from K to K. Suppose K ⊆H to be a Hopf-subalgebra. It is called normal, if for all a ∈K and all x ∈H, we have Suppose K ⊆H to be a Hopf-subalgebra. It is called normal, if for all a ∈K and all x ∈H, we have Suppose K ⊆H to be a Hopf-subalgebra. It is called normal, if for all a ∈K and all x ∈H, we have P i xui · a · xviS ∈K and P i xuiS · a · xvi ∈K . Comparison of Spectral Sequences Involving Bifunctors 725 Suppose K ⊆ H to be a normal Hopf subalgebra. Write K+ := Kern(H - ε R). By (6, 6′, 3, 4), HK+ = K+H is a Hopf ideal in H. P i xui · a · xviS ∈K and P i xuiS · a · xvi ∈K . P i xui · a · xviS ∈K and P i xuiS · a · xvi ∈K . An ideal I ⊆H is called a Hopf ideal if I∆⊆I ⊗H + H ⊗I (where we have identified I ⊗H and H ⊗I with their images in H ⊗H), Iε = 0 and IS ⊆I. In this case, the quotient H/I carries a Hopf algebra structure via An ideal I ⊆H is called a Hopf ideal if I∆⊆I ⊗H + H ⊗I (where we have identified I ⊗H and H ⊗I with their images in H ⊗H), Iε = 0 and IS ⊆I. In this case, the quotient H/I carries a Hopf algebra structure via An ideal I ⊆H is called a Hopf ideal if I∆⊆I ⊗H + H ⊗I (where we have identified I ⊗H and H ⊗I with their images in H ⊗H), Iε = 0 and IS ⊆I. g ) In this case, the quotient H/I carries a Hopf algebra structure via H/I - ε R , x + I - xε ∆ H/I - ∆ H/I ⊗H/I , x + I - P i(xui + I) ⊗(xvi + I) S H/I - ∆ H/I ⊗H/I , x + I - P i(xui + I) ⊗(xvi + I) S H/I - S H/I , x + I - xS + I . H/I - S H/I , x + I - xS + I . 8.1.4 Some remarks and a lemma Suppose given a Hopf algebra H over R and a normal Hopf-subalgebra K ⊆H. Suppose H and K to be flat as modules over R. Write ¯H := H/HK+. Given x ∈H, write ¯x := x + HK+ ∈¯H for its residue class. Suppose given a Hopf algebra H over R and a normal Hopf-subalgebra K ⊆H. Suppose H and K to be flat as modules over R. Suppose given a Hopf algebra H over R and a normal Hopf-subalgebra K ⊆H. Suppose H and K to be flat as modules over R. Write ¯H := H/HK+. Given x ∈H, write ¯x := x + HK+ ∈¯H for its residue l Suppose given a Hopf algebra H over R and a normal Hopf-subalgebra K ⊆H. Suppose H and K to be flat as modules over R. Write ¯H := H/HK+. Given x ∈H, write ¯x := x + HK+ ∈¯H for its residue class. Write ¯H := H/HK+. Given x ∈H, write ¯x := x + HK+ ∈¯H for its residue class. Let N ′, N, M, M ′ and Q be H-modules. Let P be an ¯H-module, which we also consider as an H-module via H - ¯H, x - ¯x. Let N ′, N, M, M ′ and Q be H-modules. Let P be an ¯H-module, which we also consider as an H-module via H - ¯H, x - ¯x. Let N ′, N, M, M ′ and Q be H-modules. Let P be an ¯H-module, which we also consider as an H-module via H - ¯H, x - ¯x. We write K(N, M) = K(N|K, M|K) for the R-module of K-linear maps from N to M We write K(N, M) = K(N|K, M|K) for the R-module of K-linear maps from N to M. Remark 42 Given f ∈R(N, M) and x ∈H, we define x · f ∈R(N, M) by [n](x · f) := P i xui · [xviS · n]f Remark 42 Given f ∈R(N, M) and x ∈H, we define x · f ∈R(N, M) by [n](x · f) := P i xui · [xviS · n]f Remark 42 Given f ∈R(N, M) and x ∈H, we define x · f ∈R(N, M) by [n](x · f) := P i xui · [xviS · n]f for n ∈N. This defines a left H-module structure on R(N, M). for n ∈N. This defines a left H-module structure on R(N, M). Formally, squared brackets mean the same as parentheses. Informally, squared brackets are to accentuate the arguments of certain maps. roof. We claim that x′ · (x · f) = (x′ · x) · f for x, x′ ∈H. Suppose given ∈N. We obtain Proof. We claim that x′ · (x · f) = (x′ · x) · f for x, x′ ∈H. Suppose given n ∈N. We obtain [n](x′ · (x · f)) = P i x′ui · [x′viS · n](x · f) = P i,j x′ui · xuj · [xvjS · x′viS · n]f (3) = P i,j(x′ui · xuj) · [(x′vi · xvj)S · n]f (1) = P i(x′ · x)ui · [(x′ · x)viS · n]f = [n]((x′ · x) · f) . We claim that 1H · f = f. Suppose given n ∈N. We obtain We claim that 1H · f = f. Suppose given n ∈N. We obtain [n](1H · f) = P i 1Hui · [1HviS · n]f = 1H · [1HS · n]f (2) = [n]f , marking that 1H∆= 1H ⊗1H . remarking that 1H∆= 1H ⊗1H . I owe to G. Hiß the hint to improve a previous weaker version of Corollary 45 below by means of the following Remark 43. g H H H I owe to G. Hiß the hint to improve a previous weaker version of Corollary 45 below by means of the following Remark 43. I owe to G. Hiß the hint to improve a previous weaker version of Corollary 45 below by means of the following Remark 43. Denote by M K := {m ∈M : a · m = aε · m for all a ∈K} the fixed point module of M under K. the fixed point module of M under K. the fixed point module of M under K. Remark 43 Letting ¯x · m := x · m for x ∈H and m ∈M K, we define an ¯H-module structure on M K. Remark 43 Letting ¯x · m := x · m for x ∈H and m ∈M K, we define an ¯H-module structure on M K. Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 726 Matthias K¨unzer Proof. for n ∈N. This defines a left H-module structure on R(N, M). The value of the product ¯x · m does not depend on the chosen represen- tative x of ¯x since, given y ∈H, a ∈K+ and m ∈M K, we have y · a · m = y · aε · m = 0 . It remains to be shown that given x ∈H and m ∈M K, the element x · m lies in M K. In fact, given a ∈K, we obtain It remains to be shown that given x ∈H and m ∈M K, the element x · m lies in M K. In fact, given a ∈K, we obtain a · x · m (6′) = P i xui · P j(xvi)ujS · a · (xvi)vj  · m = P i xui · P j(xvi)ujS · a · (xvi)vj  ε · m = P i,j xui · xviujSε · aε · xvivjε · m (4) = P i,j xui · xviujε · aε · xvivjε · m (ii) = P i,j xuiuj · xuivjε · aε · xviε · m (i′) = P i xui · aε · xviε · m (i′) = aε · x · m . Remark 44 We have (R(N, M))K = K(N, M), as subsets of R(N, M). Remark 44 We have (R(N, M))K = K(N, M), as subsets of R(N, M). Proof. The module ( R(N, M))K consists of the R-linear maps N - f M that satisfy P i xui · [xviS · n]f = xε · [n]f . for x ∈H and n ∈N. The module K(N, M) consists of the R-linear maps f for x ∈H and n ∈N. The module K(N, M) consists of the R-linear maps N - f M that satisfy N - f M that satisfy [x · n]f = x · [n]f for x ∈H and n ∈N. By (iii′), we have (R(N, M))K ⊇K(N, M). It remains to show that (R(N, M))K ⊆ K(N, M). Given f ∈(R(N, M))K, x ∈H and n ∈N, we obtain for x ∈H and n ∈N. By (iii′), we have (R(N, M))K ⊇K(N, M). It remains to show that (R(N, M))K ⊆ K(N, M). Given f ∈(R(N, M))K, x ∈H and n ∈N, we obtain x · [n]f (i′) = P i xui · xviε · [n]f = P i xui · [xviε · n]f (iii) = P i,j xui · [xviujS · xvivj · n]f (ii) = P i,j xuiuj · [xuivjS · xvi · n]f = P i xuiε · [xvi · n]f (i) = [x · n]f . Corollary 45 Given f ∈K(N, M) and x ∈H, we define ¯x · f ∈K(N, M) by [n](¯x · f) := P i xui · [xviS · n]f for n ∈N. This defines a left ¯H-module structure on K(N, M). Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 727 Comparison of Spectral Sequences Involving Bifunctors 727 Proof. By Remark 42, we may apply Remark 43 to R(N, M). By Remark 44, the assertion follows. Remark 46 Given f ∈ K(N, M), x ∈H, and H-linear maps N ′ - ν N, M - µ M ′, we obtain Remark 46 Given f ∈ K(N, M), x ∈H, and H-linear maps N ′ - ν N, M - µ M ′, we obtain ν(¯x · f)µ = ¯x · (νfµ) . ν(¯x · f)µ = ¯x · (νfµ) . ν(¯x · f)µ = ¯x · (νfµ) . Proof. Given n′ ∈N ′, we obtain [n′] ν(¯x · f)µ  = P i xui · [xviS · n′ν]f  µ = P i xui · [xviS · n′](νfµ) = [n′](¯x · (νfµ)). Remark 44 We have (R(N, M))K = K(N, M), as subsets of R(N, M). The following Lemma 47 has been suggested by the referee, and has been achieved with the help of G. Carnovale. It is reminiscent of [16, Cor. 4.3], but easier. It resembles a bit a Fourier inversion. Note that the right ¯H-module structure on ¯H induces a left ¯H-module structure on R( ¯H, M). Lemma 47 We have the following mutually inverse isomorphisms of ¯H-modules. K(H, M) - Φ ∼ R( ¯H, M) f - (¯x - P i xui · [xviS]f) K(H, M) Ψ ∼ R( ¯H, M) (x - P j xvj · [ xujS ]g)  g K(H, M) - Φ ∼ R( ¯H, M) f - (¯x - P i xui · [xviS]f) K(H, M) Ψ ∼ R( ¯H, M) (x - P j xvj · [ xujS ]g)  g Proof. We claim that Φ is a welldefined map. We have to show that fΦ is welldefined, i.e. that its value at ¯x does not depend on the representing element x. Suppose given y ∈H and a ∈K+. We obtain P i(ya)ui · [(ya)viS]f (1) = P i,j yui · auj · [(yvi · avj)S]f (3) = P i,j yui · auj · [avjS · yviS]f = P i,j yui · auj · avjS · [yviS]f (iii′) = P i yui · aε · [yviS]f = 0 . We claim that Φ is ¯H-linear. Suppose given y ∈H and x ∈H. We obtain [¯x]((¯yf)Φ) = P i xui · [xviS](¯yf) = P i,j xui · yuj · [yvjS · xviS]f (3) = P i,j xui · yuj · [(xvi · yvj)S]f (1) = P i(x · y)ui · [(x · y)viS]f = [¯x](¯y(fΦ)) . Documenta Mathematica 13 (2008) 677–737 728 Matthias K¨unzer We claim that Ψ is a welldefined map. We have to show that gΨ is K-linear. Suppose given a ∈K and x ∈H. Note that aui ∈K for all i, whence also auiS ∈K, and therefore auiS ≡HK+ auiSε · 1H. Remark 44 We have (R(N, M))K = K(N, M), as subsets of R(N, M). We obtain [a · x](gΨ) = P j(a · x)vj · [ (a · x)ujS ]g (1) = P i,j avi · xvj · [ (aui · xuj)S ]g (3) = P i,j avi · xvj · [ xujS · auiS ]g = P i,j avi · xvj · [ xujS · auiSε ]g (4) = P i,j auiε · avi · xvj · [ xujS ]g (i) = P j a · xvj · [ xujS ]g = a · [x](gΨ) . We claim that ΦΨ = idK(H,M). Suppose given x ∈H. We obtain We claim that ΦΨ = idK(H,M). Suppose given x ∈H. We obtain [x](fΦΨ) = P j xvj · [ xujS ](fΦ) = P i,j xvj · xujSui · [xujSviS]f (5) = P i,j xvj · xujviS · [xujuiS2]f (iv) = P i,j xvj · xujviS · [xujui]f (ii) = P i,j xvjvi · xvjuiS · [xuj]f (7) = P j xvjε · [xuj]f (i) = [x]f . We claim that ΨΦ = idR( ¯ H,M). Suppose given x ∈H. We obtain We claim that ΨΦ = idR( ¯ H,M). Suppose given x ∈H. We obtain [¯x](gΨΦ) = P i xui · [xviS](gΨ) = P i,j xui · xviSvj · [ xviSujS ]g (5) = P i,j xui · xviujS · [ xvivjS2 ]g (iv) = P i,j xui · xviujS · [ xvivj ]g (ii) = P i,j xuiuj · xuivjS · [ xvi ]g (iii′) = P i xuiε · [ xvi ]g (i) = [¯x]g . Finally, it follows by ¯H-linearity of Φ and by Ψ = Φ−1 that Ψ is ¯H-linear. The tensor product N ⊗M is an H-module via ∆. Note that R is an H-module via ε. Note that R ⊗M ≃M ≃M ⊗R as H-modules by (i, i′). Remark 48 (cf. [3, Lem. 3.5.1]) We have mutually inverse isomorphisms Remark 48 (cf. [3, Lem. 3.5.1]) We have mutually inverse isomorphisms Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 729 Comparison of Spectral Sequences Involving Bifunctors 729 of R-modules Comparison of Spectral Sequences Involving Bifunctors 729 of R-modules of R-modules ¯ H(P, K(Q, M)) - α ∼ H(P ⊗Q, M) f - (p ⊗q - [q](pf)) ¯ H(P, K(Q, M)) β ∼ H(P ⊗Q, M) (p - (q - [p ⊗q]g))  g , ¯ H(P, K(Q, M)) - α ∼ H(P ⊗Q, M) f - (p ⊗q - [q](pf)) ¯ H(P, K(Q, M)) β ∼ H(P ⊗Q, M) (p - (q - [p ⊗q]g))  g , natural in P ∈Ob ¯H-Mod, Q ∈Ob H-Mod and M ∈Ob H-Mod. Proof. We claim that α is welldefined. We have to show that fα is H-linear. Suppose given x ∈H. We obtain x · (p ⊗q) = P i xui · p ⊗xvi · q - fα P i[xvi · q]((xui · p)f) = P i[xvi · q](xui · (pf)) = P i,j xuiuj · [xuivjS · xvi · q](pf) (ii) = P i,j xui · [xviujS · xvivj · q](pf) (iii) = P i xui · [xviε · q](pf) (i′) = x · [q](pf) = x · [p ⊗q](fα) . We claim that β is welldefined. First, we have to show that [p](gβ) is K-linear. Suppose given a ∈K. We obtain We claim that β is welldefined. First, we have to show that [p](gβ) is K-linear. Suppose given a ∈K. We obtain a·q - [p](gβ) [p⊗a·q]g (i) = P i [auiε·p⊗avi·q]g = P i [aui·p⊗avi·q]g = a·[p⊗q]g . Second, we have to show that gβ is ¯H-linear. Suppose given x ∈H. We obtain β a·q - [p⊗a·q]g ( ) = P i [auiε·p⊗avi·q]g = P i [aui·p⊗avi·q]g = a·[p⊗q]g . Second, we have to show that gβ is ¯H-linear. Suppose given x ∈H. We obtain β Second, we have to show that gβ is ¯H-linear. Suppose given x ∈H. We obtai ¯x · p - gβ (q - [¯x · p ⊗q]g) (i) = (q - P i[xui · xviε · p ⊗q)]g) (iii′) = (q - P i,j[xui · p ⊗xviuj · xvivjS · q)]g) (ii) = (q - P i,j[xuiuj · p ⊗xuivj · xviS · q)]g) = (q - P i xui · [p ⊗xviS · q]g) = ¯x · (q - [p ⊗q]g) . Finally, α and β are mutually inverse. Finally, α and β are mutually inverse. Corollary 49 We have ¯ H(P, M K) ≃ ¯ H(P, K(R, M)) ≃ H(P, M) as R-modules, natural in P and M. Corollary 49 We have ¯ H(P, M K) ≃ ¯ H(P, K(R, M)) ≃ H(P, M) as R-modules, natural in P and M. Proof. Note that M ≃ R(R, M) as H-modules, whence M K ≃ K(R, M) as ¯H-modules by Remarks 43, 44. Now the assertion follows from Remark 48, letting Q = R. Proof. Note that M ≃ R(R, M) as H-modules, whence M K ≃ K(R, M) as ¯H-modules by Remarks 43, 44. Documenta Mathematica 13 (2008) 677–737 ( ¯H-Mod)◦ × ¯H-Mod - V ¯H-Mod (Y , Y ′) - V (N, M) := ¯ H(N, M) ( ¯H-Mod)◦ × ¯H-Mod - V ¯H-Mod (Y , Y ′) - V (N, M) := ¯ H(N, M) for the usual Hom-functor. Lemma 50 The ¯H-module U(H, M) is V (R, −)-acyclic. natural in P ∈Ob ¯H-Mod, Q ∈Ob H-Mod and M ∈Ob H-Mod. Now the assertion follows from Remark 48, letting Q = R. = R. Documenta Mathematica 13 (2008) 677–737 730 Matthias K¨unzer 8.2 Comparing Hochschild-Serre-Hopf with Grothendieck Let R be a commutative ring. Suppose given a Hopf algebra H over R (with involutive antipode) and a normal Hopf-subalgebra K ⊆R; cf. §8.1.3. Write ¯H := H/HK+. Suppose H, K and ¯H to be projective as modules over R. Suppose H to be projective as a module over K. Let B ∈Ob C(H-Mod) be a projective resolution of R over H. Let ¯B ∈ Ob C( ¯H-Mod) be a projective resolution of R over ¯H. Note that since ¯H is projective over R, ¯B|R ∈Ob C(R-Mod) is a projective resolution of R over R. Let M be an H-module. By Corollary 45 and by Remark 46, we have a biadditive functor (H-Mod)◦ × H-Mod - U ¯H-Mod (X , X′) - U(X, X′) := K(X, X′) . (H-Mod)◦ × H-Mod - U ¯H-Mod (X , X′) - U(X, X′) := K(X, X′) . Write Documenta Mathematica 13 (2008) 677–737 Lemma 50 The ¯H-module U(H, M) is V (R, −)-acyclic. Proof. By Lemma 47, this amounts to showing that R( ¯H, M) is V (R, −)- acyclic, which in turn amounts to showing that V ¯B , R( ¯H, M)  = ¯ H ¯B , R( ¯H, M)  has vanishing cohomology in degrees ≥1. Now, ¯ H ¯B , R( ¯H, M)  ≃ R( ¯H ⊗¯ H ¯B , M) ≃ R( ¯B , M) , whose cohomology in degree i ≥1 is Exti R(R, M) ≃0. Consider the double complex whose cohomology in degree i ≥1 is Exti R(R, M) ≃0. Consider the double complex D(M) = D−,=(M) := V ¯B−, U(B= , M)  = ¯ H ¯B−, K(B= , M)  . Note that D(M) is isomorphic in CC⌞(R-Mod) to H ¯B−⊗R B= , M  , natu- rally in M; cf. Remark 48. Note that D(M) is isomorphic in CC⌞(R-Mod) to H ¯B−⊗R B= , M  , natu- rally in M; cf. Remark 48. We have functors y ; We have functors We have functors H-Mod - U(R,−) ¯H-Mod - V (R,−) R-Mod . M - U(R, M) ≃M K P - V (R, P) ≃P ¯ H Lemma 51 Given a projective H-module P, the ¯H-module U(P, M) is V (R, −)-acyclic. Lemma 51 Given a projective H-module P, the ¯H-module U(P, M) is V (R, −)-acyclic. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 731 Comparison of Spectral Sequences Involving Bifunctors 731 Proof. It suffices to show that U(` Γ H, M) ≃Q Γ U(H, M) is V (R, −)- acyclic for any indexing set Γ. By Lemma 50, it remains to be shown that RiV (R, Q Γ Y ) is isomorphic to Q Γ RiV (R, Y ) for a given ¯H-module Y and for i ≥1. Having chosen an injective resolution J of Y , we may choose the injective resolution Q Γ J of Q Γ Y . Then RiV (R, Q Γ Y ) ≃HiV (R, Q Γ J) ≃Hi Q Γ V (R, J) ≃Q Γ HiV (R, J) ≃Q Γ RiV (R, Y ) . Theorem 52 The proper spectral sequences Theorem 52 The proper spectral sequences Theorem 52 The proper spectral sequences ˙EI(D(M)) and ˙EGr U(R,−), V (R,−)(M) ˙EI(D(M)) and ˙EGr U(R,−), V (R,−)(M) ˙EI(D(M)) and ˙EGr U(R,−), V (R,−)(M) are isomorphic (in ˙¯Z## ∞, R-Mod ), naturally in M ∈Ob H-Mod. are isomorphic (in ˙¯Z## ∞, R-Mod ), naturally in M ∈Ob H-Mod. are isomorphic (in ˙¯Z## ∞, R-Mod ), naturally in M ∈Ob H-Mod. Proof. To apply Theorem 31 with, in the notation of §5.1,  A × A′ - F B - G C  =  (H-Mod)◦× H-Mod - U ¯H-Mod - V (R,−) R-Mod  ,  A × A′ - F B - G C  and with X = R and X′ = M, we verify the conditions (a–d′) of loc. cit. in this case. and with X = R and X′ = M, we verify the conditions (a–d′) of loc. cit. in this case. Ad (c). We claim that B is a U(−, M), V (R, −)  -acyclic resolution of R. We have to show that U(Bi, M) is V (R, −)-acyclic for i ≥0; cf. §4.2. Since Bi is projective over H, this follows by Lemma 51. This proves the claim. Ad (c′). Let I be an injective resolution of M over H. We claim that I is a U(R, −), V (R, −)  -acyclic resolution of M. We have to show that U(R, Ii) is V (R, −)-acyclic for i ≥0. In fact, by Corollary 49, U(R, Ii) is an injective ¯H-module. This proves the claim. Ad (d, d′). We claim that U(Bi, −) and U(−, Ii) are exact for i ≥0; cf. §5.1. The former follows from H being projective over K. The latter is a consequence Ad (d, d′). We claim that U(Bi, −) and U(−, Ii) are exact for i ≥0; cf. §5.1. The former follows from H being projective over K. The latter is a consequence of Ii|K being injective in K-Mod by exactness of K-Mod - H⊗K−H-Mod. This proves the claim. of Ii|K being injective in K-Mod by exactness of K-Mod - H⊗K−H-Mod. This proves the claim. So an application of Theorem 31 yields ˙EGr U(R,−),V (R,−)(M) ≃˙EGr U(−,M),V (R,−)(R) . are isomorphic (in ˙¯Z## ∞, R-Mod ), naturally in M ∈Ob H-Mod. To apply Theorem 34 with, in the notation of §6.1,  A - F B′ , B × B′ - G C  =  (H-Mod)◦ - U(−,M) ¯H-Mod , ( ¯H-Mod)◦× ¯H-Mod - V C  ,   =  (H-Mod)◦ - U(−,M) ¯H-Mod , ( ¯H-Mod)◦× ¯H-Mod - V C  , =  (H-Mod)◦ - U(−,M) ¯H-Mod , ( ¯H-Mod)◦× ¯H-Mod - V C  , Documenta Mathematica 13 (2008) 677–737 Documenta Mathematica 13 (2008) 677–737 732 Matthias K¨unzer and with X = R and Y = R, we verify the conditions (a–e) of loc. cit. in this case. Ad (c). We have already remarked that B is a U(−, M), V (R, −)  -acyclic resolution of R. and with X = R and Y = R, we verify the conditions (a–e) of loc. cit. in this case. and with X = R and Y = R, we verify the conditions (a–e) of loc. cit. in this case. Ad (c). We have already remarked that B is a U(−, M), V (R, −)  -acyclic resolution of R. Ad (c). We have already remarked that B is a U(−, M), V (R, −)  -acyclic resolution of R. Ad (d). As a resolution of R over ¯H, we choose ¯B. So an application of Theorem 34 yields So an application of Theorem 34 yields ˙EGr U(−,M),V (R,−)(R) ≃˙EI  V ¯B−, U(B= , M)  . Naturality in M ∈Ob H-Mod remains to be shown. Suppose given M - m ˜ M in H-Mod. Note that the requirements of §5.2 are met. By Proposition 32, with roles of A and A′ interchanged, we have the following commutative quadrangle. ˙EGr U(R,−),V (R,−)(M) ˙EGr U(R,−),V (R,−)(m) / ˙EGr U(R,−),V (R,−)( ˜ M) ˙EGr U(−,M),V (R,−)(R) hI U(−,m)R / ≀ O ˙EGr U(−, ˜ M),V (R,−)(R) ≀ O Note that the requirements of §6.2 are met. By Lemma 36, we have the follow- ing commutative quadrangle. ˙EGr U(−,M),V (R,−)(R) hI U(−,m)R / ≀  ˙EGr U(−, ˜ M),V (R,−)(R) ≀  ˙EI  V ¯B−, U(B= , M)  ˙EI(V ( ¯ B−, U(B=,m))) / ˙EI  V ¯B−, U(B= , ˜ M)  8.3 Comparing Lyndon-Hochschild-Serre with Grothendieck Documenta Mathematica 13 (2008) 677–737 are isomorphic (in ˙¯Z## ∞, R-Mod ), naturally in M ∈Ob RG -Mod. are isomorphic (in ˙¯Z## ∞, R-Mod ), naturally in M ∈Ob RG -Mod. Beyl uses his Theorem 40 to prove Theorem 53. We shall re-derive it from Theorem 52, which in turn relies on the Theorems 31 and 34. Proof. This follows by Theorem 52. Proof. This follows by Theorem 52. Proof. This follows by Theorem 52. 8.4 Comparing Hochschild-Serre with Grothendieck Let R be a commutative ring. Let g be a Lie algebra over R that is free as an R-module. Let n ⊴g be an ideal such that n and ¯g := g/n are free as R-modules. Let M be a g-module, i.e. a U(g)-module. Write Barg;R ∈ Ob C(U(g) -Mod) for the Chevalley-Eilenberg resolution of R over U(g), having (Barg;R)i = U(g) ⊗R ∧ig for i ≥0; cf. [5, XIII.§7] or [18, Th. 7.7.2]. Note that U(g) is a Hopf algebra over R via U(g) - ∆ U(g) ⊗U(g) , g - g ⊗1 + 1 ⊗g U(g) - S U(g) , g - −g U(g) - ε R , g - 0 , where g ∈g; cf. §8.1.1. Note that U(g), U(n) and U(¯g) are projective over R, and that U(g) is projective over U(n); cf. [18, Cor. 7.3.9]. Note that U(g), U(n) and U(¯g) are projective over R, and that U(g) is projective over U(n); cf. [18, Cor. 7.3.9]. ¯ We have functors U(g) -Mod - (−)n U(¯g) -Mod - (−)¯g R-Mod, taking respective annihilated submodules; cf. [18, p. 221]. Theorem 54 The proper spectral sequences Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 733 Note that RG, RN and R ¯G are projective over R, and that RG is projective over RN. We have functors RG -Mod - (−)N R ¯G -Mod - (−) ¯ G R-Mod, taking respective fixed points. Theorem 53 (Beyl, [4, Th. 3.5]) The proper spectral sequences ˙EGr (−)N, (−) ¯ G(M) and ˙EI  RG (Bar ¯ G;R)−⊗R (BarG;R)= , M  ˙EGr (−)N, (−) ¯ G(M) and ˙EI  RG (Bar ¯ G;R)−⊗R (BarG;R)= , M  8.3 Comparing Lyndon-Hochschild-Serre with Grothendieck Let R be a commutative ring. Let G be a group and let N ⊴G be a normal subgroup. Write ¯G := G/N. Let M be an RG-module. Write BarG;R ∈Ob C(RG -Mod) for the bar resolution of R over RG, having (BarG;R)i = RG⊗(i+1) for i ≥0, the tensor product being taken over R. Note that RG is a Hopf algebra over R via RG - ∆ RG ⊗RG , g - g ⊗g RG - S RG , g - g−1 RG - ε R , g - 1 , where g ∈G; cf. §8.1.1. Moreover, RN is a normal Hopf subalgebra of RG such that RG/(RG)(RN)+ ≃R ¯G; cf. §8.1.3. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 733 are isomorphic (in ˙¯Z## ∞, R-Mod ). Proof. To apply Theorem 34, if suffices to remark that for each injective A-module I′, the B-module A(B, I′) is injective, and thus B(Y, −)-acyclic. Remark 56 The functor A(B, −) can be replaced by A(M, −), where M is an A-B-bimodule that is flat over B. Remark 56 The functor A(B, −) can be replaced by A(M, −), where M is an A-B-bimodule that is flat over B. Let X be an A-module, let Y be a B-module. Let X be an A-module, let Y be a B-module. We shall compare two spectral sequences with E2-terms Exti B(Y, Extj A(B, X)), converging to Exti+j A (Y, X). If one views X⇑B A := A(B, X) as a way to induce from A-Mod to B-Mod, this measures the failure of the Eckmann-Shapiro-type formula Exti B(Y, X⇑B A) ?≃Exti A(Y, X), which holds if B is projective over A. Let I ∈ Ob C[0(A-Mod) be an injective resolution of X. Let P ∈ Ob C[0(B-Mod) be a projective resolution of Y . Let I ∈ Ob C[0(A-Mod) be an injective resolution of X. Let P ∈ Ob C[0(B-Mod) be a projective resolution of Y . Proposition 55 The proper spectral sequences Theorem 54 The proper spectral sequences ˙EGr (−)n, (−)¯g(M) and ˙EI  U(g) (Bar¯g;R)−⊗R (Barg;R)= , M  ˙EGr (−)n, (−)¯g(M) and ˙EI  U(g) (Bar¯g;R)−⊗R (Barg;R)= , M  ˙EGr (−)n, (−)¯g(M) and ˙EI  U(g) (Bar¯g;R)−⊗R (Barg;R)= , M  are isomorphic (in ˙¯Z## ∞, R-Mod ), naturally in M ∈Ob U(g) -Mod. Cf. Barnes, [2, Sec. IV.4, Ch. VII]. Cf. Barnes, [2, Sec. IV.4, Ch. VII]. Proof. This follows by Theorem 52. 734 Matthias K¨unzer 8.5 Comparing two spectral sequences for a change of rings The following application is taken from [5, XVI.§6]. Let R be a commutative ring. Let A - φ B be a morphism of R-algebras. Consider the functors A-Mod - A(B,−) B-Mod , (B-Mod)◦× B-Mod - B(−,=) R-Mod . A-Mod - A(B,−) B-Mod , (B-Mod)◦× B-Mod - B(−,=) R-Mod . Proposition 55 The proper spectral sequences ˙EGr A(B,−), B(Y,−)(X) and ˙EI B(P−, A(B, I=))  ˙EGr A(B,−), B(Y,−)(X) and ˙EI B(P−, A(B, I=))  are isomorphic (in ˙¯Z## ∞, S -Mod ). are isomorphic (in ˙¯Z## ∞, S -Mod ). Both have E2-terms Exti R M, Extj A(X, X′)  , and both converge to Exti+j A (X ⊗R M, X′). In particular, in the situation of Example 57, both have E2-terms Exti R S, Extj RG(X, X′)  and converge to Exti+j RG(X/tk, X′). Proof of Proposition 58. To apply Theorem 31, we comment on the conditions in §5.1. Proof of Proposition 58. To apply Theorem 31, we comment on the conditions in §5.1. (c) Given a projective A-module P, we want to show that the R-module A(P, X′) is R(M, −)-acyclic. We may assume that P = A, which is to be viewed as an A-R-bimodule. Now, we have Exti R M, A(A, X′)  ≃ Exti R(M, X′) ≃0 for i ≥1 by assumption. (c′) Given an injective A-module I′, the R-module A(X, I′) is injective since X is flat over R by assumption. 8.6 Comparing two spectral sequences for Ext and ⊗ Let R be a commutative ring. Let S be a ring. Let A be an R-algebra. Let M be an R-S-bimodule. Let X and X′ be A-modules. Assume that X is flat over R. Assume that Exti R(M, X′) ≃0 for i ≥1. Example 57 Let T be a discrete valuation ring, with maximal ideal generated by t. Let R = T/tℓfor some ℓ≥1. Let S = T/tk, where 1 ≤k ≤ℓ. Let G be a finite group, and let A = RG. Let M = S. Let X and X′ be RG-modules that are both finitely generated and free over R. Consider the functors (A-Mod)◦× A-Mod - A(−,=) R-Mod - R(M,−) S -Mod Documenta Mathematica 13 (2008) 677–737 Proposition 58 The proper Grothendieck spectral sequences Proposition 58 The proper Grothendieck spectral sequences ˙EGr A(X,−), R(M,−)(X′) and ˙EGr A(−,X′), R(M,−)(X) OT -Mod - f∗ OS -Mod , (OS -Mod)◦× OS -Mod - OS((−,=)) OS -Mod . Proposition 59 The proper spectral sequences ˙EGr f∗, OS((F,−))(G) and ˙EI OS((B−, f∗A=))  ˙EGr f∗, OS((F,−))(G) and ˙EI OS((B−, f∗A=))  ˙EGr f∗, OS((F,−))(G) and ˙EI OS((B−, f∗A=))  are isomorphic (in ˙¯Z## ∞, OS -Mod ). are isomorphic (in ˙¯Z## ∞, OS -Mod ). are isomorphic (in ˙¯Z## ∞, OS -Mod ). 8.7 Comparing two spectral sequences for Ext of sheaves Let T - f S be a flat morphism of ringed spaces, i.e. suppose that OT ⊗f −1OS −: f −1OS -Mod - OT -Mod is exact. Consequently, f ∗: OS -Mod - OT -Mod is exact. y f Given OS-modules F and F′, we abbreviate OS(F, F′) := HomOS(F, F′) ∈ Ob R-Mod and OS((F, F′)) := HomOS(F, F′) ∈Ob OS -Mod. Given OS-modules F and F′, we abbreviate OS(F, F′) := HomOS(F, F′) ∈ O (( ′)) ( ′) O O S( ) S( ) Ob R-Mod and OS((F, F′)) := HomOS(F, F′) ∈Ob OS -Mod. S(( , )) S( , ) Let F be an OS-module that has a locally free resolution B ∈Ob C(OS -Mod); cf. [9, Prop. III.6.5]. Let G ∈Ob OT -Mod. Let A ∈Ob C[0(OT -Mod) be an injective resolution of G. C id th f t Consider the functors OT -Mod - f∗ OS -Mod , (OS -Mod)◦× OS -Mod - OS((−,=)) OS -Mod . OT -Mod - f∗ OS -Mod , (OS -Mod)◦× OS -Mod - OS((−,=)) OS -Mod . are isomorphic (in ˙¯Z## ∞, OS -Mod ). In particular, both spectral sequences have E2-terms Ext i OS F, (Rjf∗)(G)  and converge to (Ri+jIΓF)(G), where IΓF(−) := OS((F, f∗(−))) ≃f∗OT((f ∗F, −)). For example, if S = {∗} is a one-point-space and if we write R := OS(S), then we can identify OS -Mod = R-Mod. If, in this case, F = R/rR for some r ∈R, then IΓR/rR(G) ≃Γ(T, G)[r] := {g ∈G(T ) : rg = 0}. Proof of Proposition 59. To apply Theorem 34, we comment on the conditions in §6.1. Documenta Mathematica 13 (2008) 677–737 736 Matthias K¨unzer (c) Since f∗maps injective OT -modules to injective OS-modules by flatness of T - f S, the complex A is an f∗, OS((F, −))  -acyclic resolution of G. (c) Since f∗maps injective OT -modules to injective OS-modules by flatness of T - f S, the complex A is an f∗, OS((F, −))  -acyclic resolution of G. (e) If I is an injective OS-module and U ⊆S is an open subset, then I|U is an injective OU-module; cf. [9, Lem. III.6.1]. Hence OS((−, I)) turns a short exact sequence of OS-modules into a sequence that is short exact as a sequence of abelian presheaves, and hence a fortiori short exact as a sequence of OS-modules. In other words, the functor OS((−, I)) is exact. Documenta Mathematica 13 (2008) 677–737 [18] Weibel, C. A., An introduction to homological algebra, Cambridge Stud. Adv. Math. 38, 1995. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 737 Documenta Mathematica 13 (2008) References [1] Abe, E., Hopf algebras, Cambridge University Press, 1977. [2] Barnes, D. W., Spectral sequence constructors in algebra and topology, Mem. Am. Math. Soc. 317, 1985. [3] Benson, D. J., Representations and cohomology II, Cambridge, 1991. [4] Beyl, R., The spectral sequence of a group extension, Bull. Sc. math., 2e s´erie, 105, p. 417–434, 1981. [5] Cartan, H., Eilenberg, S., Homological Algebra, Princeton, 1956. [6] Deligne, P., D´ecompositions dans la cat´egorie d´eriv´ee, Proc. Symp. Pure Math. 55, p. 115–127, 1994. [7] Grothendieck, A., Sur quelques points d’alg`ebre homologique, Tohoku Math. J. 9, p. 119–221, 1957. [8] Haas, R., Naturality of the Grothendieck spectral sequence, Dissertation, Case Western Reserve University, Cleveland OH, available via University Microfilms International, Order No. 77-18823, 1977. [9] Hartshorne, R., Algebraic Geometry, Springer GTM 52, 1977. [10] K¨unzer, M., Heller triangulated categories, Homol. Homot. Appl. 9 (2), p. 233–320, 2007. [11] K¨unzer, M., (Co)homologie von Gruppen, lecture script, Aachen, 2006. [12] Mac Lane, S., Homology, Springer Grundlehren 114, 1975. [13] Montgomery, S., Hopf Algebras and Their Actions on Rings, CBMS Regional Conference Series in Mathematics 82, AMS, 1993. [14] Quillen, D., Higher algebraic K-theory: I, SLN 341, p. 85–147, 1973. [15] Sweedler, M., Hopf Algebras, Benjamin, 1969. [16] Schneider, H. J., Normal basis and transitivity of crossed products for Hopf algebras, J. Algebra 152 (2), p. 289–312, 1992. Documenta Mathematica 13 (2008) 677–737 Comparison of Spectral Sequences Involving Bifunctors 737 [17] Verdier, J.L., Des cat´egories d´eriv´ees des cat´egories ab´eliennes, Ast´erisque 239, 1996. [18] Weibel, C. A., An introduction to homological algebra, Cambridge Stud. Adv. Math. 38, 1995. [18] Weibel, C. A., An introduction to homological algebra, Cambridge Stud. Adv. Math. 38, 1995. Matthias K¨unzer Lehrstuhl D f¨ur Mathematik RWTH Aachen Templergraben 64 D-52062 Aachen Germany kuenzer@math.rwth- aachen.de Documenta Mathematica 13 (2008) 677–737 738
https://openalex.org/W2328198216
https://zenodo.org/records/2505092/files/article.pdf
German
null
Eine neue Stützschiene für Radialislähmung
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
1,918
public-domain
914
1. Februar 1918 1. Februar 1918 DEUTSCIIE MEDIZINISCHE WOCHENSCHRIFT 159 Eine neue Stützschiene für Radialislähmung. Von Dr E MUnch Die Wirkungsweise ist fol- gende: An einer Blechpelotte sitzen seitlich zwei Zapfen und ------- auf diesen -jè eine konisch ge- rollte Feder, an deren vorderem Ende ein Lederriemen und zwei Ringe angebracht sind. Die Blechpelotte wird durch einen Riemen am Arm befestigt. Als Daumenzug dient die auf eine Feder übergeschobene kleine Fe- der mit Ring für den Daumen. Durch diese Federkonstruktion ist es möglich, dio Schiene für jedeLähmung einzustellen. Sie kann ohne besondere Fachkenntnis stärker oder schwächer eingestellt werden. Dies ist möglich durch die eigenartige Fecierkonstruktion. Wird eine derartige Feder auf einen etwas konischen Zapfen aufgesteckt, so wird man finden, daß bei einem der sonstigen Federwirkung entgegen- gesetzten Drehen die Drahtwindungen vom Bolzen sich lösen und 1ie Feder in dieser Richtung leicht uni den Bolzen gedreht werden kann. Versucht man nn in entgegengesetzter Richtung zu drehen, so wird man finden, daß der Draht, je mehr man versucht zu drehen, desto fester sich um den Bolzen spannt und so die entsprechende Feder- kraft zum lieben der Hand abgibt. Man kann dadurch die Schiene für jeden einzelnen Fall aufs genuuste einstellen, ist die Schiene zu stark eingestellt, so müssen die Federn fast ganz um den Bolzen ge- dreht werden. Ail üb i h b h d Z i h d Ph 2 2 Die Wirkungsweise ist fol- gende: An einer Blechpelotte sitzen seitlich zwei Zapfen und ------- auf diesen -jè eine konisch ge- rollte Feder, an deren vorderem Ende ein Lederriemen und zwei Ringe angebracht sind. Die Blechpelotte wird durch einen Riemen am Arm befestigt. Als Daumenzug dient die auf eine Feder übergeschobene kleine Fe- der mit Ring für den Daumen. Durch diese Federkonstruktion ist es möglich, dio Schiene für jedeLähmung einzustellen. Sie kann ohne besondere Fachkenntnis stärker oder schwächer eingestellt werden. Dies ist möglich durch die eigenartige Fecierkonstruktion. Wird eine derartige Feder auf einen etwas konischen Zapfen aufgesteckt, so wird man finden, daß bei einem der sonstigen Federwirkung entgegen- gesetzten Drehen die Drahtwindungen vom Bolzen sich lösen und 1ie Feder in dieser Richtung leicht uni den Bolzen gedreht werden kann. Versucht man nn in entgegengesetzter Richtung zu drehen Versucht man nn in entgegengesetzter Richtung zu drehen, so wird man finden, daß der Draht, je mehr man versucht zu drehen, desto fester sich um den Bolzen spannt und so die entsprechende Feder- kraft zum lieben der Hand abgibt. Eine neue Stützschiene für Radialislähmung. Von Dr E MUnch Eine neue Stützschiene für Radialislähmung. Von Dr. E. MUnch., Spezialarzt für Chirurgie und Orthopädie in Kaiserslautern. Zur Behandlung der RaclialisliLhmungen bis zur Wiederherstellung der Nervenleitung sind eine Menge Stützschienen und andere Apparate ersonnen worden. Sie sind jedoch vielfach zu kompliziert, sodaß die Hand bei der Arbeitsleistung gestört wird. Dadurch wird leider häufig die Schiene bei Aufnahme der Arbeit von dem Träger beiseite gelegt zum Schaden der Wiederherstellungsaussichten. In den meisten Fällen ist das Tragen einer Schiene nur notwendig zur Erhaltung der Muskulatur bis zur Leitungsfähigkeit der Nerven. zur Erhaltung der Muskulatur bis zur Leitungsfähigkeit der Nerven. Da es genügt, wenn man die Hand in Streckstellung hält und die Grundglieder der Finger unterstützt, woraufhin die Extension der brigen Fingerglieder möglich ist, so soll die Stützschiene so einfach wie möglich sein. Ich führe zu diesem Zweck eine einfache Kon- struktion an, die wegen ihrer großen Vorzüge verdient, näher bekañnt zu werden. d d ------- 2 großen Vorzüge verdient, näher bekañnt zu werden. d Wie aus nebenstehender Figur ersichtlich, ist die Stütz- schiene sehr einfach und leicht, wodurch es dem Patienteft mög- lich wird, die Schiene bei allen Arbeiten zu tragen. Die Wirkungsweise ist fol- gende: An einer Blechpelotte sitzen seitlich zwei Zapfen und ------- auf diesen -jè eine konisch ge- rollte Feder, an deren vorderem Ende ein Lederriemen und zwei Ringe angebracht sind. Die Blechpelotte wird durch einen Riemen am Arm befestigt. Als Daumenzug dient die auf eine Feder übergeschobene kleine Fe- der mit Ring für den Daumen. Durch diese Federkonstruktion ist es möglich, dio Schiene für jedeLähmung einzustellen. Sie kann ohne besondere Fachkenntnis stärker oder schwächer eingestellt werden. Dies ist möglich durch die eigenartige Fecierkonstruktion. Wird eine derartige Feder auf einen etwas konischen Zapfen aufgesteckt, so wird man finden, daß bei einem der sonstigen Federwirkung entgegen- gesetzten Drehen die Drahtwindungen vom Bolzen sich lösen und 1ie Feder in dieser Richtung leicht uni den Bolzen gedreht werden kann. Versucht man nn in entgegengesetzter Richtung zu drehen, 2 Wie aus nebenstehender Figur ersichtlich, ist die Stütz- schiene sehr einfach und leicht, wodurch es dem Patienteft mög- lich wird, die Schiene bei allen Arbeiten zu tragen. schiene sehr einfach und leicht, wodurch es dem Patienteft mög- lich wird, die Schiene bei allen Arbeiten zu tragen. DEUTSCIIE MEDIZINISCHE WOCHENSCHRIFT Als weiterer Vorzug der Schiene sei erwähnt, daB der Preis sehr gering ist. g g Die Schiene ist geschützt durch D. R. G. M. Nr. 649460. 3ie wird angefertigt durch den Orthopädiemechaniker Emil Huber, Kaisers- lautem, Schillerstraße 2. urde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. Eine neue Stützschiene für Radialislähmung. Von Dr. E. MUnch., Spezialarzt für Chirurgie und Orthopädie in Kaiserslautern. Eine neue Stützschiene für Radialislähmung. Von Dr E MUnch Man kann dadurch die Schiene für jeden einzelnen Fall aufs genuuste einstellen, ist die Schiene zu stark eingestellt, so müssen die Federn fast ganz um den Bolzen ge- dreht werden. Ail b i h b h d Z i h d Ph Ailes übrige geht aus nebenstehender Zeichnung und Photo- graphie hervor.
https://openalex.org/W2611574484
https://repository.ubn.ru.nl/bitstream/handle/2066/189916/1/189916.pdf
English
null
International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases
American journal of human genetics
2,017
cc-by
11,488
International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases Boycott, K.M.; Rath, A.; Chong, J.X.; Hartley, T.; Alkuraya, F.S.; Baynam, G.; Brunner, H.G.; Bamshad, Michael J.; Lochmueller, Hanns 2017, Article / Letter to editor (American Journal of Human Genetics, 100, 5, (2017), pp. 695-705) Doi link to publisher: https://doi.org/10.1016/j.ajhg.2017.04.003 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/189916 Download date: 2024-10-24 International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases Boycott, K.M.; Rath, A.; Chong, J.X.; Hartley, T.; Alkuraya, F.S.; Baynam, G.; Brunner, H.G.; Bamshad, Michael J.; Lochmueller, Hanns 2017, Article / Letter to editor (American Journal of Human Genetics, 100, 5, (2017), pp. 695-705) Doi link to publisher: https://doi.org/10.1016/j.ajhg.2017.04.003 International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases Boycott, K.M.; Rath, A.; Chong, J.X.; Hartley, T.; Alkuraya, F.S.; Baynam, G.; Brunner, H.G.; Bamshad, Michael J.; Lochmueller, Hanns 2017, Article / Letter to editor (American Journal of Human Genetics, 100, 5, (2017), pp. 695-705) Doi link to publisher: https://doi.org/10.1016/j.ajhg.2017.04.003 Note: To cite this publication please use the final published version (if applicable). COMMENTARY International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases Kym M. Boycott,1,* Ana Rath,2 Jessica X. Chong,3 Taila Hartley,1 Fowzan S. Alkuraya,4,5 Gareth Baynam,6 Anthony J. Brookes,7 Michael Brudno,8 Angel Carracedo,9 Johan T. den Dunnen,10 Stephanie O.M. Dyke,11 Xavier Estivill,12,13 Jack Goldblatt,6 Catherine Gonthier,2 Stephen C. Groft,14 Ivo Gut,15 Ada Hamosh,16 Philip Hieter,17 Sophie Ho¨hn,2 Matthew E. Hurles,18 Petra Kaufmann,19 Bartha M. Knoppers,11 Jeffrey P. Krischer,20 Milan Macek, Jr.,21 Gert Matthijs,22 Annie Olry,2 Samantha Parker,23 Justin Paschall,18 Anthony A. Philippakis,24 Heidi L. Rehm,24 Peter N. Robinson,25,26 Pak-Chung Sham,27 Rumen Stefanov,28 Domenica Taruscio,29 Divya Unni,2 Megan R. Vanstone,1 Feng Zhang,30,31 Han Brunner,32,33 Michael J. Bamshad,3,34 and Hanns Lochmu¨ller35 Provision of a molecularly confirmed diagnosis in a timely manner for children and adults with rare genetic diseases shortens their ‘‘diag- nostic odyssey,’’ improves disease management, and fosters genetic counseling with respect to recurrence risks while assuring reproduc- tive choices. In a general clinical genetics setting, the current diagnostic rate is approximately 50%, but for those who do not receive a molecular diagnosis after the initial genetics evaluation, that rate is much lower. Diagnostic success for these more challenging affected individuals depends to a large extent on progress in the discovery of genes associated with, and mechanisms underlying, rare diseases. Thus, continued research is required for moving toward a more complete catalog of disease-related genes and variants. The International Rare Diseases Research Consortium (IRDiRC) was established in 2011 to bring together researchers and organizations invested in rare disease research to develop a means of achieving molecular diagnosis for all rare diseases. Here, we review the current and future bottle- necks to gene discovery and suggest strategies for enabling progress in this regard. Each successful discovery will define potential diagnostic, preventive, and therapeutic opportunities for the corresponding rare disease, enabling precision medicine for this patient population. the number of RGDs that exist is incomplete but is estimated to be well over 7,000 according to current medi- cal and genetic evidence4 (also see Or- phanet in the Web Resources). Despite their often chronic and progressive na- ture, long-term complications can be The American Journal of Human Genetics 100, 695–705, May 4, 2017 695 Introduction diseases are due to altered functions of single genes. Cumulatively, these rare genetic diseases (RGDs), also termed Mendelian or monogenic diseases, affect at least 1 in 50 individ- uals in the European-derived general population.3 Our understanding of Rare diseases, though individually rare, are collectively common. 1Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON K1H 8L1, Canada; 2Orphanet, Institut National de la Sante´ et de la Recherche Me´dicale US14, 75014 Paris, France; 3Department of Pediatrics, University of Washington, Seattle, WA 98195, USA; 4Department of Ge- netics, King Faisal Research Center, Riyadh 11211, Saudi Arabia; 5Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh 11442, Saudi Arabia; 6Genetic Services of Western Australia, Perth, WA 6008, Australia; 7Department of Genetics, University of Leicester, Leicester LE1 7RH, UK; 8Department of Computer Science, University of Toronto, Toronto M5S 1A1, Canada; 9Genomic Medicine Group, Galician Foundation of Genomic Medicine and University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; 10Departments of Human Genetics and Clinical Genetics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; 11Centre of Genomics and Policy, Department of Human Genetics, Faculty of Medicine, McGill University, Montreal, QC H3A 1A4, Canada; 12Experimental Division, Sidra Medical and Research Center, PO Box 26999, Doha, Qatar; 13Genetics Unit, Dexeus Woman’s Health, 08028 Barcelona, Spain; 14National Center for Advancing Translational Sciences, Na- tional Institutes of Health, Bethesda, MD 20892-4874, USA; 15Centre Nacional d’Ana`lisi Geno`mica, Center for Genomic Regulation, Barcelona Institute of Science and Technology, Universitat Pompeu Fabra, 08028 Barcelona, Spain; 16McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21286, USA; 17Michael Smith Laboratories, Department of Medical Genetics, University of British Columbia, Vancou- ver, BC V6T 1Z4, Canada; 18Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK; 19Office of Rare Diseases Research, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892-4874, USA; 20University of South Florida Health Informatics Institute, Tampa, FL 33620, USA; 21Department of Biology and Medical Genetics, Second Faculty of Medicine, Charles University and Univer- sity Hospital Motol, 150 06 Prague 5, Czech Republic; 22Center for Human Genetics, University of Leuven, 3000 Leuven, Belgium; 23Lysogene, 92 200 Neu- illy-sur-Seine, France; 24Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; 25Institut fu¨r Medizinische Genetik und Humangenetik, Charite´ Universita¨tsmdizin Berlin, 13353 Berlin, Germany; 26Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA; 27Centre for Genomic Sci- ences, University of Hong Kong, Hong Kong, China; 28Department of Social Medicine and Public Health, Faculty of Public Health, Medical University of Plovdiv, Plovdiv 4002, Bulgaria; 29National Centre for Rare Diseases, Istituto Superiore di Sanita`, Rome 299-00161, Italy; 30WuXi AppTec, Waigaoqiao Free Trade Zone, Shanghai 200131, China; 31WuXi NextCODE, Cambridge, MA 02142, USA; 32Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, the Netherlands; 33Maastricht University Medical Center, Department of Clinical Genetics, 6229 GT Maastricht, the Netherlands; 34Division of Genetic Medicine, Seattle Children’s Hospital, Seattle, WA 98105, USA; 35John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne NE1 3BZ, UK *Correspondence: kboycott@cheo.on.ca //dx.doi.org/10.1016/j.ajhg.2017.04.003. 17 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). p y http://dx.doi.org/10.1016/j.ajhg.2017.04.003. //dx.doi.org/10.1016/j.ajhg.2017.04.003. 17 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). http://dx.doi.org/10.1016/j.ajhg.2017.04.003.  2017 Th A h ( ) Thi i i l d h CC BY li (h // i /li /b /4 0/ Introduction A rare disease is defined as one that affects fewer than 200,000 people in the US1 or less than 1 in 2,000 people in Europe.2A substantive number of rare 1Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON K1H 8L1, Canada; 2Orphanet, Institut National de la Sante´ et de la Recherche Me´dicale US14, 75014 Paris, France; 3Department of Pediatrics, University of Washington, Seattle, WA 98195, USA; 4Department of Ge- netics, King Faisal Research Center, Riyadh 11211, Saudi Arabia; 5Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh 11442, Saudi Arabia; 6Genetic Services of Western Australia, Perth, WA 6008, Australia; 7Department of Genetics, University of Leicester, Leicester LE1 7RH, UK; 8Department of Computer Science, University of Toronto, Toronto M5S 1A1, Canada; 9Genomic Medicine Group, Galician Foundation of Genomic Medicine and University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; 10Departments of Human Genetics and Clinical Genetics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; 11Centre of Genomics and Policy, Department of Human Genetics, Faculty of Medicine, McGill University, Montreal, QC H3A 1A4, Canada; 12Experimental Division, Sidra Medical and Research Center, PO Box 26999, Doha, Qatar; 13Genetics Unit, Dexeus Woman’s Health, 08028 Barcelona, Spain; 14National Center for Advancing Translational Sciences, Na- tional Institutes of Health, Bethesda, MD 20892-4874, USA; 15Centre Nacional d’Ana`lisi Geno`mica, Center for Genomic Regulation, Barcelona Institute of Science and Technology, Universitat Pompeu Fabra, 08028 Barcelona, Spain; 16McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21286, USA; 17Michael Smith Laboratories, Department of Medical Genetics, University of British Columbia, Vancou- ver, BC V6T 1Z4, Canada; 18Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK; 19Office of Rare Diseases Research, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892-4874, USA; 20University of South Florida Health Informatics Institute, Tampa, FL 33620, USA; 21Department of Biology and Medical Genetics, Second Faculty of Medicine, Charles University and Univer- sity Hospital Motol, 150 06 Prague 5, Czech Republic; 22Center for Human Genetics, University of Leuven, 3000 Leuven, Belgium; 23Lysogene, 92 200 Neu- illy-sur-Seine, France; 24Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; 25Institut fu¨r Medizinische Genetik und Humangenetik, Charite´ Universita¨tsmdizin Berlin, 13353 Berlin, Germany; 26Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA; 27Centre for Genomic Sci- ences, University of Hong Kong, Hong Kong, China; 28Department of Social Medicine and Public Health, Faculty of Public Health, Medical University of Plovdiv, Plovdiv 4002, Bulgaria; 29National Centre for Rare Diseases, Istituto Superiore di Sanita`, Rome 299-00161, Italy; 30WuXi AppTec, Waigaoqiao Free Trade Zone, Shanghai 200131, China; 31WuXi NextCODE, Cambridge, MA 02142, USA; 32Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, the Netherlands; 33Maastricht University Medical Center, Department of Clinical Genetics, 6229 GT Maastricht, the Netherlands; 34Division of Genetic Medicine, Seattle Children’s Hospital, Seattle, WA 98105, USA; 35John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne NE1 3BZ, UK *Correspondence: kboycott@cheo.on.ca p y http://dx.doi.org/10.1016/j.ajhg.2017.04.003. Introduction OMIM emphasizes gene-phenotype re- lationships by cataloging the same or similar phenotypes caused by patho- genic variants in different genes as distinct entities; genetic heterogeneity is displayed through the associated Phenotypic Series. A recent analysis of OMIM (data downloaded September 5, 2016) recognized 3,209 unique genes associated with 4,550 mono- genic rare diseases. Although substantial progress has been made toward identifying the genetic basis of rare diseases, the underlying etiologies for approxi- mately half remain undiscovered. Beginning in the mid-1980s, and for the following two decades, the pri- mary approach to gene discovery was a combination of linkage analysis, po- sitional cloning, and sequencing of candidate or regionally selected genes, most of which was hypothesis driven. The subsequent introduction of next- generation sequencing (NGS) strate- gies to identify genes associated with disease, primarily based on whole- exome sequencing (WES), in 2009 accelerated the pace of discovery by enabling hypothesis-free approaches. Today, WES is routinely used as the primary technological approach to discovering disease-gene associations (Figure 1). Its favor over whole- genome sequencing (WGS) has pri- marily been due to its significantly lower cost and that the majority of pathogenic variants continue to be within the protein-coding portion of the genome. Without a doubt, as the cost of WGS decreases, clinicians and researchers will transition to its use given its more even coverage, its abil- ity to identify structural variation, and the opportunity it provides to un- cover non-exomic variants. Orphanet (see Web Resources) has maintained an inventory of both ge- netic and other rare diseases since 1997. Within Orphanet, a rare disease is defined as a recognizable and homogeneous clinical presentation, whatever the cause or the number of genes related to it. Disorders are orga- nized in a multi-hierarchical classifica- tion and can be further subdivided into subtypes, of which genetic sub- types are included. Orphanet per- forms a literature survey and curates the published literature of newly discovered genes or new gene-disease relations. As a result, a semantic rela- tion is assigned to couple the gene and disease in the database. As of September 14, 2016, Orphanet docu- mented 3,654 unique genes associ- ated with 3,551 rare diseases. 696 The American Journal of Human Genetics 100, 695–705, May 4, 2017 Current Understanding of Phenotypic and Genetic Diversity of RGDs Knowledge of the phenotypic and genetic diversity of RGDs is steadily increasing; however, substantial gaps remain. Establishing the number of RGDs is challenging for several rea- sons, not the least of which is distin- guishing between novel and known diseases to objectively segment a con- tinuum of pathologies into discrete disease entities. Two international da- tabases curate clinical and genetic data for the community: Online Mende- lian Inheritance in Man (OMIM)4 and Orphanet.6 OMIM has continu- ously provided curation and classifica- tion of Mendelian disease since it began as Mendelian Inheritance in Man, first published by Dr. V. McKusick in 1966; OMIM has been online and searchable since 1987. OMIM mines the biomedical literature and, accord- ing to expert review, curates significant new information on genes and genetic phenotypes into separate gene and phenotype entries. OMIM numbers for Mendelian diseases are incorpo- rated into the biomedical literature across many disciplines of medicine. The discrepancy in the number of rare diseases with monogenic etiology documented in each of the two databases (4,550 for OMIM and 3,551 for Orphanet) can be attributed to the way each database is structured; OMIM categorizes rare diseases on the basis of genetic etiology, whereas Orphanet groups by clinically recog- nizable diseases and can include more than one OMIM entry when the same disease is caused by variants in more than one gene. Recently, the Clinical Genome Resource (ClinGen)7 has begun defining the strength of ev- idence for published gene-disease asso- ciations. The evidence levels are scored according to semiquantitative frame- works, and the scores are posted on ClinGen’s website along with the scoring sheets that structure the evidence and sources. These scores will also soon be posted on OMIM. As ClinGen grows, it will enable a clear delineation between those genes Our analysis of OMIM documented an average of 259 ‘‘novel’’ RGD dis- coveries per year from 2012 to 2015 (Figure 1), comprising 157 new dis- ease-gene discoveries (here defined as pathogenic variants in a gene that had not been previously associated with disease) and 102 new disease- gene relations each year (defined as pathogenic variants in a gene previously associated with a different disease; data not shown).8 Orphanet documents an average of 281 novel RGD discoveries per year over the same time period: 160 new disease- gene discoveries and 121 new disease-gene relations (Figure 2). Introduction  2017 The Author(s) This is an open access art p y http://dx.doi.org/10.1016/j.ajhg.2017.04.003. p // g/ /j j g  2017 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). The American Journal of Human Genetics 100, 695–705, May 4, 2017 695 lessened or delayed for some RGDs if they are diagnosed early (e.g., via newborn screening) and optimally managed by standard and/or targeted therapies. In addition, a definitive mo- lecular diagnosis can obviate the need for further diagnostic investigations, facilitate appropriate access to health- care resources, reduce prognostic un- certainty, provide accurate recurrence- risk counseling, foster reproductive choices in affected families, and impart psychosocial benefits to the patient and their family. Importantly, under- standing the underlying genetic etiol- ogy and linking a RGD to a causative biological pathway is leading to highly effective targeted therapies for some se- vere, previously only symptomatically treatable RGDs (e.g., ivacaftor for class III CFTR [MIM: 602421] pathogenic variants).5 Ultimately, successful dep- loyment of precision medicine will be directly related to diagnostic success for patients with RGDs. lessened or delayed for some RGDs if they are diagnosed early (e.g., via newborn screening) and optimally managed by standard and/or targeted therapies. In addition, a definitive mo- lecular diagnosis can obviate the need for further diagnostic investigations, facilitate appropriate access to health- care resources, reduce prognostic un- certainty, provide accurate recurrence- risk counseling, foster reproductive choices in affected families, and impart psychosocial benefits to the patient and their family. Importantly, under- standing the underlying genetic etiol- ogy and linking a RGD to a causative biological pathway is leading to highly effective targeted therapies for some se- vere, previously only symptomatically treatable RGDs (e.g., ivacaftor for class III CFTR [MIM: 602421] pathogenic variants).5 Ultimately, successful dep- loyment of precision medicine will be directly related to diagnostic success for patients with RGDs. for which gene-disease causality is substantiated and those claims that will require further evidence for implication. OMIM emphasizes gene-phenotype re- lationships by cataloging the same or similar phenotypes caused by patho- genic variants in different genes as distinct entities; genetic heterogeneity is displayed through the associated Phenotypic Series. A recent analysis of OMIM (data downloaded September 5, 2016) recognized 3,209 unique genes associated with 4,550 mono- genic rare diseases. Current Understanding of Phenotypic and Genetic Diversity of RGDs Or- phanet and OMIM report essentially 696 The American Journal of Human Genetics 100, 695–705, May 4, 2017 Figure 1. Approximate Number of Gene Discoveries Made by WES and WGS versus Conventional Approaches since 2010 according to OMIM Data Since the introduction of WES and WGS in 2010, the pace of the discovery of genes un- derlying RGDs per year has increased, and the proportion of discoveries made by WES or WGS (blue) or by conventional approaches (red) has steadily increased. Since 2013, WES and WGS have discovered nearly three times as many genes as conventional approaches, but the rate of discovery appears to be declining. Adapted from Chong et al.8 Figure 1. Approximate Number of Gene Discoveries Made by WES and WGS versus Conventional Approaches since 2010 according to OMIM Data Since the introduction of WES and WGS in 2010, the pace of the discovery of genes un- derlying RGDs per year has increased, and the proportion of discoveries made by WES or WGS (blue) or by conventional approaches (red) has steadily increased. Since 2013, WES and WGS have discovered nearly three times as many genes as conventional approaches, but the rate of discovery appears to be declining. Adapted from Chong et al.8 researchers and organizations invested in rare disease research. Three IRDiRC Scientific Committees (Diagnostics, Interdisciplinary, and Therapies) and representation from three patient- advocacy groups (two from the US [Na- tional Organization for Rare Disorders (NORD) and Genetic Alliance] and one from Europe [Rare Diseases Europe- EURORDIS]), advise the Consortium Assembly (formerly the Executive Committee), which includes public research funders and private-sector members from pharma and biotech from 42 member institutions. Each has committed at least $10,000,000 USD to rare disease research within their jurisdiction (Figure 3; data ac- cessed January 11, 2017). Currently, rare disease research coordinated under the umbrella of IRDiRC totals more than $2,000,000,000 USD. IRDiRC aims to facilitate the under- standing of all rare genetic diseases. Figure 1. Approximate Number of Gene Discoveries Made by WES and WGS versus Conventional Approaches since 2010 according to OMIM Data Since the introduction of WES and WGS in 2010, the pace of the discovery of genes un- derlying RGDs per year has increased, and the proportion of discoveries made by WES or WGS (blue) or by conventional approaches (red) has steadily increased. Strategies for Enabling the Diagnosis of All RGDs Since the introduction of WES, many RGDs that were previously intractable to conventional gene-dis- covery approaches, largely because they were associated with a substan- tially reduced reproductive fitness, have been found to be caused by de novo pathogenic variants or to exhi- The coming years will see an expanding need for large-scale infrastructure, re- sources, and tools for completing the grand challenge: understanding the molecular pathogenesis of all RGDs. Over the past few years, our commit- tees, working groups, and task forces have identified specific areas of high Current Understanding of Phenotypic and Genetic Diversity of RGDs In addition, these RGDs are usually autosomal, X-linked recessive, or de novo domi- nant, rendering them relatively more accessible and amenable to current discovery strategies relying on WES; these RGDs represent the sweet spot of WES-based approaches. Both OMIM and Orphanet data (Figures 1 and 2) show a trend toward a de- creasing number of discoveries per year; whether this trend is real or will continue will require analysis of data from future years. However, what is clear is that recognized bottle- necks must be addressed if the current pace of discoveries is to be main- tained, or even accelerated, after the more straightforward RGDs have been solved. the same number of new disease-gene discoveries (average of 160 and 157, respectively, over the same time period), but more disease-gene rela- tions have been reported by Orphanet (121 versus 102 for OMIM). In a manual review of randomly selected discrepancies between OMIM and Orphanet, this is most likely attribut- able to differences in the process of curation; OMIM is more likely to decide that the publication reports a phenotypic expansion of an already explained RGD than a new disease- gene relation. Nevertheless, the data from OMIM and Orphanet both show that a significant proportion of RGD discoveries are new diseases asso- ciated with pathogenic variants in previously known genes (gene-disease relations): 38 and 43%, respectively. This is an interesting trend in compar- ison with a recent analysis of all of OMIM’s data, which demonstrated that nearly 25% of all genes associated with Mendelian disease underlie two or more clinically distinct disorders.8 The focus of the Diagnostics and Interdisciplinary Committees, and their associated working groups and task forces, has been identifying current and future bottlenecks to RGD discovery and suggesting strate- gies by which international coopera- tion can address them. We anticipate that several shortcomings of the pre- sent-day discovery pipeline will need to be addressed if we are to continue to make important RGD discoveries at the current pace, or even accelerate it. These include the collection and analysis of clinical and genomic data, data discovery and sharing, ge- netic and functional support for the establishment of disease causality, and the presence of disease mecha- nisms that are intractable to our cur- rent analytical and genomics-based approaches, as summarized in Table 1. Current Understanding of Phenotypic and Genetic Diversity of RGDs Since 2013, WES and WGS have discovered nearly three times as many genes as conventional approaches, but the rate of discovery appears to be declining. Adapted from Chong et al.8 bit high allelic or locus heterogeneity. These RGDs are enriched with highly recognizable clinical presentations; are often associated with early age of onset, severe phenotype, and/or clear laboratory and/or medical imaging features; and are caused by highly penetrant pathogenic, protein-coding genomic variants (i.e., in legacy termi- nology, ‘‘mutations’’). In addition, these RGDs are usually autosomal, X-linked recessive, or de novo domi- nant, rendering them relatively more accessible and amenable to current discovery strategies relying on WES; these RGDs represent the sweet spot of WES-based approaches. Both OMIM and Orphanet data (Figures 1 and 2) show a trend toward a de- creasing number of discoveries per year; whether this trend is real or will continue will require analysis of data from future years. However, what is clear is that recognized bottle- necks must be addressed if the current pace of discoveries is to be main- tained, or even accelerated, after the more straightforward RGDs have been solved. the same number of new disease-gene discoveries (average of 160 and 157, respectively, over the same time period), but more disease-gene rela- tions have been reported by Orphanet (121 versus 102 for OMIM). In a manual review of randomly selected discrepancies between OMIM and Orphanet, this is most likely attribut- able to differences in the process of curation; OMIM is more likely to decide that the publication reports a phenotypic expansion of an already explained RGD than a new disease- gene relation. Nevertheless, the data from OMIM and Orphanet both show that a significant proportion of RGD discoveries are new diseases asso- ciated with pathogenic variants in previously known genes (gene-disease relations): 38 and 43%, respectively. This is an interesting trend in compar- ison with a recent analysis of all of OMIM’s data, which demonstrated that nearly 25% of all genes associated with Mendelian disease underlie two or more clinically distinct disorders.8 bit high allelic or locus heterogeneity. These RGDs are enriched with highly recognizable clinical presentations; are often associated with early age of onset, severe phenotype, and/or clear laboratory and/or medical imaging features; and are caused by highly penetrant pathogenic, protein-coding genomic variants (i.e., in legacy termi- nology, ‘‘mutations’’). The American Journal of Human Genetics 100, 695–705, May 4, 2017 697 The International Rare Diseases Research Consortium The Human Phenotype Ontology (HPO)10,11 has been recognized as a useful annotation of phenotypic ab- normalities of RGDs, with the under- standing that other resources might be suitable in certain situations, and is being used by RGD databases such as PhenomeCentral,12 DECIPHER,13 the UK10K Project,14 and many others. The HPO has been incorporated into the United Medical Language System (UMLS), which will allow interopera- bility with an even larger range of med- ical informatics resources. The HPO is more than a clinical terminology; all terms are set in a hierarchical structure, and it is designed to allow computa- tional analysis of clinical findings for differential diagnostics,15 as well as RGD phenotypic stratification prior to WES analysis in both the clinical16 and discovery settings.17 A key area for ontological development is in- creasing the granularity and coverage of the HPO across some less well- covered rare-disease domains. Addi- tionally, enabling a means of making longitudinal assessments (onset and temporality), utilizing phenotype nega- tion (the patient does not have pheno- type X), and making quantitative spec- ifications (e.g., levels of abnormality of laboratory results) will be important. The Human Phenotype Ontology (HPO)10,11 has been recognized as a useful annotation of phenotypic ab- normalities of RGDs, with the under- standing that other resources might be suitable in certain situations, and is being used by RGD databases such as PhenomeCentral,12 DECIPHER,13 the UK10K Project,14 and many others. The HPO has been incorporated into the United Medical Language System (UMLS), which will allow interopera- bility with an even larger range of med- ical informatics resources. The HPO is more than a clinical terminology; all terms are set in a hierarchical structure, and it is designed to allow computa- tional analysis of clinical findings for differential diagnostics,15 as well as RGD phenotypic stratification prior to WES analysis in both the clinical16 and discovery settings.17 A key area for ontological development is in- creasing the granularity and coverage of the HPO across some less well- covered rare-disease domains. Addi- tionally, enabling a means of making longitudinal assessments (onset and temporality), utilizing phenotype nega- tion (the patient does not have pheno- type X), and making quantitative spec- ifications (e.g., levels of abnormality of laboratory results) will be important. priority to facilitate the achievement of this goal. 698 The American Journal of Human Genetics 100, 695–705, May 4, 2017 The International Rare Diseases Research Consortium The International Rare Diseases Re- search Consortium (IRDiRC) was es- tablished in 2011 to bring together The American Journal of Human Genetics 100, 695–705, May 4, 2017 697 Figure 2. Approximate Number of Novel Gene-Phenotype Discoveries from 2010 to 2015 according to Ophanet Data Since 2010, the proportion of discoveries that are new disease-gene relations each year (known genes associated with a new disease) has steadily increased. Since 2013, the rate of discovery of both novel genes and new disease-gene relations appears to be declining. RGDs, the newly established Interna- tional Consortium for Human Phe- notype Terminologies (ICHPT) has worked to provide the community with phenotype terminology stan- dards and definitions for the more often used phenotype terms for data- base interoperability, in particular to allow the linking of phenotype and ge- notype databases for RGDs. The ICHPT was created with input from members of several groups, including Orphanet (under the EuroGentest project; see Web Resources), HPO,18 and OMIM (Robinson et al., 2014, Am. Soc. Hum. Genet., abstract). The outcome of this effort is a set of >2,300 terms that should be present in any termi- nology through one of its synonyms. These terms have already been map- ped to a few of the major terminol- ogies, including HPO,11 PhenoDB,19 Orphanet, Elements of Morphology,20 POSSUM, SNOMED, MeSH, and MedDRA, facilitating cross-compati- bility between systems. Where ontol- ogies contain more detailed terms at a finer level of granularity, these terms will map ‘‘up’’ to the broader aligned terms. The IRDiRC recognizes and en- courages the ICHPT as the minimal set of standard terms to be used for sharing phenotypic data. Figure 2. Approximate Number of Novel Gene-Phenotype Discoveries from 2010 to 2015 according to Ophanet Data Since 2010, the proportion of discoveries that are new disease-gene relations each year (known genes associated with a new disease) has steadily increased. Since 2013, the rate of discovery of both novel genes and new disease-gene relations appears to be declining. Figure 2. Approximate Number of Novel Gene-Phenotype Discoveries from 2010 to 2015 according to Ophanet Data g p Since 2010, the proportion of discoveries that are new disease-gene relations each year (known genes associated with a new disease) has steadily increased. Since 2013, the rate of discovery of both novel genes and new disease-gene relations appears to be declining. The International Rare Diseases Research Consortium To this end, the IRDiRC has developed a quality indicator, ‘‘IRDiRC Recognized Resources,’’9 on the basis of specific criteria to highlight key re- sources (e.g., platforms, tools, stan- dards, and guidelines), which, if used more broadly, would accelerate the pace of discoveries. Ontologies, Terminologies, and Nosol- ogies for Exchanging Clinical Data g g g Understanding how genomic alter- ations result in different disease-related phenotypes is fundamental to human health research. In this endeavor, if careful phenotypic characterization is lacking, having genomic data, even from large numbers of individuals, is of limited value. Although we have made large strides toward enabling the sharing of genotype data, stan- dards are not widely used for the ex- change of phenotypic data. For undi- agnosed RGDs, the situation is even more problematic because only a few individuals in the world might have the same undiagnosed condition. Currently, numerous ontologies, termi- nologies, and nosologies are used, reflecting the disparate needs and prac- tices of different communities involved in translational research and patient care in many fields of medicine. Two complementary rare-disease no- sologies exist, the Orphanet Rare Dis- ease Ontology (ORDO)21 and OMIM.4 ORDO is a structured vocabulary for rare diseases and is derived from the Orphanet database; it captures relation- ships between diseases, genes, and other relevant features to form a useful resource for the computational analysis of rare diseases. It integrates nosologies (classifications of rare diseases), relation- ships (gene-disease relations and epide- miological data), and connections with other terminologies (MeSH, UMLS, and MedDRA), databases (OMIM, UniProtKB, HGNC, Ensembl, Reac- tome, IUPHAR, and Geneatlas), or clas- sifications (e.g., International Statistical Classification of Diseases and Related Health Problems-10 [ICD-10]). It should be noted that ICD-10 contains only 500 unique rare-disease classification codes. This deficiency is now being overcome by the development of a To bridge the compatibility gap between various systems and the lack of terminology specific enough for The IRDiRC recognizes phenotype ontologies, terminologies, and disease nosologies as critical for RGD research. 698 The American Journal of Human Genetics 100, 695–705, May 4, 2017 Figure 3. Map of the IRDiRC The IRDiRC was formally launched in 2011 and currently includes member institutions from Asia, the Middle East, Australasia, Europe, and North America. The current cumula- tive commitment from the 42 member institutions from both the public and private sec- tors is estimated at more than $2,000,000,000 USD. The International Rare Diseases Research Consortium The current cumula- tive commitment from the 42 member institutions from both the public and private sec- tors is estimated at more than $2,000,000,000 USD. bility between the rare-disease nosol- ogies ORDO and OMIM, both of which are recognized for rare-disease classification. bility between the rare-disease nosol- ogies ORDO and OMIM, both of which are recognized for rare-disease classification. hierarchical rare-disease classification and coding (Orpha numbers) scheme by Orphanet, which will become the basis for inclusion of the majority of known rare diseases into ICD. Orpha numbers are now increasingly used by European healthcare systems for informatics tracing of RGDs, and their introduction is fostered by National Action Plans and Strategies for Rare Diseases and recommended by the Eu- ropean Commission expert group on rare diseases.22 Standards, Tools, and Resources to Facilitate Genomic Data Analyses Our ability to analyze, annotate, and ultimately share genomic datasets is fundamental to the RGD research agenda. Currently, tools and methods for analysis and annotation are not standardized and lack interopera- bility; as a result, the sharing of out- puts from large genomic datasets is hampered. Pipelines for analyzing DNA sequences still have much room for improvement in terms of sequence alignment, variant calling, and functional annotation and pre- diction, especially for more complex variation such as insertions, deletions, and the wide spectrum of structural variants,23 calling for a harmonized approach. This observation is sup- ported by recent data suggesting that the limited yield of WES as re- ported in the literature, at least in the context of certain recessive dis- eases, is mostly accounted for by our limited ability to correctly call vari- ants.24 An example of such a platform has been developed by the RD-Con- nect EU project for research and diag- OMIM has also played a central role in the naming and classification of Mendelian diseases by defining recog- nizable patterns of features and highlighting those that allow one condition to be distinguished from another. In general, OMIM creates separate phenotype entries on the ba- sis of molecular etiology, that is, ge- netic heterogeneity. OMIM’s clinical synopsis for each phenotype includes only those features that have been reported in individuals with muta- tions in the disease-associated gene. Each OMIM phenotype is assigned a unique and stable identifier (MIM number) that is used in the aforemen- tioned databases and in the biomed- ical literature. The American Journal of Human Genetics 100, 695–705, May 4, 2017 699 The International Rare Diseases Research Consortium nosis, together with the EURenOmics and NeurOmics RGD research projects. Furthermore, existing tools will need to be made interoperable and widely adopted, and their curation and up- dates should be duly coordinated. Genomic data analyses for RGD dis- covery are also challenged by the identification of rare variants to be prioritized for further interpretation. Investigators studying the causes of RGDs are relying heavily on WES datasets compiled by consortia, such as the Exome Aggregation Con- sortium (ExAC; 60,000 exomes) and the NHLBI Exome Sequencing Project (ESP; 6,500 exomes), that investigate different diseases as reference datasets for analyses, and this is proving useful in decreasing the number of variants to a manageable number for certain populations. However, many of these first comparative exome datasets have been generated from popula- tions of Western European and North American origin. This limits patho- genic variant discovery, especially from populations that have been sparsely assessed, if sampled at all. The 1000 Genomes Project has made significant contributions to our un- derstanding of the architecture of the human genome as a large hete- rogeneous population dataset. Most recently, gnomAD has aggregated 15,000 genomes and 120,000 exomes, including data from the 1000 Ge- nomes Project and the ExAC and ESP exome datasets. Increasing such pop- ulation datasets and generating and sharing datasets from populations with little to no representation in ex- isting repositories that can be used by the RGD research community, as well as others investigating human health, will be of great importance in the future. The Global Alliance for Genomics and Health (GA4GH) is active in this space and is committed to enabling responsible and effective sharing of genomic and clinical data through a federated ecosystem approach; we support these efforts and their application to RGDs.25 For example, the Beacon Network, a demonstration project of GA4GH, is a global search engine for genetic Figure 3. Map of the IRDiRC The IRDiRC was formally launched in 2011 and currently includes member institutions from Asia, the Middle East, Australasia, Europe, and North America. The current cumula- tive commitment from the 42 member institutions from both the public and private sec- tors is estimated at more than $2,000,000,000 USD. Figure 3. Map of the IRDiRC Figure 3. Map of the IRDiRC The IRDiRC was formally launched in 2011 and currently includes member institutions from Asia, the Middle East, Australasia, Europe, and North America. The International Rare Diseases Research Consortium The IRDiRC strongly supports the continued interopera- The American Journal of Human Genetics 100, 695–705, May 4, 2017 699 Access Matrix (ADA-M), seek to enable systematized representation of con- sent-, legal-, and institutional-based permissions and restrictions associ- ated with research and clinical records to facilitate streamlined and appro- priate discovery, sharing, and use of extant datasets. This will also help to better standardize consent-form clauses, thereby guiding best practices in both research and ethics review committees. Just as consent practices need to become interoperable so as to enable greater data sharing, so too do data-access mechanisms. Efforts are currently underway to produce a new model that would facilitate data access (registered access) and use inter- actions with initiatives such as MME by authorizing users through a stan- dard online authentication and attes- tation process. Registered access will address different categories of poten- tial data users (researchers, clinical care professionals, and patients), as well as different levels of data depend- ing on their identifiability and sen- sitivity. Additional IRDiRC-GA4GH collaboration is underway to develop a privacy-preserving linkage system that would link data from the same individual across multiple projects while also respecting privacy. Policy for recognizing ethics review to en- courage streamlined and coherent ethics review for international pro- jects and consortia is also available. Over time, such efforts will harmonize local ethical, legal, and social policies and procedures for efficient and responsible international sharing and analysis of genomic and clinical data. Genetic Evidence to Support Gene Discovery Reports from several large-scale col- Table 1. The International Rare Diseases Research Consortium Factors Contributing to Bottlenecks in the Gene-Discovery Pipeline Clinical data d non-specific clinical presentations (e.g., developmental delay and hypotonia) d ultra-rare and unrecognized genetic diseases d lack of ontology encompassing the complete spectrum of human phenotypes d insufficient utilization of ontologies or 3D facial-gestalt analysis in phenotyping d inconsistent multidisciplinary approaches to patient evaluation d inability to account for and compare age-specific disease presen- tations Genomic data d technical limitations of WES (e.g., copy-number variants and structural variation are not captured well) d lack of standardized technical and informatics approaches d incompleteness of population-specific control datasets Data discovery and sharing d lack of a widely adopted data-sharing framework d lack of common data-sharing standards d lack of a systematic way to record data-use conditions d lack of a privacy-preserving linkage system for each research participant Genetic evidence d siloed datasets d lack of and use of data-sharing infrastructure Functional evidence d lack of standardized and moderate-throughput analyses of variant impact d lack of biological insight into the function of most human genes Novel disease mechanisms d lack of expertise in the analysis of non-coding variants d other mechanisms including tissue-specific mosaicism, methyl- ation, and di- or oligogenic inheritance Table 1. Factors Contributing to Bottlenecks in the Gene-Discovery Pipeline everyone has a right ‘‘to share in scien- tific advancement and its benefits’’ and ‘‘to the protection of the moral and material interests resulting from any scientific . production of which [a person] is the author.’’28 Recently, rec- ommendations and models for ‘‘Data Transfer Agreements’’ have been pub- lished with the ‘‘IRDiRC recognized’’ label.29 variation and connects 60 databases representing every inhabited conti- nent, enabling global discovery of ge- netic variation. Ethical Standards to Enable Data Dis- covery and Sharing 700 The American Journal of Human Genetics 100, 695–705, May 4, 2017 Ethical Standards to Enable Data Dis- covery and Sharing The launch of the MME is a major step forward, and currently PhenomeCentral,12 GeneMatcher,41 DECIPHER,13 MyGene2,54 matchbox, and Patient Archive, representing data from more than 20,000 unrelated RGD patients, are connected to one another. However, truly optimizing this type of case-based matching and enable RGD discovery on a global scale will require improvement of in- ternational data sharing, optimiza- tion, financial support, and scaling up of such infrastructure, operating procedures, and algorithms. disease-related genes in over 9,000 cases from various clinical diagnostic settings indicate an overall success rate of 30%.35–39 These latter co- horts have demonstrated that a sub- stantial fraction (25%–30%) of clin- ical diagnostic success depends on recent progress in the discovery of genes underlying disease. This obser- vation in combination with the higher solve rate in the research setting suggests that the unsolved fractions of these clinical cohorts contain many discoveries. combined with other -omics data and standardized phenotypes.55 Such initiatives need to be increased in number and made sustainable. Model Systems to Facilitate Gene Discovery. Model-systems research (in humans, yeast, flies, worms, zebrafish, mice, and other organisms) will continue to be critical in determining the functional consequences of geno- mic variants in candidate disease- related genes and in discovering and validating new drug targets, candidate drugs, and other therapeutic strate- gies. The pace of allele discovery is outstripping our ability to understand the biological consequences of indi- vidual mutations on gene, pathway, and network function. There is an opportunity for the next generation of disease modeling to address this gap in an efficient, cost-effective, and generalizable manner with higher throughput. Improved infrastructure is required for (1) allowing clinician scientists who have discovered a dis- ease-causing variant to be exposed to the full range of experimental tools available to them, (2) allowing experts in a variety of model organisms to apply their skills on pertinent ques- tions of biological and clinical inter- est, and (3) creating efficiencies so that studies are not duplicated and existing models are utilized to their full potential. Linking clinician scien- tists and basic researchers early and providing seed funds for collaborative experiments would be the ultimate goals of such an effort. y Case-Based Matching for Gene Discovery. The discovery of disease- gene associations requires confirma- tion of pathogenic genomic variation in multiple unrelated individuals affected by the same rare disease. Ethical Standards to Enable Data Dis- covery and Sharing The RGD research community is acutely and universally aware of the need for data discovery and sharing.26 Given the challenge ahead of us to un- derstand and be able to diagnose RGDs of ever increasing rarity, the ability to share clinical and genetic data maxi- mally has become of central impor- tance. In this regard, the IRDiRC is collaborating with the Human Vari- ome Project (HVP) and GA4GH to tackle major ethical, legal, and social issues and agree on standards for international data to break down exist- ing hurdles. The IRDiRC has recog- nized the Framework for Responsible Sharing of Genomic and Health- Related Data27 as a resource on the basis of international adherence to Article 27 of the UN Declaration of Human Rights, which holds that The RGD research community is acutely and universally aware of the need for data discovery and sharing.26 The IRDIRC-HVP-GA4GH collabo- ration is paving the way for interna- tional recognition of common data- sharing standards. Several critical areas of data-sharing governance are currently the focus of collaborative efforts. First, the collaboration devel- oped a ‘‘tiered’’ consent policy that is dependent on the context of data collection and use (clinical or research) and on the level of risk that the shared data will be identified; this policy is currently in use by the Matchmaker Exchange30,31(MME; see below). Two related initiatives, namely the Consent Codes32 model and the Automatable Discovery and Reports from several large-scale col- laborative research initiatives, in- cluding the FORGE Canada Con- sortium,33 US Centers for Mendelian Genomics,8 and UK Deciphering of Developmental Disorders study,34 indicate that under very select circumstances (including ascertain- ment of multiple, thoroughly pheno- typed families with the same condi- tion), the ‘‘solve rate’’ for RGDs is often >50%. Reports focusing on disease-causing variants in known 700 The American Journal of Human Genetics 100, 695–705, May 4, 2017 typic profiles through a standardized application programming interface (API) and standard operating proced- ures.40 The MME enables searches of multiple databases at once, circum- venting the need to separately search all services by depositing data in each one. Under this initial API, each server can treat any description arbitrarily: the level of similarity required (on either the genotype or phenotype level) before a match is triggered is left to the discretion of each service. The American Journal of Human Genetics 100, 695–705, May 4, 2017 701 Ethical Standards to Enable Data Dis- covery and Sharing Our collective experience suggests that it takes approximately 2–3 years to identify an additional unrelated in- dividual with likely pathogenic muta- tions in the same gene after publica- tion of a single patient or family. Thus, a central challenge is to effi- ciently identify additional and unre- lated persons with pathogenic vari- ant(s) in the same gene and an overlapping phenotype. It is difficult to gauge the number of such single surviving candidate genes (containing deleterious-appearing genetic varia- tion that remains after multiple fil- tering steps with segregation data and pathway and/or model-organism support from existing literature) that remain unpublished and/or in inac- cessible ‘‘silos’’ worldwide, but we esti- mate it to be more than 1,000. Critical Next Steps Achieving the IRDiRC’s goal of a means of diagnosing all RGDs will require the discovery of the genetic mechanism underlying every dis- order. This challenge—producing a complete catalog of the phenotypic characteristics of all RGDs and their corresponding causal variants, devel- oping successful approaches to dis- covering the underlying etiology of RGDs caused by non-traditional modes of inheritance, and establish- ing tools and resources to translate this new knowledge into patient care (e.g., harmonization and adoption of international guidelines for the clin- ical application of NGS-based ap- proaches)—is significant. This grand challenge can be achieved only with significant international cooperation and engagement of all relevant stake- holders at a scale the community has never seen before. Efforts to engage the research community, such as the IRDiRC and GA4GH, are of critical importance, and international coordi- nation and funding of activities will be necessary. Improving translation and reimbursement strategies for clin- ical genome-wide analysis of patients with rare diseases will be essential; this is particularly important for avoiding the large number of patho- genic variants identified in known genes in research projects focused on discovery and reallocating research funding to the generation and valida- tion of novel insights. Engaging clin- ical laboratories, researchers, and the patient community to share their data will be critical. W l i h It will also be important to stimu- late the establishment and validation of novel phenotyping pipelines that have correlates in other organisms by emphasizing disease relevance, patho- physiological pathways, and high efficiency. This will accelerate the evaluation of genomic variants and candidate genes, drug and drug-target testing using disease-relevant output measures, and fundamental under- standing of disease mechanisms and pathologies. Phenotyping pipelines can, in some cases, assess disease traits that resemble hallmarks of the human disorder in an obvious manner (e.g., malformations, behaviorial features, or other findings). If sufficiently specific (i.e., unique), such pheno- types can validate the relevance of a disease model. The Monarch Initiative has been working in this realm since 2009 and acts as an integrative data and analytic platform that connects phenotypes and genotypes across spe- cies. Functional Evidence to Support Gene Discovery Functional Evidence to Support Gene Discovery Integration of Genomic Data into Sys- tems Biology. Parallel to the enormous advances in gene identification through WES, other large-scale -omics approaches have been developed (e.g., proteomics, transcriptomics, and me- tabolomics) to aid RGD discovery and facilitate the validation of vari- ants of unknown significance. For instance, changes in protein levels or function help to identify the disease- causing variant if more than one plausible gene has been identified through WES. Data integration across different -omics datasets on popula- tion or individual patient levels will also be required for understanding the importance of disease-modifying variants in conditions with high phenotypic variability or incomplete penetrance and for assisting the development of diagnostics and ther- apeutic biomarkers and will play an increasing role in developing targeted therapies. For example, RD-Connect is establishing a platform where geno- mic data on rare disease patients are g One approach to accelerating col- laborations between clinicians and basic researchers is to proactively iden- tify collaborative ‘‘matches’’ and to provide seed funding to ignite col- laborative research projects. In Can- ada, a national infrastructure, the ‘‘Rare Diseases: Models and Mecha- nisms’’ network, has been established to link clinicians and basic researchers as soon as disease-related genes are discovered.56 The network is in its sec- ond year of its 3 year funding cycle and has been successful in catalyzing collaborative links for over 40 clinician and basic-scientist matches. An alterna- tive approach is through an ‘‘enabling’’ To address this challenge, several collaborative initiatives have devel- oped platforms for genotype- and phenotype-driven matching algo- rithms12,13,40–52; however, a connec- tion between these existing solutions has been lacking. Very recently, the IRDiRC Diagnostics Scientific Com- mittee, in collaboration with each participating data-sharing service, Can-SHARE, and the GA4GH, has contributed to launching a federated platform termed the MME.53 This platform facilitates the identification of unsolved patients and families with similar phenotypic and geno- The American Journal of Human Genetics 100, 695–705, May 4, 2017 701 scheme, in which national funding agencies allow investigators to jointly apply for supplemental funding to existing grants. 702 The American Journal of Human Genetics 100, 695–705, May 4, 2017 Novel Disease Mechanisms Novel Disease Mechanisms Progress toward the discovery of the genetic basis of every RGD has been substantial over the past several years. Yet, there remain a non-trivial num- ber of well-known rare diseases (e.g., Hallerman-Streiff syndrome, Dubo- witz syndrome, VACTERL, Gomez-Lo- pez-Hernandez syndrome, Aicardi syndrome, and PHACE syndrome) for which, despite multiple groups’ ef- forts to use WES and, in some cases, WGS, the causal genetic mechanism remains elusive. The reasons that such discovery efforts fail are myriad and most likely include both tech- nical limitations (e.g., annotation er- rors, missed coding and non-coding variation, and structural variation) and complex biology (e.g., extreme lo- cus heterogeneity, tissue-specific so- matic mosaicism, unusual modes of inheritance, intrafamilial allelic or lo- cus heterogeneity, and causal synony- mous variants). Approaches that over- come these barriers to RGD discovery are few in number. Moreover, the rare genetic conditions for which the genetic mechanism has yet to be identified are likely enriched with those that will not be solved easily by existing WES-based approaches. Identifying the molecular basis of conditions intractable to existing ap- proaches requires broader and in- novative application of existing dis- covery strategies (e.g., WGS, RNA sequencing of affected cells or tissues, and deep sequencing of tissues deri- ved from the three major embryonic lineages); improvement of compu- tational and statistical models for variant identification, annotation, functional prediction, and prioritiza- tion—particularly for variants in non-coding regions;59 and develop- ment of strategies for discovering causal genetic mechanisms. Also, temporally focused, multidisciplinary assessments that take advantage of cumulative expert clinician experience and preci- sion phenotyping centered around sin- Functional Evidence to Support Gene Discovery In the US, for example, administrative supplements to ‘‘R’’ and ‘‘P’’ grants are not uncommon; indeed, this model has been used by the NIH Undiagnosed Disease Program to seed research on candidate genes discovered by that effort.57 An inte- grated international virtual network allowing clinician scientists to discover relevant researchers might also be a complementary and intermediate approach. such as the validation of alleles and disease-related genes, at a scale that is urgently required in the post- genome-sequence era. gle patients, such as the Undiagnosed Diseases Network International,60 are part of a suite of approaches to support- ing the discovery of rare-disease mecha- nisms. The development and applica- tion of these strategies will further leverage investments that support genetic and functional approaches for the discovery of underlying genetic mechanisms. Critical Next Steps Alternatively, phenotyping pipe- lines can assess traits that are not linked to the disease of interest in an obvious manner but that do result from the same molecular defects un- derlying the disease phenotype in hu- mans and thus represent orthologous phenotypes (‘‘phenologs’’).58 In addi- tion, it will be important to develop and validate novel efficient and dis- ease-relevant test paradigms and phe- notypes that can be cross-compared between species (parallel phenotyp- ing). Such validated disease-relevant phenotypes across organisms could provide the required output measures for overcoming current bottlenecks, We must also recognize that as more and more genes are discovered to be associated with human disease and appropriate analytical tests are 702 The American Journal of Human Genetics 100, 695–705, May 4, 2017 of Washington Center for Mendelian Genomics though the National Human Genome Research Institute (NHGRI) and National Heart, Lung, and Blood Insti- tute (grant U54HG006493) to Drs. Debbie Nickerson, Michael Bamshad, and Su- zanne Leal. A.H. was supported by OMIM through NHGRI grant 1U41HG006627 and the Baylor-Hopkins Center for Mende- lian Genomics through NHGRI grant 1U54HG006542 to Drs. David Valle and James Lupski. M.M. was supported by funding from 00064203, 16-30880A, 15- 34904A, OPPK CZ.2.16/3.1.00/24022, and NF-CZ11-PDP-3-003-2014. H.L.R. was supported by UM1HG008900. H.L. was supported by funding from the Medical Research Council (reference G1002274, grant 98482), the Wellcome Trust (refer- ence 201064/Z/16/Z), and the European Union Seventh Framework Programme (FP7/2007-2013) under grants 305444 (RD-Connect) and 305121 (NeurOmics). Achieving a means of diagnosing all RGDs will be of great importance for patients and families. It will allow ge- netic counseling, better prognostica- tion, and identification of specific health risks to the individual and will prevent unnecessary or harmful diag- nostic interventions and treatments. Web Resources 1000 Genomes, http://www.1000genomes. org 1000 Genomes, http://www.1000genomes. org Can-SHARE, http://www.p3g.org/resources/ can-share ClinGen, https://www.clinicalgenome.org ClinVar, http://www.ncbi.nlm.nih.gov/ clinvar DECIPHER, https://decipher.sanger.ac.uk EURenOmics, http://eurenomics.eu Critical Next Steps Ultimately, such insights can be applied to genome-wide sequencing in newborns for both diagnosis and screening.64 In an increasing number of patients, effective drug treatment is available once the exact diagnosis (e.g., lysosomal-storage disorders or congenital myasthenic syndromes) has been established.65 In addition, this aim will allow more patients to participate in research cohorts for clinical trials that require a definite molecular and phenotypic diagnosis, providing potential benefit from new drugs or interventions being devel- oped by academia and the private sector.66 In our view, the understand- ing of all RGDs will be the cornerstone of precision medicine; the power of genomics to explain these rare dis- eases with concomitant fundamental insights into biological processes will rapidly transform medical care for these patients and their families. established, a significant challenge in RGD diagnosis will remain: that of in- terpreting a growing numbers of vari- ants of uncertain significance. DNA diagnostics for RGD is primarily based on shared knowledge about genes, genomic variation, and phenotypes. Currently, diagnostic data are col- lected through a multitude of ap- proaches by many different diag- nostic laboratories and are stored in a wide variety of server systems and databases, which generally lack feder- ated connections, i.e., ‘‘silos.’’ Local solutions need to be developed and implemented for storing data on ge- netic variants and their associated phenotypes in an easy and reproduc- ible way with common standards and terminologies. In addition, these local systems need to be connected worldwide to form a ‘‘genetics knowl- edge web.’’ Making this type of sharing part of the normal standard of care will require community engagement. Inte- grating existing platforms that store clinical genetic and phenotype data (e.g., ClinVar,61 Leiden Open Variation Database [LOVD],62 and DECIPHER13), linking different types of data (e.g., array and sequencing), and encom- passing small (single-nucleotide) to large (deletion, duplication, inversion, etc.) variants will be essential. These challenges are further compounded by the rate and impact of false-positive causative variant assignments63 that exist in such databases, so ultimately the curation of this knowledge by rele- vant experts will be the key to diag- nostic precision. The American Journal of Human Genetics 100, 695–705, May 4, 2017 703 Critical Next Steps Variant classification as pathogenic or benign will rely heavily on the same tools that are critically needed for RGD discovery, specifically the availability of popula- tion-specific disease and control data- bases for a diverse range of popula- tions, the use of orthogonal assays such as metabolomics, transcriptom- ics, or proteomics to clarify func- tional effect, and the systematic screening of mutations in disease- related genes in tractable models or cell systems. Clearly, the task of as- signing pathogenicity to individual variants is mission critical to infor- med patient care Acknowledgments Exome Aggregation Consortium (ExAC) We thank the present and former staff of the IRDiRC Scientific Secretariat, Se´gole`ne Ayme´, Lilian Lau, Anneliene Jonker, Anto- nia Mills, Barbara Cagniard, and members of the scientific committees, working groups, and task forces. We also thank the former chairs of the IRDiRC Therapies Sci- entific Committee (Yann Le Cam and Josep Torrent i Farnell) for helpful discussions. We thank Rachel Thompson, Emma He- slop, Victoria Hedley, and Lena Dolman for their support. Orphanet was supported in part by funding from AFM-Te´le´thon and the Joint action ‘‘677024/RD-ACTION,’’ which received funding from the European Union’s Health Programme (2014-2020). T.H. and M.R.V. were supported by the Care4Rare Canada Consortium, funded by Genome Canada, the Canadian Insti- tutes of Health Research, the Ontario Ge- nomics Institute, Ontario Research Fund, Genome Quebec, and Children’s Hospital of Eastern Ontario Foundation. J.X.C. and M.J.B. were supported by the University Browser, http://exac.broadinstitute.org GeneMatcher, https://genematcher.org Genomics England, https://www.genomics england.co.uk Genomics England, https://www.genomics england.co.uk Genotype to Mendelian Phenotype (Geno2MP), http://geno2mp.gs.washing ton.edu Genotype to Mendelian Phenotype (Geno2MP), http://geno2mp.gs.washing ton.edu Global Alliance for Genomics and Health (GA4GH), http://genomicsandhealth.org Global Alliance for Genomics and Health (GA4GH), http://genomicsandhealth.org gnomAD, http://gnomAD.broadinstitute. org gnomAD, http://gnomAD.broadinstitute. org Human Phenotype Ontology (HPO), http:// www.human-phenotype-ontology.org Human Phenotype Ontology (HPO), http:// www.human-phenotype-ontology.org Human Variome Project, http://www. humanvariomeproject.org/ International Consortium of Human Phenotype Ontologies (ICHPT), http:// www.irdirc.org/ichpt International Rare Diseases Research Con- sortium (IRDiRC), http://www.irdirc.org Leiden Open Variation Database (LOVD), http://www lovd nl/3 0/home International Rare Diseases Research Con- sortium (IRDiRC), http://www.irdirc.org Leiden Open Variation Database (LOVD), http://www.lovd.nl/3.0/home Matchbox, https://seqr.broadinstitute. Matchbox, https://seqr.broadinstitute. org The American Journal of Human Genetics 100, 695–705, May 4, 2017 703 Matchmaker Exchange (MME), http:// www.matchmakerexchange.org 18. Ko¨hler, S., Vasilevsky, N.A., Engelstad, M., Foster, E., McMurry, J., Ayme´, S., Baynam, G., Bello, S.M., Boerkoel, C.F., Boycott, K.M., et al. (2017). Nu- cleic Acids Res. 45 (D1), D865–D876. Griese, M., McKone, E.F., Wainwright, C.E., Konstan, M.W., et al.; VX08- 770-102 Study Group (2011). N. Engl. J. Med. 365, 1663–1672. Medical Subject Headings (MeSH), http:// www.ncbi.nlm.nih.gov/mesh Medical Dictionary for Regulatory Activ- ities (MedDRA), https://www.meddra.org 6. Rath, A., Olry, A., Dhombres, F., Brandt, M.M., Urbero, B., and Ayme, S. (2012). Hum. Mutat. 33, 803–808. 19. Hamosh, A., Sobreira, N., Hoover- Fong, J., Sutton, V.R., Boehm, C., Schiettecatte, F., and Valle, D. (2013). Hum. Mutat. 34, 566–571. ities (MedDRA), https://www.meddra.org Monarch Initiative, https://monarch initiative.org Monarch Initiative, https://monarch initiative.org 7. UK10K, http://www.uk10k.org 24. Shamseldin, H.E., Maddirevula, S., Faqeih, E., Ibrahim, N., Hashem, M., Shaheen, R., and Alkuraya, F.S. (2016). Genet. Med. Published October 6, 2016. http://dx.doi.org/10. 1038/gim.2016.155. 704 The American Journal of Human Genetics 100, 695–705, May 4, 2017 Acknowledgments Rehm, H.L., Berg, J.S., Brooks, L.D., Bustamante, C.D., Evans, J.P., Land- rum, M.J., Ledbetter, D.H., Maglott, D.R., Martin, C.L., Nussbaum, R.L., et al.; ClinGen (2015). N. Engl. J. Med. 372, 2235–2242. MyGene2, http://mygene2.org 20. Allanson, J.E., Biesecker, L.G., Carey, J.C., and Hennekam, R.C. (2009). Am. J. Med. Genet. A. 149A, 2–5. NeurOmics, http://rd-neuromics.eu NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs. washington.edu/EVS 21. Vasant, D., Chanas, L., Malone, J., Ha- nauer, M., Olry, A., Jupp, S., Robinson, P.N., Parkinson, H., and Rath, A. (2014). ORDO: An Ontology Connect- ing Rare Disease, Epidemiology and Genetic Data. Proceedings of the 22nd Annual International Conference on Intelligent Systems for Molecular Biology. https://www.researchgate.net/ publication/281824026_ORDO_An_ Ontology_Connecting_Rare_Disease_ Epidemiology_and_Genetic_Data. 8. Chong, J.X., Buckingham, K.J., Jhan- giani, S.N., Boehm, C., Sobreira, N., Smith, J.D., Harrell, T.M., McMillin, M.J., Wiszniewski, W., Gambin, T., et al.; Centers for Mendelian Geno- mics (2015). Am. J. Hum. Genet. 97, 199–215. OMIM, http://omim.org Orphanet, http://www.orpha.net Orphanet Rare Disease Ontology, http:// bioportal.bioontology.org/ontologies/ ORDO Orphanet RD-Action, http://www.rd- action.eu Patient Archive, http://patientarchive.org 9. Lochmu¨ller, H., Le Cam, Y., Jonker, A.H., Lau, L.P., Baynam, G., Kaufmann, P., Lasko, P., Dawkins, H.J., Austin, C.P., and Boycott, K.M. (2017). Eur. J. Hum. Genet. 25, 162–165. Patient Archive, http://patientarchive.org PhenoDB, https://phenodb.org PhenoDB, https://phenodb.org PhenomeCentral, https://www.phenome central.org POSSUM, http://www.possum.net.au 22. European Commission (2009) Na- tional plans or strategies for rare dis- eases. http://ec.europa.eu/health/rare_ diseases/national_plans/detailed_en. 10. Ko¨hler, S., Doelken, S.C., Mungall, C.J., Bauer, S., Firth, H.V., Bailleul-For- estier, I., Black, G.C., Brown, D.L., Brudno, M., Campbell, J., et al. (2014). Nucleic Acids Res. 42, D966– D974. Rare Diseases Models and Mechanisms Network (RDMM), http://rare-diseases- catalyst-network.ca 23. Alioto, T.S., Buchhalter, I., Derdak, S., Hutter, B., Eldridge, M.D., Hovig, E., Heisler, L.E., Beck, T.A., Simpson, J.T., Tonon, L., et al. (2015). Nat. Commun. 6, 10001. RD-Connect, https://platform.rd-connect.eu S O C h // h d / SNOMED CT, http://www.ihtsdo.org/ snomed-ct SNOMED CT, http://www.ihtsdo.org/ snomed-ct 11. Robinson, P.N., Ko¨hler, S., Bauer, S., Seelow, D., Horn, D., and Mundlos, S. (2008). Am. J. Hum. Genet. 83, 610–615. References Harrison, S.M., Riggs, E.R., Maglott, D.R., Lee, J.M., Azzariti, D.R., Nie- haus, A., Ramos, E.M., Martin, C.L., Landrum, M.J., and Rehm, H.L. (2016). Curr. Protoc. Hum. Genet. 89, 8.16.1–8.16.23. 49. Akle, S., Chun, S., Jordan, D.M., and Cassa, C.A. (2015). Hum. Mutat. 36, 998–1003. 36. Lee, H., Deignan, J.L., Dorrani, N., Strom, S.P., Kantarci, S., Quintero-Riv- era, F., Das, K., Toy, T., Harry, B., Your- shaw, M., et al. (2014). JAMA 312, 1880–1887. 50. Jurgens, J., Sobreira, N., Modaff, P., Reiser, C.A., Seo, S.H., Seong, M.W., Park, S.S., Kim, O.H., Cho, T.J., and Pauli, R.M. (2015). Hum. Mutat. 36, 1004–1008. 62. Fokkema, I.F., Taschner, P.E., Schaafsma, G.C., Celli, J., Laros, J.F., and den Dunnen, J.T. (2011). Hum. Mutat. 32, 557–563. 37. Saudi Mendeliome Group (2015). Genome Biol. 16, 134. 51. Au, P.Y., You, J., Caluseriu, O., Schwart- zentruber, J., Majewski, J., Bernier, F.P., Ferguson, M., Valle, D., Parboosingh, J.S., Sobreira, N., et al. (2015). Hum. Mutat. 36, 1009–1014. 63. Dorschner, M.O., Amendola, L.M., Turner, E.H., Robertson, P.D., Shirts, B.H., Gallego, C.J., Bennett, R.L., Jones, K.L., Tokita, M.J., Bennett, J.T., et al.; National Heart, Lung, and Blood Institute Grand Opportunity Exome Sequencing Project (2013). Am. J. Hum. Genet. 93, 631–640. 38. Farwell, K.D., Shahmirzadi, L., El-Khe- chen, D., Powis, Z., Chao, E.C., Tippin Davis, B., Baxter, R.M., Zeng, W., Mroske, C., Parra, M.C., et al. (2015). Genet. Med. 17, 578–586. 52. Loucks, C.M., Parboosingh, J.S., Sha- heen, R., Bernier, F.P., McLeod, D.R., Seid- ahmed, M.Z., Puffenberger, E.G., Ober, C., Hegele, R.A., Boycott, K.M., et al. (2015). Hum. Mutat. 36, 1015–1019. 39. Retterer, K., Juusola, J., Cho, M.T., Vi- tazka, P., Millan, F., Gibellini, F., Ver- tino-Bell, A., Smaoui, N., Neidich, J., Monaghan, K.G., et al. (2016). Genet. Med. 18, 696–704. 64. Berg, J.S., Agrawal, P.B., Bailey, D.B. Jr., Beggs, A.H., Brenner, S.E., Brower, A.M., Cakici, J.A., Ceyhan-Birsoy, O., Chan, K., Chen, F., et al. (2017). Pediat- rics 139, e20162252. 53. Philippakis, A.A., Azzariti, D.R., Bel- tran, S., Brookes, A.J., Brownstein, C.A., Brudno, M., Brunner, H.G., Buske, O.J., Carey, K., Doll, C., et al. (2015). Hum. Mutat. 36, 915–921. 40. Buske, O.J., Schiettecatte, F., Hutton, B., Dumitriu, S., Misyura, A., Huang, L., Hartley, T., Girdea, M., Sobreira, N., Mungall, C., and Brudno, M. (2015). Hum. Mutat. 36, 922–927. 65. Evangelista, T., Hanna, M., and Loch- mu¨ller, H. (2015). J Neuromuscul Dis 2 (Suppl 2), S21–S29. 54. References M., Boycott, K.M., Philippakis, A., and Rehm, H. (2017). Hum. Mutat., in press. Hansson, M.G., ’t Hoen, P.B., Patrinos, G.P., Dawkins, H., et al. (2014). J. Gen. Intern. Med. 29 (Suppl 3), S780–S787. 43. Lancaster, O., Beck, T., Atlan, D., Swertz, M., Thangavelu, D., Veal, C., Dalgleish, R., and Brookes, A.J. (2015). Hum. Mutat. 36, 957–964. 32. Dyke, S.O., Philippakis, A.A., Rambla De Argila, J., Paltoo, D.N., Luetkeme- ier, E.S., Knoppers, B.M., Brookes, A.J., Spalding, J.D., Thompson, M., Roos, M., et al. (2016). PLoS Genet. 12, e1005772. 56. Foley, K.E. (2015). Nat. Med. 21, 1242– 1243. 44. Lambertson, K.F., Damiani, S.A., Might, M., Shelton, R., and Terry, S.F. (2015). Hum. Mutat. 36, 965–973. 57. Gahl, W.A., Wise, A.L., and Ashley, E.A. (2015). JAMA 314, 1797–1798. 58. McGary, K.L., Park, T.J., Woods, J.O., Cha, H.J., Wallingford, J.B., and Mar- cotte, E.M. (2010). Proc. Natl. Acad. Sci. USA 107, 6544–6549. 45. Kirkpatrick, B.E., Riggs, E.R., Azzariti, D.R., Miller, V.R., Ledbetter, D.H., Miller, D.T., Rehm, H., Martin, C.L., Faucett, W.A.; and ClinGen Resource (2015). Hum. Mutat. 36, 974–978. 33. Beaulieu,C.L.,Majewski,J.,Schwartzen- truber, J., Samuels, M.E., Fernandez, B.A.,Bernier,F.P.,Brudno,M.,Knoppers, B., Marcadier, J., Dyment, D., et al.; FORGE Canada Consortium (2014). Am. J. Hum. Genet. 94, 809–817. 59. Smedley, D., Schubach, M., Jacobsen, J.O., Ko¨hler, S., Zemojtel, T., Spiel- mann, M., Ja¨ger, M., Hochheiser, H., Washington, N.L., McMurry, J.A., et al. (2016). Am. J. Hum. Genet. 99, 595–606. 46. Mungall, C.J., Washington, N.L., Nguyen-Xuan, J., Condit, C., Smedley, D., Ko¨hler, S., Groza, T., Shefchek, K., Hochheiser, H., Robinson, P.N., et al. (2015). Hum. Mutat. 36, 979–984. 34. Wright, C.F., Fitzgerald, T.W., Jones, W.D., Clayton, S., McRae, J.F., van Ko- gelenberg, M., King, D.A., Ambridge, K., Barrett, D.M., Bayzetinova, T., et al.; DDD study (2015). Lancet 385, 1305–1314. 60. Taruscio, D., Groft, S.C., Cederroth, H., Melegh, B., Lasko, P., Kosaki, K., Baynam, G., McCray, A., and Gahl, W.A. (2015). Mol. Genet. Metab. 116, 223–225. 47. Brownstein, C.A., Holm, I.A., Ramoni, R., Goldstein, D.B.; and Members of the Undiagnosed Diseases Network (2015). Hum. Mutat. 36, 985–988. 35. Yang, Y., Muzny, D.M., Xia, F., Niu, Z., Person, R., Ding, Y., Ward, P., Braxton, A., Wang, M., Buhay, C., et al. (2014). JAMA 312, 1870–1879. 48. Krawitz, P., Buske, O., Zhu, N., Brudno, M., and Robinson, P.N. (2015). Hum. Mutat. 36, 989–997. 61. The American Journal of Human Genetics 100, 695–705, May 4, 2017 705 References 12. Buske, O.J., Girdea, M., Dumitriu, S., Gallinger, B., Hartley, T., Trang, H., Misyura, A., Friedman, T., Beaulieu, C., Bone, W.P., et al. (2015). Hum. Mu- tat. 36, 931–940. 1. United States Congress. (2002). Rare Diseases Act of 2002. https://www. gpo.gov/fdsys/pkg/PLAW-107publ280/ html/PLAW-107publ280.htm. 25. Global Alliance for Genomics and Health (2016). Science 352, 1278– 1280. 2. The European Parliament and the Council of the European Union (1999). Regulation (EC) No 141/2000 of the European parliament and of the council of 16 December 1999 on orphan medicinal products. http://ec. europa.eu/health//sites/health/files/ files/eudralex/vol-1/reg_2000_141_ cons-2009-07/reg_2000_141_cons-2009- 07_en.pdf. 13. Chatzimichali, E.A., Brent, S., Hutton, B., Perrett, D., Wright, C.F., Bevan, A.P., Hurles, M.E., Firth, H.V., and Swa- minathan, G.J. (2015). Hum. Mutat. 36, 941–949. 26. Brookes, A.J., and Robinson, P.N. (2015). Nat. Rev. Genet. 16, 702–715. 27. Knoppers, B.M. (2014). HUGO J. 8, 3. 14. Peplow, M. (2016). BMJ 353, i1757. 28. United Nations (1948). Universal declaration of human rights. http:// www.un.org/en/universal-declaration- human-rights/index.html. 15. Ko¨hler, S., Schulz, M.H., Krawitz, P., Bauer, S., Do¨lken, S., Ott, C.E., Mund- los, C., Horn, D., Mundlos, S., and Rob- inson, P.N. (2009). Am. J. Hum. Genet. 85, 457–464. 29. Mascalzoni, D., Dove, E.S., Rubinstein, Y., Dawkins, H.J., Kole, A., McCor- mack, P., Woods, S., Riess, O., Schaefer, F., Lochmu¨ller, H., et al. (2015). Eur. J. Hum. Genet. 23, 721–728. 3. Ana Rath, ed. (2014). Prevalence and incidence of rare diseases: Biblio- graphic data. In Orphanet Report Se- ries: Rare Diseases collection. http:// www.orpha.net/orphacom/cahiers/ docs/GB/Prevalence_of_rare_diseases_ by_alphabetical_list.pdf. 16. Zemojtel, T., Ko¨hler, S., Mackenroth, L., Ja¨ger, M., Hecht, J., Krawitz, P., Graul-Neumann, L., Doelken, S., Ehmke, N., Spielmann, M., et al. (2014). Sci. Transl. Med. 6, 252ra123. 30. The Matchmaker Exchange (2016) Matchmaker Exchange Tiered Informed Consent Proposal. http://www.match makerexchange.org/assets/files/Match makerExchangeTieredInformedConsent Proposal.pdf. 17. Robinson, P.N., Ko¨hler, S., Oellrich, A., Wang, K., Mungall, C.J., Lewis, S.E., Washington, N., Bauer, S., Seelow, D., Krawitz, P., et al.; Sanger Mouse Ge- netics Project (2014). Genome Res. 24, 340–348. 4. Amberger, J.S., Bocchini, C.A., Schiet- tecatte, F., Scott, A.F., and Hamosh, A. (2015). Nucleic Acids Res. 43, D789– D798. 31. Dyke, S.O.M., Knoppers, B.M.K., Ha- mosh, A., Hurles, M., Firth, H., Brudno, 5. Ramsey, B.W., Davies, J., McElvaney, N.G., Tullis, E., Bell, S.C., Drevı´nek, P., 04 The American Journal of Human Genetics 100, 695–705, May 4, 2017 Hansson, M.G., ’t Hoen, P.B., Patrinos, G.P., Dawkins, H., et al. (2014). J. Gen. Intern. Med. 29 (Suppl 3), S780–S787. References Chong, J.X., Yu, J.H., Lorentzen, P., Park, K.M., Jamal, S.M., Tabor, H.K., Rauch, A., Saenz, M.S., Boltshauser, E., Patterson, K.E., et al. (2016). Genet. Med. 18, 788–795. 41. Sobreira, N., Schiettecatte, F., Valle, D., and Hamosh, A. (2015). Hum. Mutat. 36, 928–930. 66. Bladen, C.L., Rafferty, K., Straub, V., Monges, S., Moresco, A., Dawkins, H., Roy, A., Chamova, T., Guergueltcheva, V., Korngut, L., et al. (2013). Hum. Mu- tat. 34, 1449–1457. 42. Gonzalez, M., Falk, M.J., Gai, X., Post- rel, R., Schu¨le, R., and Zuchner, S. (2015). Hum. Mutat. 36, 950–956. 55. Thompson, R., Johnston, L., Taruscio, D., Monaco, L., Be´roud, C., Gut, I.G.,